Moloney leukemia virus-induced leukemogenesis in mice

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MOLONEY LEUKEMIA VIRUS-INDUCED LEUKEMOGENESIS IN MICE
Promotor: P r o f . D r . H.
Bloemendal
C o - r e f e r e n t : Or. Α.D.M. Berns
MOLONEY LEUKEMIA VIRUS-INDUCED LEUKEMOGENESIS IN MICE
PROEFSCHRIFT
TER VERKRIJGING VAN DE GRAAD VAN DOCTOR
IN DE WISKUNDE EN NATUURWETENSCHAPPEN
AAN DE KATHOLIEKE UNIVERSITEIT VAN NIJMEGEN
OP GEZAG VAN DE RECTOR MAGNIFICUS PROF. DR. P.G.A.B. WIJDEVELD
VOLGENS HET BESLUIT VAN HET COLLEGE VAN DECANEN
IN HET OPENBAAR TE VERDEDIGEN
OP DONDERDAG 5 NOVEMBER 1981
DES NAMIDDAGS OM 4 UUR
DOOR
PETRUS HERMANUS MARIA VAN DER PUTTEN
GEBOREN TE BERGEN (LIMBURG)
in opdracht van Willy an Pierre,
aan mijn ouders.
DANKuJOORD
Discussie en ыагк
m
Frans van der Hoorn
Chris Saris
τ
Afdeling ñedisohe
Fotografie
ìi
ls/im Quint
Fis Robanus Maandag
Eugenie Terulndt
Danny Min Raaij
Pater Bonants
Work and discussion:
Dr. Inder M, Uerma,
Salk Institute, San
Diego, U.S.A.;
Dr. Rudolf Dsehnisch
and Dr. Detlev Zehner
Henrich^ette institut
University of Hamburg, ω-G.
Ueefselkuieek;
Ар Groeneueld
Centraal
Dierenlaboratorium
F a c u l t e i t der
Geneeskunde
Kaft: Arno ВеГк
Instruinentmakerij,
Faculteit der
Geneeskunde
Leden van de
"Bossche B o l l e n "
Club
T h i s -jork 'ras c a r r i e d out a t t h e Department o f B i o c h e m i s t r y
(Head: Prní". D r . H. Bloemandel), F a c u l t y o f S c i e n c e , U n i v e r s i t y
of
Nijmegen, Nijmegen, The N e t h e r l a n d s , under the d i r e c t i o n o f D r . А.З.М.
Barns,
The Ph.D. Study of H.v.d.P. uas supported in part by the Foundation
for Medical Research (FUNGO), luhich is subsidized by the Netherlands
Organization for the Advancement of Pure Research (Ζ.ω.Ο.).
CONTENTS
ALGEMENE SACIENVATTING
SUnMARY
13
CHAPTER I
LEUKEMOGENESIS IN MICE
18
CHAPTER I I
THE INTEGRATION SITES OF ENDOGENOUS AND
62
EXOGENOUS nOLONEY MURINE LEUKEMIA VIRUS
CHAPTER I I I
MOLECULAR CLONING OF UNINTEGRATED AND A
PORTION OF INTEGRATED MOLONEY MURINE
LEUKEMIA VIRAL DNA IN BACTERIOPHAGE LAMBDA
CHAPTER I V
M-MuLV-INDUCED LEUKEMOGENESIS: INTEGRATION
80
AND STRUCTURE OF RECOMBINANT PROVIRUSES I N
TUMORS
CHAPTER I V APPENDICES
I:
II:
III:
MAPPING OF THE MS-PROBES
91
MONOCLONAL CHARACTER OF OUTGROa/N TUMORS
91
INTEGRATION SITES OF SOMATICALLY ACQUIRED
93
SEQUENCES IN PRELEUKEMIC TISSUES
I V : THE MOU-I LOCUS AND REINTEGRATION OF
94
AUTHENTIC M-MuLV GENOMES
V: SOMATICALLY ACQUIRED SEQUENCES IN "RJMORS
94
OF F1(AKR X Balb/Mo) MICE
CHAPTER V
DNase I SENSITIVITY AND METHYLATION OF
GENETICALLY TRANSMITTED AND SOMATICALLY
ACQUIRED PROVIRAL DNA SEQUENCES IN M-MuLVINDUCED TUTORS
96
7
ALGEMENE SAMENVATTING.
Aangezien niet usruacht mag Dorden dab е п ev/entueel geinteressaerde
lezer oog
п kennis van
вп molekulair v/iroloog heeft is hier een
'populaire' samenuatting op zijn plaats.
Ongeveer vier jaar geleden uerd in onze werkgroep een onderzoek gestart
naar de molekulair-biologische aspecten van leukemie bij muizen die door
bepaalde virussen veroorzaakt utordt. Reeds in het begin van deze eeuui had
men ontdekt dat muizeleukemie veroorzaakt kan morden door een oelvrij filtraat van bloed, afkomstig van een muis met leukemie, in een gezonde muis
in te spuiten. Later bleek dat de ziekteverwekker een virus is, dat tot
de groep van de zogenaamde C—type virussen behoort. Deze virussen worden
tot de familie van de retrovirussen gerekend, die als belangrijkste karak­
teristiek het enzym reverse transcriptase hebben, een enzym dat RNA over
kan schrijven in DNA. Een C-type partikel bestaat uit een min of meer ronde
compacte kern, bestaands uit RNA an'eiwitten, omgeven door een mantel.
De mantel is afkomstig van de plaemamambraan van ds gastheercel. De virale
envelop-eiwitten bevinden zich in deze mantel. Het belangrijkste virale
envelop-ewit, het 70k glycoproteins, vormt knopachtige structuren op de
mantel van het viruspartikel. C-type virussen onderscheiden zich van de
andere typen (B,D, en E—typen) door hun beelden in de elektronenmicros­
coop. De C-type virussen hebben een elektronen-dicht centrum. Verder be­
zitten deze virussen geen opvallende uitsteeksels aan het oppervlak van
het partikeltje. Het virale genoom van een muize-leukemievirus bestaat
uit twee identieke enkelstrengs RNA subeenhsden van ongeveer 8-10 kilobassparen lang. Elk van dezs RNA's bevat de genetische informatie over­
eenkomend met een drietal genen: 1) het gag—gen, dat codeert voor eiwitten
die т.п. in het binnenste van het virusdeeltje voorkomen, 2) het pol—gen,
dat codeert voor het enzym reverse transcriptase, en 3) het env-gen, dat
codeert voor het manteleiiuit van het virusdeeltje. Dit manteleiwit is een
glycoproteine met een molekuulgewicht van 70k, dat op de van de cel af­
komstige plasmamembraan voorkomt die het centraal gedeelte van het virus
omhult. Geen van bovengenoemde eiwitprodukten kon in het verleden aange­
wezen worden als zijnde verantwoordelijk voor de door het virus veroorzaakte
leukemie. Het glycoproteine, gecodeerd door het env-gen werd wel gekarak­
teriseerd als het eiwit dat verantwoordelijk is voor het feit of het be­
treffende leukemievirus al dan niet een bepaalde cel kan binnendringen.
De structuur van het envelop-eiwit bepaalt dus reeds a priori of een be­
paald virus een cel al dan niet kan penetreren. Alleen wanneer het envelopeiwit een interactie aangaat met de juiste receptoren op een celoppervlak
kan een infectie tot stand komen.
9
meerdere normale muizegenen b i j b e t r o k k e n , шаагиап de e r f e l i j k e i n f o r m a t i e
ingebouwd kan worden i n het M—MuLV g e n o o m . Een bepaald gedeelte van het
oorspronkelijke ПЧЧіО genoom g a a t
hierbij
virussen worden dan ook r a c o m b i n a n t e
type v i r u s l i j k t nu een n o o d z a k e l i j k e
bij muizen. De manier waarop d e z e
v e r l o r e n . De zo ontstane nieuwe
viruseen
s t a p i n de o n t w i k k e l i n g van leukemie
virussen
b e t r o k k e n zijn bij de o n t w i k k e l i n g
en het i n stand houden uan de l e u k e m i s c h e
wordt door een ongebreidelde g r o e i
van
bediscussieerd i n Hoofdstuk I
m.n·
(zie
l i t e r a t u u r g e g e v e n s geeft t e v e n s
aan,
t o e s t a n d , d i e gekarakteriseerd
b e p a a l d e l y m f a t i s c h e c e l l e n , wordt
d i s c u s s i e ) . Deze samenvatting van
d a t ondanks v e e l f e i t e n m a t e r i a a l h e t
antwoord op de basale vraag v o o r a l s n o g
tumorcel a l s autonoom zo n i e t
genoemd. De vorming van d i t
o n d u i d e l i j k i s , waarom z i c h een
anarchistisch
I n de volgende hoofdstukken w o r d t
verder
i n d i v i d u gedraagt.
een m o l e k u l a i r - b i o l o g i s c h e
analyse gegeven van de door 1*1—nuLU v e r o o r z a a k t e
leukemie i n
muizenstammen. U i t de gegewens v a n
b l i j k t , dat tijdens de o n t ­
wikkeling van leukemie
Hoofdstuk I I
г een v e r m e n i g v u l d i g i n g
p l a a t s v i n d t i n lymfatische o r g a n e n .
maken van een techniek, die h e t
die op v e r s c h i l l e n d e plaatsen
van iedere normale muizecel e e n
het
genoom van M-PluLV. Radioactief
maakt om provirussen t e d e t e c t e r e n
gastheercel—DNA
dergelijke
groot
v i r u s s e n ) , d i e op n u c l s i n e z u u r n i v e a u
van p r o v i r a l e genomen
ii/erd v a s t g e s t e l d door gebruik t e
mogelijk
van
belangrijkste obstakel voor een
Dit
verschillende
ingebouwd zijn. Het
a n a l y s e vormde het f e i t dat DNA
a a n t a l p r o v i r u s s e n bevat (endogene
e r g v e e l homologie hebben met het
gemerkt
DNA van M-FluLV werd e e r s t
gezuiverd
van die DNA stukken die homologie h e b b e n mat a l l e endogene muizevirussen
van de b e t r e f f e n d e muizenstam.
alleen nog M-MuLV provirale DNA
Vervolgens
stukken
a c t i e f merkmiddel teneinde n i e u w e
u e r d h e t overblijvende DNA, dat
k a n herkennen, g e b r u i k t a l s r a d i o ­
ΓΊ—TluLV/ DNA's op t e sporen i n c e l DNA
van a l l e r l e i weefsels en van l y m f a t i s c h e
m e a f s e l s . Teneinde nieuw i n ­
gebouwde ΓΊ-fluLV provirussen op t e
werd e e r s t het c e l DNA geknipt
sporen
i n ongeveer 1 m i l j o e n DNA f r a g m e n t e n ,
scheiden werden. Bij het knippen u e r d
die
v e r v o l g e n s naar g r o o t t e ge­
gebruik
gemaakt van bepaalde r e s t r i c t i e -
enzymen. Deze enzymen herkennen en k n i p p e n bepaalde basenvolgordes i n het
DNA. Bovendien werd voor de b e p a l i n g
van
provirussen van een r e s t r i c t i e — e n z y m
gebruik
NuLl/ DNA k n i p t maar wel in n a b i j g e l e g e n
nieuwe inbouwplaatsen van M-MuLU
gemaakt, dat n i e t i n het M-
c e l DNA. Door het r a d i o a c t i e f
gemerkte DNA t e gebruiken voor d e o p s p o r i n g van H-MuLU provirussen i n
c e l DNA fragmenten van v e r s c h i l l e n d e
g r o o t t e s kon het volgende worden
vastgesteld:
1) het ovarervende M-fluLV genoom v a n
B a l b / M o muizen i s aanwezig i n êên
cel DNA fragment van een k a r a k t e r i s t i e k e
grootte.
10
2) ДІІе п in l y m f a t i s c h e o r g a n e n ,
een vermenigvuldiging
d i e betrokken zijn bij de leukemie, kon
van M-WuLU p r o virussen worden vastgesteld,
verschillende p l a a t s e n van h e t
cel
die op
DNA kunnen worden ingebouud.
3) Een deel van deze півише i n g e b o u u j d e provirussen heeft een verandering
ondergaan in de g e n o o m s t r u c t u u r · .
4) Het erfelijk
overgedragen
Π—TOuLV/ provirus, dat in c e l l e n van lymfatische
ueefsels op dezelfde p l a a t s
i n g e b o u u d zit als in andere organen, ondergaat
geen aantoonbare v e r a n d e r i n g e n
Hoofdstuk I I I beschrijft
i n DNA structuur.
d e m o l e k u l a i r e klonering i n eer· bacteriepl v:.rus,
uit
gekloneETdE DNA f ragmen- b e i / a t
d e e e r s t e 5400 baseparen van het M-nuLU
DNA, dat afkomstig i s van h e t
d e lever van
і_-.п Balb/rio muis. Hel
van een p r o v i r a a l DNA f r a g m e n t
erfölijk
het fragment nog 35Ü0 ü a s e p a r a n ,
dia
overgedragen p r o v i r u s . Verder bevat
afkomstig zij.i vai) het c e l l u l a i r e DMA
u/aarin hsL genetisch o v p r e c v e n d
Π—MuLV ргз ігиз in Balb/Vlo muizen i s
g e l o k a l i s e e r d . In d i t h o o f d s t u k
w o r d t verder aangetoond dat het erfelijk
p r o v i r a a l M-^uLV genoom, z o a l s
g e ï s o l e e r d u i t l e v e r c e l DMA, in principe
in s t a a t i s om a c t i e f
produceren.
virus
te
M-PluLV provirus in l e v e r c e l l e n
virus worden besproken.
voor het i n a c t i e f
Tuee
geen
De redenen uiaarom het endogeen
aanleiding geeft t o t productie van
m o g e l i j k e verklaringen die gegeven worden
αv/erervende 1*1-Ч*1иИ/ genoom zijn:
zijn van h e t
a) de driedimensionale s t r u c t u u r v a n het chromatins (DMA + gebonden
e i w i t t e n ) uaarin h e t e n d o g e e n M—MuLV is gelokaliseerd, en
b) een plaatselijke c h e m i s c h e v/erandering van het DNA.
Een combinatie van b e i d e f a c t o r e n
is
umarschijnlijk veranturoordelijk voor het
n i e t a c t i e f zijn van h e t o v e r e r \ / e n d e M-fluLV genoom. Dit ii/ordt verder u i t ­
gewerkt in Hoofdstuk V.
Hoofdstuk IV beschrijft
een
a a n t a l nieuwe technieken, die het mogelijk
hebben gemaakt om de шаагп т і п д
u i t t e werken. Hierbij u e r d
п zoals in Hoofdstuk I I beschreven verder
gebruik
a c t i e f gemerkte DNA f r a g m e n t e n ,
t e n e i n d e alleen M-MuLV-type provirale
DMA stukken in c e l DNA f r a g m e n t e n
kon gebruikt worden om p r o v i r a l e
delen van het рго і г а і
gemaakt van een tweetal typen r a d i o ­
t e kunnen d e t e c t e r e n . Het eerste type
DNA stukken op t e sporen die van a l l e
g e n o o m afkomstig konden zijn. Het tweede type
r a d i o a c t i e f gemerkte DNA h e r k e n d e alleen bepaalde stukjes van het M-MuLl/
DNA. Met behulp van d i t t u e e d e
genoom DMA fragmenten op h e t
ГЛ—CluLV DMA gelokaliseerd moesten worden.
Het tweede type r a d i o a c t i e f
g e m e r k t DNA werd verder gebruikt om de
s t r u c t u u r van nieuw i n g e b o u u j d e
gedeeltelijk
bestaan u i t h e t
van A) de p l a a t s van
nieuue
t y p e konden we bepalen waar eventuele sub-
provirussen te bepalen die nog s l e c h t s
oorspronkelijke rWluLV DNA. Ter opheldering
p r o v i r a l e genomen in het cel DNA van lym-
11
fatisoha weefsels,
п В) de structuur van deze пі иш
genomen, iderd de
volgende procedure gekozen:
i) cel DNA van verschillende weefsels werd geknipt met restrictie-enzymen
zodat ongeveer 1 miljoen fragmenten werden verkregen die vervolgens naar
grootte gescheiden werden.
2) Na scheiding op grootte uerden deze fragmenten overgebracht naar een
vaste drager waaraan het DNA covalent gebonden werd.
3) De vaste drager, die het patroon van de naar grootte gescheiden cel
DNA fragmenten bevat, werd vervolgens geinkubeerd met radioactief gemerkte
DNA's die FI-PluLV genoomstukken herkennen.
Na binding van de radioactieve DNA's werd vervolgens de plaats van binding
opgespoord door op de vaste drager een rBntgenfilm te plaatsen. Na ontwikke­
len werd zo een zichtbaar beeld verkregen van die plaatsen waar het radio­
actief gemerkt DNA zich had gebonden. Daar bij scheiding van DNA fragmenten
ook fragmenten van bekends grootte meegenomen werden, kon ook de grootte
van de op de rBntgenfilm zichtbaar geworden fragmenten vastgesteld worden.
Bij het knippen van het cel DNA werd gebruik gemaakt van twee typen
restrictie-enzymen: 1) een enzym dat niet in het PI-MuH/ genoom knipt, en
2) enzymen die op de eerste plaats in de LTR knippen, welke aan beide
uiteinden van het ingebouude virus voorkomt, en vervolgens nog op andere
plaatsen in het virale DNA. Met behulp van het eerste type enzym werden
nieuwe inbouwplaatsen van M-CluLV genomen in cel DNA opgespoord. Het tweede
type enzym werd gebruikt om de interne structuur van provirale DNA's op
te helderen. Het gebruik van de bovengenoemde technieken leverde de
volgende nieuwe inzichten op die relevant lijken voor door M-PluH/ ver­
oorzaakte leukemie:
1) preleukemische weefsels (vertonen nog geen vergroting van het orgaan)
bevatten alleen normale PMIuLV genomen, die op een groot aantal min of meer
toevallige plaatsen in het cel DNA ingebouwd zijn.
2) In uitgegroeide tumoren, afkomstig van verschillende anatomische
plaatsen uit êên muis, bevinden zich de nieuw ingebouwde provirussen op
dezelfde plaatsen in het cel DNA. Dit bewijst dat tumoren binnen SSn dier
grotendeels afkomstig zijn van êSn of enkele tumorcellen en zogenaamd
klonaal zijn.
3) Uitgegroeide tumoren bevatten zowel normale M-PluLV/ genomen, als provirale genomen die nieuwe structurele informatie hebben verkregen.
4) De provirale genomen, die nieuwe structurele informatie hebben verkregen
vormen een heterogene groep van provirussen. Toch hebben al deze nieuwe
provirale DNA's een aantal gemeenschappelijke kenmerken:
a) de nieuwe genetische informatie bevindt zich bij al de nieuwe provirale
12
DNA's op ongev/θβΓ dezelfdg plaats, en usi in hst gebied dat uoor het
manteleiidit codeert.
b) De nieuu) ingebouyde informatie bezit zeer sterke overeenkomstige
kenmerken in tumor DNA van alle leukemische muizen.
De gegevens beschreven in Hoofdstuk IV suggereren dan ook dat de provirale
genomen die піеише structurele informatie hebben ingebouwd, essentieel
zijn voor de ontwikkeling van en het in stand houden van de leukemische
toestand, l/erder suggereert de homologie die er tussen al de nieuwe
recombinante provirale DMA's bestaat, dat hun nieuw ingebouwde informatie
in alle muizen afkomstig is van mogelijk êên en hetzelfde muizegen, of
van een familie van sterk gelijkende muizegenen. Er worden dan ook verschillende mogelijkheden tegen elkaar afgewogen, die kunnen verklaren hoe
de nieuwe genetische informatie in de recombinanten verantwoordelijk kan
zijn voor de kwaadaardige uitgroei van lymfatische cellen (zie hiervoor т.п.
de discussie van Hoofdstuk l ) .
Tenslotte worden in Hoofdstuk V/ bewijzen aangevoerd voor het feit dat
de recombinante provirussen zich op bepaalde plaatsen in het tumorcel DNA
bevinden. Deze plaatsen worden gekenmerkt door een driedimensionale
DNA-eiwit structuur, waarin het provirale DNA plaatselijk een hoge
gevoeligheid heeft voor een DNA—afbrekend enzym. Dit is een karakteristiek
kenmerk voor genetisch materiaal dat actief gebruikt wordt in een bepaalde
cel. Verder worden bewijzen aangevoerd voor het feit dat nieuw ingebouwde
provirale DNA·s in tumoren een lage mate van chemische veranderingen
door methylgroepen hebben ondergaan. De afwezigheid, of althans geringe
aanwezigheid van dergelijke methylgroepen in het DNA is ook een karakteris­
tiek kenmerk van actief in gebruik zijnde genetische informatie in de cel.
Verder blijkt het genetisch overgedragen M-MuLV DNA in Balb/Mo muizen
volgens de genoemde kriteria inactief te zijn in zowel tumor als niettumor weefsels.
Concluderend kan gesteld worden dat leukemie bij muizen, die door M-nuLV
wordt veroorzaakt, is gebaseerd op de vorming en activiteit van provirale
genomen, die cellulaire genetische informatie hebben ingebouwd in het
env-gen gebied, waarbij de oorspronkelijke informatie van M-PluLV uit dat
gebied is verdwenen. Deze nieuwe virussen worden waarschijnlijk geselecteerd
via chronische stimulatie van het immuunsysteem zodat de provirale
structuren van recombinante virussen in uitgegroeide tumoren sterke
overeenkomsten vertonen.
13
SUIWRY.
Noloney Murine Leukamia Virus (М-СІиЦ/) was isolated by D.B. Moloney
from a mouse tumor (3. Natl. Cancer Inst. 24: 933-947, 1960; Natl. Cancer
Inst. Monogr. 22: 139-142, 1966). M-FluLV is a replication-competent,
non-defective type-C retrovirus. The virus has bean propagated in tissue
culture and used as a model system to study the molecular biology of Ctype retroviruses including their replication cycle, their encoded polyproteins and subsequent cleavages into smaller functional and structural
subunits, and their encoded RNAs and splicing products.
M-PluLV has also been usad to study the in vivo effects of C-type virus
infections. When injected into neiuborn mice, M-PluLV gives risa to develop­
ment of mostly leukemias of T-cell origin. In exceptional events inocula­
tion of M-MuLV into animals generated transforming replication-defective
sarcoma viruses. The sarcoma viruses arose by recombination of M-MuLU
with endogenous cellular sequences of the host animal. The п шіу acquired
sequences of the sarcoma viruses are responsible for their acute trans­
forming capacity. Нош
ег, the authentic M-MuLV genome does not encode
a product which is knoun to have laukemogenic capacity. So far only three
genas have been mapped namaly from 5'- to S'-end of the genome, respectively
the gag—polyprotein which subsequently is cleaved into several smaller
proteins constituting mainly the core of the virion; further, in the
middle of the genome the gene encoding the reverse transcriptase is
located, and the S'-end of the genome encodes the envelope proteins
gp70 (gp= glycoprotein) and p15E which are located on the surface of the
virion. The gp70 determinas the host range properties of a particular
C-type virus by its capacity to bind to cell-surface receptors on certain
host calls.
One of the main characteristics of МЧЧиИ/—induced leukamogenesis is
the long latency of the disease in contrast to the acute diseases caused
by sarcoma viruses. Mice uhich carry МЧЧиІ
as an endogenous Mendelian
inherited рго ігиз (Balb/Mo mica) do not develop leukemia before around
9 months of age although virus-expression is detectable from directly
after birth and reaches maximal levala after a few weaks. Infection of
newborn mice with M-MuLU also leads to development of lymphatic leukemia
after a latency of 2.5—6 months only, dependent on the amount of infectious
particles used for injection and also on the inbred mouse strain used
as a host. Many data, gathered from a great variety of both biological
and biochemical experiments now indicate that development of M-MuLUinduced leukemogenesis in mice is dependent on a large number of genetic
14
factors of both the host animal and the particular virus used to infect
animals of a certain inbred strain of mice. Chapter I summarizes data
from literature uhich indicate that MuLV-induced leukemogenesis in mice
is dependent on host factors like the major histocompatibility complex
(H-2), the Fu-1 gene of the host, some maternally transmitted factors,
the thymic environment, the expression of endogenous viral genomes, and
also mainly on the capacity of the CluLU to generate in a particular host
animal recombinant proviruses with a general type of genomic structure.
The generation of such recombinants seems to be a prerequisite for the
development of leukemia, and the шау these viruses may be involved in
the onset and maintenance of the leukemic state is discussed in the final
part of Chapter I.
The follouing chapters describe studies uhich resolve the structures of
both authentic N-CluLV and recombinant MuLV proviral DNAs as they are found
integrated in several tissues of M-PluLV-induced leukemic Balb/no, Balb/c,
and 129 mice. Chapter II deals uith a major problem one has to face when
studying specific integrated MuLU proviral sequences in the mouse genome.
The mouse genome contains a large number of endogenous proviral sequences
uhich shoui considerable homology with sequences present in the M-CluH/
genome. Therefore, in order to establish the integration of M-MuLV spe­
cific sequences in mouse cellular DNAs, a cDNA probe uhich exclusively
detects M-MuLU specific sequences, was prepared by selection procedures.
By using this probe it was established that Pl-TluLV-induced tumors in
Balb/Mo and newborn infected Balb/c mice contain additional reintegrated
M-WuLl/ specific sequences in many neu chromosomal sites of tumor tissues.
Chapter III describes the cloning and restriction mapping of part of
the genetically transmitted H-MuLV genome (Nov-l) in Balb/PIo mice. The
clone, containing 3.5 kilobasepairs (kbp) of cellular sequences and the
first 5,-5.4 kbp sequence
of the ñov-l PWIuLV genome, was derived from
Balb/Mo mouse liver DNA. This tissue does not express PWIuLV. However,
after cloning and ligating the a.9 kbp fragment to restriction fragments
which contain the S'-part of the МЧЧиИ/ genome (these fragments were
derived from cloned unintegrated closed oicular FI-FluH/ DNA) these re­
constructed genomes were able to produce biologically active virus. The
fact that the Mov-I M-CluLV genome is not expressed in tissues like liver
(non-target-tissue) or even in tumor tissues (see last chapter) may be
ascribed to the large extent of methylation and to the structure of the
chromatin encompassing the По -І M-fluLV proviral DNA as discussed in
Chapter V.
15
Chapter IV describes tha integration and structure of recombinant
prouiruses in M-MuLU-induced tumors of Balb/Plo, Balb/c, and 129 mice.
The state of integration and amplification of proviral DNA mas established
by using tuo kinds of molecular probes to detect FI-PluLV specific sequences.
Since the integration of a certain recombinant prov/iral DNA structure
is a ubiquity observed for all M-MuLV-induced outgroun tumors this lends
credence to the notion that a certain recombinant structure is a prerequisite for development of leukemia. Also, some other observations seem
relevant to virus (N-PluHO-induced leukemogenesis:
1) authentic M-NuLV genomes are integrated randomly in the DNA of preleukemic tissues whereas such tissues do not shou detectable amounts of
recombinant proviral DMAs,
2) outgroun tumors contain both authentic FWIuLV as umll as recombinant
genomes integrated in unique sites of chromosomal DNA,
3) tumors are largely composed of descendants of one or a feu transformed
cells,
4) the recombinant proviral DMAs constitute a heterogeneous group of
proviruses but characteristic molecular markers are conserved among many
individual recombinants: a) they are all env-gena recombinants, and b)
they all have acquired endogenous-virus-like sequences at almost identical map positions, and further c) the neuly acquired sequences in all
recombinants display similar restriction sites. Therefore, the data
discussed in this chapter strongly favor a hypothesis uhich defines a
recombinant provirus as a structure essential for the onset and maintenance of the leukemic state. The large homology of all recombinant proviral DMAs integrated in DMA from outgroun tumors further suggests that
the parental cellular sequences that recombine ulth M-CluLV to generate
such recombinants are either highly related or even identical for all
different lymphomas. The possible uay by uhich the recombinants exert
their effect during leukemogenesis is discussed in the last section of
Chapter I.
Finally, the results discussed in the last chapter support the hypothesis
that the expression of a recombinant provirus is a prerequisite for the
onset and maintenance of the leukemic state. DNase I probing studies of
chromatin have provided convincing evidence that transcriptionally active
chromatin has an altered, more accessible configuration. Furthermore,
hypomethylation uas also shoun to be a characteristic, though not sufficient
marker for DNA of active genes. Chromatin comprizing proviral genomes in
outgroun tumors is divided in three different DNase I sensitivity classes
i.e. in configurations resistant, moderately sensitive, and hypersensitive
16
to DNase I. The genetically transmitted copy (flou-l) шаз found to be
extensively methylated at certain restriction endonuclease sites and
displayed a DNase I resistant configuration. These data strongly support
tha hypothesis that the Flou-I locus is probably only active in a feu
cells early in life of the Ва1Ь/Ио mouse but not in tumor and non-tumor
tissues of a mature mouse. Somatically acquired proviral genomes were
all hypomethylated. In addition most somatically acquired MuH/ sequences
displayed moderate sensitivity to DNase I whereas some proviruses dis­
played a configuration hypersensitive to DNase I. This hypersensitive
site шаз found at the S'-cellular DNA-viral LTR junction of integrated
recombinant proviral DNA. Since such hypersensitive sites are closely
associated with active genes the data strongly support the hypothesis
that an active recombinant proviral DNA structure is required during
leukemogenesis.
17
CONTENTS CHAPTER I
LEUKEMOGENESIS I N MICE
INTRODUCTION
18
ENDOGENOUS C-TYPE VIRUS RELATED SEQUENCES
21
THE AKR SYSTEM
23
Balb/Mo AND NEldBRON INFECTED Balb/c AND 129 ПІСЕ
24
HRS STRAIN
27
RADIATION-INDUCED LEUKEMIAS
THE THYMUS
a ) C57BL
NICE
28
b) Suiias
MICE
29
a ) HISTOLOGICAL CHANGES'DURING LEUKEMOGENESIS
30
b) ROLE OF THE THYMUS DURING LYMPHOMAGENESIS
31
c ) CHANGES I N CELL-SURFACE ANTIGENS
34
LATENT PERIOD OF THE DISEASE
1) ANTI-VIRAL ANTIBODIES AND CELL-PIEDIATED
39
40
IMMUNE RESPONSES
2 ) THE INFLUENCE OF ΤΉΕ H-2 HAPLOTYPE
41
3) THE INFLUENCE OF THE Fv/-1 ALLELES
42
4 ) MATERNAL EFFECTS ON SUPPRESSION OF
44
LYMPHOMAGENESIS
DISCUSSION
45
10
Chapter
I.
LEUKEMOGENESIS IN MICE.
INTRODUCTION.
M u r i n e l e u k e m i a s can be caused by s e v e r a l a g e n t s
like:
— leukemogenie ( c a r c i n o g e n i c / mutagenic) chemicals,
— radiation,
— endogenous and exogenous RNA tumor viruses.
Up till ποω the expression of C-type viruses (in contrast to A, B, D,
and E-type particles) has been mainly associated uith development of
spontaneous leukemias in mice (200). Since the discovery of leukemiatransmission by cell-free extracts from the high leukemic AKR strain,
developed by Dacob Fürth in 1933 (43), the role of C-type viruses in
mouse leukemias has been investigated.
A vast amount of evidence has been accumulated which indicates that
the expression of C—type viruses in mice is associated with leukemogenesis.
The mouse genome contains a large number of C-type related sequences.
Some of these sequences seem to contain only part of the coding information for a C-type virus whereas others comprize sequence information
for a complete endogenous MuLV (purine leukemia virus). Endogenous MuLVs
are viruses resulting from the expression of chromosomally integrated
viral genomes transmitted as Mendelian traits.
The expression of C-type provirus related sequences can result either
in the production of 1) infectious C-typs particles (encoded by nondefective proviruses), of 2) non-infectious C-type particles (encoded
by defective or mutant proviruses), or can result in 3) the expression
of virus-related proteins without production of C-type particles (21,
69,171). Most mouse strains do express virus-related proteins without
producing virus. A protein indistinguishable from a viral envelope protein
gp70 (gp= glycoprotein) is r*ften seen on the surface of cells, without
concomitant synthesis of other virus-encoded proteins; this is presumably
a consequence of the partial expression of an endogenous virus (6,22, 101,
13Θ). The prototype structure of a C-type virus, its life-cycle, and
virus-encoded RNAs and proteins have been reviewed in detail elsewere
(3,66a,212a), and will therefore not be part of the present discussion.
Env-related gp70 proteins can be expressed in complete absence of repli­
cating virus (44). Partial expression of virus-related proteins like gp70
without the presence of infectious virus particles is a generally observed
phenomenon in normal healthy mice of many strains. Other virus-related
proteins are known which appear only in the presence of replicating virus
e.g. Moloney Cell Surface Antigen (MCSA) and Gross CSA which appear only
19
in the presence of replicating Moloney (FWIuLV) or Rauscher (R-PluLV)
murine leukemia virus and AKR-^uLU respectively; FICSA is an duLV-envrelated protein (66), and GCSA is a glycosylated polyprotein containing
antigenic determinants of viral gag-proteins (98,99,205); the gag-gene
is encoding the proteins of the viral core (p15,p12,p30, and plO, in this
order from NH^-terminus to CO.H-terminus). The presence of replicating
virus and the expression of infectious C-type particles is in most cases
associated with clinical manifestations of diseases like different kinds
of T-cell leukemias (e.g. in AKR and Balb/Мо mice) or autoimmune disease
(e.g. in NZB mice; 102-104).
Experimentally one can mimic the expression of an endogenous virus by
injecting newborn mice with a certain MuLV-stock. The time of appearance
of the disease is greatly dependant on:
1) the number of infectious virus-particles used for injection, and
2) the transforming capacity of the virus in e particular host.
The exogenous virus can only infect target—tissues and generally does
not infect germ line cells or cells of non-target tissues. This phenomenon
is known as organ-tropism; organ—tropism and host—range of a particular
virus are determined by the envelope glycoprotein on the surface of the
virion. The target cell varies from one virus strain to another, e.g.
M-CluLV is known to infect and transform different classes of prothymocytes
(cells of T-cell lineage), while Rauscher PluLV exerts its effect on
erythroid cells, and AKR-^uLV acts mainly on mature T-cells.
The role of the authentic viruses like ΓΊ-ΛΙυίΙ/ and AKR-fluLU during the
onset of leukemogenesis has been questioned many times since these viruses
do not carry a transforming gene like the acute leukemia virus Abelson
MuH/ (167), and the disease is characterized by a long latency. The
discovery of certain types of recombinant viruses, first isolated from
a preleukemic thymus of an AKR mouse suggested a role for recombinant
viruses in the process of leukemogenesis (65). Recombinant viruses with
a general type of structure and phenotypic characteristics have now been
isolated from both preleukemic and leukemic tissues (thymus and also from
spleen) of several high leukemic mouse strains (169). The major charac­
teristics of these recombinants are summarized below and a general
structure is shown in Figure 1 :
1) they have been isolated from preleukemic and leukemic tissues only
(thymus and leukemic spleen; 169),
2) they are formed by somatic recombination of two types of endogenous
viruses or virus-related sequences, an ecotropic virus and probably a
xenotropic viral sequence (11): ecotropic NuLl/ can succesfully adsorb
20
to and penetrate mouse cells uhereas xenotropic viruses can only infect
heterologous cells i.e. cells from other species,
3) they are env-gene recombinants: the putative S'-end of the pol-gene
(coding for the viral reverse transcriptase) and part of the NhL-terminal
sequence but probably even the uihole gp7D encoding part of the env-gene
seems to be involved in the recombinational event (159,206) as shoun in
Figure 1,
4) they have a dualtropic host-range i.e. they can infect both mouse cells
and other types of cells (65),
5) they display MCF-nature i.e. they form cytopathic foci on mink cells
(lylCF= Nink Cell Focus-inducinq virus; 65),
6) their pISE—coding sequences seem to be mainly derived from the ecotropic
parent (206),
7) their 3·—LTR (Large Terminal Repeat) often contains xenotropic-HuLVderived (or similar cellular) sequences (110),
e«l&tUO MB
41
ECO RI ИрЛІ Эвкьр
g
a
g
|
M
ЧІ
^^^^»
p
o
i
1
е
п
Figure 1: Structure of authentic n-CluLV and recombinant proviral DNA in
M-MuLV-induced tumors.
The upper line represents the restriction map of the genetically trans­
mitted Mov-I Fl-nuLV DNA as integrated in both tumor and non-tumor tissue
DNA from Balb/rno mice. The two Іошег linas shou the structures of the
recombinant Kpn I DMA fragments from tumor DNA of Balb/do mouse Ю (5.4
kbp) and Balb/c mouse 503 (4.0 kbp), respectively; the пвшіу acquired
sequences in the mink cell focus-inducing (MCF)-type proviral DNAs are
indicated by a double line. The neuly acquired restriction endonuclease
recognition sites are also shoun, as well as the position of the recombinant-specific Bam HI (2.2 kbp) and Kpn I- Ceo RI (3,6 kbp) DNA fragments
detected in all tumors and diagnostic for recombinant proviral DNA (159,
206). The louest part of the Figure indicates the location of the envgene uhich starts at position 6.4 kbp (C. van Beveren, personal communi­
cation).
21
In contra3t to the parental eootropic viruses and different xenotropic
viruses, none of uhich does accelerate leukemia dev/elopment uhen injected
into newborn mice of high leukemic strains like AKR (so called leukemiaacceleration test), some of the recombinant viruses do accelerate leukemia
markedly in appropriate hosts (141). However, many recombinant viruses
hava ηοω been isolated which do not accelerate leukemia. Therefore
skepticism has accumulated on the role of these viruses in the onset and
maintenance of the leukemic state. The integration of defined recombinant
proviral DNAs, however, seems now directly related to clonal outgrowth of
lymphoma cells during leukemogenesis (159,206). Therefore, a direct role
of the recombinant in the switch from preleukemic to leukemic state and
probably also in the maintenance of the leukemic state has been suggested
(206). Although the recombinant viruses do not seem to play a role in the
formation of preleukemic cells, their presence in leukemic cells of many
mice probably reflects a specific property that enables the infected
cells to complete the different final steps in the metastatic process.
The formation of preleukemic cells can probably be ascribed to effects
generated by the authentic parental ecotropic viruses and the switch
from normal to preleukemic cells seems already to occur very early during
lymphocyte maturation in the bone marrow (58), at a stage when no
recombinants have yet been detected. The long latency of the disease
generally observed for leukemogenesis seems to be dependant on the
formation of recombinant viruses, and further on several host factors
like thymic environment, the major histocompatibility complex (MHC) H-2,
the Fv-1 genotype, the expression of xenotropic virus, and a chronic
immune response. These aspects will be discussed below.
ENDOGENOUS C-TYPE l/IRUS-RELATED SEQUENCES.
Figure 2 shows some examples of C—type endogenous viral sequences as
they are present in the genomes of many mouse strains. Further, different
recombinational pathways are indicated, which have been detected between
different endogenous viral sequences as well as between viral and cellular
sequences. A variety of recombinations can give rise to different recom­
binant viruses; each of these recombinant viruses seems to be associated
with typical manifestations of a disease e.g. the sarcoma virusea trans­
form fibroblasts in vitro and form fibrosarcomas in vivo (3); the dualtropic viruses and their parental viruses like M-CluLV and AKR-PluLV are
mainly associated with the formation of lymphatic leukemias of T-cell
origin (although there havs been reports that AKR-MuLV can induce B-cell
lymphomas in rare cases; 137). Ab-PluLV is associated with a typical pre-B
22
SOnATIC
ENDOGENOUS
DISEASES
Amphotropic MuLV/
(92)
Xenotropic MuLV
(26,102-104,199)
/
Defective Recombinant Erythro^
nuLU Leukemia
e.g.SFFU
(172,173,201,
202)
jaltropic nuLl/ (ПСЕ)
(30,65,110,161,169,
173,168-190,208,211)
Viruses (37,47)
)
Lymphoid
Leukemias
(45) Ecotropic NuLV
(2,38,46) N-tropic
(5,8,73) B-tropic
("louse
C-type
information
Endogenous Μ υ ί ^
acquired via germ
line integration
e.g. N-FluLV in
Balb/Mo mouse
(77)
gp70 gene
family
(37)
C-type
related
30 S RNA
encoding
genes (9a,33a,
67a,182a)
Other
mouse
genes
e.g. abl
Exogenous MuLV
e.g. M-MuLV
Sarcoma (3,181,
viruses 209)
Sarcomas
Pto-nSV
via rat
^Ha-nSV
Ki-OISl/
Ab-nuLU
(166,167 )
Null or
pre-B cell
Leukemias
Figure 2: Recombination of C-type sequences in the mouse.
Numbers indicate references. Abbreviations: Mo-MSV, Moloney murine sarcoma
virus; Ha-MSU, Harvey murine sarcoma virus; Ki-PlSV, Kirsten murine sarcoma
virus; abl indicates the cellular gene homologous to the cell-derived
sequences present in the genome of Ab-duLU. Amphotropic viruses grow both
in mouse cells as шеіі as in cells from heterologous species but do not
form foci on mink cells like dualtropic MuLV. The gp70 gene family consists
of cellular genes (both viral and non-viral) which code for 70k glyco­
proteins e.g. the gp70 found in serum. Fibroblasts, infected with N-,
B-, or NB-tropic virus are recorded by their ability to cause XC-plaques
when overlayed with XC-cells (65): l\l-tropic on NIH/SUJÍSS mouse fibroblasts,
B—tropic on Balb/c mouse fibroblasts; N-tropic viruses are restricted
in growth on B-tropic cells and vice versa, an effect mediated by the
cellular Fv-1 gene, which dictates dominant though not absolute restriction. NB-tropic viruses (e.g. M-MuLV) are not restricted and grow equally
well on cells with different Fv-1 alleles (see section on Fv-1).
23
or null c-all leukemia (166); SFFV (Splesn Focus-ForminQ Virus) is asso­
ciated with the development of erythroleukemia (172,173,201,202); Rauscher
MCF induces also erythroleukemia (162). In general the acquisition of
oncogenicity seems to be associated uiith recombination betuieen different
type-C genomes or between different type—С genomes and host genes. A
possible function of RNA tumor viruses may therefore be to act as vehicles
for the intra- or intercallular movement of genes and therefore they
could be elements of "selfish" DNA (33,147). Intercellular movement of
genes can occur uithin one organism, from one individual to another,
or even from one species to another. Furthermore, the putative capacity
of RNA tumor viruses to move genes intraoellularly implicates the
possibility that the enzyme—system of the virus might actually have
cellular counterparts which have normal physiological functions.
Many of the recombinant viral genomes were generated in exceptionally
rare events and therefore have to be classified as unique structures
e.g. SFFV, Ab-MuLV, and various sarcoma viruses (KirstenMSV/, HarveyMSV,
Moloney Mouse SV). An intriguing observation, houever, is that some
types of recombination seem to be more general phenomena associated with
the development of T-cell leukemias and myelomas in mice. The formation
of MCF-type viruses, first reported in 1977 by Hartley et al.(65), is a
generally observed phenomenon during T-cell leukemogenesis. However,
other types of recombinant molecular species have been detected in some
leukemias. Some of these examples are discussed below.
1. THE AKR SYSTEN.
nice of the AKR strain (43) express high levels of ecotropic endogenous
AKR virus (17) from soon after birth (170), and by 6 months of age most
thymus cells produce N—ecotropic virus. Different AKR substrains contain
various numbers (2-6 copies per haploid genome equivalent) of АКРЧ*1иИ/
proviral genomes as endogenous proviruses (159,159a), Xenotropic virus
is detected in the thymus at approximately 6 months of age (86). Polytropic nCF-type viruses have been isolated from preleukemic and leukemic
tissues (thymus, spleen) of AKR mice of 6 months of age and older (65).
The emergence of this neu virus, not present as an endogenous proviral
entity, seems to be of critical importance to leukemia development in
the AKR strain (30,159,190): 1) these viruses appear in close temporal
association with the development of spontaneous leukemia at 7-12 months
of age in nearly 100 % of the mice (110,169). 2) When injected into new­
born AKR mice, some of the recombinant viruses accelerate leukemia de­
velopment markedly, in contrast to the parental ecotropic and various
xenotropic viruses (30,110,169,190). 3) A certain recombinant proviral
7Λ
DNA із detscted as an integrated entity in all outgrown tumors of AKR
mice (159). 4) The structure of these recombinant oroviral DNAs is si­
milar to the structure of HCF-type viral RNA, isolated from virions or
cell lines derived from leukemic tissues (19,36,159). The general restric­
tion map for these recombinant proviral DNAs as detected in tumors from
mice of different inbred AKR sublines is similar to the map shown in
Figure 1 for recombinant proviral DNAs characteristic for ïï-CluLV-induced
leukemias (159,206); the recombination is located in the same position
on the map and also carries the tuo пеш restriction sites Bam HI and
Eco RI at similar positions (159).
Another class of retroviruses produced by spontaneous AKR leukemias
are the SL-viruses, which do not cause XC-plaques or mink cell foci.
Lymphoma cell supernatants containing these SL-viruses are pfficient in
leukemia-acceleration in young AKR hosts. Their role as potential
leukemogenic agents is still unclear although recently cloned SL-viruses
ишгв obtained which induced louk^mia but did not display a MCF-type
substitution in the env-qene; these viruses retained differences in
structure with Gross AKR passage A virus within the S'-terminal 1000
nucleotides. This suggests that sequences outside the gp70 coding sequences
and located proximal to the S'-end of the genome encode determinants of
leukemogenic activity (150). However, the structure of integrated proviral
DNAs in tumors induced by SL-viruses is not yet established so whether
additional recombinations take place during tumorgenesis is not known.
Therefore, leukemia-acceleration by such viruses might be due to their
putative enhanced capacity to recombine and form leukemoqenic recombinant
viruses.
2. Balb/Vlo AND NEWBORN INFECTED Balb/c AND 129 HICE.
3aehnisch has introduced Cl-CluLU into the germ line of Balb/c mice by
infecting preimplantation mouse embryos at the 4-8 cell stage (77). The
Balb/Mo strain carries M-PluLV as an endogenous virus at a single Hendelian
locus (Поу-1;7В) on chromosome number 6 (14)| the virus is integrated
in a characteristic Eco RI DNA fragment of 27 kbp (-207). Balb/Mo mice
are viremic from birth (7 ) and develop after a latency of 7-9 months
a thymus-dependant leukemia of viral (M-MuLV) etiology (132). On the
other hand Balb/c and 129 mice do not develop this disease spontaneously
at this age. When newborn Balb/c or 129 mice are infected with N-nuLV,
however, a thymus-dependent leukemia follows the expression of high titers
of the virus in the blood (80,153). This was also observed for M-MuLl/induced leukemias in NIH/Swiss mice (191).
25
Leukemogenesis in Balb/flo and neiubom infected ВаІЬ/с, 129, and NIH/
Swiss mice is accompanied by somatic amplification (76,79) and reinte­
gration of l*luLU DNA sequences in neu chromosomal sites of tumor tissues
(75,206,207). Spleen and thymus cells of Balb/no mice synthesize M-PluLV
specific RNA soon after birth and v/irue expression reaches high levels
at 3-6 weeks of age (79). This level remains constant throughout life
of the animal and the expression is target-specific i.e. confined to
lymphatic tissues (although many organs like liver, kidney, gut etc.
show both viral DNA and RNA amplification, due to infiltration by lym­
phatic cells at final stages of the disease; 79). The DNA amplification
seems to occur in tuto steps. A first step to approximately two copies
per haploid genome equivalent in target tissues of preleukemic mice.
A second step to approximately 3-4 copies is observed in leukemic
tissues (79). Preleukemic tissues seem to contain only authentic M-MuLV
genomes, reintegrated "randomly" in п ш chromosomal sites (75,206).
Unintegrated forms of И-ІЧиИ/ viral DNA appear in target tissues (mainly
thymus) of preleukemic Balb/Mo mice (75). The occurrence of free proviral
DNA suggested that superinfection of target cells with endogenous N-PluH/
is involved in the leukemic transformation process. The second DNA ampli­
fication step is associated with leukemic transformation (79). The suiitch
from preleukemic to leukemic stage uas shown to be associated with clonal
outgrowth of tumor cells. Outgrown tumor tissues within each animal
display a specific pattern of reintegrated proviral DNAs (75,206); although
tumors in each animal are largely monoclonal, different mice display
different proviral integration patterns (75,206), Outgrown tumors contain
both authentic FWIuLl/ genomes as well as recombinant FICF-like proviral
DNAs integrated in many different sites of the chromosomal DNA (206).
Recombinant PICF—type viruses have been isolated from thymoma cell lines
of Balb/Mo mice and seem to be present in every thymoma cell line (211).
Although the recombinants constitute a heterogeneous group of proviral
DNA structures, they are all env—gene recombinants which have acquired
"xenotropic"-like sequences in the NI-L-terminal part of the env-gene
and the putative carboxy-terminal part of the pol-gene (206). Ulhether
this has any consequence for the pol-protein is not known but since the
mature pol-protein is 84k (210), the recombination only effects a sequence
whose product has not yet been detected after processing of the gag—pol
precursor protein.
The structural similarities of integrated recombinant viral DNAs
as observed in a large number of N-TluLV-induced lymphomas in several
mouse strains strongly supports the hypothesis that the formation of
26
these recombinants is related to leukemic transformation (206). Some
possibilities for their role will be discussed later on. The recombinants
in different tumors are clearly structurally related, but are not identical.
This structural heterogeneity is probably related to the phenotypic
diversity as detected by cell-surface differentiation antigens, of thymomaa induced by H-MuLV (152,183). The diverse phenotypic patterns of
expression of differentiation antigens on cells of H-MuLV-induced
leukemias as ш іі as the structural heterogeneity among integrated
recombinant proviruses are consistent uith the hypothesis that the
putative transforming recombinants arise by random gene hic recombinabion
(35).
Balb/c mice shou a moderate incidence of lymphoreticular tumors (63,87).
Occasionally, Balb/c mice show B-tropic virus activity in their tissues
with advanced age (155), and this virus has been associated with leukemia
development (63,B7). This virus, u/hen injected into neuiborn Balb/c mice,
shoued acceleration of leukemia development (153,154). The Balb/c genome,
houraver, does not harbor a B—tropic MuLV since no such virus can be in­
duced while an N-tropic virus can be induced (1,192). The Balb/c genome
contains one N-tropic AKR—type endogenous provirus which is detectable
in an Eco RI DNA fragment of 19 kbp (9,16,94,159,165,207). The B-tropic
virus seems to be generated by a somatic recombinational event (44,165);
it harbors genetic informa bion for a xerotropic-flüLV-like рЗО prot-ein
in the gac-region (45). Since Balb/c ce]Is are relatively resistant to
N-tropic virus replication (64,157,176), the recombination leads to the
formation of a virus which can now easily grow in "alb/c cells because
of its B-tropism.
Recombinant dualtropic MCF-type viruses have not only been detected
in association with T-cell leukemias in Balb/c mice, but in addition
such viruses have been isolated from Balb/c myelomas. The viruses 1*10-21,
FL-1 (IBI), and СВ20
(23) were originally produced by different Balb/c
myelomas. The cloned preparations have a polytropic host range and exhibit
MCF activity (109). Additional features were found in common with other
MCF-type viruses e.g. normal Balb/c serum effectively neutralized the
infectivity of the two isolates 140-21 and FL-1 (41,65). Although MO-21
and FL-1 have many features of normal MCF-type viruses, some remarkable
characteristics were found. Although the viral gp70 envelope protein
had several peptides in common with ecotropic and xenotropic gp70, the
structure of the qp70 of М0-21 and FL-1 (and also of СВ20 ) seems,
however, not totally due to a reqular MCF-type env-recombination since
they contain unique peptides not found in any of the other putative
27
parental virusas testad (1ΘΘ). A remarkable feature of the tuo independent
isolates N0-21 and FL-1 uas their virtually indistinguishable gp70 peptidesf uhile previous MCF isolates were always found to display unique
peptides for each isolate. The common gp70 structure suggests that М0-21,
FL-I and CB20B were not generated by independent recombinational events
in the env-gene region as was observed for other MCF isolates, but might
have a common precursor molecule. This might well be a cellular differen­
tiation antigen which bears structural relationship with xenotropic MuLU
gp70 molecules. ΠΟ-ΣΙ and FL-1, produced by various cell lines (like the
wild mouse embryo SC-1 cells) contained also an additional gp70-like
protein distinguishable from the viral functional gp70 (1 8), while this
protein was not observed when the viruses were grown in other cell lines
(like the mink lung Nvl cells). Several possibilities for the appearance
of this env-related gp70 molecule have been postulated; it might be due
to the presence of a mixture of viruses (probably not true since after
extensive cloning of viruses by tissue culture techniques no other virus
was found), to the rescue of an endogenous virus, to the induction of
a defective virus or virus-related cellular gp70, or it might be due to
an incompletely modified or processed precursor of the viral gp70 (1B8).
Induction of an endogenous virus-related gp70 seems a very likely
possibility because this can occur in probably every cell line, even in
cell lines initially characterized as virus-negative (i.e. no virus could
be induced) like NIH (ΓΊ. Vogt, personal communication).
3. HRS STRAIN.
The HRS mouse strain carries the hairless gene (hr). Hairless homozygotes
show high incidence of spontaneous lymphoma (72%), whereas heterozygotes
show a much lower incidence (20$). Both hr/hr and hr/+ mice have high
levels of ecotropic viruses. However, only hr/hr mice express in addition
a xenotropic virus in their thymuses shortly before developing lymphoma
(67). Each cloned isolate of recombinant MCF-type virus from HRS/o mice
has been shown to contain unique oligonucleotide sequences in its envgene region and clone-specific peptides in its gp70 (51). This hetero­
geneity is also known for MCF-type recombinants of AKR origin (110).
The diversity of polytropic viral gp70 is also reflected by the extensive
phsnotypic diversity among spontaneous thymic leukemias of both HRS/D
(51,21B) and AKR/!! mice (95,114). Tumors within one strain did have
common features (218) e.g. expression of T-cell specific Thy-1.2 antigen;
a high percentage of thymocytes expressing MuLl/ antigens; a low expression
of the MHC-antigen I (HRS/D) or high expression in AKR/D (21Θ). The surface
28
phenotypas of
Иугтгатаэ induced by cloned leukemogenic ñCF-type viruses
from HRS/Э mice ы ге alujays found to be characteristic for a given clone
(51,218). Recombinant-virus-induced leukemias have been shouin to produce
the specific recombinant virus which induced them (51), although minor
modifications on genomic level can not be excluded because of limitations
of the RNA-fingerprinting technique. It seems therefore unlikely that a
п ы recombinant virus caused the leukemias. Recently, a single dominant
gene (probably encoding a thymocyte cell-surface receptor) was found to
determine susceptibility to a certain cloned recombinant virus (177):
leukemogenicity of such polytropic viruses is, however, restricted to
certain inbred strains of mica (177). The ікау by which the wild-type hr
allele can suppress xenotropic virus expression and therefore probably
lymphomagenesis is not known. A primary effect of the hr-allelss on lym­
phoid cells has been suggested since hr/hr mice also show abnormal
numbers of certain types of T-cells (164).
4. RADIATION-INDUCED LEUKEMIAS.
A) C57BL MICE (56).
Radiation-induced C57BL lymphomas do not overtly express MuH/ (only
2-45È harbor Radiation Leukemia Virus, RadLU; 52,70). The transmissible
leukemogenic agent, RadLU, isolated from such lymphomas and subsequently
passed through tissue culture for many years now, has unique env-gene
characteristics (29). This isolate has retained its leukemogenic potential.
The in vivo counterpart of RadLV can be consistently isolated from lymphoid
tumors of the thymus which develop after controlled X-irradiation of
C57BL/Ka mice (03,174); this has become possible by using very sensitive
tissue-culture techniques. RadLV is a highly oncogenic virus capable of
inducing neoplastic transformation in susceptible cells in the thymus,
bone marrow, spleen and fetal liver and transforms cells of the T-lymphocyte lineage. RNA from RadLV/VL3, a virus produced by a permanent cell
line derived from a radiation-leukemia-virus-induced thymic lymphoma
of C57BL/Ka mice consists of two components (112). Except for an 8 kb
subunit, which is also found in a non-oncogenio endogenous virus of the
same strain of mice, a 5.6 kb RNA was detected. The 5.6 kb RNA codes for
a 100k protein which can react with antibodies directed against the gag
p15 (=anti-p15) from Rauscher MuLV (the R-MuLV gag-gene protein-order is
NH„-p15-p12-p30-p10-C07H; 128) but not with anti-рЗО, and therefore contains
sequences at the S'-end coding for the viral protein p15 and p12. In
addition RadLV/VL3-virus-producing cells contain a 1.6 kb polyA
cyto-
29
plasmic RNA uhich shows very little homology to the θ kb RNA sequences
(112). The acute defectiue leukemia viruses like Ab-MuLV contain partial­
ly deleted gag and enu-qene region and completely deleted pol-gene region
idhen compared uith their helper viruses, with non-helper RNA sequences
inserted between these regions (167). The neoplastic disease induced by
RadLV is not acute since mice develop lymphomas 3 months or later after
virus inoculation. It is not clear whether all other virus- or radiationinduced thymic lymphomas display a 100k protein with serological charac­
teristics
similar to the protein translated from the 5.6 kb RNA, and
whether these lymphomas also contain both the θ kb and 5.6 kb RNAs,
Therefore, the role of the smaller RNA of 1.B kb as detected in the
RadLU-producing cell line is also still obscure.
B) Swiss NICE.
Radiation—induced leukemia development in Swiss mice does not lead
to expression of type-C viruses (71). V/irus—free X—ray—induced lymphomas
in an outbred Swiss mouse were shown to express a unique surface
recombinant-HuLU-like glycoprotein gp70 on thymoma cells
(40). A lympho­
blastic cell line derived from such a thymoma did not produce infectious
virus nor C-type particles, but showed specific viral interference with
recombinant PluLl/s. Peptide mapping of the purified gp70 revealed that it
was highly related to an endogenous xenotropic gp70, but in addition
had peptide characteristics of recombinant FICF-like gp70. The origin of
the recombinant-like gp70 is obscure for the moment. It could be encoded
by a defective or intact provirus, by a cellular gene, or it might have
resulted from internal recombination. Also, the role of the gp70 molecule
in leukemogenesis is not clear. It was postulated (40) that the recom­
binant gp70 induces a chronic immune stimulation which results in blastogenesis. This model relates very closely to the model proposed by McGrath
and Ueissman (117) who shoued the existence of receptors for recombinant
MuLV gp70s in a specific T-cell subpopulation of AKR mice (see also
discussion).
30
THE THYMUS.
A) HISTOLOGICAL CHANGES DURING LEUΚΕΠΟ GENESIS.
The thymus of a mouse reaches maximal size after a feu ueeks of age
and starts reducing in size after about 6 ш екз of age. Figure 3 shows
a schematic representation of a part of a neonatal thymus.
blood vessel
thymic lobule
___
Hassalls corpuscule
capsula
medulla
connective
tissue
septa
(separate
lobuli)
intralobular
trabeculae
Figure 3: Thymus of a neonatal mouse.
The thymus consists of tuo lobes, while each lobe consists of thousands
of lobules. Each lobule contains a cortical and medullary component. The
lymphatic tissue, unlike that of lymph nodes, is not arranged in nodules
(lymphoid follicles); such nodules contain closely packed lymphocytes
and often display a lighter staining central area, the germinal centers,
and during an active phase small lymphocytes are produced by the cells
of the germinal centers and are pushed outuard into a zone which becomes
the cortex of the nodule. In the thymus lobes are encapsulated by connec­
tive tissue and extensions from it (septa) delineate the lobules. The
reticular connective tissue of the thymus differs from that of other
lymphoid organs since it arises from entoderm rather than from mesenchyme:
it displays epithelioid characteristics. The cortex contains small
lymphocytes (thymocytes), which are very densily packed among a few
reticulum cells while the medulla contains less thymocytes and more
reticulum cells. Hassalls corpuscules consist of a concentric formation
of epithelial cells (for review· see ref. no. 120)?
The main histological changes observed in the thymus from the high
leukemia AKR strain before the onset of leukemia are:
a) the thymic cortex is reduced in size and structures resembling lymphoid
follicles and germinal centers appear in the medulla (120),
b) spontaneous leukemia arises only in one lobe of the thymus (120,185),
c) this lobe displays cortical inversion i.e. selective depletion α f
31
cortical thymocytes and enlargement of the medullary region,
d) with aging focal pockets of blast-like cells appear in the outer edge
of the thymus,
e) these cells are finally displaced by rapidly dividing leukemia cells.
Metcalf (122) concluded from these observations that progression of
leukemogenesis is associated uiith a graduated commitment of lymphoid,
preleukemic cells to the malignant phenotype. The depletion of cortical
thymocytes and the appearance of structures resembling lymphoid follicles
in the thymus medulla led people to suggest that the preleukemic thymus
may be the site of an autoimmune reaction (120). This could in some uiay
initiate the leukemogenic process. Facts consistent with the idea that
AKR mice are undergoing an immunological reaction to antigens o f thymic
origin, present on preleukemic and leukemic thymocytes are:
a) the finding of immunoglobuline with MuLV reactivity in glomeruli of
AKR kidney (145,146),
b) the observations that leukemic cells are selectively destroyed after
injecting AKR mice with complement (84), and that Ιοω levels of viral
antibody can be detected in the serum of AKR mice (72,134).
The source of the antigens could be either or both of the endogenous
viruses (ecotropic and xenotropic) which are expressed in the thymus
or a recombinant NCF-type virus that is generated in the thymus (65).
B) ROLE OF THE THYMUS DURING LYMPHONAGENESIS.
["lost leukemias discussed in this ге і ш like those induced by M-PluLV
and AKR-CluLl/ are characterized by proliferation of either prothymocytes
or mature T—cells. Therefore, some basic characteristics of T-cell
ontogeny are discussed in relation to thymus-function in normal mice
and mice prone to leukemia development.
The thymic environment is of critical importance in the differentiation
and functional maturation of T-cells. During embryogenesis stem cells
migrate into thymic epithelial remnant at about day 11 of gestation.
Precursor bone marrou cells (prothymocytes) migrate to the thymus in
post-natal animals (15,199). After processing these cells become
functionally competent and are exported into spleen, lymph nodes,
blood, and other peripheral lymphoid systems like Peyer's Patches along
the gut (14B). Changes in cell-surface antigens mark the various stages
of T-cell ontogeny (160) e.g. for the murine system the following
simplified scheme has been developed (15,118,198):
32
STCn CELL (bone marrouj)
PROTHYHOCYTES: T l . " ; T h y - 1 ~ ; L y ~
i n THYCIUS: v a s t m a j o r i t y o f l y m p h o c y t e s аг
+
+
T h y - 1 ; r L ; L y - l í " 2* 3 +
nATURATION
I
SUBPOPULATIONS
CYTOTOXIC T-CELLS
(l-A+)
AND
SUPPRESSOR T-CELLS (l-3 + ):
Thy-1+; TL"; Ly-2t 3 +
INTERMEZZO: l) The Thy-1 locus is on chromosome 9, and has tuo alleles
Thy-1
and Thy-1
which code for Thy-1.1 and Thy-1.2 respectively; Thy-1
is an antigenic marker specific for T-cells. 2) The TL-locus is described
in the next section. 3) The Ly-1 locus is on chromosome 19 and has tuo
alleles which code for 67k glycoproteins! Ly-2 and Ly-3 are two closely
linked loci on chromosome 6 and encode specificities on two closely
associated molecules or probably even on one molecule (118). Cytotoxic
T-cells are directly cytotoxic when in intimate contact with appropriate
targets. Helper T-cells help B-cells to produce IgG antibodies. Suppressor
T-cells regulate immune respons by suppressing the activities of other
Τ and B-cells. Cytotoxic T-cells express the MHC product I-A, while
suppressor T-cells express 1-3. Helper and cytotoxic T-cells can bind
to antigen only when it appears in close association with an MHC product
on the cell-surface, while suppressor cells can bind free antigen. Helper
T-cells react with I-region déterminants and cytotoxic T-cells interact
with Κ/θ products like suppressor cells (for review see ref. 49,91).
Several models have been presented, which indicate further differentiation
of thymocytes in both the thymus and periphery and which give rise to the
different peripheral thymocyte populations like helper T-cells, cytotoxic
T-cells, and suppressor T-cells (118). Tumors induced by AKR-NuLV and
N-CluLV have been typed mainly as Thy-1 , indicating a T-cell origin, but
heterogeneity appeared in expression of Ly and TL antigens. Similarly,
radiation-induced tumors in C57BL/6 or C57BL/10 mice have been shown to
be all Thy-1
but very heterogeneous with respect to the other T-cell
differentiation antigens (IIB).
33
The thymus has been considered previously to be the principal organ
in uihich both leukemic transformation and proliferation occurs (25,120)ι
a) most mice develop large thymic lymphomas; the thymus is the first site
of recognizable disease,
b) other lymphoid organs become subsequently enlarged,
c) invasion of non-hematopoietic organs (e.g. liver, kidney) is observed
during terminal stages of the disease,
d) most of these leukemias have T-cell markers indicating that the cells
have undergone some phases of differentiation in the thymus (144),
e) thymectomy in early life drastically reduces the incidence of overt
leukemia (both spontaneous and induced) development in mice (42,82),
f) subcutaneous thymus-grafting in thymectomized mice restores tumor
development; the neoplastic cells are often from host cell origin,
although leukemia develops in the thymus graft itself (56).
Tuo basic hypothesis on the role of the thymus in leukemogenesis have
been postulated:
1. The thymus is the major organ uhich provides cells that are most
susceptible to neoplastic transformation by leukemic agents (Θ3).
2. The thymus maintains the appropriate environment uherein potential
leukemic cells initially transformed in any of the hematopoietic organs
can proliferate actively into autonomous leukemic cells (25,124).
Most data ηοω clearly support the second hypothesis.
Preleukemic cells with leukemogenic potential have ηοω been detected
in bone таггош of intact and adult thymectomized irradiated C57BL/6 mice
and in irradiated bone-marrou protected mice. Similar results шег
obtained following treatment uith a chemical carcinogen. Spontaneous
occurrence of preleukemic cells has also been demonstrated in the bone
marrou of C57BL/6 mice in relation to age increase. Further, bone marrow
cells from 14 or 30 day old AKR/З mice can induce a high incidence of
leukemia of AKR/D origin, follouing transplantation into irridiated
recipients (57,59). Therefore, the initial transformation, spontaneous
or induced, into preleukemic cells often occurs in the bone marrou rather
than in the thymus (58). Although the target organ for overt leukemia
development of most spontaneous or induced murine lymphoid tumors is the
thymus (25), it does not imply that all phases occurring during thymoma
development take place in the thymus. Thymectomy prevents leukemia
development in AKR mice, but this does not a priori indicate that pre­
vention of leukemia development is caused by removing thymic lymphoid
cells susceptible to neoplastic conversion. These mice carry preleukemic
cells that are capable of repopulating and causing leukemia development
34
in subcutaneous thymus grafts. Since the normal thymus consists mainly
of turo basic components, a lymphoid component and the epithelium and
reticulum cell complex, it is very suggestive that these non-lymphoid
elements effect the conversion of preleukemic cells into leukemic celle
iidthin the environment of thymic tissue (25,61,97). Several studies
strengthen this hypothesis;
a) thymic remnants (mainly composed of epithelial reticulum cells) from
AKR mice restore a high lymphoma incidence in Gross AKR-virus-infected
thymectomized mice (54),
b) leukemic tissues of AKR mice, in contrast to non-leukemic tissues,
contain enlarged reticulum cells and large phagocytic cells derived from
reticuloendothelial cells (121); lymphoma cells in contact uith these
enlarged reticulum cells are shown to have heigher mitotic activity than
lymphoma cells situated in other sites of the thymic cortex (121). A
potential problem in these studies was the presence of small numbers of
lymphoid cells in the reticulum cell preparations that might display a
high mitotic activity and therefore could have significantly contributed
to the observed phenomenon.
In conclusion, several studies suggest that thymic epithelial reticulum
cells might be considered as a frameurork and favorable site for the
proliferation of transformed cells (25,53,151,212). The expression of
xenotropic virus, the putative parental molecules of uhich are involved
in the formation of recombinant leukemogenic MCF-type viruses, is also
confined probably exclusively to the thymus; since this xenotropic virusexpression seems also to be a neccessary step in the process of MuLVinduced leukemogenesis, its thymus-restricted appearance is also suggestive
for the framework hypothesis. The interaction of lymphoma cells with
large reticulum cells embedded in the thymic matrix might be essential
for the continued proliferation of lymphoma cells in primary lymphomas.
Whether "Thymic Nurse Cells" play an important role in this process is
not known. These epithelial cells carry large numbers of engulfed lymphocytes which are released after a certain period. TNCs also contain high
amounts of ñHC-determinants (κ/θ and I-A) and were therefore suggested
to play a role in the process of "self-recognition" by thymocytes (213;
see also discussion).
C) CHANGES IN CELL-SURFACE ANTIGENS.
Animals show an immune response to the surface of cells infected with
RNA tumor viruses. This response is due both to 1) the expression of
viral structural antigens, as well as to 2) virus-induced cell surface
35
antigens (96). The viral structural proteins proved to be markers for
a classification of different stages that can be seen during leukemo—
genesis. Noudnski and Doyle (135) divided the amplification of viral
gene expression as detected by the appearance of viral cell surface
antigens into four discrete stages:
1) loid numbers of cells from thymuses of young (2 month old) AKR mice
express p30 (less than 0.255?) and gp70 (2-75?) antigens; gp70 is only
expressed on the large cells in the subcapsular region of the thymus;
this phenomenon was also observed after inoculation of mice uiith RadLU
or other exogenous MuLl/s (2Θ).
2) Thymuses of 6 month old AKR mice shou/ a selective depletion of cortical
thymocytes uiith a concomitant increase in the medullary region of the
thymus. Increased numbers of cells express an elevated concentration of
p3D and gp70 antigens. Viral antigens are found on the cell surface of
all large cells of the subcapsular region of the thymus and in variable
numbers (2-85^) on small cells of the cortical and medullary regions.
3) Thymuses of some θ month old AKR mice show selective hypertrophy of
a single thymic lobe. This enlarged lobe contains a population of cells
that are intermediate in size between small cortical cells and the large
leukemic blast cells. These intermediate-sized cells express significant
levels of p30 and gp70 viral antigens. However, transfer of high numbers
of these cells (about 10 per injection) to syngeneic hosts did not induce
transplantable disease, so these cells are not leukemic. It was suggested
that these cells of intermediate size represent preleukemia cells.
4) Thymuses of mice with overt leukemia contain primarily large leukemic
blast cells which probably arise as clonal descendants of one or a few
preleukemic cells that have acquired transforming capacity. The large
2
3
leukemic blast cells (10 -10 per injection) are causing leukemia after
transplantation and they also express high levels of viral antigens on
their cell surface. The gp70 antigenicity was found in a 70k polypeptide
on the cell surface and corresponded to the viral envelope protein, while
the p30 antigenicity was contained in two polypeptides of 85k and 95k;
these both correspond to glycosylated forms of the polyprotein product
of the gag—gene.
The high expression of viral antigens, which is mainly detected on the
large thymocytes, occurs contemporary with the process of cortical
inversion (117,135) and this might suggest a causal relationship between
these two phenomena.
The age related antigenic changes observed in 5 to 6 month old AKR
preleukemic thymocytes (in contrast to the 2-month-old thymus) are
36
restricted to the thymus and are not found in spleen or lymph nodes.
The level of xenotropic MuLV is markedly increased (a 100 fold) and
correlates uiith the amplified expression of MuLV-relatsd antigens (25,
135), but the amount of ecotropic MuLl/ in 2 and 6 month old AKR thymus
increases only about a 10 fold (86).
Additional antigens related to nuLU-Gross (GR„DA:,» ^ Ш П 'a n d GAKSL2^
are detected on leukemias and normal lymphoid tissues of strains like
AKR, С5 , and СЗН/Гд which all display a high incidence of leukemia,
GRi.n.T-antigen uias defined by naturally occurring antibodies directed
predominantly against N-tropic viruses (140), G_DI -antigen by natural
type-specific antibodies against xenotropic virus (197), and G
. „-
antigen by natural antibodies specific for NCF-type dualtropic viruses
(197). GRflDn-r» G ι , and G
-antigens are expressed at Іош levels
on normal thymocytes of 2-month-old AKR mice like the antigens GCSA and
GIX (an antigenic determinant on the gp70 molecule; see follouing section).
All five MuLV-related cell-surface antigens ars amplified on AKR thymo­
cytes at 5-6 months of age. This age-dependent antigen—amplification is
correlated uiith increased expression of xenotropic MuLl/ and the emergence
of dualtropic NuLV in the thymus (141). Testing a variety of cloned
dualtropic viruses from AKR mice shoued three different env-gene pheno-
types: i = и х + / с Я Д 0 Д І 7 п
G
X
111= I /
G
R A D A I
7G
+
A K S L 2
;
n =
GUVG^^/G^/G^^,
~ / G E : R L D /G A | < S L 2 "/. А Н dualtropic isolates induced GIX
and G_D1 r,, probably reflecting ecotropic and xenotropic characteristics
respectively in the recombinant env-gene of dualtropic viruses. Viruses
of phenotypes I and II induced GCSA (indicating an ecotropic NuLVderived gag-gene) uhile phenotype III dualtropic viruses displayed only
partial absorption of GCSA. This indicates that these type III dualtropic
viruses contain non-ecotropic FluLV sequences which extend into the gaggene. Recently, recombinations extending into or near to the gag-gene
have also been detected by restriction mapping of proviral DMAs of
several dualtropic viruses (Chattopadhyay, S.K., personal communication).
In conclusion, AKR dualtropic NuLVs are quite heterogeneous as shoun by
their different antigen-phenotypes, restriction maps, and also by their
different infectivities on mouse and mink cells (141). Whan AKR dual­
tropic FluLVs uere tested for their ability to amplify MuLV-relatsd cell
surface antigens on thymocytes of 6-8 шеек old AKR recipients it appeared
that antigen-amplification and leukemia-acceleration are tuio distinct
phenomena. Only dualtropic viruses with phenotypes I and II (GIX /G—. _, /
G.j,-. _ ) induced antigen-amplification but not all these isolates
accelerated leukemia development. No marker has yet been found which can
distinguish between the antigen-amplification and leukemia-acceleration
37
phenomena on a molecular basis. Recently, it mas suggested that there
might be a correlation between the presence or absence of certain
restriction sites on dualtropic MuLV prov/iral DNA and leukemiaacceleration capacities of the respective viruses (Chattopadhyay, S.K.,
personal comminication). These data, however, are to preliminary; viruses
obtained after transfection of cloned dualtropic MuH/ proviral DNA have
not yet been tested for their leukemia-acceleration properties. Positive
results from such experiments would suggest that antigen-amplification
and leukemia-acceleration are phenomena which can be distinguished on
a molecular basis (i.e. the exact range which spans the recombination).
Since the dualtropic MCF-specific antigen G „
„ is also expressed in
low amounts on AKR thymus and bone marrow at two months of age (197) it
was suggested that G
ia a determinant of a differentiation product
of lymphoid cells related to a xenotropic NuLV gp70 molecule that also
contains the Gfll._. „ determinant. Recombinant dualtropic MuH/s that have
acquired such a cellular gp70 encoding sequence might display extended
host range properties and antigen-amplification, and also rarely leukemiaacceleration characteristics. O'Donnell et al., (141) have postulated
that the hypothetical AKR thymocyte gp70 could be encoded by a genetic
locus analogous to Gv-1 locus of 129 mice encoding GIX antigen (for review
see ref. 141).
2) Virus-induced cell-surface antigens.
All leukemias induced by Gross AKR virus have a common surface antigen,
GC5A, while those leukemias induced by rwiuLV and R-CluLV express NCSA
(66,8Β,Θ9). Antisera to GCSA recognize determinants expressed on the
viral internal components p30 and pi 5, and is expressed on the cell
surface in the form of a glycosylated MuLV gag-gene-coded polyprotein
(99,122,123,186,205), while antisera to I*1CSA recognize an env-related
protein distinct from the exogenous M-PluLl/ envelope protein (66). In
M-PluLV-inoculated viremic mice the target tissue for M-PluLV leukemogenesis,
the thymus primarily, is the first organ to express MCSA. NCSA positive
cells in lymph nodes or spleen occur only after overt leukemia. NCSA
positive cells (thymus) are non-malignant since these cells do not grow
as progressive tumors in syngeneic recipients (81). This indicates that
NCSA expression is associated with viral gene amplification preceding
leukemia development i.e. a high level of viral protein expression in
the preleukemic thymus of AKR mice (85,86,135).
Another MuLV-Gross associated determinant is the GIX antigen, which
was initially detected on the lymphocytes of some strains like С5 , A,
за
СЗн/Fg, AKR, NZB, and 129 (193,195). GIX is an antigenic determinant on
the gp70 molecule expressed on thymocytes, sperm, and other tissues from
several mouse strains, even in the absence of productive viral infection
(139). The expression of the antigen in the prototype 129 strain is
dependent on tuo unlinked genes: Gv-1 on chromosome 17 and Gv-2 on
chromosome 1 (196). The expression of GIX requires the presence of both
Gv-1 and Gv-2 positive alleles (193). In contrast to 129 strain, in AKR
mice the Gv-1 locus maps on chromosome 4, suggesting a rather complex
genetic linkage system (13,74). Earlier observations suggested that only
ecotropic riuLU could induce the expression of GIX in mice or cell lines
which have been characterized as shouing no GIX expression (132), but
more recent studies suggest that GIX gp7D is in fact coded by a xenotropic viral genome (37,203). The GIX specificity differs from GCSA in
the fact that GIX can be expressed in the absence of replicating virus
and productive viral infection as in strain 129 (105,138,204). Infection
by most ecotropic viruses, particularly by N-tropic viruses, generally
leads to expression of GIX antigen, even in strains classified as being
GIX
like ЭаІЬ/ü (143). The GIX antigen often app-ars on leukemic cells
even in mouse strains uhnre it is not detectable nn nomai tissue cells.
Other cell surface changes in spcntaneous and induced murine T-cell
leukemias are reduced levels of the differentiation alloantigen Thy-1
and increased levels of H-2 alloantigens (18,85). These are all surface
characteristics of the minor thymus cell subpopulation and therefore it
шаэ suggested that these transient changes in thymus subpopulations might
provide a favorable milieu for the migration of preleukemic cells from
the site of their induction in the bone таггоы into the thymus (57).
Antigens of the TL series (TL= Thymus Leukemia; 118), regulated by the
TLa locus, show irregular expression on thymic lymphoma cells. The TLantigen is probably coded for by the TLa locus adjacent to the H-2D locus
on chromosome 17 (for review see ref. 118). The TLa locus governs cellsurface differentiation alloantigens which are found on thymocytes of
some strains (TL ), but not in other strains (TL ) . Mice bearing malig­
nancies of T—cell origin do express TL antigens on leukemic cells even
if the strain is classified as TL - · TL-products resemble the К and D
products of H-2. There is no known relationship between molecules
bearing TL antigens and any virus-encoded molecule. The cell-surface
thymocyte-specific alloantigen TL is expressed on thymocytes shorlty after
irradiation of C57BL/6 mice. It was therefore suggested that activation
of the TLa locus occurs during preleuketìic phase of radiation-induced
leukemogeneais and that TL might be a marker for preleukemic changes in
•39
the thymus after X-irradiation-induced leukemogsnesis (194). The TLa
locus defines several specificities found on thymocytes and leukemic
cells, but absent from normal peripheral lymphocytes or other tissues.
The TL specificities seem to be the only ones found exclusively on
intrathymic T-cells (118). Because of the irregular expression pattern
of the TL locus during HuLV-induced leukemogenesis its role is still
obscure in this process.
LATENT PERIOD OF THE DISEASE.
The spontaneous and induced development of murine T-cell laukemias is
characterized by a long latent period up to 9 months in e.g. Balb/ño and
AKR mice. The characteristic long latency of leukemogenesis is illustrated
by the follouing examples:
a) AKR mice display overt leukemia 6 to 9 months after birth, although
this strain stems from prolonged inbreeding and selection for leukemia
(the AKR strain is a derivative from the Ak strain (ill) developed by
Jacob Fürth (43)).
b) Inoculation of the potent leukemogenic virus RadLU into newborn C57BL
mice also involves several months before overt leukemia is obvious (55).
c) Balb/rio and AKR mice, which carry and express M-MuLV and AKR-MuLV
endogenous provirus respectively, also do not develop leukemia before
9 months of age, although M-MuLV expression in Balb/Mo mice occurs from
about 3 weeks after birth (79), and AKR-PluLV expression was detected in
prenatal AKR mice already.
d) Inoculation of M-MuLl/ into nauiborn Balb/c and 129 mice also involves
a long latent period. The length of this period is grossly dependent on
the number of infectious particles used for inoculation e.g. 10
infectious
n-PluLV/ particles (аз assayed by XC-plaque formation; 171 ) yield a latent
period of about 10-12 weeks, while 10-20 infectious particles yield a
latency of about 6-
months (76,163,206).
These observations suggested that the long latency of leukemia development
may represent the time required for neoplastic transformation and proli­
feration of target cells, or it may reflect a series of changes occurring
in both target cells and host. Leukemic cells seem to undergo a series of
sequential changes defined as phases occurring during tumor development.
The expression of overt leukemia might depend on the completion of a
sequence
of events involving tumor-cell-host interactions like those
summarized in the following scheme (modified from ref. 57):
40
(Preleukemic cells not responsive
to host factors (e.g. Nude mice:
no immune respons by T-calls)
^ Overt
leukemia
Leukamogenie
treatment
(chemicals,
radiation,
viruses)
Preleukemic cells controlled by
host factors:
1) anti-viral antibodies and
cell-mediated immune responses
2) influence by H-2 haplotype
3) influence by Fv-1 alleles
4) maternal suppression
i
Cell proliferation
Differentiation
Changes in
chromosome
number
i
A l t e r a t i o n s at DNA-level
e . g . a m p l i f i c a t i o n of
p r o v i r a l genomes
Leukemic c e l l s ^
1
Progressive growth of tumor
Overt leukemia
The development of overt leukemia can Ьв markedly delayed or aven can be
suppressed because of the influence of host factors like anti-viral anti­
bodies and cell-mediated immune responses, the H-2 haplotype, the Fv-1
alleles, and certain not yet molecularly characterized maternal factors.
1) Anti-viral antibodies and cell-mediated immune responses.
Lymphocytes and antibodies that react against virus-envelope antigens
may exert beneficial effects against oncogenesis because they reduce
viremia and the number of secondary infections (32,68,175). Further,
such antibodies may kill the virus-infected cells due to the virusenvelope antigens expressed on the cell-surf ace-membrane (96).
A factor, uihich also influences the delay of leukemogenesis is the
transient immunoprevention of preleukemic cell proliferation. Ultimately,
hoiuever, this proliferation is followed by breakdown of immunity preceding
overt tumor development. Antiviral neutralizing activity is detected up
to several weeks before overt leukemia occurs in C57BL/6 mice, injected
when newborn with RadLU (59,60). The delayed appearance of leukemia in
41
these animals is H-2 linked and involves an arrest of leukemia development
at the preleukemic stage (57) f so these animals carry immunogenic preleukemic cells (59,60). The abrupt disappearance of antiviral noutralizing
activity shortly before overt leukemia is not yet inderstood. Such an
effect might be due for example to a gross imbalence between numbers of
suppressor T-cells and other T-cells (129).
Studies nn immunization of mice with syngeneic Moloney lymphoma cells
also suggested that mainly antibodies to preleukemic cells rather than
antiviral antibodies р г эе might be able to interfere with preleukemic
cell proliferation. This antibody response to preleukemic cells may prevent
or delay overt leukemia development (39,184). However, since antiviral
antibodies (anti-gp70 and anti-p15E) also neutralize surface—antigens
of virus-infected cells it is hard to discriminate the biological
consequences of a pure antiviral and anti-preleukemic-cell antibody
response. Only preleukemic AKR mice ш г
found to contain immunocompetent
lymphocytes that can participate in different immune reactions, since
only lymphoid cells from preleukemic AKR mice ш г
reactive (62).
Preleukemic AKR mice also display specific antitumor antibodies and
a non—T—cell spontaneous antilymphoma killing reaction (50).
The presence of preleukemic cells at a stage when mice do not shou
clinical manifestations of a disease has been shown in several mice (57).
Preleukemic cells present in the bone marrow shortly after X-ray induction
(61,97) as well as in some of the early spontaneously occurring preleuke­
mic cells in AKR mice have been shown by several criteria to be committed
precursor T-cells (prothymocytes; 59). Transplantation studies further
indicated that proliferation of these preleukemic cells is dependent on
specific host environments (e.g. intact thymus; H-2 haplotype) as con­
trasted to leukemic cells which can proliferate without an intact thymic
environment (57).
2) The influence of the H-2 haplotype.
The capacity of the host to mount an effective immune response is
markedly influenced by its genetic constitution with respect to the
major histocompatibility complex, H-2 on chromosome 17 (123,156,158,1B5a).
This chromosomal region carries closely linked determinants of a great
number of immunological phenomena (91). The K—end and the D-end genes
of the H-2 region influence the.occurrence of an effective immune response,
capable of destroying cells infected with or transformed by type-C viruses.
The K-end genes seem to exert their effect on the responding lymphocytes,
while the D-end genes appear to influence the immunogenicity of the virus-
42
infected cells themselves (10Θ). The latter is examplified by the follow­
ing observations. The most important immune response to virus-infection
consists of generation of a population of cytotoxic
T—lymphocytes (CTL)
capable of lysing virus-infected cells (24). The modification of these
CTLs to killer cells specific for virus-infected target cells appears
to be dependent on the recognition of tura different types of molecules
on the stimulating cells: a) certain viral molecules expressed on the
cell surface and b) molecules governed by the H-2K and/or H-2D genes
of the cell (114af217)» e.g. Friend-virus-encoded molecules become
associated with H-2D
molecules on the surface of infected cells and
this protein complex induces the CTL response and is recognized by the
effector CTL population (10).
H-2 linked genes also seem to exert an influence on the capacity of
the host to produce anti-type-C virus antibodies (133,136), Helper Tcells can recognize viral antigens in association with I-A antigen and
stimulate B-cells to produce anti-gp70 (133,136). These antibodies can
prevent virus-spreading to some extent and may therefore also be able
to delay the leukemogenic process (132,175). This was also shown
experimentally by injecting anti-gp7D antibodies into neuborn Balb/flo
(132) and AKR mice (69,175); the largest suppression was observed when
animals ware injected with antibodies within the first three days after
birth (178).
3) The influence of the Fv-1 alleles.
The non-H-2 linked host gene, Fv-1 (106), is an additional factor which
is involved in sensitivity or resistance to preleukemic but not to
leukemic cell proliferation. The Fv-1 gens is involved in the regulation
of virus-spreading (107,168) and acts at a level before integration of
viral DMA into the host genome (215). Ths viral targets for the Fv-1
restriction mechanism are probably located in the gag-proteins p30 and
p12 (for review see ref.44,27), while also a requirement for intact 355
genome RNA has been implicated (7). Evidence has been presented that a
complex between the pol-gene-product and p30 may enhance reverse trans­
criptase activity (4). Although the Fv—1 alleles regulate at a level
before integretion of viral DNA into host cell DNA, the different alleles
fall into two classes according to their effect on altered viral DNA
formation in с ЭІз with restrictive Fv-1 alleles: 1) in Fv-1
cells inoculated with B-tropi3 virus, as uell as in Fv-1
(DBA/2)
(Balt/o and
C57BL/6) cells inooulated with N-tcopic virus, the generation of two
43
CQUalüntly closed circular siip^rcoildd v/tral DNA duplex (form I) was
dnprossed but linear full-length viral DMA duplex (form III) was synthesized in normal quantities. 2) In Fu-1
(NIH Swiss mouse embryo) cells
inoculated with B-tropic virus, synthesis of fcnim III viral DNA mas
markedly inhibited. Also, B-tropic virus that is able to escape restriction in DBA cells by mutating to the NB
-phenotype, is still restric-
ted in NIH cells (106). Peptide analysis of p30 also revealed that the
change from B-tropic to NB
-tropic and to NB
-tropic is associated
with tura different changes in primary structure of p3D (44). Since the
Fv-1 locus can regulate to some extent preleukemic cell proliferation
it mas suggested that a second viral event might be involved in the
process leading to autonomous proliferation (57).
The influence of factors like H-2, Fv-1, and a maternally transmitted
resistance factor have mainly been studied by using different combinations
of mouse crosses. The Fv-1 locus on chromosome 4 is кпошп to have several
alleles, operating both in vivo and in tissue culture cells. Fv-1
defines susceptibility to B-tropic virus and relative resistance to Ntropic virus replication uihereas Fv-1
cells are relatively much more
susceptible to N-tropic virus replication. Different types of Fv-1
alleles are also кпошп which define different susceptibilities to Ntropic virus replication. The Fv-1
allele of AKR mice dictates a
very high suaceptibility to N-tropic virus replication. The Fv-1
allele
present in RF mice gives rise to similar leuœls of resistance to B-tropic
MuLV infection as the Fv-1
allele and therefore both alleles have
been designated as N-type alleles (115,157). Ноше
г, the Fv-1
allele
confers resistance to spontaneous AKR-nuLV-induced lymphoma in crosses
η
к
with AKR mice (115). AKR mice (Fv-1 ;H-2 ) show high levels of infectious
MuLV in tissues at 6 weeks of age and near 100$ leukemia at Θ-12 months
of age. In Balb/c χ AKR (Fv-1 ' ; H-2
n
n
χ Fv-l ' ; H-2 ) mice, however,
both the occurrence of virus and the incidence of spontaneous lymphoma
are suppressed to very low levels (4$ after 1 year; 107). This suppression
of AKR-MuLU-induced lymphomagenesis is mainly due to the Fv-1
allele
inherited from the Balb/c parent, since this allele grossly prevents
spreading of the endogenous N-tropic AKR virus derived from the AKR
parent. Also, the Fv-1
allele of the AKR parent prevents spread of B-
tropic viruses that might arise from recombination between endogenous
xenotropic MuLV and N-tropic AKR-MuLV (45,63,87,155,165).
nice of the Balb/flo χ AKR/FU (H-2 d | Fv-1 b ' b χ Н ^ ; Fv-l n ' n ) cross
show a high incidence of leukemia at 6-9 months of age like AKR and
'Λ
Balb/no mice. Tumor DMAs contain only reintegrated authentic M-PluLl/
and recombinant MuLV/ proviruses derived from the NB-tropic endogenous
M-PluLl/ genome (no restriction by Fv-1 ' ) in Balb/Mo mice. Therefore,
only the NB-tropic M-nuLU of the Balb/no parent is able to amplify
and yield recombinant proviral DMAs while endogenous AKR-CluLV sequences
are restricted by the Fv-1 ' genotype of the F1 mice (our unpublished
results). Studies on (Balb/c χ AKR) Χ AKR backcross mice revealed further
that there is also a marked correlation between FluLV titer and leukemia
incidence, the animals with the highest titer showing the highest and
also the earliest incidence of leukemia (107). These studies revealed
in addition a marked influence of the H-2 haplotype (107,168): the H-2
allele was found to suppress lymphomagenssis to a large extent (almost
50$ in Fv-1 ' mice). The mechanism by which the H-2 allele exerts its
suppressing activity on lymphomagenesis is probably similar to the one
discussed in section b: the generation of a population of cytotoxic
T-lymphooytas (CTL), ishich recognize virus-infected cells via an
Н-2-viral-product combination on the surface of infected cells. Leukemia
is also known to occur somewhat more frequently among females than among
males of the AKR strain, although the backcross studies mentioned above
did not reveal a significant difference between sexes. The H-2 type
was further shown to have little or no effect on the early expression
of MuLV in the backcross mice and its influence is presumably caused by
immunosurveillance of CTLs on the outgrowth of transformed cells.
4) Claternal effects on suppression of lymphomagenesis.
The maternal effect of suppressing leukemogenesis was first observed
in (Balb/c χ AKR)F1 X AKR mice crosses and other crosses with Fv-1 mice,
Suppression was only observed in F2 animals when the mother was the Fv-1
heterozygote (F1). According to the following scheme 50$ of the F2 mice
are Fv-1 ' and these mice show a marked suppression of lymphomagenesis
by maternal factor(s):
50$ F2: Fv-1 ' : suppression
by maternal
/
faotor(s)
4
(β Balb/c χ e "AKR)F1
n
Fv·'-l /
b
{
X
«/"AKR
n
'A
Fv-1 /
n
50$ F2: Fv-I
,
n/
: N-tropic
virus is
restricted
by dominant
Fv-1 allele.
45
Balb/Pio mouse strain
Γ
Endogenous virogenes
Absent"
No expression or
no replication
(Fu-I allele of
Qalb/Πο mouse
suppresses N-tropic
AKR-NuLV replication)
.Present: 1)
2)
3)
4)
n-MuLV
AKR-MuLV
xenotropic I»luLl/
gp70—encoding genes e.g. defective
xenotropic MuLV
No suppression of replication
Transient expression of Mov-I M-CluLV
BONE
MARROU
secondary infections
Infection of target cells of T-lineage
I
A m p l i f i c a t i o n o f M-fluLU рго і г э і DNA
PRELEUKEMIC CELLS: 1) contain "randomly" integrated FI-MuLl/
copies
2) no clonal expansion:
a) not yet transformed by MCF
b) immuno-surveillance
Expression of other virogenes and/or virus-related genes:
1) xenotropic MuLV at about 3 months of age
2) other gp70-encoding genes e.g. the gene coding for the
G
-determinant (defective xenotropic MuLV ?)
THYMUS
Replication of Cl-MuLV and expression and/or replication
of xenotropic information in one cell
-recombination
Chronic immune stimulation
1) a variety of MCF-type viruses are generated
2) provides a large population of putative target cells
3) immunoselection of MCF-type viruses
Result: Transformed cells by: a) recombinant gp70
b) promoter-insertion
Disappearance of anti-viral neutralizing activity shortly
before overt leukemia
Clonal expansion of transformed cells containing M-MuLl/ and
MCF
Metastasis
Outgro'in tumors: largely composed of descendants of one or a
feu transformed cells
Figure 4: Cascade model for tumor formation in Balb/Мо mice.
46
Similarly, the effect was obaerued in crosses of RF mice with AKR mice
after backcrossing with AKR in the Γ2 generation (116). RF mothers
transmit two lymphoma—suppressing factors, Fv/-1
allele and a maternal
factor(s). The effect generated by the maternal factor(s) is transmitted
from Fl mothers to F2 generation but not by Fl males, although the
inhibition is seen in both sexes. In crosses of RF mothers with AKR
fathers also a potent suppression of endogenous ecotropic MuLV expression
is passed to their offspring by RF mothers. This effect was shown not to
be transmitted to the (RF χ AKR) χ AKR backcross like the suppression of
lymphoma. This indicates that there are in fact two different maternal
effects probably mediated by two different factors. One maternal factor
was suggested to represent an antiviral antibody. It was also concluded
from such data (6,116) that high ecotropic MuLV expression seems to be
necessary though not sufficient for development of spontaneous lymphoma.
The maternal suppression of lymphoma seems to influence only the
spontaneous occurrence of the disease since it was not observed for
leukemias of RF mice induced by AKR Gross passage A virus, 3-methyl—
cholantrene, and X-ray irradiation (116).
DISCUSSION.
The data summarized in this review indicate that many genetic factors
from both host and virus are involved in the generation of MuLU-induced
lymphomas. However, one can reduce the sequence of events to basically
two steps: 1) the formation of preleukemic cells, and 2) the clonal out­
growth of preleukemic cells which have acquired leukemogenic properties.
A cascade model shown in Figure 4 contains most of the data summarized
above and places all the observed phenomena associated with leukemogenesis
in a prospective order; since the different phenomena associated with
leukemogenesis were actually observed in many different animal systems
the sequence of events might show unique features in each strain which
develops leukemia. The viral (FI-nuLV) etiology of leukemogenesis in
Balb/do mice is shown as an example although probably the same scheme
applies to Balb/c and 129 mice infected uhen newborn with M-PluLl/, to
Г1(Ва1Ь/Мо χ AKR) mice, to AKR-HuLV-induced leukemias of AKR mice, and
in general to mice which show MCF-type virus-expression during leukemo­
genesis (for review see Figure 1). Uhebher radiation-induced leukemias
follow also part of this scheme or take a different route is not yet
clear. The finding of recombinant qp70 molecules on the cell-surface
of radiation-induced leukemias in NIH Suiiss mice, without concomitant
47
expression of virus, strongly suggests thet the etiology of these leukemias
is associated u/ith the expression of dualtropic-MuLV-like gp70 molecules.
Preleukemic cell formation.
The basic molecular mechanisms which are associated with the formation
of preleukemic cells which can be potentially transformed are not known.
Preleukemic cells of N-PluLV-inoculated mice or Balb/do mice do show
random integration of authentic M-MuLV genomes. Therefore it is not very
speculative to assume that an effect caused by reintegrated authentic
M-PluLV genomes is responsible for the formation of preleukemic cells in
these mice. It might well be that preleukemic cells are in fact generated
by the integration of an authentic Cl-CluLV genome in a limited number of
chromosomal DNA sites o F lymphocytes. Such an integrated M-PluLV provirus
might exert an effect by promoter-insertion or by some other mechanism
disturbing cellular gene regulation e.g. the induction of cell-surface
receptors. Promoter-insertion (i.e. the viral promoter in the large
terminal repeat is used to transcribe a nearby cellular gene like a
potentially transforming gene) was shown for Avian leukosis virus-induced
3-":ell lymphomas in birds (130,131,149). The integration of an authentic
M-MuLV in a limited number of different chromosomal sites in cells of the
T-lineage might in fact explain the phenotypic heterogeneity of H-MuLUinduced lymphomas
(as detected by heterogeneity in cell-surface diffe-
rentiation antigens). However, this heterogeneity could also be explained
by the variation in recombinant proviral DNA structures integrated in
different tumors as discussed below. RNA-analyses of a variety of MuLVinduced lymphomas will hopefully solve the issue whether or not authentic
and/or recombinant MuLVs are involved in promoter-insertion.
The switch from preleukemic to leukemic state.
The mechanisms involved in the switch from preleukemic to leukemic
state are still speculative. Several hypotheses have been postulated.
The acquisition of leukemic potential by preleukemic cells reflected
by the property of autonomous proliferation might occur via:
1) a differentiation or dedifferentiation sequence,
2) via somatic mutations,
3) via chronic immune stimulation which is the driving force for the
selection of leukemogenic recombinant viruses.
Most data summarized in this review support the last hypothesis as
discussed below.
1) A differentiation or dedifferentiation sequence.
Since many T-cell leukemias are cheracterized by proliferation of cells
which are not fully differentiated T-cells one can imagine that a block
in differentiation or even a dedifferentiation of such cells gives rise
46
to an accumulation of cells at certain rlevelopmental stages: feed—back
control systems of mature cells or in general systems that regulate grou/th
and size of T-cell populations could be suppressed. Houeuer, the observed
v/ariation in differentiation stages involved in T-cell leukemogenesis
is probably due to secondary effects: many tumors of different origin
express antigens of embryonic origin (125) and this is probably due to
a gross imbalence in gene regulation.
2) Somatic mutations.
Mutagenic agents have been shown to confer autonomy to preleukemic cells
i.e. prcliferation in both normal and irradiated recipients (57). Different
kinds of somatic changes conferred to tumor tissues can be observed like:
a) chromosomal rearrangements or duplications e.g. trisomy 15 observed
in spontaneous T-cell leukemias of AKR mice and radiation—induced
leukemias of C570L/6 and 129 mice (31,187,214), but not in non-T-cell
lymphomas (90); deleted or translocated chromosomes among X-ray-induced
thymomas (31,214). b) Somatic amplification and reintegration of both
authentic and recombinant proviral genomes in M-fluLlZ-induced tumors (159,
206). Either шау of gene amplification (a or b) might give rise to an
imbalence in gene products which contributes to autonomous proliferation
of cells. It is also not very imaginary that the expression of dualtropic
virus-specific receptors requires first somatic mutations or gene
rearrangements in the receptor-coding sequences of the target cell; such
a mechanism uas shoun to contribute to the variation in heavy chain
variable regions in response to antigen (12).
3) Chronic immune stimulation which is the driving force for the selection
of leukemogenic recombinant virus.
It might uell be that the suitch from preleukemic to leukemic state is
determined by the results of a selection mechanism which eventually yields
a recombinant virus that causes leukemia. Selection of a recombinant
virus could probably occur via chronic immune stimulation by preleukemic
cells. Chronic immune stimulation seems to be requir^H for loul^emotjeiiesis
sir оь il IVÎS been shown that CBA/N mice, when inoculated with M-CluLU as
newborns, develop an acute viremia but do not develop leukemia or have
detectable T-cell responses against the virus (100). The hypothesis that
an immune response is required to generate appropriate target cell populations for virus-infection and/or transformation has been formulated as
follows:
a) acute viremia and expression of viral antigens leads to continuous
recruitment of antigen-reactive T-cells,
b) these T-cells produce a variety of soluble lymphokines which influence
the proliferation and differentiation of cells of the immune system
49
(macrophage activation, proliferation of early T-cells, thymocyte differentiation and maturation),
o) such a chronic state of proliferation and differentiation might increase
the probability of spontaneous events leading to transformation e.g.
trisomy 15 (31,1B7,214) and might also be the driving force for selection
of a recombinant virus. The result of such a selection mechanism seems to
be a leukemogenic recombinant virus.
The mechanism by which these recombinants are generated and selected
are not yet understood. The generation of recombinant FICF-type viruses
could be explained by molecular recombination betueen viral and cellular
RNA in certain cells resident in the thymus. For the moment it remains
unclear whether different cell types are involved in the formation of
recombinant viruses. A rather straightforuard hypothesis defines preleukemic cells as the original producers of recombinant MuLV« Preleukemic
cells do contain "randomly" integrated M-CluLV genomes in Balb/do mice
and M-MuLV RNA expression is probably restricted to these cells. The
same preleukemic cells probably express xenotropic-MuLU-like information;
its expression might be induced or enhanced by the ecotropic M-fluLV.
Subsequently, recombination on RNA level could generate a great variety
of recombinant viruses. Some of these viruses might superinfect preleukemic cells uhich contain already M-fluLV copies, or might infect other
target cells that are resident in the thymus. The thymic environment might
be essential in this respsct since it regulates differentiation of lymphocytes into various phenotypically different cell types uith many different
cell-surface receptors. Some of these receptors display affinity for MCFlike gp70. Superinfection of a preleukemic cell by a recombinant and
subsequent clonal growth of such a cell uould explain the finding of
both integrated recombinant and authentic Pl-MuH/ in DNA from cells of
outgrown tumors. Houever, pseudotype formation of authentic and recombinant n-HuLU could also explain the presence of both types of proviral
DNA in cells of outgrown tumors. Cells infected by such pseudotype virions
would contain both authentic and recombinant CluLV genomes. Since cells
in outgrown tumors of both AKR and Balb/Mo mice seem to express
simultaneously antigenic determinants characteristic for both authentic
and recombinant MuLU, it is very unlikely that different cell populations
exist which contain either authentic or recombinant MuLV.
The selection of a particular type of recombinant virus might be influenced by several factors. Since recombinant viruses have been isolated
which carry recombinations extending into the
1
gag-gene a selection a
priori on the recombinational level does no : seem to exist. The most
50
p l a u s i b l e explanation i s i n f a c t t h a t the recombinational event i s more
or l e s s random, generating a great variety o f recombinant s t r u c t u r e s .
Many d i f f e r e n t recombinant viruses are at t h i s stage produced by c e l l s
i n the thymus. At t h i s l e v e l s e l e c t i o n for c e r t a i n types of recombinant
viruses might o c c u r : 1) recombinant viruses could be selected f o r t h e i r
p r o p e r t y to i n f e c t t a r g e t c e l l s which subsequently become transformed.
Only recombinants u i t h c e r t a i n env-gene structures might be able to
i n t e r a c t w i t h thymocyte receptors (100,113,117,177,180). Chronic immune
s t i m u l a t i o n would be r e q u i r e d e i t h e r t o generate a population of thymo­
cytes heterogeneous with respect to t h e i r c e l l - s u r f a c e receptors or to
the MCF-type v i r u s which they produca. Selection f o r c e r t a i n recombinant
viruses could be i n f l u e n c e d by immuno—surveillance. P r e f e r e n t i a l s e l e c t i o n
o f recombinant v i r u s e s seems to occur uhich contain envelope antigens
t h a t are mainly derived from the h o s t . This might have two advantages
f o r the v i r u s . F i r s t , by exposing i t s e l f to the host environment via a
h o s t - d e r i v e d envelope p r o t e i n the v i r u s uould mora e a s i l y survive immunos u r v e i l l a n c e by a d a p t a t i o n . Secondly, by using a host-derived protein
which s e l e c t s f o r receptors on thymocyte c e l l - s u r f a c e the virus would be
able to i n f e c t c e l l s t h a t express c e l l - s u r f a c e receptors f o r t h i s h o s t protein.
The hypothesis based on chronic immuno-stimulation and s e l e c t i o n f o r
leukemogenic recombinant viruses explains the c o n s t i t u t i v e f i n d i n g of
a c e r t a i n type o f recombinant p r o v i r a l DNA s t r u c t u r e i n a l l outgrown
tumors (159,206). The data do n o t , Ьоше г , explain the transformed
character o f lymphocytes which carry integrated recombinant p r o v i r a l
genomes. The f o l l o w i n g remarkable features of MCF-type p r o v i r a l DNA
s t r u c t u r e s which are c o n s i s t e n t l y detected i n DNA from o u t g r c j n tumors
o f both M-MuLV and AKR-duLV induced leukemias may shed more l i g h t on t h i s
case. A l l MCF-type recombinant p r o v i r a l DNAs (both i n t e g r a t e d and uni n t e g r a t e d ) d i s p l a y a Bam HI r e s t r i c t i o n s i t e at p o s i t i o n 6.3 kbp from
the S'-end, Recently several r e s t r i c t i o n maps of endogenous xenotropic
v i r u s e s have become a v a i l a b l e . None o f the i n d u c i b l e xenotropic p r o v i r a l
genomes c a r r i e s a Bam HI s i t e at p o s i t i o n 6.3 kbp. However, such a s i t e
was detected i n cloned p r o v i m i DNAs o f the non-inducible xenotropic
MuLU-ol^ss viruses which are present i n genomes of most mice l i k e also
i n NIH Swiss mice (D.M. Young, personal communication). These noni n d u c i b l e xenotropic-MuLV-like sequences seem to represent defective
p r o v i r a l genomes. They could be c e l l u l a r genes coding f o r e . g . thymocyte
gp70 molecules which are r e l a t e d to the G .,
-determinant, the antigen
which i s d e t e c t a b l e at low l e v e l s on e.g. 2-month-old AKR thymocytes.
51
Therefore, it is likely that this class of endogenous viral sequences
provides the neu вп -д пв sequences in leukemogenic MCF-type viral
genomes. This is supported by the finding of a recombinant-CluLV-gpTO
on radiation-induced thyraomas in Suiss mice. The recombinant gp70 on
these lymphomas uas shown to contain peptide characteristics of the gp70
of the non-inducible xenotropic-fluLV-claas (40). From these data it can
be suggested that gp7Q may be a transforming protein by itself. In fact
the finding of such a recombinant gp70 on radiation-induced leukemias in
Swiss mice without concomitant virus-replication supports this hypothesis.
The high amount of recombinant gp70 molecules on the membrane of lympho­
cytes could be the cause of the transformed phenotype by changing
physiology of a cell and/or cell-to-cell interactions. It might be that
a recombinant gp70 molecule contains the information that uould be
sufficient to specify the distinctive and characteristic phenotypes of
leukemic cells (51,95,114,152,183,218). The phenotypic heterogeneity of
PluLV-induced T—cell leukemias uould explain following observations.
Certain recombinants are leukemogenic u/hile others are not (110,169);
either the gp70 of the non-leukemogenic recombinants can not bind to the
proper receptor of thymocytes (117,177) or ot lacks the transforming
character. Different inbred strains of mice might encode genetic variants
of these receptors and this would explain why a particular recombinant
virus is not leukemogenic in some strains but leukemogenic in other strains
(20,51,169,177). Also, within one mouse different preleukemia cells could
carry different receptors and might therefore display cell-specific
differences concerning susceptibility to transformation by recombinant
viruses. Since HCF-type viruses have not only been isolated from T-cell
lymphomas, but also from myelomas, erythroleukemias and recently also
from mice showing neurological diseases and non-T-cell lymphomas their
role as tumor-causing agents in general in mice becomes very likely.
First, recombinant viruses or proteins display clearly tumor-associated
appearance. Secondly, the heterogeneity in recombinant proviral DNA
structures of the env-gena could well explain the great variety in
malignancies. For example, the recombination in Moloney-MCF type viruses
clearly involves the amino-terminal part of the env-gene and probably
even the whole gp70-encoding region, Ho-PlCF-type viruses are associated
with T-cell leukemias. The AKR-PICF type viruses display similar charac­
teristics. Rauscher HCF, however, is characterized by a recombinant envgene structure that carries newly acquired sequences in the middle part
of the env-gene whereas the amino-terminal part is derived from the
ecotropic parent. R-flCF is associated with erythroleukemias.
52
Finally, um are left uith the que-tion houi a particular cell type gaines
its transformed character. Тыо main hypotheses remain still open to explain
transformation :
1) the recombinant gp7D molecules might be the actual transforming agents,
or
2) transformation is caused by newly acquired sequences in MCF-type genomes,
that are located more proximal to the S'-end of the иігэі genome; most
leukemogenic nCF-type proviral DNAs carry xenotropic-PluLV-like sequences
in their S'-large terminal repeat ii/hile the pISF-encoding sequences are
mainly derived from the ecotropic parent. The acquisition of "xsno-like"
sequencer in the LTR could be responsible for an enhanced promoter
activity of sequences in the LTR. The З'ЧТВ of en integrated MCF-type
provirus might therefore function as a strong promoter for neighbouring
cellular gequences (putative cellular transforming genes).
Although most data support the first hypothesis, additional experiments
including RNA-analyses and testing of a variety of cloned recombinant
proviruses will ultimately resolve this matter.
In conclusion, the results summarized indicate that M-fluH/ and AKR-CluLV
induced leukemogenesis is associated with clonal outgrowth of tumor cells,
containing somatically acquired [*ICF-type viruses. These MCF-type HuLVs
are probably generated via random recombination with a particular sequence
expressed in certain cells. Chronic immune stimulation is a necessary
driving force. Probably the same xenotropic-CluLlZ-like cellular sequence
is involved in the formation of different NCF-type viruses which are
derived from the same parental CluLl/. The heterogeneity of nCF-type
viruses is due to the mechanism (random rscombination) by which these
viruses probably do evolve.
53
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62
Chapter
Cell Vol 18, 109-116. September 1979. Copyright© 1979 by MIT
II.
The Integration Sites of Endogenous and Exogenous
Moloney Murine Leukemia Virus
Herman van der Putten, Eugenie Terwindt and
Anton Berns
Laboratory of Biochemistry
University of Nijmegen
Geert Grooteplein 21 Noord
Nijmegen, The Netherlands
Rudolf Jaenisch
Henrich Pette-lnstitut fur Experimentelle Virologie
und Immunologie an der Universität Hamburg
Martinistrasse 52
2 0 0 0 Hamburg 20, Federal Republic of Germany
Summary
Specific cDNA probes of Moloney and AKR murine
leukemia viruses have been prepared to characterize the provlral integration sites of these viruses in
the genomes of Balb/Mo and B a l b / c mice. The
genetically transmitted Moloney provlrus of Balb/
Mo mice was detected in a characteristic Eco RI
DNA fragment of 16 ж 1 0 e daltons. No fragment of
this size was detected in tissue DNAs from Balb/c
mice infected as newborns with Moloney virus. We
conclude that a viral Integration site, occupied in
prelmplantation mouse embryos, Is not necessarily
occupied when virus Infects cells in post-natal ani­
mals. Balb/Mo and Balb/c mice do carry the AkR
structural gene in an Eco RI DNA fragment of 12 χ
1 0 * daltons. Further restriction analysis of this frag­
ment Indicated that both mouse lines carry one
AKR-type provlrus. Leukemogenesls In Balb/Mo
and newborn infected Balb/c mice is accompanied
by reintegration of Moloney viral sequences in new
chromosomal sites of tumor tissues. Part of the
reintegrated Moloney viral sequences are of subgenomlc size. The AKR viral sequences, however,
are not found in new sites. Further restriction anal­
ysis revealed that the development of Moloney vi­
rus-induced leukemia in B a l b / M o mice does not
lead to detectable structural alteration of the ge­
netically transmitted Moloney and AKR structural
genes. Possible mechanisms of the reintegration
process are also discussed.
Introduction
A mouse line (Balb/Mo) has recently been established
which carries Moloney murine leukemia virus (MMuLV) as an endogenous virus (Jaenisch, 1976) The
virus is transmitted from parent to offspring according
to Mendelian expectations B a l b / M o mice carry the
virus at a single Mendelian locus (Jaenisch, 1977).
This locus has recently been m a p p e d on chromosome
number 6 of the Balb/Mo mouse genome (Breindl et
al., 1979).
Directly after birth, Balb/Mo mice express high
titers of M-MuLV in the blood, and after a latency of
several months they develop a thymus-dependent leu­
kemia (Jaenisch, 1977) Leukemogenesis is accom­
panied by amplification of M-MuLV sequences in tu­
mor tissues, whereas no amplification could be de­
tected in nontumor tissues (Jaenisch, 1979) This
phenomenon is characteristic for Balb/Mo mice, since
Balb/c mice normally do not spontaneously develop
thymus-dependent leukemias before two years of age.
When newborn Balb/c mice are injected with exoge­
nous M-MuLV, however, a thymus-dependent leuke­
mia follows the expression of high titers of the virus in
the blood Moloney virus-specific sequences are
found primarily in tumor tissues such as spleen and
thymus, while none could be detected in nontumor
tissues like brain and muscle (Jaenisch, Fan and
Croker, 1975) Balb/Mo and Balb/c mice also carry
an endogenous ecotropic virus which can be recog­
nized by a probe specific for AKR virus (Chattopadhyay et al , 1974; Berns and Jaenisch, 1976).
This study was undertaken to analyze, on a molecular
level, the structural relationship between the endoge­
nous M-MuLV and AKR-MuLV sequences in Balb/Mo
mice. It was also our aim to characterize the integra­
tion sites of the somatically acquired M-MuLV copies
in DNAs from tumor tissues and their relationship to
the endogenous Moloney and AKR-type sequences.
For this purpose, cDNA probes were prepared which
could distinguish specifically M-MuLV and AKR-type
sequences, respectively. These probes were used in
an experimental design which allows the detection of
single-copy genes among restriction fragments from
cellular DNAs
;
Results
Characterization of Moloney and AKR cDNA Probes
Two different cDNA probes were used in these exper­
iments to detect different sets of viral-relatea Se­
quences For both viruses, the cDNA probes were
synthesized with the endogenous polymerase reaction
(Fan and Baltimore, 1973) in the presence of random
calf thymus DNA primer (Taylor, lllmensee and Sum­
mers, 1976) which yielded DNA fragments from 1 0 0 500 nucleotides long.
The cDNAs obtained are highly representative and
protect labeled viral RNA at low DNA.RNA ratios (Tay­
lor et al , 1976). To remove sequences that crosshybndize with sequences of endogenous mouse vi­
ruses, specific cDNA probes for M-MuLV and AKRMuLV were prepared as described, for preparation of
specific M-MuLV cDNA, the total M-MuLV cDNA was
absorbed with Balb/c mouse DNA (Jaenisch, 1977),
and for preparation of cDNA specific for AKR-MuLV,
the total AKR cDNA was absorbed with 129 mouse
DNA, which does not contain the AKR-endogenous
61
Cell
110
virus (Berns and Jaenisch, 1976) The AKR-specific
probe was further selected by hybridization to mercurated Moloney 70S viral RNA to absorb sequences
which cross-hybridize with Moloney viral RNA Du­
plexes were removed by binding to an SH-agarose
column (Woo et a l , 1977) as described in Experimen­
tal Procedures The characteristics of the nonselected
and selected M-MuLV and AKR-MuLV-specific probes
are summarized in Table 1
For both specific probes, it has already been sug­
gested that these represent 30-35% of the respective
viral genomes (Berns and Jaenisch, 1976, Jaenisch,
1977) The sequences present in the Moloney-specific cDNA probe were found to recognize sequences
on restriction enzyme DNA fragments which were
mapped on the 3' and 5' end, as well as on the middle
part of the nomntegrated linear M-MuLV proviral DNA
(Yoshimura and Weinberg, 1979, A J Berns et al ,
manuscript in preparation) The probe displayed some
preferential hybridization to the fragments which were
mapped in the main loop structure of the heteroduplex
between Moloney and AKR (Chien et al , 1978, A J
Berns et al , manuscript in preparation)
M-MuLV Sequences in Nontumor Tissues
To map the Moloney proviral genome, DNA from Balb/
Mo mice was digested with Eco RI This enzyme does
not cleave in vitro synthesized, full-length M-MuLV
DNA (Verma and McKennett, 1978) Thus the size of
cellular Eco RI DNA fragments containing Moloney
proviral sequences will depend upon the occurrence
of an Eco RI recognition site in the cellular sequences
flanking the integrated provirus Proviruses integrated
in different chromosomal sites will therefore in most
cases be found in differently sized DNA fragments
Mouse DNA was prepared as described in Experimen­
tal Procedures, and the fraction of DNA with a molec­
ular weight >25 χ 10 6 daltons (>90% of the prepa­
ration) was separated from lower molecular weight
DNA by sedimentation through a sucrose cushion By
using this purified DNA as a source for restriction
enzyme analysis, we were able to rule out the possi­
bility that the detected Moloney proviral sequences
were derived from unmtegrated proviral DNA mole­
cules Liver DNA (100-150 μς) from Balb/Mo mice
Table 1 Cbaractenstlce of the Nonselected and Selected M MuLV and
was digested with Eco RI, and the fragments were
separated on 0 4% neutral agarose gels (McDonell,
Simon and Studier, 1977) DNA in gel fractions was
analyzed for the presence of viral DNA sequences, as
described in Experimental Procedures A single peak
of hybridization was detected among fragments of 16
x 10° daltons This position was localized between
the Eco RI fragments of Ad5 and Ad2 viral DNAs of
17 2 χ 10 6 and 13 6 χ 10 e daltons, respectively, and
estimated by interpolation to be 16 χ 10e daltons, as
shown in Figure 1
Moloney sequences of this size were consistently
detected in Eco RI DNA fragments from Balb/Mo
DNAs extracted from different tissues such as brain,
liver and kidney No hybridization could be detected
in Eco RI fragments isolated from DNAs of uninfected
Balb/c mice This indicates that a specific M-MuLV
cDNA probe does not recognize other endogenous
viral sequences of Balb/Mo and Balb/c mice
The detection of an Eco RI fragment carrying MMuLV-specific sequences confirms genetic studies
which indicated that a single Mendehan locus for MMuLV is present in the genome of Balb/Mo mice
(Jaenisch, 1976, 1977, Bremdl et al , 1979) Results
obtained from restriction enzyme analysis using Hind
III (see below) suggest that there is only a single Eco
RI DNA fragment of 16 χ 10 6 daltons that carries MMuLV proviral sequences As a consequence, we
conclude that the 16 χ 10 е dalton fragment corre­
sponds to the genetically defined Mov I locus on
chromosome number 6 (Bremdl et al , 1979)
Results obtained using a nonspecific Moloney
cDNA probe indicated a very extensive sequence
homology of the Moloney genome with endogenous
sequences present in the genomes of Balb/c and
Balb/Mo mice The hybridization profile obtained with
a nonspecific probe roughly followed the E260 profile
of the distributed Eco RI DNA fragments in the gel,
emphasizing the necessity of using specific probes
for the detection of M-MuLV sequences
Moloney Viral Sequences in Tumor Tissues
It has been shown that the number of M-MuLV copies
is increased from 1 up to 3-4 per haploid genome
equivalent in tumor tissues of Balb/Mo mice (Jaenisch
MuLV CDNA Probes
Viral RNA
Cell DNA
129
AKR
Balb/Mo
Mol 70S
AKR 70S
NT
60
69
98
4B
52
94
78
52
99
NT
NT
75
88
4
6
56
52
2
70
Figures are given as perceniage of hybridization Cell DNA and viral RNA were In vast excess Hybridizations were carried out as described m
Experimental Procedures (Berns and Jaenisch 1Θ76 Jaenisch 1977)
64
Integration Sites of M-MuLV
111
MWHIO
"¿tie ω ω
ECORÌ
зэ
юг?«
и
аі Мо
'
u
ti
'
I
EcoRI
TUMOR
V ? fy V V ? ¥
Д
*"
' t
à?
г
\л1
i
Ю
40
tract on number
еаіьАло
Figure 1 M MuLV Sequencee in Nontumor Tissues (Brain Liver) of
BalD/c and Balb/Mo Mice
Celtular DNA was extracted from mouse tissues purified from low
molecular weight molecules (<25 ж 10 e dallons) 150 μο were
digested with Eco RI and ttie differently sized DNA fragments were
monitored for Ihe presence of M-MuLV-type sequences as described
in Experimental Procedures The results are expressed as percentage
of hybridization (as measured by nuclease SI resistance) The mo­
lecular weights indicate the positions of fragments of the genome of
Ad2 generated by separate cleavage with the endonucleases Eco RI
20
30
40
traction number
50
MWIKT«
EcoRI
2?
iy
у
у
ι-
у
^ , ^ T UMOR
•\
»?
l'\«*«s»
a n d Bam HI (Berk and Sharp 1Θ77) run In an adiacenl channel The
sizes of these fragments indicated In units of dalton mass were used
as standards
et al , 1975. Jaenisch, 1979) We undertook this
investigation to determine whether there exist specific
integration sites for these amplified sequences Fur­
thermore, we studied the relation between the somat­
ically acquired copies and endogenous viruses
The distribution of M-MuLV-containmg fragments
from tumor DNAs was changed dramatically as com­
pared with normal tissues All tumor DNAs tested
contained Moloney-specific sequences among Eco RI
DNA fragments of 16 x 1 0 e dallons In addition,
however, many Eco RI DNA fragments containing MMuLV sequences were detected in the molecular
weight range from 4 χ 10" up to 12 χ 1 0 e daltons, as
shown in Figure 2 These additional M-MuLV se­
e
quences were not detected under 25 χ 1 0 daltons
in undigested tumor DNA preparations Trapping of
unmtegrated M-MuLV proviral sequences was ex­
cluded by loading low amounts of undigested DNA on
the gel (see Experimental Procedures) SV40 circular
DNA form I and II and Ad2 viral DNA fragments,
obtained after cleavage with Bam HI, were added to
serve as a control No trapping of these molecules
was observed (data not shown) The additional frag­
ments found in tumor tissue DNA therefore reflect
proviral sequences present in new sites, and probably
represent the somatically acquired viral copies de­
tected by hybridization kinetics (Jaenisch, 1979) Our
analysis of Eco Rl-digested DNAs shows that the viral
structure at the Mov I locus is probably not modified
as compared with that of the Mov I locus in nontumor
DNA This suggests that integration of somatically
acquired M-MuLV sequences does not occur in tan-
v
10
20
30
40
traction number
50
Figure 2 M-MuLV Sequences in Tumor Tissues of Balb/Mo Mice
As described m the legend lo Figure 1 the 23 ж 10 е dalton marKPr
corresponds to undigested Ad2 DNA The upper and lower panels
show the profiles obtained for DNAs (150 jig each) from a leukemic
thymus and a leukemic spleen respectively obtained from two dif­
ferent animals sacrificed at age 10 months
dem with the endogenous M-MuLV genome
Although the hybridization profiles obtained for in­
dividual tumors are not identical, all tumor DNAs
tested displayed high hybridization values in the mo­
lecular weight range from 4 χ 10 е up to 10 x 1 0 e
daltons The resolution of our system does not allow
firm conclusions concerning the identity of integration
sites between individual tumors It shows, however,
thai multiple integration sites are present within one
tumor and that different sets of integration sites are
present in different tumors The detection of M-MuLV
proviral sequences among Eco RI DNA fragments of
molecular weight less than full-length proviral DNA
(= 5 7 x 1 0 е daltons, Verma and McKennett, 1978)
indicates the presence of incomplete, noncontiguous
or rearranged Moloney proviral genomes in new sites
of tumor tissue DNAs Since the 16 x 1 0 e dalton Eco
RI restriction fragment was present in DNAs from both
normal and tumor tissues we performed Hind III re­
striction analyses of the 16 χ 10 е dalton fragments to
confirm that the Mov I locus was unchanged in tumor
tissues Previous restriction data on in vitro synthe­
sized Moloney proviral DNA revealed that Hind III
cleaves this DNA only once, yielding two fragments of
е
е
3 6 x 10 and 2 4 x 1 0 daltons (Verma and Mc­
Kennett 1978)
1 mg of cellular DNA was digested with Eco RI, the
CS
Cell
112
fragments were separated on a 0 4 % agarose gel,
and the DNA molecules with molecular weights be­
tween 12 x 10 6 and 18 χ 1 0 6 daltons were isolated
as described in Experimental Procedures 15 ^ g of
this DNA from both tumor and nontumor tissues were
digested with the restriction enzyme Hind III and ana­
lyzed for the presence of M-MuLV sequences in dif­
ferently sized fragments, as described above Two
fragments with molecular weights of 6 χ 105 and 2 5
χ 10 6 daltons, respectively, were detected for DNAs
from both tissues, as shown in Figure 3
These results indicate that no detectable changes
have occurred in the M-MuLV sequences of the Mov
I locus after reintegration of Moloney-type sequences
in the DNA of most of the cells in tumor tissues No
integration in tandem to the viral genome present m
the 16 x 10 6 dalton Eco RI fragment of tumor DNAs
was observed, because a tandem should have yielded
in the detection of three fragments after cleavage with
Hind III Preliminary evidence further suggests that the
6 χ 10 e dalton fragment represents a single Hind III
fragment (A Berns et al , manuscript in preparation)
Tumors In Balb/c Mice Infected as Newborns with
Moloney Virus
Injection of newborn Balb/c mice with a low dose of
Moloney CLIA virus (about 10 infectious XC particles)
leads to the development of leukemia in a few of the
injected animals (Jaemsch et al , 1975) A low dose
of virus was used to prevent primary infection of many
cells From the animals which developed leukemia,
DNA was extracted from tumor tissues (thymus and
spleen) and analyzed as described These analyses
revealed that M-MuLV-specific sequences were de­
tected in many Eco RI DNA fragments, ranging in size
from 4 χ 10 e up to 13 x 1 0 6 daltons Thus in this
respect no gross difference in the distribution of M·*
MuLV-specific sequences in tumors of newborn mBalb/Mo
^
^
fected Balb/c mice can be delected when compared
with the spontaneous tumors of Balb/Mo mice Pat­
terns obtained from tumors of several Balb/c mice
infected as newborns did not show gross differences
in the distribution of M-MuLV-specific sequences, a
typical example is shown in Figure 4 (see also Figure
5, lower panel)
However, one significant difference between DNAs
extracted from tumor s of Balb/Mo mice and newborn
infected Balb/c mice can be seen no M-MuLV se­
quences could be detected in Eco RI fragments with
a molecular weight of 16 χ I O 6 daltons This suggests
that a viral integration site occupied in preimplantation
embryos (Mov I) is not preferentially occupied when
virus infects lymphatic cells in post-natal animals
Endogenous AKR-Type Sequences
To investigate whether the endogenous M-MuLV is
integrated in or next to the ecolropic endogenous
virus, Eco RI fragments from Balb/c and B a l b / M o
DNAs were annealed with an AKR-specific probe Eco
RI digestion of in vitro synthesized full-length AKR
proviral DNA revealed that this enzyme does not
cleave this proviral genome (I Verma, personal com­
munication)
Hybridization to the ecotropic virus-specific probe
could be detected among Eco RI DNA fragments
having a molecular weight of approximately 12 x 1 0 6
daltons The presence of AKR-type MuLV sequences
in fragments of this size was detected in all normal
and tumor tissues of Balb/Mo mice, of Balb/c mice
and of Balb/c mice infected with M-MuLV as new­
borns The hybridization profiles are shown in Figure
5
The detection of a single fragment carrying eco­
tropic virus-specific sequences confirms previous o b ­
servations that Balb/c mice carry one AKR-type provirus (Robbms et al , 1977) The 12 x 1 0 e dalton
6
l
20
Newborn inlected
в
*>ь/с
MWiiO
Eco m
»« NonTUMOfì
— TUMOR
•o
Hndlll
S
·
•015
"*с
e
\
f
β?»
.l
i '-Л
10
Figure 3
20
30
-
30
(raction nuHöer
50
M-MuLV S e q u e n c e s in H i n d HI Fragments Generaled f r o m
Iracton
Figure 4
number
M - M u L V S e q u e n c e s m Tumor DNA f r o m N e w b o r n I n f e c t e d
E c o RI Fragments o l В а І Ь / М о M i c e
Balb/c Mice
A s d e s c r i b e d in Ihe l e g e n d t o F i g u r e 1 E c o RI fragments of m o l e c u l a r
As d e s c r i b e d in Ihe l e g e n d t o F i g u r e 1 E c o R l - d i g e s t e d t h y m u s D N A
w e i g h t s between 12 χ
( 1 5 0 μ9) f r o m a l e u k e m i c B a l b / c m o u s e ( i n f e c t e d w h e n n e w b o r n w i t h
1 0 ° a n d 1 8 x 1 0 * daltons were isolated f r o m
1 m g of b o t h tumor ( t h y m u s ) a n d n o n t u m o r (liver) DNA of B a l b / M o
1 0 i n f e c t i o u s XC particles of M M u L V CLIA) w a s m o n i t o r e d f o r t h e
m i c e These were d i g e s t e d w i l h H i n d III and monitored lor M - M u L V -
p r e s e n c e of M M u L V s e q u e n c e s
t y p e sequences as d e s c r i b e d in Experimental Procedures
cedures
as d e s c r i b e d in E x p e r i m e n t a l P r o ­
Integration Sites ol M-MuLV
Gtl
It3
fragment containing the endogenous AKR genome
was further digested with Hind III as described Hind
III cleaves the in vitro synthesized full-length AKR
proviral DNA once (I Verma, personal communica­
tion) The Hind III hybridization patterns seen with 12
χ 10 θ dalton fragments from tumor and nontumor
tissues of Balb/Mo and Balb/c mice were identical
and showed two fragments of about 4 x 1 0 * and 2 6
χ 10 e daltons (data not shown) This indicates that no
detectable changes have occurred in the AKR proviral
genome structure in tumor and nontumor DNAs from
Balb/Mo mice as compared with the structure in DNAs
from Balb/c mice We can conclude that development
of M-MuLV-mduced leukemia does not lead to de­
tectable structural alteration or amplification of the
endogenous AKR-type sequences
MW«10
172 tae
τ
»
ВаІЬ^Мо
-ΑΚΗ MulV
CONA..
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traction number
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BaltvW
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Discussion
In the experiments described in this paper we used a
technique which enables the detection of specific viral
sequences in restriction fragments of Balb/Mo and
Balb/c mouse DNAs We analyzed the genetically
transmitted provirus (Mov I locus on chromosome 6,
Bremdl et al , 1979) and the somatically acquired MMuLV genomes (3-4 per haploid genome, Jaemsch,
1979) in tumors of Balb/Mo mice, using a specific
cDNA probe Earlier studies revealed that the use of
nonspecific probes in restriction enzyme analysis of
integrated viral genomes in the mouse system results
In the detection of many, hard to identify fragments
that contain viral information (Steffen and Weinberg,
1978, L Bachelor, personal communication) The
specific M-MuLV cDNA probe we used represents
30-35% of the viral genome (Jaemsch, 1977) Prelim­
inary results suggest that this probe can recognize
different regions of the proviral DNA The probe does
not hybridize with other endogenous sequences of the
Balb/Mo mouse genome (see Figure 1) A second
probe specific for AKR-type sequences was used to
detect ecotropic endogenous viral sequences The
use of these two probes enabled us to characterize
and differentiate Moloney and AKR-type sequences
among other endogenous viral information in the
DNAs of Balb/Mo mice and Balb/c mice infected as
newborns with M-MuLV
Analysis of DNA digests with EcoRI, which does not
cleave Moloney and AKR proviral DNA (Verma and
McKennet, 1978,1 Verma. personal communication),
resulted in the detection of a single M-MuLV-contaming fragment of 16 χ 10* daltons No hybridization
was detected among DNA fragments from uninfected
Balb/c mice Purification of this Moloney proviruscontatntng fragment and subsequent digestion of the
fragment with Hind III generated two fragments of 6
χ 10" and 2 5 χ 10" daltons, respectively
Digestion of in vitro synthesized M-MuLV DNA with
Hind III yields two fragments of 3 6 x 10" and 2 4 χ
• M MulV CDNA.,
ν
f
V. ···
""Λ,
-AKR MulV
COJA
Spec
j
ч.""'-V-OJ I
traction number
'Newborn nlecled Balb/c
И
хіОб
23
lie
у
69
Eco R T T U M O R '
39
» M MulV c O N A S p e c
1 ^vv
'V.\
^-
10
20
30
T-S, ЪГ<Чі*Ч
40
traction number
J
'•
„v.*- ,
50
Figure 5 AKR-MuLV and M-MuLV Sequences in Balb/Mo and Balb/
с Mica
DNAs were monitored tor the presence of M-MuLV and AKR-MuLV
sequences as described In Experimental Procedures (see also Figure
1) The upper panel shows the M-MuLV· and AKR-MuLV-type se­
quences in nontumor (liver) DNA (150 μς) of Balb/Mo mice the
central panel shows both sequences In a tumor (thymus) DNA (100
μ9) Irom a Balb/Mo mouse and the lower panel shows the profiles
obtained for a tumor (thymus) DNA (150 μα) from a Bafb/c mouse
infected when newborn with 10 Infectious ХС particles (M-MuLV)
10" daltons, respectively (Verma and McKennett,
1978) The M-MuLV sequences in the 16 χ 10 е dalton
Eco RI fragments therefore represent a single unique
integration site, corresponding to the M-MuLV se­
quences of the Mov I locus Further structural analy­
ses using the restriction enzymes Hpa I, Bam HI and
Pst I (Verma and McKennett. 1978) have confirmed
this (A Berns et al , manuscript in preparation)
Balb/Mo and Balb/c mice also carry endogenous
ecotropic proviral sequences (Chattopadhyay et a l ,
Cell
114
1974) These sequences can be detected by a spe­
cific AKR-MuLV cDNA without cross-hybridization to
M-MuLV and other mouse viruses The use of this
probe resulted in the detection of a single fragment of
12 x IO 6 daltons in all Eco Rl-digested tissue DMAs
from Balb/c and Balb/Mo mice The Hind III restric­
tion map of this fragment suggested that there is only
one single Eco RI fragment of 12 χ 1 0 6 daltons
carrying the AKR-type provirus This is in agreement
with a previous observation that only one single AKRtype endogenous virus is present m Balb/c mice
(Robbms et al , 1977) The Hind III profile obtained
for the 12 χ 10 e dalton fragment from Balb/Mo mice
was identical to the one from Balb/c mice, which
strongly suggests that the Moloney provirus is not
integrated in tandem with the endogenous AKR-type
provirus Apparently, infection of preimplantation
mouse embryos with Moloney virus (Jaemsch et al ,
1975) does not necessarily result in tandem integra­
tion of the Moloney provirus with the endogenous
AKR-type provirus in this Balb/Mo mouse line, al­
though there is considerable sequence homology be­
tween the Moloney and AKR viral genomes (Chien et
al , 1978) Additional germ line integrations must be
performed to substantiate our findings
The Amplification of Viral Sequences in Tumor
Tissues
Leukemogenesis m Balb/Mo mice is accompanied by
the increase of Moloney proviral sequences from 1 up
to 3 - 4 per haploid genome equivalent (Jaemsch,
1979) Previous hybridization studies did not show
whether the additional copies found in DNAs from
tumor tissues were due to the presence of integrated
or nomntegrated proviral DNAs (Jaemsch, 1977) We
could exclude the possibility of detecting nomnte­
grated proviral DNAs by using only DNA preparations
from which DNA with molecular weights <25 χ 1 0 e
daltons was removed Integrated Moloney viral se­
quences were detected in Eco RI fragments from
tumor DNAs with molecular weights varying from 4
χ 10 e up to 12 χ 1 0 e daltons Different individual
tumors yielded similar though not identical hybridiza­
tion profiles All digested tumor DNAs displayed high
hybridization values, around 6 - 7 χ 10 6 and 9 x 10 e
daltons It is not clear whether these represent unique
integration sites per se or just specific sites for a
particular tumor It can be concluded thai multiple
integrations are present within one tumor The detec­
tion of multiple integration sites is due either to a
multiclonal origin of the tumor or to several integra­
tions in one cell
Recent data suggest that tumors within one animal
are monoclonal, tumors from three different tissues
(spleen, thymus and lymph node) within one Balb/Mo
animal displayed the same reintegration pattern for MMuLV-type sequences However, tumors from differ­
ent animals showed different hybridization patterns
67
(D Jaehner. H Stuhlmann and R Jaemsch. manu­
script in preparation)
Steffen and Weinberg (1978) suggested that the
different patterns of proviral integration are due to the
existence of multiple integration sites for proviral in­
formation in mouse as well as rat cells Multiple inte­
grations were also suggested by their observation that
three independently MuLV-mduced thymomas in NIH/
swiss mice displayed different patterns of exogenous
proviral integration Our results are compatible with
theirs in that reintegration in tumors is found in many
new sites The somatically acquired copies do not
integrate at the Mov I locus or in tandem with the
endogenous AKR provirus, since both endogenous
proviruses seem to be unchanged
The possibility cannot be excluded, however, that
M-MuLV proviral sequences integrate proximal to xenotropic viral sequences not detected by our probes
We also cannot rule out the possibility that only part
of the cells have structurally modified sequences at
the Mov I locus or in the AKR structural gene which
have escaped detection because of the high back­
ground of nonmodified proviral genomes
Amplification due to reintegration of viral sequences
is observed in tumors of leukemic AKR (Canaani and
Aaronson, 1979, our unpublished results), and Balb/
Mo- and M-MuLV-mfected Balb/c mice, as well as for
M-MTV-type sequences in mice with carcinomas (Varmus et al , 1978) The differential amplification of
specific viral sequences might be explained by a
difference in integration site, the adjacent cellular
sequences may govern the activity of a provirus
(Keshet and Temin, 1978, Keshet, О Rear and Temm,
1979)
The extra integrated Moloney sequences in tumor
DNAs might be explained by reinfection of cells by a
virus containing at least partial M-MuLV-type infor­
mation Reinfection of cells might well play a role,
since the onset of leukemia in AKR mice and in Balb/
с mice infected as newborns with M-MuLV can be
prevented by immunizing the animals with antiserum
against AKR-MuLV (Huebner et al , 1976, Schafer et
a l , 1977) and M-MuLV proteins, respectively (Ρ
Nobis and R Jaemsch, preliminary observations) Fur­
thermore, recombinants between Moloney virus and
xenotropic viruses have been isolated from lympho­
mas of leukemic Balb/Mo mice (Vogt, 1979), and
integrated recombinant viruses might provide the ex­
planation for the finding of M-MuLV-contaimng frag­
ments of subgenomic sizes in tumor tissues of Balb/
Mo mice and Balb/c mice infected as newborns with
M-MuLV, since these recombinant proviruses might
contain restriction sites for Eco RI Further restriction
analyses must be carried out to reveal the precise
structure of the integrated MuLV genomes Until now,
cDNAs prepared with the endogenous polymerase
reaction did not show enough specificity to use hy­
bridization to DNAs blotted on nitrocellulose filters
In teg rattoη Sites of M-MuLV
115
flO
Cloning of M-MuLV proviral DNA fragments is now in
progress; these fragments will be used for hybridiza­
tion to DNAs blotted on nitrocellulose filters.
ЕярегІпмпШ ProcwJurM
Mice
129 mice were obtained from ttie Jackson Laboratory All other
strains of mice were obtained from colonies maintained at the animal
care center of the Medical Faculty of the University of Nijmegen.
Holland
CeH Un— end Viru·*«
M-MuLV CLIA was propagated in monolayers of CLIA ceHs (provided
by Η Fan) m roller bottles on a Bélico autoharvester Cells were
grown in BMEM containing 2% newborn calf serum Short harvest
virus (1 5-2 hr) was concentrated from the cooled medium 50 fold
with an Amicon DC2 concentrator (containing an HP 100 filter) the
virus was subsequently aedimented through a 20% sucrose solution
on a 60% sucrose cushion in TNE buffer [10 mM Trie-HCl (pH 7 4),
100 mM NaCI 1 mM EDTA] usmg an SW27 rotor (4 hr at 20K. 4 0 C).
the material m the Interphase was resedimented through a linear 2060% sucrose gradient in TNE buffer for 16 hr al 20K in an SW27
rotor at 4 a C, the vims band was taken out, diluted and sedimentad
through a 20% glycerol solution In TNE buffer, the pellets were
suspended in TNE buffer containing 20% glycerol and stored at
~ 0 о С Virus preparations were tested for RT activity as described
by Fan and Baltimore (1 7Э) AKR virus was propagated In mono­
layers of NIH cells and isolated as described for M-MuLV CLIA Ad2
and Ad5 were propagated m suspension cultures of KB cells and
purified as described by Doerfler (1969)
Preparation and Selection of cDNA Prob··
Moloney and AKR viral cDNAs were prepared wilh the endogenous
polymerase reaction In the presence of random calf thymus DNA
primer (Taylor et a l , 1976) Reaction mixtures contained 50 mM
Trls-HCI (pH θ 3). 3 mM MgAca, 60 mM NaCI, 20 mM OTT, 2 mg/ml
calf thymus DNA primer, 50 μς/ΓηΙ actmomycin D. 0 016% NP40, 1
mM dATP, 1 mM dGTP, 2 mCI3H-dTTP (Amersham, spec act 55
Cl/mmole), 1 mCl 3 H-dCTP (25 Ci/mmole), 1-2 mg of virus In a final
volume of 1 ml, after 2 5 hr at 37 0 C, the reaction was terminated by
adding 50 μΙ 0 2 M of EDTA, 50 μΙ of 10% SOS, 50 μΙ of 6 N NaOH,
after Incubation for 16 hr at 37 0 C to degrade the RNA, the sample
was neutralized by adding HCl After chromatography on a Sephadex
G50 column equilibrated with 10 mM Trls-HCI (pH 7 6), 1 mM EDTA,
fractions containing cDNA were extracted twice with phenol (neutral­
ized with 1 M Tris-HCKpH 7)] and chloroform This procedure yielded
a b o u t o e x lO'-IO'cpm'H-cDNA^SOMgofcDNA) The M-MuLV
cDNA sequences which cross-hybridize with other endogenous
mouse viruses were removed by selection on mouse DNA from Balb/
с mice and further purified by hybridization to Moloney viral RNA
(Jaemsch, 1977)
AKR cDNA was purified from sequences which cross-hybridize
with other endogenous mouse viruses by selection on mouse DNA
from 129 mice and by hybridization to AKR viral RNA (Berns and
Jaenisch, 1976) Sequences which cross-hybridize with Moloney viral
RNA were removed by hybridization to mercurated Moloney viral RNA
s
(Dale and Ward. 1975) About 7 χ 10 cpm of AKR cDNA were
hybridized to 1 2 fig of mercurated Moloney viral RNA m hybridization
buffer [1 M NaCI. 10 mM TES (pH 7 4), 1 mM EDTA and 2 μς/ΓηΙ
polyvmylsulphate] for 3 hr at 7°С, diluted 10 fold and loaded onto
a 0 2 ml SH-agarose column (Agarose-Ethane-Thtol, Ρ L Biochemi­
cal s) The nonbound traction containing the AKR-MuLV-speclfic
cDNA was eluted with 10 mM TES (pH 7 0), 1 mM EDTA. 100 mM
NaCI and 2 дд/ттіі polyvmylsulphate, according to the method of Woo
et al (1977)
Extraction of DNA
DNA was isolated from mouse tissues according to a procedure
modified from Klrby (1968) Tissues were homogenized with a Poly-
tron homogemzer at low speed in freshly prepared solutions of 0 5%
NaCI 3% paraammosalicylate and 5 mM EDTA for 2-3 min, DNA was
extracted by two successive extractions with phenol cresol B-hydroxyquinollne (100 14 0 1) and twice with Chloroform isoamyl alco­
hol (24 1 ). I he solution was adjusted to 0 3 M NaCI and the DNA was
precipitated with 1 vol of cold ( - 20 e C) ethanol, DNA was immediately
spooled and dissolved in TE buffer [10 mM Tns-HCI (pH 7 6). 2 mM
EDTA] incubated with DNAase-tree RNAase (50-100 M g/ml) for 1 hr
at 37 0 C in the presence of 0 5% SDS, twice extracted with chloro­
form isoamyl alcohol (24 1 ) and precipitated twice with ethanol, finally
DNA was dissolved in 10 mM Tns-HCI (pH 7 6). 0 2 mM EDTA Low
molecular weight molecules (<25 χ 10 e daltons) were removed by
selective sedimentation, up to 3 mg of DNA (θ ml) were loaded on a
30 ml solution of 12 5% sucrose In TE buffer containing 300 mM
NaCI, centrlfugatran was performed for 15 hr at 24K (15 Q C) In an
SW27 rotor, the DNA pellet was dissolved in 10 mM Tns-HCI (pH
7 5). 0 2 mM EDTA and stored at 4°C These purified DNA prepa­
rations were ueed as sources for restriction analyses Viral DNAs
from Ad2 and Ad5 were isolated from purified vinons as described by
Doerfler, Lundholm and Hirsch-Kauffmann (1972)
Restriction Enzymes and Incubation Condition·
Eco RI and Hind III were obtained from Microbial Products (England)
Digestion of DNA with Eco RI was performed with 2 υ/μα DNA and
10-20 pg DNA per 50 μΙ solution [100 mM Trls-HCI (pH 7 5) 50 mM
NaCI. 5 mM MgClj, 100 м/т\ gelatine and 1 ^g/ml bovine serum
albumin) for 4 hr at 37°С Incubation solutions were adjusted with
storage buffer (9 1) which is described in the Microbial catalog Hind
III digestions were performed also usmg 2 υ/μο DNA in 10 mM TrleMC1 (pH 7 6), 50 mM NaCI. 10 mM MgCb 14 mM DTT and 5% (v/v)
storage buffer) Ad2 DNA was included In the reactions to serve as a
control for the extent of digestion
Electrophoreele, Elution end Hybridization of DNA
Fragments of digested DNAs were separated on size by electropho­
resis in 0 4 or 0 6% neutral agarose gels the buffer contained 40
mM Trls-HCI (pH 7 9). 5 mM NaAc. 1 mM EDTA (McDonell el a l .
1977) and 0 5 μς/ιηΙ ethidium bromide, gels were prepared in the
same buffer Eco Rf-dlgested DNAs were loaded at a maximal con­
centration of 1 μς/ΠΗη3 DNA, while nondigested DNAs were loaded
up to О 06 pg/mm 3 DNA DNA was eluted and concentrated from gel
slices (2-4 mm) by electrophoretic elution for 16 hr in a device similar
to that described by Alllngton et al (197Θ), 50 μα of calf thymus DNA
were added as carrier, and elution was earned out at 100 V In a
buffer containing 5 mM Tns-HCI (pH 7 0) and 1 mM EDTA, subse­
quently. samples (400 μΙ) were adjusted to 0 75 N NaOH and 1 mM
o
EDTA In Eppendorf 1 5 ml tubes, and Incubated at 100 C for 15 mm
to remove any contaminating RNA molecules and lo reduce the size
ot the DNA molecules to 200-400 nucleotides Samples were neu­
tralized with HAc and precipitated with ethanol. DNA was dissolved
m hybridization buffer containing 700 cpm of 3H-labeled M-MuLV- or
AKR-MuLV-specific cDNA probe Hybndtzalton was carried out for
72 hr at 67°C and duplex formation was monitored by SI nuclease
(Vogt, 1973) Preparative elution of DNA fragments from agarose gel
slices was performed as mentioned above except that no earner was
added Fragments were purified by two successive phenol 8-hydroxyqulnolme ( 100 0 1. phenol saturated with TE buffer) extractions,
two chloroform isoamyl alcohol (24 1) extractions and two precipita­
tions with ethanol
Acknowledgments
We are grateful to Dr H Bloemendal, m whose laboratory this
investigation was carried out This study was performed as pari of
the Ph D study of Η ν d Ρ and was supported in pari by the Foun­
dation for Medical Research (FUNGO), which Is subsidized by the
Netherlands Organization for the Advancement of Pure Research
(ZWO) R J was supported in part by a grant from the Deutsche
Forschungsgemeinschaft and by a research contract from the Na­
tional Cancer Institute
Cell
Π6
The costs ol publication of this article were defrayed m part by the
payment ol page charges This article must therefore be hereby
marked ativerftsement in accordance with 18 U S С Section 1734
solely to indicate this tact
Received April 2, 1979 revised June 1, 1979
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70
Chapter I I I .
JOURNAL OF VIBOLOCY, Oct. 1980, p. 254-263
Vol. 36, No. 1
0022-538X/80/10-0254/10$02.00/0
Molecular Cloning of Unintegrated and a Portion of Integrated
Moloney Murine Leukemia Viral DNA in Bacteriophage
Lambda
2
2
2
ANTON J. M. BERNS," M. H. T. LAI, R. A. BOSSELMAN, M. A. McKENNETT,
2
2
1
1
L. T. BACHELER, H. FAN, E. С ROBANUS MAANDAG, H. v.o. PUTTEN, AND
2
INDER M. VERMA
Tumor Virology Laboratory, The Salk Institute, San Diego, California 92138* and Laboratory of
1
Biochemistry, University of Nijmegen, 6525 EZ Nijmegen, The Netherlands
A covalently closed circular form of unintegrated viral DNA obtained from
NIH 3T3 cells freshly infected with Moloney murine leukemia virus (M-MLV)
and a portion of the endogenous M-MLV from the BALB/Mo mouse strain have
been cloned in bacteriophage lambda. The unintegrated viral DNA was cleaved
with restriction endonuclease tfmdIII and inserted into the single ifmdIII site of
lambda phage Charon 21A. Similarly high-molecular-weight DNA from BALB/
Mo mice was cleaved sequentially with restriction endonucleases £coRI and
¿/indili and separated on the basis of size, and one of the two fractions which
reacted with an M-MLV-specific complementary DNA was inserted into the
Hindlll site of Charon 21 A. Recombinant clones containing M-MLV-reacting
DNA were analyzed by restriction endonuclease mapping, heteroduplexing, and
infectivity assays. The restriction endonuclease map of the insert derived from
unintegrated viral DNA, λ-MLV-l, was comparable to published maps. Electron
microscope analysis of the hybrid formed between λ-MLV-l DNA and 35S
genomic M-MLV RNA showed a duplex structure. The molecularly cloned λ·
MLV-1 DNA contained only one copy of the long terminal repeat and was not
infectious even after end-to-end ligation of the insert DNA. The insert DNA
derived from endogenous M-MLV, X-MLV^-l, contained a DNA stretch mea­
suring 5.4 kilobase pairs in length, corresponding to the 5' part of the genomic
viral RNA, and cellular mouse DNA sequences measuring 3.5 kilobase pairs in
length. The viral part of the insert showed the typical restriction pattern of MMLV DNA except that a single restriction site, PvuIL, in the 5' long terminal
repeat wis missing. Reconstructed genomes containing the 5' half derived from
the integrated viral DNA and the 3' half derived from the unintegrated viral DNA
were able to induce XC plaques after transfection in uninfected mouse fibroblasts.
In the life cycle of RNA tumor viruses, the
viral genetic material is converted into DNA,
some of which integrates in the host chromo­
somal DNA (2, 31). Several forms of viral DNA,
ranging from linear to covalently closed circles,
have been identified in infected cells (33). How­
ever, a detailed knowledge of the structure of
the intermediates leading to the integrated
forms of viral DNA remains largely obscure. The
study of the integrated murine leukemia viruses
(MLVs) has been hampered by the large number
of related endogenous viral sequences in unin­
fected cells. Use of recombinant DNA tech­
niques, however, make it feasible to enrich and
amplify DNA fragments of single-copy genes of
eucaryotes. The BALB/Mo mouse strain (12)
contains the Moloney MLV (M-MLV) as an
endogenous virus, which has been localized in a
single £coRI fragment of 27 kilobases (kb) (27).
This fragment is derived from the Μου-1 locus
of BALB/Mo mice (5). BALB/Mo mice are
viremic from birth and develop lymphatic leu­
kemia later in life (13), indicating the presence
of a complete active M-MLV genome. In this
study, we report the molecular cloning of (i)
unintegrated circular forms of M-MLV DNA
isolated from cells freshly infected with M-MLV
(the DNA was cleaved with restriction endonu­
clease Ηindili to generate the linear form) and
(ii) a portion of the integrated form of M-MLV
present in BALB/Mo mice (the 27-kb £coRI
cell DNA fragment was cleaved with restriction
endonuclease Hinàlll to generate a fragment of
about 8.9 kb which contains M-MLV sequences).
Both the unintegrated circular M-MLV DNA
cleaved with НтаШ. and the 8.9-kb tfindlll
fragment from the mouse DNA were cloned in
the unique tfindlll site of lambda phage Charon
254
VOL 36,1980
M MLV VIRAL DNA CLONES IN BACTERIOPHAGE
21A D N A (3). T h e natures of the cloned D N A s
were examined by restriction endonuclease map­
ping, R-loop formation, and infectivity assays.
MATERIALS AND METHODS
Preparation of unintegrated closed circular
DNA. About 5 χ 10" NIH 3T3 cells were infected with
a 24-h tissue culture supernatant from M-MLV-producing clone 4A cells at a multiplicity of infection of
1 At 16 h postinfection, cells were harvested and a
Hirt supernatant was prepared After RNase treat­
ment and ÊcoRI digestion, circular DNAs were separated from linear DNA by centrifugaron in cesium
chlonde-ethidium bromide gradients (30) The material banding at the bottom one-third was collected, the
ethidmm broni'de was removed by isopropanol extraction, and the DNA was dialyzed
Preparation of mouse DNA fragmente containing M-MLV sequences. High-molecular-weight
mouse DNA was prepared from livers of 2-month-old
BALB/Mo mice as previously described (15, 27) The
DNA was digested to completion with icoRI at 2 U/
pg for 4 h, and the fragments were separated by zonal
centnfugation in a BXXIX rotor of an International
centrifuge A 50-mg amount of DNA yielded about 1
mg of 18- to 35-kb DNA fragments After cleaving
with endonuclease Hindill, the fragments were separated on agarose gels and electroeluted, and the fractions hybridizing with an M MLV-specific probe were
used for cloning
Cloning. DNA from lambda phage Charon 21A
was digested with restriction endonuclease Htndlll,
followed by treatment with bacterial alkaline phosphatase to reduce self-hgation of the phage arms (5
U/>g of DNA m 50 mM Tns-hydrochloride, pH 9 0,
for 30 mm at 560C) For cloning unintegrated M-MLV
DNA, about 10 to 20 ng of tfmdIII-cut M-MLV DNA
was mixed with 150 ng of tftndlll-cut, phosphatasetreated Charon 21A vector DNA and precipitated with
ethanol, and the pellet was incubated for 1 h at 42°C
in 3μ1 of DNA ligase buffer (66 mM Tris-hydrothloride
[pH 7 5], 10 mM dithiothreitol, 7 mM MgCU, 80 μΜ
ATP) to allow the DNA to dissolve, 0 4 U (1 μΐ) of T4
DNA ligase was added, and after incubation at room
temperature for 2 h the DNA was packaged in vitro
into infectious lambda phage particles with M-l buffer
by the protocol provided by Blattner and colleagues
The efficiency of packaging of Charon 21A DNA alone
was 3 x 10* PFU/fig After digestion with //mdlll and
treatment with bacterial alkaline phosphatase and
ligation, the efficiency of packaging of phage DNA was
±10 5 PFU//ig, ligation of the insert DNA to the
Hindill cut bacterial alkaline phosphatase-treated
Charon 21A DNA increased the efficiency of packag­
ing by a factor of 5 Generally, the efficiency of pack­
aging varied from 1 χ 10' to 5 Χ 105 PFU/fig of
substrate-vector DNA For cloning integrated M-MLV
DNA, the same protocol was used except that about
2 ^g of an 8 9-kb #mdIII fragment was ligated with 5
μξ of phosphatase-treated Charon 21A arms
Screening of recombinants. The recombinants
were plated on 14-cm plates, and the DNA from the
plaques was adsorbed onto nitrocellulose filters essen­
tially by the technique of Benton and Davis (1)
Plaques that showed hybridization to M-MLV com-
LAMBDA
255
plementary DNA (cDNA) probes (7, 27) were further
subcloned until pure recombinant plaques were ob­
tained The recombinant phages were propagated in
Escherichia coli DP50 ьирР in NZYDT broth as
described in the protocols designed by Blattner et al
(3) and Leder et al (17) AU experiments were per­
formed in a P2 laboratory facihtv, and certified EK2
or EK1CV vectors were used The National Institutes
of Health Guidelines were followed for the entire
cloning procedure
Restriction endonuclease analysis. The natures
of the cloned DNAs were examined by restnction
endonuclease mapping as described previously (31)
The restriction endonuclease digested DNA was frac­
tionated on agarose gels ranging from 0 5 to 1 2f¡ m a
buffer containing 40 mM Tris base, 1 mM EDTA, and
5 mM sodium acetate, adjusted with acetic acid to pH
7 9 (20) The fragments were identified either by etnidlum bromide staining (24) (present at 0 5 jig/ml in
both the gels and electrophoresis buffer) or by the
Southern blotting technique (26), using either a Moloney cDNA probe (7) or nick-translated DNA fragments (19) DNA fragments were nick translated with
[a-J2P]dCTP (2,000 to 3,000 Ci/mmol) from Amersham Corp , E coll polymerase I from Boehrmger
Mannheim С о ф , and DNase I from Worthington
Biochemicals Corp After incubation at 15 0 C for 90
mm, sodium dodecyl sulfate and KDTA were added to
final concentrations of 0 b% and 10 mM, respectively
Proteinase К was added at 200/ig/ml, and the mixture
was incubated for 30 mm at 37 0 C and passed over a
Sephadex G 50 column to remove unincorporated nu­
cleotides
Hybridization to nitrocellulose-bound DNA was
performed аь described previously (32) The sizes of
the restnction fragments were computed from the
standard molecular weight markers in the same gel
(lambda DNA cleaved with EcoRI 22 2, 7 3, 5 75, 4 9,
and 3 8 kb, lambda DNA cleaved with HmdIII 24 2,
9 9, 6 7, 4 4, 2 2, and 195 kb, M13 replicative form
digested with Hpatt 1 6, 0 82, 0 65, 0 54, 0 47, 0 45,
0 38, 0 18, 0 16, and 0 13 kb)
Electron microscopy. Samples of DNA and RNA
were dissolved in a solution containing 80% formamide,
0 4 M NaCl, 001 M PIPES [piperazine-iV-N'-bis(2ethanesulfomc acid)] (pH 6 3), and 0 001 M EDTA at
3 and 1 fig/ml, respectively The DNA was heated
separately at 68°C for 5 mm and then mixed with
RNA, followed by incubation at temperatures which
slowly decreased from 55 to 51 "С over a 4-h period
Molecules were prepared for electron microscopy, and
contour lengths of the molecules were measured as
described previously (4)
Infectivity assays. Infectivity of cloned M-MLV
DNA was assayed by the XC plaque assay (22) DNA
was transfected m NIH 3T3 mouse fibroblasts by the
calcium phosphate method (9) Briefly, 4 x 105 cells
were plated onto 35-mm plates the day before transfection The DNA to be assayed was coprecipitated
with calcium phosphate as described previously (16)
and added directly to the recipient cells After 5 h, the
DNA was removed, and the cells were treated briefly
with 20% dimethyl sulfoxide After overnight recovery,
cells were transferred to new plates at 10% confluency
After 5 days, the cells were UV irradiated and overlaid
with XC cells XC plaques were counted at day 9 after
72
256
BERNS ET AL.
J. VlROL.
fixing and staining the cells with hematoxylin.
RESULTS
Identification and characterization of the
clone derived from unintegrated M-MLV
DNA A«MLV-1. (i) Physical mapping by re­
striction endonuclease. Figure 1A shows the
restriction endonuclease map of in vitro-synthesized M-MLV DNA (8, 31). Since the recombi­
nant clone was made by utilizing the Htradlll
site, Fig. IB shows the physical map of λ-ML V1 with the ЯіпаІІІ cleavage site at 0 map unit.
8 Kbp
Virion RNA cap--
Q
Χ
•Û
TT
poly Α
„ „
ІЛІЛ
0.0.
О
Χ
S
-С
J
S
I
; \αi Si
EEC
10
4
12
U
8
9
10
11 12 13
FIG. 1. Characterization of \-MLVl. (A) Physical map of linear viral DNA (8, 31). The shaded areas
indicate the location of the L TR. (B) Physical map of λ · ML V-1 with HmdIII at 0 map unit. The shaded area
is the location of the LTR from the 3' end of the genome. The dashed-line triangle is the expected position of
the second LTR at the 5' end. (C) Ethidium bromide staining of restriction endonuclease-digested \-MLV-l
DNA. (D) Physical map of the insert derived from λ-MLV-l DNA. Lanes 1 to 13 are autoradiographs of
restricted endonuclease-digested λ-MLV-l DNAs, followed by Southern transfer and hybridization to total
M-MLV cDNA. λ-MLV-l was cleaved with HindJII, followed by digestion with the enzymes specified in the
figure.
73
VOL. 36,1980
M-MLV VIRAL DNA CLONES IN BACTERIOPHAGE LAMBDA
A single recombinant clone, λ-MLV-l, which
hybridized to M-MLV cDNA was obtained from
a primary screening of about 5,000 plaques. Fig­
ure 1С, lane 1, shows the size of the insert in λ·
MLV-1 to be 8.2 kb after cleavage with ffindlll.
Digestion with restriction endonucleases Sail,
Xhol, Sad, Smal, Xbal, BamHl, Kpnl, Pstl,
BgR, and Pvull revealed a pattern which is in
agreement with the map shown in Fig. 1A. How­
ever, only one of the two Pvull sites shown in
the long terminal repeat (LTR) of Fig. IA could
be detected (see below). T h e results also indi­
cated that λ-MLV-1 contains only one copy of
the LTR shown in Fig. IA and B. For instance,
digestion with H m d l l l and S a m H I should gen­
erate four fragments. The largest fragment
should be about 5.4 kb if it contained two copies
of the LTR. However, Fig. ID, lane 7, shows
that the largest fragment is only 4.8 kb. Diges­
tion with λ · MLV-1 DNAs cleaved by P W Í I I
(lane 11) and S a d (lane 4) did not show a 600bp fragment, which would be expected if two
copies of the LTR were present (Fig. IB).
(ii) Heteroduplex formation. Figure 2A
shows a heteroduplex formed between λ-MLV1 and 35S M-MLV genomic RNA. Only a single
loop structure of an average size of 8.0 ± 0.73 kb
can be seen. An enlargement of the RNA-DNA
hybrid loop of the heteroduplex is shown in Fig.
2B. The results indicate that λ · MLV-1 contains
sequences homologous to those present in the
M-MLV genomic RNA.
Identification and characterization of t h e
clone derived from integrated M-MLV
DNA, \«MLVin/-l. A single recombinant clone
which hybridized to M-MLV cDNA was ob-
О
Kbp
en
£eg
m
257
с
5
η
га
m
50-
—·
22-
7.3- ,
ι
5.7- I
4.4-
FiG. 3. Identification of the cellular origin of \·
MLVmrl- Autoradiographs of EcoRI-digested 129,
BALB/c. and BALB/Mo mouse DNAs, followed by
Southern blotting transfer and hybridization to the
nick-translated Xhol to Hpal fragment derived from
the cellular part
of\-MLVM-l.
FIG. 2. Heteroduplex mapping of\-MLVl (A) Heteroduplex formed between \-MLVl DNA and 35S MMLV genomic RNA. The arrow points to the RNA-DNA hybrid. More than 50 molecules were identified. The
average size of the hybrid molecules was 8.0 ± 0.73 kb. (B) Enlargement of the RNA-DNA hybrid shown in
(A).
1
2
J
4
5
6
7
8
9
Ю
Π
12
, 3
г
Β
=
σ
Χ
re
(0
Γ
χ
φ
СЛ
•
h
ел
Ι
F
χ
j
4 s
7 8
9
10 tl
іг
'
и
г
j
4
s
б
8
9
10
11
'?
13
Ш
M
cc
χ
Ε
го οπα
Kbp
Kbp
99
73
57
9 9
73
5 7
гг
Ι 9
ι9
ι6
ι 6
IH
m
FIG. 4. Characterization
of \-MLVMl
by restriction endonuclease mapping. (A) Ethidium bromide staining of restriction
endonuclease-dtgested
\-MLVM-l
DNA. (В) Autoradiographs
of restriction endonucleasedigested
\ • MLVmr 1 DNA, followed by Southern blotting transfer and hybridization to MMLV cDNA. In addition to the enzymes listed in each lane, all samples were treated with Hindlll to release the insert from the phage arms. (C) Same as (A),
except that hybridization was performed with a plasm id clone containing the insert from \-MLVmrl. (D) Physical map of the insert derived from
\-MLVM-I.
>
M-MLV VIRAL DNA CLONES IN BACTERIOPHAGE LAMBDA
VOL. 36,1980
259
e
il
1
I
КЬр
1
I
52
•
l i
ι
I
II
ι
/
/
'
ιΛ J£ (D ШОУ a.
I l I I II
Uil II
ÌflIIIIIIITTI
lllllllllllllllllllllllllll
lì
шχ
ι I
\
ir
о
υ
ω
ОС
та
с
I
CELL
J
ta
V
16
_L_
14
|_
12
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Kbp
0
_l
та
—L.
VIRAL
Fie. 4—continued
tained from a primary screening of approxi­
mately 12,000 plaques. The nature of the recom­
binant clone was identified as about 5.4 kb of
viral and 3.5 kb of cellular sequences. The re­
striction map of the viral sequences agreed with
that shown in Fig. 1A, except that only one
РІІЫІІ site was detected in the 5'-end LTR in­
stead of the reported two sites (8). The restric­
tion map of the cellular sequences agreed with
the map determined for the 27-kb EcoRI frag­
ment obtained from BALB/Mo mice (v.d. Putten et al., unpublished data). To ascertain that
X-MLVmt-l was derived from the DNA corre­
sponding to the Mov-1 locus of the BALB/Mo
mouse strain, we hybridized the Xhol to Hpal
cellular fragment of this clone (after labeling
with [tt-32P]dCTP by nick translation) to Southem blots of EcoRI-digested DNA from the 129,
BALB/c, and BALB/Mo mouse strains (Fig. 3).
This probe recognized an 18-kb fragment in 129
and BALB/c DNAs and a 27-kb fragment in
BALB/Mo mouse DNA, indicating that λ·
MLV„/-1 is indeed derived from the Μου-l locus
of BALB/Mo mice. Furthermore, it showed that
integration of M-MLV has occurred without
causing large deletions in the cellular DNA, as
the size difference between the EcoRI fragments
recognized in BALB/c and BALB/Mo mice cor­
responds with the genomic size of M-MLV. The
A-MLVmrl contains one copy of the LTR at its
5' end. Digestion with restriction endonucleases
Pvull and Kpnl showed the presence of a 280bp fragment, expected from the LTR (Fig. 4B,
с
ί
8
Ш
I
10
_i_
lane 10). Thus, it appears that as do avian sar­
coma viruses (11, 23) integrated M-MLV con­
tains a copy of the LTR at its 5' end. Figure 4A
to С shows the characterization of X-MLV„(-1
by utilizing several restriction endonucleases
and different probes; Fig. 4B shows an autoradiograph obtained with M-MLV cDNA as a
probe, whereas in Fig. 4C, a plasmid probe con­
taining the insert of X-MLV^-l was used.
Therefore, the fragments seen in Fig. 4C but
absent from Fig. 4B contain exclusively mouse
sequences, whereas fragments seen at a relative
higher intensity in Fig. 4C as compared with Fig.
4B comprise both mouse and viral sequences. A
detailed physical map of λ · MLV„/-1 is shown in
Fig. 4D.
Infectivity. The λ-MLV-l recombinant clone
derived from unintegrated closed circular DNA
was not infectious in any of the following forms:
(i) A-MLV-l DNA, (Ü) X-MLV-l DNA cleaved
with Hindlll, (Ш) λ-MLV-l DNA plus tfwdlll
and the insert of 8.9 kb isolated on agarose gels
(Fig. 5, lane A), and (iv) isolated insert ligated
end-to-end by T4 DNA ligase (Fig. 5, lane В).
None of these forms of DNA were infectious as
assayed by the XC plaque assay (27). The λMLV„rl lacked the 3' half of the viral genomic
information and thus remained uninfectious.
However, when λ·MLVlл^-l was supplemented
with viral DNA representing the 3' half of the
genome, it was found to be infectious. Figure 6
shows a diagrammatic sketch of how the infec­
tious DNA molecule was constructed. The λ-
76
260
BERNS ET AL.
Kbp
А
В
J.
С
D
E
I
221-
«EM
7.35.7-
3.8-
FiG. 5. Size analysis of imert ONAs used for
transfection. The lanes shown are auioradiographs
of unligated and ligated fragments after agarose gel
electrophoresis, followed by Southern bhtting trans­
fer and hybridization to M-MLV-1 cDNA. (A) Iso­
lated insert from \-MLV-l DNA; (В) same insert as
visualized in (A) but after ligation; (C) isolated
Hindlll to PstI fragment derived from \-MLV-l
DNA; (D) isolated insert derived from \-MLVmrl
DNA; and (E) Hindlll to PstI fragment (C) plus the
isolated insert shown in (D) after ligation.
MLV-1 DNA was cleaved with restriction endonucleases PstI and San. The HinálU to PstI
fragment of 4.1-kb size contained the З'-half viral
genomic sequence and about 0.51-kb sequences
from the 5' end of the genomic RNA (from Psil
to Äpnl as in Fig. 1A). The 4.1-kb Hinàlll to
P s i l fragment (Fig. 5, lane C) treated with bacterial alkaline phosphatase and the 8.9-kb λ·
MLV„,-1 insert (Fig. 5, lane D) were ligated with
T4 DNA Ugase (Fig. 5, lane E). In another
experiment, we digested the A-MLV-I DNA
with H i n d l l l and Xhol (Fig. 1A and 6). T h e 5.1kb H i n d l l l to Xhol fragment was ligated to the
8.9-kb insert of X - M L V ^ l . Table 1 shows the
specific infectivity of the in vitro-constructed
infectious DNA molecules. Unligated DNA frag­
ments were not infectious. Since M-MLV is NB
tropic, we wanted to determine the tropism of
the virus released by cells infected with in vitroconstructed DNA molecules. Table 2 shows that,
like the parental vims, the virus released from
cells infected with reconstructed DNA was NB
tropic.
DISCUSSION
An unintegrated circular form and a portion
VlROL.
of an integrated form of M-MLV DNA were
cloned in lambda phage Charon 21 A. Since MMLV DNA has a single Hindlll site (8, 31),.
unintegrated closed circular M-MLV DNA was
linearized by cleaving with restriction endonuclease H w d l l l and ligated into the unique
Hindlll site of Charon 21A phage DNA (3). An
integrated form of M-MLV DNA was obtained
from the BALB/Mo mouse strain (12), which
contains M-MLV as an endogenous virus. This
endogenous virus is present in a single Mendelian locus (Mov-1) which has been mapped ge­
netically on chromosome no. 6 (5), and M-MLV
is expressed in hematopoietic tissues from early
life on (12,13). The expression of the endogenous
virus leads to the development of a lymphatic
leukemia later in life (13), accompanied by the
amplification of M-MLV sequences in the tumor
tissues. Restriction endonuclease cleavage of
BALB/Mo mouse DNA from nontarget tissues
such as brain and liver, followed by hybridiza­
tion with M-MLV-specific probes, has indicated
that the genetically transmitted M-MLV ge­
nome can be detected in a single 27-kb .EcoRI
fragment (27). This .EcoRI fragment was isolated
and cleaved with Hindlll, generating two MMLV-containing fragments of 8.9 and 3.9 kb.
The 8.9-kb Hindlll fragment was cloned into
the Hindlll site of Charon 21A. The relative
inefficiency of the Charon 21A cloning vehicle in
accommodating DNA fragments larger than 6 to
7 kb in a unique restriction site may be respon­
sible for the identification of a single M-MLVreacting plaque obtained from either uninte­
grated or integrated M-MLV DNA. The physi­
cal map of the λ · MLV-1 was comparable to the
published maps (8, 31) except that only a single
600-bp LTR was present and one РІУМІІ site was
missing. The exact positions of the various re­
striction sites (8) on the viral genome have been
mapped more precisely, λ · MLV-1 lacks one of
the two Р ш І І sites in the LTR (Fig. 1A). The
nucleotide sequences of the 5' LTR from λ·
MLVferl (26b) and of the 3' LTR from a cloned
M-MLV cDNA transcript (26a) have been de­
termined. In both cases only one PDMII site could
be detected in the LTR (Fig. 1A). A comparison
of the LTR sequences from M-MLV with that
of murine sarcoma virus (R. Dhar, W. С. McClements, L. Enquist, and G. F. van de Woude,
Froc. Natl. Acad. Sci. U.S.A., in press) reveals a
direct duplication of 73 nucleotides in the latter
case. The direct duplication contains the addi­
tional PvuTl site. The absence of the direct re­
peat of 73 bp from the LTR of λ-MLV^-l or
λ · MLV-1 does not appear to be essential for
infectivity, as the in vitro recombinant DNA is
able to produce infectious virus (Table 1). It has
been reported that molecularly cloned retroviral
77
M-MLV VIRAL DNA CLONES IN BACTERIOPHAGE LAMBDA
VOL. 36,1980
ï I
A MLV.-1
3
Й
f
261
η
a.
X
Hind III
LTR
Cellular
Viral
Xhol
Я7Я t
В
Separation on agaroso
gels
ν
-
•о
с
Χ
I
ι
•TRANSFECTION
i
FIG. 6. Flow sheet showing the in vitro construction of infectious DNA molecules.
TABLE 1. Specific activity of in vitroconstructed
infectious DNA molecules
Nature of DNA
Amt of
DNA
used
(№)
No. of
XC
plaques
(avg of
two
plates)
XC
PFU/«
of DNA
0.24
0.21
0.18
0.18
0.500.60
0
0
0
0
0
0
λ - M L V ^ l (8.9 kb) plus
Я и а Ш to Pstl (4.1 kb)
0.02
0.06
0.18
2
7
26
100
116
144
X-MLV«-! (8.9 kb) plus
Я и м і т to Xhol (5.1 kb)
1.5
300
150
X.MLV^l(8.9kb)
AMLV^-l Qigated)
ЯіжШІ to Psil (4.1 kb)
ЯшаШ to Pstl (ligated)
HmdintoXhoKSlkb)
НтаШ to Xhol (ligated)
DNAs can lose one or two LTRs, presumably by
homologous recombination (10, 14, 28). Simi­
larly, it is possible that λ-MLV-l has lost one
LTR during molecular cloning.
The insert DNA was not infectious when
transfected in any of the following forms: (i) λ·
M-MLV DNA, (Ü) λ · M-MLV DNA cleaved with
Я т а І П , (Ш) λ-M-MLV DNA cleaved with
Hindm and the insert isolated on agarose gels,
and (iv) isolated insert, end-to-end ligated by T4
DNA ligase (Fig. 5, lane В). This result suggests
either that a mutation has occurred somewhere
in the 5' two-thirds of the viral genome or t h a t
the presence of two LTRs is essential for infec­
tion. However, when λ-MLV-l was annealed to
M-MLV genomic RNA and analyzed by S I map­
ping, only a single band of an average size of 8.5
kb could be detected on alkaline agarose gels (I.
M. Verma, unpublished data). Thus, it appears
7η
262
BERNS ET AL.
ТАЧГ E 2 Tropism of the virus released from cells
transfected with in vitro constructed infectious
DNA"
J
VIROL
generated from both strains. A more detailed
analysis will be necessary to clarify this point.
To study the biological activity of X-MLVuu1 DNA, the insert was hgated to the 4.1-kb
XC plaque-forming titers (PFU/ml)
tfmdIII to Psil fragment or the 5.1-kb Hmdlll
to Xhol fragment from the λ-MLV-l cloned
DNA. These fragments contained the З'-end ge­
nomic sequences missing from λ • MLV,,,,-! DNA.
5
5
2
In vitro
3 1 x 10 0 8 x 10 2.9 x 10
These reconstructed genomes (Fig. 6) were in­
recombinant
fectious, indicating that no defect has been in­
5
6
3
M-MLV
5.8 x 10 1.8 x IO
10
troduced in the mouse-derived clone during the
" After two transfers of NIH 3T3 cells transfected cloning procedure. The same conclusion can be
with in vitro-reconstructed DNA molecules (0.18 ßg of drawn for the 3' part of λ-MLV-l DNA. The
DNA, Table 1), 24-h tissue culture fluid was assayed specific infectivity of the reconstructed mole­
on NIH 3T3 cells (N-tropic), BALB/3T3 cells (Bcules was similar to that observed for in vitrotropic), and F 2408 (rat) cells. For a comparison, 24-h
synthesized
genome-length DNA (16) but much
tissue culture fluid from mouse fibroblasts chronically
lower than that reported for unintegrated Minfected with M-MLV was also assayed m parallel.
MLV (25) or integrated, molecularly cloned
AKR MLV DNA (18). It is, however, difficult to
that λ-MLV-l has no major deletion. A similar
get an exact estimate as the hgated molecules of
phenomenon is observed with cloned Moloney
the right length were not individually isolated.
murine sarcoma virus DNA; only insert DNAs
It is interesting to note that a molecular clone
containing two copies of the LTR gave rise to
derived from an integrated viral genome, which
focus formation (30). In contrast. Oliff et al. (21)
is not expressed in the tissue from which the
have recently shown that molecularly cloned
DNA was isolated (mouse liver), is infectious.
Friend MLV containing one copy of the LTR is
Therefore, we will focus our attention on the
able to infect cells and produce Fnend MLV.
regulatory functions, specified by the cellular
The reason for this discrepancy remains obscure
sequences present in this clone, as well as on the
for the moment. The clone containing the insert
extent of base modification present in the inte­
derived from the integrated M-MLV DNA has
grated M-MLV, which is expected to be related
been characterized by restriction endonuclease
to the expression of the M-MLV genome speci­
mapping (Fig. 4). The viral portion of the insert
fied by the Mov-1 locus (6).
was comparable by restriction endonuclease
mapping from in vitro- and in vivo-synthesized
ACKNOWLEDGMENTS
M-MLV viral DNAs (8, 31), except for the ab­
We
thank
R
Jaerusch
providing the BALB/Mo mouse
sence of one Pvull site from the 5' LTR. The strain and F R Blattner for
and colleagues for the EK2 vectors
restriction fragments characteristic for the Mov- and strains used in this work
1 integration site (fragments containing both
This investigation was supported by Public Health Service
viral and cellular sequences) could also be gen­ research grants CA-16561 and CA-22408 from the National
Cancer Institute, grant VC-297 from the American Cancer
erated from this clone, with the exception of Society,
and the Foundation for Medical Research (FUNGO),
some fragments which were generated by en­ which is subsidized by the Netherlands Organization for the
zymes sensitive for DNA methylation. Further­ Advancement of Pure Research (ZWO) A J Μ В was sup­
more, a ^P-labeled fragment (Xhol to Hpal) ported by a travel allowance from the Netherlands Organiza­
derived from the cellular part of this clone rec­ tion for the Advancement of Pure Research Μ Η Τ L was a
special fellow of the Leukemia Society of America
ognized specifically a 27-kb ЕсоШ fragment in
DNA from BALB/Mo mice, whereas an 18-kb
LITERATURE CITED
fragment was detected in DNA from the BALB/
1 Benton, D., and R. W. Davie. 1977 Screening of recom­
с and 129 mice, which were the parental strains
binant clones by hybridization of single plaques "in
of the BALB/Mo mouse strain (12). The ob­
situ " Science 196:180-182
served difference in size of 9 kb between BALB/
2 Bishop, J. M. 1978 Retrovirusee Annu Rev Biochem.
Mo and its parental strain (Fig. 3) is in agree­
47:37-88
3 Blattner, F. IL, В. G. Williams, A. Biechi, К. Denniement with the insertion of a single M-MLV
ton-Thompson, Η. E. Faber, L· Furlong, D. J.
genome and suggests that no major deletions
Gninwald, D. O. Keifer, D. D. Moore, J. W.
have occurred in the cellular DNA during the
Schunun, E. L. Sheldon, and О. Smithies. 1977
integration process. Analysis with a variety of
Charon phages safer'denvatives of bacteriophage
lambda for DNA cloning Science 196:161-169
restriction enzymes did not allow us to deter­
4 Boseelman, R. Α., L. J. L. D. van Griensven, M. Vogt,
mine whether the germ line integration of Mand I. M. Verma. 1979 Genome organization of retroMLV took place into the BALB/c or 129 ge­
vmises VI Heteroduplex analysis of ecotropic and xennome, since the same restriction fragments were
otropic sequences of Moloney mink cell focus-mducing
79
VOL. 36,1980
M-MLV VIRAL DNA CLONES IN BACTERIOPHAGE LAMBDA
viral RNA obtained from either a cloned isolate or a
thymoma cell line J Virol 32:968-978
5 Bremdl, M., J. Doehmer, K. Willecke, J. Daueman,
and R. Jaeniech. 1979 Germ line integration of Mo­
loney leukemia virus Identification of the chromosomal
integration site by somatic cell genetics Proc Natl.
Acad Sci U S A 78:1938-1942
β Cohen, J. С 1980 Methylation of milk-borne and genet­
ically transmitted mouse mammary tumor virus proviral DNA Cell 19:653-662
7 Fan, H., and D. Baltimore. 1973 RNA metabolism of
munne leukemia virus Detection of vinis-specific RNA
sequences m infected and uninfected cells and identifi­
cation of vmis-specific messenger RNA J Mol Biol.
80:93-117
8 Gilboa, E., S. Goff, A. Shields, F. Yoshlmura, S. Mi­
tra, and D. Baltimore. 1979 "In vitro" synthesis of a
9 kbp terminally redundant DNA carrying the mfectivity of Moloney murine leukemia virus Cell 16:863-874
9 Graham, F. L-, and A. J. v a n der Eb. 1973 A new
technique for the assay of infectmty of human adeno­
virus 5 DNA Virology 52:456-461
10 Hager, G. L., E. H. Chang, H. W. Chan, С. F. Garon,
M. A. Ureal, M. A. Martin, E. M. Scolnick, and D.
R. Lowy. 1979 Molecular cloning of the Harvey sar­
coma virus closed circular DNA intermediates initial
structural and biological characterization J Virol 3 1 :
795-809
11 Hughes, S. H , P. R. Shank, D. H. Spector, H. J. Kung,
J. M. Bishop, H. E. Varmus, P. K. Vogt, and M. L.
Breitman. 1978 Proviruses of avian sarcoma virus are
terminally redundant, co extensive with unintegrated
linear DNA and integrated at many sites. Cell 15:13971410
12 Jaeniech, R. 1976 Germ line integration and Mendelian
transmission of the endogenous Moloney leukemia vi­
rus Proc Natl Acad Sci U S A 73:1260-1264
13 Jaeniech, R. 1979 Moloney leukemia virus gene expres­
sion and gene amplification in preleukemic and leu­
kemic Balb/Mo mice Virology 93:80-90
14 Ju, G., L. Boone, and A. M. Skalka. 1980 Isolation and
characterization of recombinant DNA clones of avian
retroviruses size heterogeneity and instabüity of the
direct repeat J Virol 33:1026-1033
15 Kirby, Κ. 196Θ Isolation of nucleic acids with phenolic
solvents Methods Enzymol 12:87-99
16 Lai, M. H. T., and I. M. Verma. 1980 Genome organi­
zation of retroviruses VII Infection by ds DNA synthe­
sized "in vitro" from Moloney munne leukemia virus
generates a virus indistinguishable from the original
virus used in reverse transcription Virology 100:194198
17 Leder, Ρ , D. Tiemeier, and L Enquist. 1977 EK-2
dérivâtes of bacteriophage lambda useful m the cloning
of DNA from higher organisms the Agt WES system
Science 196:175-177
18 Lowy, D. R., E. Rands, S. K. Chattopadhyay, С F.
Garon, and G. L. Hager. 1980 Molecular cloning of
infectious integrated munne leukemia virus DNA from
infected mouse cells Proc Natl Acad Sci U S A 77:
614-618
19 Maniatis, T., A. Jeffrey, and D. Kleid. 1975 Nucleotide
sequence of the nghtward operator of phage λ Proc
Natl Acad Sci U S A 72:1184-1188
20 McDonnel, M W., M. N. Simon, and F. W. Studier.
1977 Analysis of restnction fragments of T7 DNA and
determination of molecular weights by electrophoresis
263
in neutral and alkaline gels J Mol Biol 110:119-146
21 Oliff, A. I., G. L·. Hager, Ε. H. Chang, E. M. Scolnick,
H. W. Chan, and D. R. Lowy. 1980 Transfection of
molecularly cloned Fnend murine leukemia virus DNA
yields a highly leukemogenic helper-independent type
С virus J Virol 33:475-486
22 Rowe, W. P., W. Pugh, and I. W. Hartley. 1970 Plaque
assay techniques for murin? leukemia viruses Virology
42:1136-1139
23 Sabrán, J. L , T. W. Hsu, С Yeater, A Kaji, W. S.
Mason, and J. M. Taylor. 1979 Analysis of integrated
avian RNA tumor virus DNA in transformed chicken,
duck, and quad fibroblasts J Virol 29:170-178
24 Sharp, P., B. Sugden, and J. Sambrook. 1973 Detec­
tion of two restnction endonuclease activities in Hae
mophilus parainfluenzae
using analytical agaroseethidium bromide electrophoresis Biochemistry 12:
3055-3063
25 Smotkin, D., A. M. Gianni, S. RozenMatt, and R. A.
Weinberg. 1975 Infectious viral DNA of munne leu­
kemia virus Proc Natl Acad Sci U S A 72:4910-4915
26 Southern, W. M. 1975 Detection of specific sequences
among DNA fragments separated by gel electrophore­
sis J Mol Biol 98:503-517
26a Sutcliffe, J. G., T. M. Shmick, I. M. Verma, and R. A.
Lemer. 1980 Nucleotide sequence of Moloney leuke­
mia virus the 3 end reveals details of replication anal­
ogy to bacterial transposons and an unexpected gene
Proc Natl Acad Sci U S A 77.3302-3306
26b v a n Beveren, C , J. G. Goddard, A. Berns, and I. M.
Verma. 1980 Stiicture of Moloney murine leukemia
viral DNA nucleotide sequence of the 5' long terminal
repeat and adjacent cellular sequences Proc Natl
Acad Sci U S A 77:3307-3311
27 van der Putten, H., E. Terwindt, A. Berne, and R.
Jaenieh. 1979 The integration sites of endogenous and
exogenous Moloney munne leukemia virus Cell 18:
109-116
28 van de Woude, G. F., M. Oekarsson, L. W. Enquist,
S. Nomura, M. Sullivan, and P. J. Fischinger. 1979
Cloning of integrated Moloney sarcoma proviral DNA
sequences in bactenophage Proc Natl Acad Sci
U S A 76:4464-4468
29 Verma, I. M. 1977 The reverse transcriptase Biochim
Biophys Acta 473:1-37
30 Verma, I. M., M. H. T. Lai, R. A. Bosselman, M. A.
McKennett, H. Fan, and A. Berns. 1980 Molecular
cloning of unintegrated Moloney mouse sarcoma viral
DNA in bactenophage lambda Proc Natl Acad Sci
U S A 77:1773-1777
31 Verma, I. M., and M. A. McKennett. 1978 Genome
organization of RNA tumor viruses II Physical maps
of in vitro synthesized Moloney munne leukemia virus
double-stranded DNA by restnction endonucleases J
Virol 26:630-645
32 Wahl, G. M., M. Stern, and G. R. Stark. 1979 Efficient
transfer of large DNA fragments from agarose gels to
diazo benzyloxy methyl-paper and rapid hybridization
by using dextran sulfate Proc Natl Acad Sci U S A
76:3683-3687
33 Weinberg, R. 1977 Structure of the intermediates lead­
ing to the integrated provirus Biochim Biophys Acta
473:3^-15
34 Yoshimura, F. K., and R. A. Weinberg. 1979 Restric­
tion endonuclease cleavage of linear and closed circular
munne leukemia viral DNAs discovery of a smaller
form Cell 16:323-332
Cell Vol 24 729-739 June 19β1 Copyright © 1981 by MIT
Г^арІРГ
^
IV.
M-MuLV-lnduced Leukemogenesis: Integration and
Structure of Recombinant Proviruses in Tumors
Herman van der Putten, Wim Quint,
Janny van Raaij, Els Robanue Maandag,
Inder M. Verena* and Anton Berns
Department of Biochemistry
University of Nijmegen
Geert Grooteplein N21
6525 EZ Nijmegen, The Netherlands
* Salk Institute for Biological Studies
P O Box 8 5 8 0 0
San Diego, California 9 2 1 3 8
Summary
M-MuLV-speclfic DNA probes were used to estab­
lish the state of integration and amplification of
recombinant proviral sequences in Moloney virusinduced tumors of Balb/Mo, Balb/c and 129 mice.
The somatically acquired viral sequences contain
both authentic M-MuLV genomes and recombinants
of M-MuLV with endogenous viral sequences. All
reintegrated genomes carry long terminal repeat
(LTR) sequences at both termini of their genome. In
the preleukemic stage a large population of cells
exhibiting a random distribution of reintegrated MMuLV genomes are seen, but during outgrowth of
the tumor, selection of cells occurs leaving one or
a few clonal descendants In the outgrown tumor. In
this latter stage recombinant genomes can be de­
tected. Although these recombinants constitute a
heterogeneous group of proviruses, characteristic
molecular markers are conserved among many in­
dividual proviral recombinants, lending credence to
the notion that a certain recombinant structure Is a
prerequisite for the onset of neoplasia. The struc­
ture of these recombinants shows close structural
similarities to the previously described mink cell
focus-inducing (MCFHype viruses.
Introduction
Murine leukemia viruses can induce leukemogenesis
in a wide variety of mice The endogenous C-type
virus is part of the mouse genome and is transmitted
from parent to offspring In contrast, the exogenous
murine retroviruses are acquired horizontally and so
are normally not part of the germ line However it is
possible to introduce Moloney murine leukemia virus
into the germ line of mice by infecting preimplantation
embryos at the 4 - 8 cell stage (Jaemsch, 1976, 1977,
Jaemsch et al , 1981) The Balb/Mo mouse stram
carries Moloney murine leukemia virus (M-MuLV) as
an endogenous virus (Jaemsch, 1976, 1977) in a
single Eco RI DNA fragment of 27 kb (van der Putten
et a l , 1979, Jaehner et a l , 1980) at a single Mendehan locus (Mov-1 ) on chromosome number 6 (Breindl
et al , 1979) While these Balb/Mo mice are viremic
from birth and develop a thymus-dependent leukemia
of viral (M-MuLV) etiology (Nobis and Jaemson, 1980)
after a latency of several months, Balb/c and 129
mice do not develop this disease spontaneously at
this age (Peters et al , 1973, Jolicoeur et al , 1978)
However, when newborn Balb/c or 129 mice are
injected with M-MuLV, a thymus-dependent leukemia
follows the expression of high titers of the virus in the
blood (Jaemsch et al , 1975, Reddy et al , 19Θ0)
Leukemogenesis in Balb/Mo and newborn-infected
Balb/c and 129 mice is accompanied by somatic
amplification (Jaemsch et al , 1975, Jaemsch, 1979)
and reintegration of Moloney viral sequences in new
chromosomal sites of tumor tissues (van der Putten et
a l , 1979, Jaehner et al , 1980) The endogenous virai
integration site occupied in preimplantation embryos
is not preferentially occupied when newborn Balb/c
mice are infected with M-MuLV Some of the reinte­
grated Moloney sequences do not contain the contig­
uous genomic information characteristic for M-MuLV
but are composed of incomplete, rearranged or recombmed Moloney proviral genomes present in new
sites in the chromosomal DNA (van der Putten et al ,
1979, Jaehner et al , 1980)
Recombinants between Moloney virus and xenotropic viruses have been isolated from lymphomas of
leukemic Balb/Mo mice (Vogt, 1979) Heteroduplex
studies revealed that these are env-gene recombi­
nants of M-MuLV with xenotropic viruses (Bosselman
et al , 1979) This type of virus has been implicated in
malignant transformation of erythroid and lymphoid
cells in several mouse strains, and the presence of a
recombinant virus with such a distinct structure in the
DNA of every virus-induced lymphoma would suggest
a role for recombinant viruses in the leukemogemc
process Therefore we have searched for common
features of integrated recombinant viral genomes in
Moloney virus-induced tumors of leukemic Balb/Mo,
Balb/c and 129 mice
Until now this kind of analysis has been severely
hampered by extensive nucleotide sequence homol­
ogy between the endogenous viral DNA sequences
and the nucleic acids of M-MuLV and other exogenous
retroviruses To avoid this problem we prepared two
kinds of molecular probes that recognize M-MuLV
without interacting with other endogenous viruses MMuLV-specific cDNA probes prepared by absorption
of the sequences that M-MuLV has in common with
other retroviruses and M-MuLV-specific DNA frag­
ments molecularly cloned in the plasmid pBR322 We
report the physical mapping of M-MuLV-related ge­
nomes integrated in the DNA of a series of preleu­
kemic and leukemic tissues of Balb/Mo, Balb/c and
129 mice
Results
Characterization of M-MuLV-Specific DNA Probes
M-MuLV-Specific cDNA
The M-MuLV-specific cDNA probe was prepared by
a procedure slightly modified from those described
81
Cell
730
pBR322 by (dC-dG) tailing method as described in
Experimental Procedures. Three of the M-MuLV-specific probes (MS-2, MS-3 and MS-4) were mapped at
different positions of the M-MuLV genome is shown in
Figure 1D. The size of the inserts was rather small
(from 150 to 200 nucleotides). Both the selected
cDNA probe and the MS probes recognize M-MuLVspecific sequences distributed throughout the MMuLV genome. More importantly, the results in Figure
1С show clearly that these specific probes are capa­
ble of recognizing M-MuLV-specific sequences
among the background of endogenous viral genomes.
previously (van der Putten et al., 1979; Bacheler and
Fan, 1981; Jaehner et ai., 1980) and is outlined in
Experimental Procedures. The resulting M-MuLVspecific cDNA probe detects only M-MuLV se­
quences; other endogenous viral sequences (Keshet
et al., 1980) in Balb/Mo, Balb/c and 129 mice were
not recognized (Figure 1A). Furthermore, the MMuLV-specific cDNA probe displayed hybridization
with genomic fragments derived from different loca­
tions of cloned M-MuLV proviral DNA (Gilboa et al.,
1979; Yoshlmura and Weinberg, 1979; Berns et al.,
1980; С. Shoemaker, personal communication) as
shown in Figure IB. Some preferential hybridization
was seen to fragments which map in the substitution
loop structures of heteroduplexes between M-MuLV
full-length cDNA and AKR (Chien et al., 1978) and
Moloney mink cell focus-inducing (MCF) viral RNA
(Bosselman et al., 1979). The results indicate that MMuLV cDNA-specific sequences are present at many
locations on the M-MuLV genome and the cDNA
probes can be used successfully to detect M-MuLV
subgenomic fragments in cellular DNA.
Molecularly Cloned M-MuL V- Specific DNA
Fragments
A random population of in vitro synthesized M-MuLV
double-stranded DNA was cloned in the plasmid
Integration Sites of Somatically Acquired
Sequences in Tumors
Earlier studies have shown that somatically acquired
M-MuLV sequences (Jaenisch et al., 1975; Jaenisch,
1979) are found in many sites of the mouse genome
(van der Putten et al., 1979). A distinct pattern of
reintegrated M-MuLV genomes is seen in outgrown
tumors of individual animals indicating a monoclonal
origin of the tumors (Jaehner et al., 1980).
Study of the patterns of reintegrated viruses, how­
ever, have provided little insight into the structure of
reintegrated M-MuLV sequences both in the early and
later stages of the disease. Therefore, DNAs from a
с
в
CDNA
• 2ШМ-МЦ/сІопе
M S РГОЬяа
Figure 1. Characterization of the M-MuLVSpectfic DNA Probes
(A) 10 μβ of 129. Balb/c and Balb/Mo brain
DNA were cleaved by Eco RI and analyzed for
M-MuLV-specific sequences by a selected
η а
cDNA probe as described in Experimental Pro­
=» л те
£: ш m kbp
cedures. The M-MuLV-spectfic cDNA probe
<cDNAJ recognizes specifically the Mov-1 MMuLV proviral DNA among other endogenous
viral sequences, while a M-MuLV total cDNA
probe (cDNA) recognizes many endogenous
viral sequences
(B) DNA from M-MuLV proviral DNA (contain­
ing one LTR) cloned Into phage Charon 21A
(Berns et al.. 1980) was cleaved with Hind III,
Htnd Hi-Bam Hi and Hind Hl-Kpn I; each lane
represents an equlmolar amount of M-MuLV
DNA as present in tissue DNA from a homo­
zygous Balb/Mo mouse (2 copies/diploid ge­
nome equivalent). Fragments were transferred
to nitrocellulose sheets and hybridized to MMS-2
MS-3
MuLV-specific cDNA as described in Experi­
mental Procedures The line figure shows the
restriction map of molecularly cloned lamda,
MuLV-1 (Berns et al.. 1980) Since the MMuLV viral DNA was cleaved at the unique
i 1 il
Hind III site and cloned in the unique Hind Ml
pol
gag
I env
site of Charon 21 A. the tine diagram shows
the structure of the permuted M-MuLV DNA. (Broken line at 8.2 kb indicates the 5' end of the nonpermuted M-MuLV genome ) The molecular
weights of the M-MuLV-specific restriction fragments are indicated in the figure. Marker DNA fragments that have been used for these and
subsequent analyses were Eco RI. Hind III. Bam HI and Kpn I digests of Ad2 viral DNA and phage lambda DNA: molecular weights Indicated in this
and subsequent figures were determined from log molecular weight versus mobility curves.
(С) Юмд of 129. Balb/c and Balb/Mo brain DNA cleaved by Eco RI, were analyzed by hybridization to MS-2. MS-3 and MS-4 probe. All these
probes yielded the same result: no hybridization to endogenous viral sequences in 129 and Balb/c mouse DNAs and specific recognition of the
Mov-1 M-MuLV proviral DNA in the characteristic 27 kb Eco RI DNA fragment of Balb/Mo brain DNA.
ÍD) The positions of the MS probes on the linear map of the M-MuLV genome are shown The plasmid MS-3 mapped in the gag gene region, while
MS-4mappedin the putative carboxy terminal part of the po/gene (Chien et a!.. 1978. Sutciiffeet al.. 1980) MS-2 mapped m the LTR indicating
that terminally redundant sequences of different MutVs display unique sequences
oS
CDNAB
о
al
ili
D
inu
#
II
Iff
Ui
R2
M-MuLV-Induced Leukemogenesis
731
series of tumors of Balb/Mo and Balb/c mice (the
latter infected with M-MuLV when newborn) were an­
alyzed in more detail by restriction endonuclease map­
ping using a selected cDNA probe and M-MuLV-specific (MS) plasmid probes. High molecular weight
DMAs (van der Putten et al., 1979) were cleaved with
Eco RI and analyzed by Southern blotting (Figure 2).
The MS-2 plasmid probe, which recognizes M-MuLVspeciflc sequences In the large terminal repeat (LTR)
hybridized to the same array of Eco RI fragments as
the selected cDNA probe. This Indicates that each
reintegrated M-MuLV genome or subgenomic frag­
ment contains M-MuLV-speclflc LTR sequences. In
contrast, the plasmid probes MS-3 and MS-4, which
correspond to regions in gag and pol, respectively,
hybridized to about half the fragments detected by
MS-2 or the selected cDNA probe. The detection of
fragments smaller than 8.8 kb suggests the involve­
ment of internal rearrangements of the proviruses
(Figure 2). Apparently the somatically acquired MMuLV genomes are often modified, deleted or recomblned. More detailed analyses of the integrated MMuLV genomes revealed a rather specific recombi­
nation event leading to the acquisition of an Eco RI
site within the recombinant genomes. Therefore the
detection of multiple M-MuLV-contalnlng Eco RI DNA
fragments Is not due to a large variety of structural
rearrangements within provlral genomes but rather to
integrations In different sites of the host genome. No
preferential sites seem to exist, since outgrown tumors
of different animals show a distinct Integration pattern
specific for each individual animal (Jaehner et al.,
1980; data not shown).
In contrast, preleukemic Balb/Mo (Jaehner et al.,
1980; our unpublished results) and Balb/c mice (in­
fected with M-MuLV when newborn) do not display
this pattern of Integrated recomblned M-MuLV se­
quences in the DNA of their target tissues. However,
these tissues do contain 'randomly' integrated MMuLV genomes as shown by analyses with restriction
TOTAL
с *4Л
SPECIFIC cDNA
MS ,
"b
endonucleases Ava I and Kpn I. For example, restric­
tion endonuclease Ava I cleaves M-MuLV DNA ten
times to generate 11 fragments of 2.45, 1.85, 1.5,
1.2 kb and a number of fragments ranging from 80 to
600 nucleotides. The four major Ava I fragments and
the Kpn I fragments characteristic for M-MuLV proviral
DMA (Figure 4B) can be detected in all preleukemic
target tissues as well as In Balb/Mo and Balb/c tumor
DNAs.
Molecular Structure of M-MuLV Sequences In
Tumors
Most recombinant viruses isolated from leukemic
Balb/Mo mice (Vogt, 1979) and from other strains
with a high incidence of leukemia are env gene recom­
binants (Elder et al., 1977; Hartly et al., 1977; Chien
et al., 1978; Donoghue et al., 1978; Faller et al.,
1978; Rommelaere et al., 1978; Shih et al., 1978;
Bosselman et al., 1979; Lung et al., 1980; Rowe et
al., 1980). They are composed mainly of ecotropic
MuLV parental sequences, and therefore differences
in restriction endonuclease patterns with M-MuLV can
be expected to be derived mainly from the env gene
region. To detect recombinant M-MuLV sequences,
tumor DNAs were cleaved by restriction endonuclease
Kpn I which cleaves M-MuLV proviral DNA in the LTR
at map positions 0.5 and 8.7 kb, and within the coding
region at position 3.4, 6.05 and 7.35 k b ( G i l b o a e t a l . ,
1979; Berns et al., 1980; Shoemaker et al., 1980).
The genetically transmitted Mov-1 M-MuLV DNA in
both tumor and nontumor DNA of BALB/Mo mice is
cleaved at nearly identical positions (Figure 3).
All BALB/Mo, BALB/c and 129 tumor DNAs dis­
played one or more new Kpn I DNA fragments in
addition to those derived from the endogenous MMuLV (BALB/Mo) or the exogenous M-MuLV clone
1A provlral DNA (BALB/c and 129) as shown in Figure
4. Since unlntegrated provlral DNAs were removed
from our DNA preparations (van der Putten et al.,
1979), the new Kpn I DNA fragments must be derived
Figure 2 M-MuLV Sequences in Eco RI Frag­
ments of Cellular DNAs
Each panel represents an individual gel with
five lanes containing, respectively, Eco RI
fragments of Balb/c brain DNA. Balb/Mo
brain DNA. two Balb/Mo (mouse M9 and M10)
tumor DNAs. and a newborn-infected Balb/c
mouse (503; infected with 10 XC plaques of
M-MuLV clone 1 A) tumor DNA Southern blots
were screened with a representative M-MuLV
cDNA probe, a selected M-MuLV-specific
cDNA probe, and nick-translated plasmid
probes of MS-2. MS-3 and MS-4. respectively,
as described in Experimental Procedures 129
mouse DNA displays the same characteristic
features when hybridized to a total or selected
cDNA probe as Balb/c DNA, the major differ­
ence is the presence of the endogenous Akv
genome in Balb/c DNA. which is not present
In the 129 mouse genome (see also Quint et
a l , 1981)
Cell
732
Balb/Mo Mov-1
-4НШІ
Figure 3 Restriction Map of the Mov 1 M MuLV DNA and Flanking Cellular Sequences
This map was constructed by combining restriction data from the cloned 5' viral DNA-eellular DNA 8 9 kb Hmd til fragment (Berns et al 1960)
and additional restriction analysis of Balb/Mo tissue DNAs (brain) and Isolated Mov-1 M MuLV DNAs from both tumor and nontumor tissues (see
also Figure 5) Balb/Mo DNA was cleaved with several restriction enzymes and screened with a M MuLV-specific cDNA probe as described in
Experimental Procedures The presence of the Sma I site at position β θ kb and the Pvu II alte at position θ 32 kb are still presumptive these sites
have been mapped in cloned M-MuLV proviral DNA (our unpubllahed resulta Shoemaker et al 19Θ0) but not yet in the integrated Mov 1 M MuLV
DNA Also the 3 Kpn I fragment (7 Э5- 7 kb) contains a small deletion (60-BO nucleotides see for example Figure 4A) when compared to MMuLV clone 1A proviral DNA (see for example Figure 4E)
from integrated vtral DNAs Although a variety of Kpn
I fragments could be observed in some tumors (Figure
4A, lanes 5, 7, 9,10) all tumor DNAs contained a 5 25 6 (5 4) kb and/or 4 0-4 θ (4 4) kb Kpn I DNA
fragment It thus appears that outgrown tumors within
each mouse (tumors from different anatomical loca­
tions showed the same pattern) contain a specific
subset of M-MuLV-denved sequences, integrated in
different chromosomal sites In the preleukemic tis­
sues (thymus and spleen) the additional Kpn I frag­
ments (Figure 4B) observed in DNA from outgrown
tumor tissues were not detected (unless after ex­
tremely long exposures, Figure 4B lanes 9-13)
Therefore, the appearance of these new Kpn I DNA
fragments coincides with the outgrowth of 'umor cells
All new Kpn I DNA fragments around 5 kb, identified
in DNA from tumors of Balb/Mo mice or newborninfected Balb/c and 129 mice, could be cleaved by
Eco RI (Figure 4C), resulting in the generation of a 3 6
kb fragment and one or more fragments of approxi­
mately 1 7 kbp, in two instances a 4 kb fragment was
found in addition (Figure 4C, lanes 6 and 9) The
presence of an Eco RI site in these Kpn I DNA frag­
ments indicates that non-M-MuLV sequences consti­
tute part of these fragments (Verma and McKennett,
1978, Smotkm et al , 1976)
The Kpn I DNA fragments around 5 kb as well as
the 3 6 kb fragment (Kpn l-Eco RI) are all recognized
by the MS-4 plasmid probe (Figure 4D) which maps In
the putative carboxy-termmal region of the poi gene
(Figure 1D)
Hybridization studies with MS-2 and MS-3 were
performed to determine whether sequences derived
from the gag and LTR regions were contained in the
5 4 and 4 4 kb Kpn I DNA fragments Kpn l-digested
tumor DNA was hybridized to MS-3 probe, which had
been localized in the gag gene region (Figure 1)
Figure 4E shows that while M-MuLV-speciftc cDNA
recognizes the additional Kpn I fragments in tumor
DNA from three different 129 mice, the MS-3 probe
only recognizes the M-MuLV-specific 2 9 kb Kpn I
DNA fragment MS-2 (which maps in the LTR region)
recognized the 5 4 kb tumor-specific Kpn I fragments
whereas the 4 4 kb fragments were not detected (data
not shown) Based upon these hybridization results
with MS-2, MS-3 and MS-4 probes, the location of the
recombinant Kpn I 5 4 kb fragments extends from
map position 3 4 toward the 3' end and the 4 0-4 θ
kb fragments from 3-3 5 to 7 4 kbp Therefore both
contain the information for a recombinant em gene
In animal 14 (Figure 4D, lane b) Eco RI + Kpn I
digestion generated, in addition to the 3 6 kb frag­
ment, a fragment of 4 0 kb also recognized by MS-4,
suggesting the presence of yet another organization
of recombinant proviral DNA Since this fragment can
also be obtained by cleaving the 4 4 kb Kpn I fragment
by Eco RI it is probably derived from position 3-7 35
on the corresponding M-MuLV map Apparently re­
combinations deviating from the one described above
can be fixed in tumor DNA although with much lower
frequency We want to emphasize, however, that they
are only formed in addition to the recombinant de­
scribed earlier and never constitute the single recom­
binant genome type in tumor DNA Upon digestion of
tumor DNA with Eco Rl-Kpn I, besides a homogene­
ous 3 6 kb fragment, heterogeneous smaller frag­
ments of around 1 7 kb can be detected which are
not seen in nontumor DNA (Figure 4C) These small
fragments correspond to the region 3' terminal of the
Eco RI site in the recombinant viral sequences The
heterogeneity in fragment sizes observed seems
mainly to be due to variation in LTR sequences of the
recombinant proviral DNAs (data not shown) The
variation in size of these smaller fragments is also
reflected In the heterogeneity of the 5 4 kb Kpn I DNA
fragments (Figure 4C)
The variation in size of the 4-4 8 kb Kpn I fragments
might be caused by the position of the recombination
site around map position 7 35 kb Several Kpn I sites
probably are present in the parental genomes around
m
M-MuLV-lnduced Leukemogenests
733
A
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Figure 4. M-MuLV-Specific and Recombinant M-MuLV Sequences In Baib/Mo and Balb/c Mouse Tumors
(A) 10 μβ of Balb/Mo tissue DNAs were cleaved by Kpn I, fractionated on 1 % agarose gel and screened with a selected cDNA probe as described
in Experimental Procedures. Lanes represent DNAs from Balb/Mo tumors from mouse M9 (spleen), MIO (spleen). M28 (spleen). M27 (tnymus).
Μ26 (thymus). M23 (thymus). M l 9 (spleen). M18 (spleen). M14 (spleen). M13 (spleen). M l 2 (spleen). M i l (thymus), and Balb/Mo brain DNA
The differences in hybridizations between tumor DNAs do not reflect differences in amount of DNA loaded in each lane; they merely reflect
differences In numbers and amounts of reintegrated M-MuLV and recombinant sequences in individual tumors.
(B) Balb/c preleukemic and leukemic tissues. Lanes represent DNAs analyzed as in A. Balb/Mo and Balb/c DNAs are shown for reference (lanes
1 and 2). Lanes 3-13 represent tissue DNAs from Balb/c mouse 503 (spleen: infected with 10 XC plaques of M-MuLV when newborn, sacrificed
at 6 months of age), from 5 Balb/c mice (infected with 10' XC plaques of M-MuLV when newborn; sacrificed at 3-6 weeks of age; lanes 4-8
contain either spleen or thymus DNA) at preleukemic stage and from 5 Balb/c mice (10 e XC plaques) sacrificed at 10-12 weeks of age (lanes 9 13. either thymus or spleen), when leukemic. No additional Kpn I fragments can be detected in preleukemic tissue DNAs: leukemic tissue DNAs
do display new fragments characteristic of integrated recombinant M-MuLV genomes in addition to fragments specific for authentic M-MuLV
genomes. The lower part of the autoradiogram is not shown (see Figure 4E) and was identical to Figure 4E (only a doublet of 1.3 kb).
(C) 10 pg of Balb/Mo DNAs. cleaved by Kpn I + Eco RI, were screened with a selected cDNA probe as described In Experimental Procedures
Lanes represent the same tissue DNAs as shown in A The lower part of the x-ray film was exposed for a longer period in order to visualize the
smaller fragments.
(D) Each set of lanes represents a Kpn I (lane a) and Kpn l-Eco Bl (lane b) digest of Balb/Mo mouse tumor DNA as shown in A, and hybridized
to the MS-4 Plasmid probe which maps In the putative carboxy terminal part of the pol gene (Figure 1 ). Both the Kpn I DNA fragments (5 4 and 4 4
kb) and the Kpn I-Eco RI DNA fragments (3 6 and 4 0 kb) diagnostic for recombinant M-MuLV genomes hybridize to MS-4 probe. The 2 9 kb
fragment derived from integrated authentic M-MuLV genomes is also detected by MS-4 probe
(E) 10 μg of 129 tumor DNAs (obtained from three individual 129 mice, injected with about 10 XC plaques of M-MuLV clone 1A and sacrificed at
6 months of age) were cleaved by Kpn I and analyzed with a selected cDNA probe and the M5-3 Plasmid probe as described in Experimental
Procedures The recombinant Kpn I DNA fragments do not hybridize to the MS-3 Plasmid probe.
this position, giving rise to a variety of recombinant
Kpn I fragments depending on the exact site of recom­
bination. So far we have not determined the genomic
location of the Kpn I fragments of around 1.3 kb seen
in tumors of some Balb/Mo mice (Figure 4A, lanes 5,
9, 10).
To establish a fine structure map of the rearranged
M-MuLV genomes, the detailed restriction map of the
Cell
734
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Figure 5 Structures of the Mov-1 M-MuLV and Recombinant Proviral DNA
(A) Lanes represent Moy-1 M-MuLV DNA (20-100 kb Eco RI DNA fraction from Balb/Mo brain: [van der Putten et al., 1979P. equivalent to 10
μο of Balb/Mo brain DNA and cleaved with restriction endonucleases as indicated. Lanes 2-5 show the Mov-1 M-MuLV DNA fraction cleaved with
Kpn I and an additional enzyme, while lanes 7-10 represent similar analyses with Sac I and another enzyme as indicated. Fragments were
separated on 1 % agarose gel and analyzed with a selected M-MuLV-speciflc cDNA probe as described in Experimental Procedures (the upper
band in the Bgl I digestion is a partial cleavage product).
(B) Lanes represent 10 ;ig of tumor DNA from Balb/Mo mouse M9. analyzed as in A. Both the Mov-1 M-MuLV-speciflc restriction fragments and
the new fragments specific for recombinant M-MuLVs ere shown In this map. No hybridization was detected >5 8 kb. Sma I does cleave the Mov1 M-MuLV DNA from Balb/Mo tumor M9 (this was also confirmed by cleaving the isolafed Mov-1 M-MuLV DNA in the 20-100 kb Eco Hl DNA
fraction from Balb/Mo tumor DNA), but no from brain due to tissue-specific methylation.
(C) 10 fig of tumor DNA from Balb/c mouse 503, infected with M-MuLV when newborn, was analyzed In each lane as In B. Lane 2 shows two Bam
HI DNA fragments of 2 2 and 2 5 kb, respectively, diagnostic for recombinant M-MuLV genomes in addition to the 3 kb fragment characteristic for
authentic M-MuLV proviral DNA
'. (D) Lanes represent 10 дд of 129 tumor DNA, isolated from three animals with a distinct integration pattern of M-MuLV sequences (same animals
as analyzed in Figure 4E) after cleavage by 8am HI The first three lanes show the analysis with a selected M-MuLV-speciflc cDNA probe while
M-MuLV-lnduced Leukemogenesis
735
genetically transmitted M-MuLV (Figure 3) was com­
pared with the physical map of somatically acquired
M-MuLV genomes Cleavage of tumor DNA from both
Balb/Mo and newborn-infected Balb/c and 129 mice
revealed the presence of internal fragments charac­
teristic of authentic M-MuLV in addition to fragments
derived from integrated recombinant viruses (Figure
5C, lane 2 shows two additional Bam HI fragments of
2 2 and 2 5 kb compared to Figure 5A, lane 7) Figure
5C lane 4 shows, in addition to the Kpn l-Sac I
fragments of 2 9, 2 65, 1 3 and 1 25 kb characteristic
for M-MuLV, the 4 4 kb Kpn I fragment diagnostic for
the presence of a recombinant M-MuLV genome The
2 2 kb Bam HI fragment was found in every tumor
DNA, like the 3 6 kb Eco Rl-Kpn I fragment Also, the
2 2 kb Bam HI fragment was recognized by the MS-4
probe For example, the DNA of three M-MuLV-mduced tumors of different 129 mice with each a dis­
tinct integration pattern of M-MuLV sequences (Figure
5D, lanes 1,2,3) contained the same 2 2 kb Bam HI
recombinant DNA fragment in addition to the 3 kb
fragment characteristic for M-MuLV The resulting
physical maps of the two slightly different integrated
recombinant viruses is depicted in Figure 5E
Discussion
Amplification of viral sequences is observed in tumors
of Moloney virus-induced leukemias of Balb/Mo,
Balb/c and 129 mice (van der Putten et a l , 1979,
Jaenisch et a l , 1975, Jaenisch, 1979, Jaehner et a l ,
1980) Similar phenomena have been reported for
MuLV-mduced thymomas in NIH/Swiss mice (Steffen
and Weinberg, 197Θ) and in tumors of leukemic AKR
mice of several inbred sublines and Akv congenie
mice (Canaam and Aaronson 1979, Steffen et al ,
1979 Quint et al , 1981), in M-MTV-mduced carci­
nomas (Varmus et al , 1978, Morris et al , 1980), in
bovine leukemia virus (BLVHnduced lymphocytosis
(Kettmann et al , 1980), and avian leukosis virus
(ALVHnduced metastasizing lymphomas (Neiman et
al , 1980) Somatic acquisition of viral sequences
seems therefore to be a frequently observed phenom­
enon dunng virus-induced tumor development
M-MuLV-induced leukemias of Balb/Mo, Balb/c
and 129 mice display a unique pattern of reintegrated
M-MuLV sequences for each animal and no common
ИГ,
reintegration sites are detected (Jaehner et a l , 1980)
Hybridization studies with M-MuLV-specific Plasmid
probes and a selected cDNA probe show that all
outgrown tumors contain both somatically acquired
authentic M-MuLV genomes and recombinant provi ral
genomes, each integrated in a different site of the
chromosomal DNA These recombinants carry an Eco
RI site, cleavage with Eco RI generates fragments of
subgenomic size, all of which hybridize to a probe
recognizing the LTR region (MS-2) The smaller frag­
ments do not, however, hybridize to a 5'-speciftc
probe (MS-3) or to a probe recognizing a region
proximal to the recombinant region (MS-4) This sug­
gests that the majority of these smaller fragments
represent 3' portions of recombinant proviruses How­
ever, the fragments that hybridize to MS-3 also hy­
bridize to MS-4 and are almost exclusively larger than
6 9 kb indicating that deletions in the 5' half of inte­
grated recombinant viruses (with an Eco RI site at 6 9
kb) are rare The relative simple patterns of proviral
restriction fragments confirm this notion A strong
selection pressure during the outgrowth of the tumor
might give rise to tumor cells with relatively low num­
bers of defective recombinants Alternatively, if defec­
tive recombinants are generated later in tumor devel­
opment they might escape detection by our analysis
since they will be Integrated in multiple sites at low
copy number However, heteroduplexes formed be­
tween genome-length M-MuLV cDNA and Balb/Mo
thymoma RNA showed only one major substitution
loop in the п region, suggesting the absence of
aberrant species of viral RNA in addition to nondefective recombinant viral genomes (Bosselman et a l ,
1979) whose structure we have identified here at the
DNA level
Preleukemic tissues only contain authentic contig­
uous M-MuLV genomes integrated randomly in the
host genome (see also Jaehner et al , 1980) Leu­
kemic tissues, however, harbor specifically integrated
M-MuLV as well as recombinant proviruses Clonal
selection of cells (McGrath et a l , 1980) must therefore
take place after the preleukemic phase, giving rise to
the distinct integration pattern seen in the outgrown
tumor
The molecular structure of two recombinant proviral
genomes in Balb/Mo, M9 and Balb/c 503 tumor
DMAs, respectively, is characterized by a recombma-
ttie last Ihree lanes show the hybridization results ol MS-4 probe with the same filler The M-MuLV-specltlc selected cDNA probe recognizes both
the cellular-viral DNA lunctlon fragments and the Internal M-MuLV (3 kb) and recombinant (2 2 kb) specific Barn HI DNA fragments MS-4
recognizes only the authentic M-MuLV-speciflc 3 kb Bam HI DNA fragment and the maior recombinant specific 2 2 kb Bam HI DNA fragment The
faint hybridization of MS-4 to a 6 and 1 75 kb Bam HI DNA fragment cannot yet be explained although these might represent other recombinant
proviral organizations like detective recombinant
(E) The upper line represents the map of the Mov-1 M-MuLV DNA as derived for both tumor and nontumor DNA The two lower lines show the
structures of the recombmanl Kpn I DNA fragments from tumor DNA of Balb/Mo mouse M9 (5 4 kb) and Balb/c mouse 503 (4 0 kb) respectively
the newly acquired sequences in the mink cell focus-inducing (MCFVtype proviral DNAs are Indicated by a double line The newly acquired
restnctlon-endonuclease recognition sites are also shown as well as the position of the recombinant specific Bam HI (2 2 kb) and Kpn l-Eco RI
(3 6 kb) DNA fragments detected in all tumors and diagnostic for recombinant provbral DNA The presence ol the Bgl I and Bgl II sites In the ΜΘ
recombinant and their absence m the 503 recombinant have been established by adntional digestions The lowest part of the Figure Indicates the
location of the п gene which starts at position 6 4 kb (C van Beveren personal ccjmmunicatlon)
Cell
736
Hon with cellular sequences between map positions
5 9 and 7 35 kb from the 5' end of the M-MuLV
genome (Figure 5E) Although the recombination at
around map position 7 3 5 kb seems to vary slightly in
different recombinant proviral organizations, the re­
gion coding for the carboxy terminus of gp70 and the
nucleotide sequence for p i 5E (Sutcliffe et al , 1980a,
1980b) are probably not affected by the recombmational event and are derived from the ecotropic paren­
tal genome (in agreement with heteroduplex data,
Bosselman, 1979) The newly acquired genetic infor­
mation has nearly the same length as the substituted
M-MuLV sequences and contains several new restric­
tion sites at fairly similar positions in different recom­
binants isolated from different strains of mice A Bam
HI DNA fragment of 2 2 kb (detected by MS-4, Figure
5D), specific for somatically acquired recombinant
sequences, is detected in all Balb/Mo, Balb/c and
129 mouse tumor DMAs analyzed so far Tumors of
AKR mice also display Bam HI DNA fragments of 2 2
kb and contain similar recombinants (Quint et al ,
1 9 8 1 , W Herr, personal communication) Proviral
DNA of the leukemogenic Akv recombinant virus MCF
247 isolated from AKR thymus, also carries an Eco RI
site 6 9 kb from the 5' end (Pedersen et al , 1980)
Therefore, these recombinants almost certainly rep­
resent MCF proviruses The location of the substitu­
tion in the two analyzed recombinants agrees well with
a recombination in the env gene The newly acquired
sequences probably are derived from endogenous
xenotropic viral sequences since it was shown that
substitution loops disappear by hybridization of MCF
viral RNA to New Zealand Black (NZB) or Balb virus2 cDNA (Bosselman et al , 1979) The heterogeneity
in length of the 5 4 kb recombinant Kpn I DNA frag­
ments from tumors of different mice (Figure 4A) prob­
ably reflects variation in the LTR sequences of the
recombinant M-MuLV genomes (van Beveren et al ,
1980, Rands et al , 1 9 8 1 , Ju et al , 1980, Chueng et
al , 1980) The variation in size of the 4 0 - 4 В kb
recombinant Kpn I DNA fragment can be explained by
the presence of several Kpn I sites in the parental
genomes at around map position 7 35, the exact site
of recombination determines the length of the Kpn I
fragments Despite variations in site of recombination,
all tumor DNAs contain integrated recombinant proviral DNAs with the same type of recombination with
the N H ? terminus of the env gene (which starts at
position 6 4 kb [C van Beveren, personal communi­
cation]) derived from the xenotropic parent
Our data show lhat molecular probes can be pre­
pared that can reveal the structure of rearranged
MuLV sequences among a background of related
endogenous viruses m mouse DNAs This seems a
prerequisite for revealing changes in viral structures
mtrinsicly related to the leukemogenic process The
relative inefficiency of cloned MCF viruses in the leu­
kemia acceleration test has led to doubt concerning
BV
the role of these env recombinant viruses in the leu­
kemogenic process Because of their general occur­
rence some of our observations seem relevant to virus
induced leukemogenesis Authentic M-MuLV ge­
nomes are integrated randomly in the DNA of preleukemic tissues Outgrown tumors contain authentic MMuLV genomes as well as recombinant genomes in­
tegrated in unique sites of the chromosomal DNA The
recombinants integrated in multiple copies in the dif­
ferent tumor tissues (we analyzed more than 30) show
a striking similarity in structure all are env gene
recombinants with a genomic size similar to the pa­
rental M-MuLV, which have acquired xenotropiclike ' sequences approximately between map posi­
tions 6 kb and 7 5 kb from the 5' end corresponding
to the NH2-terTTiina1 port of the envelope glycoprotein
Every lymphoma induced by M-MuLV (or AKR, Quint
et al , 1981 ) in a variety of mouse strains harbors the
same type of recombinant virus integrated in its DNA,
suggesting that similar xenotropic-hke sequences
are available for recombination in different mouse
strains, although no xenotropic virus can be induced
from some of these strains
The structural similarities of integrated recombinant
viral DNAs observed in a large number of lymphomas
induced by MuLV in several mouse strains strongly
support the hypothesis that the formation of defined
recombinants is related to the leukemogenic transfor­
mation The coincidence of the formation of these
recombinants with the clonal outgrowth of trans­
formed cells is clearly illustrated, the progenitor cell
of the outgrown tumor carries this recombinant virus
in addition to the ecotropic virus integrated in its DNA,
whereas the preleukemic target tissues contain almost
exclusively the parental ecotropic provirus These ob­
servations suggest a double role for the MuLV in
lymphomagenesis generating lymphomagenic re­
combinants (Cloyd et al , 1980, Vogt, 1979) and
acting as a helper by facilitating infection of MCF
genomes by genomic masking (Haas and Patch,
1980) or by another helper function still obscure
Since integration of defined recombinants seems
directly related to clonal outgrowth of transformed
cells, we favor a direct role of the recombinant genes
in the transformation event It might reflect a specific
property that enables the infected cells to complete
the different final steps in the metastatic process
(Poste and Fidler, 1980) Testing a variety of cloned
recombinant viruses identified first by the type of
approaches described in this paper will shed more
light on their generation and exact role in the leuke­
mogenic process
Experimental Procedures
Mice, СеИ Line· end Viruses
The origin of mice cell lines and Isolation procedures of M-MuLV
clone 1A AKR-MuLV and Balb virus-2 have been described previ­
ously (Bosselman el al 1979 van der Pullen et al 1979)
M-MuLV-Induced Leuhemogenesis
737
8H
The injection of newborn Balb/c and 129 mice was performed as
described by Jaenisch et al (1975)
Selection of cDNA Prob«
a-32P-labeled M-MuLV cDNA was prepared ae described for 3H-cDNA
(van der Putten et a l , 1979), a-^P-dCTP (Amersham, spec act
2000-3000 Ci/mmole) was used as precursor About 5 x 10 e cpm
^P-cDNA were obtained m typical reactions containing 250 μΟι a·
"P-dCTP. 1 mM dTTP. 1 mM dGTP. 1 mM dATP, 2 mg/ml calf
thymus DNA pnmer, 50 mM Trls-HCI (pH θ 3), 3 mM MgAc3. 60 mM
NaCI, 20 mM DTT, 50 μfl/ml Aclmomyclne D. 0 016% NP-40 and
0 15-0 2 mg of virus m a final volume of 150 μ\
M-MuLV cDNA sequences that crosshybndize with other mouse
viruses were removed by selectron on a mixture of AKR viral RNA ( 2 3 μ9) and RNA (200 μg) from livers of NZB mice which express
xenotropic virus information (Levy, 1973, Levy et a l , 1Θ75) This
procedure was used to absorb the major part of M-MuLV sequences
that reacted with AKR and xenotropic viral sequences as shown by
heteroduplex analysis (Chien et a l , 1976. Bosselman et a l , 1979),
5 x 10 8 cpm cDNA were hybridized to the mixture of RNAe m 10 mM
TES (pH 7 4), 1 mM EDTA. 2 д д / т і polyvlnytaulfate and 0 5 M NaCI
for 3-5 hr at 65 C C Hybrid formation was assayed by SI nuclease,
50-55% was usually Si resistant at maximal hybridization, and
hybrids were removed by chromatography on hydroxy-apatlte The
M-MuLV cDNA probe was further depleted of sequences present In
the Balb/c mouse genome by hybridization to an excess of Balb/c
mouse DNA followed by hydroxy-apatlte chromatography (Jaenisch,
1977, van der Putten et al , 1979), the remaining single-stranded
DNA traction (about 1 5 x 1 0 " cpm) was used as a M-MuLV-spectfic
cDNA probe
Restriction Analysis
Preparation of high molecular weight DNAs from tissues, restriction
analysis, electrophoresis and elutlon of DNA fragments from agarose
gels has been described (van der Putten et a t , 1979, Quint et al.,
1ΘΘ1) Phage λ DNA was included in the reactions to serve as a
control for the extent of digestion Reaction conditions were as
described In the manufactures protocols (New England BioLabs,
Boehrmger-Mannheim)
Blotting Hybridization
After electrophoresis the DNA was transferred with Ю х or 20x SSC
to nitrocellulose filters (Southern. 1975)
cDNA Hybridization»
Before hybridization, the fitters were incubated for 2 hr at 42*C in a
volume of 100 μΙ/cm 3 prehybrldlzatlon mix (50% formamide, 5 x
SSC. 5 x Denhardt s solution. 250/ig/ml of denatured salmon sperm
DNA and 0 05 M sodium phosphate (pH 7,0) Hybridization was
performed m 50% formamide, 5 x SSC, 1 X Denhardt s solution,
0 02 M sodium phosphate, 10% dextran sulfate, 100 мд/ті of de­
natured salmon sperm DNA (Wahl et al , 1979) and 0 5-1 x 10 e
cpm/ml M-MuLV cDNA for 16 hr at 42 0 C After hybridization, Alters
were washed with hybridization mix without probe and dextran sulfate
for 2 hr at 42*0. once for 15 mm with 2 x SSC, 0 1% SDS at 50 o C,
once with 0 1 χ SSC, 0 1 % SDS at 50°С. and finally 15 min at 65 0 C
Autoradiography was performed as described (Quint et a l , 1961)
Plasmid Probe»
Conditions were as described for the cDNA probe except for prehybndizatlon was for at least 4 hr, hybridizations were for at least 32 hr
without dextran sulfate and with 1-15 x 10 e cpm/ml denatured
Plasmid probe, filters were washed with hybridization mix three times
for 1 hr each in freshly prepared solutions The SSC washing proce­
dures were carried out at 45 0 C and 50 a C-55 0 C, respectively, for 15
min each
Cloning of Moloney Virus-Specific (MS) Plasmid Probes
A random population of (n vitro synthesized double-stranded M-MuLV
DNA (Verma, 1976) was cloned In the Pst I site of pBR322 by the dGdC tailing method (Chang et al . 1978) Transformants resistant to
tetracycline but sensitive to ampicillin were screened with M-MuLV,
AKR-MuLV and Balb virus-2 cDNA probes, prepared by the endoge­
nous polymerase reaction Clones displaying hybridization with MMuLV but not with AKR or Balb virus-2 cDNA were analyzed for their
ability Lo recognize M-MuLV proviral DNAs Several M-MиLV-specific
(MS) probes were obtained which detected exclusively M-MuLV se­
quences Details on their preparation and identification will be de­
scribed elsewhere
Acknowledgment·
We thank Dr R Jaenisch for providing the Balb/Mo mouse strain
and A Groeneveld tor growing the viruses We are grateful to Dr H
Bloemendal, m whose laboratory this investigation was earned out
This study was performed as a part of the Ph D study of Η ν d Ρ ,
and was supported In part by the foundation for Medical Research
(FUNGO), which is subsidized by the Netherlands Organization for
the Advancement of Pure Research (ZWO) This investigation was
also supported by research grants from the National Cancer Institute
The costs of publication of this article were defrayed m part by the
payment of page charges This article must therefore be hereby
marked advertisement' in accordance with 16 U S С Section 1734
solely to Indicate thts fact
Received November 18, 1980, revised March 12, 1981
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91
APPENDICES CHAPTER IU.
AI. NAPPING OF THE NS-PROBES.
DNA from Charon гіА/п-ГПиИ/ clone
d e s c r i b e d i n t h B le
^ n d to
Figure 1 шаз cleaved with restric­
США
tion enzymes as indicated and an
amount of each digest equimolar to
J I η та
! I $
α. ie
5x10 /ig of M-PluLV DNA was loaded
- ЧЬр
in
each lane and subsequently
m
«x^iSi
*
Kto
4 HS
_ -t-aí? * t
«ρ
screened with a HS-plasmid probe
•
î
»
as indicated. The rightuard part
Ш
-i»
4»
«• 18
zl^
of the Figure shous restriction
4
fragments of the clone as detected
«*
•»-tr
m •f - ( «іл«5
й
•m
by a representative cDNA probe of
-ш
-1
M-MuLV. The positions of the MSprobes on the linear map of the
. -0«,
PWIuLl/ genome are shown in the
*.
-U4S
-045
lower part of Figure 1. 1*15-3 was
mapped more precisely to the 3'end of the Pst I site (at position 1.15 kbp from the S'-end); 1*15-3 recognizes
the Pst I-Sal I fragment.which ranges from position 1.15 to 4.2 kbp from the
S'-end of the M-MuLl/ proviral DNA and is therefore localized to the З'-епа
of the Pst I site (at 1.15 kbp) and to the 5»-end of the Pvu II site (at 2.5
kbp) as shown on the linear map in Figure 1. 1*15-2 is localized on both sites
of the Pvu II site at position 0.12 kbp in the S'-LTR (and .6В kbp in the
S'-LTR of M-MuLV proviral DNAs containing two LTR sequences) as both a 1.
and a 0,2 kbp fragment can be detected by this probe. Therefore the 1*15-2
probe recognizes both 3' and 5' LTR sequences of integrated M-PluLV proviral
DNAs.
Charon 21A/M-MLV+HmdlII
a s
ì
m
A2. MONOCLONAL CHARACTER OF OUTGROUN TUMORS.
Figure A2a shows the integration patterns of PWluLV sequences in tumors
from various anatomical locations of three Balb/cio mice. Although the pat­
terns of reintegrated l*luLU sequences in tumors of different animals are
clearly unique for each animal, the patterns of reintegrations in tumors
from different anatomical sites of the same animal are identical. This
indicates that tumors within one animal are largely derived from one or
a few transformed cells as also observed by others (Oaehner et al., 19B0;
Varmus et al., 197 ). Figure A2b shows that tumor DNAs isolated from
different anatomical sites of a single animal display identical Kpn I
patterns. This indicates that outgrown tumors within each mouse contain
a specific subset of M-MuLV derived recombinant l*luLV sequences integrated
in different chromosomal sites as shown in Figure A2a.
Although one can define tumors as being largely monoclonal it seems not
unlikely that tumor tissues are in fact composed of a few clonal cell popu­
lations each composed of different numbers of calls. For example it is not
known whether the two tumor-specific Kpn I fragments in tumor DNA from
mouse 1*18 (see A2b) are present in each tumor cell or whether each fragment
is characteristic for one population of tumor cells containing only that
particular recombinant proviral DNA.
92
kb
P ω ω S Τ L-N L S LL-NK S Τ
54_
37.5m
22-
^
151
'—
86.54Д—
brau
ci
Вя.Ь/Мо
MS
M9
a s¡ ι. і.г τ s к -Ρ L s
Ц
kbp
54¿s4~
3.7аэ-
Z82.31.8!25-
Figure А2а! The integration sites of M-fluLU in Balb/lOo mouse tumors.
10 ug of tissue DMA uas cleaved by Eco RI, fractionated on a 0.6 % agarose
gel and analyzed for M-MuLU specific sequences by a selected cDNA probe
as described in Experimental Procedures. Lanes represent DMAs from Balb/c
and Balb/no brain; Balb/Mo mouse MB spleen, thymus, peripheral and visceral
lymph-nodes, and liver highly infiltrated by lymphatic cells; Balb/Mo mouse
Ю spleen, liver, lymph-nodes, and infiltrated kidney; Balb/Vlo mouse 1*110
spleen, and thymus·. Other tumor tissues that have been screened were gutassociated lymph-nodes (Payets Patches) and large lymphatic tumors along
the digestive tract; a largely monoclonal character uias observed for outgroun tumors in each animal.
A2b; Balb/Mo tumors: different anatomical locations.
As described for DNAs in A2a, but nou cleaved with Kpn I. Lanes represent
DNAs from tumors of two Ва1Ь/|*1о mice (1*18 and Ю);
Balb/c and Balb/do brain
DNAs are shown for reference. L= liver; l-n= lymph-nodes; S= spleen; T=
thymus; K= kidney.
93
A3.
INTEGRATION SITES OF SOHATICALLY ACQUIRED SEQUENCES IN PRELEUKEMIC
TISSUES.
Injection of one-day-old Balb/c mice uith about 10 XC-plaques of n-MuLU
leads to thymic enlargement and splenomegaly at 10-12 ueaks of age. All
the leukemic animals carried several integrated M-CluLV genomes in their
tumor DNA with a unique distribution for each individual animal (Figure
A3a), while identical patterns were found in tumors from different ana­
tomical locations of a single animal as shown in Figure A2a. In contrast,
preleukemic Balb/c mice at 3-6 weeks of age did not display a distinct
pattern of integrated PWluLU sequences in the DNA of their target tissues
as shown by Eco RI analysis (Figure A3a). However, these tissues do contain
integrated CI-MuLU genomes as shown by analysis with restriction enzyme
Ava I (Figure A3b) and Kpn I (Figure 48): Ava I cleaves M-nuLU DNA ten
times to generate 11 fragments of size 2.45, 1.85, 1.5, 1.2 kbp, and a
number of fragments ranging from 80-600 nucleotides. Figure A3b shows
that the 4 major Ava I fragments can be detected in all preleukemic
target tissues as well as in Balb/c tumor DNA. DNA from Balb/c tumor
503 and ВаІЬ/Мо brain showed partial cleavage products of M-CluLU DNA by
Ava li the Ava I recognition sequence contains a CpG base pair, and
methylation prevents cleavage by Ava I. These results therefore reflect
differences in the extend of base methylation of n-CluLU genomes integrated
in different chromosomal sites and-also shows that the Clov-I M-WuLU DNA
in Balb/no brain is methylated at many Ava I recognition sequences. The
basic conclusions from these studies summarized are: 1) PI-PluLl/ genomes
are randomly integrated in preleukemic tissues, and 2) outgrown tumors
within each individual are composed largely of clonal descendants of one
or a few transformed cells. Similar results have been obtained for
preleukemic tissues and tumors of Balb/По mice (3aehner et al,,1980; our
unpublished results).
gJBsttMiìMBfi ^ ц ^ ^
ίΟ·ί< 4 !> S 7 S 810111213
Figure A3: The integration sites
of PWluLl/ in preleukemic and
leukemic tissues of newborn in­
fected Balb/c mice.
A3a: Lane 1 and 2 represent Eco
RI digestions of ВаІЬ/По and
Balb/c brain DNA for reference.
Lane 3: tumor DNA from Balb/o
mouse 503, infected when newborn
with about 10 XC-plaque equivalents
of PI-MuLU clone 1A and sacrificed
at 6 months of age. Lanes 4-8 repre­
sent thymus DNAs from 5 individual
Balb/c mice, infected with 10 XCplaque equivalents of M-MuLl/ clone
1A and sacrificed at 3-6 weeks of
age (preleukemic mice). Lanes 9-12
represent DNAs from Balb/c mice,
also infected with 10 XC-plaque
equivalents of the same virus-stock
but sacrificed at 10-12 weeks of age (leukemic). All lanes represent Eco RI
digestions. The preleukemic animals did not show enlargement of spleen or
thymus whereas these organs were greatly enlarged in the leukemic mice,
PreleuBemic tissues show randomly integrated n-WuLU genomes while outgrown
tumors within each individual show a distinct integration pattern for each
animal. The lower part of the X-ray film was exposed for a longer period in
order to visualize the smaller fragments, АЗЬ: lanes represent Ava I digest­
ions of the preleukemic tissue DNAs as analyzed in A3a (lanes 4-8), The
presence of characteristic M-fluLl/ proviral DNA fragments indicates integra­
tion of n-PluLU sequences in preleukemic tissue DNAs,
kbp
23-
94
A4. THE MOU-I LOCUS AND REINTEGRATION OF AUTHENTIC n-HuLl/ GENOMES.
The Kpn I fragment proximal to the S'-end of the Mov-I H-TOuLV DNA is
60-Θ0 nucleotides smaller that the З'-Крп I fragments of cloned, unintegrated n-MuLV DNA of mass-infected 3T6 cells (Bacheler and Fan, 1980) or
tumors of Balb/c and 129 mice infected uihen пеыЬогп with n-PluLU. This
deletion is localized either in or directly next to the LTR and does not
interfere udth the biological activity of the virus produced by the Πον-Ι
locus since high titers of infectious n-MuLV are expressed in these animals
already before amplification of proviral copies is detectable, and further­
more the offspring virus retains the same deletion (unpublished results),
Restriction analysis using Pvu II revealed that the deletion is localized
to the S'-end of the Pvu II site at position Θ.1 kbp In M-MuLl/ proviral
DNA| therefore the deletion is almost certainly localized uithin the LTR
sequence. Heterogeneity in LTR sequences of retroviruses has been observed
previously (Du et al., 1980; Rands et al., 1981). Differences in terminal­
ly redundant sequences of a DNA virus (AAV) have also been detected uhen
comparing early and late passage, infected cell cultures (Chueng et al.,
1980). Therefore, the observed deletion at the 3'—end can almost certainly
be classified under frequently occurring rearrangements often seen in
redundant sequences.
The 1.25 kbp Kpn I DNA fragment, characteristic for the S'-end of the
Ио -1 M-nuLl/ proviral DNA can also be detected in digests of somatically
acquired proviral DNAs (after removal of the Mov-I M-MuLV containing
Eco RI DNA fraction of 20-100 kbp: Eco RI dugested tumor DNA шаз fractio­
nated on size and the fragments smaller than 20 kbp (the Mov-I containing
FI-MuLV proviral DNA is present in a 27 kbp Eco RI DNA fragment) pooled
and cleaved uith a variety of restriction endonucleases among which also
Kpn I ) . All n-FluLV specific fragments шеге detectable in this fraction
whereas no junction fragments diagnostic for the Hov-I locus were
detectable. Also, the S'-end 1.25 kbp Kpn I DNA fragment uas detected in
this fraction and could be identified as S'-terminal fragment by its
hybridization with NS-2 probe. Therefore, the tumors harbor both somatical­
ly acquired recombinant as well as authentic M-MuLV genomes as observed
for newborn infected mice, and the deletion in the S'-terminal Kpn I
fragment can be inherited by somatically acquired copies.
A5. SOMATICALLY ACQUIRED SEQUENCES IN TUTORS OF F1(AKRxBalb/ио) MICE.
Since the endogenous M-MuLV of /Balb/Мо mice is a ΝΒτ-tropic virus (i.e.
grows equally well in both Fv-1 ' e.g NIH and Fv-1 ' e.g Balb/c mice
and cell lines) the amplification of these viral sequences should also
occur/during, leukemogenesis in miQi derived from crosses between AKR (
F v - 1 n / n ; H-2 ) and Balb/Мо (Fv-1 ' ; H-2 ) mice. F1 mice develop
spontaneous thymus-dependant leukemias with high frequency similar to
Balb/Мо and AKR mice. The endogenous AKR-MuLV sequences· however, would
not be able to replicate due to restriction by the Fv-1 allele of the
F1 mice, which is/a dominant trait. Therefore tumor DNA from Fl(Balb/Mo
xAKR) mice (Fv-1n' ; H-2 ' ) was screened for amplification of both
M-MuLV and AKR-PluLV specific sequences. The results (data not shown)
indicate that only M-CluLV type sequences are amplified while the endo­
genous AKR-MuLV sequences did not reintegrate. Furthermore, new restric­
tion fragments were detected in tumor DNAs, diagnostic for the presence
of recombinant proviral DNAs that display a characteristic structure as
shown to be present in other M-MuLU-induced tumors (Figure 5 ) . These
95
results indicate that M-PluLV-induced Івиквтодвпезіз in Fl(ВаІЬ/noxAKR)
mice is also associated with the emergency of characteristic recombinant
proviral DNAs as observed in tumor tissues from Balb/Mo, Balb/c and 129
mice, and confirm the ubiquity of these recombinants in M-PluLV-induced
Isukemias. The amplification of AKR-CluLV sequences as observed in AKRMuLU-induced leukemias of AKR mice is restricted in F1(Balb/floxAKR) mice;
this is probably the result of Fw-1 restriction in vivo. Other factors
that might be involved in determining the latency of the disease in
similar crosses have been discussed in the introduction (Chapter I ) .
Chapter \l,
96
DNase I Sensitivity and Clathylation of Genetically Transmitted and
Somatically Acquired Proviral DMA Sequences in ІЧ-МиИ/ Induced Tumors.
1
1
2
1
Herman van der Putten, Wim Quint, Inder M. l/erma, and Anton Berns.
1
Laboratory o f Biochemistry,
U n i v e r s i t y of Nijmegen,
Geert Grooteplein N21,
6525 EZ Nijmegen,
The Netherlands.
2
Tumor l/irology Laboratory,
The Salk I n s t i t u t e ,
San Diego,
C a l . 9213B, U.S.A.
ABSTRACT.
The DNase I sensitivity of chromosomal DNA regions, carrying integrated
proviral genomes of FI-fluLU, AKR-PluLV and the cellular homologue of the mosgene of FISI/ (c-mos), шаге studied in tumor tissues of leukemic mice. Using
DNase I sensitivity to probe for structural alterations of chromatin, potentially
active in transcription, it appeared that the genetically transmitted genomes
of M-FluLl/ and AKR-MuLU and also the c-mos gene are all in DNase I resistant
chromatin conformations in M-FluLV induced tumors. A feu of the somatically
acquired M-MuLl/ related sequences mere hypersensitive to DNase I uhereas the
other reintegrated proviral sequences displayed a configuration with a moderate
sensitivity to DNase I. The hypersensitive sites ш гв found in the cellular
sequences
juxtaposed to the S'-LTR. Both the coding sequences as uell as the
31—flanking sequences of recombinant proviruses did not reveal such hyper­
sensitive sites. The extent of base methylation at the Hpa II/ Изр I
recognition sequences of somatically acquired integrated MuLU sequences in
tumors urns greatly deminished as compared to the genetically transmitted
Mov-I M-MuLV DNA in Balb/Πο mice; this proviral DNA is extensively methylated
at the Hpa II/ Msp I sites in both tumor and non-tumor tissues. No gross
differences in methylation ш г observed betueen reintegrated MuLV DNAs
moderately sensitive to DNase I or FluLVs displaying a DNase I hypersensitive
configuration at their 5'-end.
INTRODUCTION.
Moloney (N-MuLV) and AKR (AKR-MuLV) murine leukemia viruses can induce
leukemogenesis in a variety of mouse strains. Leukemogenesis is accompanied
by somatic amplification and reintegration of n-FluLl/ (9-11,35,36) and AKR-fluLW
(1,4,24,30,31) sequences respectively in new chromosomal sites of tumor tissues.
M-CluLl/ and AKR-FluLl/ induce predominantly thymus-dependent leukemias in
appropriate hoste. Leukemia development follous the expression of high titers
of the virus in the blood (10,26). The thymus-dependsnt leukemia of viral
(M-PluLl/) etiology in Balb/do mice (22) is probably due to the transient
expression of the endogenous M-CluLU genome (Mov-I; 9,35,36) on chromosome
number 6 (2), which leads to infection of the lymphatic target tissues u/here
virus-specific RNA can be detected (11) concomitantly uith proviral DNA
amplification (9,11,35,36).
Although Balb/no, Balb/c and AKR mice carry AKR-MuLV as an endogenous provirus
(24,30,31,35), the expression of this virus is only seen occasionally later
in life of Balb/c mice (12). Apparently, the expression of the AKR endogenous
provirus is under strong host control in these mice in mhich the Fv-1 locus
could well play a major role (13,17),
97
Previous studies haue shouin characteristic features of H-MuLV (9,35,36) and
AKR-MuLV (4,24,30,31) induced leukemias. Otgroum tumors, largely composed of
one or a few clonal cell populations, harbor recombinant proviral genomes
integrated in multiple unique sites of chromosomal DMA. The recombinant
proviral DMAs are all env-qene recombinants, ufhich have acquired cellular
sequences in the 3-portion of the viral genome comprizing information for
the amino-terminal part of the env-glycoprotein. Every lymphoma induced by
M-PluLV or AKR-PluH/ harbors this same type of recombinant provirus, although the
copy number varies in different tumors (1G,36). This uould favor a model which
defines a recombinant provirus as a prerequisite for the onset and maintenance
of the transformed state. No common integration sites, providing a promoter for
neighbouring cellular sequences (as observed for ALI/ in ALV-induced tumors in
birds; 8,21,23) have been detected so far.
If the expression of a recombinant proviral genome is required for the
maintenance of the transformed state then at least some of the reintegrated
genomes are expected to be localized in active chromatin regions. Several
DNase I probing studies of chromatin have provided convincing evidence that
"active" chromatin has an altered, more accessible configuration (28,29, for
ге іеш see 20). The sensitivity to DNase I has been correlated with the
commitment or competence to synthesize a given gene product (29,40a). Furthermore,
chromosomal domains active in transcription have been identified as contiguous
chromosomal structures exhibiting high and moderate DNase I sensitivity (28,29).
DNase I hypersensitive sites have been detected almost exclusively at the 5 1 ends of potentially active genes (6a,15,27,29,40a,41) and probably reflect
exposed DNA sequences which are essential for gene activation (40,40a). Lie
have analyzed the preferential sensitivity of viral genomes for DNase I. It
appears that chromatin comprizing proviral genomes in outgrown tumors can be
divided in three different DNase I sensitivity classes i.e. in configurations
resistant, moderately sensitive and hypersensitive to DNase I. Somatically
acquired proviral genomes, displaying moderate sensitivity or configurations
hypersensitive to DNase I, are hypomethylated whereas the genetically
transmitted copy (Mov-l) is extensively methylated at the Hpa II/ Msp I
recognition sequence.
MATERIALS AND METHODS.
Mice, cell lines, viruses, oDNA probes and hybridizations.
nice, cell lines and isolation procedures of ñ-MuLV clone 1A and AKR-MuLV
have been described previously (35). The injection of newborn Balb/c mice with
M-FluLU clone 1A was performed intraperitoneally (10,35,36),
The selection of M-fluLl/ and AKR-PluLV specific cDNA probes, and hybridization
procedures have also been outlined previously (24,35,36).
Preparation of cell nuclei.
Nuclei were isolated by a procedure modified from those described before (3,
28), in order to reduce the activity of endogenous DNases. All manipulations
were carried out at 0 to 4 C. Either frozen (-80 "c) or fresh mouse tissues
(0.2-0.8 g) were homogenized in 10 ml PAP l\l buffer (17.1 % sucrosej 15 ml*]
Tris-HCl, pH 7.5; 0.5 mM spermidine; 1 ml*! EDTA; 0.1 mN EGTA; 25 ml»! KCl) by
douncing Ю х in a dounce homogenizer (Pistol A); 5 ml PAP IV + 0.2 % NP-40
were added in two portions and dounced 1x and 2x respectively; the homogenate
was filtered through cheese cloth and centrifuged 5 min at ЗОООхд; the nuclei
were washed twice with PAP IV buffer without NP-40 and resuspended in 5 ml
PAP IV buffer; the suspension was filtered through scrynell nylon and E260
was determined by sampling into 1 % SOS, 0.2 Π NaOH. Nuclei were pelleted
98
M-MLV cDNA S p ec
ÜNabcS
f-
йй
ІЬ >
( t l
,
AKR-MLV cDNA s p ec
_»_
pMSV 31
¿
Kbp
2 Í5 -у
94—
e.-è
^ і ^ Щ И ^ ^ ^ ^ ^ ^ ' ^ w ^ ^ H M № ЧИР яц^ ей·
ттшщ
4 3-
2330~
M MLV c D N A S D p r
AKR-MLVcDNA 4Dec
pMSV-31
Kfap
4,3-
2.320-
Sensitiyity to DNass I of Mov-I, Aku, c-mos, and somatically
acquired MuLUs in M-fluLU-induced tumors.
Nuclei were isolated from tumor tissues of a Balb/no mouse (СШ; IA) and
a newborn infected Balb/c mouse (1B) as described in naterials and
Methods and incubated ujith different concentrations of DNase I as
indicated (0-2,4^jg/ml). DNA from each sample uas subsequently purified,
10 ^jg restricted uith Eco RI and screened uith different probes after
Southern blotting. И-ИиИ/ and AKR-MuLU specific probes шеге prepared
as described previously (24,36). For detection of cellular mos-sequences
(c-mos) a nick-translated probe of рП5и-31 mas used (14). The arrous
indicate reintegrated ItuLU sequences hypersensitive to DNase I, The
fragment indicated by a small arrow indicates the genetically
transmitted Που-Ι n-CluLU DNA in Balb/Mo mice. The fragment, neuily
emerging after DNase I digestion and subsequent cleavage by Eco RI ia
indicated by an open arrou.
99
once mora, uiashed in 5 mi RSB,- buffer (10 mi"! Tris-HCl, pH 7.5; 10 mi"! NaCl;
5 mM ПдС1„) and finally resuspended in RSB buffer at a DNA concentration of
1 mg/ml, EGTA mas added to a final concentration of 0.2 ml*!. Exogenous DNase I
,
(Sigma) digestions were carried out for 2 min at 25 С in Eppendorf tubas
containing 1.4 mM CaCl« and 0.3-5 mg/ml DNase Τ (1505 Kunitz/mg protein).
DNase I storage buffer contained: DNase I, 1 mg/ml; 0.15 mfl NaCl; 1 mg/ml
BSA. Reactions шеге terminated by adding 1 volume 1 % SDS; 5 mM EDTA; 20 mM
Tris-HCl, pH B.0, and incubated for 1 min at 60 "e. DNA uas purified by Proteinase
К (50-100 mg/ml) treatment, subsequent extractions uith phenol and chloroform
and tuio precipitations uith ethanol.
Restriction analysis.
Restriction analysis of high molecular weight DNAs from tissues ш г carried
out as described previously (24,35,36). Elution of DNA fragments from agarose
gel slices u/as performed either by electroelution (24,35) or by adsorption to
glass beads (З ) . Marker DNA fragments for size determination as shou/n in Fig.
1,3 and 4 represent Hind III fragments of phage lambda DNA.
RESULTS.
Chromatin conformation of Hov-I, Akv, c-mos, and somatically acquired MuLVs.
Previous studies revealed an increase in M-CluLV specific RNA sequences,
concomitantly uith proviral DNA amplification, in target tissues of Balb/flo
and newborn infected Balb/c mice (10,11). Although it has been reported that
the majority of the new somatically acquired sequences in tumors are
preferentially digestible by DNase I (3), these studies did not indicate
whether a difference in sensitivity to DNase I existed between the individual
reintegrated and genetically transmitted viral copies. Furthermore, it remained
unclear whether DNase I sensitivity is confined to specific proviral copies
(authentic or recombinant MuLVs) or to specific sequences of proviral DNA.
Therefore the DNase I sensitivity of chromosomal DNA regjons, carrying
integrated proviral genomes of authentic and recombinant H-MuLUs were studied
in tumor tissues of leukemic mice. The DNase I sensitivity cf two cellular
sequences, thm AKR-MuLl/ proviral genome (24,35) and the cellular hcmoloyue of
the mos-gene of MSV (c-mos; 14), were taken for reference,
Nuclei were isolated from both tumor and поп-tumor tissues of individual
mine (both Balb/ño and Balb/o). The differential sensitivity of th4 individual
proviral n-MuLV genomes for DNase I was determined after incubating nuclei
with different concentrations of DNase I: DNA was extracted, digested with
Ecn RI, DNA fragmenbs separated on siza and after Southern transfer hybridized
to a N-CliO specific cDNA probe (Fig. 1 ) . The results show that some of the
reintegrated MuH/ sequences are hypersensitive to DNase I (open arrows). The
Mov-I M-MuLV proviral locus in both non-tumor (brain) and tumor tissues is not
sensitive to DNase I. Even at considerably heighar DNase I concentrations
(2.5-5 fiq/ml)
the Mov-I M-fluLl/ DNA was relatively resistant whereas the
somatically acquired MuLV copies were all more sensitive, liihen nakod DNA was
probed with DNase I both the Mov-1 M-MuLV DNA and the reintegratnd MuLV genomes
were not preferentially digested with DNase I (data rot shown). These analyses
show that the different MuLU sequences in M-MuLU-induced tumors show a
differential sensitivity to DNase I. All reintafjrateil genomes are more
sensitive to DNase I than the genetically transmitted genome.
In order to relate the DNase I sensitivity of the Mov-1 M-MuLl/ DNA and
reintegrated MuLl/ DNAs with other proviral and cellular genes the blots shown
in Fig. 1 were hybridized with an AKR-MuLV specific cDNA probe (24) and a
mos-specific plasmid probe (14). The single genetically transmitted ecotropic
100
MS-2
• Kpnl
Isacl
MS-2
MS-4
l
MS-3
Kpnl
Sacl
Pstl I
ill H l
BamHI
Kpnl
Tig. 2 A)
Ipíasmid
CUNAgp ее
O
O
œ
0)
2
m m
Kbp
27-
4J
a
ι
CM
Ζ
α
+
(β
2
ЩЦ^іШі M»
^
Fig, 2 B)
Prob es
I
m
<*
(
(h
5
£/)
S
Kpnl
Recombinant MuLU DNA in tumors,
The position (in kbp) of some relevant
restriction sites are indicated on the
linear map of authentic and recombinant
Г-ПиИ/ prov/iral DIMA. Black boxes represent
LTR sequences. The dashed area indicates
sequences that are recombined in Pl-CluLU
deriued recombinant prouiral DNAs. Sites
marked by one asterisk denote пешіу
acquired restriction sites in recombinant
MuLUs; the new Bam HI site (at 6.3 kbp)
and Ceo RI site (at 6.9 kbp) are
characteristic for recombinant MuLU
DNA, found in every lymphoma (24,36).
These neu sites are responsible for the
detection of tumor-specific Bam HI
(2.2 kbp) and Kpn I-Eco RI (3.6 kbp)
DNA fragments diagnostic for M-HuLUinduced tumors. In acditian, recombinant
ñuLU DIMA in the Balb/no tumor П9 carries
also a Sac I site at 7,6 kbp (36),
Sites below the line are M-PluLU prouiral
DNA specific sites that are lost in
recombinant HuLl/ DNA; the Kpn 1* site
is only occasionally lost. Regions
v
MS-2, 1*13-3, and llS-4 represent those
sequences that are recognized by the
rs^pective M-TOuLU specific plasmid
probes (36),
PI-fluLU specific sequences in DNA fragments sensitive to DNase I,
Lanes 1-7 contain 10 ^jg of Eco RI restricted DNA from Balb/c brain (lane 1),
Balb/Mo brain (lane 2), Balb/Mo М9 tumor (lanes 3-7), Southern blots were
hybridized to a rWluLU specific cDNA probe (lanes 1-4) or to M-fluLU specific
plasmid probes as indicated. Lane 3 contains an Eco RI digest of М9 tumor DNA,
whereas lane 4 contains an Eco RI digest of П9 tumor DNA, isolated from nuclei
treated with DNase I (2,1 iig/ml) as shown in Гід, 1A, The 27 kbp fragment
represents the По -І PI-MuLU genome. The arrows indicate the two DNA fragments
located in chromatin hypersensitive to DNase I, The newly emerging DNA fragment
at 7,0 kbp (lane 4) is marked by an open arrow. The upper DNA fragment (>27 kbp)
hypersensitive to DNase I, hybridized to 1*15-2, MS—3, and И5-4 probes, but was
only visible after long exposures (not shown),
101
AKR proviral DNA (in a 20 kbp Eoo RI DNA fragments; 24) appeared in a DNaae I
resistant chromatin conformation in both tumor and non-tumor (not shown) tissues
from Balb/no and Balb/c mice. Other endogenous uiral sequences can cross^-react
uiith AKR-HuLV/ cDNA probe using hybridization conditions allouing some base-pair
mismatching (24) (Fig. 1, panel 2 ) . These cross-reacting endogenous prov/iral
sequences also appeared rather resistant to DNase I, In addition ше probed the
DNase I sensitivity of the cellular homologue of Ио-РІЗ specific mos-ssquences
as an internal reference of a cellular non—viral gene. These sequences uere
previously identified in a 14 kbp Eco RI DNA fragment from uninfected mouse
cells (14). The cloned cellular DNA fragment contains an uninterrupted stretch
of about 1.1 kbp homologous to no-ClSV-specific sequences; a clone, рМЗ -ЗІ (14),
containing this mos-specific DNA fragment was used as a probe. These analyses
revealed that also the cellular homologue of the viral mos-sequences is in a
DNase I resistant chromatin conformation in n-MuLV-induced tumors.
In conclusion, proviral genomes in M-MuLV-induced tumors were detected in
three different DNase I sensitive classes of chromatin; the genetically
transmitted AKR-PluLV and Mov-1 M-nuLV DNA nere found in DNase I resistant
configurations like the с-чпоз gene and proviral genomes cross-reacting ujith
AKR-duLV cDNA probe. Somatically acquired FI-MuH/ related sequences in tumors,
houever, fall into two classes, one displaying moderate sensitivity, and one
showing hypersensitive configurations to DNase I.
Location of DNase I sensitive sites uiith respect to proviral genomes.
Previously it was established that PI-CluLV-induced tumors contain both
authentic and recombinant MuLU proviral sequences that display a general
structure as shown in Fig. 2A (36). The recombinant ñuLV proviral DNAs display
some very characteristic restriction sites not present in authentic ("l-MuLV
proviral DNA: an Eco RI site at 6.9 kbp and a Bam HI site at 6.3 kbp. To
determine whether fragments hypersensitive to DNase I comprize S'-ends of
recombinant MuLV genomes or sequence information for authentic PI-fluLV and/or
a S'-part of a recombinant MuLl/, Eco RI DNA fragments of the tumor (Fig. 1A)
were hybridized to three rWluLV specific plasmid probes (36). These probes
recognize different parts of the M-nuLV/ genome (Fig. 2A). Fig. 2B shows that
fragments that are hypersensitive to DNase I all hybridize to 1*15-2, МЗ-З and
1*15-4. This indicates that these fragments contain either an authentic M-CluLV
genome and/or a S'-part of a recombinant MuLV DNA (up to the Eco RI site at
6.9 kbp). Furthermore, the intensities of hybridization of some Eco RI DNA
fragments indicates the presence of at least two proviral copies among such
fractions. Additional restriction analyses (see last section) revealed that
the two DNase I hypersensitive Eco RI fractions (Fig. 1A) contain only a S'-end
of a recombinant MuLV provirus. None of the fragments which only hybridized to
1*15-2 and therefore contain S'-parts of reintegrated recombinant proviruses
were hypersensitive to DNase I.
A striking feature of the DNase I analyses of tumor tissue chromatin from
both Balb/l*lo and Balb/c mice was the appearance of a new fragment of 7.0 kbp
after DNase I digestion of nuclei and subsequent cleavage by Eco RI. This
fragment (Fig. 1 A and B, and Fig. 2B, open arrows) was only detected after
Eco RI digestion of DNA uhich was isolated from nuclei treated with DNase I
and could not be detected after DNase I treatment of chromatin without
subsequent restriction or after low-level digestion of pure DNA with DNase I.
To elucidate the structure of this new fragment DNA fractions were isolated
after Eco RI digestion of DNA which was isolated from nuclei treated with
DNase I at a concentration of 2.1 pg/ml as shown in Fig. 1A. Subsequently,
DNA from each fraction was cleaved by Bam HI, Pst I, and Sac I (Fig. 3 ) .
Lane В represents a fraction containing predominantly 7.0 kbp Eco RI DNA
fragments. The newly emerging 7.0 kbp DNA fragment appeared to be derived
102
BamHf
Кой
гз7-
Pst I
іг345в гt · β ι
ι ¿ ι ··. -
Sad
иіфгздьегявюі?
9 4-
«
'_5.3 5.7-4.0
2.9-2.2
6 7-
-3.9
г-2.6
-Π
Fig. 3
M-CluLU sequences in the neuly emerging 7.0 kbp fragment.
300 ^jg DNA, isolated from nuclei (Balb/pio tumor Πθ) treated ujith 2.1 /Jg/ml
DNase I (rig. ΙΑ), was restricted uith Eco RI, fragments separated on a
0.6 % agarose gel and the gel part containing the 6.5-7.5 kbp Eco RI DNA
fractions was sliced into 11 fractions (lane 1= 6.5 kbp; lane 11= 7.5 kbp);
each fraction uas subsequently analyzed by further restriction uith Bam HI,
Pst I and Sac I respectively, and Southern blots were screened with a M-fluLl/
specific cDNA probe. Lanes 12 contain an Eco RI digest of tumor N9 DNA,
isolated from DNase I treated (2.1 jjg/ml) nuclei. Large numbers indicate
the length of fragments hybridizing to the M-MuH/ specific cDNA probe in
each gel slice.
Fig. 4
Restriction map and location of DNase I hypersensitive site on
recombinant MuLU proviral DNA.
Lanes 1 and 2 show the blot-hybridization results of Eco RI restricted DNA
from ВаІЬ/Ио tumor 149 without (lane 1) and with (lane 2) prior treatment
of nuclei with DNase I. Fragments ara marked as in Fig. 1 and 28. The
restriction maps obtained for the 7,0 and 6.7 kbp fragments are shown; the
dashed area indicates the recombined sequences.
94—
67І
43-
!
103
from the S'-part of one or several reintegrated recombinant proviruses since
only restriction fragments diagnostic for recombinant WuLV DMA (Гід. 2Д) were
detected in this neu fragment (Fig. 4 ) . Therefore, the specific degradation
of some recombinant proviral DNAs in chromatin by DNase I must be ascribed to
a DNase I hypersensitive site around the junction of the S'-LTR and cellular
DMA sequences (Fig. 4 ) . The location of this DNase I hypersensitive configuration
was further confirmed by hybridizing the Pst I blot (Fig. 3) with 1*15-2 probe
which is complementary to a region at the start of ths LTR; in lane θ a single
fragment of 1.1 kbp uas detected (not shown). The coding sequences of these
recombinant HuH/s, however, are not hypersensitive to DNase I but show moderate
sensitivity like most somatically acquired MuLV proviral sequences.
Lane 3 (Fig. 3) represents the fraction containing predominantly 6.7 kbp
Eco RI DNA fragments. The 6.7 kbp Eco RI DNA fragment represents a S'-part of
a recombinant FluLV genome as shown by the differential hybridization
characteristics against the MS-plasmid probes (Fig. 2B). Cleavage by Bam HI,
Pst I,and Sac I (Fig. 3) revealed a restriction map for this S'-part of a
recombinant MuLV (Fig. 4) which shows that no DNase I hypersensitive site
could be mapped in the S'-part of this recombinant DNA and also not in the
4.В kbp of cellular sequences following the S'-LTR sequence. Ide could not find
any evidence for other З'-Eco RI DNA fragments of recombinants hypersensitive
to DNase I which suggests that only the S'-ends of some reintegrated recombinant
proviruses are in a "active" chromatin conformation.
DNase I sensitivity and methylation of proviral DNAs in tumors.
Differences in methylation patterns of both genetically transmitted and
somatically acquired PI-nuLV sequences have been observed. In tumor tissues the
Ptov-I M-MuLV DNA has a different methylation patterns e.g. Sma I and Sal I cleave
the ΓΊον-Ι M-MuLV DNA from tumor tissues but not from brain (36) and also other
tissue-specific differences in methylation of the Mov-I M-PluLV DNA have been
observed using other enzymes (Ava I, Xho I) for target and non-target tissues
(data not shown). Further analyses of tumor DNAs by Hpa II/ Wsp I , Hha I,
Sal I, Ava I, Sma I, and Xho I revealed that somatically acquired proviruses
are hypomethylated at these sites when compared to the genetically transmitted
Mov-I M-fluLV DNA from both tumor and non-tumor tissues.
In this study we analyzed whether hypomethylation of somatically acquired
CluLU genomes is correlated with moderate or hypersensitivity to DNase I. Also
the extent of methylation at Hpa II/ Clsp I sites was determined for the
genetically transmitted Clov-I M-MuLV DNA. Therefore, Eco RI DNA fractions
containing several of the reintegrated proviral sequences were isolated and
cleaved by Kpn I. This enzyme yields differently sized fragments for authentic
and recombinant proviral genomes (Fig. 2A)j Eco RI-Kpn I fragments of 3.6 and
1.Θ kbp are diagnostic for recombinant MuLV DNA whereas authentic genomes
yield fragments of 2,6 and 1.3 kbp respectively (Fig. 2A; 36), In addition each
Kpn I digested Eco RI DNA fraction was cleaved by Hpa II and Nsp I respectively
(Fig. 5 ) . Hpa II and Msp I are isoschizomeres from which Nsp I cleaves the
recognition sequence independent whether this site is methylated or not (34).
Our analyses revealed that both authentic and recombinant reintegrated M-fluLU
genomes are largely hypomethylated at the Hpa II/ Msp I recognition sequences
when compared to the Plov-I M-CluLl/ genome (Fig. 5, panel A) which is extensively
methylated at these sites.
Although only some of the reintegrated proviral genomes display flanking
chromatin regions hypersensitive to DNase I, these proviral DNAs display grossly
a similar extent' of hypomethylation at their Hpa II/ Msp I sites when compared
to the proviral copies moderately sensitive to DNase I. Similar results were
obtained after analyzing Eco RI DNA fractions directly with Hpa II/ Msp I
(data not shown). Since all somatically acquired MuLU sequences are moderately
104
sensitive to DNase I (Fig. I) the data might suggest a correlation of this
moderate sensitivity to DNase I and hypomethylation. However, there is no
direct correlation between hypersensitivity to DNase I and hypomethylation
of reintegrated proviral genomes at the Hsp l/ Hpa II sites.
••
•
f i| > *
13-^
Fig. 5
ц
Methylation at Hpa II/ Hsp I restriction sites in tumor tissue DNA.
Several Eco RI DNA fractions, containing Mov-I H-fluLU DNA or reintegrated
proviral sequences шаге analyzed by Southern blotting after cleavage with
Kpn I (lanes 1), Kpn I + Plsp I (lanes 2), and Kpn I + Hpa II (lanes 3), by
hybridization to a M-^uLU specific cDNA probe. Panel A shows the results for
the По -І M-nuLU containing fraction (indicated by the small arrow); this
fraction also contains a S'-end of a recombinant provirus (displaying a
hypersensitive configuration to DNase I), indicated by the characteristic
3.6 kbp Eco RI-Kpn I DNA fragment (Fig. 2A). The faint hybridizing fragment
of 2.5 kbp represents the S'-virus-cellular DNA junction fragment of the
Mov-I Pl-CluLU DNA, whereas the fragments around 2,9 and 1.3 kbp are characteristic
internal fragments of authentic n-CluLU proviral DNA (Fig. 2A; 36). Panels B-F
show analyses of reintegrated proviral DNAs; the 1.8 kbp fragment (Panels C,
E, and F, lanes 1) is derived from the S'-end of recombinant proviral DNA
(Flg. 2 A ) . Fragments are further indicated as in Fig. 2B.
105
DISCUSSION.
Several DNase I probing studies of chromatin haue provided convincing
evidence that "active" chromatin has an altered, more accessible configu­
ration (39). Such DNase I sensitive conformations have bean observed for
the globin gene sequences in nuclei of cells actively expressing these
genes (28,29), for the ovalbumin gene in the hen oviduct (16), for rDNA in
various organisms, and for integrated viral genomes of both DNA and RNA (6a)
tumor viruses (for г івш see 20), for heat-shock loci in Drosophila tissue
culture cells (15,41), and for chromatin sequences complementary to a total
population of nuclear RNA (20).
This more accessible configuration results in a more DNase I sensitive
chromatin structure when compared ujith overall nontranscribed chromatin.
More recent studies have demonstrated that there are sites in chromatin
idhich are hypersensitive to DNase I. Such sites have been mapped at the 5'ends of several heat-shock genes in Drosophila melanogaster (15,41). Also,
embryonic chick-red-blood-cells display a DNase I hypersensitive site close
to the beginning of the embryonic p-globin (29) and presumed o(—type U—
gene (40a); both embryonic and definitive red cells show a DNase I hyper­
sensitive site near the S'-end of both o(-type globin genes (40a). Further,
the Drosophila melanogaster histone gene repeat also exposes DNA segments
near the 5'-terminus of all five histone genes (27), Recently it шаз found
that the transcriptionally active endogenous retrovirus locus ev—3 in chick
cells also contains DNase I hypersensitive sites in each of its tuo long
terminal repeats ujhile the inactive ev-1 locus does not display such sites
(6a). These results suggest that mainly sequences around promoter regions
of actively transcribed genes are hypersensitive to DNase I uhereas the
coding sequences are not. Coding sequences of actively transcribed genes,
houever, are always found in chromatin conformations more sensitive to
DNase I than non-transcribed genes.
In order to probe for potentially active proviruses ше have determined the
DNase I sensitivity of genetically transmitted and somatically acquired
proviruses in M-fluLV-induced tumors. These studies revealed that the
genetically transmitted Mov-I Fl-MuLl/ genome in both tumor and non-tumor
tissues of Balb/No mice is not in a DNase I sensitive conformation. The
genetically transmitted endogenous AKR-MuLV genome (24,35) and other
endogenous viral sequences that can cross—react with AKR-WuLl/ cDNA (24)
are also not in DNase I sensitive conformations in these tissues from both
Balb/Mo and Balb/c mice. The non-viral, cellular homologue of Mo-PlSV specific
mos-sequences (14) was also found in a DNase I resistant conformation in
chromatin from M-MuLV-induced tumors. The somatically acquired MuLV sequences
in n-MuLU-induced tumors of Balb/ño and Balb/c mice can be divided in two
classes according to their sensitivity to DNase I: most proviral DMAs
display a moderately sensitive conformation to DNase I whereas some expose
a DNase I hypersensitive configuration at their S'-end. A new fragment of
7.0 kbp arises after DNase I digestion of chromatin and subsequent cleavage
with Eco RI, This fragment contains sequences derived from the S'-part of
recombinant proviruses: from the S'-LTR up to the Eco RI site at 6.9 kbp.
A DNase I hypersensitive configuration is located probably slightly in front
of the S'-LTR. No S'-ends of recombinant proviruses and their S'-flanking
cellular sequences have bssn found to be hypersensitive to DNase I. Also,
the analyses did not reveal whether reintegrated authentic N-HuLl/ genomes
display DNase I hypersensitive configurations since none of the tumors
analyzed contained an Eco RI DNA fraction comprizing exclusively an authentic
proviral genome. Other restriction enzymes are currently being tested to
solve this issue. The results indicate that some reintegrations occur near
DNase I hypersensitive chromatin regions or these reintegrations generate
in some instances DNase I hypersensitive configurations in chromatin at
their site of insertion.
106
The function of the exposed sequences at the 51—ends of proviral DNA is
still obscure. Previously, exposed sequences have been mapped at the initial
portion of the mRNA leader sequences of the histone genes H3, H4, H2A, and
H2B in Drosophila melanogaster (19). Therefore, the exposed sequences might
represent interaction sites uith specific regulatory proteins or present
exposed DNA sequences for binding of RNA-polymerase. Weintraub (19Θ0; 40)
postulated that regions of DNase I hypersensitivity may be involved in
orienting regulation signals on DNA with respect to their associated
nucleosomes and also uith respect to the coiling of the DNA in the chromosome
fiber· A remarkable feature of the DNase I hypersensitive sites juxtaposing
the recombinant proviruses is their location: 400-500 nucleotides before the
TATA-box and Cap-site in the 5»-LTR (33). A subdomain of chromatin defined
by a DNase I hypersensitive site approximately mithin 500 bp before the 5 1 end of the first oi-globin gene in the active ^-gene cluster in definitive
erythrocytes of chickens has been reported recently (40a),
The relative broadness of the band, representing the neuily emerging
fragment at around 7,0 kbp after DNase I digestion and Eco RI cleavage
(Fig, 1) suggests the presence of several closely positioned DNase I
hypersensitive sites in front of or at the start of the S'-LTR of some
recombinant proviruses. Such closely positioned sites, uhich can span up to
300 bp, have also been found at S'-ends of heat-shock genes in Drosophila
melanogaster (15,41),
Since hypomethylation has also been postulated as a characteristic though
not sufficient marker associated uith active genes (6,6a,19,25,32,37,40a) uie
have also determined the exten*", of base methylation at Hpa II/ Msp I
recognition sequences. All somatically acquired authentic and recombinant
MuLV genomes are hypomethylated (as observed previously for somatically
acquired FI-PITV sequences in carcinomas; 5) uhereas the genetically trans­
mitted M-MuLV copy is extensively methylated at the Hpa II/ Msp I recog­
nition sequences, MuLV proviral copies that display a hypersensitive site
to DNase I at their S'-end display grossly the same extent of mthylation at
their Hpa II/ Map I recognition sequences as compared to copies moderately
sensitive to DNase I. All reintei_patec proviral DNAs are at least moderately
sensitive to DNase I; this might therefore suggest a correlation between
moderately DNase I sensitive sequences and hypomethylation. It has been
reported for other genes that sequences sensitive to DNase I are hypomethyl­
ated, whereas Ιοω-І
і methylation does not a priori imply DNase I sensi­
tivity (16,19), The lack of extensive methylation of somatically acquired
sequences might simply be due to inefficient de novo methylation and not be
related to chromatin conformation, lile have also observed that during aging
of a Balb/c mouse the extend of methylation at Hpa II/ Msp I sites of
endogenous viral sequences (those that can cross-react with AKR-MuLV cDNA)
is deminished while these sequences are not preferentially digested by
DNase I.
Previously it was shown that the Nov-I Fl-WuLV DNA is hypomethylated at
Sal I and Sma I sites in target tissues as compared to non-target tissues
(36), although this proviral DNA is extensively methylated at the Hpa II/
Msp I sites in tumor tissues. Also, the По -1 ГМЧиИ/ proviral DNA is not
preferentially digested by DNase I in tumor tissues. These data suggest that
this locus is inactive in transcription in tumor tissues.
Tumors of different mice contain variable numbers of reintegrated recombinant
and authentic M-fluLV proviral genomes, A certain recombinant proviral genome
(Fig. 2A) seems to be required for transformation (24,36). Whether these
proviruses exert their transforming activity by promoter-insertion or via
a recombinant env-gene product is still obscure. However, those recombinant
FluLV copies, displaying a flanking cellular DNA region hypersensitive to
DNase I are probably the best candidates for proviruses active in transcrip­
tion in each tumor. Also, these copies are therefore most likely responsible
for leukemic transformation.
107
Cloning of these proviral sequences, selected by virtue of their DNaee I
hypersensitive configuration, uill hopefully enable us to study the role
of these recombinant genomes and flanking cellular sequences in the
transformation process.
ACKNOklLEDGTOTS.
Ы are grateful to Prof, Dr. H. Bloemendal, in uhosa laboratory these
investigations ыег carried out. Further, ше wish to thank Mrs, Els
Hulsebos for excellent technical assitance and Mr. Arnold Bosch for
setting up the isolation procedure for nuclei. This study шаэ performed
as part of the Ph.D. study of H.v.d.P., and uas supported in part by the
Foundation for Medical Research (FUNGO), uhich is subsidized by the
Netherlands Organization for the Advancement of Pure Research (ZU/O).
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CURRICULUM VITAC
P. Herman ñ. van der Putten uerd geboren op 23 januari 1954 te Afferden
(Limburg; gemeente Bergen).
In 1971 behaalde hij het H.B.5.-B diploma aan het Sint Chrysostomus Lyceum
(het huidige Elzendaal College) te Boxmeer.
In september 1971 maakte hij een aanvang met de studie Biologie aan de
Katholieke Universiteit te Nijmegen. In juni 1974 behaalde hij het kandidaats examen Biologie B1G (bijvak Geologie) met de aantekeningen Organische Chemie (B4) en Chemische Binding (B4). Het doctoraal examen Biologie
met als hoofdvak Moleculaire Biologie (Prof. dr. 3.G.G. Schoenmakers) en
als bijvakken Genetica (mijlen Prof. dr. H.D« Berendes) en Biochemie (Prof.
dr. H. Bloemendal) uerd in september 1977 afgelegd (cum laude).
Vanaf oktober 1977 mas hij als wetenschappelijk medewerker werkzaam op het
laboratorium voor Biochemie aan de Katholieke Universiteit te Nijmegen,
In deze periode werd het onderzoek aan de integratie van RNA-tumorvirussen
(т.п. het Moloney Murine Leukemie Virus) verricht in de werkgroep van
Dr. A.3.M. Berns, Tevens begeleidde hij in het kader van zijn onderwijstaak
gedurende vier jaar de praktica Biochemie voor eerste en tweede jaars
studenten Medicijnen. Tijdens de promotieperiode werd twee maal (1978 en
1980) de RNA-tumorvirus vergadering te Cold Spring Harbor (New York)
bijgewoond met financiële steun verleend door de Faculteit der Geneeskunde
van de Universiteit van Nijmegen en de Nederlandse organisatie voor zuiverwetenschappelijk onderzoek (Z.üJ.O.). Met financiSle steun van beiden en
van het laboratorium te Cold Spring Harbor werd in juli 1979 de RNAtumorvirus cursus gevolgd op het Cold Spring Harbor laboratorium (New
York). In september 1980 nam hij deel aan de E.M.B.O. cursus "Cellulaire
en molekulaire aspecten van de vroege zoogdierembryogenese" op Kreta
met financiële steun van zowel E.M.B.O., de Europese Organisatie voor
Molekulaire Biologie, als van Z.U.O.
Uanaf december 1981 zal hij als post-doc op,basis van een Z.U.O.-stagebeurs 10 maanden verbonden zijn aan het "Laboratoire de différenciation
Cellulaire, Departement de Biologie Animale" te Geneve in Zwitserland,
o.l.v. dr. Karl IJlmensee. Hierna zal hij verder verbonden, zijn aan dit
laboratorium als post-doc op basia van een verkregen "Long term fellowship" van E.M.B.O,. Gedurende deze periodes zal onderzoek verricht
worden aan de integratie en expressie van gekloneerde eukaryotische genen
die geïnjecteerd worden in preimplantatie muizenembryo's.
STELLINGEN
1.
De conclusie dat de leukemogene potentie uan muizeleukemie virussRn
geïsolaard uit SL3 cellijnen niet toe te schrijven is aan een gemodificeerd
env-gen is vooralsnog niet gerechtvaardigd.
Pedersen, F.S., Crouther, R.L., Tenngy, D.Y., Reimold, A.n., and Haseltine,
U.A. 19Я1. Nature 292: 167-170.
2.
De bewering door Linemeyer et al. dat de env-gerelateerde gensequentie
van SFFU als enigste verantuoordalijk is voor de ontuikkeling van erythroleuksmie is vanwege mogelijke additionele recombinaties aan tui;fel onder­
hevig.
Linemeyer, D.L., Ruscetti, S.K., Scolnick, F.П., Evans, L.H., and Duesbern,
P.H. 1981. Proc. Natl. Acad. Sci. U.S.A. 78: 1401-1405.
3.
Oe overerving en het voortbestaan van op het eerste gezicht minder gunstige
allelen kan een oorzaak zijn van het verschijnsel dat bepaalde genen ge­
koppeld o vererven terwijl deze genen niet fysisch gekoppeld zijn ("quasilinkage").
Boyse, E.A. 1977. Immunological Reviews 33: 125-145.
4.
Alhoewel Bachelor et al. in de titel van hun artikel een Ploloney HuLVapecifieke cDNA probe vermelden blijkt deze probe niet specifiek te zijn
onder de gebruikte condities zoals uit hun eigen foto's blijkt die ge­
toond worden in het betreffende artikel.
Bacheler, L.T., and Fan, Η. 19 0. 3. Virol. 33: 1074-10 2.
5.
Oe door Breindl et al. uitgevoerde kinetische analyses die de DNase I
gevoeligheid weergeven van provirale genen in chromatine zonder gebruik
te maken van de blotting-techniek zijn niet betrouwbaar genoeg om uit­
spraken te doen over de "actieve" structuur van individuele provirale
genomen in tumor DNA.
Breindl, Cl., Bacheler, L., Fan, H«, and Jaehnisch, R. 1980. D. Virol. 34:
373-ЗЯ2.
6.
De aanuezlgheid van san DNase I hypergewoelige plaats in de S'-lange
tsrminale "repsat" uan het endogene retrovirus locus eu—3 uordt in de
samenvatting van het artikel door Groudine et al. wel vermeld, maar urardt
onvoldoende beuezen door het getoonde feitenmateriaal.
Groudine, П., Eisenman, R., and u/eintraub, H. 1981. Natura 292: 311-317.
7.
De meest waarschijnlijke functie van Ь Ьа-г-тісгодІоЬиІіп -РІНС-апіідеп
dimeran is te dienen als algemene ankerplaatsen voor eiwitten die Organo­
genese reguleren. Het gp70 van een recombinant virus kan in dit model
gezien urarden als een receptor; overproductie van dergelijke receptoren
met als gevolg onderbezetting door eiultten die Organogenese reguleren
leidt tot kwaadaardige groei van de betreffende cel.
Ohno, S. 1977. Immunological Я
Dit proefschrift, hoofdstuk I.
і шз 33: 59-59.
8.
Verschillende recombinante flCF-type virussen kunnen worden gevormd via
ean algemeen recombinatie-mechanisme in de muizecel. De heterogeniteit
in structuur van deze recombinante virusganomen kan de grote variatie
in celtypen verklaren dis door dergelijke virussen getransformeerd kunnen
worden.
Dit priafschrift, hoofdstuk I en IV.
9.
Поіоп у an AKR murine leukemievirus-gsinduceerde leukemie gaat gepaard
mat da vorming en expressie van recombinante virussen: aan chronische
immuunrespons ia een belangrijke drijvende kracht voor dit proces.
Dit proefschrift, hoofdstuk I en IV.
Quint, Ui., Quax, 'i., van der Putten, H., and Berns, Α. 1981. Э. Virol.
39: 1-10.
Lee, 3.C., and Ihle, Ü.N. 1981. Nature 2B9: 407-409.
10.
Op grond van de gepresanteerda gegevens van Steffen et al. kan niet
geconcludeard worden dat het Akv-2 locus in de congene NIH/Swiss Як -2
stam afkomstig is van of identiek is aan een van de drie loci in da
A K R / N muis.
S t e f f e n , D . , B i r d , S . , Rok'e, Ы.Р.,
Aca.J. S c i . U . S . A . 7 6 : 4 5 5 4 - 4 5 5 8 .
and u l e i i b a r g , R.A.
1979. Ргос.
Natl.
11.
Het transГогт гвпсіе gen "R" beschrtvan door SutoliFFe et al. schijnt ook
de t r a n s f o m a U n beuerkatelliqcJ te hcïbben uan de betrouwbaarheid v.-in ds
uietenscha^pel'ijke elgensohappen uan de auteurs.
Sut;liffe, T.G., Shinnick, T.C., Green, N. t Liu, F.-T., Ni^an, H.L., and
Lerner, R.A. 19BQ. Nature 2 7: 801-605.
Lnrner, R.A., gutcliffe, 3.G,, and Shinnick, Т.П. 1481. Cell 23: 309-310.
12.
Gezien het verbod van dg kerkel'jke aiitoritaitan, ^е ouderdom van de lijk­
wade van Turijn te bepalen middels de C-14 mathode, is het de vraag of de
kerk in deze zaak naar waarheid streeft.
De lijkuiada getuigt. Rodney Ноагв. 1979. E.C.I. Utrecht.
13.
De aanduiding promotor suggereert een functie als promoter voor een
meerjarig transcript (proefschrift) maar is misleidend daar de functie
van de promotor in de praktijk neerkomt op die van een terminator.
14.
Het feit dat discriminatie van bepaalde groepen zoals homofielen n'.et
meer wordt toegestaan maar ook niet wordt gestraft verandert niets aan
de oude situatie en is aan voorbeeld van bureaucratische democratie.
15.
Gezien de aanzienlijke verlaging van de vergoeding ten behoeve van het
drukken van proefschriften moeten concessies gedaan worden aan de
kwaliteit van het drukwerk.
Dit proefschrift, pag. 92,93.
16.
Filosofisch gezien is de gedachte dat een kankercel zich gedraagt als
een anarchist verantwoord.
17.
Aangezien de mogelijke toepassingen van gen-therapie van utopie naar prak­
tijk verschuiven dient het onderzoek naar gen-transplantatie in Nederland
sterk bevorderd te worden.
Nijmegen, 5 november, 1991,
Herman van der Putten.