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MURINE LEUKEMIA VIRUS
INDUCED
LEUKEMOGENESIS
Proviral activation of cellular oncogenes
H
^ н
^
• і ^ ч « Gerard Selten
MURINE LEUKEMIA VIRUS
INDUCED
LEUKEMOGENESIS
Proviral activation of cellular oncogenes
Promotor : Prof. Dr. H. Bloemendal Co-referent: Dr. A. Berns
MURINE LEUKEMIA VIRUS
INDUCED
LEUKEMOGENESIS
Proviral activation of cellular oncogenes
PROEFSCHRIFT
ter verkrijging van de graad van doctor in de wiskunde en natuurwetenschappen
aan de Katholieke Universiteit te Nijmegen op gezag van de Rector Magnificus
Prof. Dr. J.H.G.I. Giesbers volgens besluit van het College van Decanen
in het openbaar te verdedigen op donderdag 16 oktober 1986
des namiddags te 1.30 uur precies.
door
GERARDUS CORNELIS MARIA SELTEN
GEBOREN te MILL
NIJMEGEN 1986
Gaarne wil ik allen bedanken die op enigerlei wijze hebben
bijgedragen tot de totstandkoming van dit proefschrift.
In het bijzonder ben ik dank verschuldigd aan Theo Cuypers voor de
vele wetenschappelijke discussies en voor zijn onuitputtelijke inzet,
Els Hulsebos voor de bekwaamheid en zorgvuldigheid waarmee zij haar
experimenten omringde, Annemiete van de Kemp voor de precisie waarmee
zij embryonale weefsels isoleerde, Wilbert Boelens voor zijn accurate
bijdrage aan het "sequence" werk, Paul Krimpenfort, Wim Quint en Peter
van Wezenbeek voor de waardevolle discussies en Sjef Verbeek voor zijn
hulp gedurende de laatste maanden.
Dank gaat ook uit naar Ron de Ruyter, Fons Haenraets, Jos Rewinkel,
Hans Nottet en Nico Verbeek, die in het kader van hun doctoraal stage
eveneens een bijdrage hebben geleverd aan het onderzoek.
Alle medewerkers van de afdeling Biochemie ben ik zeer erkentelijk
voor de prettige samenwerking in de afgelopen jaren.
Dr. M. Zijlstra en Dr. C. Melief van het Centraal Laboratorium van de
Bloedtransfusie Dienst te Amsterdam wil ik danken voor de uitzonder
lijke samenwerking en het ter beschikking stellen van vele tumoren.
De medewerkers van het Centraal Dierenlaboratorium dank ik voor hun
technische bijdrage en het verzorgen van de talloze muizen.
Dank ook aan Jack Leunlssen voor de goede adviezen, die voor het
verwerken van de tekst voor dit proefschrift onontbeerlijk waren.
Mijn dank gaat ook uit naar Arthur Slmonetti van wiens hand het
ontwerp op de omslag afkomstig is.
Aan de nagedachtenis van raijn vader
Aan mijn moeder, Anne-Marie en Paul
The investigations described in this thesis were prepared in the
Department of Biochemistry (head; Prof. Dr. H. Bloemendal), Faculty of
Science, University of Nijmegen, Nijmegen, The Netherlands, under the
direction of Dr. A. Berns.
The studies presented in this thesis were supported by grant NUKC 83-7
of the Koningin Wilhelmina Fonds, the Netherlands Cancer Foundation.
CONTENTS
CHAPTER 1 In t roduct ion 9
1. Proto-oncogenes and tumorigenesis
2. General c h a r a t e r i s t i c s of r e t r o v i r u s e s
3. Proto-oncogenes as t a r g e t s i t e for p rov l ra l
In teg ra t ion
4. Mode of p rov i ra l a c t i v a t i o n of proto-oncogenes
CHAPTER 2 Murine Leukemia virus- induced T-ce l l lymphomagenesis: 29
I n t e g r a t i o n of proviruses in a d i s t i n c t chromosomal
r eg ion .
CHAPTER 3 Involvement of c-myc in MuLV-lnduced T-ce l l lymphomas Hi
in mice: frequency and mechanisms of a c t i v a t i o n .
CHAPTER 1 Provi ra l a c t i va t i on of the pu ta t ive oncogene Pim-1 in 51
MuLV-induced T-ce l l lymphomas.
CHAPTER 5 The primary s t r u c t u r e of the puta t ive oncogene Pim-1 59
shows extensive homology with Pro te in Kinases .
CHAPTER 6 The oncogene Pim-1 i s predominantly expressed in lym- 87
phoid t i s s u e s during pre- and pos tna ta l development.
SUMMARY 99
SAMENVATTING 103
LIST OF PUBLICATIONS 107
CURRICULUM VITAE 109
7
CHAPTER 1
INTRODUCTION
INTRODUCTION
1. Proto-oncogenes and tumorIgenesls.
A large number of malignancies. Including carcinomas, sarcomas,
leukemlas and lymphomas can be Induced by RNA tumor viruses In various
vertebrates (Klein, 19Θ0). The study of the pathogenicity of
transforming retroviruses has provided basic Information on the
mechanisms underlying tumor development, which Is also relevant for
non-vlrally Induced tumors. A distinct number of genes, originally
Identified In "acute" transforming retroviruses have been implicated
In the development of cancer (Bishop and Varmus, 19 Ч). These on
cogenes are not only part of the "acute" transforming retroviruses,
but are an Integral part of the DNA complement of vertebrates, playing
an essential role In the regulation of growth. Most cellulair on
cogenes or proto-oncogenes are Identified by sequence homology with
viral transforming genes (v-onc genes), present as dominant deter
minants of cancer in "acute" transforming retroviruses (Bishop, 1983).
Additional oncogenes are Isolated using procedures of gene transfer of
tumor DNA to transform NIH3T3 cells or became evident by insertional
mutagenesis of "non-acute" transforming retroviruses (Cooper and Lane,
1984). Non-cellular oncogenes have been found and characterized in
DNA tumor viruses (Bishop, 1985).
As already mentioned, proteins of proto-oncogenes possess proper
ties essential for normal cellular functions. Up till now, cellular
functions of more then 20 proto-oncogenes are partly identified.
These proto-oncogenes can be derived into four classes on basis of the
nature of the encoded protein and their cellular localization: 1. the
three ras genes, whose products are likely controlling elements of
adenylate cyclase activity within the plasma membrane (Toda et al.,
1985; Levlnson, 1986); 2. the genes myc , myb , fos , ets , and ski ,
whose proteins are localized within the nucleus modulating gene ex
pression (Abrams et al., 1982; Klempnauer et al., 1984; Curran et al.,
1984; Bishop, 1985; Bishop, 1985a) 3. the sis protein, which shows ex
tensive homology with the platelet derived growth factor (Doollttle,
1983) and 4. the largest group of oncogenes encode proteins which ca-
11
talyse the phosphorylation of proteins (Hunter and Cooper, 1985).
Within this class, distinction with respect to amino acid specificity
for protein kinase activity is observed. The oncogenes src , fma ,
erbB , abl , fgr , fps/fes , ros , yes and kit , initially identified
In "acute" transforming retroviruses and the oncogenes neu and met ,
Identified by DNA transfectlon experiments (Schechter, ІЭб'*; Cooper,
1984), encode protein kinases phosphorylating exclusively the tyrosine
residues (Hunter and Cooper, 1985; Dean, 1985¡ Besmer et al., 1986).
The products of the oncogenes mos and raf/mll show kinase activity
more specific for serine and/or threonine residues (Maxwell, 1985;
Moelllng, 1984). During tumorlgenesls, these proto-oncogenes can be
activated either by transduction (Bishop, 1983), translocation (Klein,
1983; Perry, 1983), amplification (Alitalo, 1984) or by proviral
insertion (Hayward et al.,1981; Cooper and Lane, 1984) and act as dom
inant transforming elements. However, a significant higher expression
level of an oncogenic protein certainly cannot completely explain the
onset of tumorlgenesls.
Quite frequently, activated proto-oncogenes have been found either
to be truncated or to have undergone mutations, suggesting that the
products of activated proto-oncogenes gain transforming activity
through structural alterations. For example, the transduced oncogenes
in "acute" transforming retroviruses show generally single base sub
stitutions, deletions or rearrangements as compared to their cellular
homologues, the proto-oncogenes. Transduction probably truncates
proto-oncogenes so that their oncogenicity is considerably increased
(Bishop, 1983).
In tumors Induced by agents other than retroviruses, chromosomal
abnormalities such as point mutations, translocations and gene amplif
ications are mainly connected with altered oncogenetic loci.
For example, the observed point mutations in the газ genes in human
bladder carcinoma cell lines (Reddy et al.,1982; Chapon étal., 1983a)
and In human colon- and lung carcinomas (Chapon et al., 1983b; Der and
Cooper, 1983), the frequently observed chromosomal translocations of о
myc to the immuno-globulin domain in Burkitt lymphomas (Taub et al.,
1982; Dalla-Favera et al., 1983; Erikson et al., 1983; Leder et al.,
1983; Croce et al., 1983), In murine plasmacytomas (Shen-Ong et al.,
1982; Crews et al., 1982; Stanton et al., 1983; Mushlnskl et al.,
12
1983; Leder étal., 1983; Adams et al., 1983) and the amplification of
several oncogenes Including c-myc , c-myb , c-Ha- and Kl-ras , c-abl ,
c-erbB and N»myc Is observed In a variety of (human) cell lines and
tumors (Collins and Groudlne, 1982; Dalla-Favera et al., 1982; Alitalo
et al., 1984; Brodeur et al., 1984; Rabbits, 1985 and Yokota et al.,
1986).
Another mechanism, which can activate proto-oncogenes, Is frequent
ly observed In leukemogenesls Induced by the "non-acute" or slow
transforming RNA tumor viruses. These "non-acute" RNA tumor viruses,
lacking oncogenes in their genomes exert their oncogenic action by
Insertion of a provlrus adjacent to cellular oncogenes (Hayward et
al., 1981; Nusse and Varmus, 1982; this thesis).
This thesis describes the Identification, isolation and characterl·1
zatlon of the plm-1 gene which becomes, likes the с-шус oncogene, fre
quently activated by provlral Integration in lymphomas induced by the
slow transforming murine leukemia viruses (MuLVs) in mice.
To get more insight into viral induced lymphomagenesis I will
briefly discuss 1) some general characteristics of retroviruses, in
particular MuLVs 2) (putative) cellular oncogenes as target site for
proviral Integration and 3) mechanisms involved in provlral activation
of cellular oncogenes.
2. General characteristics of retroviruses.
Features, common to all replication competent retroviruses regard
less of their origin or pathogenic potential, are the RNA genome and
the strategy to replicate through a DNA intermediate. The RNA genome
encodes 3 primary gene products: structural proteins (gag), an RNA
dependent DNA polymerase or reverse transcriptase (pol) and envelope
glycoproteins (env). RNA termini carry a short sequence (R) and U5 or
U3 sequences specific to the 5' and 3' termini, repectlvely, encom
passing all regulatory sequences necessary for proviral transcription.
After Infection, RNA genomes serve as a template for the viral encoded
reverse transcriptase, resulting in the generation of double-stranded
linear DNA bearing two copies of a long terminal repeat (LTR), which
13
Is derived from sequences at both ends of the viral RNA (Varmus,
1982). These proviruses Integrate Into the host genome by a circular
DNA Intermediate with two tandem LTRs (Panganiban and Temln, 1984).
Sequencing data of LTRs cloned from several strains of retroviruses
show that LTRs contain regulatory sequences like CAAT, TATAA and the
polyadenylatIon sequence AATAAA as observed in many eukaryotic genes
(Dahr et al., 1980; van Beveren et al., 1980; Hishlnuma et al., 1980;
Shiraototohno et al., 1980; Swanstrora et al., 1981). Proviruses in
tegrate at many (random) sites into the host genome. When provirai in
tegration has occurred in the DNA of germ cells, proviruses will be
transmitted as Mendelian genes (endogenous proviruses). In tumori-
genesls, integrated proviruses can physically interrupt specific cel
lular genes (proto*-oncogene3), altering their expression pattern by
the inserted LTR or generate transducing proviruses by rare recombina
tion events (Bishop and Varmus, 1984). The transduced cellular on
cogenes (v-onc) will be transmitted, transcribed and expressed as
viral genes.
Based upon their oncogenic potential RNA tumor viruses can be de-
vided into two classes. The first class, the highly oncogenic "acute"
transforming retroviruses develop neplasla in vivo after a relatively
short latency period (within weeks). Moreover, "acute" transforming
retroviruses are able to transform cultured cells in vitro. The
"acute" transforming retroviruses have incorporated v-oncogenes, which
are derived from cellular proto-oncogenes by transduction (Bishop,
1983). They are mostly replication deficient, as part of the genes
essential for replication are replaced by the oncogene and for propa
gation they require the presence of helper viruses. The expression of
the v-onc gene products enables the "acute" transforming retroviruses
to Induce and maintain neoplasia (Bishop, 1983, 1983a).
The second class of RNA tumor viruses, known as "non-acute" or slow
transforming retroviruses gives rise to leukemlas and lymphomas after
a latent period of several months to more than a year (Reddy et al.,
1980). These retroviruses harbor no oncogenes in their viral genome,
they are replication competent and In contrast to the "acute"
transforming retroviruses, are not able to transform cultured cells in
vitro. MuLVs studied In this thesis, like Moloney" and AKR murine
leukemia viruses (MoMuLVs and AKR-MuLVs), belong to the class of slow
14
transforming retroviruses. MuLVs are C-type retroviruses on basis of
their morphology and Induce lymphomas within 3~12 months after infec
tion of new-born mice (Reddy et al., 1980). Present In certain mouse
strains as endogenous virus, they might give rise to spontaneous lym
phomas (Rowe and Hartley, 1971; Jaenlsch, 1976),
MuLVs possess a specific host range. Classification of MuLVs in
ecotropic, xenotropic and amphotroplc viruses is based on this host
range specificity. For example, AKR- and Moloney MuLVs are ecotropic
retroviruses according to their ability to infect and replicate only
In mouse cells. This host range specificity of MuLVs is associated
with the viral gp70 envelope protein, which Interacts with mouse cel
lular receptors (Rein, 1982; Cloyd et al., 1985). MuLVs which Infect
and replicate efficiently only In non-mouse species are called xeno
tropic. They replicate poorly In mouse cells, but are present in mouse
strains as germline transmitted proviruses. Amphotroplc viruses are
able to Infect and replicate in both murine-1 and non-murlne cells. Im
portant members of this class are the MCF-viruses (Mink Cell Focus
forming). They originate by recombination events between eco- and xe
notropic viral sequences and are most altered in the env gene and 3'
LTR sequences (Hartley et al., 1977).
Recombinant viruses have been isolated from lymphoma tissues of
mouse strains with a high lymphoma Incidence. The formation of MCF-
viruses in leukemic tissues is thought to be essential for the onset
of leukemia by these MuLVs. Although MCF-viruses can accelerate the
leukemogenlc process when inocculated together with ecotropic viruses
in new born mice (Nowinski and Hays, 1978; Cloyd et al., 1980;
O'Donnell et al., 1981), the recombinant glycoprotein cannot be con
sidered as an oncogene.
The precise role of MCF viruses in the induction of lymphomas Is still
a matter of speculation. It might be required for the infection of a
target cell which is normally refractory to infection by an ecotropic
virus (Cloyd, 1983). It could also provide an immunogenic stimulus, or
more specifically interfere with communication between lymphocytes,
thereby eliciting a proliferative respons (Lee and Ihle, 1981; Mellef
et al., 1980). This proliferative respons might result in a large po
pulation of cells which serve as targets for insertlonal mutagenesis
by MuLVs.
15
Since proto-oncogenes can be activated by retroviral insertion
(Hayward et al., 1981; Neel et al., 1981), it may be expected that the
analysis of a number of provlral Integrations in MuLV-induced leukemo-
genesis might result in the Identification of genes, which are in
volved in the leukemogenic process. This principle of provlrus tag
ging has successfully been used to identify a number of proto"
oncogenes (Nusse and Varmus, 1982; Cuypers et al., 1984).
3. Proto-oncogenes as target site for provlral integration.
The mechanisms by which the slow transforming retroviruses induce
neoplastic diseases have remained obscure for a long time. However,
there Is substantial evidence now that leukemia viruses can initiate
tumorigenesis by acting as insertion mutagens. First evidence that
retroviruses trigger cellular oncogenes by insertional mutagenesis was
provided by Hayward and Neel In 1981 In avian leukosis (ALV) induced
B-cell lymphomas.
Observations, leading to the detection of с-my с as a target site
for proviral insertion in ALV-lnduced lymphomas are the following: (1)
Several tumors have been found lacking infectious virions, intact
viral mRNAs or Intact proviruses (Payne et al., 1981; Neel et al.,
1981), making It unlikely that a viral gene product was required for
neoplastic transformation. (2) Detailed mapping studies using probes
specific for LTR sequences and viral genes showed that in some tumors
only the 3' LTR sequences were retained or that deletions In the 5'
provlral LTR had occurred (Payne et al., 1981). (3) Many tumors con
tained RNA species detectable with cDNA probes specific for the U5 LTR
region, but not with cDNA transcribed from other regions of the ALV
genome (Neel et al., 1981). It has been shown that the 3' LTR could
serve as a promoter to initiate transcription of flanking cellular se
quences, as proposed earlier (Quintrell et al., 1980; Tsichlis and
Coffin, 1980). (1) Detailed mapping studies suggested that one or more
ALV proviruses in each tumor might be located in a distinct region of
the host genome (Fung et al., 1981; Neel et al., 1981; Payne et al.,
1981). (5) The defenltive proof, showing that ALV proviruses in dif
ferent tumors Integrated into the same chromosomal region was provided
16
by Hayward et al.(1981). Using probes specific for several known viral
oncogenes, it appeared that ALV provlruses frequently Integrated Into
the vicinity of the с-шус gene, the cellular homologue of vmyc.
Proviral Integration near с-myс was always associated with enhanced c·
my с mRNA levels. Later studies provided convincing evidence that the
activation of c-myc is due to the Insertion of ALV sequences in the
vicinity of с-щс (Neel et al., 1981, 1982; Fung et al., 1981; Payne
et al., 1981, 1982; Westaway et al., 19811).
Involvement of c-myc is not limited to ALV induced EHcell lympho
mas. Provlral activation of the с » myc oncogene has also been
described In chicken syncitial virus (CSV) induced lymphomas (Noorl-
Dahll et al., 1981; Swift etal., 1985), in MuLV Induced T-cell lympho
mas in mice (Corcoran et al., 198t; Selten et al., 1981; LI et al.,
1984; Steffen, 1981; 0'Donneil et al., 1985) and in Feline Leukemia
Virus (FeLV) induced lymphomas In cats (Neil et al., 1984).
Other known cellular oncogenes have also been identified as target
sites for provlral Integration. In ALV-induced erythroblastosis pro-
viral integrations were frequently observed near c-erbB , a gene ini
tially defined by homology to the transforming gene v-erbB in avian
erythroblastosis virus (AEV). This resulted in consistently elevated
expression of c-erbB related mRNAs (Fung et al., 1983; Raines et al.,
1985; Nllson et al., 1985; Miles and Robinson 1985). The reported
rearrangements of с myb loci (with the concomitant Increased c«myb
transcription levels) in mouse plasmacytoid lymphosarcomas appeared
also to be caused by the nearby insertion of murine leukemia pro-
viruses (Mushlnski et al., 1983a; Shen-Ong et al., 1984, 1986). Pro
viral Insertion within the oncogenes c-Ha-ras ,с-К1-газ and с-mos
resulting In aberrant mRNA levels have been seen In a myeloblastosls-
assoclated vlrally Induced nephroblastoma (Westaway et al., 1986), in
a mouse bone marrow-derived, early myeloid cell line (George et al.,
1986) and in a mouse plasmacytoma (Rechavi et al., 1982; Canaanl et
al., 1983), respectively.
In many cases, common provlral integration sites were detected by
probes derived from oncogenes found In acutely transforming retro
viruses. Another approach to determine common provlral integration
sites Is applying the transposen tagging technique. A provirus to
gether with flanking cellular host DNA sequences is cloned and the
17
flanking cellular DNA sequences used as probe to detect rearrangements
within the corresponding chromosomal region In independently generated
tumors. Using this strategy, two common proviral Integration regions
were identified and characterized in mouse mammary tumor virus (MMTV)
Induced mammary carcinomas. Two independent genes, designated as lnt-1
(Nusse and Varmus, 1982; van Ooyen and Nusse, 1984) and lnt-2 (Peters
et al., 1983) appeared frequently to be targets for MMTV integration.
Moreover, proviral integration near both int genes was always associ
ated with int mRNA transcription (Nusse et al., 1984; Peters et al.,
1981). More recently, a new site of integration for MMTVs was ob
served in mammary- and kldney-adenocarcinomas, called Int-ll, express
ing altered int- Ί1 mRNA transcripts as a consequence of integrated
MMTV proviruses (Garcia et al., 1986).
In early T-cell lymphomas induced in mice by MuLV infection, simi
lar observations were made. Besides the already mentioned Involvement
of c»myc , the plm-1 gene was discovered as a target for provlral in
tegration in approximately 50$ of the tumors (Cuypers et al., 1984,
this thesis). Proviral integration near plm-1 is also Invariable asso
ciated with enhanced plm-1 mRNA levels (Selten et al., 1985, 1986
(this thesis)).
Over the last years, a discrete number of common domains for pro-
viral integration sites has been identified by the transposen tagging
technique, predominantly in tumors induced by MuLVs: in rats the loci
MLvt-1,-2, and-3 (Tsichlls et al., 1983, 1983a, 1985) and mis-l (Lemay
and Jolicoeur, 1984), in mice the loci fis-l (Silver et al., 1986),
gin -1 (Jolicoeur, pers. comm.), pvt-1 (Webb et al., 1984; Graham et
al., 1985) and in MMTV Induced carcinomas the locus lnt-3 (Callahan,
pers. comm.). All loci showing frequently rearrangements by provlral
insertions are sumnarlzed In table 1. As mentioned earlier, integrat
ed proviruses exert their oncogenic nature by activation of adjacent
cellular oncogenes. For the above-'mentioned loci, however, transcrip
tional activation of adjacent cellular (onco) genes has not been shown
yet.
More recently, another approach to detect and isolate new cellular
oncogenes has been described. The method is based upon the observation
that many oncogenes show protein kinase (PK) activity. In LSTRA cells,
derived from a Moloney MuLV-lnduced T-cell lymphoma, the tck gene was
18
Identified, giving rise to high levels p56tck tyrosln protein kinase.
Elevated p56tck encoding mRNA levels were the consequence of the In*·
tegratlon of proviral sequences upstream from this locus ( Marth et
al., 1985; Voronova and Sefton, 1986).
1. Mode of provlral activation of cellular oncogenes.
Retroviruses, lacking oncogenes In their genome have been shown to
activate transcription of a particular cellular oncogene by integra
tion adjacent to the structural gene. From recent data it has become
evident that Integrated provlruses can activate adjacent cellular
genes by two mechanisms: either by transcription from the viral pro
moter (Hayward et al, 1981) or by virtue of the enhancer activity of
proviral sequences on the adjacent cellular promoter (Payne et al.,
1982). The position and orientation of the integrated provirus with
respect to the flanking gene, as well as the oncogene in question
determines to a large extend which activation mechanism can be opera
tional.
Provlral activation of the cellular oncogenes с»myс and c-erbB by
promoter Insertion has been well documented in ALV- and CSV-'lnduced
B-cell lymphomas and erythroblastomas (Neel et al., 1981; Fung et al.,
1983; Swift et al., 1985) Provlruses were positioned exclusively
upstream from both genes with the same transcriptional orientation.
Transcription was initiated at the viral promoter and proceeded down
stream into the oncogene. Proviral activation of cellular oncogenes
by virtue of enhancer sequences present in the viral LTRs is described
for the Int genes (Nusse et al., 1984; Peters et al., 1983, 1984), and
for plm-1 and c-myc In MuLV-induced T-cell lymphomas (Selten et al.,
1984, 1985). The transcriptional direction of the provlruses In these
lymphomas is mostly oriented away from the protein encoding domain of
the oncogene. Differences In the position of Integrated provlruses
with respect to the oncogene seem to depend predominantly on the na
ture of the cellular oncogene. Strlnklngly, there appears to exist
"hot spots" for proviral Integration as observed in the 5' region of c·
myс and c-erbB (Selten et al., 1984; O'Donnell et al., 1985; Raines et
al., 1985; Swift et al., 1985) and in the 3' region of the £lm-1 gene
19
TABLE 1: PROVIRAL INTEGRATION SITES IN VIRAL INDUCED TUMORIGENESIS.
(Putative) Nature of Oncogene Integrated
Рго ігизез Tumors
Frequency Enhanced Species of mRNA
Integrationen) Levels
References
c-myc ALVs Висе11 lymphomas birds 90
c^myc MuLVs T* and В*»се11 lymphomas mice/rats 10*65
c-myc
c-erbB
с-чпуЬ
c-Ha-ras
с-К1-газ
c-mos
FeLVs
ALVs
MuLVs
ALVs
MuLVs
A-type
Lymphosarcomas
erythroblastosis
lymphosarcomas
nephroblastomas
myeloid cell line
myeloma
cats
birds
mice
birds
mice
mice
20
80-90
n.d.
2 cases
-
1 case
+ Payne,1982i Neel.igSl; Swift,
1985; Noorl-Daloll,198l.
+ Corcoran,1984; Selten,1984;
Steffen,1984; Li,1984;
0'Donnell,1985.
n.d Nell,1984.
+ Fung,1983; Raines,1985;
Nilson,1985; Mlles,1985.
+ Muschlnskl,1983a;
Shen-Ong, 1984,1986.
+ We3taway,1986.
+ George,1986.
+ Rechavl,1982; Canaanl,1983.
lnt-1
lnt-2
int-a
mt-1^!
plrn-l
MMTVs
MMTVs
MMTVs
MMTVs
MuLVs
mammacarclnomas
mammacarclnomas
mammacarclnomas
mamma" and kidney carcinomas mice
T-cell lymphomas
mice
mice
mice
mice
mice
70-80
40
10
2 cases
15
n.d.
MLvl-1,2,3
fis-1
mis-l
gln-1
pvt-1
Isk/tck
MuLVS
MuLVs
MuLVs
MuLVs
MuLVs
MuLVS
thymomas
hematopoietic neoplasms
thymomas
thymomas
T*»cell lymphomas
cell line LSTRA
rats
mice
rats
mice
mice
mice
35-60
10
35
25
30
•
n.d
n.d
n.d
n.d
-
+
Nu3se,19B2; Низзе,198Ч.
Peters,1983,1984.
Callahan,pers. comm.
Garcia,1986.
Cuypers,1984; Selten,1985;
O*Donnell,1985;
Muschlnskl,pers.comm.
Tslchlls,1983,1983a,1985.
Silver,1986.
Lemay,1984.
Jolicoeur,pers. comm.
Webb,1984; Graham,1985.
Marth,1985; Voronova,1986.
Abréviations: ALVs, Avian Leukosis Viruses; MuLVs, Murine Leukemia Viruses; FeLVs, Feline Leukemia Viruses; MMTVs, Mouse Mammary Tumor Viruses; n.d., not documented.
(Selten et al., 1985). Apparently, disruption of sequences controlling
transcription of the oncogene might be necessary for provlral activa
tion of cellular oncogenes, as proposed for the translocation of c-myo
In plasmacytomas (Leder et al., 1983). Alternatively, the observed
"hot spots" for proviral Integration might Just reflect a structure
which is preferred by the Integration machinery of the virus.
Although provlruses in retrovlrally induced tumors Integrate Into
the transcriptional unit of cellular oncogenes resulting in truncated
transcripts, the protein encoding domain of cellular oncogenes is not
affected (van Ooyen et al., 1984; Selten et al., 19Θ1, 1986), except
for the oncogene c-erbB where truncation is likely Instrumental for
oncogenesis. Therefore enhanced expression of an Intact oncogene pro
duct can be sufficient to confer a selective advantage to the tumor
cell. In two cases of ALV-lnduced erythroblastosis the oncogene c-erbB
expresses a truncated EGF receptor protein with close resemblance to
the v-erbB oncogene product of avian erthroblastosls virus (Lax et
al., 1985).
Hence, "acute" and slow transforming retroviruses can use the same
genetic information to exert their transforming activity.
22
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28
CHAPTER 2
Murine Leukemia Virus-Induced T-Cell Lymphomagenesis: Integration of Proviruses in a Distinct Chromosomal Region
Cal. Vol. 37. 141-150, May 1984
Cel. Vol 37. 141-150, May 19Θ4
Murine Leukemia Virus-induced T-Cell Lymphomagenesis: Integration of Proviruses in a Distinct Chromosomal Region
H. Theo Cuypen,* Gerard Selten,* Wim Quint,* Maarten Zijlstra,' Els Robanu« Maandag,* Wilbert Boelens,* Peter van Wezenbeek,* Cees Melief,' and Anton Bern·* "Department o( Biochemistry University of Nijmegen Geert Grooteplein N 21 6525 EZ Niimegen, The Netherlands •Central Laboratory ot the Netherlands Red Cross Blood Transfusion Service PO Box 9406 1006 AK Amsterdam. The Netherlands
Summary
A number ot mink cell focus-forming (MCF) proviruses was moleculatly cloned from mouse lymphoma DNA. From each clone, flanking probes were prepared to detect common integration regions in other MuLV-induced lymphomas. One clone frequently revealed variations in the molecular structure of the corresponding region (Pim-1) in other lymphomas. The results show the following. Changes in the Pirn region are seen in 24 out of 93 lymphomas tested. Over 50% of the early T-cell lymphomas show integration in the Pim-1 region. The alterations are seen in different mouse strains and with various MuLVs. The observed variations are caused by the integration of predominantly MCF genomes. AH integrations occur m a region spanning less than 20 kb and are associated with the transcriptional activation of a distinct region within the Pim-1 domain. The activated region does not show any homology with 13 known and three putative oncogenes.
Introduction
RNA tumor viruses can be divided into two categories the acute transforming viruses (sarcoma and acute leukemia viruses) and the viruses that cause neoplasms after a long latency (e g , avian leukosis virus [ALV], mouse mammary tumor virus [MMTV], and murine leukemia virus [MuLV]) The slow transforming viruses differ from the acute transforming viruses in three important properties first, they cause a long-term wemia in inoculated animals, which eventually (after 3 to 12 months) leads to neoplastic growth In contrast, the acute transforming viruses cause lymphomas within a few weeks Second, they induce no morphological transformation in fibroblasts in tissue culture Third, their genomes do not contain transforming genes
The neoplasms induced by slow transforming viruses are generally of monoclonal origin (Neel et al, 1981, Hayward et al., 1981, Payne et al, 1981, Noon Dalo» et ai.. 1981, Cohen et a l , 1979, van der Putten et al, 1981,
Quint et al, 1981) Insertional mutagenesis is the most tenable model to explain Ihe induction of oncogenesis by these retroviruses (Noon-Daloii el al, 1981 Jenkins et al, 1981, Varmus et al, 1981, Payne el al. 1982 Nusse and Varmus, 1982, Tsichlis el al. 1983. Pelers et al, 1983) The besl documented examples are represented by ALV-induced В cell lymphomas and erythroblastosis in birds, in which the transcription ol the cellular oncogenes c-myc and с erbB is frequently augmented by msertional mutagenesis (Neel et al, 1981, Hayward et al, 1981, Payne et al, 1981, Payne et al, 1982 Fung et al, 1983) Another example is that of MMTV-mduced mamma carcinomas in which the proviral insertion of MMTV in distinct chromosomal domains has been described (Nusse and Varmus.
1982, Peters et al, 1983) The proviral integration in the int 1 region is accompanied by the transcriptional activation of the mt 1 gene (Nusse and Varmus, 1982, Nusse et al, 1984) A third example is Moloney MuLV induced lymphomas in rats, where frequently a MuLV provirus is integrated in a distinct chromosomal site (Tsichlis et al, 1983)
Moloney (M-MuLV) and AKR (AKR-MuLV) murine leu kemia viruses can induce lymphomas in a variety of mouse strains Previous studies have shown lhat outgrown lym phomas, largely composed of one or a few clonal cell populations, harbor recombinant proviral genomes (mink cell focus forming virus [MCF]) integrated in multiple siles of chromosomal DNA (van der Pulten et al, 1981. Jaehner et al, 1980, Quint el al, 1981. Herr and Gilbert, 1983) The aim ol the present investigation is to determine whether virus-induced lymphomagenesis in mice is initialed through the viral activation of dislmcl cellular genes, which can induce cell prolifération and/or transformation One predicts that if MuLV mediales Iranslormation by the activation (or modification) of one or a few cellular genes, it should be possible to define distinct chromosomal DNA regions, which serve as targets for viral insertions If integration in a distinct domain ol chromosomal DNA is relevant to the transformation process, integration in the region is only expected in lymphoma tissue and not in nontrans formed cells, which are infected by MuLV Therefore dif ferent virus-host junction fragments Irom MCF proviruses were mdecularly cloned from lymphomas of BALB/Mo and AKR mice, and single copy flanking cellular probes were derived These probes were used to determine whether in other MuLV-induced lymphomas an MCF pro-virus is integrated in Ihe same chromosomal domain
We descnbe here the characterization of a domain (Pim-1 ) in which the integration ol MCF proviruses is frequently observed Integration in this Pirn 1 domain is associated with the transcriptional activation of part of this region The data strongly suggest that this Pirn 1 region might play an important role in the generation of early T-cell lymphomas
Results
Cloning of Virus-Cell Junctions Proviruses were cloned from lymphoma DNAs with a limited number ot proviral integrations It appeared unfeas-
31
ible lo clone all proviral integrations wilhin one tumor Therefore 5' wal-cell junctions were preselected for don ing on the basis of the presence of DNAase l-hypersensi-tive sites near their 5' ends (van der Putten et al, 1982), whereas 3' virus-cell junctions were chosen randomly The MCF prowuses denved from Moloney and AKR MuLV carry an Eco RI recognition site at map position 6 9, and each has a single Hind IH site at positions 5 4 and 3 0. respectively Upon digestion with Eco RI or Hind IH, each proviral MCF integration yields two characteristic fragments, representing the 5' and 3' virus-cell junctions the sizes of which will be determined by the position of the neighboring Eco RI or Hind III sites in the cellular DNA The Eco RI or Hind III fragments, which reacted with MuLV-specific probes, were molecularty cloned in the appropriate vectors We obtained five authentic clones of MCF virus-cell junctions Each of the phage inserts was transferred into pBR322 and further characterized by restrrclion enzyme analyses Two clones represented 5' parts of MCF prowuses, whereas three clones contained 3' parts of MCF genomes Single copy hybridization probes could be derived from four ol the five flanking cellular DNA regions in these clones These probes were used to search for
common integration regions in other lymphomas For this purpose DNA extracted Irom 93 different lymphomas was digested with the appropriate restriction endonucleases, separated on agarose gels, and blot hybridized (Southern, 1975) Hybridization with three of the four probes did not reveal any integration of proviral genomes in the corre spending region ol other lymphomas (data not shown) However, probes from one clone (C5C) showed a significant number of alterations in olher lymphomas The chromosomal domain, in which frequent integration of MuLV could be observed, was denominated Pim 1 (Proviral in tegration site MuLV)
Characterization ol the Pim-1 Region Four different cellular subclones (encoded l-IV), spanning 10 3 kb of Hanking cellular sequences were derived from the C5C clone (Figure 1c) The use of the probes A-E, denved from these subclones on chromosomal DNA blots, as well as on blots containing digests of the C5C phage clone, yielded the physical map of the preinlegration site (Figure la) The region thai corresponds to the C5C clone is shown in Figure 1b In order to show thai C5C was derived from the original proviral integration, probe A was
Figure 1 Physical Map of Ihe Ρνπ 1 Region
The reslncton endonuclease sites presented m (a) were determined both in chromosomal DNA from the AKfl/FufìdA slran and m the various plasmKj clones C5C 'b) and I up to IV (c) The probes used lor ihe Southern plot analysis ol chromosomal DMA are shown η (с) (Probe A a Bam HI Bam HI Iragment trom
position - 1 9 to - 0 9 probe Β β Bam Hl-Pvu D Iragment from position - 0 9 to approximately 0 probe С a Htnd lU-Sac I fragment from posrtion - 7 θ to
- 6 6 probe D a Sac l-Sac I tragmenl from position - 6 6 to - 4 1 probe E a Sac l-Bam HI Iragment from position - 4 1 to - 1 9) Posrtion 0 corresponds lo
the integration site ot the Moloney MCF provmis in the tumor from which the C5C clone was obtained The drection of integration n this tumor was arbrtranty
chosen to be 5 3'
Open boxes Long terminal repeals ol the MoMCF No restriction enzyme sites could be determined beyond the sites marked with an asterisk Abbreviations
used lor restnclnn endonucleases В Bam и Η Hmd III κ Κρη ι Ρ Pvu I Ps Pst I RI Eco RI Rv Eco RV Sc Sac I Sm Sma I and X Xba I
32
MuLV Induced Τ Cell LymphoTiaqcnesis
nybridized with Soulhern Ыоіь containing Eco RI digests of lympnoma DNA trom which Ihe provirus was cloned This probe recognized a 12 kl- fragment representing Ihe preintegralion site and an 18 kb band which corre sponded to the CiC clone (Table 1)
Screening of Lymphomas A lotdl of 93 DNAs Irom virally induced lymphomas were analyzed for the presen -e of com non intégration sites We used restriction endonucleases Eco RI and Eco RV separately on every lymphoma DNA preparation Eco RI cleaves ihe MCF genome once at map position 6 9 (Quint el al 1981 van der Putten el al 1981) whereas Eco RV has a recognition site in the LTR 0 14 kb from the 5' boundary of the U3 LTR (van Beveren el al 1980) If mlegration occurs wilhm the 22 kb Eco RV fragment
(Figure 1) this snould result in a reduction in size ol the hybridizing fragment unless integration occurred close lo Ihe Eco RV recognition siles in Ihe cellular DNA If integra Іюп occurs within Ihe 12 kb Eco RI fragment this could result in eithf an increase or a decrease of the hybridizing fragment depending on Ihe exact integration site In all instances an mtegralion delected after Eco RI analysis should .ifso be seen with Eco RV
Analysis ol ihe 9J Ero RI digested DNAs with probe A (Figure 1 c) revealed novel bands in 18 cases (Table 1 and Figure 2a) When Eco RV was used lo cleave the chro mosomal DNA 24 alterations .«ere detected in the Pirn 1 region (e g see Figure 3) In all cases changes seen with Eco RI were also observed after digestion with Eco RV A survey of the observed variations is presented in Table 1
Table l
NO
1
2
3
4
4
6
7
8
9
0
11
12
13
14
.5
16
17
В
iq
•·,
2i
22
23
24
25
Charactenslics ol the Poviral Intogrations mio the Pim 1 Locu:
Mouse SUaiO
BALB/Mo'
BALB/Mo
AKR/Fu
BALB/C
BALB/C
BAL8/C
BALB/C
BALB/C
BALB/r
BALB/C
BALB/C
BALB/C
BALB/C
BAIB/C
BALB/C
BALB/C
C57BL
C5TBL
'•57BL
: 5 7 B I
AKR/Fu
AKR/Fu
C57BI
C57BL
b,Le/c
MoJe Type ol
Transmis MuLV
Ь Т
C T
G '
NBI
NBI
NBI
1 «
NBI
NBI
NBI
NBI
NBI
NBI
NBI
NBI
NBI
NBI
NBI
N",
NBI
NBI
NBI
NBI
NBI
NBI
Used abtxevialions G Τ r , 1 Mouse (rom wtnrh Hanking ι
MoMuLV
MoMuLV
AKV
MoMuLV
MoMuLV
MoMuLV
MoMuLV
MoMiLV
MoMuLi/
MoMuLV
MoMuLV
MoMuLV
MoMuLV
MoMuLV
MoMuLV
MoMuLV
MoMuLV
MoMuLV
MoMuLV
MoMuLV
I/1CF247
MCF247
MCFI?33
MCF 1233
MCF1233
τ ι lino iransfr 'ted
Tissue
ьріесп
spleen
spleen
spleen
lymphn
spleen
spleen
soteen
spleen
lymphn
spleen
spleen
spleen
spleen
spleen
spleen
spleen
spleen
spleen
spleen
spleen
spteen
lymphn
spleen
spleen
Age (wks)
35
29
4β
10
17
21
17
17
12
19
10
13
13
13
12
12
19
26
27
31
13
24
26
22
2D
i in Independent Turra
Fragment Size (kb)
Probe A
Eco И
Normal
12
12
12
12
12
12
12
12
12
12
12
12
12
12
12
12
12
12
12
12
12
12
12
12
12
Tumor
16
17
22
20
195
18 5
22
20
165
22 17
9 0
175
19
19
22
9 2
22 17 5
17S
17
NBI Newborn mleUed nd not determmpd
Ulular sequences (C5C) were obtained
ys
EcoRV
Normal
22
22
22
22
22
22
22
22
22
22
22
22
22
22
22
22
22
22
22
22
22
22
22
22
22
Tumor
105
10 5
105
115
11
11
2 0 5
105
20
105
17 5
10
17
16
18
175
105
11
185
115
105
11 5
105
9 5
10 5
Probe В
Ceo η ν
Normal
35 22
35 22
35 22
35 22
35 22
35 22
35 22
35 22
35 22
35 22
35 22
35 22
35 22
35 22
nd
nd
35 22
35 22
35 22
35 22
35 22
35 22
35 22
nd nd
35 22
Tumor
105
12 10 >
105
115
12 11
12 11
205
125 105
20
12 105
17 5
11 5 10
17
16
nd
nd
1?5 105
11 5 11
18 5
115
125 105
115
12
nd
125 105
Probe С
Eco RI
Ni rmal
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
12
nd
nd
nd
12
12
nd
nd
nd
nd
nd
nd
nd
nd
nd
— Tumor
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
165
nd
nd
nd
6 2
6 2
nd
nd
nd
nd
nd
nd
nd
nd
nd
33
probe A R-I
' 2 3 4 5 6 / 8 9 10 1 I 12
2 3 —
2 0 —
MoUÌI TR R.j
< X ) 4 5 β 7 В 9 IO I t IS
Г
m
2 3—<
7 0—
Structural Organization of MuLV Proviral Integrations in the Pim-1 Region The alterations observed with specific cellular probes should also be recognized by virus specific probes Therefore. the nitrocellulose filters that were used in Figure 2a and for the results listed in Table 1 were hybridized with a specific Moloney U3 LTR probe (MoLOLTR). in case of Moloney MuLV induced lymphomas and with a mixture of the AKV pl5E probe (AkMCp15E) and the AKV U3 LTR probe (AkU3LTR) in case of AKV and MCF1233 induced lymphomas (Quint et al. 1984) An example of the hybrid ization pattern obtained with the MoLOLTR probe on Eco RI digests of various lymphoma DMAs is presented in Figure 2b The hybridization profiles indicate that each lymphoma contains multiple, newly acquired proviral ge nomes Novel bands detected with probe A (Figure 1 c) in Eco RI digested DNA (Figure 2a. lanes 3, 6. 9. 10 and 11 ) also hybridized with the MoU3LTR probe (Figure 2b. arrows) Similar results were obtained for the Eco RI digests of the other lymphoma DNAs with rearrangements in the Rim 1 region with the exception of lymphoma 22. which was induced by AKR-MCF247 AKR MCF247 carries an Eco RI recognition site 0 1 kb from the 5' boundary of the U3 LTR (Khan et al 1982) This interferes with the recognition by the AkLOLTR probe Therefore in case an AKR MCF247 provirus is integrated in the Pim-1 region, we would not be able to detect the ¡unction fragment with the AkLOLTR probe after digestion with Eco RI
A more accurate determination of the site of integration was possible In some instances by using the cellular fragment В as hybridization probe (Figure 1c) Probe В recognizes two fragments in Eco RV digests of normal BALB/c and C57BL liver DNA the 22 kb fragment, corre spondmg to the Pim-1 region, and the 35 kb fragment, which comprises a region with strong sequence homology with a portion of the Pim 1 region (Figure 3, lanes 1 and 2) If the MCP proviral integrations in the Pim-I region occur between map positions - 0 9 and 0 (Figure la), probe В should recognize the 5' and 3'-flanking cellular se quences separately since the MCF proviral genome har bors an Eco RV recognition site in both LTRs Examples are shown in lanes 6 9. and 11 of Figure 3 Whereas probe A (Figure 3) recognizes one additional band probe В identified two additional fragments, one of which is
Figure 2 CharactenzatKin of Eco RI Digests ol Lymphoma DNAs by Probe
A and ine MOU3LTR Probe
DNA samples (10 мд) were digested with Eco RI separated on agarose
gels and transferred to nitrocellulose Filters were first incubated with probe
A (see Figure 1c) washed under stringent conditions (0 1 χ SSC 0 1 %
SDS 650C) and exposed to Χ ray film (a) Subsequently the filters were
nnsed with alkali to remove residual label incubated with the M0U3LTR
probe washed (0 1 χ SSC 0 1 % SDS 60°C) and reexposed (b) The
proviral integrations m (b) that correspond to insertions in the Pim 1 region
are marked by arrows
Lane 1 liver DNA from young C57BL mouse lane 2 liver DNA trom a
young BALB/c mouse lanes 3-9 lymphoma DNAs from MoMuLV newborn
infected C578L mice lanes 10-12 lymphoma DNAs from MoMuLV new
born infected BALB/c mice Lane 3 corresponds to lymphoma 20 in Table
1 l a n e s 6 t o l 8 l a n e s 9 t o l 7 lanes 10 to 11 and lanes 11 to 10
34
MuLV-lndtjced Т-СеИ Lymphomageneas
e ' e s
Figtye 3 Charactenzatton of Eco RV Digests of
Lymphoma DNAs with Probe A and Probe В
The DNA samples desenbed η Figure 2, were
digested with Eco RV and btot-hybridized as de
scribed in the legend to Figure 2 with probes A
and В The distribution o( DNA samples ts as
described in the legend to Figure 2 Lane 5 cor
responds to lymphoma 19 in Table 1. and lane 12
to tymphoma 9
identical with the fragment recognized by probe A Ten out of the 24 lymphomas with an integration in the Pim-1 region showed this phenomenon The fragment sizes are summarized in Table 1
From the observed fragment sizes determined with probes A. B. and the viral LTR probe on digests with Eco RI and Eco RV, the precise tntegration position and proviral orientation could be derived A schematic representation is given in Figure 4 (lymphomas 2, 4, 5, 6, 10, 17, 18, 20, 24 and 25) The analysis of lymphomas 10. 17, 18, and 20 are depicted in Figures 2a and 3 in lanes 11, 9, 6, and 3, respectively In these lymphomas, the MCF promises are positioned in the same orientation as the integration in the lymphoma of the mouse from which the flanking cellular sequences were cloned (Figure la)
The additional band of 22 kb in the Eco RI digest of lymphoma 21 (Table 1), which was induced by AKR MCF247, cannot be explained by the integration of an MCF proviral genome. However, the AkU3LTR and Ak-MCplSE probes recognized this 22 kb fragment There-lore, the 9 kb increase in fragment size most likely has to be ascribed to the integration of a complete ecotropic MuLV genome The same explanation is probably appli cable to lymphomas 3 8. 12 and 23 (Table 1). The detection of two additional Eco RI fragments in lymphomas 12 and 23 could be explained by either a dual-clonal origin ot the lymphoma or the occurrence of a deletion in the proviral genome in part ot the lymphoma cells. The integration sites of these presumptive ecotropic proviral genomes all map in the region between - 0 9 and +1 1 on the physical map of the Pim 1 region (Figure 4) The orientation of these proviral integrations was derived from the hybridization patterns of Eco RV digested DNAs with the MoLOLTR probe (data not shown)
Proviral integrations in the Pim-1 region in lymphomas 15 and 16 (Table 1) are positioned around map position - 6 Their structure and orientation will be described elsewhere (Selten et a l . unpublished) In lymphoma DNA
ïi
O ir Л Я ñ τ s i í а
24
а 22 го З" 4 M 13 « 9 7
1 \ ili •f-r
Figure 4. Location and Orientation of the Prowal Integrations m the Pim 1 Region
Vertical arrows indicate the position of prowal integrations withm the Рвл-
1 region The numbenng of the lymphomas refers to the listing m Table 2
The small horizontal arrows indicate the direction of integration 5' to 3'
7, 9,13,14, and 19 (Table 1), only Eco RV digestion gave rise to additional fragments Representative examples of this group are presented in lane 5 (lymphoma 19) and lane 12 (lymphoma 9) of Figures 2a and 3 These proviral integrations are situated downstream from the Eco RI site between map position + 1 1 and +10 on the physical map of Pim 1 (Figure 4) The orientation of these proviral genomes cannot be determined with available probes and has to await the molecular cloning of the Pim 1 region further to the right Also the position and orientation of the integration in lymphoma 11 has yet to be established.
Proviral Integration in the Pim-1 Region Is Associated with Transcriptional Activation of Neighboring Host Sequences Pim 1 specific transcripts were detected in lymphomas with proviral integrations in the Pim-1 region; for this purpose, polyadenylated mRNA was prepared from lymphomas, immobilized on nitrocellulose filters, and subsequently hybridized with four different probes derived from
35
Cel
A •
•
•
•
•
В •
Figure 5 Expression of Pirr 1 Specific Sequences in Lymphomas with and wifhouf Рго лаІ Insertions in the Pim 1 Domain
Polyadenylated RNA from fymphomas was isolated Two migrams of it was applied to nitrocellulose filters as described in Experimental Procedures The filters were hybridized with probe A, washed under stringent conditions (0 1 χ SSC 0 1 % SDS 65°C) and exposed Ιο X ray film Lymphomas of
row A have a proviral insertion in the Pim 1 region in lymphomas of row В no atterations were found within fhe 22 kb region bounded by the Eco RV sites
the Pim-1 region, probes A С D and E An example is shown in Figure 5
Insertion of a provirus in the Pim-1 region is associated with the presence of polyadenylated RNA. which hybridizes with comparable efficiency with probes A, E, and D, but not with probe С Preliminary results show the presence of distinct RNA transcripts on Northern blots (Selten et al, unpublished) In lymphomas without insertions in the Pim-1 domain, only minor hybridization was observed The difference in expression between lymphomas with and without a proviral integration in the Pim-1 region is estimated to be 10 to 50 fold Further analyses are in progress to determine the nature of the trarbcript{s).
The Pim-1 Region Shows No Homology with Known Oncogenes In order to verity whether the Pim 1 region harbors an already identified oncogene, we screened the Pim-1 clone with available viral and cellular oncogenes Cloned DNAs containing part or all information for abl, src. fes, fms, Ki-ras, Ha-ras, N-ras, с myb (human), c-myc (mouse) fos, sis, mos, rel, (Coffin et al, 1981), were denatured and immobilized on nitrocellulose filters and hybridized with a ^P-labeled C5C phage clone (Figure 1b) In no instance
was any annealing observed between C5C and one of the oncogenes mentioned Southern blot analysis performed with rat DNAs revealed that although Pim 1 sequences are easily detected in rat DNA, the region does not show any homology with the preferential integration site for Moloney MuLV in rat lymphoma: (Tsichlis et al, 1983, data not shown) A similar analysis Oh mouse DNAs also re vealed no homology with the preferentie integration sites for MMTVj int-1 and mt 2 (Nusse and Varmus, 1982, Peters et al, 1983)
High Frequency of Proviral Integration in the Pim-1 Region is Associated with Early T-Cell Lymphomagenesis Early T-cell lymphomas induced by the highly leukemo genie viruses, show a high frequency of proviral integration in the Pim-1 region We can discern three groups (Tahte 2) Group 1 lymphomas in BALB/c and C57BL mice inoculated as newborn with Moloney MuLV and in AKR mice, infected with AKR-MCF247 These mice developed lymphomas within 30 weeks and showed integration in the Pim 1 domain in 40%-65% of the lymphomas Group 2 lymphomas in BALB/c and C57BL mice, inoculated as newborns with the MCF1233 isolate (Zijlstra et al, 1983), in spontaneous lymphomas in AKR (Hartley et al, 1977 Cloyd et al, 1980, Quint et al, 1981 Herr and Gilbert 1983), and BALB/Mo mice (Jaenisch, 1976, Nobis and Jaenisch, 1980) The latency of lymphoma development varies in this group between 30 and 40 weeks and in less than 20% of the lymphomas, proviral integration In the Pim 1 region is found Group 3 lymphomas in BALB/c and C57BL mice, infected as newborns with the poorly oncogenic MCF1130 isolate (Zijlstra et al, 1983) and lymphomas induced by the milk-transmitted Btropic C57BL-MULV (Melief et al, 1980) These lymphomas, which occurred after a latency of more than a year, do not show proviral integrations in the Pim-1 region In all instances, where the lymphomas with integrations in the Pim 1 region were characterized with respect to their cell-surface-specific antigens, it appeared that they were from T-cell origins (16 of the lymphomas listed in Table 1 were tested) There was no correlation between the integration of a provirus in the Pim-1 region and the expression of Lyt-1, Lyt-2, or TL antigens by the Τ cell lymphomas (data not shown) In groups two and three, a significant portion of the lymphomas belonged to the В cell or non T/non B-cell category (Melief et al 1980, Zijlstra et al, unpublished)
The data of Table 2 show that proviral integration in the Pim-1 region occurs in all the mouse strains tested The induction is not restricted to one virus but is seen with different MuLVs Proviral integration in the Pim-1 region is more common in tumors that arose after a short latency In order to verify whether rapidly developing lymphomas have more viral integrations in their lymphoma DNAs than the lymphomas arisen after a long latency, viral-specific probes were hybridized to Southern blots containing DNA from groups one and two No obvious difference was seen
36
Tatote 2 Frequency oí Integralion of ЬЛА-Vs tn Oie Pim-1 Regon and Cofrelatton яві Latency of Lymphoma Development
Vtus Stram Induction Mode Mouse S&ain Latency·
in the number of proviraJ integration in either group (data not shown)
Discussion
Cellular oncogenes can be activated alter translocation to other chromosomes (Taub et al, 1982, de Klein et al, 1982, Mushinski et al, 1983, Enkson et al 1983, Dalla-Pavera et al, 1983) by gene amplification (Collins and Groudme, 1982. Chattopadhyay et al, 1982, Schwab el a l , 1983), by msertional mutagenesis (Hayward et al, 1981, Payne et al, 1982, Nusse and Varmus, 1982, Peters et al, 1983, Rechavi et al, 1982, Kuff et al, 1983, Fung et al, 1983), and by point mutations (Taparowski et al 1982, 1983, Reddy el al, 1982, Der and Cooper, 1983, Capon et al, 1983) In lymphomas induced by viruses, which do not harbor oncogenes, msertional mutagenesis (Hayward et al, 1981, Payne et al, 1982, Fung el al, 1983, Nusse and Varmus, 1982) seems the favonte mech anism to activate cellular oncogenes The phenomenon we observe in MuLV induced lymphomas shows a resem blance to both the activation of the int-1 gene in MMTV induced mammacarcinomas (Nusse and Varmus, 1982, Nusse et al, 1984) and с myc activation by AVL in bursal lymphomas in birds (Hayward et al, 1981, Payne et al, 1982)
Induction of Т-с ІІ lymphomas by MuLV appears to be a complicated process in which the generation of recombinant MCF viruses seems essential (Hartley et al, 1977, Cloyd et al, 1980, ZHjstra et al, 1983) One of the models for lymphomagenesis assumes that the generation of MCF viruses is needed ю order to infect a specific target cell, which is refractory for infection by ecolropic MuLV (Cloyd, 1983) Рго гаІ activation of cellular genes, like с myc, could then initiate the transformation process Alternatively, the exposure of recombinant envelope glycoprotein on the
Nirnbef Nunber Inleg
Tested
20
7
5
11
7
11
16
4
5
7
P m l
13
4
2
2
1
1
1
0
0
0
Latency» in Weeks
14 ± 4
2 5 ± 5
19 ± 8
24 ± 3
26
47
29
-— _
K.
65
57
40
18
14
9
6
0
0
0
Hip (data cell surface could evoke cell proliferation by interfering with the communication between lymphocytes, or by eliciting a chronic immune stimulation (McGrath and Weissman, 1979)
We provide evidence that in a certain class of MuLV cation to induced Τ cell lymphomas a distinct host gene is activated ¡in et al, by the integration of predominantly MCF proviruses in its Й, DaNa- vicinity We have denominated this region Pim-1 (prowal dims and integration site MuLV) :hwab el d et al, Arrangement and Structure of MuLV Proviruses in 12, Peters the Pim-1 Region 83, Fung The proviral integrations in the Pim-1 region are predomi-зкі et al nantly found in the - 1 to +1 region The other integrations er, 1983, are found dispersed in the +1 to +10 region and around ι viruses, the - 6 region Not a single provirus was found in the agenesis region between —1 and —5 5 This agrees with the theory ig el al, that the gene that becomes activated is located in this ite mech region As far as we have determined the transcnptional nomenon orientation of the integrated proviruses, all proviruses in a resem the - 1 to +1 position showed the 5'-3' onenlation with
η MMTV respect to the Pirn 1 region as depicted in Figures 1 and js, 1982, 4 The cellular gene that becomes activated m these in bursal lymphomas is located upstream from these proviruses
ne et al, The 5' LTR could function here as an enhancer element We observed both MCF and ectropic-like genomes in this
ars to be region Apparently the presence of a recombinant provirus Df recom- is not required for the activation of the Pim-1 region One al, 1977, could speculate that the MCF vtus is only needed for the e models infection of the appropriate target cet, and therefore that fiofMCF pseudotyped MuLV (MCF envelope, ecotropic genome) irget cell, can activate the Pirn 1 gene equally weU V (Cloyd, Furthermore, the Pirn 1 region does not show any ho-e с myc, mology with sis, Ki-ras, Ha ras, N-ras, rel, src, alb, c-myb, •rnatively, mos, tos, fes, fms, and c-myc No homology was found an on the with int-1 (Nusse and Varmus, 1982), mt 2 (Peters et al,
MoMuLV
MoMuLV
AKR MCF247
MCF 1233
MCF 1233
AKV
MoMuLV
MCF 1 1 X
MCF1130
CSTBL-MuLV
NBF
NBI
NB
NBI
rei
G T "
G T
NBI
№
M T ·
BALB/c
C57BL
AKR
CSJBL
BALB/c
AKR
BALB/Mo
C57/B1
BALB/c
C57BL
15 ± 3
24 ± 6
1 θ ± 5
Э 8 ± 1 1
37 ± 0
3 9 ± 7
3 5 ± 1 2
6 2 ± 9
57 ± 5
67 ± 11
• Latency (mean ± standard deviation) o) the total number ol tumors tested ь Latency (mean ± standard deviation) of the tunors with integration in the Pim 1 locus 'NBI Newborn elected * G Τ Gemvkne transmtled
' M T ЬМк transmrtled(Mebelet a l , 1980)
37
1983), and the preferential integration site described tor Moloney MuLV in rats (Tsichlis et al, 1983)
Relation to Disease Pattern In mice infected as newborns with Moloney MuLV, the majonty of the lymphomas develop wilhm 30 weeks after inoculation About 50% of these early Τ cell lymphomas wrthm different mouse strains contain a prowrus in Ihe Pirn-1 гедюп In contrast, BALB/Mo and AKR mice, which spontaneously develop a lymphatic leukemia after 35-40 weeks (see Table 2), show a low incidence of integration in the Pim-1 region Since the average number of proviral integrations in lymphoma DNAs from BALB/Mo mice (in cidence 6%) is very similar to the numbers seen in mice infected as newborn with MoMuLV or AKR-MCF247 (incidence 50%, see Table 2), the difference in incidence of integrations in the Pim-1 region between these groups cannot be ascribed to more integrations per cell Rather, the availability of more or different target cells at the time the MCF viruses are generated in newborn infected animals increases the chance of MuLV integration in transformation-sensitive regions ol chromosomal DNA
Not all early Τ cell lymphomas show integration in Ihe Pim-1 region Therefore, it activation of the Pirn 1 region is involved in the tumongemc process, there are other routes, as well, to induce lymphomagenesis In this respect, it is interesting to note that m a significant portion of the early T-cell lymphomas, MuLV integration is observed near c-myc (Selten et a l , unpublished)
Integration Is Associated with Transcriptional Activation of a Region within the Pim-1 Domain We have analyzed lymphomas with integrations in the Pim-1 domain, and lymphomas without integrations in the Pim-1 гедюп, for the presence of polyA+ RNAs homologous to Pim-1 sequences A clear difference was observed be tween these two groups of tumors Lymphomas with an integration in the Pim-1 region contained polyA* RNA homologous to part of Ihe Pirn 1 region, whereas lympho mas without integrations in this region contained only minor amounts of Pim-1 mRNA The diflerence in the level of expression between the two types of lymphomas is esti mated to be 10 to 50 fold The гедюп that becomes activated is located between the integration regions, around positions —6 and - 1 to +10 on the physical map (see Figure 4) Although we cannot formally exclude the possibility that integration occurred in a region thai was already transcnptionally active, it is more likely thai Ihe transcriptional activity is the result of the integration of a provirus in this гедюп Experiments are in progress to determine the exact nature of this activated transcript
It is tempting to speculate about Ihe remarkable asso ciation of MuLV integrations in the Pirn 1 region with early T-cell lymphomas The observations of others and our own results might suggest the following model Tumongenesis can be induced by the activalion ol many differenl genes genes thai can initiate the Iransformation process, and
genes that actually transform This has been elegantly shown in ALV-mduced lymphomagenesis. where the activation of с myc seems a crucial step (Hayward el al. 1981, Payne et al 1982) in the process Nevertheless the trans forming activity is found in a diflereni gene (Goubm el al, 1983)) The same observations were made in cell lines derived from human hematopoietic tumors (Murray et al, 1983) One could speculate that in early MuLV induced Τ cell lymphomas a potentially transforming gene is activated directly through msertional mutagenesis by a proviral ge nome Most likely, a restricted number ol cellular onco genes can induce lymphomas upon activation, e g , by msertional mutagenesis Al least 'Λ of Ihe early Τ cell lymphomas show proviral mtegralions in Ihe Pim-1 region or near с myc In the lymphomas negalive for Pirn 1 and myc expression, other cellular genes may become activated through a similar mechanism The observed relationship between Ihe mode of infection and frequency of mlegrations in the Pirn 1 region could be explained by assuming that the Pim 1 domain is far from a preferential integration site, only a large number ol integration events may be necessary in order to achieve insertion of a provirus in this region The generation ol MCF viruses early in life might be essential in this respect This model makes a number of predictions that can be tested experimentally The Pim 1 region should harbor an oncogene itself, or be able to elicit transformation through a limited number of steps Experiments are in progress to verify Ihe validity of this model
Е ж р е п т е л Ы Procedures
Mice, Ced Lines, and Vhuses
The origin ol mice cell lines and isolation procedures of Moloney MuLV
clone 1A and ol me ПиаКгорю МСП130 and MCF1233 viruses have been
described previously (van der Putten et al 1979 Quint et al 19 1 Zijlstra
eia] 1983) AKR MCF247 was obtained trom Dr F Jensen
Source of Lymphomas and Preparation o l High Molecular Weight
DNA
Spontaneously developing lymphomas from BALB/Mo and AKR/Fu m c e
were obtained as described previously (van der Putten et al 1979 Qumt
el al 1981) The isolation o( lymphomas induced m C57BL BALB/c and
AKR/Fu mice by Ihe duaJIropc MCF 1233 MCF 1130 AKRMCF247 and
IhemilktransmittedB tropic MuLV has been described (Meliet el al 1980
Zijisfra et af 1983) The injection o l newborn BALB/c and C57BL with
Moloney MuLV done 1A and the isolation ol the induced lymphomas was
performed as deserted by Jaenisch et al (1975) High molecular weigh!
DNA trom lymphoma and nonlymphoma (issues was prepared as described
(van der Putten el a l . 1979)
Molecular Cloning
Virus cefl functions were cloned η charon phages according to established
procedures DNA fragments hybridizing with MuLV specific probes were
isolated trom agarose gels hgated mio the appropriate λ vector and in
vitro packaged Recombinant phage plaques were hybridized with cDNA
from MoMuLV or AKV made by the endogenous polymerase reaction
(Taylor el al 1976) Mini phage isolates from positively reading plaques
were analyzed by restnclion mapping Phage isolates that contained the
characteristic MuLV or MCF tragments were plaque punfied and the inserts
translerred mio р Я322
38
MuLV-Induced Τ Cel Lymphomagenesis
Rettotcbon Enzyme А л а і у н ·
DNA samples (10 ^g) were digested m a volume ol 50 μΙ under cornibons
recommended by ihe supptere (Amersham BRL Воеі юдег) with 100 ^g/ ml ol bovine serum albumm m addrbon Gel eleclrophoresis transfer to retroceWose fitere and hybndtzahon with ^Ф labeled probes were earned oui as deserted previously (Qunt et aJ 1981) Fragments of phage lambda generated by restriction endonuclease Hind M were used as motecüa; weight markers
Preparation oí RNA For the preparation ot total cytoplasmic RNA from tymphomas frozen tissues wore homogenized m 50 mM Ins HCl buHer (pH 7 4) containing 25 mM NaCl 5 mM magnesium chlonde 0 25 M sucrose 3 mM Gtutall^one and 10 U/ml RNasm Alter centnfugabon at 9000 x g lor 10 mm lolaJ RNA was isolated from the supemalanl as described (Selten et al 19 2) Poly A* RNA was selected by one cycle ol oligo(dT) ceBulose chromatography and apphed lo nrtrocellutose paper as desenbed by Muller et al (1982)
Prepanbon of Specific Probe· The hybndizalion probe specilic lor the U 3 region ol the Moloney MuLV LTR (MoUXTR) was prepared usmg a subdoned fragment of 180 nucleotides from the CSCctone it spans a г дюп from ал Hpa H site 10 nucleotides upstream from the 5 LTR η the cellular DNA to the tirsi Hpa D site in the
U3 region at positon 168 This fragment was inserted mto M13mp9
(Messing and Vieira 1982) The probe was prepared by the pnmer
extens*on reaction with one dideoxynucleobde induced in the reaction
(Sanger et al 1977) Filters hybridized with the MoUXTR probe were
washed with 0 1 χ SSC 0 1 % SOS at 60"С lor 20 mm under these conditions exctusn/ety Moloney LTR sequences were recognized The ecotropc specific hybnehzahon probes corresponding to the middle and С lermmaJ region of pISE (AkMCplSE) and for the U3 гедюп of the AKV LTR (AkU3LTR) were subdoned from Ihe recombmant phage AKR 623 (courtesy of О Lowy) (Qunt et al 1984;
Probes lor cettular DNA flanking the integrated provmjs in the C5C clone (Figure 1c) were as desenbed m the lexi In most instances subdoned fragments were excised from the plasmd vector the insert separated from the vector DNA by agarose gel electrophoresis and the DNA was recovered by binding to glass powder according to the method ol Vogelstem and Gillespie (1979) Hybridization ol the nitrocellulose Mers with the transferred DNA or RNA was pertormed as desenbed earlier (Oumt et al 1981 van der Putten et al 1981) using 1-2 χ 108 cpm of ^ Р ruck translated probe ю the hybndizalion mixture Routinely filters were washed m 0 1 x SSC and 0 1 % SOS at eS'C tor 20 mm
Acknowtodgmenti
We wish to thank Dr H Bloemendal π whose laboratory this work was
performed We appreciate the assistance ol Mat Schuurmans We are
gratefJ to the coworkers of the central animal lacrtity and to A van de
Kemp We also thank Drs Bishop Chen Cole Lowy Nusse Sherr
Stehehn van de Ven Verme and Wigier lor provichng cloned oncogene
probes Th« work was supported by grants from the Когипдю Wilhetmna Fonds (G S and Μ Ζ ) and by the Nethertands Organization for the
Advancement of Pure Research (ZWO) through the Foundation for Funda
mental Medicad Research (FUNGO)
The costs of publication of (his article were defrayed л part by the payment of page charges This arbde must therefore be hereby marked
aOverttsement m accordance with 18 U S C Section 1734 solely to indicate this fact
Received December 23 19 Э revised February 13 19Θ4
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Ι M and Bems A (1981) M MU.V nduced teukemogenesis integration
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40
CHAPTER 3
Involvement of c-myc in MuLV-induced Τ cell lymphomas in mice: frequency and mechanisms of activation
The UMBO Journal vol.3 no.l3pp.321J-3222. 1984
ТЬг ИЧВО Journal vol I n o 13 pp 3215-3222 1984
Involvement of c-myc in M uLV-induced Τ cell lymphomas in mice: frequency and mechanisms of activation
Gerard Selten, Η. Theo Cuvpers, Maarten Zijlsira1, Cees Melief1 and Anton Berns
DeparliTiLnl ol Bioi-hcmistry University οΓ Nijmegen Geert Grootcplcin N21 б ^ Г 7 Nijmigui and Central I aboralory of the Netherlands ReO С ross Uluod Translusion Servite PO Bo\ 9406 1006 ЛК Amsterdam The Nitlisrlands
С ommunitati by H Bloemendal
In — 45σ?ο с the murine leukemia virus (Mul V) induced early developm Τ cell lymphomas in mice, intégration of pro-viruses occui near c-myc. From the 33 lymphomas with pro-viral intégrât ns in the c-myc domain, 29 insertions were localized upsli am of the first exon in a region spanning < 2 kbp, and four ..itegralions occurred within the first exon. In 90% of the lymphomas the transcriptional onenlation of the proviruses was opposite to the transcriptional direction of C-//IVC. In 20% of the early Τ cell lymphomas, proviral integrations were delected both near c-mvc and lhe/7/m-l gene. They comprise both lymphomas in which integration near c-mvc and pim-l occurred m separate tumor cell populations, as well as tumors in which proviral integration near c-myc and pun-1 occurred m the same cell clone. Provirai integration m the c-mvc domain is associated with increased myc mRINA levels (up to 30-fold). The size and nature of the Q-invL mR!S Λ precursors and processed transenpts depend on the position and orientation of the integrated proviruses. Key words с mvc/insertional mutagcncsis/munnc leukemia virus/p»?;-1 /T cell l>mphomas
Introduction
c-Myc has been reported to be involved in a vanei> of malignant neoplasms Translocation, amplification, viral transduction, as well as insertional mutagenesis by integration of retroviruses can cause derangement of normal mvc expression The translocation of c-mvc to the immunoglobulin domain in Burkm lymphomas (Taub el al, 1982, Dalla-Favera et al, 1983, Enkson et al, 1983, Leder et al, 1983, Croce et al, 1983), and murine plasmacvtomas (Crews et al, 1982, Shen-Ong et al, 1982, Stanton et al, 1983, Mushinski et al, 1983, Leder et al, 1983, Adams et al, 1983) is well documented Amplification of ¡.-mye gene sequences has been reported (Collins and Groudine, 1982, Dalla-Favera et al, 1982), and like the frequently observed trisomy of chromosome 15 in mouse Τ cell lymphomas, has been implicated in enhanced myc expression (Klein, 1981) The transduction of c-mvc by feline leukemia occurs in Τ cell lymphomas in cats (Neil et al, 1984, Mullins et al, 1984, Levy et al, 1984) Modification of the c-myc locus bv proviral insertion has been described in avian leukemia virus (ALV)-induced lym phomas (Hayward et al, 1981, Neel et al, 1981, Payne et al, 1982, Westaway et al, 1984), and in MuLV induced Τ cell lymphomas in mice (Corcoran et al, 1984), rats (Steffen, 1984), and cats (Neil et al, 1984) A similar mechanism underlies the activation of c-erb in ALV-induced erythroblas tosis (Fung et al, 1983), the activation of the mt-l (Nusse and
Varmus, 1982, Nusse et al, 1984) and inl-2 gene (Peters et al, 1983, Dickson et al, 1984) in mouse mammary tumor virus (MMTV) induced mammacarcinomas in mice, and the activation of the pirn-1 gene in MuLV-induced Τ cell lymphomas in mice (Cuypers et al, 1984) Other common integration regions have been reported in MuLV-induced lymphomas in rats (Tsichhs et al, 1983, Lemay and Jolicoeur, 1984) However, the mechanisms of gene activation by insertional mutagenesis of proviruses appear to differ in ALV-induced lymphomas, c-myc is transcribed predominantly from the promoter of the upstream integrated, frequently deleted, ALV provirus (Payne et al, 1982, Hayward et al,
1981, Neel et al, 1981, Westaway et al, 1984), while m MuLV-induced lymphomas in mice (Corcoran et al, 1984) enhancement of transcription from cellular promoters by mostly intact proviruses seems the predominant mechanism of activation Also the activation of int-\ and int 2 in MMTV induced mammacarcinomas and the activation of pim-i in mouse Τ cell lymphomas seems to be mediated predominantly through activation of cellular promoters (Nusse et al, 1984, Dickson et al, 1984, Cuypers et al, 1984)
Here we present evidence showing that in MuLV-induced early developing Τ cell lymphomas in mice, c-myc activation is mediated predominantly through proviruses integrated upstream of с myc, which have a transcriptional orientation opposite to c-mvc The frequency and mechanisms of activation of с mvc is compared with the proviral activation of the pim 1 gene in the same group of lymphomas
Results
Alterations in c-mvc locus bv proviral insertion DNA from i30 lymphomas induced by MuLVs in different mouse strains was analwed for proviral insertions in the c-myc domain Lymphoma DNA was digested with ZfcoRI, £coRV, Kpn\ and in some instances with Hindlll, and analyzed by Southern blots (Southern, 1975) Figure la shows a map of the c-mvc region with the positions of the probe and relevant restriction endonuclease sites Analysis of Kpnl and/or FcoRl digestions of 130 lymphoma DNAs revealed novel bands in 33 cases (Table 1) The sizes of the additional bands in these 33 lymphomas depended on the position and nature of the integrated provirus Both ecotropic as well as mink cell focus forming viruses (MCF) were found to be integrated near c-myc MCF viruses, which are generated in vivo by recombination between ecotropic MuLV and endogenous xenotropic like sequences (Hartley et al, 1977, van der Putten et al, 1981, Quint et al, 1984) contain an £coRI cleavage site at position 6 9 on the genomic map (Quint et al, 1982, van der Putten et al, 1981), whereas ecotropic viruses lack this site (Verma and Mckenneth, 1978, Lowy et al, 1980) Normally sized proviruses ( - 8 8 kbp) lacking the EcoRl site were assumed to be ecotropic Approximately 50% of the proviruses integrated in the c-myc showed an MCF like structure, whereas 80% of the proviruses in the
43
G Selten et at
Table I Characieristics of proviral integrations in the с туе locus in independent lymphomas
Tumor *
4 5 7 8
9 13 17
26 27 28 31 32 33 34 36 37 18 39 40 41 42
43 44 44 46 47 4« 49 SO M
S2 53 S4
D N A Tragnient size hybridizing bands
& o R I £ r o R \
30 30 30 21 21 21 19 30 16 21 21 nt 30 21 30 10 21 27 21 27 30 10 2S 21 17 10 10 21 21 2j
20 nl nt
3 7 ni 3 2 3 5 3 1 nt nt 3 6 3 2 1 0 2 4 nt 1 3 3 7 2 1 3 0 1 6 2 9 1 8 2 1 1 6 4 2 1 1 1 0 nt nt nt nt nt m nt nt nt
(kbp) of addii
Kpnl
8 0 6 2 7 8 8 2 7 7 8 0 8 0 8 0 7 0 7 5 7 0 8 1 7 7 8 2 5 8 7 2 8 2 7 6 8 1 6 0 7 9 8 8 •м 7 4 7 1 7 7 7 6 9 2 8 4 8 3 •> t
8"' 8 5
tonally
Hmdin
nt nl nt nl nt nl nt absent absent nt absent nt nt nl
7 5 nt nl nl nl 115
nl nl nt nt nt nt nt nt nt nt nl nl nl
Provn
Fco Eco Fco M C F M C F M C F M C F beo M C P M C F MC Г nt Fco M C F Fco beo
M C l · U o Fco fcco Fco Ceo beo М П M C I Ceo beo МСЬ M C F M C F M C F nt nl
DNA fragment sizes of normal allele after digestion with £ ÍOR1 £ ioR\ kpnl and Hmdlll arc 21 22 * 21 11 and 4 4 kbp respcvlivtls Abbrevia lions Lto txolropit MC Ь mink Lcll locus inducing virus nt not teslcu
pim-\ domain (Cuypers et al, 1984) were of MCF origin (Table 1)
fcoRV digestions revealed additional bands in the same tumors which were positive after £coRI analysis (Table I) Since £coRV has a recognition sue inside the ime probe (Figure U), two fragments (22 and 23 kbp) were observed in normal ussues An icoRV recognition site is also present at 0 14 kbp from the boundarv of the 5 long terminal repeal (1 TR) (van Beveren et al, 1980) The site of proviral integra lion can be estimated quite accurately from the si7cs of the additionally hvbndmng fragments Digestions with kpnl confirmed the results and allowed the determination of the orientation of the proviruses after hvbridization with UTLTR probes (Cuypers et al, 1984) Since a Kpnl site is onlv present in the U's portion of the LTR (van Beveren et al, 1980), hybridization of fragmems to both the UILTR and /uve probe is only seen if the transcriptional orientation of the pro-virus is directed away from the region corresponding with the myc probe Alternativeh, novel míe reactive £coRl fragments should hvbridize to the U3LTR probe in dependently of the orientation of the integrated provirus ^n example of the hybridization pattern with the mvc and MoU3LTR probe on Kpnl digests of 14 lymphoma DNAs is
presented in Figure 1 b and c, respectively Tumors 39, 42, 44, 26 and 33 show additional bands of reduced size which are recognized both by the c-myc as well as the U3LTR probe A survey of the analyses performed with different mouse strains and various MuLV is presented in Table I Proviral integration occurred in all cases upstream of the c-myc coding sequence in a region of - 2 kbp More precisely, 29 tumors showed proviral integration in a region - 1 5 kbp upstream from exon 1, whereas in four lymphomas the provirus integrated in the non-coding first exon of с туе (Figure 2) In 24 lymphomas the transcriptional orientation of the pro-viruses was opposite to the transcriptional direction of c-myc, in three lymphomas the same orientation was observed, and in six cases the proviral orientation was not determined In the lymphomas with integration in exon 1, proviruses were found both upstream (lymphoma 31) and downstream (lymphomas 5, 36 and 41) of promoter 2, in different transenp-tional orientations (Figures 2 and 4, Table 1)
Frequency ofproviral integration near c-myc depends on the latency of lymphoma development Lymphomas were divided into three groups on the basis of the latency of tumor development (Table II) The first group, which gave rise to lymphomas within 6 months, comprised BAI B/c, C57BL and AKR mice, inoculated as newborn with Moloney Mul V and AKR MCF247, respectively This group showed an incidence of proviral integration in the с туе domain in -40% of the lymphomas Slightly higher percentages of proviral integration near the pirn-1 gene were observed (Table II) In 1Ί oui of the 66 lymphomas in this group, proviral integration was detected both in the c-myc as well as the pim-\ domain (see below) In this group a considerable portion of the tumors were oligoclonal as judged by the low hybridizing intensities of the modified allele(s), and the occurrence of different integrations near/?im-l or c-myc within the same tumor (e g , Figure 1, tumor 44)
The second group consists of lymphomas of BALB/c and C57BI mice, infected as newborns with MCF1233 (Zijlstra et al, 198J), and spontaneously arising lymphomas in AKR (Hartley et al, 1977) and BALB/Mo mice (Jaemsch, 1976) The incidence of proviral integration near pirn-1 or c-myc in this group, which developed lymphomas after 6 - 12 months, ranged from 0 to 20% No proviral integration in both domains were detected within lymphoma tissues of the same animal Most of these tumors appeared monoclonal
Group 3 represents l>mphomas in BALB/c and C57BL mice, infected as newborns with MCF1I30 (Zijlstra et al, 1983) and tumors in C57BL mice which transmit the B-tropic CS7BL MuLV through the milk (Mehef et al, 1980) No proviral integration near pim-l or с mvc were observed here All Kmphomas in group 1 and 2 with proviral insertions near с /me and/or pim-l were identified as genuine Τ cell lymphomas on the basis of their cell surface markers (7ijlstra et al, unpublished results) Some of the hmphomas in group 2, and a considerable number of the tumors in group 3 were of В or non T/non-B cell origin
Proxiral integration near с mvc is associated nith enhanced nnc mR\A le\els V/ic mRNA levels were estimated by dot blot hybridization of poly (A) ' RNA, isolated from hmphomas and normal tissues Figure 3 shows the hjbridization of pol>(A)" RNA of 12 l>mphomas with the myc and actm probe In lymphoma tissues, m which a provirus was integrated near c-myc (Figure 3, marked by an asterisk) /mc RNA levels were in
44
ІПМІІМ-ІШ-МІ of c-mvc in MuLV-induced Τ cell hmphomas in mice
2S - J — ^
•10 5 0 ι ι ι ι I l ι ι 1 1 -
J - ^ — L
•10 -2$ Kbp _ j 1 1 1, 1
Η X R„ Η К X R, Rv
myc probe kpn digests MoUJLTR probe
2 3
20
Fig. I. Charactenzaiion of Kpn\ digests of lymphoma DNAs uuh c-mvc and M0U3LTR probes (a) Fh>sicai map of the c-m^c region with the position of the probe {Xba\ Hindlll fragment). DNA samples (10 μg) from 14 different lymphomas were digested with Kpni, separated on 0 6% agarose gels and transferred to nitrocellulose filters. Filters were hybridized with !!P-labeled myc probe, washed (0.1 χ SSC. 0 m SDS. fO C) and exposed to X ray film (panel b). After treatment with alkali, the filter was hybridized with a i z P labeled MoU3LTR probe, washed and re-exposed (panel c) Bands marked with an arrow in (c). correspond to the rearranged DNA fragments shown in (b) Numbers correlate to listing in Table I Abbreviations: Rl. £coRI. RV £coRV; K. Kpni: H. Hmdin. X. АТмІ
creased 5- to 30-fold as compared with myc mRNA levels of nonral adult tissues. In tumors with proviral integrations near c-myc and pirn-1, increased mRNA levels of both genes were detected (data not shown). However, in lymphoma 29 and 30 increased myc mRNA levels were found without concomittant provirus insertion within the vicinity of c-myc. Either proviral integration in these tumors occurred outside the region of 45 kbp which was examined for proviral integrations or other mechanisms for c-myc activation are operational in these lymphomas. In addition, in lymphomas 15 and 71, which harbor no proviruses near c-myc, 10- to 50-fold less myc mRNA was detected as seen in normal adult thymus and/or spleen. Over 60% of the early developing Τ cell lymphomas (29 randomly chosen tumors were tested for RNA expression) showed significantly enhanced myc expression, whereas increased myc and/or/wn-l expression was detected in >80% of the lymphomas. Obviously, the expression of c-myc and/or jOT/n-l might be increased in only a subpopulation of the tumor cells, since many of the early Τ cell lymphomas are oligoclonal.
Size and nature of myc mRNA transcripts depends on position and orientation of the inserted provirus
Northern blot analysis of poly(A) ' RNA from lymphomas with proviral integrations upstream of promoter 1 of c-/ryc (Figure 4. lanes A and B) revealed two myc mRNA transcripts of -2.4 and 2.3 kb in size. These two Anyc mRNA transcripts are probably initiated from promoters 1 and 2, respectively (Bernard et al., 1983: ar-Rushdi et al., 1983; Leder et al., 1983; Corcoran et al., 1984) The use of promoters 1 and 2 for myc mRNA synthesis is also reflected in the sizes of the myc mRNA precursors. Two bands can be detected in lanes A and В (Figure 4a) at 5.0 kb (reflecting precursors containing both introns, initiated from promoter 1 and 2, respectively) and at 3.5 kb (precursor without intron 1) Hybridization of the same blot with a myc intron I probe (Xbal-Pvull fragment near the border of exon 2) shows that myc mRNA precursors around 5.0 kb contain intron 1 se-
45
G. Sdten et al
quences, whereas precursors around 3.5 kbp lack intron 1 sequences (Figure 4b). The RNA bands with estimated lengths of ~4 kb most likely reflect transcripts initiated from promoters in intron 1 (Bernard et al., 1983). The patterns of both precursors as well as processed myc mRNAs shown in lanes A and В of Figure 4a, suggest that both promoter 1 and 2 are used in lymphomas with proviral integrations upstream from exon 1.
Sequence analysis of mouse c-myc (Bernard e! al., 1983) revealed a Hindlll recognition site directly downstream of promoter 2. In lymphoma 31 (Figure 4a, lane C) integration of a provirus occurred just upstream of the HindlU site between promoter 1 and 2 (Table 1). A single myc mRNA species of 2.3 kb was detected in this tumor. Accordingly, only the
- 4
L
-1 0 Kbp
X R v
26 34 40
Í3 49 50 53 51 54
27 'S? "5T 5 39 46 36 45 52 41
j^ 7 8 9 13 17 ¿8 33 38 42 44 47
48 32
Fig. 2. Position and orientation of integrated proviruses in the c-mvc region of 33 lymphomas Localization of integrated proviruses are shown by arrows or blocks. 1 ymphoma numbers correspond to the listing in Table I. Horizontal arrows above the tumor numbers denote the orientation of the provirus (5 ' - 3 ' ). The orientation for lymphomas 32. 47. 48. 49. 53 and 54 was not determined. For abbreviations of restriction en-donucleases see legend to Figure I.
PROBE
д д poly A'RNA —
T U M O R No
71
* 39
4 0 *
2 6 *
* 27 29
30
* 31
lb
16
76
75
' Ψ
•
MYC
o ; ooj • •
φ |
Щ
•
φ -
m
OOOÖ •
ACTIN
' •
Ψ f •
004 ΟΟΟβ • •
•
*
•
• • •
•
»
A D U L T '
t i l y
spi
hu
kid
* proviral integration
Fifi. 3. с-Мус mRNA levels In normal and lymphomas hssucv Several dilutions of poly(A) " RNA. ranging irom I ^g lo 8 ng. Irom 12 lymphomas and Irom normal aduli BAI lì/c tissues were spoiled on nitrotcllulosc iilters jnd hybndi/cd (o ^P-labcled c-ww probe or attin probe, which was used as a control for the guanlily and integrity ol the RNA samples 1 ym-phomas marked with an asterisk contained a provirus m Ihc с mvt region (see also Tabic I) Abbreviations: Thy. thymus; Spi. spleen; I iv, liver; Kid. kidney.
Table II. Frequency of integration of MuLVs in the c-myc and/or pirn 1 region. Correlation with latency of lymphoma developmeni
Group Virus strain Mode
inf.a
Mouse strain Latency
weeksh
Number
tested
Number and percentage wiili proviral integration in:
c-myc pim-1
MCH233 MCF1233 AKV MoMul V
M i l M i l
α τ t r i
C57BL BALB/c
AKR BALB/Mo
3 8 ± l l 37 ±8 37±8 35 ±12
11 7
14 16
0 0 2(14%) 3(19%)
2(18||/о) I (14%) I ( 7%) I ( 6%)
MCFII30 NBI C57BL 6 2 ± 9 4 MCFII30 NBI BALB/c 57 ±5 5 G57-MLV M T . C57BL 6 7 ± l l 7
Total numbers of lymphomas
c-myc *рііп-ì 'total
MoMuLV NBI BALB/c 13 ±3 54 27(50%) 25(4«%) 14(26%) 38(7(1%) MoMuLV NBI C57BL 24 ± 6 7 1(14%) 4(57%) 1(14%) 4(57%) MCF247 NBI AKR I8±5 5 0 2(40%) 0 2(40%)
2 (18%) 1 (14%) 3 (21%) 4 (25%)
aNBI. newborn-infected; G.T., germ-line transmitted; MT, , milk transmuted (Mehef et al., bLatency (mean ± standard deviation) of the total number of lymphomas tested.
1980)
46
Involvement of c-mvc in Mu ΓΛ -induced Τ cell lymphomas in mice
S MYC probe
kb А В С D 6.7-
4.a-
b INTRON probe
23-
2Ω-
C MYC probe
A B C D k b k b E F G H -67
0 Kbp
I? R H F?. X R u
^ i¡?
îî îît A B G С D < LANE
40 26 2 7 3 I 4 Î < TUMOR No
. _ · ХОП2
INTRON probe
- 2 0
Flg. 4. Northern blot analysis of lymphoma RNAs. Glyoxal-treated poly(A)* RNA was separated on 1% agarose gels, transferred to nitrocellulose filters and hybridized to c-myc probes. Lanes A,B,C,D and G contain 5 μί poly(A)* RNA from lymphomas 40,26,31,41 and 27, respectively. Lane E, 10 ßg poly(A)* RNA from tumor 15; lane F, 10 iig poly(A)* RNA from newborn BALB/c spleen; lane H, 10 „g poly(A)+ from tumor 29. (a) and (c), hybridization to the Xbal/HmdlU probe, (b) Hybndization of blot (a) to Xbal/PvuU intron probe Drawing shows position of c-myc promoters 1 and 2 , and intron probe. The position and orientation of integrated proviruses are indicated with vertical and horizontal (5' - 3 ' ) arrows, respectively Abbreviations: H, НтЛП; Pv, Pvull; X, Xbal, RV, EcoRV.
smaller sized precursors were detected. Since the transcriptional orientation of the provirus was opposite to that of c-myc, transcription probably initiated from promoter 2 in this lymphoma.
Analysis of RNA from lymphoma 41 (Figure 4a, lane D) revealed a myc transcript of -2.2 kb and concomitant smaller precursors. A provirus, with the same transcriptional orientation as c-myc, is integrated to the right of the Hindlll site, as was evident from Southern blot analysis after cleavage with Hindlll (Table I). Therefore, myc promoter 1 and 2 are no longer available for the initiation of myc mRNAs in this tumor. Presumably, c-myc transcription in lymphoma 41 is initiated from the viral promoter in the 3 ' LTR as seems the case for part of the transcripts in lymphoma 27 (Figure 4c, lane G). The provirus in lymphoma 27 is integrated -500 bp upstream from the first c-myc promoter in the same transcriptional orientation as the c-myc gene (Figures 2 and 4). The additional mRNA precursor of 5.7 kb, detected in this tumor (Figure 4c, lane G) probably represents a c-myc precursor initiated from the proviral promoter in the 3' LTR, whereas the additional hybridizing band around 3 kb most likely represents the processed mRNA. Remarkably, also the normal c-myc promoters appear to be used, as can be deduced from the presence of normal sized myc mRNA precursors.
Figure 4c also shows c-myc mRNA and precursors from normal tissue (lane F), and lymphomas without proviral integration near c-myc. The latter comprise both lymphomas with an enhanced c-myc mRNA level (lane H, tumor 29), as
well as lymphomas with a strongly reduced c-myc mRNA level (lane E, tumor 15).
Evidence for proviral activation of pim-I and c-myc within the same tumor cell population In 23% of the early developing Τ cell lymphomas, we observed proviral integration both nearpim-1 and c-myc in the same lymphoma DNA. Cells from three different primary lymphomas (8, 9 and 17) with proviral integrations near both genes, were injected i.p. into syngeneic recipient mice. Lymphomas developed after 2 - 4 weeks. Southern blots containing DNAs from the primary lymphomas and transplanted tumors were hybridized with thep/m-l and c-mjvc probes. An example of the hybridization patterns seen with DNAs of the primary and transplanted lymphomas is shown in Figure 5. The original tumors 8, 9 and 17 showed proviral integration in a fraction of the tumor cells both in thep/m-l as well as in the c-myc domain (lanes 1, 3 and 5). However, upon transplantation of tumor 9 into five different receipients, lymphomas developed which showed the same additional bands as the parental lymphoma with hybridizing intensities which were identical to the intensities of the normal alleles of pim-l and c-myc (lanes 2, only one receipient is shown).
In the other transplanted lymphomas (8 and 17) only the rearranged myc (tumor 8, lanes 4) or pim-\ (tumor 17, lanes 6) was inherited from the original tumor, indicating that the primary tumor was oligoclonal and clones were selected upon transplantation. The transplants of tumor 17 still seemed
47
G. Sehtn я al.
LYMPHOMA 9 8 17
1 2 3 4 5 6
k b p -
23.7- L _ -
a piM
9 5 -
MYC 9 5 -
6 7 -
Fig. 5. Segregation of proviral integrations in transplanted lymphomas. EcoRV (panel a, lanes 1 and 2). £coRl (panel a, lanes 3,4,5 and 6) and Kpn\ (panel b) digests of 10 μg lymphoma DNA from mouse 9,8 and 17 (lanes 1,3 and 5 respectively), and the corresponding transplanted tumors (lanes 2,4 and 6, respectively), (a) Hybridization to pim-i probe (b) Hybridization to c-myc probe. The normal pim-i allele corresponds to the 22 kbp (EcoRV) or the 12 kbp (£coRl) fragment (panel a); the normal c-myc allele corresponds to the 11-kbp fragment (panel b).
oligoclonal, although we cannot exclude that in this case, contaminating normal tissue reduced the relative hybridizing intensity of the modified allele. The oligoclonality of many of the primary tumors was also confirmed by comparing the 'multiple proviral integration' pattern between the primary and transplated lymphomas: in several instances the latter harbored a subset of the proviral integration sites, present in the original lymphoma (data not shown).
Discussion
Activation of cellular (onco)genes by insertional mutagenesis is observed in ALV, MuLV and MMTV-induced tumors (Hayward et al., 1981; Neel et al., 1981; Payne et al., 1982; Nusse et al., 1984; Dickson et al., 1984; Fung el al., 1983; Corcoran et al., 1984; Cuypers el al., 1984). Integrated pro-viruses can activate flanking cellular genes either by transcription from the viral promoter (Neel el al., 1981; Payne et al., 1982), by virtue of the enhancer activity of proviral sequences on adjacent cellular promoters (Nusse et al., 1984; Corcoran et al., 1984; Cuypers et al., 1984; Dickson el al., 1984), or by disruption of cis-controlling elements, as suggested for c-myc activation upon translocation (Leder et al., 1983).
Obviously, the position and orientation of the integrated provirus with respect to the flanking gene determines to a large extent which activation mechanism can be operational.
Proviral activation of c-myc might require integration in a distinct region. Such a region specificity has also been observed for the breakpoint near c-myc in plasmacytomas. The need to disrupt a tightly controlling c;5-acting element from the structural gene has been suggested as an explanation for this observation (Leder el al., 1983). We found the proviruses integrated near c-myc were all localized upstream from the coding region. This still allows activation by any of the three mechanisms described above. The site and orientation of pro-viruses with respect to the flanking activated gene, appears to differ from system to system. In this study proviral integrations near c-myc were all found upstream of the coding region, predominantly in a transcriptional orientation opposite to c-myc, whereas the majority of the proviruses in the vicinity of pim-\ were located near or in the 3 ' portion of the gene in the same transcriptional orientation (Cuypers el al., 1984). In contrast promoter insertion appears the predominant mechanism of c-myc activation in ALV-induced bursal lymphomas (Neel el al., 1981; Payne et al., 1982). These differences in integration sites and orientations with respect to the activated (onco)genes can be caused by specific requirements for the activation of distinct genes or reflect characteristic features of the virus or the target cell, e.g., the proviral activation of c-myc might require the dislocation of upstream located regulatory elements (Leder el al., 1983; Siebenlist el al., 1984) or alternatively the observed integration region might just exhibit a structure which is preferred by the integration machinery of the virus.
Frequency of integration near c-myc in Τ cell lymphomas Proviral integration near c-myc is observed more frequently in early developing Τ cell lymphomas (28 out of 66) than in lymphomas which develop after a latency between 7 and 12 months (5 out of 48). Similar results were obtained for the pim-l gene (Table II). This difference in incidence could have been caused by the availability of more or different target cells for virus infection in the development of early Τ cell lymphomas, increasing the chance of proviral integration in transformation-sensitive regions of chromosomal DNA. This could explain the frequently observed oligoclonality of these early Τ cell lymphomas as compared with the predominantly monoclonal origin of the spontaneously developing tumors in BALB/Mo and AKR mice.
Remarkably, >80% of the proviruses near pim-i showed an MCF-like structure (Cuypers el al., 1984), whereas only 50% of the proviruses near c-myc belonged to this class, as judged by the presence or absence of an £coRl cleavage site at map position 6.9 of the proviral genome. MCF viruses show a different target cell specificity than their ecotropic parents (Hartley et al., 1977; Cloyd, 1983; O'Donnel el al., 1984). Interestingly, after infection of newborn mice with MCF viruses, proviral integration seemed to occur nearp/m-l rather than near c-myc (see Table II).
Nature and expression of c-myc mRNA Proviral integration near c-myc is associated with the presence of increased levels (up to 30-fold) of myc mRNA. Enhanced myc mRNA levels were also found in some lymphomas without proviral integration near c-myc (Figure 3). This could be the result of integration of proviruses outside the region we analysed, be caused by other structural changes which escaped detection, or point to alternative mechanisms formic activation (Kelly ei a/., 1983; Keathe/a/., 1984). Ina few lymphomas without proviral integration near c-myc, myc mRNA transcripts were hardly detectable (Figure 3, lym-
4 8
phoma 15 and 71). The original target cells giving rise to these lymphomas might have produced only minute amounts of myc mRNA or the level of myc expression was reduced during the progressive growth of the lymphomas. In any event, this observation shows the relative value of normal thymus and spleen, like any other organ, as reference tissues for myc expression. Identification of myc mRNA precursors on Northern blots clearly shows the involvement of myc promoters 1 and 2. When a provirus is inserted in the 'promoter insertion orientation' upstream from promoter 1 (Figure 4, tumor 27), transcription starts at three different sites: at myc promoter 1 and 2 and probably at the promoter in the U3LTR of the integrated provirus. In a tumor with a provirus integration between promoter 1 and 2 (lymphoma 31), transcription seemed exclusively to be initiated from promoter 2. In tumors with proviral integrations downstream of promoter 2 (lymphomas 5, 36, 41), transcription most likely starts from a proviral promoter or from promoters in intron 1 (Bernard et al., 1983). Oligoclonalily of virus-induced Τ cell lymphomas Analysis of transplanted lymphomas revealed that the primary lymphomas were often oligoclonal, confirming the conclusions drawn from comparison of the hybridization intensities of the rearranged and normal alleles on Southern blots. Injection of cell suspensions from the primary lymphomas 8 and 17, which showed proviral integrations both near myc and pirn-1, resulted in lymphomas in which none or only one of the rearranged myc and pim-\ alleles were retained, illustrating that the original lymphomas were composed of at least two (tumor 8) or three (tumor 17) different cell clones. Some transplanted lymphomas seemed to remain oligoclonal as revealed by the persistence of the reduced hybridizing intensity of the band corresponding with the rearranged allele in the transplants (Figure 5, lane 6). However, transplantation of lymphoma 9, which also showed proviral integration near c-myc and pirn-l, resulted in tumors which inherited in all instances the rearranged c-myc and pim-l allele. Although lymphoma 9 was probably oligoclonal, as judged from the intensities of the additional hybridizing bands, the transplanted tumors appeared monoclonal: the normal and altered allele showed equal hybridizing intensities in five independent transplants (Figure 5, lane 2), suggesting the presence (by proviral insertion) of both a modified c-myc and pim-l allele within the same cell clone. It is likely that proviral integration near c-myc or pirn-1 is detectable because of the selective advantage conferred by the activation of these genes. Therefore, proviral integration near both c-myc and pim-\ within the same tumor cell population suggests a transformation process in which c-myc and pim-l might act cooperatively. One could imagine that the first insertion resulted in the massive proliferation of the cell and provided a large target population for the second insertion, which conferred additional growth advantage. We are currently testing whether synergism between c-/wvc and pim-l can also be shown in 'in vitro' transformation assays.
Materials and methods
Mit?, vinises and lymphomas
Lymphomas were induced by injection of newborn BALB. e. C57BL or AKR mice with various Mul.V strains as described by Jacnisch el αι., (1975). The origin of Moloney MuLV clone 1A. MCF1233. MCF1130. AKR-MCF247. and the milk-transmitted B-tropic Mul.V were described previously (Cuypers et at.. 1984). Spontaneously developing lymphomas from UALB/Mo and AKR/Fu mice were obtained as reported (van der Putten et ai.. 1979; Quint ei ai.. 1981). Transplantation of lymphomas was performed by i.p. injection of
Invofrement of c<m.wr in MuLV-lnduced Τ cvfl lymphomas In mkx
- 5 χ 10· viable primary lymphoma cells imo syngeneic recipient mice, which
developed lymphomas in 2—4 weeks.
DNA isolation and restriction enzyme analysis
Preparation of high mol. wt. DNA was as described (van der Putten et al., 1979). DNA samples (10 μg) were digested under conditions recommended by the suppliers (Boehringer, Amersham, BRL). Gd electrophoresis, transfer to nitrocellulose filter and hybridization to ^P-labeled probes was carried out as described (Quint et al., 1981). As mol. wt. markers //mrflll-digcstcd phage λ DNA was used. Preparation of RS A
For RNA isolation, frozen tissues were homogenized with a polytron (2 min, full speed) at 0CC in 3 M LiCl, 6 M urea, and maintained overnight at 4 0C as described by Auffray and Rougeon (1980). After centrifugation at 16 000 g for 30 min, the supernatant was discarded and the pellets dissolved in 10 mM Tris-HCl, pH 7.4. 0.5^0 SDS. deproleini7ed by two successive phenol: chlorophormiisoamylalcohol (50:48:2) extractions, and precipitated with ethanol. Poly(A)+ RNA was selected by one cycle of oligoid'I^-chroma-tography, after heating the RNA samples at 650C for 5 min. Analysis of polyadenylated RNA
For dot-blot analysis, poly(A) ' RNA was dissolved in water and applied to nitrocellulose filters as described by Muller el al. (1982). Glyoxal treatment and Nonhern blot analysis of pol>lA) + RNA was carried out as described (Carmichael and McMaster, 1980; Thomas, 1980). As mol. wt. markers /y/mrtll-digested λ-DNA fragments were used. Denaturation was performed as described for RNA. Hybridizaiion probes
For the detection of myc sequences, the XbaVHindill fragment, covering ex-on2, intron 2 and the major part of exon 3. w-as used (Shen-Ong er α/., 1982). A probe for intron 1 of c-mvc was prepared by subcloning a Pvull/Xbal DNA fragment, located near the border of exon 2 (see Figure 4). The MoU3l.TR probe and the probe for pirn-1 (probe A) were described previously (Cuypers el al., 1984). The aclin cDNA clone was described by Dodemont ero/. (1982).
Acknowledgement
This work was performed in the laboratory' of Dr. H, Bloemendal, to whom we are particularly indebted. We appreciated the contributions of Ron de Ruyier and the co-workers of tlic Central Animal Laboratory. We wish to thank Drs. Cole and Quax for providing the c-m.vc (mouse) and actin clones. This work was supported by grants from the Koningin Wilhelmina Fonds (G.S. and M.Z.) and by the Netherlands Organization for the Advancement of Pure Research (ZWO) through the Foundation for Fundamenta) Medical Research (FUNGO).
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50
CHAPTER 4
Proviral activation of the putative oncogene Pim-1 in MuLV induced T-cell lymphomas
The EMBO Journal vol.4 no 7 pp. 1793-1798. 198S
The EMBO Journal vol 4 no 7 pp 1793- 1798, 1985
Proviral activation of the putative oncogene MuLV induced T-cell lymphomas
Gerard Selten, H. Theo Cu>pers and Anton Berns
Dcpanment of Bio-hcmistry University of Nijmegen Осел Grootcplem N21 652S E7 Nijmegen The Netherlands
Communicated hy H Bloemendal
Proviral integration near the ftm-1 gene is frequently observed in murine leukemia virus induced T-cell lymphomas in mice. Integration in the Pim-l domain is associated with the presence of enhanced levels of a Pim-l mRNA, which is normally expressed as a predominant 2.8 kb species at low levels in lymphoid tissues. The majority of integrations occurred in the 3' region of the Pim-l transcription unit. This resulted in transcripts ranging in size from 2.0 to 2.6 kb, which were terminated in the 5' proviral LTR. Dependent on the site of integration up to 1300 bases of Pim-l specific sequences were missing from the modified ftm-1 mRNA in these lymphomas. Ke\ words insertional mutagenesis/murine leukemia virus/ Pim-1/Т-сеІІ lymphomas
Introduction Murine leukemia viruses (MuLVs) arc replication-competent retroviruses which lack transforming genes but cause lymphomas after a long latent period (Rowe and Pincus, 1972, Pcderson el al . 1980, Nobis and Jaemsch, 1980. Steffen and Weinberg, 1978) Tumor induction by the slow transforming retroviruses most likely depends on activation of cellular genes by integrated proviruses Activation of the c-m\c proto-oncogene by proviral DNA insertion has been described in avian leukosis virus (ALV) induced B-cell lymphomas in birds (Hay ward e! al, 1981. Nccl et al, 1981. Payne el al , 1982, Westaway ei al , 1984), in MuLV induced T-cell lymphomas in mice (Corcoran el al, 1984, Selten ei al, 1984) and in feline leukemia virus (FcLV) induced lymphomas in cats (Neil el al , 1984) A similar mechanism underlies the activation of c-erb in ALV induced erythroblastosis (Fung et al, 1983), and the activation of the ml-1 and ш;-2 genes in mouse mammary tumor virus (MMTV) induced mamma-carcinomas in mice (Nusse and Varmus, 1982, Nusse et al , 1984, Peters el al . 1983, Dickson el al , 1984)
Integrated proviruses can activate flanking cellular genes cither by transcription from the viral promoter (Hay ward el al . 1981, Neel et al, 1981, Payne el al , 1982 Westaway el al , 1984, Cullen et al, 1984), by virtue of the enhancer activity of pro-viral sequences on adjacent cellular promoters (Corcoran et al. 1984, Nusse et al , 1984, Dickson el al , 1984, Cuypers et al , 1984, Selten el al 1984), or by disruption of rij-controlling elements (Leder el al , 1983) Furthermore, integration of proviruses within the transcription unit may modify the structure, and thereby the biological characteristics, of the encoded protein The activation of c-mu by ALV proviruses occurs predominantly by deleted proviruses inserted upstream from the m\c gene in the same transcriptional orientation (Neel et al , 1981 Payne el al 1982) In contrast, the activation of с /me in MuLV induced T-cell lymphomas is mediated predominantly
ft'ra-1 in
by proviruses integrated upstream of c-mv< in the opposite transcriptional orientation (Corcoran et al. 1984. Selten et al , 1984), whereas the activation of ml 1 and mr-2 in MMTV induced mammacarcinomas involves proviruses integrated either upstream or downstream of the Ш/-1 and ml 2 genes with a transcriptional orientation which is directed away from the ml genes (Nusse et al , 1984, Dickson et al, 1984) Recently wc have described the frequent integration of proviruses in the Pim-\ domain in MuLV induced T-cell lymphomas (Cuypers el al, 1984) Here we describe the orientation and site of integration of proviruses in additional lymphomas, the tissue specific expression pattern of the Pim-l gene, the mode of activation of Pirn-1 and the effect of the site of proviral integration on the size and nature of ftm-l mRNA transcripts
Results Іуосаіцапоп of the Pim-l transirtptwn unii The ftm-l gene is exoressed at low levels in normal thymus and spleen, and at elevated levels in lymphomas with proviral integration near ftm-l (see below) To locate the ftm-1 transcription unit and to determine the direction of transcription, polyA+ RNA derived from normal and lymphoma tissues was hybridized with several probes from the ftm-1 region Hybridization was found with probes A, B, D and b, but not with probe С (see Figure lg for location of probes) The use of single strand M13 probes from the ftm 1 region revealed that the direction of transcription was from left to right on the ftm-l map Furthermore, SI nuclease analyses, using subclones from the ftm 1 region, indicated that at least 80% of the ftm-1 mRNA sequence is encoded in the region between position —6 and +1 on the ftm-1 map The location and transcriptional direction of the ftm 1 transcription unit is visualized by the large horizontal arrow in Figure Ig The dotted lines indicate that the initiation- and termination-site(s) of the ftm-1 transcription unit arc still unsettled Position and orientation of proviruses m the ftm-У region Proviral integration near ftm-1 or с mu is observed with high frequency in MuLV induced T-cell lymphomas which develop after a latency of less than six months (Cuypers et al , 1984) Wc have extended our earlier study by analyzing additional fast developing lymphomas induced in BALB/c mice by infection of newborns with Moloney MuLV A total of 31 out of 66 mice which developed lymphomas within six months, contained a pro-virus integrated in the ftm-1 region, whereas only five out of 64 mice which developed disease after more than six months, had a pros irai insertion near ftm-1 The sites of proviral integration as well as the transcriptional orientation of the proviruses with respect to the Pim-l gene were determined by Southern blot analysis of lymphoma DNAs digested with £roRI, £coRV and P\u\\. using probes specific for ftm-l and the Moloney IßLFR In Figure 1 the proviral integration sites and the transcription orientations of proviruses with respect to the ftm-1 gene arc shown in detail for a number of lymphomas Southern blot analysis of EcoRV digested lymphoma DNA with specific ftm-l
53
G.í>elten. H.T.Cmpers and A.Bern·»
/ j отеле С digest R,
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Fiß. 1. Southern blot analyse of lymphoma DNA with specific Pirn I and proural probes 10 μ£ ol Kiiiphoiiia DNA. obtained trom spleen tsplì thvnuis (thy), peripheral lymphnodes (Ly) or normal mouse (BALB/c) tissue (N) were digested with restriction endonucleases bdRV (a.b.c.f). ¿KIRI (d) or Pmll (e). separated on 0 6% agarose gels, transferred to nitrocellulose filters and annealed to probe A (a.e.f). probe С (b.d). or to the Molones U.ÏLTR (MoU3LTR) probe Autoradiographs a,b and с were obtained from the same filter with the probes indicated abose the panels The location ot Pmi I specific probes A-Ε is shown in g A physical map of the Pim-\ region, as well as the position and orientation of the provinises in the Ριηι-\ region ot Iviiiphoinas analyzed in panels a - e . are presented in g The integration site of the prosirus in tumor 1 is defined as position 0 Small horizontal arrows abose tumor numbers indicate the direction of transcription of the integrated provirus (5 • > 3 ) Localization of Pun i transcription unit and direction ol transcription is indicated by the large horizontal arrow Dotted line reflects the fact that the beginning and end ot the Pun \ transcription unit is still uncertain Abbres talions used RV, EroRV, RI. &oRI. H. «тоЯІІ. Sc. Siici. P. Р иП. Β. BarnHÌ
probes (Figure lg) results in the recognition of a 22 kb fragment, corresponding with the unaltered allele, and the detection of an additonal fragment (e.g. lymphomas 31, 15 and 16, Figure la and lb), corresponding with the allele in which the provirus is inserted between position - 11 and + 11 on the physical map of Pim-1 The size of the additional fragments detected after blot hybridization using various restriction endonucleases and different Pim-l probes establish the proviral localization within the ftm-1 domain. Since proviruses harbor £coRV recognition sites in their LTRs, Southern blot analyses of EcoRV digested DNA from lymphoma 15, 16 and 31 with Pim-\ probes A and С (Figure la and lb) map the provirus in these lymphomas at position —5.9. —5.7 and +7.5, respectively. Hybridization of ¿coRl digested DNAs from the same lymphomas with probe С confirms the pro viral integration sites in lymphomas 15 and 16 (Figure Id). Besides the 11 kbp EcoRI fragment from the unaltered allele, an additional £coRI fragment was observed in DNA from lymphoma 15 and 16. In lymphoma 31 no additional EcoRl fragment was detected by probe C, as the provirus in this tumor was inserted outside the region corresponding with the 11 kb £<oRI fragment
Evidence that the observed alterations in the Pin:-1 region arc indeed the result of proviral integrations was obtained b\ hybridizing the same Southern blots with a specific Moloney U3LTR pro be Additional fragments detected by hybridization with probe A and С (Figure la and lb) also annealed with the MoU3LTR probe (see arrows in Figure le). A more detailed mapping of proviruses integrated in the 3' region of the Pim-\ gene is shown in Figure le Southern blot analysis of/Ίι/ΙΙ digested lymphoma DNAs with probe A results both in the recognition of a 2.1 kbp fragment corresponding with the unaltered allele, as well as the detection of novel DNA fragments of 1.7, 1.0, 1.0, 18 and I 1 kbp in lymphoma 34, 32. 29. 27 and 2. respectively Since these proviruses contain a Р иП site in their LTR (Van Beveren ci al.. 1982). the fragment sizes observed after /VuII digestion map these proviruses as depicted in Figure lg.
In DNA of 32 lymphomas, proviruses were found in the 3' domain of the Wm-I gene (Figure 2). and in three lymphomas the proviruses were localized in the 5' region of the Pim-\ gene A total of 24 out of the 32 proviruses. which were integrated near the 3' end of the ftm-l gene, were localized between map position — 1 and +1 and possessed the same transcriptional orten-
54
Oncogene Pimi activation in Т-МІ1 lymphomas
33
16
35 54'
25 г?2?ЭзгЗГ 7г Τ? ίβ К 23 24
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Fig 2 Locdiion and üntnldlion of proviral integrations in the Λ/π 1 region (a) Hortfontal arrows above lumor numbers denote the orientation of the intcgralcd proviras (S > 1 1 boxes show the position of integrated provinises within the Pirn 1 region In those instances in which the orientation of the provirus could not be confirmed b> blot hybridization after cleavage with a different enzyme no orientation is indicated Localization of Pim 1 mRNA sequences and direction of transcription arc indicated by the large horizontal arrow (5 > 3 ) In case the proviral integration pattern differed among lymphoma tissues within the same animal the tissue is indicated t thymus s spleen (b) A detailed restriction cndonuclease map of the integrated provirus ol lumor 15 al posinun 5 9 on the Pim I map Abbreviations see legend 10 Figure 1 and S Smal К Kpnl Ps Λ/1 Χ Xbal
Ution as Pirn 1 , in eight lymphomas the provirus was inserted downstream from the Pim 1 gene between map position +5 and +9 Three lymphomas (15 16 and 34) contained proviruses close to map position -6 in the 5 region of the Pim 1 gene In nearly all instances the orientation of the pro\irus could be established unambiguously However in lymphoma 16, the U3LTR probe recognized identical sized fragments as Pim 1 probes A and С (f-igure 1c) Also analysis with other enzymes did not allow the determination of the proviral orientation in tumor 16 Since the proviruscs which were integrated downstream from map posi tion 1 3 (£ÍORI) could only be detected after digestion with EioKV the orientation is not given although hybridization analyses using a U3LTR probe indicated that probably all these provimses have the same transcriptional orientation as Pim 1 (data not shown) Figure 2a presents an overview of the integration sites and transcriptional orientation of the proviruses in the Pim 1 region In some lymphomas the anatomical location of the tumor is indicated as in these instances the integration pattern differed between tissues as the result of the oligoclonal origin of these tumors (e g Figure If, lymphoma 26 and 54, see also Selten el al 1984)
To search for possible proviral integrations further upstream (beyond position -10) specific cellular probes were prepared from genomic DNA clones comprising the region between -30 and - 10 kbp on the map of Pim I No additional integrations or alterations were found in the - 10 to -30 kbp region in any of the 130 lymphomas analyzed No Pim 1 probes are available so far lo delect proviral integrations downstream trom position + 11
Siruaure of integrated prourusei Both mink cell focus inducing (MCF) provimses as well as ecotropic proviruses were found integrated in the Pm-\ domain Approximalely 80% of the proviruses exhibited an MCF slruc ture as revealed by the presence of an EcoRI site at position 6 9 on the proviral genomic map (Quint el al 1981 van der Putten ί tal 1981) In lymphomas 3 8 12 23 32 and 33 the
EcoRl site was absent, suggesting integration of ecotropic pro-viruses near Pim 1 in these lymphomas
Proviruses in lymphomas 15 and 16, which are integrated in the 5 region of the Pun 1 gene, showed internal deletions ot 4 5 and 3 kbp, respectively The provirus in lymphoma 15 show ed the same transcriptional orientation as the Pim-\ transcript, and therefore might represent an example of promoter insertion This provirus with adjacent PirnA sequences was molecularly cloned in phage lambda In Figure 2b the physical map of the cloned provirus is shown The proviral integration in lymphoma 15 was mapped at position - 5 9 The restriction sites /VuII, Xbah Sill, Smal, £coRV and Kpnl were found to be present in both LTRs (van Bevercn et al, 1980) The LTR-specific fragments generated by these enzymes indicated that as in one of the proviruses cloned from the 3' region (Berns el al , 1983, van Beveren el al, 1982), this provirus contained two intact LTRs with duplicated enhancer sequences in the U3 region Xbal and Kpnl digestion gave rise to a single internal proviral fragment of 3 7 kbp, indicating the presence of a deletion of 4 5 kbp in this provirus The absence of recognition sites for restriction endonucleases Xhol, BamHl, Bgill and НішПІІ located this dele tion between map position 1 8 (Pmll) and 6 8 (Smal) on the physical map of Moloney MCF (Bossclman et al, 1982) Further analysis of lymphoma 16 showed that the provirus in this tumor contained a deletion ~3 kbp (data not shown) Ргомгаі insertion enchances Pim 1 transcription To investigate whether proviral insertion near or in the Pim-1 gene affects the level of Pim-l mRNA poly A+ RNA was isolated from lymphoma tissues and the amount of Pim-\ mRNA estimated by the dot-blot hybridization technique Examples of Pim 1 mRNA levels in normal and tumor tissues are shown in Figure 3 As a control for the integrity and amount of spotted poly A + RNA, hybridization with an actin probe is included In poly A+ RNA samples of lymphomas bearing a provirus near Pun 1 (lymphomas 2 15, 16 26, 27t, 28 29, 30, 31, 32 and 541 marked with an astcrix) the Pirn-1 mRNA levels were
55
G.Seiten, H.T.Cu>pers and A.Berns
PROBE -
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Fig. 3. Expression of Pim-1 sequences in lymphoma and normal tissues Several dilutions ranging from 1 to 0 04 /ig pol) A* RNA from 24 randomly chosen lymphomas (t thymus, s spleen) and from newborn and adult tissues were applied to nurocellulose fillers and hybndtzed to " P labeled probe A An actin probe was used as a control for the quantity and integrity of the RNA samples Tumor numbers marked by an astenx denote lymphomas with a proviral integration in the P¡m-\ region (see Figure 2} The lower panels of the figure show expression levels of Pim I in poly A * RNA from normal thymus (thy), spleen (spi), liver (lis) and kidney (kid) of newborn ( 1 - 4 days) and adult (3 months) BALB/c mice
significantly elevated as compared with the levels of Pimi mRNA in lymphomas without proviral integration near the Pim-1 gene. In a few tumors increased levels of Pim·] mRNA was observed without the concomittant integration of a provirus (e.g., lymphoma 73) In these lymphomas either provirai integration occurred outside the examined 40 kbp region (-30 to +11 kbp on the Pim-l map), or other mechanisms for the activation of Pim-\ are involved.
The amount of Pim-1 mRNA appeared dependent on the site of proviral integration In tumors with a provirus in the — 1 to 0 region, Pim-\ mRNA levels were significantly higher (e.g., lymphomas 2, 26, 27t, 29, 32 and 54t) than in tumors in which the provirus was integrated in the 5' region (lymphomas 15 and 16) or far downstream of the Pim-l gene ( e g , lymphomas 28, 30 and 31). This effect was even more pronounced when the expression level was adjusted by taking into consideration the intensity of the additional hybridizing bands on Southern blots, or by comparing the expression levels of monoclonal tumors obtained after transplantation of primary lymphomas (data not shown).
The level of expression varies in tumor tissues obtained from different anatomical sites (compare tumors 27, 29 and 74, Figure 3). A higher level of expression in these tumors is associated with a more pronounced hybridizing modified Pim-\ allele. In lymphoma 27 enhanced Pim-1 expression was seen in the thymus but not in the spleen. In accordance with this observation pro-viral integration was only detectable in the thymic lymphoma. In almost all lymphomas without proviral integration near Pim- ί, the Pim-l mRNA contents were comparable with the levels in normal lymphatic tissues. Exceptions are lymphomas 27s and 74t, in which expression was hardly detectable
a
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Fig. 4. Analysis of Pmt-\ transcripts (a) Northern blot analysis of pol\A* RNA from newborn spleen (N. 3 /ig), and lymphomas 31 (5 μg). 15 (5 /ig). 27 (2 /ig) and 32 (2 /tg) Glyoxal treated RNAs and marker DNAs (Яші/Ш digested λ DNA) were separated on 1 rr agarose gels, transferred to nitrocellulose filters and hybridized to 3 2 P labeled probe A (b) Demonstration of covalent linkage Ol U3LTR sequences to Pim-\ transcripts Filters containing EaiRI and BainHX digested probe Λ plasmid were hybridized to 32P-labeled plasmid probe A ( -". left panel) Filters incubated wilh poh A " RNA from lymphomas 27. 31 or 32 were subsequently annealed to " P labeled MoUM.TR probe (right panel) (c) Drawing showing position ol provimses in lyniphomas 27 and 32 in the 3 region of the Ptm I gene (position - 0 2 and - 0 8 on the Pim 1 map. respectively) Details concerning lymphomas 15 and 31 are shown m Figure 1 Abbreviations see legend to Figure I
In RNA isolated from newborn and adult mice, P/m 1 mRNA transenpts are predominantly found in spleen and thymus Minor amounts are detectable in liver and no Pim-l transcripts are present in kidney RNA (Figure 3, lower panels). No Pim 1 mRNA transcripts were detected in RNA from newborn and adult brain. lung, heart, ovaries and testes. During embryonic development Pim-1 expression is mostly restricted to liver, thymus and spleen (data not shown) In liver the highest levels are seen around day 16— 19 of gestation, for the thymus around birth, whereas Pmi-l mRNA levels in spleen increase steadily up to 14 days alter birth. Also in the 10— 12 days old placenta a high expression level is observed.
Nature of Pim-l transcripts Northern blot analysis of polyA+ RNA. isolated from normal tissues or lymphomas with a proviral integration around position —6 ( e g , lymphoma 13) or downstream of position +1 ( e g , lymphoma 31) on the Pim-l map, showed a 2 8 kb (the predominant species) and a 2 45 kb Pim-1 mRNA (see Figure 4a). Because total cellular RNA was isolated, Pim 1 precursors were also detected.
Proviral integrations in the 0 to - 1 region of the Pim-1 domain affected the size of the Pim-l mRNA Northern blot analyses of polyA+ RNA of these lymphomas showed smaller transcripts ranging in size from 2.0 to 2 6 kb (eg . lymphomas 27 and 32 m Figure 4a) Also larger (up to 10 kb) transcripts were found
56
in some of these tumors (lymphoma 32 Figure 4a) They most likely represent read through products in which proviral RNA is covalently linked to the 3' end of the Ptm-l mRNA
Integration of a provims in the 3 region of the Pun 1 gene not only results in a reduction in size of the Л/и-1 mRNA but also in the linking of LTR sequences to the Pint 1 transcript Sandwich hybridization with RNA from lymphomas 27 and 32 showed that Pirn 1 transcripts were terminated in the 5 LTR of the integrated provirus Nitrocellulose filters containing a mixture of BamHl and EroRI cleaved probe A plasmid giving rise to fragments of 5 2 kbp (pBR322 + Pint I) 4 3 kbp (pBR322) and 0 9 kbp (Pun 1) were successively hybridized with lym phoma poly A + RNA and a 32P-labeled Moloney UlLTR probe (Figure 4b) The results indicate that in lymphomas 27 and 32, but not in lymphoma 31 viral sequences are covalently linked to Pim 1 mRNA transcripts Disruption of Pint I mRNA se quences by termination in the integrated proviral LTR is observed in over 60% of the lymphomas with proviral integration in the Pim-1 region
Integrations around position - 6 did not affect the size of the Pim-l mRNA In tumor 33 a provirus was integrated at position -6 2 with a transcriptional orientation opposite to Pim-l Activation of Ptm-l transcription in this tumor is likely to be mediated by enhancement Therefore the normal functional Pim-\ pro moter is expected to be located to the right of position - 6 Also the mature Pint-1 mRNAs in tumor 15, in which the provims was integrated in the 'promoter insertion' orientation were of similar size as the mRNAs in normal spleen and lymphoma 31 (Figure 4a) To verify whether transcription was initiated from a proviral LTR sandwich hybridization was performed, as described for lymphomas with inserted proviruscs in the 3' region of the Pim 1 gene (Figure 4b) For this analysis a Moloney U5LTR probe was used However, no U5LTR sequences were found to be linked to Ptm-l transcripts in tumor 15 (data not shown) This observation, and the fact that lymphomas 33 15 and 16 have identical sized Ptm 1 transcripts (despite their opposite proviral orientation), suggests that the Pim 1 promoter is probably located downstream from position —6 This was fur ther supported by the observation that promoter-like sequences were found in this region (data not shown) However, we have not formally excluded the possibility that the integration region around position - 6 is part of a Pint 1 intron
Discussion
Position of provtruses m the Ptm-l domain The activation of с туе by ALV in bursal lymphomas is mediated predominantly by insertion of a viral promoter upstream of the с mvc coding sequence (Hayward et al , 1981, 1981. Neel et at , 1981, Payne et al , 1982. Wcstaway et al , 1984, Cullen et al , 1984) In contrast, in mice the proviral activation of c-myc (Corcoran et al , 1984, Selten et al , 1984) as well as the activation of mil (Nusse et al , 1984), tnt 2 (Dickson et al , 1984), and Pim-l occurs almost exclusively by enhancement The transcriptional orientation of the majority of the proviruses in these mouse tumors is directed away from the activated gene Even the few proviral integrations in the 5' region of с туе and Pim-l, which have the 'promoter insertion' orientation seem to activate c-m>c and Ptm-1 predominantly by enhancement (Selten et al , 1984)
The position of the integrated proviruscs with respect to the cellular promoters differs in the various systems (Corcoran et αϊ , 1984, Selten et al , 1984, Nussc et al, 1984, Dickson et
Oncogene Pim-l fictivation In T<ell bmpbomas
al 1984 Shih et al, 1984) Whereas in mouse T-cell lymphomas proviral integration near с /me is almost exclusively found in the promoter region of the gene, near Ptm 1 most integrations occur within the 3' region of the transcription unit Several factors may contribute to this integration pattern Firstly the preference of the proviral integration machinery for distinct sequences of chromatin structures (Breindl et al, 1984) Secondly the alterations needed to potentiate a particular proto-oncogene The second aspect might require the displacement of m-conlrolling elements as suggested for the activation of c-m\c upon translocation (Leder et al. 1983). the removal of control elements which might interefere with efficient translation (Miller et al 1984), or alteration of the gene product as observed for the ras proto oncogene (Tabin et al , 1982. Reddy eral , 1982) These factors could explain the observed clustering of integrations in the Яіт-l domain
Le\el and nature of Pini I mRNA m hmphomas Proviral integration near fim-l is always associated with increased Ptm 1 mRNA levels In most lymphomas without a proviral insertion near Pim-l, mRNA levels were comparable with the levels in normal lymphatic tissues In a few instances (tumors 73 and 40) enhanced expression was seen without proviral integration near Ptm-l Incontrasi in the splenic lymphoma of mouse 27, and the thymic lymphoma of mouse 74, Ptm-l mRNA was hardly detectable The primary target cell giving rise to these lymphomas might have belonged to a cell lineage which normally produces only minute amounts of Pim-l mRNA or the level of Pim-l mRNA expression was reduced during the progressive growth of these lymphomas Similar observations were made for с т\с mRNA expression in MuLV induced lymphomas (Selten et al , 1984)
In -60% of the lymphomas with an activated Pim-l gene, the provims is integrated in the 3 ' region of the Pim 1 transcription unit This results in higher Pim-l mRNA levels than seen in tumors with integrations in other regions of the Pim-1 domain These mRNAs arc missing up to 1300 nucleotides of ЛІМ-1 specific sequences and contain -500 nucleotides of LTR sequences ai their 3' ends Integration in this region could have scscral effects (i) it could alter the Pim-l encoded protein, possibly increasing its oncogenic potenti.il Since proviruses are found integrated throughout this region and outside the Pirn-l gene, truncation of the Pint 1 protein by proviral insertion is certainly not essential for the potentiation ol Pim-1 The absence of integrations in the -1 to - 5 5 region suggests that the integrity of that part of the Pim-l gene is required to encode a biological active pro'ein (u) It might enhance more effectively transcription from the Pim-l promoter, or increase Pim 1 mRNA stability The significant higher level of Pim-1 mRNA transenpts present in lymphomas w ith an integration in this region of Pim-1 as compared with lymphomas with an integration outside the Pim-1 gene supports this explanation (in) The removal of 3' se quences could affect the efficiency of translation Such an effect has recently been described for the fos mRNA (Miller et al , 1984) Protein expression studies have to be performed to resolve this issue
Remarkably, in lymphomas with an altered Pim-1 allele (e g , Figure 4a tumors 27 and 32) transcription from the normal allele was not observed Either allelic exclusion prevents transcription from the normal allele as has been suggested for c-mvr expres Sion (Leder et al , 1983), or expression of the normal allele is inhibited by other mechanisms (compare low levels of Pim-1 mRNA in lymphomas 74t and 27s in Figure 3)
57
G.Seiten, Η Τ Cuypers and A Berns
The increased level of Pim-l mRNA in tumors with proviral integrations near Pim-1 corroborates the notion that a high expression level of a proto-oncogene can confer a selective growth advantage to a cell The proviral activation of Pim-l in a high percentage of MuLV induced early Τ-cell lymphomas, and its expression pattern during development, reminiscent for cell lineages of the haemopoictic system, suggest that Am-l might belong to a specific class of proto-oncogenes with a regulatory role in lympoid cells
Materials and methods
Afííf umses and lymphomas
1 ymphomas were generated by injeuing newborn BALB/c mice with Moloney MuLV clone 1A as described (Jaenischrt al 1974 Cuypers et al 1984) Spon laneously deseloping lymphomas from BALB/Mo and AKR/Fu mice were obtained as described previously (van der Punen el at 1979 Quint eí al 1981 Cuypers et al 1984)
Molecular cloning
A 12 kb ИікШ DNA fragment localized between position - 8 and 0 on the Pint I map comprising the intcgratuJ (deleted) provtrus with adjacent cellular Ptm I sequences of lymphoma 1 't was molccularly cloned in baacnophagc lambda
To obtain Ptm 1 specific probes comprising the region between map position - 1 0 a n d - 3 0 WO/<g of high mol wt Iiser DNA (BALB/c) wis panially digested with reslnclion endonuclease SauW DNA fragments were separated on size and the 12-20 kbp fragments were cloned in Charon phage 28 Recombinant phage plaques were selected which hybridized to probe С From a positive react )ng dont a single eopy probe was obtained which was located bclween position -17 and -18 on the Ptm 1 map
DNA tsolatton and reslrtilton enz\me o/ia/utt
High mol wt DNA from lymphoma tissues was prepared as described (van der Putten et al 1979) DNA samples of 10 μ% were digested as recommended by the suppliers (Amersham BRL Bochnnger) separated on 0 ft*? agarose gels transfened to nitrocellulose filters and hybridized to 3 Ϊ Ρ labeled probes as described prcMOuslv (Quint el al 1981) After hybridization filters were washed in 0 I χ SSC 0 I % SDS at 6S'C After hybridization with the Moloney L'I TR probe the final wash was performed at 600C To remove hybridized probe fillers were rinsed with 0 2 M alkali As mol wt markers Hindlil digested phage К DNA was used
Preparation of RS Л
Frozen tissues were homogenized at 0 e C in 3 M LlCI 6 M urea and maintained overnight at 4 eC as described (Auffray and Rougcon 1980 Sellen el at 1984) Total cellular RiNA was obtained after centnfugation at 16 000 χ RNA was depm-teimzed bv two successive phenol chloroform isoamylalcohol СЮ 48 2) estrac lions and prceipildtcd with ethanol RSA samples were heated at 65 С for five minutes before poly A ~ selection on oligo(dT) chromatography \vnhem and dot blot anahut
For dot blol anaKsis polvA RNA was dissolved in distilled water and applied to nnrocellulosc lillers as described by Muller et al (1982) Glvoxal trcaiment and Northern blot analysis were carried out as described (Carmichael and McMaster 1980 Thomas 1980) Filters were hybridized with " P labeled probes as desenbed above for DNA containing filters including the washing procedures (last washing 0 1 χ SSC 0 I 9, SDS at 65=C) and exposed to X ray film As mol wl markers Httidill digested λ DNA was used Denaturation was performed as described for RNA Sandwich hybridization
2 /ig of plasmid DNA containing probe A insen was cleaved with ÍÍWRI and BrwiHI respectively The fragments were mixed separated on a 0 6Я agarose gel and transferred to nitrocellulose Three fragments were thus obtained a 5 2 kbp fragment representing the linearized plasmid a 4 1 khp pBRt22 fragment and a 0 9 kbp fragment representing the Pim 1 specific insen Filters were incu hated overnight with 5 ßg polvA RNA under conditions as described (Susse a al 1984) washed twice in 0 I χ SSC 0 15! SDS at 53 С blotted dry and subsequcntlv annealed to " P labeled Mol TR specific probes for 16 h Filters were washed twice with 0 1 χ SSC 0 Ì7< SDS for I h at 53 С dried and autoradiographed overnight
H\bridi anon probes
Isolation of Pun 1 spécifie probes A E as well as the MoL'I TR probe was per 1 -med as described (Cuvpers et al 1984) The MoL5LTR probe comprises a ipall fragment from the MSV 12 LTR (van Beveren a at 1982) subtloncd m МИ The actin cDNA clone is described bv Dodemonl el al (1982)
Acknowledgements
This work was performed in the laboratory of Dr H Bloemendal to whom we are particularly indebted We appreciated the assistance of the coworkers ol the Central Animal Laboratory We wish to thank Drs /ijlslra and Melici ol the Central laboratory of the Netherlands Red Cross Blood Transfusion Service lor their contributions This work was supponed by grants Irom the Koningin Wilhclmina Fonds (G S ) and by the Netherlands Orgjni/jnon lor the Advance ment of Pure Research (/WO) through the Foundation lor funadmcntal Medical Research (FUNGO)
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Groenen M A Bernardi G and IMocmcndal H (1982) hMHOJ I 167 171 Fung Y Τ l-cwisWG Cnttcndcn L В andKungll (1983)07/ 33 357 368 HaywardWS Neel В G and Aslrm S (1981) Wurr 290 475^180 Jaemsch R Fan H and Craker В (1975) Pm Mail /liad WÍ UU 72
4008 4012 l-cilcr Ρ Вапсу J Lenoir G Mould ng С Murjihy W Poner H Stewart Τ
and Taub R (1983) •»teme Wash) 222 765 770 Miller A D Curran Τ and Vcrma I M (I9H4) tell 36 51 60 Muller R SlamonDJ Tremblay J M Cime M J and Vcrma I M (1982)
nature 299 640 644 NcclBG HaywardWS Robinson H 1 Fang J and Aslnn S M (1981) Cr«
23 323 334 Neil J С Hughes D McFarljnc R Wilkic N M Onions D fc Lees G and
JarrctO (1984) Calure 308 814 820 Nobis Ρ and Jaemsch R (1980) Prot r«all Atad Vi USA 77 3677 3681 Nüsse R and Vamius H Ь (1982) Cell 31 99 109 Susse R van Ooven A Cox D Fung Υ Κ Τ and Varmus Η (1984) Watun
307 131 136 Payne G S Bishop J M and Varmus H t (1982) Vaiare 295 209 214 PcdcrsonFS Buchhagcn D I ChcnCY Hass fc F and Haseltinc W A
(1980) J Viral 3^ 211 218 Peters G Brookes S Smith R and Dickson С (1983) Cell 33 369 377 Quint W Quax W van der Punen H and Berns A (1981) У Virol 39 I 10 Rcddv F Ρ Reynolds R К Santos E and Barbacid M (1982) Sature 300
149 152 RowL W Ρ andPincusT (1972) У f.t/¡ Med 135 429 436 Seilend СиурегчНТ ¿i|lstra M Melici С and Berns Л (1984) FMM) J
3 3215 3222 ShihCK Limai M Goodenow M M and Hay ward W S (1984) Рпн Suil
Acad Sa ЬЧЛ 81 4697 4701 ЫсІІсп D and Weinberg R A (1978) Cr« 15 1003 1010 Tabin С J Bradlcv S M Hargmann С I Weinberg R A Papageorge A G
Seolniek E M Dhar R I owy D R and Chang F H (1982) Vururr 300 143 149
Thomas Ρ S (1980) Pm \ail Aiad Sci t/S/( 77 52015205 van Beveren С Goddard J G Berns A and Vcrma I (1980) Рпи Salt Attui
5π OU 77 3307 3311 van Beveren С Rands E E Chattopadhyay S К Lowy D R and Vcrma I
(1982) J Virol 41 542 556 van der Pullen H Tcrwindt F Berns A and Jacniseh R (1979) C<// 18
109 116 van der Putten H Quint W vanRaavJ Robanus Maandag Г Vermal and
Berns A (1981) UU 24 729 739 Weslawav D Pavnc G and Varmus H F (1984) Prot Sali liad Sa (SA
81 843 84"
Reitned ni 4 April IWi
58
CHAPTER 5
The Primary Structure of the Putative Oncogene p/m-1 Shows Extensive Homology with Protein Kinases
Cell 46: in press
THE PRIMARY STRUCTURE OF THE PUTATIVE ONCOGENE ΡΙΜ-Ί SHOWS EXTENSIVE
HOMOLOGY WITH PROTEIN KINASES.
Gerard Selten , Η. Theo Cuypers , Wilbert Boelens, Els Robanus-+ + #
Maandag, Jos Verbeek , Jos Domen , Charles van Beveren and Anton *+
Berns .
Department of Biochemistry, University of Nijmegen, Geert
Grooteplein Ν 21, 6525 EZ Nijmegen, The Netherlands.
# The Salk Institute for Biological Studies, P.O. Box 85800, San
Diego, Cal. 92138, U.S.A.
Section for Molecular Genetics of the Netherlands Cancer Institute
and Department of Biochemistry of the University of Amsterdam,
Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands.
Present address: Gist-Brocades N.V., P.O. Box 1, 2600 MA Delft, The
Netherlands.
61
SUMMARY
We have shown previously that the putative oncogene plm-1 Is fre
quently activated by provlrus Insertion in murine leukemia virus1-
induced Τ cell lymphomas. Here we describe the structure of the plm-1
gene as determined by sequencing genomic and cDNA clones. The gene
has an open reading frame, encoding a protein of 313 amino acids, ex
tending over six exons and preceeded and followed by stopcodons In all
reading frames. Proviruses always integrate outside the protein-
encoding domain, showing a high preference for a small region in the
3' terminal exon; Integration in the 3' exon results in relatively
high levels of plm-1 mRNA. Computer search reveals homology between
plm-1 and protein kinases: All the domains characteristic of protein
kinases are conserved in the plm^l amino acid sequence. The highest
homologies were observed with the protein- serine kinases.
INTRODUCTION
Oncogenic transformation by the non-acute transforming retero-
viruses probably depends on the activation of cellular genes by in
tegrated proviruses. Activation of the с myс proto-oncogene by pro-
viral DNA insertion has been described in avian leukosis virus (ALV)
and chicken syncytial virus (CSV) induced B-cell lymphomas In birds
(Hayward et al., 1981¡ Neel et al., 1981¡ Payne et al., 1981; Westaway
et al., 1984; Swift et al., 1985), in murine leukemia virus (MuLV) in
duced T-cell lymphomas in mice (Corcoran et al., 1981; Selten et al.,
1984; Li et al., 1984; Steffen et al., 1984; O'Donnell et al., 1985)
and in feline leukemia virus (FeLV) induced lymphomas in cats (Neil et
al., 1984). A similar mechanism underlies the activation of с erb in
ALV induced erythroblastosis (Fung et al., 1983; Nllsen et al., 1985),
the activation of с myb in Abelson and Moloney murine leukemia virus
induced plasmacytoid lymphosarcomas (Sheng-Ong et al., 1984), the ac
tivation of lnt-1 (Nusse and Varmus, 1982; Nusse et al., 1984) and
lnt-2 (Peters et al., 1983) in mouse mammary tumor virus (MMTV) in
duced maramacarcinomas, and the activation of plm-1 in MuLV Induced T-
62
cell lymphomas (Cuypers et al., 1984; Selten et al., 1985; 0'Donneil
et al., 1985). Other common integration regions include Mlvl-1,
Mlvl-2, Mlvl-3 (Tsichlis et al., 1985), mis-I (Lemay et al., 1984),
pvt-1 (Webb et al., 1984), and gln-1 (Jolicoeur, personal communica
tion) .
Integrated proviruses can activate cellular genes either by tran1-
scrlptlon from the viral promotor (Neel et al., 1981; Voronova and
Sefton, 1986) or by virtue of the enhancer activity of the proviral
long terminal repeat (LTR) (Payne et al., 1982; Nüsse et al., 1984;
Corcoran et al., 1984; Dickson et al., 1984; Selten et al., 1985). In
addition, Integrations within the transcription unit"might modify the
stability or translatablllty of the mRNA, or give rise to a truncated
protein product with altered biological characteristics.
Here we describe the structure of the putative oncogene plm-l,
which is frequently activated In MuLV Induced T-cell lymphomas. We re1-
port the nucleotide sequence of the plm-1 gene and the effects of the
site of proviral Integration on the level of mRNA expression. The de
duced amino acid sequence Indicates that the plm-1 protein Is a member
of the class of proteins with protein kinase activity.
RESULTS
The pf-Ί Dosa in.
A schematic overview of the distribution of integrated proviruses
within the plm-1 domain in 36 Independent lymphomas is shown in Figure
1 (see also Cuypers et al., 1984; Selten et al., 1985). Proviral in
tegrations are found within a region of 15 kb, showing a strong
preference for the 3' part of the plm-1 transcription unit. From a
total of 36 lymphomas with a provirally activated plm-1 gene, 25 con
tained a provlrus within the 3' region of the transcription unit, in 8
lymphomas the provlrus was Inserted downstream of plm-1 and In 3 in
stances the provirus was Integrated upstream of the plm-1 transcrip
tion unit. Proviral insertion is associated with increased plm-1 mRNA
levels. Integration upstream of map position -6 or downstream of posi
tion +0.5 (see Figure 1) does not alter the size of the 2.8 kb plm-1
63
mRNA transcript on Northern blots (Selten et al., 1985). In lymphomas
containing a provirus around position -0.5 of the plmJ1 map (approxi
mately 70J of the lymphomas), modified transcripts ranging in size
from 2.0 to 2.6 kb, covalently linked to viral LTR sequences, were
detected (Selten et al., 1985). The nucleotide sequences of the plm-1
genomic DNA and of cDNAs synthesized from plm-1 mRNAs obtained from
three independent MuLV induced lymphomas, were determined.
Genomic plm^l Sequences.
The 11 kb EcoRI fragment, corresponding to the region between -9.6
and +1.3 on the plm-1 map (Figure 1), was molecularly cloned from nor
mal BALB/c DNA. The nucleotide sequence betweem position -6.6 (Sad
site) and +1.3 (EcoRI site) was determined from both strands and with
overlapping regions across every restriction site. A partial sequence
of this region, containing 7993 nucleotides, is shown in figure 2.
Plm-1 cDdA Clones.
To determine the plm-1 gene structure, рІш-Ί cDNA's were molecular"
ly cloned and sequenced. mRNA from lymphomas with provlruses within
the plm-1 transcription unit (tumors 10 and 5*1) and downstream of the
plm-1 gene (lymphoma 9) were used as starting material. Proviral
insertion as observed in lymphomas 10 and 54 gives rise to a plm-1
mRNA of approximately 2.0 kb. Plm-1 transcription in these lymphomas
is terminated at the polyadenylation site of the Moloney MuLV long
terminal repeat (Selten et al., 1985). Integration of provlruses
downstream of the plm-1 gene, as in lymphoma 9, does not affect the
size of the plm-1 mRNA transcript (Selten et al., 1985). cDNAs were
cloned in Xgtll using established protocols. The largest ollgo(dT)-
primed plm-1 cDNA clone was obtained from lymphoma 10. The cDNA clone
was mio bp long, including 508 bp of LTR sequences. This clone
reached to position 2711 in the fourth exon. Using a synthetic primer
on this mRNA preparation (position 4652 to 4667 in the fifth exon), we
obtained 2 cDNA clones of 918 bp that reached to map position 1719 in
the first exon. 011go(dT)-primed cDNA synthesis using a 2.8 kb plm-1
mRNA preparation as template resulted in a series of relatively small
64
•IO
ν к
•5
К Ρ Ρ Ρ
0
Ρ κ Ρ
5 10 kbe
R-V
Β Η Β R.
NUMBER • - ^ ^ LYMPHOMAS • 3 25
PIMmRNA ^ 5'^^^^^^^м^в^з'
1 1 1
Figure 1. Proviral Integration Sites within the plm-1 Domain. Vertical large arrows Indicate the regions in which proviral in
tegrations were detected in the independently induced lymphomas. Numbers beneath the arrows give the number of lymphomas having an integrated provirus within these regions. The localization as well as transcriptional direction of the plm-1 gene is indicated by the horizontal arrow. Restriction sites: R V, EcoRV; RI, EcoRI; K, Kpnl; H, Hindlll; Sc, Sad; P, PstI; and B, BamHI. Map coordinates (in kb) are shown above.
cDNA clones. The largest clone In this series was 550 nucleotides
long, reaching to map position 6444. These and several other plm-1
cDNA clones were sequenced by the M13 dldeoxyribonucleotide chain ter
mination method (Sanger et al., 1980) and the sequences were compared
with that of plm-1 genomic DNA. This revealed both the structure of
the plm-1 gene as well as proviral integration sites in lymphomas 10
and 54.
Structure of the pl«-1 Gene.
The plm-1 gene contains at least 6 exons, as indicated in Figure 2.
These 6 exons are 419 (or slightly larger), 107, 51, 367, 177, and
(probably) 1499 bp in size. They cover in total 2620 nucleotides of
plm-1 mRNA, which was estimated to be 2.8 kb long by Northern blot
analysis (Selten et al., 1985). We cannot completely exclude the pos
sibility that the 3' exon of 1499 bp is interrupted around position
6500 (Fig. 2) by a small intron of variable size. This is based upon
the following observations. Fragments of approximately 1000, 440, 390,
and 380 nucleotides corresponding to the region between positions 5496
and 7000 , were protected from SI nuclease digestion after hybridiza
tion of the 2.8 kb plm-1 mRNA to an 11 kb genomic clone (-9.7 to
+ 1.3)· Hybridization with small Ml 3 probes showed that the 1000 nu
cleotide fragment could be assigned to the region between positions
65
Зло I H 1 totg»«<»icte«gt«go«g»*tttmoc»ttotctctg<]tatcgtitg<ccctgtgtcatccc»gtgacgagtcatttt i ;eUtctgcaa«jmM»agtMttetccttcccct
t t f t t t t teteett tagto*bcaga»«t i>«tt»gttagt 0.520 kb cgetcaegaett t tcctgctcctcccgccctctceeMee 720
«etei ieegoâtttgiCMgBgMagcteaogtttgtgtcgtgatttootctctcagecectcgeacatBBaccgBgggeatcaggtaccccggetcccttoteattctctctgcitt 8*0
te tuAttgBf iatat t tgtcaoouotootcgcetccce 0.320 kb ctfBCCgcgaegcgagageegeegcecgc«*cgccttet| 1 ΊίΟ
o»— UfHO«tettetMttMCtg««gcgcgooeg»gggcggggocggggagaggggtgtagccgcg»gggggcggagoggagggggag-ggccctggtcecgccgcctccccgcccc ISM
ototaaKOootacgo-goegagocagoooggotccccacctcg-c-tcccggagaegccoogecccBtccccgcccgccgccgccotcoooagcaacBCgtccgeecctbtactcctggc 1680
ggCggMCMgCCgggCgtCtgcegtlgeggCCgtgge GGAlKAGGCTCCAGCGCAGTC&^AGTœCGCCGGCGCGATCCÛCAGTAGCAœ 1800 '-> ежоп 1
AOCCCTOMTCCCOCA«(KCTCTCíXCCTCCCGCCTCCCGCACTGCCTGACCCA0CCO«GAACCCGCCCCKGGCCAGCCCCGCCAGCGCCCTCAGTCGTCCTCCGACTC 1920
CnGCGCGCCAGCCGCAGCCAGAGCCCCAACGCCACCCGCAGTCTCAGTCCCGGCCOTCGCTCAGCH 20l l0 1 10 20
HttUuLeuSerLfSllaAenSerLeuAlaHlaLeuArgAlaArgProCysAanAepLeuHlaAlaThrLysLeuAlaProG ACCCTTni№TO(№:ATWTCCTtlTCCAAGATCAACTCCCTGGCCCACCTGCGCGCCCGCCCCTGCAACGACCTGCAC(K;CACCAAGCTGGCGCCGGgtgagtgtOOOcac 2160
e n n 1 «J 30 10
IrLysCLuLysGluProLeuGluSerGlnTjrrGlnValGlyPro igöeeeo*fft«fttggg»cgccggg»g»CCcecgcgtgggtcgagcgcgctgagtgtcccetgcctctttcrtagpCAAAGAGAAGGAGCCCCTCGAGTCGCAGTACCAGGTGGGCCCG 2280
T-> βion 2 SO 60
UuUuGlrSarGlyGlyPhaGlySerValTypSerGlyl laArgValAlaAapAenLeuPro CTGTTGGGCAGCtMmXKTTCOGCTCGUTCTACTCTGGCATCCGCGTCGCCGACAACrrGCCGgtgagteggcaacgccgcetcggegagggegcgcggaeggatgctttgBgcaggagg 2400
aron 2 «J 70 80
ValAlalleLyeHlaValGluLyaAapArglleSerAspTrpGlyGluLeu ftfltflategagactOggeggtcttcttOOatfTGGCCArrAAGCACGTGGAGAAGCACCGGATTTCCGATTGGGGAGAACTGgteBetgAgCCggagceagagBOOOtOtCatgggtegg 2520
T-» a m n 3 e m n 3 4^ 90 100
RroAsnGlyTlirArgValProHetCluValValLeuLeuLyaLyaValSerSerAapPtiaSerG tg|gtgr)B»oaeeOBtgOOegaaccggcgcgctaaccctogocetcaotctgcagCCCAATOGCACCCGAGTGCCCATGGAACTGGTCCTGTTGAAGAAGGTÜia£I£GCACTTCTCOG 2640
^-l· eion 4 Sac I no іго 130 mo
irValllaArgLauLeuAspTrpPhaOluArgProAepSarPhaValLauIleLeuOluArgproGluProValGlnAapLeuPheAepPhelle'nirGluArgGlyAlaleuClnGluA OCGTCAnAGACTTCTGGACTGGTTCGAGAGCCCCGATAGrnCGTGCTGATCCTGGAGAGGCCCGAACCGCTCCAACACCTCnrGACTTTATCACCGAACCAGGAGCCCTACAGGAGG 2760
150 160 170 180 •pLeuAlaAriGlyPhaRiaTrpGlnValLauGluAlaValArgHlaCyaHlaAsnCysGlyVBlLeuHlsArgAspIleLyaAspGluAanlleLeuneAapLeuSerArgGlyGLuI ACCTGGCCCGAGGATTCrrCTG«ACGT«T«AG«CGTCCGCCATTCCCACAA 2880
190 200 ItLysUuIlaAapPtMGlySerGlyAlaLauLeuLyaAapThrValTyrTTu-AspPheAspG TCAAACTCATCGACnCGGGTCGGGGGCOCTGCTCAAGCACACAGTCTACACGGACTTTGATGgtgBgoeaggccccggggagggagctgcocaaggtgccggcctggaagcctecggcg 3000
ежоп 4 4·* gootoggotocMccctttgtaaaggtuattgggcogect 1.1Ô0 kb gaaactttggaggagaaaaaaaaaattaaaagaagegget 4560
210 220 lyThPArgValTyrSerProProGluTrpIleArgTyrHIsArgTyrHlsGly
oatactagetgatttgttaaotttttttttgctaatgctggtttctttctattccctetgotggtagGGACCCGAGTGTACAGTCCTCCAGAGTGGATTCGCTACCATCGCTACCACGCC 4680 ^-» axon 5
230 240 250 260 Ar(S«rAlaAlaValTrpSerL*uGlyIl«LeuLeuTyrA3pHeCValCysGlyA9pIl«ProPhcGluHl9AspGluGluIleIleLysGlyGlnValPhePheArgGlnTru-Val^er AGCTC(»CAGCTGTCTGGTCCCTTC^ÎiI£CTGCTCTATGACATGGTCTGCGGAGATATTCCGTTTGAGCACGATGAAGAGATCATCAAGGGCCAAGTGTTCTTCACœ 4 8 0 0
Baa HI екоп 5 «-S«rG TCAGgtaacttcectcagacaeocccaatccttcocaaac 0.520 kb acogggcagtgtttggtataaaaagtaccgtgcctcttta 5400
' 269 luCy5GlrHIsLeuIl«LysTi-pCy
aaaacecoagaotttgtaggtctggggaagaagaoctttgcattacaaaaaoaagtteaetcfttctttaacctctgtcteccccgccttctgcaftAGTGTCAGCACCTTAnAAATGGTG 5520 '-l· exon 6
280 290 300 309 •UuSarLauArgProSerAapATBProSerPheGluGluIleArgAsnHlsProTrpMetGlnGlyAspUuLeuFYoGlnAlaAlaSerGluIleHlsLeuHlaSerLeuSepproGl CCTGTCCCTOAGACCGTCAGATCCKXCCTCCTTTGAAGAAATCCGGAACCATCCGTGGATGCAGGGTGACCTCCTaCCCAGCCAGCTTCTGAGATCCATCTGCACAGTCTGTCACC^ai 5 6 4 0
3 1 3 y S a r S a r L y s * * 1
llECaOCAACTAGCAGCCTTTCTGCTaTGTCTGTCAGACCCTCCAGCGAGGGAGAXnGTCTTCTGÍKCTCCAACAGGACCCTGCGTGACGATCKAGGCACACKUTGACAACTCAT^ 5 7 6 0 tam Hl L u u D o r 54 CCACACTCCAMTCCCTGGAGGACCCTCCCACAAGGGAAAGAGACTACTTCACTGGTCCTGCGCCCCTCTTTGCOCCCCCCGCCCCCAGACTCAGCGTCTAGTGTTGCTGGGCTCCCC^^ 5 β θ 0
ι · > tuaoi* 10 UTCCATCCCTGGTTGCGOOTCGGAGTCCATCAGAGCCCTCTCGATCGAACTTTAGTCACCATGGAGACT<KGTCACCAACATGCGCCGGGGGGGAGAGGAAAACAGTCTGGGGGGTGGG 6 0 0 0
аТАААІІСТаССКСАТАТПААСТСССТСТСАССТСТТССААСТССТСАСТСССТТСТСТССССАСТСССССТСТОСТСССАСАААТАСГГСААСПТАССТСТТАССТТС 6 1 2 0
UACTCTaCTGGGTTTTTKTTCCTTCCTCTCCTCTCCnTCCCATCCCCCACTTCCTATGAAAMTGCCATGGGAGAGGCTACAGGTCCAACTGCTGAÍXCAC 6 2 4 0
TCCT^AGTAAAATTCCCAACCAAACTOCTCTTCCTCTTTGGTTTCCACTTAACTGTTTCAAAACCAAGACCTCACACAGAACGCACAACCAATGCAAAACAAAAAACAAAAAAAACAAA 636О
UâAAAACCAGACAAGCTCTAAATGTCTGTATAGTTCCGGCATOCTAGCCTGCAAAAGGACTGTAGATGATAACAAnTTAAACAAGAAAAAAAAATGCCTTTAAGnATTTTATGTGTT 6 4 8 0
o c c G o n r o o ' i ι г о ш ι г т л п т г и т т г г г т ι п с ш І П А І I t ! i m I I X T T G T T T T T G A A A G A T G G C A C G T T C T A G C C C G G A A G T T T T A A T C T A T T T A T T T A T T T A T T T A T 66O0
nOCrrCCCnCCTGnXAAGCTTCCACTGACTCTTGCCCCCGGTTTATTGTCATGrTCTGCCrTTTnrm^TTTCCTTCCTCCTGACTT 6 7 2 0
CTTCCTGGGOTOITGGGGCTCCGGTÎHWKAœTGCTGTAGCCCCClTÎ^^ 6 8 4 0
СТОСТСТОССТТССІИЩаТСТСТСССаТОТССТП-САССАОССТХАССССОСТССТТПАТСТСАСТАААСТАТАСТСТАСАСССОААТААААСАСАТСГ^^ 6 9 6 0
Ваи HI ψ- and o f RNA AtT liAfcAl 1111 іСДЩ^ААССІ ITlUJLTtiïïcctttgagttttccattcattcatacaaagatgtatgcttgetacagagccaggtagactgggcactttcagactae^tctB* ΤΟβΟ
a «on 6 4-1 tttcctgctggegcac*gag"gtnccaggetttctgaac О.ТбокЬ gggaactaagaggatgagagtcaggagttcaaggteagtc 7920
tcattetgggataotoctgactggtctaatoagctggaeagatgcaotctggtagacaggaceaaaggaatts 7993 Eco RI
66
5446 and 6150, whereas the U10, 390, and 380 nucleotide fragments
could be located between map positions 6550 and 7000. This was unex
pected, since the cDNA clones starting at the second poly(A) addition
site at position 6995 were collnear over their complete length with
the genomic sequence; they thereby cover the region between map posi-1
tlon, 6444 and 6994. This discrepancy might have resulted from insta
bility In the SI nuclease protection assay of the AT-rich stretches
around map position 6500 or may be the result of differential splic
ing. The cloning of additional cDNAs Is required to resolve this іэа
sue. At the 5' end the presence of one (small) exon located upstream
of exon 1 is not yet completely excluded. SI nuclease protection as
says showed a heterogeneous pattern for exon 1, with protected frag
ments of sizes 450, 430 and 360 nucleotides. Since an acceptor se
quence is found around position 1706 ( a position that would
correspond to an exon of 431 nucleotides), plm-1 raRNA transcription
might start further upstream. However, pSP6 RNAase protection assays
did not reveal a distinct exon between positions 0 and 1705. Further
more, two Independent cDNA clones reached to position 1719, suggesting
that this position might be an initiation site for RNA transcription.
Most likely, transcription can initiate in the region around map coor
dinate 1720 at several different positions, as has been observed for
other genes. Primer extension studies have to be performed to deter
mine the various start positions more accurately. Upstream of position
1720 the GC rich motif CCGCCC is repeated 8 times. In addition, the
Figure 2. Sequence of the ріш^І Gene and the Predicted plinti Protein Sequence
The "plus" strand of the nucleotide sequence is shown. Numbering starts at the Sad restriction site at position -6.6 on the physical map (see Figure 1). Exon sequences are given In capital letters, starting with exon 1. Portions of large (sequenced) introns are replaced by abbreviations (e.g. —0.520 k b — ) , referring to the exact number of nucleotides In kb. Restriction sites for Sad, BamHI, and EcoRI as well as the polyadenylation signal AATAAA and the CG-rich motifs In the promotor region are underlined. The ATGCAAAT octanucleo-tide motif is double underlined. Six independent cDNA clones end within the AAA triplet at position 6995. Provlral integration sites of MuLV, which were derived by sequencing cloned host-virus Junction DNA fragments (tumor 15) or cloned cDNAs (tumors 10 and 54) are marked by arrows. The proposed amino acid sequence for the plm-1 protein is given above the nucleotide sequence. Numbering starts at the methionine within exon 1.
67
lymphoid specific octamer motif ATGCAAAT is present at position 1438.
RNA Processing Signals.
Comparison of the cDNA and genomic DNA nucleotide sequences showed
consensus donor and acceptor site sequences (Breathnach and Chambón,
198I ; Mount, 1982) for all exon-intron boundaries in the plm-1 gene
(see Figure 2). At the 3' end of the plm-1 gene, two sites with the
consensus polyadenylatIon sequence AATAAA were found at positions 6931
and 6976, respectively. Although there was no evidence for utilization
of the site at position 6931. the sequence of 6 independently derived
plm-1 cDNA clones revealed poly(A) tracts starting exclusively at po
sition 6995, 19 nucleotides downstream of the polyadenylation signal
at position 6976. Since three A residues are present in the genomic
sequence between positions 6995 and 6997, the exact polyadenylation
site could be situated between nucleotides 6995 and 6997.
The Predicted ріи-і Protein.
A single long open reading frame (ORF), extending over six exons
was found. In the 5' region of exon 1, stop codons in all three read
ing frames are present. A single Initiation codon is present In exon 1
at position 2056 downstream, of these stop signals. The AUG Initiator
codon is followed by the ORF , which ends in the large exon at posi
tion 5650. The nucleotide sequence within this ORF corresponds to a
plm-1 polypeptide of 313 amino acid residues and a molecular mass of
-35.5 kd.
Provlruses Are Integrated outslte the Protein Encoding Doaaln.
The three provlruses found at the 5' end of the plm-1 gene were lo
cated approximately 1 kb upstream of exon 1 (Selten et al., 1985). The
integration site in tumor 15 was determined precisely by sequencing
the provlrua-host DNA Junction fragment from a genomic clone. The pro-
virus in this tumor was Integrated 900 bp upstream of exon 1 (see Fig
ure I), and It showed the same transcriptional orientation as plm-1.
At the 3' end of the gene, the majority of the provlruses were insert-4
68
Figure 3. Deteralnation of Proviral Integration Sites by an RNAase Protection, Assay.
A P-labeled antisense RNA fragment corresponding to the region between positions -0.95 and +0.3 on the pim-1 map (positions 5639 to 6860 in F|g. 2) was annealed with poly(A) RNA and treated with RNAase as described in Experimental Procedures. The size of the protected fragment is a measure of the distance between the start of the BamHI recognition sequence at position 5639 and the proviral integration site. Proviral integration sites for lymphomas 17, 32, and 34 were determined by this assay. mRNA from lymhpomas 10 and 54, for which the proviral integration sites were determined by cDNA sequencing, served as reference. Rna from lymphoma 9, which harbors a provlrus downstream of the pim-1 gene at map position +9, was used as a control. The 92 long fragment present in all lanes is caused by nonspecific hybridization.
ed in a small region within the 3' exon (Selten et al., 1985). To
verify that truncation of the plm-1 protein occurred by proviral
Insertion in this exon, the proviral integration sites in six lympho
mas were investigated in more detail (tumors 1, 10, 17, 32, 34, and
54). Proviral positions within the plm-1 gene in these lymphomas were
determined by sequencing of genomic (lymphoma 1) and cDNA clones (lym
phomas 10 and 5*1) or by an RNAase protection assay (lymphomas 17, 32,
and 34) in which labeled antisense RNA was protected from RNAase
digestion by hybridization to plm-1 mRNA (Figure 3)- The precise in
tegration sites of the proviruses in these lymphomas are shown In Fig
ure 4. The data show that proviruses in the 3' exon were clustered in
a small region. The provirus in lymphoma 32 was located 7J* nucleotides
downstream of the plm-1 termination codon, and that in lymphoma 54 was
located 5 bases further downstream (Figures 2, 3. and 4). Integration
in lymphomas 10 and 17 occurred about 140 nucleotides downstream of
the plm-1 coding region at the same nucleotide position (Figures 3 and
4), whereas the provirus in lymphoma 34 was integrated 3 nucleotides
TUMOR No » 32 54 9 10 17 34
154-^ 151 —
9 4 ^ *
89 , 4 ·
69
-«GíCCCTQCGT(iC(MTGCíCO0a^>IX*ATDAC*»CTCATTCC«ACTCC*0GTCCaGGAGGAOCCTCC O C MCCGA»»-
р»оя'иі m tumor I j j 5J Ю r 34
Figure 4. Structure of the pirn*! Gene. The intron/exon structure as determined by comparison of cDNA and
genomic DNA sequences Is shown. Exons are indicated by blocks; coding sequences are filled in. Horizontal arrows show the direction as well as the position of integrated provlruses in the numbered lymphomas. Proviral Integration sites that were determined by sequencing of cloned vlrushost DNA Junctions (lymphomas 1 and 15), by sequencing cDNA clones (lymphomas 10 and 54), or by RNAase mapping (lymphomas 17, 32, 3
1)) are shown here in detail. Abbreviations are the same as In
Figure 1, except that E represents EcoRI restriction sites.
Site ol "tegration • DOWNSTREAM
^мд poly"RNA» ' 0 2 1 02 1 02
TUMOR No» is · « >o Α Φ 7 m
16 · * 1 7 φ « 9 φ ·
3 3 . 2 6 « · 3 , .
2 7 # «
32 # «
54 # ·
Figure 5. Рів-Ч шНИА Levels Relate to Proviral Integration Sites.
Poly(A)+ RNA (1 and 0.2 pg)
from 12 lymphomas was bound to nitrocellulose filters. The Integration regions denoted as 5', 3' and downstream are as Indicated In Figure 1 by vertical arrows. The filter was hybridized first to a plm-1 specific probe (upper), then washed and subsequently hybridized to an actin probe as a control for the quantity of the mRNA in the samples (lower).
70
further downstream from this latter alte (Figures 3 and 4). This
result shows that within this region Integration Is nonrandom. All
provlruses were Integrated outside the ORF, although In at least 25
out of 36 lymphomas the provlral Integration event occurred within the
plm-1 transcription unit. This suggests strongly that the Intact plnM
protein Is required to confer a selective advantage to the tumor cell.
Remarkably, the steady state level of plm"1 mRNA appeared to be depen
dent on the provlral Integration site (Figure 5). Lymphomas bearing a
provlrus within the 3' region of the plm-1 transcription unit exhibit
ed significantly higher plm-1 mRNA levels than lymphomas having a pro"
viral Insertion upstream or downstream of the £lm-1 gene. In the lym
phomas with high plm-1 expression levels, the plm4 mRNA sequences are
covalently linked to viral LTR sequences (Selten et al., 1985). We
have not yet determined whether these differences in plm-1 mRNA level
are caused by difference in transcription rate or result from an al
tered mRNA stability, as observed for myc in murine plasmacytomas
(Plechaczyk et al., 1985).
РІИ-1 Shows Hoeology with Protein Kinases.
Computer analysis shows that the predicted amino acid sequence of
the plm'-l protein is not Identical to that corresponding to any of the
(onco)genes described to date. However, the plm-1 protein sequence re
veals all the domains characteristic of protein kinases. Figure 6
shows a comparison between the predicted amino acid sequence of plm-l
and the amino acid sequences of protein kinases encoded by v-src
(Czernllofsky et al.,1980), v-abl (Reddy et al., 1983), ν-mos (Van
Beveren et al., 1981; Donoghue, 1982), and tck (Voronova and Sefton,
1986), as well as the Ύ-subunlt of Phosphorylase kinase (Reimann et
al., 1984). All protein kinases show a 30 kd catalytic domain (Levin-
son et al., 1981; Brugge and Dar row, 1981; Weinmaster et al., 1983;
Hunter and Cooper, 1985; Van Beveren and Verma, 1985), and this domain
is also found within the plm-1 protein (e.g. compare the plm-1 se-
V —SPC
quence with residues 261-512 in pp60 , Figure 6). The consensus
sequences Gly-X-Gly-X-Phe-Cly-X-Val-X-X-Gly [residues 271-284 for pp60
v-src ] and Val-Ala-X-Lys [residues 292-295 in pp60 v-src ] are com
pletely conserved In the plm-Ί protein. In this region the amino acid
71
«MIMO АСІЭ SEQUENCE CWPtRISCM BETVEEM p H - 1 . t o . ¡ O S , i b i . STO, Ш > T - I u t K U I l t Of РПк
3 0 5 0 7 0 9 0 p l m HATKLAPCKEKEPL£SQTQVGPH.CSCCFGSVTSCIRVAOHL PVAIKHVEKDRI SDWCEL PNC TRVPHEVVLLKKVSSDFSC
Ph4 RD»LPaSHSTHCFtEIIIEPKEILCRCVSSVVRRClHKEPrCKEIAVK[IDVTaOÛS FSAtEV QEL REATLKEVPILRKVSGHI,4 a o s A^CLeRRLAWFSIDHEgVCL'WRLGSCGFaSVÏKATÏHCV PVAIKaVNKCTEDLRASQRSFWAELNIACLnHDNIVRVVAASTRTPE3SNSi.CTn4EFüG а Ы И 0 5 Р 1 ЗКЫЕМЕЯТ0ІтЧКНИ.0ООЗІОЕ ІЕО 1ІКК>ЗІТ—VAVKTLKESTME/E—EFLKEAAVnKEIl!-h'>NLVQI.LGV:TREPPFIIITEF4IÏ t c t P^-KPWrtEJEAE/PRETLK^VERL'ÏAGijrGErfJMCÏÏNGHTK VArfKSLKQGSHSPD—AFLAEANLMKQLa-HPRLVRLTAVVTQrP-m ІТЕГНЕМ s r e POTQGUKDAWEIPRES-RLEAKLCQG-FGEVdH^moTTH VAIKTLKPGTHSPE—AFLGEAQVMKKLR-HEKUQLYAVVSEEP-IÏIVIEÏMSK
¿ 6 0 2 8 0 3 0 0 3¿3 3-Ό
KL Е Е Qt LGSGGFÇ5VÏ GI L PVAIK V KD S fi EL βΝ 'Я Ρ V L SS F О
η : ч ; '50 i-o ч.' Ρ " .'IRLLDlirERPDSFVLILER^EPVi^.rariTERGAbQEOLAHGFFrfQVLEAVRHCHNCGVLHFDIKDENILIDLSrG-:KLIEFGSGALLIC-tfï-OFDG Τ
a*í* IIiaLK2Ty:"IÍTFFFL;rDL4KKGE--F0YLTElCVTLSEKETRKr4nAL-EVICALHKLMVH4D KPENILLDC0M-,j:KLTDF^,-i-.,l-:&GEÍÍb4EVCG Τ
=03 N V T L H C V I Y ^ A T R S P E P L SCRK0LSLGKCLKÏSLD\VNGLLFL-£3SILHL:;LKPAM.,ISEÎLi-*::KI$DFGCS.^LaDLRGR0ASsPHIC*J7 a t ) ! -G4LL:ïLRE CNR(;EVSAVVLLÏHA-Q:SSA4EYLErtKNFI4RrLAA4NCLVGENH-_VKVADFGu5RL4rc:>TÏTA4AGAKF--B
t e k -GSLVDF^KT Ρ50Ιΐα.ΝνΝΚίίΟΗΑΑ0ΐΑεΐ>ΙΑΡ1ΕΕ3ΝΥΙΗΗ214ΑΑ4Ι-ΐ;3Ετ--5ςΜΑβΓΟι.ΑΗί1Ε^Νΐ"ΥΤΑ ;1ΓαΑ'(Γ--Γ s r c -CSLLOF-KG EÏ^GKYLRtaPQLVÛ^«AQlSAGHAYVE^Ì·mYVHHD^RДANILVGEHL-VCK^ADFGL·ДЧ_IEl^EÏTAЧύGAKF--P
350 360 350 •.CO ~20
Ζ LLD Ξ F L L L t D TER L E _R 3VLEA ri LHRD К E N I L n IKL DFG LL D ÏT G
210 2 3 0 2 5 0 270 291 ρ ί τ RVÏSPPErfIRYHR ÏHGRSAAVWSLGILLYDMVC-GDIPFEHDEEIIK GQVFFRffTVSSECaHLIKWCbSLRPSDRPSFEEIRNHPW^J
Phic PSYLAPEIIECSMNDNHPGYGKEVDMkSTGVIMYTLLA-GSPPFHHRKQHLH—LRHIMSC3)YQFGSPEWDDYSDTVKDLVSRFL..'QPgiiftYTAEEALAHPFFQ TOS ITHQAPEILKGEI ATPKADI YSFGITLWQMTT-REVPTSGEPqvVQYAVVAYNLHPSLAGAVFTASLTGKALqNMQSCWEARGLGBPSAELLQRDLKAF a b l IKWTAPESLAÏHK FSIKSDVJAFGVLLWEIATYGMSPYPGIDLSOVÏELLEK—DYRHERPEGCPEKVYE LMRACHQ^NPSDRPSFAEIHÛAFET4
t c ^ ÏKVTAPEAINYGT FTIKSDVWSFGIbLTEIVTHGRIPÏPWTNPEÎ'IliNLER—GYRMVRPDNCPEELÏH LMMLCWKERPEDRPTFDYLRSVLDDF s r c 1KHTAPEAALÏGR mKSÛVl.SFGILLTELTTKGHVPYPGKVNREVLDOVER~CYRMPCPPECPESLHD LMCQCmKDPEERPTFKÏLQAgLLPA
UliO U60 UBO 500 519
Y _PE I Y R G A _ ; H S GILLÏ_MV G IPF H С VF VS О LI CL RPSDRPSFEEIR HP w
Figure 6. Homologies between the Amino Acid sequence of pim-1 and Pro
tein Kinases.
The plm-1 protein sequence, residues 21 to 291, is aligned with se
quences from the γ subunit of Phosphorylase kinase (PhiO and from the
protein kinases encoded by v-mos, v-abl, tck, and v-src. The single-
letter amino acid code is used. The numbering below indicates v-src
sequences from residue 250 to the carboxy terminus (residue 519).
Homology of plm-1 with any of the other sequences is indicated below;
underlined residues are highly conserved in protein kinases (van
Beveren and Verma, 1985).
sequence homology is closest between plm-1 and v-mos. Other highly
conserved sequences present in protein kinases as well as in pim-1 are
Hls-Arg-Asp-X-X-X-X-Asn-X [residues 384-392] and Lys-X-X-Asp-Phe-Gly
[Д01-Ц06 in pp60 v-src ]. The tyrosine at position 415 In pp60 ν src
that Is a target for autophospho^ylation and that is conserved In the
catalytic domain of all tyrosine kinases (Patschinsky et al.,1982;
Snyder ani Bishop, 1981) is also present in the pim-l protein at amino
acid redidue 198 (Figure 6). The consensus sequence Asp-Val-Trp-Ser-
X^Gly-Ile-^eu-Leu-X-X-X-X-X-X-Cly-X-X-Pro-Tyr seen in tyrosine kinases
[residues і|Чі|-і)63 in pp60 v-src ] is almost completely retained in
plm-1 [residues пгЧ-гЧЗ]. At the carboxy terminus the homology із
greatest between plm-1 [residues 275-284] and v-abl. The overall
homology of plm-1 with the various protein kinases is reflected by the
72
Sequence
зге
Ï£S
fgr
LSTRA
abl
fps
Р56
fes (ST)
ros
fms
erbB
trk
mil
raf
mos
cAPK
cGPK
MLCK
PhK
Alignment
Versus
5.5
6.9
5.4
8.1
7.3
7.2
9.0
3.9
5.3
3.7
2.H
5.1
U.!»
Θ.1
10.3
11.1
Θ.3
11.8
plm-
scores
•1 Versus
103.1
89.1
72.6
46.0
33.0
35.0
35.2
25.7
24.4
23.6
21.1
19.1
11.1
4.9
6.9
10.9
4.9
Table 1. Сошрагізоп of Amino acid Sequences of pirn·*! and Protein Kinases.
Comparisons were made using the ALIGN program (Dayhoff, 1979) with a unitary matrix, with the exception that cysteine was scored as 2. The gap penalty was 3 and 100 random runs were carried out for each comparison. The average standard deviation for the group of random comparisons was 2.011 + 0.33. The alignment score is the real score (the number of identities minus penalties for gaps) minus the mean random score divided by the average standard deviation. Scores greater than two plus the average standard deviation are considered significant. Sequences were compared in the region between amino acid residues 250-519 (for arc) (see also Figure 6 ) . Sequences for comparison were obtained from the following sources: arc, yes, fgr, abl, fps, fea (ST), ros, fms, erb-B, mll/raf, mos (Weiss et al., 198577 LSTRA p56 (tck) (Voro*· nova and Sefton, 1986); trk, (Martin Zanca et al., 1986) cAPK (Shojl et al., 1981) cGPK (Takio et al., 1984) MLCK (Takio et al., 1985)! PhK (Relmann et al., 1984). Abbreviations: cAPK, cAMP-dependent protein kinase; cGPK, cGMP-dependent protein kinase; MLCK, myosin light chain kinase; PhK, Phosphorylase kinase.
alignment score shown In Table 1. From this alignment It appears that
plm-1 has the highest homology with protein-aerlne kinases. The con
servation of amino acids in the plm-1 protein at positions that are
required for the enzymatic activity of protein kinases stongly aug-
geats that plm-1 belongs to the class of proto-Oncogenes encoding pro-
73
teln kinases. Experiments are In progress to determine whether the
plm-l protein has serine/threonine kinase or tyrosine kinase activity.
DISCUSSION
The Structure of the pla^l Gene.
The results reported here show that the plm-1 gene contains 6 cod"
ing exons. The total length of these 6 exons is approximately 2600 nu
cleotides, close to the mRNA size of 2.8 kb, which Includes the
poly(A) tall (Selten et al., 1985). The splice sites conform to the
typical canonical sequences (Breathnach and Chambón, 1981; Mount,
1982). The 3' exon is rauch larger than the others and 1314 nucleotides
of this exon are noncoding. Although two poly(A) addition signals are
found at the 3' end, the sequences of a number of independently
derived plm-1 cDNAs revealed that only the second poly(A) addition
signal, at position 6976, was used. The 5' end of the plm-1 gene
remains less welldeflned. Two oligonucleotide primed cDNAs ended at
position 1719; thus we predict a 5' exon of И 9 nucleotides. Exons of
slightly larger size as well as somewhat smaller size were found by SI
nuclease protection analysis, suggesting the presence of multiple Ini
tiation sites for mRNA transcription. No TATA consensus sequence was
found upstream of this region. However, 8 CCGCCC motifs were present
in the 250 nucleotides preceding one of the putative transcription in
itiation sites. This sequence, which is found in multiple copies in a
number of promoters, such as the human and mouse hypoxanthine rlbosyl-
transferase genes (Patel et al., 1986; Melton et al., 1986), the human
Harvey газ proto-oncogene (Ishil et al., 1985a), the epidermal growth
factor receptor gene (Ishll et al., 1985b), and the adenosine deam
inase gene (Valerlo et al., 1985), has been shown to bind specifically
the transcription factor SP-1 (reviewed by Dynan and TJlan, 1985).
Transcription from these promoters lacking TATA box sequences has been
shown to start at multiple sites. The octamer motif ATGCAAAT, which Is
present upstream of the promoter of the immunoglobulin genes (Falkner
and Zachau, 1984; Parslow et al., 1984) and which interacts with lym
phoid specific transacting factors (Singh et al., 1986), is also found
74
approx. 280 bp upstream of ρ Inri exon 1 at position 1138. This Is
normally found approximately 70 bp upstream of the RNA cap site. How
ever, in some Immunoglobulin genes this motif is present at a position
similar to the one it has in the plm-l gene (Neuberger, 1983). In ad
dition, cruciform structures with significant stability can be formed
in this region. Although these characteristics are Indicative of a
promoter region, we still have to prove that this region has promoter
activity and is used for plm-1 transcription. Furthermore, it is Im
portant to recall that the cDNAs used to Identify the 5' region were
obtained from mRNAs present In tumors in which plm-1 was transcrip
tionally activated by Integrated provlruses. Therefore, it is possible
that in normal cells transcription is initiated at different sites.
The first exon contains a single AUG codon, which is followed by a
313 amino acid long ORF extending over б exons. The AUG codon is pre-*
ceded by stop codons In all three reading frames. The noncoding рог-*
tlon of exon 1 is at least 337 nucleotides long (based on cDNA
clones). Thus, In spite of the uncertainties concerning the 5' end of
the plm-1 gene, there Is little doubt about the deduced amino acid se
quence of the corresponding protein.
Transcriptional Activation.
The primary effect of provlral Insertion appears to be enhanced
plm-l expression. Although the majority of provlral Integrations are
clustered within the 3' exon, the plm-1 coding sequence Itself is
spared in all Integrations examined to date. All provlruses Integrated
in this 3' exon are oriented away from the body of the plma1 tran
scription unit, in accordance with the enhancement model of gene a c
tlvation. This situation is reminiscent of the activation of lnt-1 ,
in which Integration in the non-coding region of the 3' exon Is also
observed (Van Ooyen and Nusse, 1984). Tumors in which provlruses were
Integrated within the 3' exon showed the highest plm-1 mRNA levels.
Provlral integration in a transcription unit is likely to affect the
transcription rate, and It may also affect the stability of the mRNA.
The contribution of each of these factors to the steady state level of
plm-l mRNA has not been determined. The high plmtt1 expression level
seen In tumors with provlruses In the 3' exon might have provided the
75
driving force for their selective outgrowth, and might therefore
responsible for the high Incidence of these provlral integrations In
outgrown tumors.
Provlruses upstream of exon 1, which were found In both transcrip
tional orientations, gave rise to only a moderately enhanced expres
sion level. The plm-Ί mRNA In these tumors was Indistinguishable from
the normal plm-1 mRNA and no hybrid transcripts were detected (Selten
et al., 1985).
The рІш-Ί Protein.
The deduced amino acid sequence of the plm-1 gene shows that the
plm-1 polypeptide is 313 amino acids long. A hydrophobicity plot does
not reveal a long hydrophobic amino terminus or any other signal pep
tide that might direct the gene product to a membrane within the cell.
Plra-1 shows a remarkable amino acid sequence homology with protein
kinases. The highest homologies were observed with the Y-subunlt of
Phosphorylase kinase (Reimann et al.,1984) and with cGMP dependent
protein kinase (Takio et al., 1984; see Figure 6 and Table 1). In the
amino-termlnal region of the catalytic domain the homology with mos
was remarkably high, whereas at the carboxytermlnus close resemblance
with abl was observed. All the domains conserved in protein kinases
are found In plmH. At present we cannot predict whether plm-1 Is a
serine/threonine kinase or a tyrosine kinase. The tyrosine present at
position 41 б In v-src , which can serve as an autophosphorylatlon site
in all tyrosine kinases. Is also conserved In plm-1• Plm-1 probably
belongs to a class of lymphoid specific protein kinases that fulfills
a role in the Intracellular transduction of signals Involved in the
proliferative stimulation of lymphocytes. The recently Identified T-
cell specific tyrosine protein kinase from LSTRA cells (tck) might
serve a similar function (Voronova et al., 1984; Marth etal., 1985).
Both plm-l and tck are activated by provlral Insertions that spare the
protein-encoding domains (Voronova and Sefton, 1986, this work); nei
ther gene has undergone mutations affecting the amino acid sequence.
Apparently, the Increased expression of plm-1 and tck is sufficient to
confer a selective (growth) advantage to the cell. Experiments are in
progress to identify the enzymatic activity and cellular localization
76
of the plm-1 gene product.
EXPERIMENTAL PROCEDURES
MuLV-Induced Ьушрііоааз
The origin of MuLV-induced lymphomas was decrlbed previously (Cuypers
et al., ІЭв^; Selten et al., 198U, 1985).
Molecular Cloning of Genoalc DNA Fragments
An 11 kb EcoRI plm-1 DNA fragment, located between positions *9.6 and
+1.3 on the plm-Ί map (see Figure 1) was molecularly cloned from nor
mal BALB/c DNA. From the cloned plm-1 DNA fragment, subclones were
derived in PBR322 and/or рЗРбЧ/бб Plasmids. The cloning strategy for
virus-host DNA Junctions of lymphomas 1 and 15 has been described pre
viously (Cuypers et al., 1984; Selten et al., 1985).
Hybridization Probes
Plm-1 specific probes were described previously (Cuypers et al., 1984;
Selten et al., 1985). The actin cDNA clone was described by Dodemont
et al. (1982).
DNA Sequence Analysis
From the plm-1 region, 8000 bp was sequenced from both strands, with
overlapping sequences across every restriction site, plm-1 DNA frag
ments, subcloned in pBR322 covering the SacI/EcoRI DNA fragment
between positions -6.6 and +1.3 on the plm-1 map, were digested with
Sau3A, TaqI, or Hpall, and were then subcloned in phage M13· DNA
sequencing was performed by the dideoxynucleotlde chain termination
method (Sanger et al., 1980).
Synthesis and Molecular Cloning of cDNAs
DNAs complementary to poly (A) RNA from lymphomas 9, 10, and 54 were
synthesized as follows. 10 yg of polyA RNA, treated with methylraercu-
ric hydroxide, was neutralized with ß-mercaptoethanol. This mixture
was adjusted to 50 mM Tris-HCl (pH 8.3). 40 mM KCl, 6 гаМ MgCl , 10 mM
DTT, 3000 U RNasln/ral, 4 mM NaLP 0 , 50 pg actinomycln D/ml, 0.1 mg
77
ollgo(dT) (12-l8)/ml or 0.1 mg synthetic plmH specific ollgomer/ml,
0.5 шМ dGTP, 0.5mM dATP, 0.5mM dTTP, 0.25 шМ dCTP and 100 μϋΐ o3 2P-
dCTP/ml, all In a final volume of 100 μΐ. The mixture was put on ice
for 10 minutes, heated for 2 minutes at 12 С, and the synthesis was
started by addition of 150 U AMV reverse transcriptase; Incubation was
performed for 1 hr at Ц20С. Second'-strand synthesis was performed by
adding DNA polymerase and RNAase Η according to Gubler and Hoffman
(1983). After treatment of the ds DNA with ТЧ DNA polymerase to obtain
blunt ends, decamerlc EcoRI linkers were ligated to the dsDNA frag
ments. After digestion with EcoRI, double stranded cDNA fragments were
separated from EcoRI linkers by Biogel A15m chromatography. Approxi
mately 200 ng EcoRI linker containing double-stranded cDNA was ligated
with 10 yg of EcoRI digested and calf Intestine-phosphatase treated
Xgt 11 vector DNA by T4 DNA ligase (Huynh et al., 1985). An average of
1.5 x 10 plaque forming units of phage per 200 ng cDNA was obtained
after in vitro packaging of the ligation mixture. Libraries were
32 screened with P-labeled plm-1 probes. Clones containing plm-1 EcoRI inserts were subcloned in pSPôt.
Preparation of RNA
Total cellular RNA was obtained as described previously (Selten et
al., 1985). To obtain poly(A) RNA, total cellular RNA samples were
heated at 65 С for 5 minutes prior to poly(A) selection by oligo(dT)
chromatography.
Dot-Blot RNA Analysis
For dot-blot analysis, poly(A) RNA was dissolved in distilled water
and then applied to nitrocellulose filters as described by Muller et
al. (1982). Before exposure to X-ray film, filters were washed as
previously described (Selten et al., 1985).
RNAase Happing
First, 0.2 vg of a plm-1 BamHI DNA fragment (probe B, between posi
tion, 5642 and 6858 in Figure 2), subcloned in pSP64 and linearized at
32 the EcoRI site (in the polylinker), was transcribed to
J P-labeled
32 plm-1 RNA by RNA polymerase using [a- P]-CTP as labeled rlbonucleo'·
tide under conditions indicated by the supplier (Promega Biotec). The
78
reaction mixture was treated with DNAase, extracted with a
phenol/chloroform mixture, passed over a Sephadex G-50 column, and
precipitated with ethanol. Next, 1-2 ng P·4 labeled plm-Ί RNA was
mixed with 3 ug lymphoma poly(A) RNA In 30 μΐ of a hybridization mix
containing 80S formamlde, 10 mM Pipes (pH 6.1), 1 mM EDTA and 400 mM
NaCl. After heating to 85 С for 10 minutes, samples were Incubated for
3-6 hours at 370C and 10-12 hours at 30 C. Samples were treated with
RNAase A and RNAase T., deprotelnlzed, and precipitated twice with
ethanol In the presence of 10 ug tRNA and IM aomoniumacetate. Protect-
ed RNA fragments were separated on a 6$ Polyacrylamide gel containing
8 M urea and were analyzed by exposure to X"ray film.
Acknowledgaents
This work was performed in the laboratory of Dr. H. Bloemendal, to
whom we are particularly Indebted. We appreciate the assistance of Jos
Rewinkel in the sequencing of the plm-1 region. We thank Dr. C.
Klaassen for the preparation of plm-1 specific oligonucleotides and
Mrs. M.H. Sonne-Gooren and Mrs. P.E.T. van Nuland" van Wljngaarde for
typing the manuscript. This work was supported by grants from the
Koningin Wilhelmina Fonds (G. S.) and by the Netherlands Organization
for the Advancement of Pure Research (ZWO) through the Foundation for
Fundamental Medical Research (FUNGO).
Received March 17, 1986; revised May 12, 1986.
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86
CHAPTER 6
THE ONCOGENE PIM-1 IS PREDOMINANTLY
EXPRESSED IN LYMPHOID TISSUES DURING
PRE- AND POSTNATAL DEVELOPMENT
THE ONCOGENE PIH-1 IS PREDOMINANTLY EXPRESSED IN LYMPHOID
TISSUES DURING PRE«· AND POSTNATAL DEVELOPMENT.
# #* + #* Gerard Selten , H. Theo Cuypers , Maarten Zylstra and Anton Berns
Department of Biochemistry, University of Nijmegen, Geert Grooteplein
Noord 21, 6525 EZ Nijmegen, The Netherlands.
t Section of Molecular Genetica of the Netherlands Cancer Institute and
Department of Biochemistry of the University of Amsterdam, Plesmanlaan
121, 1066 CX Amsterdam, The Netherlands.
Central Laboratory of the Netherlands Red Cross Transfusion Service,
P.O. Box 9406, 1006 AK Amsterdam, The Netherlands.
89
ABSTRACT
The expression of plm "1, which was Identified by the nearby lnu
tegratlon of provlruses In MuLV Induced T*cell lymphomas, was studied
In normal mouse tissues and cell lines. Expression of plm-1 was
predominantly found In hematopoietic cells and followed the migration
of these cells during development. High plm-1 mflNA levels were also
frequently found In lymphoid amd myeloid tissue culture cell lines.
The plm-1 gene Is an oncogene discovered as a target for frequent
provlral Integration In Murine Leukemia Virus (MuLV) Induced lymphomas
(Cuypers et al., 1981; Selten et al., 1985). MuLVs belong to the
class of slow transforming retroviruses, lacking transforming on-«
cogenes in their genomes, in contrast to their counterparts, the acute
transforming retroviruses. Insertional mutagenesis by provirai in
tegration near proto-Oncogenes in the host genome which become tran-1
scrlptionally activated, is commonly accepted as a mechanism for the
transforming potential of the slow transforming retroviruses (Hayward
et al., 1981; Nusse and Varmus, 1982; Peters et al., 1983; Fung et
al., 1983; Corcoran et al., 1984; Cuypers et al., 1981). In MuLV-
Induced T-*cell lymphomas in mice the oncogenes plm-1 (Cuypers et al.,
1984; Selten et al., 1985) and Cmyc (Corcoran et al., 1984; Selten et
al., 1985) were activated by adjacent proviral insertions. Integra*-
tion into the рІш-Ч locus occurred at three different domains and In
all tumors, except in one with a provlrus Integrated upstream from the
gene, the provlrus had the same transcriptional orientation as plm-1
(figure 1; Selten et al., 1985).
A 2.8 kb sized plm-1 mRNA transcript is observed In lymphomas with
an Integrated provirus positioned upstream or downstream (e.g. lympho
ma 9 in figure 3) the ploM gene. Truncated ріш-і mRNAs were detected
in 25 lymphomas (e.g. Lymphoma 54 in figure 3). In all these 25 lym
phomas, the provirus was integrated in the 3' region of the plm-1
90
β S 4 3 2 1 0 1 8 КЬр
I • ! — Ρ — - Я С PSc Ρ В PB
Figure 1 : Physical map of the pim-1 locus.
Exons are indicated by black (coding) and open (non-coding sequences) boxes. Large horizontal arrows shows the sites as well as transcription direction of integrated proviruses in the plm-1 locus. Transcription direction of the plm-1 gene is from left to the right on this map. Figures Indicate number independent lymphomas harboring a provirus at that position. Abbreviations used for restriction endonu-cleases: B, BamHI ; E, EcoRI ; P, Pstl ; К Kpnl ; Sc, Sad.
transcription unit (Selten et al., 1985). However, the plm-1 protein
encoding domain was always spared (figure 1; Selten et al., 1986).
This Indicates that enhanced expression of plm-1 contributes to tumor-
genesis.
To obtain Insight in the functional role of plm-1 , we have studied
the tissue distribution and level of plm-1 mRNA in mice during
embryonic- and postnatal development.
An appropriate number of embryonic organs or tissues (strain 129)
were pooled and total RNA was isolated from liver on day 16 and from
thymus, spleen, liver, hart, lung, kidney and brain on day 19 of ges
tation. RNA was also isolated from placental tissues on day 12, 16 and
19. The same organs were collected after 4 days, 2 weeks, 4 weeks and
3 months after birth. RNA from bone marrow and lymphatic nodes was
only obtained from adult mice. PolyA RNA was selected by ollgo(dT)
chromatography and applied to nitrocellulose filters. The quantity of
spotted RNA was determined by hybridization with a ' P-labeled cDNA
actin probe (figure 2). Hybridization with a J P-labeled plm-1
specific probe showed a distinct expresssion pattern of plm-1 during
development (figure 2). Plm-1 mRNA expression is predominantly res
tricted to hematopoietic tissues, namely spleen, thymus, lymphatic
nodes, bone marrow and embryonic liver. Only minor or no plm-1 mRNA
91
a PIM-1 EXPRESSION
tissue
^gpolyA^RNA - 4
gestation
newborn
adult
day 12
day 16
day 19
day 4
day 14
week 4
Ψ month 3
thymus
1 02
spleen
1 02
• Ш
liver
1 0.2
• «
placenta
1 02
adult lymph nodes
bone marrow
b PIM-1
yitq poly+RNA> 1 0 2
ACTIN
1 0 2
SP-2 (myeloid)
YAC (Τ-type)
2M3 (B-type)
WEH 3 (myeloid)
RADA-1 (Τ-type)
13-4 (B-type)
CTLL (T-type)
• «
#
• · • ·
• · • -
LYMPH NODES
BONE MARROW %
THYMUS
SPLEEN
92
Figure 2: Pim-1 expression In
developing tissues and cell lines.
+
a. 1 and 0.2 yg polyA RNA iso
lated from developing tissues at
Indicated stages were applied to
nitrocellulose filter and hybri
dized to a P-labeled specific
plm-1 probe (gelten et al., 1985).
Spotted polyA RNA from lymphatic
nodes and bone marrow are of adult
origin. No RNA was spotted on dots
Indicated by "-".
b.+Hybridlzation of 1 and 0.2 yg
polyA RNA containing dot 3pota?of
indicated cell lines with a P-
labeled specific pira-1 probe are
shown. Between brackets the nature
of cell lines is indicated. After
autoradiography, all dot blots were
washed and hybridized to an cDNA
actin probe as control for amount
spotted RNA (Selten et al., 1985).
Only actin hybridization assay of
blot (b) is shown аз example.
expression was seen in hart, kidney, lung and brain at any developmen
tal stage and in adult liver. Only organs with significant amounts of
plm-1 mRNA are shown in figure 2. Pim-1 mRNA levels varied among
these lymphoid tissues and depend on the developmental stage. In
thymus, the highest levels were observed around birth and in spleen
shortly after birth. In both tissues the plm-1 mRNA levels decrease in
later life, but are still detectable in the adult. The plm-1 expres"
Sion is seen in both T- and B-lymphocytes. Separation of splenic cells
In T- and B-cells showed a similar plm-1 expression in both cell types
(not shown). The expression of plm-1 in lymphatic nodules and bone
marrow cells was comparable to plm-1 mRNA levels in splenic and thymic
tissue (figure 2a).
Highest plm-1 mRNA levels in the liver were detected at day 16 to 19
of gestation and decreased later on with exception of the Ч-week old
liver, in which slighty higher amounts plnM mRNA was found. In adult
liver plm-1 mRNA levels were extremely low (figure 2a). In placenta
the plm-1 mRNA level was high at day 12 of gestation and decreased
later on (figure 2a).
These data clearly indicate that the pim-1 gene is predominantly tran*·
scribed in tissues involved in hematopoiesis and that the level
changes during development.
Plm-1 expression in vitro was studied in a number of lymphoid (T-
and B-type), аз well аз myeloid cell lines. Myeloid cell lines SP-2
and WEH-3 cells show high plm-1 mRNA levels (figure 2b). However,
highest plm-1 mRNA levels in vitro were measured in B-Туре cell lines
as shown in figure 2b for 2M3 and 13-4 cells. A low plm-1 expression
level is observed in the T-type cell lines Yac and CTLL. In Rada-1
cells (T-type) no plm-1 mRNA could be detected.
A 2.8 kb plm-1 transcript was observed in normal adult tissues. In
MuLV-lnduced lymphomas, however, normal sized plm-1 mRNAs (e.g. lym
phoma 9 in figure 3) as well as truncated 2.0 kb transcripts were ob
served (e.g. lymphoma 5^ in figure 3). The cell lines analysed also
showed the predominant 2.8 kb plm-1 transcript (figure 3). Since total
RNA was isolated, also plm-1 mRNA precursors are detected. In normal
tissues predominantly a 2.8 kb sized plm-1 transcript Is determined.
In polyadenylated RNA of placenta, however, an aberrant plm-1 mRNA is
determined. So far, no data are available whether alternative Initia-
93
CELL-LINES TUMORS NORMAL TISSUES
SP-2 2M3 WEH· 13-4 9 54 THY SPL LIV PLA LYM
Figure 3: Northern analyses of pim-lmRNAs.
Total cellular polyA RNA (7.5 ug formaldehyde treated) were separated on a 1.2$ agarose gel, transferred to nitrocellulose filter and hybridized to a ^ P-labeled specific pim-1 probe. Type of cell lines are indicated in the legend of figure 2, origin of tumors 9 and 51 are described ealier (Selten et al., 1985). The stage containing the highest plm'-l mRNA levels in developing tissues were chosen for northern analysis (see fig. 2): THY, thymus at day Ц; SPL, spleen at day Hj LIV, 16 day old embryonic liver; PLA, placenta at day 12 of gestation; LYM, adult lymphatic nodes. As markers, HindXII digested lambda DNA fragments were treated as RNA samples.
tion, termination or splicing is involved.
The observed restriction of pira-1 expression to hematopoietic tis
sues during embryogenesis and postnatal development, as well as the
high expression levels in lymphoid cell lines and viral induced lym
phomas suggest a regulatory role of the pim-1 gene in the hematopoiet
ic system. The observation of pim-1 expression in T- and B-lymphoid as
well as in myeloid cell lines might indicate that ріш-1 is involved in
the differentiation or proliferation process of hematopoietic precur-
94
sors common to myeloid and lymphoid lineages (Keller et al., 1985).
Nagarajan et al.(1986) showed that also in many human cell lines of a
hematopoietic lineage hplm-1 (human plm"1 ) Is also expressed at vari
ous levels. Aspeclally In the К 562 erythroleukemla cell line, which
contains a rearrangement In the 6p21 region, a considerable amount of
hplm-1 mRNA was detectable. The observation that the human homolog of
plm-1 is localized within this 6p21 region (Nagarajan et al., 1966;
Cuypers et al., 19Θ6) suggest that in the frequently observed translo
cation t(6;9)(p21 ;q33) in human erythro-* and myeloid leukemlas could
Involve the hplnM gene.
ACnOHLEDGEMENTS
We thank Dr. H. Bloemendal, in whose laboratory this work was per"
formed and A. van de Kemp for preparing embryonic tissues. G.S. was
supported by the Netherlands Cancer Society (Koningin Wilhelmina
Fonds).
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mice: frequency and mechanisms of activation. EMBO J. 3: 3215-3222.
- Selten, G., Cuypers, H.T. and Berns, Α. (1985). Proviral activation
of the putative oncogene plm-1 in MuLV^lnduced T-cell lymphomas.
EMBO J. 1: 1793·*1798.
- Selten, G., Cuypers, H.T., Boelens, W., Robanus-Maandag, E., Ver
beek, J., Domen, J., van Beveren, C. and Berns, A. (1986). The pri
mary structure of the putative oncogene plm*4 shows extensive
homology with Protein Kinases. Cell 46, in press.
96
SUMMARY
In the last few years substantial progress Is made In cancer
research by studying RNA tumor viruses Induced lymphomagenesls. RNA
tumor viruses Induce predominantly leukemlas, lymphomas and sarcomas
dependent on retroviral origin. The "acute" transforming retroviruses
develop malignant tumors within several weeks. It appeared that this
highly oncogenic potential Is due to the Incorporation of oncogenes
(v-onc) In the retroviral genome. Expression of v-oncogene proteins
enables "acute" transforming retroviruses to Induce and maintain neo
plasia.
"Non-acute" or slow transforming retroviruses, like Murine Leukemia
Viruses (MuLVs), lack transforming genes and develop leukemlas and
lymphomas only after a long latency period of Infection (several
months). The "non-acute" RNA tumor viruses exert their oncogenic ac~
tlon by proviral insertion. Analysis of retroviral induced lymphomas
revealed that provlruses Integrate within or In the vicinity of cellu
lar (onco)genes, which become activated by virtue of enhancer and/or
promoter sequences present In the LTR.
Identification, Isolation and characterization of provlrally ac
tivated cellular oncogenes in MuLV-induced leukemogenesis in mice is
the scope of this thesis.
Chapter 1_ gives a brief outline of cellular- or proto'Oncogenes in
volved in tumorigenesis and describes how RNA tumor viruses can in
teract with the cellular oncogenes. Additionally, general charac
teristics of RNA tumor viruses are described.
In chapter 2, the identification and Isolation of a common chromo
somal provlral Integration region in MuLV-induced lymphomas is
described. Different virus-host DNA Junctions were molecularly cloned
from lymphoma DNAs and flanking cellular probes were derived. These
probes were used to determine whether in other MuLV-induced lymphomas
a provirus was Integrated in the same chromosomal domain. In one
domain, which we named Pim-1, rearrangements by provlral Integration
were observed in approximately 50Í of early T-cell lymphomas. The fre
quency of provlral integration appeared to be strongly dependent on
the latency of tumor development. These alterations were observed in
99
tumors Induced by various MuLVs and in different mouse strains. More
over, Plm-1 rearrangements were associated with the transcriptional
activation of a distinct region within the Plm-1 domain, suggesting an
important role in the generation of early TBcell lymphomas.
Chapter ¿ describes the Involvement of the cellular myc gene In
MuLV-lnduced lymphomas. Proviral Insertion mutagenesis of the c-myc
gene was observed in approximately 45% of the tumors, with concomit
tant enhanced c-myc mRNA levels. Additionally, In nearly 20$ of the
early T-cell lymphomas provlral integration occurred near c-myc as
well as near Pim-1. The presented data show that proviruses were in
tegrated exclusively upstream the coding domain of the c-myc gene with
a transcriptional orientation predominantly opposite to the transcrip
tion direction of c-myc. The activation of c-myc in MuLV-lnduced lym
phomas occurred predominantly by virtue of the enhancer activity of
Inserted proviral LTR sequences. This in contrast to the promoter
insertion mode of c-myc activation as observed in avian leukosis virus
Induced lymphomas. Obviously, the position and orientation of the in
tegrated provlrus with respect to the flanking gene determine to a
large extend the mechanism of c-myc activation.
In chapter ¡\_ data are reported concerning the structure and posi
tion of integrated proviruses within the Pirn·*! region as well as the
nature of the enhanced Pim-Ч mRNA transcripts. Predominantly intact
MCF proviruses were Integrated at three distinct domains within the
Pim-1 region. The majority of provlral integrations occurred in the 3'
region of the Pim-1 transcription unit with the same transcriptional
orientation as Plm-1. Activation of plm-1, observed in all lymphomas
showing an Inserted provirus, occurred almost exclusively by enhance
ment. Normal sized 2.8 kb Pirn1-! mRNA transcripts were only detected In
a minor population of lymphomas. As a result of a provlral integration
into the 3' Pim-1 transcription unit, the majority of MuLV-lnduced
lymphomas showed Pim-1 transcripts ranging in size from 2.0 to 2.6 kb,
which were covalently linked to viral LTR sequences.
Chapter 5 describes the structure of the Plm-1 gene as well as the
homology of the Pim-1 protein with oncogenic encoded protein kinaeses.
The pim-1 gene encoding domain extends over 6 exons and encodes a pro
tein of approximately 35.5 KD. Proviruses were integrated preferen
tially In the 3' terminal exon, resulting in relatively high Pira-1
100
mRNA levels. However, proviral Integrations never affected the pro
tein encoding domain. Apparently, the Increased level of expression of
Plm-1 Is sufficient to confer a selective (growth) advantage to the
cell. Computer analysis revealed that all the domains, characteristic
for protein kinases are also found In the proposed Plm-1 amino acid
sequence. However, the Plm-1 protein Is not identical to any of the
oncogenes described to date.
Chapter 6 deals with the expression of Plmu1 In tissues of normal
mice during embryonic and postnatal development. Expression of Plm-1
was predominantly found In hematopoietic cells and followed the migra
tion of these cells during development. Moreover, high Plm-1 mRNA lev
els were frequently found in T- and B-lymphold as well as in myeloid
tissue culture cell lines. The presented data indicate that Pira-1
might be involved In the differentiation or proliferation of hemato
poietic cell lineages.
101
SAMENVATTING
In de afgelopen Jaren Is het Inzicht In het on3taan van kanker be
langrijk toegenomen. Daar heeft ook de bestudering van de door RNA
tumorvirussen geïnduceerde lymfomen toe bijgedragen. RNA tumor
virussen Induceren, afhankelijk van hun retrovlrale origine, voor
namelijk leukemleën, lymfomen en sarcomen. De "acuut" transformerende
retrovirussen ontwikkelen in enkele weken maligne tumoren. Hun sterk
oncogene karakter is toe te schrijven aan de aanwezigheid van on-
cogenen (v-onc) In het retrovlrale genoom. Expressie van v-onc eiwit
ten resulteert in de inductie van neoplasmas.
"ΝΙβΙ-ΒΟΗΐθ" of langzaam transformerende tumorvirussen, zoals de
muizeleukemlevlrussen (HuLVs) bezitten geen transformerende genen,
maar zijn toch in staat om lymfomen te ontwikkelen na een lange
periode van viremie (enkele maanden). Deze RNA tumorvirussen veroor
zaken tumoren door provlrale Insertie mutagenese. Uit de analyse van
retroviraal geïnduceerde lymfomen bleek, dat provlrussen Integreren
in, of nabij cellulaire (onco)genen en dat deze geactiveerd worden
door enhancer/promoter sequenties, welke aanwezig zijn in de LTR.
Identificatie, isolatie en karakterisering van proviraal geac
tiveerde cellulaire oncogenen vormen de essentie van dit proefschrift.
Hoofdstuk 1_ geeft een korte samenvatting van de cellulaire on
cogenen of proto-oncogenen, die betrokken zijn in de tumorgenese en
beschrijft op welke wijze RNA tomorvirussen interacties aangaan met
die cellulaire oncogenen. Tevens komen in dit hoofdstuk een aantal
algemene aspecten van RNA tumorvirussen aan de orde.
Hoofdstuk 2 beschrijft de identificatie en isolatie van een gemeen
schappelijke chromosomale provlrale Integratie plaats in MuLV geïndu
ceerde lymfomen. Verschillende virus-gastheer DNA sequenties werden
gekloneerd uit lymfoom DNAs. Flankerende cellulaire probes werden
geïsoleerd en gebruikt om na te gaan of ook in andere MuLV geïndu
ceerde lymfomen provlrussen In hetzelfde chromosomale gebied gelnter-
greerd waren. In het gebied, dat we Ріш-Ч genoemd hebben, werd een
verandering door provirale integratie gedetecteerd in ongeveer 50% van
de vroege T-cel lymfomen. De frequentie van provlrale integratie
bleek sterk afhankelijk te zijn van de latentietijd van tumorontwikk-
103
ellng. Deze veranderingen werden geconstateerd in tumoren geïnduceerd
door verscheidene MuLVs en In verschillende mulzestammen. Bovendien
hadden de veranderingen In het Pim-1 gebied tot gevolg, dat een
bepaald gebied van het Pim-1 locus transcriptloneel geactiveerd werd.
Deze activering suggereert dat dit gebied een belangrijke rol zou kun-*
nen spelen bij het veroorzaken van vroege T-cel lymfomen.
In hoofdstuk 3 wordt beschreven op welke wijze het cellulaire myc
gen door MuLV kan worden geactiveerd in vroege T-cel lymfomen. Pro
virale insertie-mutagenese van het c-myc gen werd waargenomen in
ongeveer 45% van de lymfomen, vergezeld van verhoogde c-myc mRNA
niveaus. In bijna 20$ van deze vroege T-cel lymfomen trad niet alleen
provirale integratie In het счпус gen op, maar ook in het Plm-1 gen.
De gegevens laten zien dat de provirussen stroomopwaarts van het
coderende gedeelte van c-myc waren geïntegreerd, hoofdzakelijk met een
transcrlptionele oriëntatie, die tegengesteld was t.o.v. de c-myc
transcriptie-richting. De activering van c-myc in MuLV geïnduceerde
lymfomen geschiedde voornamelijk door "enhancer" activiteit van geln-
serteerde provirale sequenties. Dit in tegenstelling tot de c-myc ac
tivering door promoter-insertie zoals beschreven voor lymfomen geïndu
ceerd door "avian" leukemie-vlrus. Blijkbaar bepaalt de positie en
oriëntatie van het geïntegreerde provlrus t.a.v. het flankerende c-myc
gen, in belangrijke mate het mechanisme van activering.
In hoofdstuk k worden gegevens gepresenteerd betreffende de struk«-
tuur en positie van geïntegreerde provirussen In het Pim-1 locus,
alsmede de aard van de verhoogde plra-1 mRNA transcripten. Integratie
van hoofdzakelijk intacte provirussen vond plaats In 3 karakteristieke
domeinen. De meerderheid van de provirale integraties werd gedetec-1
teerd in het 3' deel van het Plm-1 transcriptiegebied, met dezelfde
oriëntatie als plm-1. Activering van Plnrl, waargenomen in alle tu
moren met een geinserteerd provirus, geschiedde voornamelijk door
"enhancement". Normale Pim-1 mRNA transcripten ter grootte van 2.8 kb
werden slechts in een kleine populatie lymfomen waargenomen. Pro1-
virale Integratie in het 3' transcriptie gedeelte, had tot gevolg dat
de meerderheid van de MuLV geïnduceerde tumoren Pira-1 mRNA transcrip
ten vertoonden die covalent gebonden waren aan virale LTR sequenties
en varleerden in grootte van 2,0 tot 2,6 kb.
104
Hoofdstuk 5 beschrijft naast de struktuur van het Plm-1 gen, de
horaologle van het Pim-1 eiwit met proteine kinases. Het Pim-1
coderende domein is verdeeld over 6 exonen en codeert een eiwit van
35,5 KD. Vele provirussen waren preferentieel geïntegreerd In het 3'
terminale exon. Dit resulteerde in relatief hoge Plm-1 mRNA niveaus.
Echter, provlrale integraties werden nooit waargenomen in het eiwit
coderende domein. Blijkbaar is een verhoogde expressie van Plm-1 vol
doende, om de cel een selectief (groei)voordeel te geven. Uit compu-*
teranalyses bleek dat alle domeinen, karakteristiek voor proteine ki
nases, ook aanwezig zijn in de aminozuur volgorde van Plm-1. Het Plm'-I
eiwit is echter niet Identiek aan een van de tot nu toe beschreven on-
cogenen.
In Hoofdstuk 6 is de expressie van Plm-1 in weefsels van normale
muizen tijdens de embryonale- en postnatale ontwikkeling bestudeerd.
Expressie van Plm-1 werd hoofdzakelijk gevonden in hematopoletlsche
cellen en volgde ook de migratie van deze cellen gedurende de ontwikk
eling. Eveneens werden hoge Plm-1 mRNA niveaus gemeten in een aantal
T- en B- en myelolde cellijnen. Deze gegevens duiden er op, dat Plm-I
een regulerende rol zou kunnen spelen bij de differentiatie of proli
feratie van hematopoletlsche cellen.
105
LIST OF PUBLICATIONS
- Seutter-Berlage, F., Selten, G., Oostendorp, S. and Hoog AntInk, J.
The modified Thlo*-ether Test. In: Chemical Porphyria In Man, El
sevier Biomedical Press, pp 233"*236, 1979.
- Selten, G.C.M., Selten^Versteegen, A.M.E. and Yap, S.H.. Expression
of alpha-fetoproteln and albumin genes In a rat hepatoma cell line
SÏ/1/βΟ: Evidence for the need of species specific serum factors.
Blochem. Blophys. Res. Comm. 103· 278-281, 1981.
- Selten, G.C.M., Prlncen, H.M.G., Selten-Versteegen, A.M.E., Mol-
Backx, G.P.В.M. and ïap, S.H.. Sequence content of AFP, albumin and
fibrinogen polypeptide mRNAs In different organs, developing tis
sues and In liver during carcinogenesis In rats. Biochimica et
Biophyslca Acta 699, 131-137, 1982.
* Prlncen, H.M.G., Selten, G.C.M., Selten-Versteegen, A.M.E., Mol-
Backx, G.P.В.M., Nieuwenhulzen, W. and Yap, S.H.. Distribution of
mRNAs of fibrinogen polypeptides and albumin In free and membrane-
bound polyribosomes and Induction of AFPmRNA synthesis during
llverregeneratlon after partial hepatectomy. Biochimica et Biophy
slca Acta 699, 121-130, 1982.
*- Prlncen, H.M.G., Selten, G.C.M., Nieuwenhulzen, W. and Yap, S.H..
Changes of synthesis of fibrinogen polypeptide and albumin mRNAs In
rat liver after turpentine and after partial hepatectomy or laparo
tomy. In: Fibrinogen Structure, Functional Aspects, Metabolism, 2,
263-277, 1983.
- Cuypers, H.T., Selten, G., Quint, W., Zljlstra, M., Robanus-
Maandag, E., Boelens, H., van Wezenbeek, P., Mellef, C. and Berns,
Α.. Murine Leukemia Virus Induced T-cell lymphoraagenesls: Integra
tion of proviruses In a distinct chromosomal region. Cell 37, Ill-
ISO, 1984.
·* Quint, W., Boelens, W., van Wezenbeek, P., Cuypers, H.T., Robanus-
Maandag, E., Selten, G. and Berns, Α.. Generation of AKR mink cell
focus-forming viruses: a conserved single copy xenotrope like pro-
virus provides recombinant long terminal repeat sequences. Journal
of Virology 50, 432-438, 1984.
107
- Selten, G., Cuypers, H.T., Zljlstra, M., Mellef, C. and Berns, Α..
Involvement of C-myc In MuLVinduced T'-cell lymphomas In mice: fre-1
quency and mechanisms of activation. EMBO J. 3, 3215-3222, 1984.
- Dltzhuysen van, T.J.M., Selten, G.С.M., van Loon, A.M., Wolters,
G., Matthyssen, L. and Yap, S.H.. Detection of Hepatitis В Virus
DNA In serum and relation with the IgM class antl HBc titers In
Hepatitis В Virus Infection. Journal of Medical Virology 15, ЧЭ-'Ъб,
1985.
-< Selten, G., Cuypers, H.T. and Berns, Α.. Proviral activation of the
putative oncogene Plm-1 in MuLV Induced T-cell lymphomas. EMBO J.
1, 1793-1798, 1985.
- Hllkens, J., Cuypers, H.T., Selten, G., Kroezen, V., Hilgers, J.
and Berns, Α.. Genetic mapping of Pinf-I putative oncogene to mouse
chromosome 17. Somatic Cell and Molecular Genetics 12, 81-88, 1986.
•· Cuypers, H.T., Selten, G., Berns, Α. and Geurts van Kessel, Α..
Assignment of the human homolog of Plm-1, a mouse gene Implicated
in leukemogenesis, to the pter-q12 region of chromosome 6. Human
Genetics 72, 262-265, 1986.
- Selten, G., Cuypers, H.T., Boelens, Vf., Robanus-Maandag, E., Ver"
beek, J., Domen, J., van Beveren, C. and Berns, Α.. The primary
structure of the putative oncogene Plm l shows extensive homology
with Protein Kinases. Cell 46, in press, 1986.
-• Cuypers, H.T. Selten, G. Zijlstra, M., de Goede, R., Mellef, С and
Berns, Α.. Tumor progression in MuLV Induced T-cell lymphomas:
monitoring of clonal selections with viral and cellular probes. J.
of Virology, in press, 1986.
- Berns, Α., Selten, G. and Cuypers, H.T.. The putative oncogene
Pim-I. In: The handbook of oncogenes, In press, 1986.
CURRICULUM VITAE
Gerard Selten werd geboren op 17 september 1950 te Mill en St. Hu-
bert (N.Br.). In 1966 behaalde hij het Mulo A en В diploma te Etten-
Leur, waarna hIJ de avondopleldlng voor biochemisch analist volgde aan
de Katholieke Universiteit van Nijmegen en het HBO-В diploma In 1971
behaalde. Als analist was hij van februari 1967 tot september 1973
werkzaam op de afdeling Farmacologie (Prof. Dr. E.J. Ariens) en de аГ-
dellng Microbiologie (Prof. Dr. Ir. G.D. Vogels) van de Katholieke
Universiteit te Nijmegen, In dienst van de Nederlandse Organisatie
voor Zuiver Wetenschappelijk Onderzoek.
Dit dienstverband werd van maart 1972 tot maart 1973 onderbroken
ter vervulling van de militaire dienstplicht.
In september 1973 begon hij zijn studie scheikunde aan de Katho
lieke Universiteit van Nijmegen. Na het colloquium doctum in oktober
1974 werd het kandidaatsexamen (S2) afgelegd In Juni 1976. Het doc
toraalexamen met als hoofdvak Farmacochemie (Dr. F. SeutterftBerlage;
Prof. Dr. J.M. van Rossum) en ala bijvakken Biochemie (Dr. A. Berns;
Prof. Dr. H. Bloemendal) en Klinische Chemie (Prof. Dr. A.P. Jansen)
werd afgelegd in februari 1979. Zijn onderwijsbevoegdheid scheikunde
behaalde hij in 1978.
Van september 1978 tot februari 1979 was hij als docent scheikunde
verbonden aan de (avond)anallstenoplelding 0.L.A.N, te Arnhem/
Nijmegen.
Vanaf maart 1979 tot maart 1983 was hij als wetenschappelijk
medewerker verbonden aan het laboratorium voor maag-, darm- en lever-
ziekten (Dr. S.H. Yap; Prof. Dr. J.H.M, van Tongeren) van het St.
Radboudzlekenhuis te Nijmegen, waar hij onderzoek heeft verricht aan
de regulatie van α-foetoprotelne (AFP) en albumine genen in hepatocel-
lulalre carclnomen en aan de detectie van Hepatitis В Virus DNA in
serum en levertumorbiopten. Verslag van dit onderzoek werd o.a. uit
gebracht bij het congres van "The European Association for the Study
of the Liver" in 1982 te Goteborg, Zweden.
Vanaf maart 1983 tot december 1985 was hij verbonden aan het la
boratorium voor Biochemie (Prof. Dr. H. Bloemendal) aan de Katholieke
Universiteit van Nijmegen. In deze periode werd in de werkgroep van
Dr. A. Berns het onderzoek verricht dat in dit proefschrift beschreven
109
Staat. Voordrachten betreffende de Inhoud van dit proefschrift werden
gehouden o.a. op de "Cold Spring Harbor" congressen (New York, U.S.A.)
over "RNA Tumor Viruses" In mei 1981) en 1985.
Sinds december 1985 Is hij werkzaam als moleculair bloloog bij
Glst-Brocades N.V. te Delft.
110
Stellingen
1 Doordat Townes et al. de ехргэззіе van het humane S-globlne-
gen bestudeerd hebben in op grootte geselekteerde transgene
muizen, is de conclusie, dat metallothionine en/of humane
groeihormoon nucleotide-sequentles de expressie van het
humane B-globine-gen beïnvloeden, onjuist.
Townes, T.M., Chen, H.Y., Lingrel, J.B., Palmlter, H.D. к
Brinster, R.L. (1985). Mol. Cell. Biol. 5, 1977-1983.
2 De bewering van Graham et al., dat in T-cel lymfomen pro-
virale inserties in het pvt-1 locus het c-myc gen activeren
en daardoor bijdragen tot tumorontwikkeling, mist elk bewijs.
Graham, M., Adams, J.M. к Cory, S. (1985). Nature 314, 710-
743.
3 De door Yuuki et al. beschreven nucleotlde-sequentle van het
Bacillus Llchenlformls α-amylase-gen, moet met een zekere
terughoudendheid worden gehanteerd, gezien de vele, nood·4
zakelijke correcties in de door de dezelfde auteurs eerder
gepubliceerde nucleotlde-sequentle van het Bacillus Stearo-
thermophilus a-amylase-gen.
Yuuki, T., Nomura, T., Tezuka, H., Tsuboi, Α., Yamagata, Η.,
Tsukagoshi, N. к Udaka, S. (1985). J. Blochem. 98, І1іі7-115б.
Ihara, H., Sasaki, T., Tsuboi, Α., Yamagata, H., Tsukagoshi,
N. к Udaka, S. (1985). J. Biochem. 98, 95-103.
4 De door Garcia et al. gepresenteerde gegevens bevatten onvol
doende bewijs voor de aanwezigheid van MMTV/LTR-int-1)! mRNA
fusietranscripten in een met dexamethason geïnduceerde nier-
turaorcellijn.
Garcia, M., Wellinger, R.p Vessaz, A. & Diggelmann, H.
(19B6). The EMBO J. 5, ігТ-ІЗ1».
5 Detectie van gemeenschappelijke provirale integratleplaatsen
in het genoom van onafhankelijke tumoren, waarvan het expres
siepatroon nog niet is bestudeerd, rechtvaardigt niet de con
clusie dat deze loei een rol spelen in de leukemogenese.
Lemay, G. & Jollcoeur, P. (1984). Proe. Natl. Acad. Sci. USA
81, 38-42.
Tsichlls, P., Strauss, P. i Hu, L. (1983). Nature 302, 1(45-
449.
Silver, J. & Kozak, С. (1986). J. of Virology 57, 526-533.
6 Het Is aanvechtbaar om op basis van de door Jannière et al.
gepresenteerde gegevens te spreken van stabiele genampllflca-
tie in het chromosoom van Bacillus Subtilis.
Jannière, L., Niaudet, В., Pierre, E. & Ehrlich, S.D. (1985).
Gene 40, 47-55.
7 De economische waarde van een micro-organisme lijkt omgekeerd
evenredig met de frequentie van transformatie.
8 Het Is wenselijk dat voor de aanvang van een tertiaire
opleiding de militaire dienstplicht vervuld moet zijn.
9 De verschuiving van de term "belastingontduiking" naar "be
lastingontwijking" maakt duidelijk dat het hier niet meer om
een incidentele maar om een structurele aangelegenheid gaat.
10 Het toenemend gebruik van tekstverwerkers bij het opstellen
van publikaties, zal de druk op de wetenschappelijke pers om
een uniform literatuur-referentiesysteem in te voeren, ver
groten.
Nijmegen, 16 oktober 1986
C.C.M. Selten
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