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SV40 mutants with an altered small-t protein are tumorigenic in newborn hamsters
VIROLOGY
(1981)
111,341-350
SV40 Mutants
with an Altered Small-t Protein Are Tumorigenic Newborn Hamsters
W. C. TOPP,’ D. B. RIFKIN,* Cold
Harbor Laboratory, New York University
Spring
AND
in
M. J. SLEIGH*
Cold Sprig Harbor, New York lli’.%& and *Department of Cell Biology, School of Medicine, 550 First Avenue, New York, New York 10016 Accepted November 24, 1980
Mutants of Simian virus 40 which have suffered deletions in one of the two early viral gene products, the small-t protein (dl54/59 mutants), induce tumors following subcutaneous injection into newborn LHC inbred Syrian hamsters. The tumors, mainly fibrosarcomas, are histologically identical to those induced by WT (strain 776) SV40 and are indistinguishable by a number of criteria once explanted to culture. However, the dl54/59 SV40 mutants induce tumors with only 50% the efficiency of WT virus and with tumor latencies twice as long. The viral large-T protein is primarily responsible for SV40 oncogenicity while the small-t protein may function as a tumor promoter.
plantation rejection antigen (the SV40-specific TSTA (Tevethia and Rapp, 1965; Tevethia and Tevethia, 1975, 1976)). The viral infection itself is only weakly mutagenic, increasing the mutational frequency at several loci 2- to lo-fold over the spontaneous rate (Marshak et al., 1975; Theile et al., 1976), and highly malignant in vitro transformants, once reverted from transformation through the loss of viral sequences, are no longer tumorigenic (Steinberg et al., 1979). Therefore it is likely that the oncogenicity of the virus results from the expression of the viral genome. The virus itself is very well characterized: the genome, a single molecule of circular double-stranded DNA, has been sequenced in toto (Fiers et al., 1978; Reddy et al., 1978), accurate transcriptional maps of the transforming portion of the virus (Graham et al., 1974) are available (Berk and Sharp, 1978) and the two proteins encoded by this region (Prives et al., 1977), the socalled large-T (MWSOK) and small-t (MW17K) proteins, have been purified virtually to homogeneity (Tjian, 1978; Tenen et aZ., 1977; Tegtmeyer, personal communication). The biological activities of these two proteins are currently the subject of intense molecular biological scrutiny, and it is not unlikely that in the near future we
INTRODUCTION
Simian virus 40 (SV40,3 Sweet and Hilleman, 1960) induces a variety of malignant tumors when injected intravenously into weanling hamsters (Diamandopolous 1972, 1973, 1978; Diamandopolous and McLane, 1975). Subcutaneous injection results principally in fibrosarcomas arising at the sight of injection of the hamster (Eddy et al., 1961; Girardi et al., 1962; Allison et al., 1967). It is difficult to assess the true oncogenie potential of the virus. The virus is overall a weak oncogen, inducing tumors only in Syrian hamsters (or mastomys) where tumors appear 5 to 10 months after injection of very high titer virus (approximately 10’ plaque-forming units). However the problem is complicated both by the inefficiency with which the infecting virus initially forms a stable cell/viral interaction (Todaro and Green, 1964) and by the expression on the surfaces of infected cells of an extremely strong virus-specific trans1 To whom reprint requests should be sent. * Present address: Laboratory of Molecular Biology, CSIRO, Box 123, Epping, New South Wales, 2121, Australia. 3 Abbreviations used: SV40, simian virus 40; TSTA, tumor-specific transplantation antigen; WT, wild type; DME, Dulbecco’s modified Eagle’s medium. 341
0042-6822/81/080341-10$02.00/O Copyright 0 1981 by Academic Press, Inc. All rights of reproduction in any form reserved.
TOPP,
342
RIFKIN,
will have a good idea of at least some of the pathways by which these proteins alter the regulatory state of the cell. Both proteins are involved in the establishment of transformation in vitro and many of these transformants are tumorigenic. It is therefore important to examine the relative role of the two viral proteins in direct viral tumorigenicity. The small-t protein is not required for viral growth in cultured African green monkey kidney cells and a series of viable deletion mutants in this region (dl 54/59 mutants) have been isolated (Shenk et al., 1976; Sleigh et al., 1978). These viruses have substantially deleted sequences (between 0.54 and 0.59 map unit on the viral genome) encoding the small-t protein (Sleigh et al., 1978; Crawford et al., 1978; Khoury et al., 1979) so that the residual viral influence in cells infected with these mutants is due to the large-T protein alone. We report here that subcutaneous injection of these small-t defective viruses induces tumors histologically identical to those induced by WT (strain 776) SV40, albeit with increased latencies and reduced efficiencies. While the small-t protein may play some role in tumor progression it is not required for tumor induction in hamsters nor does it determine the ultimate nature of the tumor. It is therefore the large-T protein which is primarily responsible for SV40 oncogenesis. This is in agreement with reports that small-t antigen does not seem to be involved in the induction of either transformation or of cellular tumorigenicity of hamster cells in culture (Frisque et al., 1980; Martin et al., 1979a), but is in contrast to the viral interaction with rat cells in which the small-t protein is required for the expression of the in vitro phenotypes which most closely correlated with tumorigenicity in rat cells (Sleigh et uZ., 1978; Frisque et al., 1980; Topp and Rifkin, 1981). MATERIALS
AND
METHODS
WT strain 776 and dl 54/59 mutant viruses were plaque purified, grown, and titered on CV-1 cells, an established line of African green monkey kidney cells. dl 884 and dl890 were the gift of Tom Shenk. All
AND
SLEIGH
viral stocks were doubly plaque purified and titered l-2 x 10B PFU/ml. Viral DNA was prepared by the procedure of Hirt (1967) and all viruses appeared to consist of a single homogeneous species on gel electrophoresis. Viral DNA fragments were isolated by cleavage with restriction endonucleases (BRL) followed by electrophoresis through horizontal agarose slab gels (Tris-acetate buffer). DNA bands were located by uv illumination of ethidium bromide-stained gels, cut out, Dounce hoand repeatedly extracted mogenized, against phenol with buffer (10 mM Tris, pH 7.9, 10 n&f EDTA, 0.1 M NaCl). DNA fragments thus extracted were concentrated by ethanol precipitation and found to be reduced at least three orders of magnitude in infectivity from WT SV40 DNA as assayed by DEAE-Dextran facilitated uptake into CV-1 cells (McCutchan and Pagano, 1968). Timed pregnant LHC inbred Syrian hamsters were purchased (Charles River) allowing the injection of baby hamsters within 6-12 hr of birth. Hamsters were injected subcutaneously over the right shoulder with either 0.2 cc of virus stock or 1 clg viral DNA suspended in 0.1 cc phosphatebuffered saline solution. Hamsters were examined weekly for a period of 1 l/2 years. Tumors once palpable grew rapidly to several centimeters in diameter within 2 to 3 weeks before regressing. Tumors were removed prior to necrosis, formaldehyde tlxed, paraffin embedded, and sectioned. Slides were stained with hematoxylineosin. At the time of excision the tumors were coded and after sectioning were examined blind at one sitting by one observer and scored dichotomously for a number of histologic or cytologic features (absence of necrosis, monomorphic cell population consisting of spindle cells, small round cells, or large cells, the presence of giant cells, and the presence of two or more histologic patterns), and on a scale of one to five for the degree of admixture of cells, for nuclear-cytoplasmic ratios, and for the relative proportion of giant cells and other bizarre forms. After the histologic study was completed the slides were uncoded and possible correlations between type of virus and his-
TUMORIGENICITY
OF SMALL-t
tologic and cytologic patterns were tested. Tumor lines were established in culture by physical disaggregation of nonnecrotic tumor tissue followed by trypsinization. Tumor-derived lines were only passaged long enough to be frozen in bulk. All experiments were performed on replica thawed vials within 2 weeks of revival. The derivation of the LHC hamster embryo fibroblast SV40 transformants was described in Frisque et al., (1980). All cultures expressed SV40 T-antigen(s) as assayed by immunofluorescence (Botchan et al., 1976) in at least 95% of the cells. Cells were propagated on plastic culture plates (Falcon) at 37.5”C in a humidified atmosphere at 10% COdair. Growth medium was DME (Gibco) containing penicillin and streptomycin supplemented with 10% fetal calf serum (Irvine). Growth in methocel, immunofluorescent determination of cytoplasmic actin cable networks, and measurement of plasminogen activator synthesis were determined exactly as in Frisque et al. (1980). Rabbit anti-a&in serum was the gift of Keith Burridge. The determination of the SV40 integration patterns in the tumor derived and SV40-transformed lines was performed essentially as in Botchan et al. (1976). Highmolecular-weight cellular DNA was purified from two roller bottles of cells. Five micrograms of DNA from each line was restricted with an enzyme which does not cleave within the SV40 sequences (e.g., BgZII or S&I, BRL) and electrophoresed through a 0.7% horizontal agarose slab gels (Tris -phosphate buffer). The DNA was deTABLE
MUTANTS
343
OF SV40 TABLE
2
Virus
Number of animals
Tumor incidence 66)
Average latency (months)
WT d12000 dl2001 d12004 di2007 d1890 dl2003 d12005 dl2006 d1884
14 4 11 6 4 5 4 4 7 8
106 50 73 83 100 40 0 75 28 38
7 l/2 13 l/2 12 13 12 10 12 13 14
Note. 6- to 12-hr-old LHC inbred Syrian hamsters were injected subcutaneously over the right shoulder with 0.2 cc virus stock containing 2-4 x 10’ PFU virus. At least six animals were injected with each virus although a few died during the course (18 months) of the experiment due to causes apparently unrelated to the injected virus (fight wounds, exposure to the elements during a heating failure, etc.).
natured in situ and transferred to nitrocellulose filters by “blotting.” The filters were hybridized to 0.33~pg “nick translated” SV40 (cloned in the bacterial plasmid pBR322) in 15 ml of hybridization solution. The specific activity of the probe was routinely lo* cpm/pg DNA. Films (Kodak XRl) were exposed to the hybridized blots for 7 - 10 days using intensifier screens (DuPont) for enhanced sensitivity. Recovery of virus was by polyethylene glycol enhanced DNA infection of permissive CV-1 cells essentially as described in Graessmann et al. (1979). RESULTS
1
SV40 DNA
Number of animals
Tumor incidence (8)
Uncut Linear (RI)
9 8
89 a7
6 II2 5 l/2
BamlHpaII
7
29
9
Pst I “A”
7
0
Average latency (months)
-
Note. 6 to 12hr old LHC inbred hamsters were injected subcutaneously over the right shoulder with 1 pg DNA suspended in 0.1 cc phosphate-buffered saline solution.
Tumorigenicity of WT SV4.0 DNA DNA Fragments
and
It is known that the DNA of SV40 is infectious in cultured cells (Gerber, 1962). More recently it was reported that viral DNA is also tumorigenic when injected subcutaneously into newborn hamsters (Sol and van der Noordaa, 1977). The results of injection of various SV40 DNA sequences into inbred LHC Syrian hamsters are shown in Table 1. Both closed circular and linear (generated by cleavage with endonu-
TOPP,
344 TABLE
RIFKIN,
3
Virus
Animals injected
Tumor incidence (%I
Average latency (months)
WT dl54/59
14 55
100 51
7 l/2 12.5
Note. averaged.
Results
in Table
2 for
dl 54/59
viruses
are
clease EcoRI which cuts the SV40 circular genome once within the region not expressed in nonpermissive cells) viral DNA are tumorigenic, tumors appearing with latencies similar to those induced by whole virus (Table 2). Not all the viral DNA sequences are required for tumor induction. The fragment of DNA generated by double cleavage with restriction endonucleases BumHI and HpaII is capable of inducing tumors albeit with a reduced frequency (Table l), consistent with the fact that this fragment will induce transformation in vitro with a similarly decreased efficiency (Graham et al., 1974). This fragment (roughly 55% of the viral genome) contains the entire region normally transcribed in transformed cells and will direct the synthesis of normal large-l‘ and small-t protein but has deleted sequences encoding all other (known) viral proteins. A fragment (P&I “A” fragment) which contains roughly 75% of the genome but which has lost approximately 170 amino acids at the carboxy terminus of the large-T protein is not tumorigenic consistent with the observation that all SV40-transformed cells examined have been found to synthesize a large-T protein with an electrophoretic mobility very similar to that observed in a lytic infection. It is likely that the minimum colinear region of WT SV40 which will induce tumors is that which encodes the entire early region. Tumorigenicity of WT and dl 54159 SV4.0 The viruses studied, the number of animals injected, the tumor incidence (within 18 months of injection), and the average tumor latency are listed in Table 2. The averaged results are in Table 3. It can be
AND
SLEIGH
seen that all the dl 54159 viruses with the possible,exception of dl2003 are capable of inducing tumors. However, the average tumor latencies are close to twice those observed in the animals injected with similar doses of WT SV40. Also the average tumor incidences are significantly reduced. Tumor Histology
Ten Wt- and fifteen dl 54i59 SV40-induced tumors were excised before necrosis, fixed with formaldehyde, embedded, and sectioned. It was impossible within this sampling to distinguish between the two classes of tumor by any criterion tested. Two WT and two mutant-induced tumors appeared to be highly cellular small-cell tumors and perhaps represented stem cells. The remainder of the tumors were fibrosarcomas roughly one-half of which were myxoid variants. Although most tumors appeared healthy, areas of focal necrosis were occasionally observed and roughly one-third of the sections showed substantial numbers of giant cells and other bizarre forms. Properties of Tumor Culture
Lines Explanted
to
Ten tumors induced by WT and twenty tumors induced by dl54i59 SV40 were explanted to culture by physical and enzymatic disaggregation. These uncloned tumor-derived lines were examined for the ability to grow suspended in methylcellulose, the loss of cytoplasmic actin-containing cable networks, and the synthesis of plasminogen activator. These properties have been found to be good in vitro markers of cellular tumorigenicity for rat cells (Pollack et al., 1975). The results are summarized in Table 4. It can be seen that a substantial majority of the tumor-derived lines, both WT and dl 54159 induced, are transformed by these criteria. Moreover an examination of the data (not shown) shows that there is a correlation between the expression of these three properties; i.e., cells which fail to clone in methylcellulose also tend to retain actin cable networks and to synthesize low levels of plasminogen activator. This is in contrast to LHC hamster
TUMORIGENICITY
OF SMALL-t
embryo cells transformed in culture which appear to show no correlation among these properties and in which growth in methylcellulose is the only good in vitro marker of cellular tumorigenicity (Frisque et al., 1980). We do now know if the rare (approximately 10%) tumor-derived lines which fail these criteria of in vitro transformation will yield secondary tumors with the same properties upon in vivo passage. As with the histological sections we can detect no difference on the basis of these criteria between the tumors induced by Wt or dl54/59 virus. The patterns of viral DNA integration within five WT- and five dl 54/59-induced tumors were examined by cleavage of whole cell DNA with restriction endonucleases followed by gel electrophoresis. DNA was transferred to nitrocellulose filters and viral sequences detected by hybridization to radioactive labeled SV40. Because the cell DNA was restricted with an enzyme, SstI or BglII, which does not cleave SV40 each band hybridizing to radioactive viral DNA corresponds to an insertion of SV40 flanked on either side by host sequences (Botchan et al., 1976). The total number of bands in each track represents the minimum number of unique insertions of SV40 in each line. Most tumorderived lines, both WT and dl 54l59 SV40 induced, contain several viral insertions, and there is no obvious difference between the patterns of WT and dl54/59 viruses. In addition all lines appear to be carrying free viral DNA at a level of one to two copies/ cell, although this may be due to a rare cell containing several hundred to a thousand copies while the majority are free of unintegrated sequences. Cleavage with enzymes cutting SV40 only once results in free linear viral DNA as well as host-viral “junction bands.” An autoradiogram of one gel resulting from restriction endonuclease digestion of the mutant-induced tumor DNA with Sst I is shown in Fig. 1. We have not monitored these lines for release of infectious virus. The integration patterns in several lines of LHC hamster embryo fibroblast cells transformed in vitro by WT or dl 54t59 virus (Frisque et al., 1980) were also exam-
MUTANTS
OF SV40
345
TABLE
4
IN VITRO PROPERTIES OF VIRALLY INDUCED TUMOR LINES
Virus WT SV40 dl 54159 sv40
Fraction of lines tested growing in suspension
Fraction of lines tested losing actin cables
Fraction of lines tested synthesizing plasminogen activator
9/10
719
617
17119
lO/lO
16/19
ined (data not shown). Again most lines show several unique insertions and all carry free viral DNA at the average level of one to two copies/cell. It would appear that at the DNA level the virus/cell interaction is similar whether established in vivo or in vitro, and by WT or by dl 54159 mutant virus. By a similar procedure we were able to determine that the integrated viral sequences possessed at least one feature characteristic of the infecting virus; resistance to the restriction endonuclease Tag1 (Shenk et al., 1976; Sleigh et al., 1978) which cleaves SV40 once at 0.57 on the standard map. Cleavage of a number of tumor cell DNAs by Tug1 produced results consistent with a “no-cut” enzyme including the continued presence of form II SV40 similar to that in Fig. 1 (data not shown). Further, virus was recovered from 8 of 15 lines of dl 54/59-induced tumor cells which were fused to permissive CV-1 cells. All recovered viruses were identical to those originally injected by restriction enzyme analysis. Although we have not analyzed these recovered viruses at the sequence level it is very likely that the dl 54159 viruses were responsible for the induced tumors. DISCUSSION
There are two virally encoded proteins expressed in nonpermissive cells infected with SV40 (Prives et al., 1977). The results reported here indicate that SV40 variants which have suffered deletions in sequences encoding one of these two proteins, the
346
TOPP, RIFKIN,
AND SLEIGH
It III I
FIG. 1. DNA from SV40-induced tumor-derived lines cleaved with restriction endonuelease SstI electrophoresed through a 0.7% agarose slab gel, transferred to a nitrocellulose filter, and hybridized to radioactive SV40 as described under Materials and Methods. SstI does not cleave within SV40 so each band represents a unique insertion of SV40. Shown are five tumors induced by dl54/59 mutants, one induced by TsA209, an in vitro SV40-transformed rat cell with multiple insertions, and 1O-5 pg SV40. Forms II and III SV40 are blurred by overexposure.
viral “small-t,” are still tumor-inducing viruses. Many of these viral mutants (e.g., dl 884 SV40) not only have deleted sequences encoding protein but also have lost sequences necessary for the proper splicing and subsequent maturation of the small-t mRNA. Thus only the large-T protein is expressed in cells infected with these mutants (Khoury et al., 1979). It is therefore significant that these small-t defective viruses induce tumors that are by all criteria examined identical to those induced by WT virus. Within our sample the dl 54/59- and Wt SV40-induced tumors: (1) are histologically similar, mainly fibrosarcomas, (2) grow at roughly the same rates after appearance, (3) possess the same phenotypes in vitro, i.e., the majority grow suspended in methocel, have lost actin cable networks, and synthesize plasminogen activator, (4) show similar patterns of viral integra-
tion into the cellular chromosomal DNA, i.e., one to several copies of the viral genome are integrated at unique sites along with a low level (one copy/cell) of free viral DNA. Based on these results it is likely that it is the large-l‘ protein alone which is primarily responsible for the oncogenic potential of SV40 in hamsters. Similar results have recently been reported from another lab (Lewis and Martin, 1979). This result is in contrast to the requirement for the small-t protein for the development of the fully transformed phenotype in rat cells, a difference upon which we have remarked elsewhere (Frisque et al., 1980). We cannot rule out the formal possibility that either of the two proteins can function as an oncogene. Viral mutants to test this hypothesis lacking only large-T activity are only now becoming available (Gluzman et al., 1980). The phenotypic “transformation” of cultured cells which follows infection with
TUMORIGENICITY
OF SMALL-t
SV40 has been studied as an in vitro model of viral tumorigenicity. The small-t protein is not required for either the initiation of the maintenance of the transformed state in cultured hamster cells (Martin et al., 1979a; Frisque et al., 1980) except under certain restricted circumstances (Martin et al., 19’79b), in good agreement with the tumorigenicity of the dl 54/59 (small-t defective) SV40 mutants. On the other hand the viral large-T protein has been found to be required for the maintenance of transformation in all cells examined (Kimura and Dulbecco, 197’7; Tegtmeyer, 1975; Osborn and Weber, 1975a; Martin and Chou, 1975; Brugge and Butel, 1975; Kimura and Itagaki, 1975). Large-T is a self-aggregating (Potter et al., 1969; Osborn and Weber, 1975b; Kuchino and Yamaguchi, 1975) phosphoprotein (Tegtmeyer et al., 1975) with a monomer molecular weight as determined by SDS-acrylamide gel electrophoresis of 9OK daltons (Prives et al., 1977) although the true molecular weight is somewhat lower (Griffin et al., 1978). Much is known about the interaction of large-T with infected cells in which this protein which expresses several enzymatic activities plays a role in the control of both host and viral DNA and RNA synthesis (Table 5). As yet there is no clue as to which, if any, of these large-T-related properties are relevant either to viral transformation or tumorigenicity. While all the evidence is that the WTand dl 54/59-induced tumors are qualitatively similar the reduced frequency and greater latency of the mutant-induced tumors may indicate a role for small-t protein at the aggregate rather than the cellular level, perhaps as a tumor promoter. Tevethia et al., (1980) have shown that small-t defective viruses are not significantly different in the induction of transplant rejection antigens so the effect is unlikely to be due to differential immune surveillance. Interestingly, it has been reported that agents which act as tumor promoters can in some ways compensate for a small-t defect for transformation in vitro (Topp et al., 1979; Martin et al., 197913) and it is an intriguing possibility that the mechanisms of
MUTANTS
TABLE BIOLOGICAL
347
OF SV40 5
PROPERTIES OF SV40 PROTEIN
Property DNA
binding
LARGE-T
Reference protein
Required for SV40 DNA synthesis Has three high-affinity DNA binding sites at the origin of viral DNA replication Stimulates host cell DNA synthesis Required for initiation of late viral transcription Regulates level of early viral transcription
Induces the synthesis of and binds to a cellular protein of MW 50-60K Possesses ATPase activity
Carrol et al., 1974; Jesse1 et al., 1975 Tegtmeyer,
1972
Tjian, 1978; 1975;
Reed
Tijan
et
al.,
et al., 1978
Tegtmeyer and Ozer, 1971 Tegtmeyer et al., 1975; Aiwine et al., 1975; Reed et al., 1976; Khoury and May, 1977 Lane and Crawford, 1979; Linzer and Levine, 1979 Tjian and Robbins, 1979; Tjian et al., 1980; Griffin et al., 1979, 1980
action of the small-t protein and of tumor promoters such as the phorbol esters may be related.4 Several mechanisms have been proposed to explain the action of tumor promoters (Slaga et al., 1978). Two of these are based on the expression of a normally recessive transforming mutation and on the generation of a critical tumor cell mass (Blumberg, 1980). The expression of a recessive trait may arise through the production of cells homozygous for a mutation by induction of sister chromatid exchange. SV40 does induce chromosomal abnormalities in infected cells (Bartsch, 1970; Lehman, 1974) although it is not known if the viral small-t protein is involved. The second hypothesis is that of a minimum clone size. Adherants of this theory would contend that there is a minimum number of potential tumor cells which must 4 A similar possibility has been suggested for the of Rous sarcoma virus (Bissell et al., 1979).
SRC gene
348
TOPP, RIFKIN,
be present for tumor growth. This might be due to either the production by surrounding normal tissues of factors (chalones) which control growth, from which the inner mass of the tumor must be isolated in order to grow (Bell, 1976) or to the production by the tumor cells of a growth factor (Todaro et al., 1976) which, at low tumor cell densities, diffuses away. The tumor promoter (small-t) wouold act as a mitogen, enhancing the rate of division of the tumor cells and bringing the tumor more quickly to critical mass. We are looking at the affect of small-t on chromosomal abnormalities and are characterizing the TD,, of cells transformed either in vivo or in vitro by the dl 54/59 and WT viruses. It will be interesting to see if the small-t protein is implicated in either of these two phenomena. Tumor promotion is not the only possible explanation for the role of small-t in viral tumorigenesis. The similarity of integration patterns of the dl M/59 and WT virus tentatively rules out a strict gene dosage effect. However it is possible that WT SV40 initially becomes stably integrated within a greater number of cells. This may be due to an increase in effective viral inoculum by a limited proliferation in vivo of only nondefective virus leading to the spread of WT virus to a larger population of cells. If the probability is constant for a given virally initiated cell to begin growth as a tumor, the greater number of cells initiated by nondefective SV40 would produce tumors with decreased average latency and increased average frequency. We cannot distinguish between this and the other hypotheses on the basis of our data. ACKNOWLEDGMENTS We would like to thank Chris Paul and Ruth Crowe for excellent technical assistance and Bob Martin, Peter Blumberg, and particularly Bernard Lane for helpful discussions during the course of the work. This work was supported by Grants CA24803, CA23753, and HL22266 from the NIH. REFERENCES ALLISON, A. C., PHESTERMAN, F. C., and BARON, S. (196’7). Induction of tumors in adult hamsters with simian virus 40. J. Nat. Cancer Inst. 38, 567-572. ALLISON, A. C., and TAYU)R, R. B. (1967). Observa-
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GIRARDI, A. J., SWEET, B. H., and SLOTNICK, V. B. (1962). Development of tumors in hamsters inoculated in the neonatal period with vacuolating virus SV40. Proc. Sot. Exp. Biol. Med. 109,649-660. GLUZMAN, Y., FRISQUE, R. J., and SAMBROOK, J. (1980). Origin-defective mutants of SV40. Cold Spring Harbor Symp. Quant. Biol. 44,293-300. GRAESSMANN, A., GRAESSMANN, M., TOPP, W. C., and BOTCHAN, M. (1979). Retransformation of a Simian Virus 40 revertant cell line which is resistant to viral and DNA infections, by microinjection of viral DNA. J. Viral. 32, 989-994. GRAHAM, F. L., ABRAHAMS, P. J., MULDES, P., HEIJUEKER, H. L., WARNAAR, S. O., DEVRIES, E. A. J., FIERS, W., and VAN DER EB, A. J. (1974). Studies on in vitro transformation by DNA and DNA fragments of human adenovirus and simian virus 40. Cold Spring Harbor Symp. Quant. Biol. 39, 637-650. GRIFFIN, J. D., LIGHT S., and LIVINGSTON, D. M. (1978). Measurements of the molecular size of the simian virus 40 Large-T antigen. J. Viral. 27, 218-226. GRIFFIN, J. D., SPANGLER, G., and LIVINGSTON, D. M. (1979). Protein kinase activity associated with simian virus 40 T-antigen. Proc. Nat. Acad. Sci. USA 76, 2610-2614. GRIFFIN, J. D., SPANGLER, G., and LIVINGSTON, D. M. (1980). Enzymatic activities associated with the SV40 Large-T antigen. Cold Spring Harbor Symp. Quant. Biol. 44, in press. HIRT, B. (1967). Selective extraction of polyoma DNA from infected mouse cell cultures. J. Mol. Biol. 26, 365 -369. JESSEL, D., HUDSON, J., LANDAN, T., TENEN, D., and LIVINGSTON, D. M. (1975). Interaction of partially purified simian virus 40 T antigen with circu-
MUTANTS
OF SV40
lar viral DNA molecules. Proc. USA
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KHOURY, G., GRUSS, P., DHAR, R., and LAI, C. J. (1979). Processing and expression of early SV40 mRNA: A role for RNA conformation in splicing. Cell
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KUCHINO, T., and YAMAGUCHI, N. (1975). Characterization of T-antigen in cells infected with a temperature sensitive mutant of simian virus 40. J. Virol. 15, 1302-1307.
LANE, D. P., and CRAWM)RD, L. V. (1979). T-antigen is bound to a host protein in SV40 transformed cells. Nature (London) 278, 261-263. LEHMAN, J. M. (1974). Early chromosomal changes in diploid Chinese hamster cells after infection with simian virus 40. Znt. J. Canad. 13, 164-172. LEWIS, A. M., and MARTIN, R. G. (1979). The oncogenicity of simian virus 40 deletion mutants that induce altered 17K t-proteins. Proc. Nat. Acad. Sci. USA,
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LINZER, D. I. H., and LEVINE, A. J. (1979). Characterization of a 54,000 MW cellular SV40 tumor antigen present in SV40 transformed cells and uninfected embryonal carcinoma cells. Cell 17, 43-52. MARSHAK, M. I., VARSHAVER, N. B., and SHAPIRO, N. I. (1975). Induction of gene mutations and chromosomal aberrations by simian virus 40 in cultured mammalian cells. Mutat. Res. 30, 383-386. MARTIN, R. G., and CHOW, J. Y. (1975). Simian virus 40 functions required for the establishment and maintenance of malignant transformation. J. Virol. 15, 599-612.
MARTIN, R. G., SETLOW, V. P., EDWARDS, C. A. F., and VEMBU, D. (1979a). The roles of the simian virus 40 tumor antigens in transformation of Chinese hamster lung cells. Cell 17, 635-643. MARTIN, R. G., SETLOW, V. P., and EDWARDS, C. A. F. (197913). Roles of the simian virus 40 tumor antigens in transformation of Chinese hamster lung cells: Studies with simian virus 40 double mutants. J. Virol.
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MCCUTCHAN, J. H., and PAGANO, J. S. (1968). Enhancement of the infectivity of simian virus 40 deoxyribonucleic acid with diethylaminoethyldextran. J. Nat.
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OSBORN, M., and WEBER, K. (1975a). Simian virus 40
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SWEET, B. H., and HILLEMAN, M. R. (1960). The vacuolating virus, SV40. Proc. Sot. Exp. Bio. Med. 105, 420-427.
TEGTMEYER, P. *Simian virus 40 Deoxyribonucleic acid synthesis: The viral replicas. J. Viral. 10, 591-59s.
TEGTMEYER, P. (1975). Function of simian virus 40 gene A in transforming infection. J. Virol. 15, 613-618.
AND SLEIGH TEGTIKEYER, P., and OZER, H. L. (1971). Temperature-sensitive mutants of simian virus 40: Infection of permissive cells. J. Viral. 8, 516-524. TEGTMEYER, P., SCHWARTZ, M., COLLINS, J. K., and RUNDELL, K. (1975). Regulation of tumor antigen synthesis by simian virus 40 gene A. J. Virol. 16, 168-178.
TENEN, D. G., GAREWAL, H., HAINES, L. L., HUD SON, J., WOODARD, V., LIGHT, S., and LIVING STON, D. M. (1977). Purification of simian virus 40 tumor antigen from a line of simian virus IO-transformed cells. Proc. Nat. Acad. Sci. USA 74, 37453749. TEVETHIA, S. S., FLYER, D. C., TEVETHIA, M. J., and TOPP, W. C. (1980). Biology of Simian Virus 40 (SV40) Transplantation antigen (TrAg). VII. Induction of SV40 TrAg in nonpermissive mouse cells by early viable SV40 deletion (0.54ZO.59) mutants. Virology,
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TEVETHIA, S., and RAPP, F. (1965). Demonstration of new surface antigens in cells transformed by papovavirus SV40 by cytotoxic tests. Proc. Sot. Exp. Biol.
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TEVETHIA, S. S., and TEVETHIA, M. J. (1975). DNA virus (SV40) induced antigens. In “Cancer: A Comprehensive Treatise” (F. Becker, ed.). Plenum, New York. TEVETHIA, M. J., and TEVETHIA, S. S. (1976). Biology of SV40 transplantation antigen (TrAg) I. Demonstration of SV40 TrAg on glutaradehyde hxed SV40-infected African green monkey kidney cells. Virology
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THEILE, M., SCHERNECK, S., and GEISSLER, E. (1976). Mutagenesis by simian virus 40 I. Detection of mutations in Chinese hamster cell lines using different resistance markers. Mutat. Res. 37, lll124. TJIAN, R. (1978). The binding site on SV40 DNA for a T antigen-related protein. Cell 13, 165-179. TJIAN, R., FEY, G., and GRAESSMANN, A. (1978). Biological activity of purified simian virus 40 T-antigen proteins. Proc. Nat. Acad. Sci. USA 75, 12791283. TJIAN, R., and ROBBINS, A. (1979). Enzymatic activities associated with a purified simian virus T antigen-related protein. Proc. Nat. Acad. Sci. USA 76, 610-614.
TJIAN, R., ROBBINS, A., and CLARK, R. (1980). Catalytic Properties of the SV40 Tumor Antigen. Cold Spring Harbor Sgmp. Quant. Biol. 44,103-112. TODARO, G. J., DELARCO, J. E., and COHEN, S. (1976). Transformation by Murine and Feline sarcoma viruses specifically blocks binding of epiderma1 growth factor to cells. Nature (London) 264, 26-31. TODARO, G. J., and GREEN, H. (1364). An assay for cellular transformation by SV40. Virology 23, 117119.
(1981)
111,341-350
SV40 Mutants
with an Altered Small-t Protein Are Tumorigenic Newborn Hamsters
W. C. TOPP,’ D. B. RIFKIN,* Cold
Harbor Laboratory, New York University
Spring
AND
in
M. J. SLEIGH*
Cold Sprig Harbor, New York lli’.%& and *Department of Cell Biology, School of Medicine, 550 First Avenue, New York, New York 10016 Accepted November 24, 1980
Mutants of Simian virus 40 which have suffered deletions in one of the two early viral gene products, the small-t protein (dl54/59 mutants), induce tumors following subcutaneous injection into newborn LHC inbred Syrian hamsters. The tumors, mainly fibrosarcomas, are histologically identical to those induced by WT (strain 776) SV40 and are indistinguishable by a number of criteria once explanted to culture. However, the dl54/59 SV40 mutants induce tumors with only 50% the efficiency of WT virus and with tumor latencies twice as long. The viral large-T protein is primarily responsible for SV40 oncogenicity while the small-t protein may function as a tumor promoter.
plantation rejection antigen (the SV40-specific TSTA (Tevethia and Rapp, 1965; Tevethia and Tevethia, 1975, 1976)). The viral infection itself is only weakly mutagenic, increasing the mutational frequency at several loci 2- to lo-fold over the spontaneous rate (Marshak et al., 1975; Theile et al., 1976), and highly malignant in vitro transformants, once reverted from transformation through the loss of viral sequences, are no longer tumorigenic (Steinberg et al., 1979). Therefore it is likely that the oncogenicity of the virus results from the expression of the viral genome. The virus itself is very well characterized: the genome, a single molecule of circular double-stranded DNA, has been sequenced in toto (Fiers et al., 1978; Reddy et al., 1978), accurate transcriptional maps of the transforming portion of the virus (Graham et al., 1974) are available (Berk and Sharp, 1978) and the two proteins encoded by this region (Prives et al., 1977), the socalled large-T (MWSOK) and small-t (MW17K) proteins, have been purified virtually to homogeneity (Tjian, 1978; Tenen et aZ., 1977; Tegtmeyer, personal communication). The biological activities of these two proteins are currently the subject of intense molecular biological scrutiny, and it is not unlikely that in the near future we
INTRODUCTION
Simian virus 40 (SV40,3 Sweet and Hilleman, 1960) induces a variety of malignant tumors when injected intravenously into weanling hamsters (Diamandopolous 1972, 1973, 1978; Diamandopolous and McLane, 1975). Subcutaneous injection results principally in fibrosarcomas arising at the sight of injection of the hamster (Eddy et al., 1961; Girardi et al., 1962; Allison et al., 1967). It is difficult to assess the true oncogenie potential of the virus. The virus is overall a weak oncogen, inducing tumors only in Syrian hamsters (or mastomys) where tumors appear 5 to 10 months after injection of very high titer virus (approximately 10’ plaque-forming units). However the problem is complicated both by the inefficiency with which the infecting virus initially forms a stable cell/viral interaction (Todaro and Green, 1964) and by the expression on the surfaces of infected cells of an extremely strong virus-specific trans1 To whom reprint requests should be sent. * Present address: Laboratory of Molecular Biology, CSIRO, Box 123, Epping, New South Wales, 2121, Australia. 3 Abbreviations used: SV40, simian virus 40; TSTA, tumor-specific transplantation antigen; WT, wild type; DME, Dulbecco’s modified Eagle’s medium. 341
0042-6822/81/080341-10$02.00/O Copyright 0 1981 by Academic Press, Inc. All rights of reproduction in any form reserved.
TOPP,
342
RIFKIN,
will have a good idea of at least some of the pathways by which these proteins alter the regulatory state of the cell. Both proteins are involved in the establishment of transformation in vitro and many of these transformants are tumorigenic. It is therefore important to examine the relative role of the two viral proteins in direct viral tumorigenicity. The small-t protein is not required for viral growth in cultured African green monkey kidney cells and a series of viable deletion mutants in this region (dl 54/59 mutants) have been isolated (Shenk et al., 1976; Sleigh et al., 1978). These viruses have substantially deleted sequences (between 0.54 and 0.59 map unit on the viral genome) encoding the small-t protein (Sleigh et al., 1978; Crawford et al., 1978; Khoury et al., 1979) so that the residual viral influence in cells infected with these mutants is due to the large-T protein alone. We report here that subcutaneous injection of these small-t defective viruses induces tumors histologically identical to those induced by WT (strain 776) SV40, albeit with increased latencies and reduced efficiencies. While the small-t protein may play some role in tumor progression it is not required for tumor induction in hamsters nor does it determine the ultimate nature of the tumor. It is therefore the large-T protein which is primarily responsible for SV40 oncogenesis. This is in agreement with reports that small-t antigen does not seem to be involved in the induction of either transformation or of cellular tumorigenicity of hamster cells in culture (Frisque et al., 1980; Martin et al., 1979a), but is in contrast to the viral interaction with rat cells in which the small-t protein is required for the expression of the in vitro phenotypes which most closely correlated with tumorigenicity in rat cells (Sleigh et uZ., 1978; Frisque et al., 1980; Topp and Rifkin, 1981). MATERIALS
AND
METHODS
WT strain 776 and dl 54/59 mutant viruses were plaque purified, grown, and titered on CV-1 cells, an established line of African green monkey kidney cells. dl 884 and dl890 were the gift of Tom Shenk. All
AND
SLEIGH
viral stocks were doubly plaque purified and titered l-2 x 10B PFU/ml. Viral DNA was prepared by the procedure of Hirt (1967) and all viruses appeared to consist of a single homogeneous species on gel electrophoresis. Viral DNA fragments were isolated by cleavage with restriction endonucleases (BRL) followed by electrophoresis through horizontal agarose slab gels (Tris-acetate buffer). DNA bands were located by uv illumination of ethidium bromide-stained gels, cut out, Dounce hoand repeatedly extracted mogenized, against phenol with buffer (10 mM Tris, pH 7.9, 10 n&f EDTA, 0.1 M NaCl). DNA fragments thus extracted were concentrated by ethanol precipitation and found to be reduced at least three orders of magnitude in infectivity from WT SV40 DNA as assayed by DEAE-Dextran facilitated uptake into CV-1 cells (McCutchan and Pagano, 1968). Timed pregnant LHC inbred Syrian hamsters were purchased (Charles River) allowing the injection of baby hamsters within 6-12 hr of birth. Hamsters were injected subcutaneously over the right shoulder with either 0.2 cc of virus stock or 1 clg viral DNA suspended in 0.1 cc phosphatebuffered saline solution. Hamsters were examined weekly for a period of 1 l/2 years. Tumors once palpable grew rapidly to several centimeters in diameter within 2 to 3 weeks before regressing. Tumors were removed prior to necrosis, formaldehyde tlxed, paraffin embedded, and sectioned. Slides were stained with hematoxylineosin. At the time of excision the tumors were coded and after sectioning were examined blind at one sitting by one observer and scored dichotomously for a number of histologic or cytologic features (absence of necrosis, monomorphic cell population consisting of spindle cells, small round cells, or large cells, the presence of giant cells, and the presence of two or more histologic patterns), and on a scale of one to five for the degree of admixture of cells, for nuclear-cytoplasmic ratios, and for the relative proportion of giant cells and other bizarre forms. After the histologic study was completed the slides were uncoded and possible correlations between type of virus and his-
TUMORIGENICITY
OF SMALL-t
tologic and cytologic patterns were tested. Tumor lines were established in culture by physical disaggregation of nonnecrotic tumor tissue followed by trypsinization. Tumor-derived lines were only passaged long enough to be frozen in bulk. All experiments were performed on replica thawed vials within 2 weeks of revival. The derivation of the LHC hamster embryo fibroblast SV40 transformants was described in Frisque et al., (1980). All cultures expressed SV40 T-antigen(s) as assayed by immunofluorescence (Botchan et al., 1976) in at least 95% of the cells. Cells were propagated on plastic culture plates (Falcon) at 37.5”C in a humidified atmosphere at 10% COdair. Growth medium was DME (Gibco) containing penicillin and streptomycin supplemented with 10% fetal calf serum (Irvine). Growth in methocel, immunofluorescent determination of cytoplasmic actin cable networks, and measurement of plasminogen activator synthesis were determined exactly as in Frisque et al. (1980). Rabbit anti-a&in serum was the gift of Keith Burridge. The determination of the SV40 integration patterns in the tumor derived and SV40-transformed lines was performed essentially as in Botchan et al. (1976). Highmolecular-weight cellular DNA was purified from two roller bottles of cells. Five micrograms of DNA from each line was restricted with an enzyme which does not cleave within the SV40 sequences (e.g., BgZII or S&I, BRL) and electrophoresed through a 0.7% horizontal agarose slab gels (Tris -phosphate buffer). The DNA was deTABLE
MUTANTS
343
OF SV40 TABLE
2
Virus
Number of animals
Tumor incidence 66)
Average latency (months)
WT d12000 dl2001 d12004 di2007 d1890 dl2003 d12005 dl2006 d1884
14 4 11 6 4 5 4 4 7 8
106 50 73 83 100 40 0 75 28 38
7 l/2 13 l/2 12 13 12 10 12 13 14
Note. 6- to 12-hr-old LHC inbred Syrian hamsters were injected subcutaneously over the right shoulder with 0.2 cc virus stock containing 2-4 x 10’ PFU virus. At least six animals were injected with each virus although a few died during the course (18 months) of the experiment due to causes apparently unrelated to the injected virus (fight wounds, exposure to the elements during a heating failure, etc.).
natured in situ and transferred to nitrocellulose filters by “blotting.” The filters were hybridized to 0.33~pg “nick translated” SV40 (cloned in the bacterial plasmid pBR322) in 15 ml of hybridization solution. The specific activity of the probe was routinely lo* cpm/pg DNA. Films (Kodak XRl) were exposed to the hybridized blots for 7 - 10 days using intensifier screens (DuPont) for enhanced sensitivity. Recovery of virus was by polyethylene glycol enhanced DNA infection of permissive CV-1 cells essentially as described in Graessmann et al. (1979). RESULTS
1
SV40 DNA
Number of animals
Tumor incidence (8)
Uncut Linear (RI)
9 8
89 a7
6 II2 5 l/2
BamlHpaII
7
29
9
Pst I “A”
7
0
Average latency (months)
-
Note. 6 to 12hr old LHC inbred hamsters were injected subcutaneously over the right shoulder with 1 pg DNA suspended in 0.1 cc phosphate-buffered saline solution.
Tumorigenicity of WT SV4.0 DNA DNA Fragments
and
It is known that the DNA of SV40 is infectious in cultured cells (Gerber, 1962). More recently it was reported that viral DNA is also tumorigenic when injected subcutaneously into newborn hamsters (Sol and van der Noordaa, 1977). The results of injection of various SV40 DNA sequences into inbred LHC Syrian hamsters are shown in Table 1. Both closed circular and linear (generated by cleavage with endonu-
TOPP,
344 TABLE
RIFKIN,
3
Virus
Animals injected
Tumor incidence (%I
Average latency (months)
WT dl54/59
14 55
100 51
7 l/2 12.5
Note. averaged.
Results
in Table
2 for
dl 54/59
viruses
are
clease EcoRI which cuts the SV40 circular genome once within the region not expressed in nonpermissive cells) viral DNA are tumorigenic, tumors appearing with latencies similar to those induced by whole virus (Table 2). Not all the viral DNA sequences are required for tumor induction. The fragment of DNA generated by double cleavage with restriction endonucleases BumHI and HpaII is capable of inducing tumors albeit with a reduced frequency (Table l), consistent with the fact that this fragment will induce transformation in vitro with a similarly decreased efficiency (Graham et al., 1974). This fragment (roughly 55% of the viral genome) contains the entire region normally transcribed in transformed cells and will direct the synthesis of normal large-l‘ and small-t protein but has deleted sequences encoding all other (known) viral proteins. A fragment (P&I “A” fragment) which contains roughly 75% of the genome but which has lost approximately 170 amino acids at the carboxy terminus of the large-T protein is not tumorigenic consistent with the observation that all SV40-transformed cells examined have been found to synthesize a large-T protein with an electrophoretic mobility very similar to that observed in a lytic infection. It is likely that the minimum colinear region of WT SV40 which will induce tumors is that which encodes the entire early region. Tumorigenicity of WT and dl 54159 SV4.0 The viruses studied, the number of animals injected, the tumor incidence (within 18 months of injection), and the average tumor latency are listed in Table 2. The averaged results are in Table 3. It can be
AND
SLEIGH
seen that all the dl 54159 viruses with the possible,exception of dl2003 are capable of inducing tumors. However, the average tumor latencies are close to twice those observed in the animals injected with similar doses of WT SV40. Also the average tumor incidences are significantly reduced. Tumor Histology
Ten Wt- and fifteen dl 54i59 SV40-induced tumors were excised before necrosis, fixed with formaldehyde, embedded, and sectioned. It was impossible within this sampling to distinguish between the two classes of tumor by any criterion tested. Two WT and two mutant-induced tumors appeared to be highly cellular small-cell tumors and perhaps represented stem cells. The remainder of the tumors were fibrosarcomas roughly one-half of which were myxoid variants. Although most tumors appeared healthy, areas of focal necrosis were occasionally observed and roughly one-third of the sections showed substantial numbers of giant cells and other bizarre forms. Properties of Tumor Culture
Lines Explanted
to
Ten tumors induced by WT and twenty tumors induced by dl54i59 SV40 were explanted to culture by physical and enzymatic disaggregation. These uncloned tumor-derived lines were examined for the ability to grow suspended in methylcellulose, the loss of cytoplasmic actin-containing cable networks, and the synthesis of plasminogen activator. These properties have been found to be good in vitro markers of cellular tumorigenicity for rat cells (Pollack et al., 1975). The results are summarized in Table 4. It can be seen that a substantial majority of the tumor-derived lines, both WT and dl 54159 induced, are transformed by these criteria. Moreover an examination of the data (not shown) shows that there is a correlation between the expression of these three properties; i.e., cells which fail to clone in methylcellulose also tend to retain actin cable networks and to synthesize low levels of plasminogen activator. This is in contrast to LHC hamster
TUMORIGENICITY
OF SMALL-t
embryo cells transformed in culture which appear to show no correlation among these properties and in which growth in methylcellulose is the only good in vitro marker of cellular tumorigenicity (Frisque et al., 1980). We do now know if the rare (approximately 10%) tumor-derived lines which fail these criteria of in vitro transformation will yield secondary tumors with the same properties upon in vivo passage. As with the histological sections we can detect no difference on the basis of these criteria between the tumors induced by Wt or dl54/59 virus. The patterns of viral DNA integration within five WT- and five dl 54/59-induced tumors were examined by cleavage of whole cell DNA with restriction endonucleases followed by gel electrophoresis. DNA was transferred to nitrocellulose filters and viral sequences detected by hybridization to radioactive labeled SV40. Because the cell DNA was restricted with an enzyme, SstI or BglII, which does not cleave SV40 each band hybridizing to radioactive viral DNA corresponds to an insertion of SV40 flanked on either side by host sequences (Botchan et al., 1976). The total number of bands in each track represents the minimum number of unique insertions of SV40 in each line. Most tumorderived lines, both WT and dl 54l59 SV40 induced, contain several viral insertions, and there is no obvious difference between the patterns of WT and dl54/59 viruses. In addition all lines appear to be carrying free viral DNA at a level of one to two copies/ cell, although this may be due to a rare cell containing several hundred to a thousand copies while the majority are free of unintegrated sequences. Cleavage with enzymes cutting SV40 only once results in free linear viral DNA as well as host-viral “junction bands.” An autoradiogram of one gel resulting from restriction endonuclease digestion of the mutant-induced tumor DNA with Sst I is shown in Fig. 1. We have not monitored these lines for release of infectious virus. The integration patterns in several lines of LHC hamster embryo fibroblast cells transformed in vitro by WT or dl 54t59 virus (Frisque et al., 1980) were also exam-
MUTANTS
OF SV40
345
TABLE
4
IN VITRO PROPERTIES OF VIRALLY INDUCED TUMOR LINES
Virus WT SV40 dl 54159 sv40
Fraction of lines tested growing in suspension
Fraction of lines tested losing actin cables
Fraction of lines tested synthesizing plasminogen activator
9/10
719
617
17119
lO/lO
16/19
ined (data not shown). Again most lines show several unique insertions and all carry free viral DNA at the average level of one to two copies/cell. It would appear that at the DNA level the virus/cell interaction is similar whether established in vivo or in vitro, and by WT or by dl 54159 mutant virus. By a similar procedure we were able to determine that the integrated viral sequences possessed at least one feature characteristic of the infecting virus; resistance to the restriction endonuclease Tag1 (Shenk et al., 1976; Sleigh et al., 1978) which cleaves SV40 once at 0.57 on the standard map. Cleavage of a number of tumor cell DNAs by Tug1 produced results consistent with a “no-cut” enzyme including the continued presence of form II SV40 similar to that in Fig. 1 (data not shown). Further, virus was recovered from 8 of 15 lines of dl 54/59-induced tumor cells which were fused to permissive CV-1 cells. All recovered viruses were identical to those originally injected by restriction enzyme analysis. Although we have not analyzed these recovered viruses at the sequence level it is very likely that the dl 54159 viruses were responsible for the induced tumors. DISCUSSION
There are two virally encoded proteins expressed in nonpermissive cells infected with SV40 (Prives et al., 1977). The results reported here indicate that SV40 variants which have suffered deletions in sequences encoding one of these two proteins, the
346
TOPP, RIFKIN,
AND SLEIGH
It III I
FIG. 1. DNA from SV40-induced tumor-derived lines cleaved with restriction endonuelease SstI electrophoresed through a 0.7% agarose slab gel, transferred to a nitrocellulose filter, and hybridized to radioactive SV40 as described under Materials and Methods. SstI does not cleave within SV40 so each band represents a unique insertion of SV40. Shown are five tumors induced by dl54/59 mutants, one induced by TsA209, an in vitro SV40-transformed rat cell with multiple insertions, and 1O-5 pg SV40. Forms II and III SV40 are blurred by overexposure.
viral “small-t,” are still tumor-inducing viruses. Many of these viral mutants (e.g., dl 884 SV40) not only have deleted sequences encoding protein but also have lost sequences necessary for the proper splicing and subsequent maturation of the small-t mRNA. Thus only the large-T protein is expressed in cells infected with these mutants (Khoury et al., 1979). It is therefore significant that these small-t defective viruses induce tumors that are by all criteria examined identical to those induced by WT virus. Within our sample the dl 54/59- and Wt SV40-induced tumors: (1) are histologically similar, mainly fibrosarcomas, (2) grow at roughly the same rates after appearance, (3) possess the same phenotypes in vitro, i.e., the majority grow suspended in methocel, have lost actin cable networks, and synthesize plasminogen activator, (4) show similar patterns of viral integra-
tion into the cellular chromosomal DNA, i.e., one to several copies of the viral genome are integrated at unique sites along with a low level (one copy/cell) of free viral DNA. Based on these results it is likely that it is the large-l‘ protein alone which is primarily responsible for the oncogenic potential of SV40 in hamsters. Similar results have recently been reported from another lab (Lewis and Martin, 1979). This result is in contrast to the requirement for the small-t protein for the development of the fully transformed phenotype in rat cells, a difference upon which we have remarked elsewhere (Frisque et al., 1980). We cannot rule out the formal possibility that either of the two proteins can function as an oncogene. Viral mutants to test this hypothesis lacking only large-T activity are only now becoming available (Gluzman et al., 1980). The phenotypic “transformation” of cultured cells which follows infection with
TUMORIGENICITY
OF SMALL-t
SV40 has been studied as an in vitro model of viral tumorigenicity. The small-t protein is not required for either the initiation of the maintenance of the transformed state in cultured hamster cells (Martin et al., 1979a; Frisque et al., 1980) except under certain restricted circumstances (Martin et al., 19’79b), in good agreement with the tumorigenicity of the dl 54/59 (small-t defective) SV40 mutants. On the other hand the viral large-T protein has been found to be required for the maintenance of transformation in all cells examined (Kimura and Dulbecco, 197’7; Tegtmeyer, 1975; Osborn and Weber, 1975a; Martin and Chou, 1975; Brugge and Butel, 1975; Kimura and Itagaki, 1975). Large-T is a self-aggregating (Potter et al., 1969; Osborn and Weber, 1975b; Kuchino and Yamaguchi, 1975) phosphoprotein (Tegtmeyer et al., 1975) with a monomer molecular weight as determined by SDS-acrylamide gel electrophoresis of 9OK daltons (Prives et al., 1977) although the true molecular weight is somewhat lower (Griffin et al., 1978). Much is known about the interaction of large-T with infected cells in which this protein which expresses several enzymatic activities plays a role in the control of both host and viral DNA and RNA synthesis (Table 5). As yet there is no clue as to which, if any, of these large-T-related properties are relevant either to viral transformation or tumorigenicity. While all the evidence is that the WTand dl 54/59-induced tumors are qualitatively similar the reduced frequency and greater latency of the mutant-induced tumors may indicate a role for small-t protein at the aggregate rather than the cellular level, perhaps as a tumor promoter. Tevethia et al., (1980) have shown that small-t defective viruses are not significantly different in the induction of transplant rejection antigens so the effect is unlikely to be due to differential immune surveillance. Interestingly, it has been reported that agents which act as tumor promoters can in some ways compensate for a small-t defect for transformation in vitro (Topp et al., 1979; Martin et al., 197913) and it is an intriguing possibility that the mechanisms of
MUTANTS
TABLE BIOLOGICAL
347
OF SV40 5
PROPERTIES OF SV40 PROTEIN
Property DNA
binding
LARGE-T
Reference protein
Required for SV40 DNA synthesis Has three high-affinity DNA binding sites at the origin of viral DNA replication Stimulates host cell DNA synthesis Required for initiation of late viral transcription Regulates level of early viral transcription
Induces the synthesis of and binds to a cellular protein of MW 50-60K Possesses ATPase activity
Carrol et al., 1974; Jesse1 et al., 1975 Tegtmeyer,
1972
Tjian, 1978; 1975;
Reed
Tijan
et
al.,
et al., 1978
Tegtmeyer and Ozer, 1971 Tegtmeyer et al., 1975; Aiwine et al., 1975; Reed et al., 1976; Khoury and May, 1977 Lane and Crawford, 1979; Linzer and Levine, 1979 Tjian and Robbins, 1979; Tjian et al., 1980; Griffin et al., 1979, 1980
action of the small-t protein and of tumor promoters such as the phorbol esters may be related.4 Several mechanisms have been proposed to explain the action of tumor promoters (Slaga et al., 1978). Two of these are based on the expression of a normally recessive transforming mutation and on the generation of a critical tumor cell mass (Blumberg, 1980). The expression of a recessive trait may arise through the production of cells homozygous for a mutation by induction of sister chromatid exchange. SV40 does induce chromosomal abnormalities in infected cells (Bartsch, 1970; Lehman, 1974) although it is not known if the viral small-t protein is involved. The second hypothesis is that of a minimum clone size. Adherants of this theory would contend that there is a minimum number of potential tumor cells which must 4 A similar possibility has been suggested for the of Rous sarcoma virus (Bissell et al., 1979).
SRC gene
348
TOPP, RIFKIN,
be present for tumor growth. This might be due to either the production by surrounding normal tissues of factors (chalones) which control growth, from which the inner mass of the tumor must be isolated in order to grow (Bell, 1976) or to the production by the tumor cells of a growth factor (Todaro et al., 1976) which, at low tumor cell densities, diffuses away. The tumor promoter (small-t) wouold act as a mitogen, enhancing the rate of division of the tumor cells and bringing the tumor more quickly to critical mass. We are looking at the affect of small-t on chromosomal abnormalities and are characterizing the TD,, of cells transformed either in vivo or in vitro by the dl 54/59 and WT viruses. It will be interesting to see if the small-t protein is implicated in either of these two phenomena. Tumor promotion is not the only possible explanation for the role of small-t in viral tumorigenesis. The similarity of integration patterns of the dl M/59 and WT virus tentatively rules out a strict gene dosage effect. However it is possible that WT SV40 initially becomes stably integrated within a greater number of cells. This may be due to an increase in effective viral inoculum by a limited proliferation in vivo of only nondefective virus leading to the spread of WT virus to a larger population of cells. If the probability is constant for a given virally initiated cell to begin growth as a tumor, the greater number of cells initiated by nondefective SV40 would produce tumors with decreased average latency and increased average frequency. We cannot distinguish between this and the other hypotheses on the basis of our data. ACKNOWLEDGMENTS We would like to thank Chris Paul and Ruth Crowe for excellent technical assistance and Bob Martin, Peter Blumberg, and particularly Bernard Lane for helpful discussions during the course of the work. This work was supported by Grants CA24803, CA23753, and HL22266 from the NIH. REFERENCES ALLISON, A. C., PHESTERMAN, F. C., and BARON, S. (196’7). Induction of tumors in adult hamsters with simian virus 40. J. Nat. Cancer Inst. 38, 567-572. ALLISON, A. C., and TAYU)R, R. B. (1967). Observa-
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