Synergy between Apc min and an activated ras mutation is sufficient to induce colon carcinomas.

Colon carcinomas appear to arise from the cumulative effect of mutations to several genes (APC, DCC, p53, ras, hMLH1, and hMSH2). By using novel colonic epithelial cell lines derived from the Immorto mouse, named the YAMC (young adult mouse colon) cell line, and an Immorto-Min mouse hybrid, named the IMCE (Immorto-Min colonic epithelial) cell line, carrying the Apc min mutation, we investigated the effect of an activated v-Ha-ras gene on tumor progression. The YAMC and IMCE cell lines are normal colonic epithelial cell lines which are conditionally immortalized by virtue of expression of a temperature-sensitive simian virus 40 (SV40) large T antigen. Under conditions which permit expression of a functional SV40 large T antigen (33 degrees C plus gamma interferon), neither the YAMC nor the IMCE cell line grows in soft agar or is tumorigenic in nude mice. In vitro, when the SV40 large T antigen is inactivated (39 degrees C without gamma interferon), the cells stop proliferating and die. By infecting the YAMC and IMCE cell lines with a replication-defective psi2-v-Ha-ras virus, we derived cell lines which overexpress the v-Ha-ras gene (YAMC-Ras and IMCE-Ras). In contrast to the parental cell lines, under conditions in which the SV40 large T antigen is inactive, both the YAMC-Ras and IMCE-Ras cell lines continue to proliferate. Initally YAMC-Ras cells do not form tumors; however, tumors are visible after 90 days of incubation. IMCE-Ras cells form colonies in soft agar under both permissive and nonpermissive culture conditions. Furthermore, IMCE-Ras cells form tumors in nude mice within 3 weeks. The phenotype of the IMCE-Ras cell line thus clearly demonstrates that a defective Apc allele and an activated ras gene are sufficient to transform normal colonic epithelial cells and render them tumorigenic.     

Recently activated T cells are costimulation-dependent in vitro.

While the prominent role of B7-mediated signaling in the activation of naive and resting T cells has been exhaustively demonstrated, it is unclear whether costimulation is required in the amplification of an initiated immune response. In this study we have developed a multistep culture system to investigate the costimulation requirements of recently activated alloreactive CD4(+) T cells and the outcome of allorecognition of B7-deficient, MHC class-II-expressing epithelial cells. The results show that following in vitro "priming" with allogeneic costimulation rich antigen presenting cells, T cells can be reactivated to proliferate only if B7-mediated costimulation is provided. Furthermore, recognition of antigen on B7-negative epithelial cells induced allospecific nonresponsiveness in the responder T cells. Finally, the nonresponsive state was not accompanied by IL-4 secretion and appeared to be reversible, since T cell reactivity could be restored by short-term culture in the presence of IL-2. These observations suggest that "primed" T cells remain B7-dependent in vitro and are susceptible to functional inactivation following costimulation-deficient antigen presentation.     

Middle T antigen as primary inducer of full expression of the phenotype of transformation by polyoma virus.

A large number of polyoma virus-transformed cells of rat, mouse, and hamster origin were examined for presence of T-antigen species. The results showed that all lines of cells contained middle and small T antigens, but not all contained a full-sized large T antigen, in some cell lines large T antigen was absent, whereas in others it was present as truncated forms lacking various lengths of the carboxy-terminal part of the protein. Cells transformed by the new viable deletion mutants of polyoma virus, dl-8 and dl-23, formed larger and smaller colonies or foci, respectively, when they were suspended in semisolid medium or plated as monolayers together with untransformed cells on a plastic surface. The deletions in the DNA of these mutants resulted in the shortening of the large and middle T antigens simultaneously without affecting the size of the small T antigen. Variation of large T-related proteins in dl-8 and dl-23-transformed cells seemed to be the same as that observed in wild-type-transformed cells. Regardless of the amount and size of large T-related protein in mutant-transformed cells, the phenotype of the cells was entirely dependent on the mutant used. The results suggest that (i) persistence of large T antigen is not universally required for the maintenance of the transformation phenotype, (ii) small T antigen alone may not be sufficient for inducing the full expression of the transformation phenotype, and (iii) middle T antigen is implicated as being primarily responsible for the full expression of the phenotype of transformation. The results also provide the evidence that the carboxy-terminal region of middle T antigen and a part of large T antigen are encoded in the genome in the same DNA segment around map units 88 to 94 in different reading frames.     

Comparison of a T cell clone and of T cells from a TCR transgenic mouse: TCR transgenic T cells specific for self-antigen are atypical

It has been widely assumed that T cells from TCR-transgenic (Tg) mice better represent the behavior of T cells from normal mice than do in vitro cultures of T cell clones. We have found that autoreactive T cells arising in the presumably more physiological environment of the BDC-2.5 TCR-Tg mouse, despite being apparently "naive" in surface phenotype, are highly activated functionally and do not resemble CD4(+) T cells from a spontaneously diabetic nonobese diabetic (NOD) mouse or the NOD-derived, diabetogenic CD4(+) T cell clone of origin, BDC-2.5. Our results suggest that autoreactive T cells cloned from the spontaneously diabetic NOD mouse more closely resemble effector T cells arising during the natural disease process.     

Antibody response to simian virus 40 tumor antigen in nude mice reconstituted with T cells.

Athymic BALB/c nude mice (nu/nu) fail to generate circulating antibodies to simian virus 40 (SV40) tumor (T) antigen when immunized with SV40-transformed mouse cells or with T antigen positive somatic cell hybrids derived from SV40-transformed human and normal mouse parental cells. However, normal BALB/c mice readily produce antibodies to SV40 T antigen. When nude mice were reconstituted with normal syngeneic T lymphocytes from spleen or thymus source, the humoral immune responsiveness to SV40 T antigen was restored.     

Properties of cells transformed by the middle T-antigen-coding region of polyomavirus.

A series of 10 Fischer rat transformed clonal cell lines were independently obtained in infections with a defective polyomavirus containing a scrambled genome except for an intact middle and small T-antigen-coding region. These cells synthesize middle and small T antigens; no fragment of large T antigen can be detected in any of them. The transformed phenotype of this set of cell lines (designated LT-) has been studied with respect to serum dependence, saturation density, and anchorage independence and compared with the phenotype of a set of six transformants (designated LT+) which synthesize detectable to high levels of shortened or normal-sized large T antigen. Both the LT+ and the LT- groups of polyomavirus transformants display a range of transformed phenotypes. These ranges overlap, and the variations within each group are larger than the variations between the two groups. Thus, the results suggest that, for established Fischer rat fibroblasts, the maintenance of any of the three phenotypes tested and, in particular, of serum independence is not necessarily correlated with the levels of large T antigen or fragments thereof.     

Untransformed rat cells containing free and integrated DNA of a polyoma nontransforming (Hr-t) mutant.

The polyoma virus hr-t deletion mutant A185, when compared to wild-type (Py) virus, is at least 10⁵ fold inhibited in its transforming ability. Total cellular DNA from 50 cell lines derived from individual colonies formed after infection of Rat-1 cells with A185 virus was analyzed for the presence of viral sequences by “blot” hybridization (Southern, 1975). Viral sequences were detected in two of these cellular DNAs. One positive cell line (18–37) was studied in detail. The viral sequences present in 18–37 cells as well as the viral sequences present in virus rescued from 18–37 after fusion with permissive mouse cells were identified as A185 and not Py sequences. The A185 viral sequences in 18–37 cells were found to exist both covalently linked to host DNA sequences (integrated) and as free forms. The integrated A185 viral sequences were present in a partial head-to-tail tandem array, as has been observed for Py sequences in transformed rat cells (Birg et al., 1979). Both integrated and free forms of A185 viral sequences were retained in subclones of the parental 18–37 cell line although a simplification of the integrated viral sequence was observed. In the 18–37 cells the 100K large T antigen was synthesized but the 55K middle and 22K small T antigen species were not detected. The 18–37 cells had a normal morphology, were density-sensitive, anchorage-dependent and did not form tumors when injected into syngeneic animals. This normal phenotype of the 18–37 cells was not a result of the inability of the cells to express the transformed phenotype, since the 18–37 cells could be transformed at a high frequency upon infection with Py virus. These results show that integration of viral sequences per se or the presence of the 100K large T antigen is not sufficient for the transformed phenotype to be expressed, and strongly suggest that Py-induced transformation is mediated by the 55K middle and/or 22K small T antigens.     

Conditional immortalization of normal and dysgenic mouse muscle cells by the SV40 large T antigen under the vimentin promoter control.

We have created new mouse muscle cell lines of an immortalized type, expressing normal differentiation at the myotube stage: sarcomeric organization, functional excitation-contraction coupling, and triadic differentiation. The DNA immortalizing recombinant utilizes a deletion mutant of the regulatory region of the human vimentin promoter controlling the expression of a SV40 thermosensitive large T antigen, in which the small t sequence has been deleted. Skeletal mouse replicative myoblasts synthesized predominantly vimentin. After myoblast fusion the vimentin gene is strongly repressed in multinucleated syncytia. Furthermore, the normal activity of the vimentin promoter in myoblasts is increased in the large T antigen-expressing cells. We observed that continuous and rapid division of myoblasts occurs at permissive temperature, suggesting that immortalization is achieved even though the small t antigen is absent. When fusion is induced by changing media conditions, large T antigen expression is totally repressed by the vimentin promoter. When the temperature is elevated to 39 degrees C, the preexisting large T antigen is inactivated. The resulting myotubes from normal mouse differentiate totally normally as indicated by their morphology, ultrastructure, and electrophysiological properties. Mutant (muscular dysgenesis) immortalized cells express the same properties as mutant primary counterparts with no contraction, no slow Ca2+ current, and no triadic differentiation. These immortalized cell lines are potentially very useful for further pharmacology, transplantation, and cell biology studies. The vimentin promoter control of immortalizing recombinant DNA can be used for any mammalian normal and mutant muscle cell lines.     

Cooperation of p53 and polyoma virus middle T antigen in the transformation of primary rat embryo fibroblasts.

Cell transformation in vivo seems to be a multistep process. In in vitro studies certain combinations of two oncogenes, a cytoplasmic gene product together with a nuclear gene product, are sufficient to transform primary rodent cells. Polyoma virus large T antigen can immortalize and, in cooperation with polyoma virus middle T antigen, transform primary cells. On the other hand mutant mouse p53 can also immortalize and, in cooperation with an activated Ha-ras oncogene, transform primary cells. In the present study we analyzed whether mutant p53 can replace polyoma virus large T antigen in a cell transformation assay with polyoma virus middle T antigen. Transfection of mutant p53 alone resulted in a cell line which had retained the actin cable network, grew poorly in medium with low concentration of serum, and failed to grow in semisolid agar. Cotransfection of mutant p53 together with polyoma virus middle T led to cells which grew in medium containing low serum concentration, grew well in semisolid agar, and displayed an altered morphology with the tendency to overgrow the normal monolayer. By these criteria these cells were considered fully transformed. The rate of p53 synthesis was similar in both cell lines. However, only p53 from the transformed cell line turned out to be stable. Cells transformed by mutant p53 and polyoma virus middle T expressed nearly the same amount of the c-src-encoded pp60c-src protein as cells transformed by the same p53 and cotransfected activated Ha-ras oncogene. However, only the polyoma virus middle T/p53-transformed cells exhibited an elevated level of pp60c-src-specific tyrosine kinase activity. Thus, despite different mechanisms leading to cell transformation, mutant p53 can replace polyoma virus large T antigen and polyoma virus middle T can replace the activated Ha-ras oncogene in cell transformation.     

Analysis of a large-T-antigen variant expressed in simian virus 40-transformed mouse cell line mKS-A.

Earlier reports had suggested that the large T antigen expressed in simian virus 40 (SV40)-transformed mKS-A cells may be replication defective. Our experiments support these earlier observations showing that the mKS-A T antigen has a reduced DNA-unwinding activity in vitro. To investigate the molecular basis for this defect, we have isolated from an mKS-A genomic library an EMBL-3 bacteriophage clone carrying in its insert a full-length SV40 DNA element that most likely encodes the expressed T-antigen variant. DNA sequencing revealed only one nonconservative amino acid exchange, Asp to Asn at residue 636. Surprisingly, when a plasmid clone carrying the mKS-A T-antigen-coding sequence was transfected into monkey cells, we found that it replicated quite efficiently, probably suggesting that a high nuclear concentration of the variant T-antigen form compensates for the partial biochemical defect. However, a high nuclear concentration of T antigen was also found in mKS-A T-antigen-transformed mouse cells, yet a fusion of these cells to permissive monkey cells failed to induce in situ replication and excision of integrated SV40 DNA. We discuss possible reasons for the different behavior of T antigen in monkey cells and in mouse cells and suggest that one possibility for the replication-negative phenotype in transformed cells may be related to the fact that T antigen forms a tight complex with the cellular p53 protein in mouse cells but not in monkey cells.     

The role of activated accessory cells in preventing immunosuppression by hydrocortisone.

The generation of humoral immunity in vitro by normal and antigen-primed mouse spleen cells was suppressed by in vitro treatment with hydrocortisone. Functions of normal and antigen-activated helper T lymphocytes and of accessory cells were inhibited by the corticosteroids. Spleen cells cultured overnight in medium containing fetal bovine serum became highly resistant to the effects of hydrocortisone. Similar resistance was found to occur when spleen cells were cultured with accessory cells that previously had been activated with bacterial lipopolysaccharide. These studies show that immunologically nonspecific processes significantly alter the effects of the steroids on specific immune responses and suggest that accessory cell products modulate T cells in ways which differ from antigen induction.     

Active ras and myc oncogenes can be compatible, but Sv40 large T antigen is specifically suppressed with normal differentiation of mouse embryonic stem cells

The pathobiological effects of oncogenes on normal differentiation of mouse embryonic stem cells from 4-day embryos were examined by introducing active ras, myc, and SV40 large T genes, all driven by mouse metallothionein I enhancer and promoter. Stem cell clones R5, M3, and T2 for ras, myc, and SV40 T genes, respectively, were particularly chosen for analyses because of their higher levels of transgene expression and their diploid chromosomal constitutions. These stem cells were then introduced into host 4-day embryos and the embryos were allowed to develop in the uterus of foster mothers. The stem cells colonized the tissues as extensively as the parent cells and gave rise to adult chimera with no apparent loss or abnormality of the embryos. The active ras and myc oncogenes introduced were expressed not only in the stem cells, but also in the developing embryos and in a variety of tissues of adult chimeras. However, although T antigen was originally expressed in the stem cells, it was not expressed in either developing embryos or tissues of adult chimeras. Induced by retinoic acid treatment in vitro or by subcutaneous grafting, this suppression of T-gene expression was also confirmed in differentiated progeny cells from several stem cell clones expressing T antigen. Permanent lines of fibroblast-like cells could be established at higher frequency from primary cultures of tissues of chimera, subcutaneous differentiated cells, and in vitro differentiated cells derived from T2 cells, and all these clones reexpressed T antigen. The results suggest that active myc and ras genes can be compatible with normal differentiation of the stem cells, but the expression of T antigen is specifically suppressed with recognition of its coding domain.     

Expression of human IFN-gamma genomic DNA in transgenic mice

We have introduced an 8.6-kb fragment of human genomic DNA containing the full length IFN-gamma gene into the mouse germline. The transgenic animals had no biologic or developmental defects as human IFN-gamma does not bind to the mouse IFN R. Regulation of the transgene paralleled that of the endogenous murine IFN-gamma gene in that: 1) it is not expressed constitutively in any tissue examined thus far, 2) it can be induced in thymus and spleen cells by T cell mitogens, 3) it is not expressed in B cells stimulated by LPS, and 4) it produces normal mRNA and biologically active IFN protein. Whereas expression of the transgene is likely restricted to T cells, we had observed that both fibroblasts and B cell lines could express the same DNA when transfected in vitro; this indicates that in vivo, developmental factors restrict expression of the IFN-gamma gene to T cells. These findings also indicate that the 8.6-kb fragment contains the regulatory elements necessary for normal tissue specific expression in vivo. Moreover, they indicate that the regulatory elements for this gene are completely preserved over the phylogenetic distance separating mouse and man, even though substantial drift has occurred in the structural gene, and probably in the IFN-gamma R as well.     

Biological and biochemical studies of cells transformed by simian virus 40 temperature-sensitive gene A mutants and A mutant revertants.

The growth properties of hamster cells transformed by wild-type Simian virus 40 (SV40), by early SV40 temperature-sensitive mutants of the A complementation group, and by spontaneous revertants of these mutants were studied. All of the tsA mutant-transformed cells were temperature sensitive in their ability to form clones in soft agar and on monolayers of normal cells except for CHLA-30L1, which was not temperature sensitive in the latter property. All cells transformed by stable revertants of well-characterized tsA mutants possessed certain growth properties in common with wild-type-transformed cells at both temperatures. Virus rescued from tsA transformants including CHLA30L1 was temperature sensitive for viral DNA replication, whereas that rescued from revertant and wild-type transformants was not thermolabile in this regard. T antigen present in crude extracts of tsA-transformed cells including CHLA30L1, grown at 33 degreeC, was temperature sensitive by in vitro immunoassay, whereas that from wild-type-transformed cells was relatively stable. T antigen from revertant transformants was more stable than the tsA protein. Partially purified T antigen from revertant-transformed cells was nearly as stable as wild-type antigen in its ability to bind DNA after heating at 44 degrees C, whereas T antigen from tsA30 mutant-transformed cells was relatively thermolabile. These results further indicate that T antigen is a product of the SV40 A gene. Significantly more T antigen was found in extracts of CHLA30L1 grown to high density at the nonpermissive temperature than in any other tsA-transformed cell similarly grown. This is consistent with the suggestion that the amount of T antigen synthesized in CHLA30L1 is large enoughto allow partial expression of the transformed phenotype at the restrictive temperature. Alternatively, the increase in T antigen concentration may be secondary to one or more genetic alterations that independently affect the transformed phenotype of these cells.     

Phosphorylation of polyoma T antigens.

The T antigens of polyoma virus have been examined for phosphorylation in vivo and associated protein kinase activities in vitro. The 100K "large" T antigen is the major phosphoprotein among the T antigen species in vivo as determined by labeling virus-infected cells with 32P-orthophosphate. Hr-t mutants show normal phosphorylation of their 100K T antigens. The wild-type 56K plasma membrane-associated "middle" T antigen is also phosphorylated in the cell, but to a lesser extent than the 100K; this low level phosphorylation is also observed in the presumably altered 56K protein induced by hr-t mutant NG59 and in the 50K truncated "middle" T of hr-t mutant SD15. Addition of dibutyryl cyclic AMP to the medium does not affect labeling of either large or middle T antigens in wild-type- or mutant-infected cells. Thus no differences are observed in T antigen phosphorylation in vivo between wild-type virus and hr-t mutants. Hr-t mutants are defective in a protein kinase activity assayed in vitro by adding gamma-32P-ATP to T antigen immunoprecipitates. In the case of wild-type virus, the 56K protein is the major phosphate acceptor in the in vitro kinase reaction, with a somewhat lower level of phosphorylation observed in the 100K band. Hr-t mutants NG59 and SD15 show no labeling of the altered 56K or 50K, respectively, but do show detectable levels of 32P in the 100K bands. A wild-type virus carrying a small deletion affecting the 100K and 56k bands shows a normal level of kinase activity associated with the truncated T antigens. Ts-a mutants appear to be normal with respect to the middle T antigen-associated kinase. Photoaffinity labeling of infected cell extracts with 8-azido cyclic AMP shows that the two major classes of regulatory subunits of cyclic AMP-dependent protein kinases are present in the immunoprecipitates. Phosphorylation of histone H1 occurs when this substrate is added to immunoprecipitates of either mock-infected or virus-infected cells, again demonstrating the presence of cellular kinases. Further experiments will be required to determine whether the middle T antigen of polyoma virus is itself a protein kinase or simply a substrate for one or more cellular kinases.     

High affinity binding of the large T protein of polyoma virus to a genomic mouse DNA sequence

We purified a fragment of mouse DNA to which the large T protein of polyoma virus was bound in chromatin prepared from transformed mouse cells. This sequence, which is not repeated to a measurable extent within the mouse genome, does not show any significant homology to the viral ori region, except in a short region, which comprises a sequence related to the consensus for recognition by large T proteins ((A,T)GPuGGC). This region of pCG4 was confirmed by in vitro binding assays to be essential for T antigen binding.     

Maturation and function of mouse T-cells with a transgenic TCR positively selected by highly disparate xenogeneic porcine MHC.

Remarkably normal cellular immune function, along with specific T-cell tolerance to highly disparate xenogeneic donors, can be achieved by grafting fetal pig thymus (FP THY) tissue to T and NK cell-depleted, thymectomized (ATX) mice. Porcine MHC can mediate positive selection of mouse CD4+ T-cells with a mouse MHC-restricted TCR in FP THY-grafted, T- and NK cell-depleted, ATX TCR-transgenic "AND" mice. However, functional studies were not performed on transgenic mouse T-cells selected in a FP THY graft. We have now performed further studies to confirm the ability of porcine MHC to mediate the positive selection of mouse T-cells with a mouse MHC-restricted TCR, and to exclude the possibility that the maturation of mouse T-cells with a mouse MHC-restricted TCR in FP THY grafts in ATX "AND" mice is a special case. For this purpose, TCR-transgenic mice with an unrelated transgenic TCR ["3A9", specific for hen egg lysozyme (HEL) peptide 46-61 presented by I-Ak] were employed. Similar to FP THY-grafted ATX "AND" mice, large numbers of mouse CD4 single positive thymocytes expressing the transgenic TCR (Vbeta8.2) and expressing a mature phenotype (Qa-2high and heat stable antigen, HSAlow) were detected in FP THY grafts. Porcine thymus grafting led to a high level of peripheral repopulation with mouse naive-type (CD44low CD45RBhigh CD62Lhigh) CD4+ cells expressing the transgenic TCR in T and NK cell-depleted ATX "3A9" mice, regardless of whether the recipients had a positive selecting or a non-selecting, class II deficient MHC background. The mouse CD4+ T-cells expressing the "3A9" TCR showed efficient primary proliferative responses to the protein antigen (HEL) when it was presented by mouse class II+ antigen presenting cells (APC) in vitro. These results, collectively, support the general conclusion that discordant xenogeneic porcine MHC can mediate positive selection of mouse T-cells with mouse MHC-restricted TCR. This study has implications for the potential clinical use of xenogeneic thymus transplantation to reconstitute cellular immunity in the setting of thymic insufficiency or thymectomy, and hence for its applicability to the induction of xenograft tolerance and in the treatment of immunodeficiency diseases.     

Characterization of flat revertant cells isolated from simian virus 40-transformed mouse and rat cells which contain multiple copies of viral genomes.

Eight clones of flat revertants were isolated by negative selection from simian virus 40 (SV40)-transformed mouse and rat cell lines in which two and six viral genome equivalents per cell were integrated, respectively. These revertants showed either a normal cell phenotype or a phenotype intermediate between normal and transformed cells as to cellular morphology and saturation density and were unable to grow in soft agar medium. One revertant derived from SV40-transformed mouse cells was T antigen positive, whereas the other seven revertants were T antigen negative. SV40 could be rescued only from the T-antigen-positive revertant by fusion with permissive monkey cells. The susceptibility of the revertants to retransformation by wild-type SV40 was variable among these revertants. T-antigen-negative revertants from SV40-transformed mouse cells were retransformed at a frequency of 3 to 10 times higher than their grandparental untransformed cells. In contrast, T-antigen-negative revertants from SV40-transformed rat cells could not be retransformed. The arrangement of viral genomes was analyzed by digestion of cellular DNA with restriction enzymes of different specificity, followed by detection of DNA fragments containing a viral sequence and rat cells were serially arranged within the length of about 30 kilobases, with at least two intervening cellular sequences. A head-to-tail tandem array of unit length viral genomes was present in at least one insertion site in the transformed rat cells. All of the revertants had undergone a deletion(s), and only a part of the viral genome was retained in T-antigen-negative revertants. A relatively high frequency of reversion in the transformed rat cells suggests that reversion occurs by homologous recombination between the integrated viral genomes.     

Analysis of a transgenic mouse containing simian virus 40 and v-myc sequences.

Two plasmids, one containing the simian virus 40 (SV40) genome and the mouse metallothionein I gene and one containing the v-myc gene of avian myelocytomatosis virus MC29, were coinjected into mouse embryos. Of the 13 surviving mice, one, designated M13, contained both myc and SV40 sequences. This mouse developed a cranial bulge identified as a choroid plexus papilloma at 13 weeks and was subsequently sacrificed; tissue samples were taken for further analysis. Primary cell lines derived from these tissues contained both myc and SV40 DNA. No v-myc mRNA could be detected, although SV40 mRNA was present in all of the cell lines tested. T antigen also was expressed in all of the cell lines analyzed. These data suggest that SV40 expression was involved in the abnormalities of mouse M13 and was responsible for the transformed phenotype of the primary cell lines. Primary cell lines from this mouse were atypical in that the population rapidly became progressively more transformed with time in culture based on the following criteria: morphology, growth rate, and the ability to grow in soft agar and in serum-free medium. The data also suggest that factors present in the mouse regulated the ability of SV40 to oncogenically transform most cells and that in vitro culture of cells allowed them to escape those factors.