Transforming viruses directly reduce the cellular growth requirement for a platelet derived growth factor

Early passage mouse embryo fibroblasts, mouse 3T3 cell lines, and early passage diploid human fibroblasts grew to higher cell densities in tissue culture medium supplemented with serum than in medium supplemented with defibrinogenated platelet-poor plasma (PPP). Unlike the mouse cells, the human fibroblasts displayed this differential growth response only in the presence of hypophysiologic concentrations of calcium. The addition of heat-treated extracts of human platelets to PPP-supplemented medium stimulated the replication of both the normal mouse cells and early passage human embryo fibroblasts. Human or mouse fibroblasts transformed by either retroviruses or by SV40, including SV40 infected “serum revertants” and “flat transformants,” grew to equal cell densities in medium supplemented with either serum or PPP. Infection of Balb/c-3T3 cells with SV40 rapidly induced them to grow in PPP-supplemented medium demonstrating that the ability of SV40-transformed cell lines to proliferate in PPP-supplemented medium does not arise from the cell culture selection procedures usually employed to obtain stable virus-transformed cell lines. 3T3 cells infected but not transformed by retroviruses do not replicate in PPP-supplemented medium demonstrating that reduction of the growth requirement for the platelet growth factor(s) by retroviruses is a transformation-specific response. Cell cultures that did not proliferate well in PPP-supplemented medium did not form tumors when inoculated into athymic nude mice. Many, although not all, of the lines which grew well in PPP medium were tumorigenic in nude mice. Together, these findings indicate that: (1) normal fibroblast-like cells display a growth requirement for factor(s) present in serum but not found in PPP; (2) this serum specific growth factor is derived from platelets; (3) a primary response to viral transforming genes is a reduction in the growth requirement for these platelet-derived factors; and (4) cells that have a reduced requirement for the platelet-derived growth factor are often tumorigenic.     

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.     

Evidence that a target-derived soluble factor is necessary for the selective lysis of SV40-transformed fibroblasts by activated mouse macrophages

Our laboratory is investigating the basis for the selective recognition of transformed cells by activated mouse macrophages. As targets we are using a panel of SV40-transformed, C3H.OL fibroblast cell lines (SV-COL) that display widely different levels of sensitivity to lysis, from highly sensitive to completely resistant. Our results show that adding conditioned medium from the macrophage-sensitive target SV-COL-E8 (CM(E8] to a cytolysis assay with the macrophage-resistant target SV-COL-F5f causes the macrophages to kill the resistant targets in a contact dependent fashion. We have termed this activity "macrophage cell lysis factor" (MCLF). MCLF activity was not detected in conditioned media from cells not killed by activated macrophages (i.e., 3T3-like cell lines or embryo fibroblasts) but was present in conditioned media from six other SV-COL cell lines at levels that were directly proportional to the sensitivity of those targets (r = 0.98). These data suggest that MCLF plays a key role in determining the lytic sensitivity of SV40-transformed fibroblasts. Finally, to ask whether the production of MCLF is sufficient to explain the selective recognition of SV40-transformed fibroblasts by activated macrophages we have tested whether CM(E8) will cause macrophages to kill normal cells. Our results show that in the presence of CM(E8) macrophages will kill immortalized, 3T3-like fibroblasts but will not kill normal embryo fibroblasts. These results suggest that the production of MCLF, or a similar activity, is necessary but not sufficient for macrophage cytolysis to occur and that a change in target cell phenotype that occurs during the process of immortalization is also required.     

Isolation of novel cDNA transformation markers from SV40-transformed human keratinocytes

Differential screening of a cDNA library was used to isolate probes for mRNAs that are induced in simian virus 40 (SV40)-transformed human keratinocytes. Several of these cDNAs hybrid select mRNAs which encode transformation-induced proteins found in the cytoskeletal component of SV40-transformed keratinocytes. One of these cDNAs was used to study the phenotype of normal and transformed cell lines derived from various tissues. We found that mRNA encoding the novel transformation-induced proteins is expressed in two squamous carcinoma cell lines derived from the oral epithelium, four SV40-transformed keratinocyte cell lines, and two SV40-transformed fibroblasts. Normal or transformed lymphoid cells or cell lines derived from carcinoma of the cervix do not express mRNAs which hybridize to these probes. The results from this study suggest that these probes may be used to detect markers of transformation in certain cell types.     

Fluctuation of simian virus 40 (SV40) super T-antigen expression in tumors induced by SV40-transformed mouse mammary epithelial cells.

Higher-molecular-weight forms of the simian virus 40 (SV40) large tumor antigen (T-Ag), designated super T-Ag, are commonly found in SV40-transformed rodent cells. We examined the potential role of super T-Ag in neoplastic progression by using a series of clonal SV40-transformed mouse mammary epithelial cell lines. We confirmed an association between the presence of super T-Ag and cellular anchorage-independent growth in methylcellulose. However, tumorigenicity in nude mice did not correlate with the expression of super T-Ag. In the tumors that developed in nude mice, super T-Ag expression fluctuated almost randomly. Cell surface iodination showed that super T-Ag molecules were transported to the epithelial cell surface. The biological functions of super T-Ag remain obscure, but it is clear that it is not important for tumorigenicity by SV40-transformed mouse mammary epithelial cells. Super T-Ag may be most important as a marker of genomic rearrangements by the resident viral genes in transformed cells.     

Cells transformed by a wide variety of agents express higher abundance levels of some cellular RNA species

A cDNA-cloned library was prepared from mRNA synthesized by SV40-transformed mouse cells. Eleven cDNA clones were selected based on their ability to hybridize higher levels of mRNA in SV40-transformed 3T3 cells than in 3T3 cells. These cDNA clones were employed to screen the steady-state levels of cytoplasmic RNAs in a wide variety of viral (SV40, polyoma, adenovirus, and Rous sarcoma virus) and nonviral (methylcholanthrene, embryonal carcinoma) transformed cell lines. Two of the cDNA clones—A17 and 104—detected greater than 40–100-fold higher levels of mRNA in all the transformed cell lines tested when compared to nontransformed cells (3T3, C3HEF). The levels of mRNA complementary to these two cDNAs were regulated in a temperature-sensitive fashion (87–100-fold) in both SV40tsA- and RSV ts-src-transformed murine cell lines. These two cDNA clones detected greater than 100-fold, higher levels of complementary RNA derived from SV40 tumor tissue than in normal mouse liver. RNA species complementary to cDNA clones A17 or 104 were not detected in either actively growing nontransformed cells or in serum-stimulated 3T3 cells. The abundance levels of mRNAs detected by these two cDNA clones appear to be regulated 100-fold or greater by the transformed state, independent of the transforming agent. The higher levels of these RNA species detected in transformed mouse cells appear not to be solely regulated by the state of growth of nontransformed cells.     

Virogenic Properties of Bromodeoxyuridine-sensitive and Bromodeoxyuridine-resistant Simian Virus 40-transformed Mouse Kidney Cells

When simian virus 40 (SV40)-transformed mouse kidney cells (mKS) were grown in the presence of susceptible indicator cells, SV40 was readily recovered from: (i) 15 transformed cell lines, (ii) transformed cells subcultured 45 times over a 7-month period in medium containing antiviral serum and bromodeoxyuridine (dBU), (iii) 45 of 46 clonal lines isolated in the presence of antiviral serum, (iv) 19 of 19 secondary clones isolated from two clonal lines, and (v) dBU-resistant transformed cell lines. dBU-resistant SV40-transformed mouse kidney cell lines were selected and shown to contain the T antigen and to have normal levels of thymidylate kinase and deoxyribonucleic acid (DNA) polymerase, but to be deficient in thymidine (dT) kinase. Radioautographic and biochemical experiments demonstrated that very little (3)H-dT was incorporated into DNA of dBU-resistant cells during a 6-hr labeling period. After infection of dT kinase-deficient mKS cells with vaccinia virus, high levels of dT kinase were induced. The properties of SV40 recovered from dBU-sensitive and dBU-resistant cells were studied. SV40 recovered from transformed cells was shown to express in CV-1 cells at least six functions characteristic of parental virus: synthesis of capsid antigen, synthesis of T antigen, synthesis of viral DNA, induction of dT kinase, induction of DNA polymerase, and induction of host cell DNA synthesis. In addition, SV40 recovered from the transformed cells induced T antigen, dT kinase, deoxycytidylate deaminase, thymidylate kinase, and DNA polymerase in abortively infected mouse kidney cultures, and the virus was also capable of transforming primary cultures of mouse kidney cells.     

Rearrangement of integrated viral DNA sequences in mouse cells transformed by simian virus 40.

The organization of viral DNA sequences in several cell lines derived from a primary colony of simian virus 40 (SV40)-transformed mouse cells was analyzed to examine the origin of the various distinctive patterns of SV40 sequence arrangement present in transformed cells. This analysis revealed a complex arrangement of viral sequences in the uncloned transformed cells but simplified arrangements in cloned derivatives of the primary transformant. The cell lines studied had certain SV40 sequence arrangements in common, but the cloned lines had lost some parental arrangements and acquired new arrangements. These results indicate that the arrangement of viral sequences in some SV40-transformed cells is not fixed but that alterations occur after integration, creating a heterogeneous population of transformants. In the process, expression of viral genes may be altered. Possible causes for and implications of this genetic instability are discussed.     

Tumor formation by SV40-transformed human cells in nude mice: the role of SV40 T antigens

Human cells transformed in vitro by SV40 rarely form tumors in nude mice. We examined whether these cells as a group are inherently nontumorigenic or whether they are potentially tumorigenic but rejected by the athymic host, possibly by nonspecific immune mechanisms. SV80 and NG8 are SV40-transformed human cell lines that express all of the transformed properties, including anchorage-independent growth, but do not form tumors in adult nude mice after injection of as many as 10(8) cells. Both the SV80 and NG8 cell lines have SV40-specific transplantation antigens which crossreact with those present on SV40-transformed (but tumorigenic) rodent cells. We found that SV80 cells, though not NG8 cells, induced progressively growing lethal tumors if the cells are injected repeatedly into neonatal nude mice. Somatic cell hybrids between SV80 or NG8 cells and a highly tumorigenic cell line derived from a human tumor continue to express the virus-induced antigens and fail to form tumors in adult nude mice. These results strongly suggest that at least for some SV40-transformed human cells, the failure to form tumors in nude mice may be due to their expression of virus-induced transplantation antigens rather than the absence of tumorigenic potential.     

Antigenic and Immunogenic Characteristics of Nuclear and Membrane-Associated Simian Virus 40 Tumor Antigen

Antisera were prepared in syngeneic hosts against subcellular fractions of simian virus 40 (SV40)-transformed cells (MoalphaPM, MoalphaNuc), glutaraldehydefixed SV40-transformed cells (HaalphaH-50-G, MoalphaVLM-G), and electrophoretically purified denatured SV40 tumor antigen (T-ag) (RaalphaT). Immune sera were also collected from animals bearing tumors induced by SV40-transformed cells (HaalphaT, MoalphaT, HAF) and from SV40-immunized animals that had rejected a transplant of SV40-transformed cells (HaalphaS, MoalphaS). Immunological reagents prepared against cell surface (MoalphaPM, HaalphaS, MoalphaS, HaalphaH-50-G, MoalphaVLM-G) reacted exclusively with the surface of SV40-transformed cells by indirect immunofluorescence or protein A surface antigen radioimmunoassay. Immunological reagents prepared against the nuclear fraction (MoalphaNuc) or whole-cell determinants (HaalphaT, MoalphaT, HAF, RaalphaT) reacted with both the nuclei and surface of SV40-transformed or -infected cells. All reagents were capable of immunoprecipitating 96,000-molecular weight large T-ag from solubilized whole cell extracts of SV40-transformed cells. The exclusive surface reactivity of HaalphaS exhibited in immunofluorescence tests was abolished by solubilization of subcellular fractions, which then allowed immunoprecipitation of T-ag by HaalphaS from both nuclear and plasma membrane preparations. Specificity was established by the fact that all T-reactive reagents failed to react in serological tests against chemically transformed mouse cells, and sera from mice bearing transplants chemically transformed mouse cells (MoalphaDMBA-2) failed to react with SV40-transformed mouse or hamster cells. Reagents demonstrating positive surface immunofluorescence and protein A radioimmunoassay reactions against SV40-transformed cells were capable of blocking the surface binding of RaalphaT to SV40-transformed cells in a double-antibody surface antigen radioimmunoassay. This blocking ability demonstrated directly that a component specificity of each surface-reactive reagent is directed against SV40 T-ag. A model is presented which postulates that the differential detection of T-ag by the various serological reagents is a reflection of immunogenic and antigenic differences between T-ag polypeptides localized in nuclei and plasma membranes.     

Radioimmunoassay of the cellular protein p53 in mouse and human cell lines

We have developed quantitative radioimmunological solid phase assays for the host protein p53 from mouse cells and from human cells. The first assay, for mouse p53, depends on having two monoclonal antibodies reacting with different determinants on the p53 molecule. With this assay we have shown that SV40-transformed cells have approximately 100-fold more p53 than untransformed mouse cells and that other transformed cells have intermediate levels. Embryonal carcinoma cell lines have approximately 50-fold less p53 than SV40-transformed cells. This is in contrast to the high levels of incorporation of [35S]methionine into p53 in these cells and indicates that metabolic labelling is not a valid approach for measuring p53 levels. The second assay, for human p53, required a different approach and made use of the anti-p53 antibodies detected in the sera of some breast cancer patients. Human tumour cell lines contained amounts of p53 varying from the high level seen in SV40-transformed human fibroblasts down to less than one hundredth of this amount. Normal human cells showed low levels of p53. The data confirm that many, but not all, human tumour cell lines contain more p53 than normal cells.     

Transformation of isolated rat hepatocytes with simian virus 40

Rat hepatocytes were transformed by simian virus 40 (SV40). Hepatocytes from two different strains of rats and a temperature-sensitive mutant (SV40tsA 1609), as well as wild-type virus were used. In all cases, transformed cells arose from approximately 50% of the cultures containing hepatocytes on collagen gels or a collagen gel-nylon mesh substratum. Cells did not proliferate in mock-infected cultures. SV40-transformed hepatocytes were epithelial in morphology, retained large numbers of mitochondria, acquired an increased nucleus to cytoplasm ratio, and contained cytoplasmic vacuoles. Evidence that these cells were transformed by SV40 came from the findings that transformants were 100% positive for SV40 tumor antigen expression, and that SV40 was rescued when transformed hepatocytes were fused with monkey cells. All SV40-transformed cell lines tested formed clones in soft agarose. Several cell lines transformed by SV40tsA 1609 were temperature dependent for colony formation on plastic dishes. Transformants were diverse in the expression of characteristic liver gene functions. Of eight cell lines tested, one secreted 24% of total protein as albumin, which was comparable to albumin production by freshly plated hepatocytes; two other cell lines produced 4.2 and 5.7%, respectively. Tyrosine aminotransferase activity was present in five cell lines tested but was inducible by dexamethasone treatment in only two. We conclude from these studies that adult, nonproliferating rat hepatocytes are competent for virus transformation.     

Sensitivity of simian virus 40-transformed C57BL/6 mouse embryo fibroblasts to lysis by murine natural killer cells

The susceptibility of mouse cells expressing full-length or truncated transforming protein (T antigen) of simian virus 40 (SV40) to lysis by murine natural killer (NK) cells was assessed. For these studies, C57BL/6 mouse embryo fibroblasts (B6/MEF) were transformed by transfection with SV40 DNA encoding the entire T antigen. The transformed cell lines were tested for susceptibility to lysis by nonimmune CBA splenocytes as a source of NK cells and to lysis by C57BL/6, SV40-specific cytolytic T cells (CTL). It was found that 13 of 15 clonally derived, SV40-transformed H-2b cell lines were susceptible to lysis by NK cells. However, there was some variation in their susceptibility to lysis by NK cells. There was no correlation between susceptibility to lysis by SV40-specific CTL and to lysis by NK cells. Cells transfected with a plasmid which encodes only the N-terminal half of the SV40 T antigen were consistently less susceptible to lysis by NK cells, suggesting that expression of only the N-terminus of the T antigen was insufficient for optimal susceptibility to lysis by NK cells. Primary mouse embryo fibroblasts transformed by human adenovirus type 5 E1 region DNA were also found to be susceptible to NK cell-mediated lysis. Lysis of SV40-transformed cells by nonimmune CBA splenocytes was mediated by NK cells because: lysis was augmented when the effector cells were treated with interferon before assay; and lysis was abrogated when the effector cells were obtained from mice that had been depleted of NK activity by treatment with antiserum against the asialo GM1 surface marker. These results indicate that primary mouse cells which are transformed by SV40 and which express the native T antigen are susceptible to lysis by mouse NK cells. Conversely, cells transformed by a plasmid encoding only the N-terminal half of the T antigen express reduced susceptibility to lysis by NK cells.     

Demonstration of T antigens on the surface of cells transformed with primate polyoma viruses

Primate polyoma virus-transformed hamster, mouse, and rat cell lines were examined by indirect immunofluorescence staining for cell surface-associated T antigens, by using a rabbit antiserum prepared against sodium dodecyl sulfate-denatured large T antigen of simian virus 40 (anti-SV40-SDS-T serum). Positive surface staining was shown not only on SV40-transformed cells, but also on BK and JC virus-transformed cells. In contrast, normal cells and cells transformed with mouse polyoma-, human adeno-, and murine sarcoma viruses were negative. The data on SV40-transformed cells confirmed the reports of others demonstrating the cell surface location of SV40 large T antigen, and the data on BK and JC virus-transformed cells proved that these cells have cell-surface T antigens that cross-react with anti-SV40-SDS-T serum.     

Characterization of a 54K dalton cellular SV40 tumor antigen present in SV40-transformed cells and uninfected embryonal carcinoma cells.

SV40 infection or transformation of murine cells stimulated the production of a 54K dalton protein that was specifically immunoprecipitated, along with SV40 large T and small t antigens, with sera from mice or hamsters bearing SV40-induced tumors. The same SV40 anti-T sera immunoprecipitated a 54K dalton protein from two different, uninfected murine embryonal carcinoma cell lines. These 54K proteins from SV40-transformed mouse cells and the uninfected embryonal carcinomas cells had identical partial peptide maps which were completely different from the partial peptide map of SV40 large T antigen. An Ad2+ND4-transformed hamster cell line also expressed a 54K protein that was specifically immunoprecipitated by SV40 T sera. The partial peptide maps of the mouse and hamster 54K protein were different, showing the host cell species specificity of these proteins. The 54K hamster protein was also unrelated to the Ad2+ND4 SV40 T antigen. Analogous proteins immunoprecipitated by SV40 T sera, ranging in molecular weight from 44K to 60K, were detected in human and monkey SV40-infected or -transformed cells. A wide variety of sera from hamsters and mice bearing SV40-induced tumors immunoprecipitated the 54K protein of SV40-transformed cells and murine embryonal carcinoma cells. Antibody produced by somatic cell hybrids between a B cell and a myeloma cell (hybridoma) against SV40 large T antigen also immunoprecipitated the 54K protein in virus-infected and -transformed cells, but did not do so in the embryonal carcinoma cell lines. We conclude that SV40 infection or transformation of mouse cells stimulates the synthesis or enhances the stability of a 54K protein. This protein appears to be associated with SV40 T antigen in SV40-infected and -transformed cells, and is co-immunoprecipitated by hybridomas sera to SV40 large T antigen. The 54K protein either shares antigenic determinants with SV40 T antigen or is itself immunogenic when in association with SV40 large T antigen. The protein varies with host cell species, and analogous proteins were observed in hamster, monkey and human cells. The role of this protein in transformation is unclear at present.     

Species-specific phosphorylation of mouse and rat p53 in simian virus 40-transformed cells.

We have analyzed in detail the phosphorylation of p53 from normal (3T3) and simian virus 40 (SV40)-transformed (SV3T3) BALB/c mouse cells and from normal (F111) and SV40-transformed [FR(wt648)] rat cells by two-dimensional tryptic peptide mapping and phosphoamino acid analyses. To accommodate the different half-lives of p53 in normal (half-life, 15 min) and transformed (half-life, 20 h) cells and possible differences in the rates of turnover of phosphate at specific sites, cells were labeled for 2 h (short-term labeling) or 18 h (long-term labeling). Depending on the labeling conditions, either close similarities or marked differences were observed in the phosphorylation patterns of p53 from normal and transformed cells. After the 2-h labeling, the phosphorylation patterns of p53 from normal and transformed mouse cells were quite similar. In contrast, p53 from normal and transformed rat cells exhibited dramatic quantitative and qualitative differences under these labeling conditions. The reverse was found after an 18-h label leading to steady-state phosphorylation of p53 in transformed cells: while p53 in transformed mouse cells revealed a marked quantitative increase in phosphorylation compared with p53 from normal cells, the corresponding patterns of p53 from normal and transformed rat cells were similar. Our data thus indicate species-specific differences in the phosphorylation of mouse and rat p53 in SV40-transformed cells, reflected by (i) different turnover rates at specific sites in mouse and rat p53 and (ii) phosphorylation of nonhomologous serine and threonine residues in rat p53, as revealed by indirect assignment of phosphorylation sites to the phosphopeptides of rat p53. Analyses of p53 from the SV40 tsA58 mutant-transformed F111 cell lines FR(tsA58)A (N type) and FR(tsA58)57 (A type) yielded no conclusive evidence for a direct correlation between phosphorylation of p53, the metabolic stabilization of p53, and expression of the transformed phenotype.