Transformation of BALB/c-3T3 cells by tsA mutants of simian virus 40: effect of transformation technique on the transformed phenotype.

Simian virus 40 tsA-transformed BALB/c-3T3 cells isolated as foci of overgrowth in liquid medium were compared with those isolated as colonies in soft agar. Efficiencies of transformation were equivalent in the two procedures. Cells isolated as foci were able to grow in agar and vice versa. No difference in temperature sensitivity of the transformed phenotype was detected when tsA transformants selected in agar were compared with those selected as foci. The use of the two different transformation procedures, then, did not form the basis for generation of different transformed phenotypes, and transformants generated in both ways were dependent upon expression of the A gene for maintenance of the transformed state.     

Transformation of BALB/c-3T3 cells by tsA mutants of simian virus 40: temperature sensitivity of the transformed phenotype and retransofrmation by wild-type virus.

The function of the A gene of simian virus 40 (SV40) in transformation of BALB/c-3T3 cells was investigated by infecting at the permissive temperature with wild-type SV40 and with six tsA mutants whose mutation sites map at different positions in the early region of the SV40 genome. Cloned transformants were then characterized as to the temperature sensitivity of the transformed phenotype. Of 16 tsA transformants, 15 were temperature sensitive for the ability to overgrow a monolayer of normal cells, whereas three of three wild-type transformants were not. This pattern of temperature sensitivity of the transformed phenotype was also observed when selected clones were assessed for the ability to grow in soft agar and in medium containing low concentration of serum. The temperature resistance of the one exceptional tsA transformant could be attributed neither to the location of the mutation site in the transforming virus nor to transformation by a revertant virus. This temperature-resistant tsA transformant, however, was demonstrated to contain a higher intracellular concentration of SV40 T antigen than a temperature-sensitive line transformed by the same tsA mutant. A tsA transformant displaying the untransformed phenotype at the nonpermissive temperature was found to be susceptible to retransformation by wild-type virus at this temperature, demonstrating that the temperature sensitivity of the tsA transformants is due to the viral mutation and not to a cellular defect. These results indicate that continuous expression of the product of the SV40 A gene is required to maintain the transformed phenotype in BALB/c-3T3 cells.     

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.     

Growth control in simian virus 40-transformed rat cells: temperature-independent expression of the transformed phenotype in tsA transformants derived by agar selection.

Fisher rat fibroblasts (FR 3T3), transformed with the tsA30 mutant of simian virus 40 and selected by colony formation in soft agar, maintained the transformed phenotype at high temperature, whereas most transformants isolated from foci were found to undergo a phenotypic reversion toward the normal state in their saturation density, ability to grow in soft agar, and rate of 2-deoxyglucose transport. The temperature-independent phenotype observed in agar-selected transformants was not due to a reversion of the viral mutation. These results, similar to those previously obtained with polyoma virus tsa mutants, further suggest that two distinct mechanisms may operate in both cases for maintaining the transformed phenotype. Immunofluorescence studies suggested a different regulation of T antigen synthesis in these two classes of transformants.     

Separation of lytic and transforming functions of the simian virus 40 A region: two mutants which are temperature sensitive for lytic functions have opposite effects on transformation.

Thirty-six of 40 rat cell clones transformed to anchorage independence at low multiplicity of infection by simian virus 40 tsA58 were heat sensitive for continued expression of the transformed phenotype. tsA1499 is an 81-base-pair deletion at 21 map units which is like tsA58 in that it is also heat sensitive for lytic growth, belongs to the A complementation group, and produces rat cell transformants which contain a thermolabile T antigen. Unlike tsA58, however, tsA1499 generated rat cell transformants efficiently at the temperature at which it was lytically defective, and 10 of 17 clones transformed by tsA1499 were cold rather than heat sensitive for the continued maintenance of the transformed phenotype. The lytic and transforming activities of the A region thus appeared to function independently in mutant tsA1499.     

Simian virus 40 gene A function and maintenance of transformation.

Transformants have been isolated after infection of rat embryo cells at 33 C with either wild-type simian virus 40 or with the temperature-sensitive gene A mutants, tsA7 and tsA28. Examination of properties usually associated with transformation such as growth in 1% serum, growth rate, saturation density, and morphology show that these properties are temperature dependent in the tsA transformants characterized, but are not temperature dependent in the wild-type transformants that have been examined. In the most thoroughly characterized tsA transformants the expression of T antigen also appears to be temperature dependent. These data suggest that an active A function is required for the maintenance of transformation in these cells. In the lytic cycle, the A function is involved in the initiation of DNA synthesis. Thus transformation by simian virus 40 may be the direct consequence of the introduction of the simian virus 40 replicon and the presence of its DNA initiator function, which causes the cell to express a transformed phenotype.     

Simian virus 40 small t antigen is not required for the maintenance of transformation but may act as a promoter (cocarcinogen) during establishment of transformation in resting rat cells.

Simian virus 40 deletion mutants affecting the 20,000-dalton (20K) t antigen and tsA mutants rendering the 90K T antigen temperature sensitive, as well as double mutants containing both mutations, induced host DNA synthesis in resting rat cells at the restrictive temperature. Nonetheless, the deletion mutants and double mutants did not induce transformation in resting cells even at the permissive temperature. On the other hand, the deletion mutants did induce full transformants when actively growing rat cells were infected; the transformants grew efficiently in agar and to high saturation densities on platic. The double mutants did not induce T-antigen-independent (temperature-insensitive) transformants which were shown previously to arise preferentially from resting cells. Thus, small t antigen was dispensable for the maintenance of the transformed phenotype in T-antigen-dependent rat transformants (transformants derived from growing cells) and may play a role in the establishment of T-antigen-independent transformants. We attempt to establish a parallel between transformation induced by chemical carcinogens and simian virus 40-induced transformation.     

Effects of large and small T antigens on DNA synthesis and cell division in simian virus 40-transformed BALB/c 3T3 cells.

The roles of the large T and small t antigens of simian virus 40 in cellular DNA synthesis and cell division were analyzed in BALB/c 3T3 mouse cells transformed by wild-type, temperature-sensitive A (tsA), or tsA-deletion (tsA/dl) double mutants. Assessment of DNA replication and cell cycle distribution by radioautography of [3H]thymidine-labeled nuclei and by flow microfluorimetry indicate that tsA transformants do not synthesize DNA or divide at the restrictive temperature to the same extent as they do at the permissive temperature or as wild-type transformants do at the restrictive temperature. This confirms earlier studies suggesting that large T induces DNA synthesis and mitosis in transformed cells. Inhibition of replication in tsA transformants at the restrictive temperature, however, is not complete. Some residual cell division does occur but is in large part offset by cell detachment and death. This failure to revert completely to the parental 3T3 phenotype, as indicated by residual cell cycling at the restrictive temperature, was also observed in cells transformed by tsA/dl double mutants which, in addition to producing a ts large T, make no small t protein. Small t, therefore, does not appear to be responsible for the residual cell cycling and plays no demonstrable role in the induction of DNA synthesis or cell division in stably transformed BALB/c 3T3 cells. Comparison of cell cycling in tsA and tsA/dl transformants, normal 3T3 cells, and a transformation revertant suggests that the failure of tsA transformants to revert completely may be due to leakiness of the tsA mutation as well as to a permanent cellular alteration induced during viral transformation. Finally, analysis of cells transformed by tsA/dl double mutants indicates that small t is not required for full expression of growth properties characteristic of transformed cells.     

Distinct transformation phenotypes induced by polyoma virus and simian virus 40 in rat fibroblasts and their control by an early viral gene function.

Several transformed cell lines established from Fisher rat cells (FR 3T3) infected with wild-type polyoma virus or simian virus 40 or early temperature-sensitive mutants (polyoma tsa and simian virus 40 tsA30) were studied for their transformation phenotypes. The distinct patterns which were obtained for polyoma and simian virus 40 transformants led to the conclusion that these two viruses express different transforming abilities in rat cells. The results obtained with temperature-sensitive mutant-derived transformants indicate that all of the transformation characteristics studied so far may be under the control of a viral function in polyoma tsa-transformed cells.     

Differentiation of human epidermal cells transformed by SV40

Human epidermal cells were transformed with DNA from wild-type SV40 virus or with DNA from a temperature-sensitive A mutant (tsA209). The SV40-transformed cells differed from nontransformed cells in their morphologic appearance, growth properties, and expression of certain characteristics associated with differentiation. The transformed cells were more variable in size and shape than their nontransformed counterparts and were less stratified and less keratinized. While the growth properties of the cells were similar under optimal growth conditions, the transformed cells could be propagated under stringent growth conditions that did not support the growth of nontransformed human epidermal cells. The transformants still required a 3T3 feeder layer for growth, remained anchorage dependent as assayed in soft agar, and were not tumorigenic in athymic nude mice. The expression of certain differentiated functions of the human epidermal cell, the presence of keratins and cross-linked envelopes, was decreased in the transformed cells, and these functions could be restored at the nonpermissive temperature in the tsA209 transformed cells.     

Structural prerequisites of simian virus 40 large T antigen for the maintenance of cell transformation.

We have investigated the functional roles of two structural subsets of simian virus 40 (SV40) large T antigen, namely homo-oligomers and complexes with the host cellular p53 protein, for the transformed phenotype. We examined T antigen produced in cells transformed by temperature-sensitive SV40 large T mutants: heat-sensitive or unrestricted SV40 tsA58-transformed rat cells and unrestricted tsA1499 transformants. In both unrestricted cell lines, T antigen was temperature-sensitive only for the formation of fast sedimenting homo-oligomers. Corresponding to our recent observations obtained with tsA1499-infected monkey cells, in tsA1499 transformants large T was competent to form stable T-p53 complexes independently of the temperature. However, T antigen coded for by tsA58, which is heat-sensitive for binding to p53, occurred in stable complexes with this protein in unrestricted tsA58 transformants under all conditions. Furthermore, in both unrestricted transformants T-p53 complexes arise in the absence of homo-oligomers of T antigen. In conclusion, T antigen homo-oligomers are not involved in cell transformation, whereas T-p53 complexes may be involved in the maintenance of this phenotype.     

Role of the simian virus 40 gene A product in regulation of DNA synthesis in transformed cells.

Cells transformed by tsA mutants of simian virus 40 (SV40) are temperature sensitive for the maintenance of the transformed phenotype. The kinetics of induction of DNA synthesis were determined for hamster cell transformants shifted to the permissive temperature after a 48-h serum arrest at the nonpermissive temperature. DNAsynthesis was initiated in the tsA transformants by 8 h after shiftdown was maximal by 12 h. The presence or absence of fetal bovine serum at the time of temperature shift had no effect on the kinetics of initiation of DNA synthesis. Analysis of TTP in tsA transformants revealed similar levels of incorporation of [3H]thymidine into TTP at both permissive and nonpermissive temperatures. Autoradiography revealed that by 12 h after a shift to the permissive temperature, approximately 50% of the cells exhibited labeled nuclei after a 60-min pulse with [3H]thymidine, indicating that a majority of the cells were actively synthesizing DNA. By 8 to 12 h after a shiftup of confluent tsA transformants to the nonpermissive temperature, the number of labeled nuclei was reduced to approximately 16%, regardless of serum concentration. These data indicate that the SV40 gene A product, either directly or indirectly, regulates cellular DNA synthesis in transformed cells.     

Phenotype-specific phosphorylation of simian virus 40 tsA mutant large T antigens in tsA N-type and A-type transformants.

To identify molecular differences between simian virus 40 (SV40) tsA58 mutant large tumor antigen (large T) in cells of tsA58 N-type transformants [FR(tsA58)A cells], which revert to the normal phenotype after the cells are shifted to the nonpermissive growth temperature, and mutant large T in tsA58 A-type transformants [FR(tsA58)57 cells], which maintain their transformed phenotype after the temperature shift, we asked whether the biological activity of these mutant large T antigens at the nonpermissive growth temperature might correlate with phosphorylation at specific sites. At the permissive growth temperature, the phosphorylation patterns of the mutant large T proteins in FR(tsA58)A (N-type) cells and in FR(tsA58)57 (A-type) cells were largely indistinguishable from that of wild-type large T in FR(wt648) cells. After a shift to the nonpermissive growth temperature, no significant changes in the phosphorylation patterns of wild-type large T in FR(wt648) or of mutant large T in FR(tsA58)57 (A-type) cells were observed. In contrast, the phosphorylation pattern of mutant large T in FR(tsA58)A (N-type) cells changed in a characteristic manner, leading to an apparent underphosphorylation at specific sites. Phosphorylation of the cellular protein p53 was analyzed in parallel. Characteristic differences in the phosphorylation pattern of p53 were observed when cells of N-type and A-type transformants were kept at 39 degrees C as opposed to 32 degrees C. However, these differences did not relate to the different phenotypes of FR(tsA58)A (N-type) and FR(tsA58)57 (A-type) cells at the nonpermissive growth temperature. Our results, therefore, suggest that phosphorylation of large T at specific sites correlates with the transforming activity of tsA mutant large T in SV40 N-type and A-type transformants. This conclusion was substantiated by demonstrating that the biological properties as well as the phosphorylation patterns of SV40 tsA28 mutant large T in cells of SV40 tsA28 N-type and A-type transformants were similar to those in FR(tsA58)A (N-type) and in FR(tsA58)57 (A-type) cells, respectively. The phenotype-specific phosphorylation of tsA mutant large T in tsA A-type transformants probably is a cellular process induced during establishment of SV40 tsA A-type transformants, since tsA28 A-type transformant cells could be obtained by a large-T-dependent in vitro progression of cells of the tsA28 N-type transformant tsA28.3 (M. Osborn and K. Weber, J. Virol. 15:636-644, 1975).     

Polyoma virus middle t antigen: a tumor progression factor.

Polyoma virus (PyV) deletion mutant dl23 (affecting both large T and middle t but not small t antigens) was used to study transformation of 3T3 rat cells. This mutant generated stable transformants in the agar assay at a frequency similar to that of wild-type virus (WT). However, WT-induced transformants were detected 3 weeks after infection, whereas those induced by the mutant could not be detected until 6 to 8 weeks after infection. In this respect, dl23 PyV behaved like WT simian virus 40 (SV40). Cells transformed by WT SV40 or by dl23 PyV were similar in all their transformed properties. Those transformed by WT PyV were different from the others on the basis of morphology, cell adhesion to the substrate, release of protease activity, efficiency of doubling in agar, growth rate, and time required for tumor formation. Saturation density, the ability to grow in agar, the serum requirement for cloning, and the ability to grow on a cell monolayer were similar for all transformants. Middle t antigen enhanced membrane alterations and growth rate of the transformed cells, shortening the time required for tumor formation in rats.     

Study of liver differentiation in vitro

A clonal rat fetal liver cell line that expresses the functions of differentiated liver cells under controllable conditions has been established. Normal fetal liver cells were transformed by a temperature-sensitive A (tsA) mutant (tsA209) of simian virus 40. At the permissive temperature (33 degrees C), the tsA209-transformed liver cell line (RLA209-15) can be cultured indefinitely and cloned readily. The RLA209-15 cells were temperature sensitive for maintenance of the transformed phenotype. These transformed liver cells selectively lost four characteristics of the transformed phenotype at the restrictive temperature (40 degrees C): generation time of the cells increased, the saturation density decreased, the efficiency of growth on nontransformed cell layers decreased, and the ability to clone in soft agar was lost. The transformation can be reversed simply by a shift in temperature. RLA209-15 fetal liver cells synthesized alpha-fetoprotein albumin, and transferrin. At 33 degrees C, the levels of these liver proteins were relatively low. At 40 degrees C the transformed phenotype was lost and the levels of alpha-fetoprotein, albumin, and transferrin were greatly increased. At the restrictive temperature, maximal induction of the synthesis of alpha-fetoprotein, albumin, and transferrin was achieved 3-4 d after the upward shift in temperature. The synthesis of alpha-fetoprotein then decreased; the synthesis of albumin and transferrin, however, was maintained. A second phase of albumin and transferrin synthesis was observed in all cultures after 6 d or more at 40 degrees C. Alpha-Fetoprotein, albumin, and transferrin secreted by RLA209-15 cells were immunologically indistinguishable from authentic alpha-fetoprotein, albumin, and transferrin, respectively. RLA209-15 cells, like primary cultures of hepatocytes and a simian virus 40 tsA255-transformed fetal liver cell line (RLA255-4) reported earlier from this laboratory, responded to glucagon with markedly elevated levels of cyclic AMP. Thus, it appears that glucagon receptors characteristic of hepatocytes are retained in the simian virus 40 tsA-transformed fetal liver cells.     

Large-T-antigen-p53 complex formation is not cold sensitive in a cold-sensitive transformant induced by simian virus 40 mutant tsA1499

F111 rat cells transformed by simian virus 40 mutant tsA1499 are cold sensitive for the expression of transformation. Yet, unlike F111 cells transformed by tsA58, they do not lose the ability to stabilize the transformation-associated host cell protein p53 at the temperature at which transformation is extinguished.     

Growth of immortal simian virus 40 tsA-transformed human fibroblasts is temperature dependent.

Simian virus 40 (SV40)-mediated transformation of human fibroblasts offers an experimental system for studying both carcinogenesis and cellular aging, since such transformants show the typical features of altered cellular growth but still have a limited life span in culture and undergo senescence. We have previously demonstrated (D. S. Neufeld, S. Ripley, A. Henderson, and H. L. Ozer, Mol. Cell. Biol. 7:2794-2802, 1987) that transformants generated with origin-defective mutants of SV40 show an increased frequency of overcoming senescence and becoming immortal. To clarify further the role of large T antigen, we have generated immortalized transformants by using origin-defective mutants of SV40 encoding a heat-labile large T antigen (tsA58 transformants). At a temperature permissive for large-T-antigen function (35 degrees C), the cell line AR5 had properties resembling those of cell lines transformed with wild-type SV40. However, the AR5 cells were unable to proliferate or form colonies at temperatures restrictive for large-T-antigen function (39 degrees C), demonstrating a continuous need for large T antigen even in immortalized human fibroblasts. Such immortal temperature-dependent transformants should be useful cell lines for the identification of other cellular or viral gene products that induce cell proliferation in human cells.     

Temperature-sensitive growth regulation in one type of transformed rat cells induced by the tsa mutant of polyoma virus.

A fibroblast line of the 3T3 type with a low saturation density was established from Fisher rat embryo cells. After infection with either wild-type or tsa mutant polyoma virus, transformants were isolated and cloned at 33 degrees C on the basis of their ability either to grow as dense foci on plastic in liquid medium (type N) or to form colonies in soft agar (type A). Polyoma T antigen was detected in all of the transformed lines. The following growth characteristics were studied for both types at 33 and 41 degrees C: saturation density, growth in soft agar and at a low serum concentration, colony-forming ability, and generation time. tsa-N transformants behaved at 33 degrees C similarly to transformed cells, but reverted at 41 degrees C to the nontransformed phenotype for all of these characters. tsa-A transformants and all of the wild-type transformants exhibited the transformed phenotype at both low and high temperatures. These results led us to distinguish at least two types of virus-induced transformants. In one of them, the activity of the protein affected by the tsa mutation appears to be necessary for the expression of several of the characters defining the transformed state.     

Roles of the simian virus 40 tumor antigens in transformation of Chinese hamster lung cells: studies with simian virus 40 double mutants.

Simian virus 40 mutants containing both a tsA mutation (rendering the 90,000 molecular weight [90K] T-antigen thermolabile) and a deletion between 0.54 and 0.59 map units (reducing the size and the amount of the 20K t-antigen) were used to transform Chinese hamster lung cells. The frequencies of transformation by the double mutants were comparable to that of the tsA mutant alone by both the focus and agar assays except when the cells were serum depleted before infection. Growth-arrested cells were transformed (using the agar assay) by the deletion mutants at less than 2% the frequency found when the 20K t-antigen was normal. Growth arrest had very little effect on the temperature sensitivity of the resultant transformed cell lines whether or not the deletion was present.     

Rat cells transformed by simian virus 40 give rise to tumor cells which contain no viral proteins and often no viral DNA.

Rat 3T3 cells transformed by simian virus 40 were injected into rats to examine their capacity to develop into tumors. Both large T-dependent (N) transformants and large T-independent (A) transformants were used. All the transformed cell lines contained large T and small t and could multiply efficiently in agar. Only some transformants could develop into tumors. All tumor cells examined had lost both large T and small t. Tumor cells in which the viral genome could still be detected were found together with tumor cells in which the simian virus 40 DNA could no longer be detected. N transformants which displayed the transformed phenotype in a temperature-sensitive manner became temperature insensitive during tumor formation.