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.     

Altered patterns of keratin synthesis in human epidermal keratinocytes transformed by SV40

Transformation of human epidermal keratinocytes by the oncogenic virus SV40 is a stage-specific process in which normal patterns of differentiation are progressively altered over time following infection. Within the context of this scheme, we examined the keratins produced by the infected cells. Immunofluorescence studies indicated that viral infection led to the formation of variant cells visibly lacking the normal keratin cytoskeleton after about 10-15 serial passages (60-90 cell generations) post infection. Analyses of variant cell formation in clonal populations grown on palladium islands revealed that the variants were derived within 2-3 cell divisions from cells containing an apparently normal keratin cytoskeleton, but that variant formation depended upon cell density. Immunoprecipitation of 35S-methionine labelled keratins from the infected keratinocytes revealed a gradual loss of the normal 46, 50, 56 and 58Kd keratin species over a period of many months after infection. The loss of the normal keratins was accompanied by the appearance of at least two species in the 48-52Kd size range not present in uninfected cells and the enhancement of a third, 40Kd, protein quite early after infection. Analysis of the altered keratin patterns on two-dimensional acrylamide gels using either isoelectric focusing (IEF) or non-equilibrium pH gradient electrophoresis (NEPHG) along the first dimension showed that the infected cells produced basic keratins which increased in relative abundance as cells became more transformed with serial passage including at least five isoelectric forms not seen in uninfected cells. Translation of poly A+ RNAs from the infected cells indicated that the altered keratin synthesis probably reflects changes in the translatable mRNA pool.     

The location of SV40 specific sequences in the heterogeneous nuclear RNA of transformed mouse cells

Summary Rapidly labelled nuclear RNA from an SV40 transformed mouse 3T3 line was isolated, and different molecular size classes pooled. The fractions were treated with Ehrlich ascites exonuclease III for various lengths of time, and the digestion of the 3 ends of the RNA measured by the appearance of TCA-soluble material. The resulting partially degraded RNA populations were then hybridised to SV40 DNA. The data suggest that a high proportion of the viral specific sequences are located near the 3 ends of heterogeneous nuclear RNA.     

Quiescent SV40 virus transformed 3T3 cells in culture

Serum counteracts low nutrient concentrations in the culture medium in SV40 virus transformed 3T3 (SV3T3) cells. The transport of [³H]-leucine into TCA soluble material in SV3T3 cells is stimulated by serum and inhibited by But₂-cAMP. When SV3T3 cells are cultured in low leucine concentrations (? 8 × 10^?6 M), the cell's morphology is similar to the one of cells incubated in complete medium in the presence of But₂-cAMP and cells become quiescent. Cells become arrested throughout the cell cycle. The results suggest that the mechanism by which But₂-cAMP inhibits growth of SV3T3 cells is by inhibiting the transport of leucine in SV3T3 cells.     

Expression of SV40 tumor antigen in SV40 transformed teratocarcinoma-derived cell lines

The relative importance of viral tumor antigen expression and the cellular background in the maintenance of a transformation phenotype was examined in five SV40-transformed teratocarcinoma-derived cell lines. These cell lines show qualitative differences in growth characteristics associated with transformation, and vary in their state of differentiation. Viral T antigen expression was evaluated by two criteria: 1) the amount of immunoprecipitated antigen in growing cells, and 2) the amount and rate of antigen synthesis in density-inhibited cells. There was no direct correlation found between retention, or rate of synthesis, of the viral T antigen and the degree of transformation. These findings imply that the cellular environment has a more important influence on the growth properties of a stably transformed cell than the quantitative levels of viral T antigen expression.     

Variation in the appearance of SV40 tumor antigen in transformed cells

Five types of variation in the appearance of SV40 T antigen have been observed by immunofluorescence in wild-type and tsD^*101 transformed BALB and Swiss/3T3 cells: (1) clones having more than 95% uniformly stained nuclei; (2) clones having variable stained nuclei; (3) clones having T-negative nuclei at the periphery at 39 °C, but not at 33 °C; (4) D101 cells having T-negative nuclei at 39 °C, but not at 33 °C, during growth and after saturation; and (5) wild type cells having T-negative nuclei after confluence at both 33 and 39 °C. This fifth type of variation may involve an immuno-specificity change in the T antigen molecule. Any models proposed for the regulation of SV40 T antigen in transformed cells will have to explain these variations.     

Patterns of cell communication and differentiation in SV40 transformed human keratinocytes

Fluorescein dye microinjection was used to demonstrate changes in communication between human epidermal keratinocytes grown in vitro after infection by the oncogenic virus, SV40. Whereas keratinocytes are normally fully coupled to each other, dye spread becomes progressively restricted to small cell subpopulations after infection, although dye coupling is increased when the infected cells attain high densities. Reduction or enhancement of dye coupling is correlated with similar changes in the extent of cytochemical differentiation and cornified envelope formation.