Simian virus 40 A gene function: DNA content analysis of Chinese hamster cells transformed by an early temperature-sensitive virus mutant.

Abstract

Replication of two Chinese hamster embryo cell lines transformed by an early temperature-sensitive mutant of simian virus 40, tsAS8, was examined by flow microfluorometry and autoradiography of [3H~thymidine-labeled cells in order to determine whether transformed cell DNA synthesis is initiated by the virus A gene. At the permissive temperature (370), cells transformed by the mutant were like the wild-type virus transformants in appearance, colony-forming ability, high saturation density, and rapid replication. At One approach to understanding neoplasia has been to study cells transformed by temperature-sensitive virus mutants at temperatures permissive and nonpermissive for the function of specific viral genes. In this way, the A gene of simian virus 40 (SV40) has been tentatively identified as the gene responsible for initiation and maintenance of transformation in mammalan cells (1-6). In the lytic system, the A gene initiates viral and perhaps cellular DNA synthesis (6-8). It has therefore been suggested that transformation of nonpermissive cells is maintained by the A gene product continuously initiating cell DNA synthesis, which would free cell replication from its normal control mechanisms (1-5, 9, 10).If this "initiator" hypothesis is correct, cells transformed by SV40 mutants that specify a temperature-sensitive A gene product (tsA mutants) should not only lose the transformed phenotype at the nonpermissive temperature, but also should return to normal growth control, and arrest in the G1 (Go) phase of the cell cycle at confluence as normal cells do. When grown at the permissive temperature, the same cells should resume cell cycle traverse in response to the functional A gene. This "initiator" hypothesis was tested by analyzing the cell cycle of Chinese hamster embryo (ChH) cells transformed by wild-type (WT) SV40 and the SV40 tsA mutant, tsA58. Several independent methods of cell cycle analysis, including flow microfluorometric determination of cell DNA content, were used. The results of our studies suggest that the loss of the A gene function represented by the tsA58 mutation does not allow SV40-transformed ChH cells to arrest in the G1 (Go) phase of the cell cycle.MATERIALS AND METHODS Viruses and Cell Cultures. WT SV40 and the temperature-sensitive group A SV40 mutant, tsA58, were obtained from Peter Tegtmeyer. Virus pools were prepared and were plaque assayed on CV-1 monkey kidney cells at 330 and 40.5°(11). The presence of SV40-induced T or V antigen in infected cells was demonstrated by indirect immunofluorescence (12).Confluent secondary cultures of normal ChH fibroblasts (12) were infected with 2-5 plaque-forming units of SV40 per cell at 370 and subcultured frequently until morphologic transformation was observed (five to six passages). This uncloned population, selected only for rapid growth on plastic, was designated ChH A58. Another embryo cell culture was similarly infected with WT or tsA58 SV40, but the cells were seeded into soft agar (13) after morphologic transformation was o...

Simian virus 40 a gene function: Further characterization and growth of tsA transformed Chinese hamster cells

“…It was therefore of interest to examine the thermolability of their T antigen even though we had previously rescued only tsA SV,, from these cells (Robinson and Lehman, 1978).…”

“…In fact, when the distribution about the cell cycle was assayed, no changes in G,, s, Gz + M phases were observed at 40.5"C. Therefore, the tsA transformed cells cycled at uniform rates at both temperatures. However, a lengthening of the doubling time at 40.5 "C was noted, possibly owing to the cycling and dying at the nonpermissive temperatures (Robinson and Lehman, 1978). Further, these tsA transformed cells did not arrest in the G, phase of the cell cycle in reduced glutamine, isoleucine, or serum.…”

“…Coordinate control of transformation and cell DNA synthesis by temperature has been reported in tsA mutant transformed Chinese hamster lung fibroblasts (Martin and Stein, 1976;Butel and Soule, 1978), Syrian hamster embryo cells (Butel and Soule, 1978), mouse 3T3 cells (Brockman, 1978), and mouse embryo cells (Hiscott and Defendi, 1979;O'Neill et al, 1980). However, in the Chinese hamster embryo cell system, we found dissociation of the two processes (Robinson and Lehman, 1978). At the restrictive temperature, tsA mutant transformed Chinese hamster embryo cells lost many characteristics of transformation, yet unlike normal cells, they continued to incorporate 3H-thymidine at confluence and did not arrest in the G, phase of the cell cycle.…”

Hormonal regulation of the transformation phenotype in simian virus 40-transformed rat embryonic preadipose cell lines.

“…The cellular response of REC-A16 at the nonpermissive temperature, where lipid accumulation did not occur in all cells after the growth arrest, might be the result of such a nonspecific cell cycle restriction as a consequence of an inactivated large T (24,36,49).…”

Analysis of intracellular antigens by flow cytometry

“…The cultures were maintained at 37°C in a humidified 5% COz atmosphere. In some experiments, primary CHE cells were used as negative controls, and a transformed CHE cell line (A58), which has been described (29), as the positive population. The diploid wild-type SV40 transformant 12383-91-3 was isolated and cloned from infected CHE cells.…”