TRANSFORMATION BY V-H-RAS DOES NOT RESTORE PROLIFERATION OF A SET OF TEMPERATURE-SENSITIVE CELL-CYCLE MUTANTS OF RAT 3Y1 FIBROBLASTS

Three temperature-sensitive cellcycle mutants of rat 3Y1 fibroblasts (3Y1tsD123, 3Y1tsG125, and 3Y1tsH203, each belonging to distinct complementation groups) were transformed with plasmid DNA carrying Harvey murine sarcoma virus cDNA. The criteria for transformation were increase in saturation cell density, capability to clone in soft agar, and alteration in the cellular morphology. At 39.8°C (restrictive temperature of the parental cell lines), all the transformed sublines of each mutant ceased to proliferate and were arrested rever-sibly in the G1 phase of the cell cycle like the parental lines. At both 39.8°C and 33.8°C (permissive temperature for the parental lines), all the untransformed parental lines synthesized p21ras at low rate. At 33.8°C, all the transformed sublines synthesized p21ras at much higher rate and expressed the morphological phenotype characteristic to v-H-ras-induced transformation. At 39.8°C, the rate of p21ras synthesis was not changed in the transformed sublines of 3YltsD123 and 3Y1tsG125, and the morphology of transformed phenotype also remained intact. In the transformed subline of 3Y1tsH203, the rate of p21ras synthesis was lowered at 39.8°C to that seen in the untransformed parental line, and the transformed phenotype in morphology disappeared. In all of the transformed sublines, the amount of v-H -ras mRNA markedly expressed at both 33.8°C and 39.8°C. We conclude that in both v-H-ras-tranfornied sublines of 3Y1tsD123 and 3Y1tsG125 the temperature arrest is not unlocked by the process specifically causing the transformed phenotype, and that in the v-H-ras-transformed subline of 3YltsH203, the process specifically causing the transformed phenotype is blocked by the mutation after transcription of the v-H-ras oncogene. © 1990, Japan Society for Cell Biology. All rights reserved.