Entrance of SV40-transformed Cells into G0 Phase as Revealed by a Study Using the Go-specific is Mutant

The purpose of this study is to distinguish two possibilities that the transformed cells are blocked to enter a resting state (G0) and that they enter but are persistently stimulated to return to a growing cycle, using SV40-transformed r.v.l'l60 cells as a model system, rv.ll 60 is a temper ature-sensitive (r\) mutant of a Fischer rat cell line (3Y1), which is classified as a G0 mutant; i.e., the Is defect is not expressed within the growing cell cycle but is expressed only between (.„ and S phases. We assumed that if the former possibility were the case, the transformed cells would not show any is phenotype; and if the latter case, they might be ts. All SV'40-transformed rU 160 clones grew at 34°Cas well as SV40transformed 3Y1 cells did at both temperatures. At 39.5°C, some SV40transformed rsJT60 clones grew (not ts) but others did not (rv) under conditions adequate for growth of untransformed cells. When clones that grew at 39.5°Cwere cultured under conditions inadequate for growth of untransformed cells such as serum restriction or high cell density, they were ts for growth or even cytocidal at 39.5°C.These results indicate that all clones of SV'40-transformed rv.l160 cells are ts and the latter possibility is the case. an exponential growth phase it grows normally at both permis sive and nonpermissive temperatures; and (b) when stimulated by serum to leave G0 it enters S phase at the permissive temperature, 34°C,but fails at the nonpermissive temperature, 39.5°C.fsJToO cells enter G0 at both temperatures when they reach a confluent stage. The presence of such a mutant postu lates the presence of a unique biochemical step between G0 and S phase which is not involved in the progression of G, phase in growing cycle. The purpose of the studies in this paper is to distinguish two possibilities, that the transformed cells are unable to enter G0 phase and that they enter G0 but are persistently stimulated to reenter a growth cycle, by using ttJT60 cells transformed with SV40 virus or with DNA containing SV40 early gene as a model system. We simply assumed that if the former were the case, the transformed cells would not show any is phenotype, and if the latter were the case, they would. The results indicate that the latter is the case.