Restriction point control of cell growth by a labile protein: evidence for increased stability in transformed cells.

Abstract

Animal cells apparently must accumulate a labile protein(s) before they can pass the restriction (R) point in the G1 phase of the cell cycle. This R protein may acquire increased stability in transformed 3T3 cells, thereby allowing these cells to continue growth under conditions that arrest untransformed cells. Low doses of cycloheximide or histidinol drastically reduced the rate at which normal 3T3 (A31) fibroblasts in early G1 could enter DNA synthesis. These drugs had less effect on entry of 2 tumorigenic A31 derivatives, BP[benzo[a]pyrene-transformed]A31 and SV[SV40-transformed]A31, into S, although measurement of [3H]leucine incorporation showed that the inhibitors were equally effective in the 3 cell lines. The transformed lines appears to be less sensitive because moderate inhibition of their R protein synthesis is compensated by lower rates of protein degradation. To test this idea, cytoplasmic protein synthesis was completely inhibited for several hours shortly before A31 and BPA31 cells had reached the R point. After removal of inhibitor, A31 cells showed delays in the onset of S that were in excess of the inhibitor pulse, consistent with decay of labile protein during the pulse. BPA31 cells showed no excess delays, suggesting a much more stable R protein. The half-life of the R protein was estimated as 2.5 h in A31 cells, indicating that R protein synthesis starts at the beginning of G1. In BPA31 cells, the R protein showed no signs of decay for at least 8 h.