ftJT16, a Cell Cycle ts Mutant of Rat Fibroblast Defective in Early G0/G1 Transition, Fails to Induce Gl-cyclin and cdk2 Genes after Serum Stimulation at the NonpermissiveTemperature

taJT16 is a cell-cycle temperature-sensitive its) mutant derived from rat fibroblasts whose functional defect appears soon after the growth stimulation from GOphase. In addition to c-fos, c-myc and ornithine decarboxylase gene, 7 primarily inducible genes, c-jun9 KC9 JE, 2F1, 2A99 egr-l9 and egr-2, were further shown to be expressed after serum stimulation at both permissive and nonpermissive temperatures. However, expression of secondarily inducible genes, cyclin Dl and D3 and cdk2, was ts and was cycloheximide sensitive. Expression of cyclin C was not inhibited by cycloheximide but it was ts. Failure in expression of Gl cyclins and Cdk2 is suggested to be a causal event for inability of growth induction of tsJT16 at the nonpermissive temperature. Different growth factors activate signal transduction pathways via different second messenger molecules and result in an induction of several cell-cycle-dependent genes whose products drive GO-arrested cells toward the S phase. MT16 is a G0/G1 temperature-sensitive (ts) mutant of the cell cycle (12). It has a functional defect that appears soon after the growth stimulation from the GOphase and is a mutant of early signal transduction process assumed to be characterized as follows. 1) WhenMT16is stimulated from GOphase with serum, it enters S phase at the permissive temperature (34°C) but fails to do so at the nonpermissive temperature (40°C) (12). 2) The execution point after which the cells become temperature-insensitive for the progression towards S phase is within 4 h or less after the serum stimulation (12). 3) After serum stimulation from GOphase, the accumulation of mRNAs from such cell-cycle-dependent genes as c-fos and c-myc occurs at the same level at both temperatures (12). 4) The synthesis of a nuclear labile protein with molecular weight of 70 kD and apparent pi of 7.5 (tentatively referred to as p70) is induced at 34°C but not at 40°C following serum stimulation (13). 5) The synthesis of p70 is at maximal level around 2h and continues for about 5h after serum stimulation and diminishes before entering S phase (13). 6) p70 is a low abundance protein and is likely to be a product from a primarily inducible gene (13). 7) p70 is exclusively synthesized at early GO/S transition process and not synthesized in growing cell cycle (14). 8) mRNAof p70 was supposed to be synthesized exclusively within 2h of growth stimulation at 34°C (13). 9) Defect in tsJT\6 to induce p70 is likely to be located at the commondownstream of protein kinase C-dependent and -independent pathways (7). It is suggested that p70 is a nuclear protein possibly responsible for the early transition stage between GOand S phase and is induced via a signal transduction sequence different from that for the other primarily inducible genes such as cfos and c-myc. Recent biochemical and genetic data have demonstrated that the Gl cyclin/Cdk complex plays a positive role in promoting cell cycle transition controlling passage through the restriction point in mammaliancells, after which cells become committed to a round of cell division (for review, see ref. 10). The sequence of induction processes and regulation mechanisms of these genes still remain obscure. However, it is reasonable to assume that tsJT16 stimulated at 40°C could not express some primarily inducible genes such as p70 and resulted in a failure in induction of secondarily inducible genes such as cyclins and Cdks. In the present paper, we describe that the expression of Gl cyclins and Cdk2 is inhibited when tsJT16 is stimulated from GO phase at the nonpermissive temperature. T To whomcorrespondence and reprint request should be addressed. * Present address: Corporate Rresearch and Development Laboratory, Tonen Corporation, 1-3-1 Nishitsurugaoka, Ohimachi, Irumagun, Saitama 358, Japan.