EFFECTS OF C-MYC EXPRESSION ON CELL-CYCLE PROGRESSION

We used targeted homologous recombination to disrupt one c-myc gene co py in a diploid fibroblast cell line and found that a twofold reductio n in Myc expression resulted in loner exponential growth rates and a l engthening of the G(0)-to-S-phase transition (hi. Shichiri, Ii;. D. Ha nson and J. hi. Sedivy, Cell Growth Differ. 4:93-104, 1993). Myc is a transcription factor, and the number of target genes whose regulation could result in differential growth rates may be very large. We have a pproached this problem by examining effects of reduced c-myc expressio n in three broad areas: (i) secretion of growth factors, (ii) expressi on of growth factor receptors, and (iii) intracellular signal transduc tion between Myc and components of the intrinsic cell cycle clock. We have found no evidence that differential medium conditioning can accou nt for the growth phenotypes.. Likewise, the expression of receptors f or platelet-derived growth factor, epidermal growth factor, basic fibr oblast growth factor, and insulin-like growth factor I was the same in diploid and heterozygous cells (platelet-derived growth factor, epide rmal growth factor, fibroblast growth factor, and insulin-like growth factor are the sole growth factors required by these cells for growth in serum-free medium). In contrast, expression of cyclin E, cyclin A, and Rb phosphorylation were delayed when quiescent c-myc heterozygous cells were stimulated to enter the cell cycle. Expression of cyclin D1 , cyclin D3, and Cdk2 was not affected. The timing of cyclin E inducti on was the earliest observable effect of reduced Myc expression. Our d ata indicate that Myc contributes to regulation of proliferation by a cell-autonomous mechanism that involves the modulation of cyclin E exp ression and, consequently, progression through the restriction point o f the cell cycle.