DISSECTION OF C-MYC DOMAINS INVOLVED IN S-PHASE INDUCTION OF NIH3T3 FIBROBLASTS

The product of the c-myc proto-oncogene is an important regulator of c ell proliferation and apoptosis in murine fibroblasts. Addition of the tumor promoter, phorbol myristate acetate (PMA), prevents apoptotic c ell death induced by low serum concentrations in NIH3T3 cells that con stitutively express and are transformed by v-myc. The protective effec t of PMA allowed us to analyse the ability of normal c-Myc and Myc del etion mutants to induce serum starved, untransformed NIH3T3 cells to e nter S phase. By microinjecting these quiescent cells with wild type a nd mutant human c-myc plasmids, we showed that full length c-myc is ab le to induce S phase entry in presence of PMA, but that c-Myc mutants that delete amino acids Delta 7/91, Delta 41/53, Delta 56/103, Delta 1 06/143, Delta 265/317 and Delta 414/433 are totally inactive. c-Myc di d not shorten the period before entry into S phase, since Myc overexpr essing cells entered S phase with the same kinetics as control cells w hen both were stimulated with 20% fetal calf serum (FCS). However, c-M yc overexpression did increase the percentage of cells entering S phas e when these cells were stimulated with 2% fetal calf serum. Interesti ngly, this ability to enhance stimulation by a suboptimal concentratio n of FCS was retained to a significant degree by Myc mutants that dele te amino acids Delta 41/53, Delta 56/103 or Delta 265/317. Finally, My c mutants that delete Delta 106/143 or Delta 414/433 exerted a dominan t negative effect on S phase entry both in quiescent cells stimulated with 2% FCS and in unsynchronized, cycling cells.