Timing of proto-oncogene replication: a possible determinant of early S phase sensitivity of C3H 10T1/2 cells to transformation by chemical carcinogens.

The temporal order of replication of several genes was studied in 10T1/2 cells synchronized by release from confluence-induced arrest of proliferation followed by treatment with 2 micrograms/mL aphidicolin for 24 h. DNA subjected to bromodeoxyuridine substitution for 1- or 2-h intervals spanning the S phase was separated from the remaining DNA in cesium chloride gradients, filtered onto nitrocellulose in a slot-blot apparatus, and hybridized with various 32P-labeled probes. Ha-ras was among the first genes replicated at the onset of the S phase. The myc proto-oncogene replicated later but within the first hour of the S phase. The replication of Ki-ras, raf, and mos was detected between hour 1 and 2 of the S phase. The dihydrofolate reductase gene replicated early (0-2 h) and the myb proto-oncogene replicated in mid-S phase (2-4 h). An immunoglobulin VH sequence and the beta-globin gene replicated late in 10T1/2 cells, 4-6 h after removal of aphidicolin. Replicating DNA is preferentially adducted by chemical carcinogens, and replication of damaged proto-oncogenes before they are repaired may activate their transforming potential. Therefore, the observed replication of proto-oncogenes during the early S phase may underlie the enhanced sensitivity of 10T1/2 cells to chemically induced transformation at this point in the cell cycle.