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 μg/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 β‐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 protooncogenes 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.

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