Expression of the p12 subunit of human DNA polymerase δ (Pol δ), CDK inhibitor p21WAF1, Cdt1, cyclin A, PCNA and Ki-67 in relation to DNA replication in individual cells

We recently reported that the p12 subunit of human DNA polymerase (Pol 4) is degraded by CRL4(Cdt2) which regulates the licensing factor Cdt1 and p21(WAF1) during the G(1) to S transition. Presently, we performed multiparameter laser scanning cytometric analyses of changes in levels of p12, Cdt1 and p21(WAF1), detected immunocytochemically in individual cells, vis-a-vis the initiation and completion of DNA replication. The latter was assessed by pulse-labeling A549 cells with the DNA precursor ethynyl-2-deoxyribose (EdU). The loss of p12 preceded the initiation of DNA replication and essentially all cells incorporating EdU were p12 negative. Completion of DNA replication and transition to G(2) phase coincided with the re-appearance and rapid rise of p12 levels. Similar to p12 a decline of p21(WAF1) and Cdt1 was seen at the end of G(1) phase and all DNA replicating cells were p21(WAF1) and Cdt1 negative. The loss of p21(WAF1) preceded that of Cdt1 and p12 and the disappearance of the latter coincided with the onset of DNA replication. Loss of p12 leads to conversion of Pol 4 to its trimeric form, Pol 3, so that the results provide strong support to the notion that Pol 3 is engaged in DNA replication during unperturbed progression through the S phase of cell cycle. Also assessed was a correlation between EdU incorporation, likely reflecting the rate of DNA replication in individual cells, and the level of expression of positive biomarkers of replication cyclin A, PCNA and Ki-67 in these cells. Of interest was the observation of stronger correlation between EdU incorporation and expression of PCNA (r = 0.73) than expression of cyclin A (r = 0.47) or Ki-67 (r = 0.47).