title: Expression of the p12 subunit of human DNA polymerase δ (Pol δ), CDK inhibitor p21(WAF1), Cdt1, cyclin A, PCNA and Ki-67 in relation to DNA replication in individual cells.
abstract: We recently reported that the p12 subunit of human DNA polymerase δ (Pol δ4) is degraded by CRL4(Cdt2) which regulates the licensing factor Cdt1 and p21(WAF1) during the G1 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-à-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 G2 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 G1 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 δ4 to its trimeric form, Pol δ3, so that the results provide strong support to the notion that Pol δ3 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).
authors.affiliation: a Brander Cancer Research Institute; Department of Pathology; New York Medical College ; Valhalla , NY USA.
authors.forename: Hong
authors.initials: H
authors.lastname: Zhao
authors.forename: Sufang
authors.initials: S
authors.lastname: Zhang
authors.forename: Dazhong
authors.initials: D
authors.lastname: Xu
authors.forename: Marietta Ywt
authors.initials: MY
authors.lastname: Lee
authors.forename: Zhongtao
authors.initials: Z
authors.lastname: Zhang
authors.forename: Ernest Yc
authors.initials: EY
authors.lastname: Lee
authors.forename: Zbigniew
authors.initials: Z
authors.lastname: Darzynkiewicz
mesh_terms: D002453:Cell Cycle / Q000235:genetics; D018797:Cell Cycle Proteins / Q000235:genetics*; D019925:Cyclin A / Q000235:genetics*; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000235:genetics*; D004258:DNA Polymerase III / Q000235:genetics*; D004261:DNA Replication / Q000235:genetics*; D016193:G1 Phase / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D019394:Ki-67 Antigen / Q000235:genetics*; D018809:Proliferating Cell Nuclear Antigen / Q000235:genetics*; D034622:RNA Interference; D016196:S Phase / Q000235:genetics; D054875:Ubiquitination
chemical_list: C493956:CDKN1A protein, human; C467199:CDT1 protein, human; D018797:Cell Cycle Proteins; D019925:Cyclin A; D050759:Cyclin-Dependent Kinase Inhibitor p21; D019394:Ki-67 Antigen; D018809:Proliferating Cell Nuclear Antigen; C521101:POLD4 protein, human; D004258:DNA Polymerase III
keywords: CDK inhibitor p21WAF1, imaging cytometry; CRL, cullin-ring ligase; CRL4Cdt2; Cdk, cyclin-dependent kinase; Cdt1; Cdt1, Cdc10-dependent transcript 1; Cdt2, Cdc10-dependent transcript 2; DNA replication; EdU labeling; PCNA, proliferating cell nuclear antigen; Pol δ, DNA polymerase δ; S-phase; cell cycle; cell proliferation; laser scanning cytometry; p12; p21; polymerase δ