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DNA replication was investigated in isolated HeLa cell nuclei from different phases of the cell cycle. The addition of S-phase cytoplasm to S-phase nuclei restored the ability of these nuclei to synthesize DNA. The S-phase cytoplasm requirement could be replaced by partially purified calfthymus DNA polymerases A and B. The activity of both the S-phase cytoplasm and purified DNA polymerases in the nuclear system was ATP-dependent. Using nuclei prelabelied with bromodeoxyuridine and CsCl equilibrium centrifugation, the DNA synthesis in vitro was shown to be of the replicative, not repair type when supported by either S-phase cytoplasm or DNA polymerase A. DNA polymerase B, but not DNA polymerase A, was capable of using G1 nuclei to synthesize DNA; however, this synthesis was not ATP-dependent. Gel-filtration chromatography of HeLa cell-free extracts have shown that DNA polymerases B and C are present in S-phase and missing in G1-phase cells. [ABSTRACT FROM AUTHOR]

Background: Alkylating N-nitroso compounds can interact directly with DNA, forming O(6)-alkylguanine, a DNA adduct proved to be mutagenic and carcinogenic if not sufficiently repaired. A specific DNA repair enzyme, O(6)-methylguanine-DNA methyltransferase (MGMT), can remove the alkyl group from the O(6)-position of the guanine, thereby preventing its mutagenic and carcinogenic effects. Inactivation of the MGMT gene in association with promoter hypermethylation results in persistence of O(6)-alkylguanine in DNA, leading to G:C to A:T transition mutation and these G:C to A:T transition mutations can inactivate p53 tumor suppressor gene or activate ras proto-oncogene., Methods: We analyzed MGMT promoter hypermethylation and protein expression patterns in 94 cases of primary head and neck squamous cell carcinoma (HNSCC) by methylation-specific PCR (MSP) and immunohistochemical staining. The results were then correlated with clinical follow-up data., Results: MGMT promoter hypermethylation was present in 17 of 94 patients (18.1%) and apparent loss of protein expression was seen in 19 of 93 HNSCC patients (20.4%). The presence of MGMT promoter hypermethylation was significantly correlated with loss of MGMT protein expression in HNSCC. Both MGMT promoter hypermethylation and loss of protein expression were significantly correlated to increased tumor recurrences and decreased patient survival, independent of other risk factors, such as tumor site, tumor size, nodal status, age, and chemoradiation therapy., Conclusions: MGMT promoter hypermethylation and apparent loss of protein expression are reliable and independent prognostic factors in HNSCC. The above study may also provide guideline or basis for applying alkylating antitumor agents to patients with HNSCC that display MGMT promoter hypermethylation and/or loss of MGMT protein expression.