The cell cycle phase specificity of DNA damage induced by radiation, peroxide and chemotherapeutic drugs targeting topoisomerase II, and CD4 and CD8 receptor expression on apoptotic human lymphocytes.

摘要

Drugs targeting topoisomerase II (topoII) are widely used to treat human cancers. Although cytostasis and cytotoxicity induced by these drugs arise in specific cell cycle phases, it is unknown whether the induced DNA damage triggering these responses varies with cell cycle phase. A flow cytometric DNA alkaline unwinding assay was optimized to measure DNA damage (strand breaks and alkali-labile sites) and its repair in each cell cycle compartment of human tumor cell lines treated with clinically-relevant drug concentrations. To determine the assay sensitivity and reproducibility, it was applied to cells exposed to model DNA damage agents. The lowest γ radiation dose and the lowest H₂O₂ concentration that induced detectable damage in each cell cycle compartment was 10cGy and 0.5–2.5μM, respectively, comparable to the detection limits of standard assays. DNA damage in each cell cycle compartment increased approximately linearly with increasing treatment. Although untreated cells in S phase exhibited greater DNA damage than did cells in G₀/G₁, or G₂/M phase (presumably due to DNA strand breaks associated with replication forks), there was no differential susceptibility of G₀/G₁, S and G₂ /M phase cells to the induced DNA damage or in the rate of damage repair. In contrast, cells treated with topoII-stabilizing drugs (doxorubicin, daunomycin, etoposide, and mitoxantrone) exhibited the greatest SB in G₂/M phase and the least in G₀/G₁ phase, with SB in S phase generally being intermediate. The cell cycle phase specificity of the DNA damage appeared to be predictive of the cell cycle phase of growth arrest and to be dependent on topoIIα expression. The rate of repair of SB induced by some of the drugs differed between cell types and between cell cycle compartments. These data indicate that the cell cycle phase specificity of cytostasis and cytotoxicity induced in tumor cells by these drugs may be directly related to the cell cycle phase specificity of drug-induced DNA damage and its repair. In a related study, cytotoxicity induced by these and additional agents was evaluated in immunophenotyped human blood lymphocytes. Diminished expression of CD4 and CD8 surface receptors was found to be a common feature of apoptotic human T lymphocytes.