Abstract
ObjectivesDifferences in the incidences and types of DNA damage induced by antitumor agents for clear cell carcinoma (CCC) were determined in 2 ovarian CCC cell lines using γH2AX.Material and methodsThe antitumor activity of anticancer agents, CDDP, CBDCA, PTX and SN-38, was examined using ovarian clear cell carcinoma cultured cell lines (OVISE and RMG-I). After culture, each cell line was treated with each anticancer agent, the cells were collected, fixed, and then reacted with the anti-γH2AX antibody. γH2AX and nuclear DNA were then simultaneously detected by flow cytometry using FITC and propidium iodide, respectively, to determine γH2AX in each cell cycle phase.ResultsAfter administration of CDDP, DNA damage was frequent in S-phase cells, while cell-cycle arrest occurred in the G1 and G2/M phases and γH2AX did not increase in CDDP-resistant cells. Sensitivities to CDDP and CBDCA differed between the two cell lines. The antitumor effect of PTX is induced by G2/M arrest, and combination treatment with CBDCA, inducing DNA damage in G2/M-phase cells, might be effective.ConclusionsThis is the first study in Japan to evaluate the antitumor activity of anticancer agents by focusing on the relationship between the cell cycle and DNA damage using γH2AX as an indicator. The immunocytochemical method used in this study detects γH2AX, which indicates DNA damage even at very low concentrations and with high sensitivity. Therefore, a promising method of easily and rapidly identifying agents potentially effective against CCC.
Highlights
Clear cell adenocarcinoma (CCC), a subtype of epithelial ovarian cancer, is less sensitive to chemotherapy and is classified as a refractory ovarian cancer [1]
The Japanese Gynecologic Oncology Group (JGOG) started an international randomized controlled trial (RCT) of cisplatin (CDDP)/CPT-11 therapy with a control arm of CBDCA/TXL (TC) therapy (JGOG3017/GCIG); patient accrual is ongoing and approximately 560 patients had been enrolled in the trial as of July 2010
Platinum Agents CDDP OVISE cells showed an increase in the number of distinct green dots after exposure to 10 μg/ml CDDP for 24 h, which indicates that CDDP caused DNA damage (Figure 1)
Summary
Clear cell adenocarcinoma (CCC), a subtype of epithelial ovarian cancer, is less sensitive to chemotherapy and is classified as a refractory ovarian cancer [1]. Dot γH2AX, which is detectable using γH2AXspecific antibody, is considered to correspond to specific DNA damage. It has become apparent that phosphorylation of histone H2AX, one of the variants of the nucleosome core histone H2A, can provide a sensitive and reliable marker of DNA damage. The presence of γH2AX in cells can be detected immunocytochemically in the form of distinct nuclear γH2AX immunofluorescent foci and each focus is considered to correspond to a single DSB. This immunocytochemical approach has made it possible to assay DNA damage and in situ repair of the chromatin of individual cells. Determination of the cell-cycle phase targeted by the drug is of importance in elucidating the mechanism of antitumor drug activity
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