Are platinum agents, paclitaxel and irinotecan effective for clear cell carcinoma of the ovary? DNA damage detected with γH2AX induced by anticancer agents
- Eriko Takatori†1,
- Tadahiro Shoji†1Email author,
- Seisuke Kumagai†1,
- Takashi Sawai†2,
- Akira Kurose†3 and
- Toru Sugiyama†1
© Takatori et al.;licensee BioMed Central Ltd. 2012
Received: 20 April 2012
Accepted: 12 June 2012
Published: 12 June 2012
Differences 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 methods
The 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.
After 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.
This 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.
Clear cell adenocarcinoma (CCC), a subtype of epithelial ovarian cancer, is less sensitive to chemotherapy and is thus classified as a refractory ovarian cancer . It has been shown that a combination of carboplatin (CBDCA) and paclitaxel (PTX ), a standard therapy for ovarian cancer [2, 3], is effective against serous adenocarcinoma and endometrioid adenocarcinoma, with a response rate of approximately 75%, while CCC has lower response rates ranging from 18% to 50% . The incidence of CCC has been increasing and is now 25% in Japan, while that in Europe is 5-6%. As yet, no treatment for this histological subtype of ovarian cancer has been established. Histopathology remains the gold standard for classifying epithelial ovarian cancer subgroups; however, there is emerging evidence indicating different genetic and molecular profiles. Consequently, there is no international consensus regarding the necessity of establishing treatment strategies based on histological subtypes. In fact, global clinical trials of CCC and mucinous adenocarcinoma have already begun. Although which cytotoxic agents have true efficacy against CCC remains unknown, small trials in Japan and basic studies have suggested the efficacy of irinotecan (CPT-11) [5–7]. 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. In addition, an ongoing translational study, as part of the JGOG3017/GCIG trial, also aims to establish an updated treatment strategy.
Nucleosomes, units of chromatin, consist of core histones wrapped in 146 bp of DNA and linker DNA. Core histones are octamers designated H2A, H2B, H3 and H4. Histone H2AX is a variant of histone H2A and accounts for 10-15% of all variants. When DNA damage occurs, serine 139 of histone H2AX in chromatins on both sides of a damaged site is phosphorylated by two enzymes: ataxia telangiectasia mutated (ATM) protein kinase and by ATM and Rad3 related (ATR) protein kinase [8, 9]. Phosphorylated histone H2AX is called γH2AX. Dot γH2AX, which is detectable using γH2AX-specific antibody, is considered to correspond to specific DNA damage. Therefore, DNA damage can be immunocytochemically detected . DNA damage in individual cells has been detected employing a single-cell DNA gel electrophoresis technique (comet assay), in which the extent and length of the comet’s tail indicate the severity of DNA damage. Recently, however, 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. More specifically, DNA damage, particularly that involving the formation of DNA double-strand breaks (DSBs), induces phosphorylation of histone H2AX on Ser-139; phosphorylated H2AX is defined as γH2AX. The phosphorylation takes place on H2AX molecules on both sides of DSBs along a megabase length of DNA. Although DSBs generated during DNA fragmentation in the course of apoptosis also induce γH2AX, the degree of γH2AX induction in apoptotic cells is much greater than that in primary DSBs induced by antitumor drugs or radiation. 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. The immunocytochemical approach is significantly more sensitive than the comet assay. The use of multi-parameter flow cytometry in measurements of γH2AX immunofluorescence allows DNA damage to be correlated with cellular DNA content and, therefore, the cell-cycle phase. Determination of the cell-cycle phase targeted by the drug is of importance in elucidating the mechanism of antitumor drug activity.
In the present study, we conducted flow cytometric bivariate analyses of γH2AX and DNA contents in two different cell lines of CCC treated with CDDP, CBDCA, PTX or CPT-11 (SN-38), which have been used in the aforementioned international clinical randomized trial targeting CCC, and examined effects of these drugs with regard to the induction of DNA damage, apoptosis and cell-cycle progression vis-à-vis the cell-cycle phase.
Materials and methods
Clinical biological characteristics of the cell line
Median doubling time
CAP × 6 courses
Minimum effective concentration (MEC)
Both cells floating in the medium and the cells that remained attached after trypsinization were collected and fixed with 1% methanol-free formaldehyde (Polysciences Inc., Warrington, PA, USA) in PBS at 0 °C for 15 minutes and post-fixed with 80% ethanol for at least 2 hours at −20 °C. The fixed cells were washed twice in PBS and suspended in a 1% (w/v) solution of bovine serum albumin (BSA) (Sigma) in PBS to suppress non-specific antibody binding. The cells were then incubated in 100 μl of 1% BSA containing 1:100 diluted anti-phosphohistone H2AX (Ser-139) monoclonal antibody (Upstate, Lake Placid, NY, USA) for 2 hours at room temperature, washed twice with PBS and resuspended in 100 μl of 1:20 diluted fluorescein isothiocyanate (FITC)-conjugated F(ab’)2 fragment of goat anti-mouse immunoglobulin (Dako, Glostrup, Denmark) for 30 minutes at room temperature in the dark. The cells were then counterstained with 5 μg/ml propidium iodide (PI) (Sigma) in the presence of 100 μl of RNaseA (Sigma) for 30 minutes.
Fluorescence measurements by flow cytometry
The FITC (green) and PI (red) fluorescence of individual cells in suspension induced by excitation with a 488-nm argon ion laser was measured using a FACScan flow cytometer (Becton-Dickinson, San Jose, CA, USA). The green and red fluorescence from each cell was separated and quantified using standard optics and Cell Quest software (Becton-Dickinson). Ten thousand cells were measured per sample. All experiments were repeated at least three times.
After γH2AX and DNA staining, the DNA content was represented on the x axis and the γH2AX content on the y axis using flow cytometry. The γH2AX in each cell cycle was determined, thereby allowing the relationships between cell kinetics and DNA damage induced by antitumor agents to be examined.
After CDDP administration, DNA damage was observed mainly in the S phase. It is reasonable to assume that the DNA was structurally altered by CDDP, leading to DNA replication fork arrest and ultimately resulting in apoptosis. This result was consistent with a known pharmacological effect of CDDP . In RMG-I, apoptotic cells were minimally increased in the S phase, moreover the cells showing arrest in the G1 and G2/M phases without DNA damage were increased as compared with OVISE. Therefore, the results of the present study support the clinical experience that RMG-I is CDDP-resistant [11, 13]. After CBDCA administration, DNA damage was seen in the S and G2/M phases in both cell lines. OVISE contained a remarkable cell population rescued from apoptosis and surviving with DNA damage. On the other hand, most RMG-I cells with DNA damage underwent apoptosis. These results suggest that cell lines respond differently to platinum agents, i.e., RMG-I was CDDP-resistant but responded to CBDCA. PTX directly induced apoptosis in M-phase cells but not via DNA damage, an observation consistent with a known pharmacological effect of PTX, i.e. microtubule inhibition . PTX was confirmed to induce apoptosis through a p53-independent pathway; it was, therefore, expected to have an effect on CCC, in which the p53 mutation is rare [15, 16]. The mechanism underlying the antitumor effect of PTX is G2/M arrest. Therefore, the combination with CBDCA, an agent inducing DNA damage, in G2/M-phase arrested cells might be effective, at least theoretically. As shown in this study, it is noteworthy that sensitivities to CDDP and CBDCA differed between the CCC cell lines. In practice, CCC is less sensitive to CBDCA/PTX treatment [4, 6, 17], which is the standard regimen for ovarian cancer. Since the effect of PTX was independent of both the concentration and the response time, these results raise the possibility that repeated administration of PTX at a low dose increases the antitumor effect more than a single administration. These findings support the results of the JGOG3016, i.e. that weekly CBDCA (AUC6)/PTX (80 mg/m2, weekly × 3) is more effective than tri-weekly CBDCA (AUC 6)/PTX (175 mg/m2) treatment .
On the other hand, after administration of SN-38, DNA damage occurred in S-phase cells, followed by apoptosis. This confirmed that SN-38 acts as a type I topoisomerase inhibitor . Furthermore, it appears that SN-38 had an effect on the cell cycle because S-phase arrest continued for more than 120 hours. It is, therefore, possible that the improved administration method for SN-38 increases its antitumor effect. Cells rescued from apoptosis remained in S phase with DNA damage; consequently, the efficacy of combining SN-38 with CDDP, which induces DNA damage mainly in S-phase cells, was supported.
In conclusion, the present results suggest that an effective treatment for CCC with a slow growth rate and a low ratio of S-phase cells would be a combination of agents arresting the cell cycle, thereby causing accumulation of cells in the S phase or the G2/M phase, and agents specifically inducing DNA damage in S-phase cells. The method used in this study allows immunocytochemical detection of γH2AX, which indicates DNA damage even at very low concentrations and has high sensitivity in comparison with the comet assay. Employing this method, we were able to analyze relationships between anti-tumor effects and cell cycle perturbations. Therefore, γH2AX detection is a promising method of simply and rapidly identifying agents potentially effective against CCC.
Eriko Takatori, Tadahiro Shoji, Seisuke Kumagai, Takashi Sawai, Akira Kurose, Toru Sugiyama contributed equally to this work.
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