Correction: Aberrant DNA Damage Response Pathways May Predict the Outcome of Platinum Chemotherapy in Ovarian Cancer.

[This corrects the article DOI: 10.1371/journal.pone.0117654.].

Aberrant DNA damage response pathways may predict the outcome of platinum chemotherapy in ovarian cancer.

Ovarian carcinoma (OC) is the most lethal gynecological malignancy. Despite the advances in the treatment of OC with combinatorial regimens, including surgery and platinum-based chemotherapy, patients generally exhibit poor prognosis due to high chemotherapy resistance. Herein, we tested the hypothesis that DNA damage response (DDR) pathways are involved in resistance of OC patients to platinum chemotherapy. Selected DDR signals were evaluated in two human ovarian carcinoma cell lines, one sensitive (A2780) and one resistant (A2780/C30) to platinum treatment as well as in peripheral blood mononuclear cells (PBMCs) from OC patients, sensitive (n = 7) or resistant (n = 4) to subsequent chemotherapy. PBMCs from healthy volunteers (n = 9) were studied in parallel. DNA damage was evaluated by immunofluorescence γH2AX staining and comet assay. Higher levels of intrinsic DNA damage were found in A2780 than in A2780/C30 cells. Moreover, the intrinsic DNA damage levels were significantly higher in OC patients relative to healthy volunteers, as well as in platinum-sensitive patients relative to platinum-resistant ones (all P<0.05). Following carboplatin treatment, A2780 cells showed lower DNA repair efficiency than A2780/C30 cells. Also, following carboplatin treatment of PBMCs ex vivo, the DNA repair efficiency was significantly higher in healthy volunteers than in platinum-resistant patients and lowest in platinum-sensitive ones (t1/2 for loss of γH2AX foci: 2.7±0.5h, 8.8±1.9h and 15.4±3.2h, respectively; using comet assay, t1/2 of platinum-induced damage repair: 4.8±1.4h, 12.9±1.9h and 21.4±2.6h, respectively; all P<0.03). Additionally, the carboplatin-induced apoptosis rate was higher in A2780 than in A2780/C30 cells. In PBMCs, apoptosis rates were inversely correlated with DNA repair efficiencies of these cells, being significantly higher in platinum-sensitive than in platinum-resistant patients and lowest in healthy volunteers (all P<0.05). We conclude that perturbations of DNA repair pathways as measured in PBMCs from OC patients correlate with the drug sensitivity of these cells and reflect the individualized response to platinum-based chemotherapy.

Development and validation of a PCR-based assay for the selection of patients more likely to benefit from therapeutic treatment with alkylating drugs.

WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT: Previous studies have indicated that the levels of DNA damage induced in peripheral blood mononuclear cells by the alkylating drugs melphalan, cisplatin and carboplatin can serve as useful biomarkers predictive of the therapeutic response of cancer patients to these drugs. WHAT THIS STUDY ADDS: In the present study we developed a quantitative PCR-based assay, for the measurement of DNA damage. The advantages of this methodology are based on: its far greater sensitivity (about 250 times) than the traditional Southern blot-based method (the detection limit is ~10-20 lesions/10(6) nucleotides from the equivalent DNA of ~8000 cells); its simplicity and speed (results obtained within ~8h); its excellent reproducibility, with a coefficient of variance of 10-15% for different DNA preparations from similarly treated cells; its requirement for only minute amounts of material, and; the avoidance of radioisotope labeling. Moreover, emphasis was given to translate basic research findings into clinical practice through the validation of this assay for prediction of clinical outcome in multiple myeloma patients. AIM: In order to develop and validate a simple, sensitive and rapid method for the quantitation of alkylating drug-induced DNA damage. METHODS: HepG2 cells and blood samples were treated with alkylating drugs (melphalan, cisplatin, carboplatin). Gene-specific damage was examined using Southern blot and a multiplex long quantitative PCR (QPCR) carried out in a 7 kb fragment (part of the p53 gene) and a 0.5 kb fragment (part of the IFN-ß1 sequence; internal standard). RESULTS: The extent of PCR amplification of a p53 fragment was inversely proportional to the treatment concentrations of all anticancer drugs examined, indicating a dose-related inhibition by the DNA adducts formed. Parallel analysis of the same samples using both Southern blot and QPCR showed that the DNA adducts measured by QPCR corresponded to the interstrand cross-links in the case of melphalan, and to total drug-induced lesions in the case of the platinum drugs. The detection limit was ~10-20 lesions/10(6) nucleotides using DNA from ~8000 cells. The method is about 250 times more sensitive than the Southern blot-based method and the reproducibility is excellent, with an intraday coefficient of variance (CV) of 5-9% and an interday CV of 4-12%. Application of the QPCR assay to ex vivo melphalan-treated peripheral blood mononuclear cells from multiple myeloma patients, showed that the positive predictive value of this assay for clinical response to melphalan therapy was 92.9%. CONCLUSION: The PCR-based assay developed in this study can be used for the selection of cancer patients more likely to benefit from therapeutic treatment with alkylating drugs.

The repair of melphalan-induced DNA adducts in the transcribed strand of active genes is subject to a strong polarity effect.

To investigate the mechanisms of the therapeutic action and drug resistance to the nitrogen mustard melphalan, melphalan-induced DNA damage repair and chromatin structure were examined along the p53, N-ras and d-globin gene loci in cells carrying different repair activities. In nucleotide excision repair-deficient XP-A cells, similar levels of adducts were found in all fragments examined, indicating uniform distribution of DNA damage. In both, repair-proficient CS-B and XP-C cells, faster repair was observed in regions inside the transcribed N-ras and p53 genes, compared to regions on both sides outside of the genes, while no such difference was observed for the inactive d-globin gene. Moreover, very fast adduct repair on the transcribed strand of the active genes was seen immediately downstream of the transcription start site, together with a steeply decreasing gradient of repair efficiency along the gene towards the 3'-end. In all cells analyzed, the above variation in DNA repair efficiency was paralleled exactly by the variation in the degree of local chromatin condensation, more relaxed chromatin being associated with faster repair. Similar results were obtained using peripheral blood mononuclear cells from healthy volunteers, suggesting that the existence of a repair gradient along transcribed genes may be a universal phenomenon. In conclusion, these findings demonstrate that the repair of melphalan adducts in the transcribed strand of active genes is subject to a strong polarity effect arising from variations in the chromatin structure.

Association between transcriptional activity, local chromatin structure, and the efficiencies of both subpathways of nucleotide excision repair of melphalan adducts.

The repair of melphalan-induced N-alkylpurine monoadducts and interstrand cross-links was examined in different repair backgrounds, focusing on four genes (beta-actin, p53, N-ras, and delta-globin) with dissimilar transcription activities. Adducts were found to be substrates for both global genome repair (GGR) and transcription-coupled repair (TCR), with TCR being less efficient than GGR. In nucleotide excision repair-deficient cells, adducts accumulated to similar levels in all four genes. The repair efficiency in different gene loci varied in a qualitatively and quantitatively similar way in both GGR-deficient and TCR-deficient backgrounds and correlated with transcriptional activity and local chromatin condensation. No strand-specific repair was found in GGR(+)/TCR(+) cells, implying that GGR dominated. Adducts were lost over two sharply demarcated phases: a rapid phase resulting in the removal within 1 hour of up to approximately 80% of the adducts, and a subsequent phase with t(1/2) approximately 36 to 48 hours. Following pretreatment of cells with alpha-amanitin, the rate of transcription, the state of chromatin condensation, and the repair efficiencies (both TCR and GGR) of the transcribed beta-actin, p53, and N-ras genes became similar to those of the nontranscribed delta-globin gene. In conclusion, a continuous, parallel variation of the state of transcription and local chromatin condensation, on one hand, and the rates of both GGR and TCR, on the other hand, have been shown.

Preferential in vivo DNA repair of melphalan-induced damage in human genes is greatly affected by the local chromatin structure.

To investigate the molecular mechanisms of action of the nitrogen mustard melphalan in patients treated for multiple myeloma, the in vivo induction and repair of melphalan-induced DNA damage was measured in genes with different transcriptional activity (b-actin>p53>N-ras>d-globin) from leukocytes of 20 multiple myeloma patients following chemotherapeutic administration of high-dose melphalan (200mg/m(2)) and autologous blood stem cell transplantation. Heterogeneous repair was found among the studied genes. The extent of repair was always in the order: b-actin>p53>N-ras>d-globin, correlating with the gene transcriptional state. Similar findings were obtained using peripheral blood mononuclear cells (PBMC) from healthy volunteers following in vitro treatment with melphalan, indicating that these results are not malignant disease-specific. Following in vitro treatment of PBMC from healthy volunteers with alpha-amanitin, an inhibitor of RNA polymerase II that can also induce condensation of chromatin structure, a significant inhibition of the removal of melphalan-induced damage in the three active genes but not in the silent d-globin gene was found, suggesting that transcription and/or chromatin structure may play important roles in the preferential DNA repair. When the in vivo DNA damage formation and repair in multiple myeloma patients following chemotherapeutic administration of melphalan was measured in the two strands of the active genes, no strand bias was found, indicating that the global genome repair subpathway of nucleotide excision repair may play a crucial role in the repair of these adducts. These results were also confirmed in PBMC from healthy volunteers following in vitro treatment with melphalan. Using micrococcal nuclease digestion of nuclei isolated from PBMC of multiple myeloma patients before the chemotherapeutic treatment, as well as from PBMC of healthy volunteers, we probed the chromatin structure in each gene and found that the "looseness" of the chromatin structure correlated with the levels of the gene-specific repair, being again in the order: b-actin>p53>N-ras>d-globin. To conclude, the in vivo gene-specific repair of melphalan-induced damage in humans is greatly affected by the local chromatin structure.