Purpose: This study determined the effects of cis-diamminedichloroplatinum(II) on radiation-induced foci formation of γ-H2AX and Rad51 in lymphocytes.
Experimental Design: Twenty-eight cancer patients were irradiated for intrathoracic, pelvic, or head and neck tumors and received simultaneous cisplatin containing chemotherapy. The effect of cisplatin on radiation-induced γ-H2AX and Rad51 foci as a response to ionizing radiation–induced DNA double-strand breaks was measured in lymphocytes after in vivo and in vitro radiochemotherapy. The role of DNA-dependent protein kinase and ataxia-telangiectasia mutated kinase in γ-H2AX signaling, the consequences of altered γ-H2AX foci formation on double-strand break end joining, was studied.
Results: Cisplatin decreased the number of induced γ-H2AX foci in lymphocytes after in vivo or in vitro irradiation by 34% ± 6% at days 0 to 3 after cisplatin (P < 0.0001) and remained significant until day 6. The variation in this cisplatin effect from patient to patient was larger than the retest error within the same patient (P = 0.01). The cisplatin effect was not accompanied by an inhibition of end joining of double-strand break as analyzed using gel electrophoresis of DNA under neutral conditions. Cisplatin also decreased radiation induced Rad51 foci formation in lymphocytes after stimulation of proliferation with phytohemagglutinin by 47% ± 6% (P < 0.0001).
Conclusion: Cisplatin has long-term effects on the early double-strand break response of γ-H2AX and Rad51 foci formation after ionizing radiation. Inhibition of sensing and processing of double-strand break by γ-H2AX and Rad51 foci formation are important mechanisms by which cisplatin can alter the radiation response.
Inhibition of radiation-induced γ-H2AX and Rad51 foci formation (IRIFF) by cisplatin is a novel mechanism by which cisplatin can alter downstream radiation responses. Translational relevance is as follows. (a) Cisplatin containing treatment schedules can be evaluated on the rapid end point of IRIFF. In lymphocytes, we found full IRIFF over several days for weekly cisplatin schedules in the clinic. (b) Cisplatin resistance in tumors can be evaluated by IRIFF. (c) Cisplatin-dependent IRIFF in lymphocytes and tumor cells after in vivo or in vitro exposure has the potential of a predictive test for hematotoxicity and tumor response that has to be confirmed in further prospective studies. Here, we observed significant interpatient differences of cisplatin dependent IRIFF. (d) The strength of an association between cisplatin-dependent IRIFF and modifications of double-strand break repair or cell death has to be further studied.
Cisplatin is one of the most successfully used chemotherapeutic drugs for the treatment of solid tumors. Concurrent radiotherapy and cisplatin treatment is more effective than radiotherapy alone in non–small cell lung cancer (1), carcinomas of the cervix uteri (2), head and neck cancer (3), and other tumors such as esophageal carcinomas (4). Cisplatin enhances the effect of concurrent radiotherapy given in daily, weekly, and thrice weekly schedules (3, 5). There is large experience on side effects of concurrent cisplatin and radiotherapy in normal tissues. The acute hematologic toxicity and mucosal reactions are usually increased by concurrent cisplatin and radiotherapy in comparison to the same chemotherapy or radiotherapy alone (6, 7). The normal tissue late effects in soft tissues, lung, bone, and spinal cord are not altered (3, 5).
The primary mode of cisplatin action is formation of covalent intrastrand (Pt-GpG; Pt-AG) and to a lesser extent interstrand cross-links (G-Pt-G). These adducts modify the DNA structure, thereby inhibiting key cellular processes (e.g. transcription, replication, and repair) and can lead to cell death (8). Cisplatin adducts are usually repaired by the nucleotide excision repair or homologous recombination (8, 9). The mechanism by which a pretreatment with cisplatin can alter the sensing and repair of ionizing radiation–induced double-strand break is not well understood. Nonhomologous end joining has been suggested as one candidate for the mechanism of radiosensitization by cisplatin (10). Synergistic effects of cisplatin and radiation were observed in tumor cell lines with intact nonhomologous end joining repair but not in DNA-dependent protein kinase or Ku80-deficient cell lines (10). Ionizing radiation induces double-strand break as the primary form of damage. A number of chemotherapeutics can also generate double-strand break to a varying extent during repair (e.g., cisplatin, alkylating agents, and topoisomerase I inhibitors; ref. 11).
One of the critical determinants of the signaling from DNA double-strand break is rapid phosphorylation of histone H2AX (γ-H2AX) at or near the site of the lesion. Double-strand break–dependent phosphorylation of H2AX was found either after treatment with exogenous DNA-damaging agents such as ionizing radiation, topotecan, etoposide, bleomycin, and doxorubicin (12–14) or generated endogenously during programmed DNA rearrangements (15). The γ-H2AX assay is sensitive enough to measure radiation-induced double-strand break and its repair at low doses after in vitro and in vivo irradiation (12, 13). γ-H2AX foci are important platforms to concentrate repair proteins [e.g., Nbs, Mre11, Rad50, Mdc1, 53BP1, and ataxia-telangiectasia mutated kinase (ATM)] to double-strand break–flanking chromatin, which are also involved in the homologous recombination process (16). A distinct role of γ-H2AX in Rad51 foci formation, a key protein of homologous recombination, has also been shown (17).
The aim of the present study was to evaluate the in vivo and in vitro combination effects of ionizing radiation and cisplatin on γ-H2AX foci formation in peripheral lymphocytes from cancer patients undergoing standard radiochemotherapy and the interindividual heterogeneity in the response to the combined treatment. γ-H2AX foci are formed by phosphoinositol 3-kinase–related kinases, of which ATM, ataxia telangiecasia and Rad3 related, and DNA-dependent protein kinase were identified as the most important ones (18, 19). Activity of these proteins was modulated with specific inhibitors. In addition, formation of Rad51 foci in stimulated lymphocytes was used to evaluate the effect of cisplatin on homologous recombination of double-strand break.
Materials and Methods
Patients. Twenty-eight cancer patients were included in this study and were treated with locoregional standard radiotherapy and simultaneous cisplatin-containing chemotherapy. They gave their written informed consent to be included in this study, which was approved by the local ethics committee. Patients were irradiated for intrathoracic, pelvic, or head and neck tumors in 21, 4, and 3 cases, respectively. All patients received cisplatin-containing chemotherapy concurrent with radiotherapy at weekly or longer intervals. The cisplatin doses per application ranged from 20 to 50 mg/m2 and were given at days 0 and 7 of each 21- to 28-d cycle. Cisplatin was given according to three schedules, either as cisplatin monochemotherapy or, for non–small cell lung cancer patients (n = 14), as cisplatin 50 mg/m2 and navelbine 15 mg/m2 doublet at days 0 and 7 after the start of radiotherapy (day −1). For small-cell lung cancer patients (n = 4), it was given as cisplatin 45 to 50 mg/m2 at days 0 and 7 combined with 100 mg/m2 etoposide at days 2, 3, and 4 after the start of radiotherapy (day −1). All patients had WBC counts at or >2,000/μL at the times of blood sampling for γ-H2AX foci determination. Patients were irradiated with doses of 1.5 to 2.0 Gy per fraction to the target volume using 6 or 15 MV photons from a linear accelerator (Varian, Inc.). Three-dimensional conformal treatment was given to all patients. The applied high dose rate in the treatment volume varied from 1.5 to 4.8 Gy/min for the different treatment fields.
Separation, irradiation, and treatment of isolated lymphocytes. Blood samples of about 5 mL were taken from the cubital vein and collected in heparin-containing tubes. Mononuclear cells were isolated as previously described (13). For the in vitro drug exposure and irradiation experiments, lymphocytes from nontreated patients were incubated with 0, 2, 5, 10, 20, and 50 μg/mL cisplatin (Medac) over 1 h in supplemented RPMI 1640 (10% FCS and 100 units/mL penicillin/streptomycin, Invitrogen) before irradiation. In addition, inhibitors of DNA-dependent protein kinase (Nu7026, Calbiochem), ATM (Ku55933, Calbiochem), ATM/ataxia telangiecasia and Rad3 related (CGK733, Calbiochem), and the more unspecific kinase inhibitors caffeine and wortmannin (Sigma) were used. Cells were exposed to the kinase inhibitors over 1 h before irradiation during as well as after irradiation until fixation. Cells were in vitro irradiated 1 h after cisplatin or inhibitor treatment without medium change at room temperature using a Co-60 γ-ray machine (Philips) with a dose rate of 1.7 Gy/min. For the in vivo cisplatin exposure and in vitro irradiation experiments, blood samples were collected at the indicated days since in vivo cisplatin exposure but before radiotherapy at the respective day. This ensured that the time interval from the last in vivo irradiation until blood sampling was longer than 20 h. For the in vivo cisplatin exposure and in vivo irradiation, blood samples were taken at the indicated days since cisplatin application just before and 30 min after irradiation. Cisplatin was given 1 to 1.5 h before in vivo irradiation on the same day. For the measurement of Rad51 foci, lymphocytes had to be stimulated for proliferation to increase the fraction of cells in the S/G2 cell cycle phases. Lymphocytes were incubated with 5 μg/mL phytohemagglutinin (PHA)-L (Sigma) in supplemented RPMI 1640 for 72 h. Cell cycle distributions were measured using the Galaxy flow cytometry system (Partec) after 4′,6-diamidino-2′-phenylindole dihydrochloride staining of ethanol-fixed lymphocytes. The non–small cell lung carcinoma cell line H460 was obtained from American Type Culture Collection and was grown in supplemented RPMI 1640 under an atmosphere of 5% CO2 and 95% air at 37°C.
Immunofluorescence analysis. Samples were fixed for immunofluorescence analysis at 0.5 and 4 h after irradiation as previously described (13). The following primary antibodies were used: mouse anti–γ-H2AX (3F2, Abcam) at a dilution of 1:400, rabbit anti–Rad51 (Ab-1, Oncogene Research Products) at a dilution of 1:500, and Alexa 488–labeled secondary antibody, either goat to rabbit or goat to mouse (Invitrogen) at a dilution of 1:500. γ-H2AX and Rad51 foci were counted in 30 to 70 cell nuclei per blood sample.
Cell extracts and Western blot analysis. Lymphocytes were treated with 0 and 20 μg/mL cisplatin and 20 μmol/L ATM inhibitor Ku55933 for 1 h before irradiation with 10 Gy. At about 2 h after irradiation, lymphocytes were washed twice with cold phosphate-buffered salt solution resuspended in one pellet volume of extraction buffer (50 mmol/L NaF; 450 mmol/L NaCl; 20 mmol/L HEPES, pH 7.6; 25% w/v glycerol; 0.2 mmol/L EDTA; 0.5 mmol/L DTT (1,4-Dithio-DL-threitol); 0.5 mmol/L phenylmethylsulfonylfluoride; 0.5 mg/mL pepstatin A; 1 mg/mL trypsin inhibitor; 0.5 mg/mL aprotinin; 40 mg/mL bestatin; all proteinase inhibitors were from Roche). The swollen cells were disrupted by incubation alternatively on liquid nitrogen and 30°C (four times) for 1 min each. The resulting suspension was sedimented by centrifugation (15,000 × g; 10 mins; 4°C). Concentrations in the supernatant were determined by DC protein assay (Bio-Rad) analysis using bovine serum albumin as the standard. Aliquots (40 μg) of whole cell extracts were resolved in 4% to 12% precasted polyacrylamide-SDS gels and subjected to Western blot analysis using phospho-p53 (Ser15, Cell Signaling Technology) and glyceraldehyde-3-phosphate dehydrogenase (Ambion). Bound antibodies were detected by incubation with horseradish peroxidase–conjugated secondary antibody, followed by enhanced chemiluminescence (Amersham) and autoradiography.
Gel electrophoresis of double-stranded DNA. Lymphocytes from seven control persons were exposed to cisplatin in vitro over 1 h at 20 μg/mL and in vitro irradiated with a single dose of 8 or 15 Gy. In addition, the inhibitors wortmannin (20 μmol/L), Nu7026 (50 μmol/L), Ku55933 (50 μmol/L), CGK733 (50 μmol/L), and caffeine (20 mmol/L) were used. Cells were exposed to the kinase inhibitors over 1 h before irradiation during as well as after irradiation. Cells were serially sampled after a repair incubation of 0 and 60 min at 37°C. Thereafter, 3,000 cells/μL were casted into plug moulds of 0.7% InCert agarose (Biozym Diagnostik). Cell lysis and gel electrophoresis were done under conditions of neutral pH as previously described (20). The fraction of DNA released into the gel (FDR) as a measure of double-strand break was calculated according to the formula FDR = (intensity in the lane) / (intensity in the lane + intensity in the well).
Data analysis. A linear model was used to describe the dependence of the average number of radiation-induced γ-H2AX foci per nucleus (NmeanH2AX) on radiation dose and the effect of the time of cisplatin application and other classification variables on the slope of the dose-response curve after in vitro irradiation (13, 21). Blood samples that were taken at days −3 to 0 before in vivo cisplatin treatment and had not received cisplatin containing chemotherapy during the last 6 d before the day of blood sampling were classified as d-1. Those taken 1 to 2 h after cisplatin administration were classified as d0, those taken at days 1 to 6 after cisplatin administration as d1 to d6, and at day 7 or later for no further cisplatin administration as d≥7. On each day, blood samples were taken just before irradiation and 30 min after irradiation. The blood samples were taken around the first cisplatin administration during concurrent radiochemotherapy and in some patients in addition also around the second administration. No significant residual foci from the last radiation fraction 24 h before the actual irradiation dose and no accumulation of foci throughout radiotherapy were observed (13). The model was fitted to the foci counts after irradiation, with an intercept variable that estimates the background foci at a dose of 0 Gy. These background levels were comparable with the number of background foci per lymphocyte measured just before irradiation. The effect of the following classification variables on the slopes of the radiation dose–γ-H2AX response curves was analyzed: (a) cisplatin dose (≤30 or >30 mg/m2), (b) inhibitors of DNA-dependent protein kinase and ATM or DMSO control, (c) the respective number of the cisplatin administration during radiotherapy (1 or 2), (d) the type of chemotherapy (cisplatin mono, cisplatin/navelbine, cisplatin/etoposide), and (e) time of blood sampling from cisplatin exposure. A repeated measurement analysis was used to evaluate the intrapatient effects of time since cisplatin exposure on the radiation-induced γ-H2AX foci, taking the respective γ-H2AX dose response in blood samples at d-1 before cisplatin exposure as the reference for each patient. Weighted regression was used, and the weights were taken as reciprocals of the square of the SE of NmeanH2AX in the respective blood sample. This analysis was done with the general linear model procedure of the SAS software system (21).
After in vivo irradiation, the number of radiation-induced γ-H2AX foci was determined by the number of γ-H2AX foci in the blood samples taken 30 min after in vivo irradiation minus the average number of background foci before irradiation over all patients, that is, 0.11 foci per lymphocyte. The radiation induced number of γ-H2AX foci per lymphocyte after in vivo radiotherapy is at first approximation proportional to the received integral body dose (13). Because of the individualized radiation dose distributions applied to the patients in the clinic using standard radiotherapy, the number of radiation-induced foci varied from patient to patient and was normalized by the average number of γ-H2AX foci at d-1, d6, and d≥7 as the reference value of the patient. On these days, no significant cisplatin effect was detectable. Analysis of the variance was done using the normalized mean numbers of radiation-induced γ-H2AX foci in the blood samples as the dependent variable and the time since cisplatin exposure, cisplatin dose, the number of the previous cisplatin applications, and the type of chemotherapy as independent variables.
The dependence of the distributions of the number of Rad51 foci in lymphocytes on cisplatin and radiation exposure, as well as the effects of cisplatin administration on radiation-induced double-strand break measured by the gel electrophoresis assay, were also analyzed by ANOVA.
γ-H2AX foci after in vivo cisplatin exposure and in vitro irradiation. Lymphocytes from the peripheral blood were sampled at different days between d-1 and d≥7 relative to in vivo cisplatin administration and before the daily ionizing radiation fraction. The mean background foci per lymphocyte in the various samples ranged from 0.00 to 0.70 foci per nucleus; the grand mean over all samples was 0.11 foci per nucleus. There was no significant influence of the sampling day in relation to cisplatin administration, the type of cisplatin containing chemotherapy, or the ordinal number of cisplatin administration on the background levels (P > 0.1; F-test on foci number ranks).
Figure 1A shows the in vitro radiation dose γ-H2AX foci response data at d-1 until d≥7 in relation to the in vivo cisplatin exposure. The background numbers of foci in the unirradiated samples did not change over time or exceeded 10% of the foci at a dose of 1 Gy. The individual slopes of the in vitro γ-H2AX dose–response curves of the different patients at d-1 to d≥7 in relation to cisplatin administration are shown in Fig. 1B. The mean slope at d-1 before cisplatin exposure was 9.9 ± 0.1 foci/Gy. There was a highly significant decrease in slope at d0, d1, d2, d3, and d4 after cisplatin exposure by −3.0 + 0.2, −3.5 ± 0.2, −3.2 ± 0.3, −3.3 ± 0.3, and −2.6 ± 0.3 foci/Gy, respectively (P < 0.0001; F-test). A detectable decrease in slope by −1.2 ± 0.4 and −0.7 ± 0.2 foci/Gy still remained significant at d5 and d6, respectively (P < 0.05; F-test).
By analyzing different classification variables, it became apparent that the effects of the in vivo cisplatin dose in the given range and the number of previous cisplatin applications were not significant (P < 0.11; F-test), but the effect of the type of chemotherapy was (P = 0.006; F-test). At d0 and d1, the decreases in foci formation after cisplatin monotherapy were by 1.9 ± 0.8 and 1.2 ± 0.5 foci/Gy, slightly smaller than after cisplatin/etoposide alone. No significant differences were observed from the other days. There were also no significant differences between cisplatin/navelbine and cisplatin/etoposide doublets. In addition, subgroup analysis of the nine patients receiving cisplatin monotherapy had enough discrimination to reveal a significant decrease in foci formation after cisplatin monotreatment (P < 0.0001; F-test), with significant decreases in slope of −1.2 ± 0.6 foci/Gy at d0, −2.9 ± 0.4 foci/Gy at d1, −2.6 ± 0.8 foci/Gy at d2, and −3.3 ± 0.5 foci/Gy at d3 in comparison with d-1, respectively.
Repeated analysis of samples at d-1 and d≥7, days on which no chemotherapy effect was present, detected no significant interpatient variability of the γ-H2AX–irradiation dose slopes after in vitro irradiation (P = 0.69; F-test). In addition, we looked at the interpatient variability of the in vivo cisplatin effect on the slope of the radiation dose–response curves after in vitro irradiation. As an estimate of the intrapatient retest error, we used the cisplatin-dependent decreases of the slopes from the γ-H2AX–radiation dose response between d-1 and d1 and between d7 and d3. These days were used because the mean slopes were the same at d-1 and d7, as well as at d1 and d3. We found a significant greater variability of the cisplatin effect from patient to patient than for repeated measurements within a patient with a coefficient of variation of 24% versus 11% (P = 0.01).
In vivo cisplatin treatment and in vivo irradiation. The normalized γ-H2AX foci numbers after in vivo irradiation in the blood samples of the different patients at d0 to d≥7 in relation to cisplatin administration were compared with respective values at d-1 before cisplatin exposure. In agreement with the data after in vitro irradiation, there was a significant decrease in the normalized γ-H2AX foci at days 0 to 4 after cisplatin therapy by 28% to 46%, but no significant effect remained at d5 or later. The respective percentage decreases at d0, d1, d2, d3, and d4 were −28% ± 8%, −40% ± 8%, −37% ± 10%, −46% ± 8%, and −36% ± 9%, respectively. Although the effect of cisplatin dose (P = 0.66; F-test) and the number of the cisplatin application (P = 0.49; F-test) were not statistically significant, the effect of the type of chemotherapy was (P = 0.006; F-test). At d0, the decrease after cisplatin monotherapy was smaller than after cisplatin/etoposide, whereas no significant differences were observed at the other days.
In vitro cisplatin treatment and in vitro irradiation.Figure 2 shows the number of radiation induced γ-H2AX foci in lymphocytes after in vitro exposure to cisplatin. There is no influence on the number of background foci of cisplatin up to the highest concentration of 50 μg/mL in unirradiated cells as studied by ANOVA (P = 0.94; F-test on ranks). However, cisplatin led to a highly significant decrease in the number of γ-H2AX foci after irradiation in a concentration-dependent manner, with a half-maximal effect on radiation-induced γ-H2AX foci at about 5 μg/mL.
Influence of cisplatin on radiation-induced γ-H2AX foci formation in the presence of DNA-dependent protein kinase and ATM inhibitors. Specific inhibitors of DNA-dependent protein kinase (Nu7026), ATM (Ku55933), ATM/ataxia telangiecasia and Rad3 related (CGK733), and more unspecific inhibitors (wortmannin and caffeine) were used to evaluate the role of both kinases for γ-H2AX foci formation in lymphocytes (Fig. 3). All modifiers significantly decreased the number of γ-H2AX foci at 30 minutes after irradiation at 1 Gy in comparison with the DMSO controls. The respective decreases induced by wortmannin (20 μmol/L), Nu7026 (50 μmol/L), caffeine (20 mmol/L), CGK733 (50 μmol/L), Ku55933 (50 μmol/L), Nu7026 + CGK733 (50 μmol/L each), and Nu7026 + Ku55933 (50 μmol/L each) were −2.9 ± 0.4, −4.1 ± 0.3, −2.1 ± 0.8, −2.9 ± 0.4, −4.2 ± 0.4, −5.4 ± 0.4, and −5.7 ± 0.4 foci/Gy, respectively.
The cisplatin effect was small in the samples exposed to DNA-dependent protein kinase and ATM inhibitors. The respective cisplatin-dependent decreases in the wortmannin, Nu7026, caffeine, CGK733, Ku55933, Nu7026 + CGK733, and Nu7026 + Ku55933 groups were −1.0 ± 0.4, −0.8 ± 0.4, −0.4 ± 0.8, −1.0 ± 0.5, −0.2 ± 0.4, −0.8 ± 0.5, and −0.5 ± 0.5 foci/Gy, respectively.
At 4 hours after irradiation, the mean number of γ-H2AX foci remaining was 1.7 ± 0.2 foci/Gy for the DMSO-treated controls. All kinase inhibitors significantly increased the number of remaining foci at 4 hours in comparison with the controls. This is a reflection of the inhibition of double-strand break repair by DNA-dependent protein kinase and ATM inhibitors. The disappearance of γ-H2AX foci between 30 minutes and 4 hours after irradiation was almost completely inhibited in the caffeine and Nu7026 + Ku55933 groups (Fig. 3). In contrast, cisplatin had no significant effect on the number of remaining foci observed at 4 hours after irradiation. We also measured the effect of cisplatin on the kinase activity of ATM by an ATM-dependent activation assay for p53 phosphorylation at serine 15. Although treatment of lymphocytes with Ku55933 fully abrogates p53-ser15 phosphorylation, cisplatin had no effect (Fig. 4A).
Effect of cisplatin on induction and rejoining of double-strand break. Induction and rejoining of double-strand break were measured by gel electrophoresis of DNA from lymphocytes taken from seven healthy persons after in vitro irradiation. The experiments studying the effects of the kinase inhibitors and cisplatin on the mobile DNA fraction were done at the steep part of the dose-response relation at 8 and 15 Gy without repair incubation and at 15 Gy for repair incubations of 60 minutes. The fractions of mobile DNA isolated immediately after irradiation on ice were 24.9% ± 1.4% and 48.2% ± 1.6% after 8 and 15 Gy, respectively. The mobile fractions after irradiation with 15 Gy and repair incubation for 60 minutes at 37°C were 21.3% ± 1.4%. The kinase inhibitors Ku55933, CGK733, caffeine, wortmannin, and Nu7026 resulted in an increase in the mobile fraction by 8% ± 2%, 11% ± 2%, 13% ± 2%, 6% ± 2%, and 17% ± 2%, respectively at 60 minutes after 15 Gy in comparison with a mobile fraction of 21% for the DMSO control (Fig. 4B). The overall modulator effect (P < 0.0001; F-test) and the effects of all single modulators were significant (P < 0.01; F.-test). Thus, the kinase inhibitors led to a significant increase in the mobile fraction because of repair inhibition.
In addition, there was a significant effect of cisplatin on the mobile fraction in the gel electrophoresis assay. Cisplatin did not increase the mobile fraction after repair incubation but led to a slight decrease in the mobile fraction of DNA immediately after irradiation and after repair incubation for 60 minutes. However, the measured decrease due to cisplatin was −3% ± 1% at 0 minute and −5% ± 1% at 60 minutes of repair incubation in comparison with the DMSO controls without cisplatin (P = 0.0006; F-test). The respective relative decreases in the mobile fraction were 10% and 13%. There was no significant difference of this cisplatin effect across the kinase modulators or the DMSO control used (P = 0.55; F-test).
Effect of cisplatin on radiation-induced Rad51 foci formation. Rad51 is a key protein of homologous recombination. Rad51 foci formation was therefore used to study the effect of cisplatin on radiation-induced double-strand break. Homologous recombination is predominately active in the S/G2 phase of the cell cycle after irradiation. To increase the percentage of S/G2 cells, lymphocytes were stimulated with PHA-L. The fraction of cells in S/G2 in unstimulated lymphocytes was 9% ± 5% and increased after stimulation with PHA-L to 44% ± 5%. Stimulated and unstimulated lymphocytes were in vitro irradiated with 10 Gy, and Rad51 foci formation was analyzed 4 hours thereafter. At 4 hours, foci formation reached its maximum level. The percentage of cells with ≥5 Rad51 foci was found to be of similar magnitude as the percentage of S/G2 cells and increased from 6% ± 3% in the unstimulated and irradiated samples to 44% ± 6% after PHA-L stimulation and irradiation. Analyzing the radiation effect on Rad51 foci formation in unstimulated lymphocytes, the mean number of Rad51 foci per cell increased only slightly but significantly over background level from 1.2 ± 0.1 foci in unirradiated probes to 1.8 ± 0.1 after 10 Gy irradiation (P < 0.0001; F-test on ranked foci numbers; seven blood samples). After PHA-L stimulation, the mean number of Rad51 foci increased much markedly from 1.8 ± 0.1 in unirradiated samples to 4.4 ± 0.2 foci at 4 hours after 10 Gy (P < 0.0001; F-test on ranks; four blood samples). Cisplatin at 5, 10, and 20 μg/mL decreased radiation induced Rad51 foci in stimulated lymphocytes from 4.4 ± 0.2 foci by 2% ± 8%, 51% ± 8%, and 47% ± 6% (P < 0.0001), respectively. Cisplatin alone did not alter the number Rad51 foci in PHA-L–stimulated lymphocytes. The mean number of Rad51 foci in PHA-L–stimulated but sham-irradiated lymphocytes with or without 20 μg/mL cisplatin exposure alone was 1.1 ± 0.1 and 0.9 ± 0.1 foci/cell, respectively (P = 0.10; F-test on ranked foci numbers; four blood samples). In addition, cisplatin did not change the cell cycle distribution in PHA-L–stimulated lymphocytes.
A similar effect of 20 μg/mL cisplatin on Rad51 foci formation after irradiation was observed in exponentially growing H460 lung tumor cells with a S/G2 content of 51% ± 2%. The decrease of Rad51 foci formation was 52% ± 4% in that model after irradiation with 4 Gy.
We observed a significant decrease of the number of radiation-induced γ-H2AX foci by about 30% in cisplatin-pretreated lymphocytes as a novel mechanism of interaction between cisplatin-containing chemotherapy and radiation. The cisplatin effect on γ-H2AX foci induction after irradiation showed significant interindividual differences, a prerequisite for a predictive assay. The in vivo effect of cisplatin administration given in the clinic within a weekly schedule simultaneously with radiotherapy on radiation-induced γ-H2AX foci formation was as high as the maximum effect seen after in vitro exposure at high cisplatin concentrations over 1 hour. Although the cisplatin effect on γ-H2AX foci remained relatively constant during the first 3 to 4 days after cisplatin exposure, a significant decrease of cisplatin adducts was found in other studies within this time span (22). Thus, the effect of cisplatin on radiation-induced γ-H2AX foci formation must not parallel the concentration of cisplatin adducts and might be saturated at a certain adduct level.
The γ-H2AX foci are formed by phosphorylating the histone H2AX at serine 139 at sites of radiation-induced double-strand break by phosphoinositol 3-kinase–related kinases. Burma et al. (18) found in mouse embryonic fibroblasts from wild type, DNA-dependent protein kinase catalytic subunit (DNA-PKcs)-/-, and ATM-/- mice that ATM is the major kinase involved in the phosphorylation of H2AX, whereas Stiff et al. (19) showed that inactivation of ATM and DNA-PKcs is required to ablate γ-H2AX formation after irradiation. In this study, we were able to inhibit the number of radiation-induced γ-H2AX foci by 23% to 47% using the ATM inhibitors Ku55933, caffeine, and CKG733 and by 32% to 46% using the DNA-dependent protein kinase inhibitors NU7026 and wortmannin. The effect of these kinase inhibitors on radiation-induced γ-H2AX foci formation was therefore of the same magnitude as the cisplatin effect. An increased inhibition of radiation-induced γ-H2AX foci by 60% was seen using ATM and DNA-dependent protein kinase inhibitors. The small molecule compounds NU7027, Ku55933, and CGK733 used in the present study are potent and specific kinase inhibitors for DNA-dependent protein kinase (23), ATM (24), and ATM/ataxia telangiecasia and Rad3 related (25), respectively. The results indicate that ATM and DNA-dependent protein kinase are involved in radiation-induced γ-H2AX phosphorylation in resting human lymphocytes in a partly overlapping and competing manner.
ATM and DNA-dependent protein kinase inhibition but not cisplatin led to a marked increase in the mobile fraction of DNA in a gel electrophoresis assay after a repair incubation of 60 minutes as a measure for nonhomologous end joining. In contrast, cisplatin slightly decreased the mobile fraction of double-stranded DNA immediately after irradiation and after repair incubation for 60 minutes by about 10% in comparison with cisplatin-unexposed lymphocytes. The latter finding is in accordance with that of Groen et al. (26), who also found a decrease in the mobile fraction of double stranded DNA by cisplatin immediately after irradiation in the pulsed-field gel electrophoresis assay. This was interpreted as an effect of cisplatin on the mobile fraction of DNA due to interstrand cross-links and not as reduced induction of double-strand break. It has to be concluded from the gel electrophoresis experiments that cisplatin does not directly impair nonhomologous end joining in lymphocytes in contrast to ATM or DNA-dependent protein kinase inhibition. Direct cisplatin effects on radiation induced ATM activation were not found in this study. This is in accordance with the safety of a concurrent cisplatin and radiation treatments in the clinic. In clinical trials, normal tissue late effects in soft tissues, lung, bone, and spinal cord were not found to be increased after simultaneous cisplatin and radiation therapy in comparison with radiotherapy alone (3, 5).
The observation that cisplatin did not affect double-strand break end joining kinetics in the gel electrophoresis assay despite the marked negative effect on radiation induced γ-H2AX foci formation is in accordance with experiments using H2AX-deficient cells. Celeste et al. (27) did not find an influence of H2AX deficiency on end joining of double-strand break. On the other hand, Turchi et al (28) found that a single cisplatin adduct within a very near range of about 20 bp to the DNA ends of a double-strand break can result in a reduced activation of DNA-dependent protein kinase by inhibition of translocation of the Ku subunits (28). The DNA-dependent protein kinase activity could be gradually decreased with decreasing distance of the cisplatin adducts from the DNA termini (29). However, under the assumption of a completely random distribution of radiation-induced double-strand break, the probability of such a near colocalization of cisplatin adducts and radiation-induced double-strand break ends would be very low (10, 29).
The inhibitors of ATM or DNA-dependent protein kinase directly inhibited radiation-induced γ-H2AX focus formation and also decreased the cisplatin effect on focus formation. However, the cisplatin effect on focus formation was not totally abolished at least after wortmannin, Nu726, and CGK733 incubation. The mechanism by which cisplatin inhibits radiation-induced focus formation remains to be identified. One possible mechanism is perturbation of higher-order chromatin organization by cisplatin cross-links. It has been shown that cisplatin adducts alter the rotational setting of DNA around the histone core (30, 31). Berkovich et al. (32) found a time-dependent loss of histone 2B from the double-strand break site by measuring protein recruitment and loss around double-strand break at defined endogenous sites, indicating nucleosomal disruption. This was paralleled by accumulation of ATM at the double-strand break site measured by chromatin immunoprecipitation assay. Such nucleosomal disruption processes may be altered by chromatin changes introduced by cisplatin cross-links (31).
In the present study, we also found that cisplatin efficiently reduced radiation-induced Rad51 foci formation in PHA-L–stimulated lymphocytes. Rad51 initiates strand invasion and promotes homologous pairing and strand exchange within a regular nucleoprotein filament during homologous recombination, which predominately takes place in the S/G2 phases of the cell cycle (33, 34). Studies on cells lacking H2AX showed that H2AX is not required for Rad51 focus formation (17, 27). A similar cisplatin effect on Rad51 and on γ-H2AX foci formation points to an inhibition of common upstream events. However, γ-H2AX foci measurement is the more sensitive assay for the evaluation of the cisplatin effect than Rad51 foci measurements because of the higher signal-to-background ratio at clinically relevant radiation doses of <2 Gy. Reduction of homologous recombination by a decrease of Rad51 foci formation (35) is a mechanism by which cisplatin could radiosensitize proliferating cells. Thus, synergistic effects of cisplatin and radiation on cell death were observed in some proliferating tumor cell lines in previous studies (36).
In conclusion, cisplatin reduces radiation-induced γ-H2AX foci formation in lymphocytes. Marked inhibition was achieved by cisplatin regimens used in the clinic and was detectable up to day 6 after cisplatin exposure. In addition, cisplatin led to a reduction in Rad51 foci formation after irradiation in proliferating cells. γ-H2AX and Rad51 foci are important events during radiation-induced double-strand break processing. Further studies on the mechanisms of cisplatin-dependent inhibition of irradiation-induced foci formation and the relations between reduced γ-H2AX and Rad51 foci formation and the interaction effects of cisplatin and irradiation on cell death in tumor and normal tissue models are indicated according to the observations of this study.
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