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Purpose: To evaluate the relationship between an estimated integral total body radiation dose delivered and phosphorylated histone H2AX protein (γ-H2AX) foci formation in peripheral blood lymphocytes of cancer patients. Material and methods: γ-H2AX formation was quantified as the mean number of foci per lymphocyte (NmeanH2AX) and the percentage of lymphocytes with ≥n foci. The integrated total body radiation dose was estimated from the dose volume histogram of patient's body corrected for the proportion of the body scanned by computed tomography for 3D treatment planning. Results: There was a strong linear correlation between the mean number of γ-H2AX foci per lymphocyte in the peripheral blood sample and integrated total body radiation dose (r = 0.83, p < 0.0001). The slope of the relationship was dependent on the site of body irradiated. In comparison to chest irradiation with a slope of 8.7 ± 0.8 foci Gy-1, the slopes for brain, upper leg and pelvic sites were significantly shallower by -4.7, -4.3, and -3.8 Gy-1, respectively (p < 0.0001), while the slope for upper abdomen irradiation was significantly larger by 9.1 ± 2.6 Gy-1 (p = 0.0007). There was a slight time effect since the start of radiotherapy on the slopes of the in vivo dose responses leading to shallower slopes (-1.5 ± 0.7 Gy-1, p = 0.03) later (≥10 day) during radiotherapy. After in vitro irradiation, lymphocytes showed 10.41 ± 0.12 foci per Gy with no evidence of inter-individual heterogeneity. Conclusions: γ-H2AX measurements in peripheral lymphocytes after local radiotherapy allow the estimation of the applied integral body dose. The site and time dependence have to be considered. [ABSTRACT FROM AUTHOR]

Lung cancer is difficult to control locally by radiotherapy and is known to have frequently p53 mutations. Previous results have shown that non-small-cell lung cancer (NSCLC) cell lines with nonfunctional p53 have a higher fraction of radiation-induced apoptosis and that apoptosis follows after the release from the G2/M arrest. The aim of the present work was to study whether inhibition of the p53 response in NSCLC cell lines can modulate the G2/M arrest and the induction of apoptosis after ionizing radiation. Antisense oligodeoxynucleotides (As-ODNs) were used to inhibit the p53 response in the cell lines H460 and A549 with functional p53. In addition, H661 with nonfunctional p53 was used. The results have shown that As-ODNs targeting mRNA of p53 and p21 downregulate radiation-induced expression of p53 and p21WAF1/CIP1. Delayed apoptosis (35.7±4.2% in H460, 1.2±0.4% in A549 and 72.2±6.5% in H661) was observed after cell cycle progression beyond the G2 block, either in the late G2 phase of the same cell cycle being irradiated (H661) or in the G1 phase of the subsequent cell cycle (H460, A549). As-p53 significantly decreased the fraction of G2/M-arrested cells in H460 cells and increased radiation-induced apoptosis at 96?hours by 17.9±8.5 and 9.1±3.3% to 53.6±7.4 and 10.8±2.9% in H460 and A549 cells (P<.01), respectively, but had no effect in H661 cells with nonfunctional p53. In addition, As-p21 decreased the fraction of G2-arrested A549 and H460 cells and increased apoptosis by 23.8±5.2 and 31.6±7.3% to 59.4±3.1 and 32.8±7.3%, respectively (P<.01). In conclusion, these data show that radiation-induced G2 arrest is decreased in NSCLC cells and radiation-induced apoptosis is increased when p53-responsive pathways are blocked via As-ODN targeting p53 or p21WAF1/CIP1 mRNA. In view of the fact that p53 and p21 As-ODN had similar effects on radiation-induced apoptosis normalized by their ability to inhibit radiation-induced p21 expression, we concluded that p21 is an important trigger of late ionizing radiation-induced apoptosis.Cancer Gene Therapy (2003) 10, 926-934. doi:10.1038/sj.cgt.7700649 [ABSTRACT FROM AUTHOR]

Lung cancer is difficult to control locally by radiotherapy and is known to have frequently p53 mutations. Previous results have shown that non-small-cell lung cancer (NSCLC) cell lines with nonfunctional p53 have a higher fraction of radiation-induced apoptosis and that apoptosis follows after the release from the G2/M arrest. The aim of the present work was to study whether inhibition of the p53 response in NSCLC cell lines can modulate the G2/M arrest and the induction of apoptosis after ionizing radiation. Antisense oligodeoxynucleotides (As-ODNs) were used to inhibit the p53 response in the cell lines H460 and A549 with functional p53. In addition, H661 with nonfunctional p53 was used. The results have shown that As-ODNs targeting mRNA of p53 and p21 downregulate radiation-induced expression of p53 and p21WAF1/CIP1. Delayed apoptosis (35.7±4.2% in H460, 1.2±0.4% in A549 and 72.2±6.5% in H661) was observed after cell cycle progression beyond the G2 block, either in the late G2 phase of the same cell cycle being irradiated (H661) or in the G1 phase of the subsequent cell cycle (H460, A549). As-p53 significantly decreased the fraction of G2/M-arrested cells in H460 cells and increased radiation-induced apoptosis at 96?hours by 17.9±8.5 and 9.1±3.3% to 53.6±7.4 and 10.8±2.9% in H460 and A549 cells (P<.01), respectively, but had no effect in H661 cells with nonfunctional p53. In addition, As-p21 decreased the fraction of G2-arrested A549 and H460 cells and increased apoptosis by 23.8±5.2 and 31.6±7.3% to 59.4±3.1 and 32.8±7.3%, respectively (P<.01). In conclusion, these data show that radiation-induced G2 arrest is decreased in NSCLC cells and radiation-induced apoptosis is increased when p53-responsive pathways are blocked via As-ODN targeting p53 or p21WAF1/CIP1 mRNA. In view of the fact that p53 and p21 As-ODN had similar effects on radiation-induced apoptosis normalized by their ability to inhibit radiation-induced p21 expression, we concluded that p21 is an important trigger of late ionizing radiation-induced apoptosis.Cancer Gene Therapy (2003) 10, 926-934. doi:10.1038/sj.cgt.7700649 [ABSTRACT FROM AUTHOR]

Purpose: This study determined the effects of cis-diamminedichloroplatinum(II) on radiation-induced foci formation of gamma-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 gamma-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 gamma-H2AX signaling, the consequences of altered gamma-H2AX foci formation on double-strand break end joining, was studied., Results: Cisplatin decreased the number of induced gamma-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 gamma-H2AX and Rad51 foci formation after ionizing radiation. Inhibition of sensing and processing of double-strand break by gamma-H2AX and Rad51 foci formation are important mechanisms by which cisplatin can alter the radiation response.

The T-allele of a common C825T single nucleotide polymorphism (SNP) in the gene GNB3, encoding the G3 subunit of heterotrimeric G-proteins, is associated with a truncated form of the G3 protein that imparts a greater signaling capacity than the alternative C-allele encoding a nontruncated protein. We analyzed the C825T-allele status with regard to disease progression in patients with head and neck squamous cell carcinoma (HNSCC). The prognostic value of the SNP was evaluated in an unselected series of 341 patients treated with curative intent for HNSCC including all tumor stages with different therapeutic regimens. Genotype analysis was done by Pyrosequencing using DNA from paraffin-embedded tissue samples. Genotypes were correlated with relapse-free and overall survival. Proportions of 5-year relapse-free intervals were 62% for CC, 60% for TC, and 42% for TT genotypes. Kaplan-Meier curves revealed a significant genotype-dependent relapse-free interval (P = 0.036). In multivariate analysis with stage, localization, grade, gender, and smoking habits as covariates, GNB3 825T homozygous patients displayed a higher risk for relapse than C825 homozygous patients (TT versus CC, hazard ratio; 95% confidence interval, 1.4-4.8; P = 0.002). The same genotype effect was found for overall survival, TT genotypes were at higher risk for death compared with CC genotypes (hazard ratio, 2.6; 95% confidence interval, 1.6-4.3; P < 0.001), and 5-year survival proportions were 60% for CC, 52% for TC, and 33% for TT. The GNB3 C825T SNP thus represents a host derived prognostic marker in HNSCC, which allows identifying high-risk patients, which could benefit from novel and/or more aggressive therapeutic regimes.

Purpose: In previous studies, we have shown that the T allele of a specific single-nucleotide polymorphism (SNP) in the Galphas gene (T393C) correlates with increased Galphas expression and hence apoptosis. The T allele was associated with a favorable outcome in a variety of human cancers, e.g., carcinoma of the urinary bladder, kidney, and colorectum., Experimental Design: The prognostic value of the T393C SNP was evaluated in an unselected series of patients treated with curative intent for oropharyngeal and hypopharyngeal squamous cell carcinomas, including all tumor stages with different therapeutic regimens. Genotype analysis was done using DNA from paraffin-embedded tissue samples from 202 patients (162 men, 40 women) with a median follow-up of 38 months (1-133 months). The various genotypes were correlated with relapse-free and overall survival., Results: GNAS1 393C homozygous patients displayed a higher risk for disease progression than T393 homozygous patients (hazard ratio CC versus TT, 1.9; 95% confidence interval, 1.1-3.2; P = 0.019). The same genotype effect was observed for overall survival with CC genotypes at higher risk for death compared with TT genotypes (hazard ratio, 1.7; 95% confidence interval, 1.1-2.9; P = 0.015). Multivariate analysis showed that, besides American Joint Committee on Cancer stage, tumor localization, and gender, the T393C polymorphism was an independent prognostic factor for disease progression and death., Conclusion: The T393C SNP could be considered as a genetic marker to predict the clinical course of patients suffering from oropharyngeal and hypopharyngeal cancer.