DNA methylation changes following DNA damage in prostate cancer cells.

Many cancer therapies operate by inducing double-strand breaks (DSBs) in cancer cells, however treatment-resistant cells rapidly initiate mechanisms to repair damage enabling survival. While the DNA repair mechanisms responsible for cancer cell survival following DNA damaging treatments are becoming better understood, less is known about the role of the epigenome in this process. Using prostate cancer cell lines with differing sensitivities to radiation treatment, we analysed the DNA methylation profiles prior to and following a single dose of radiotherapy (RT) using the Illumina Infinium HumanMethylation450 BeadChip platform. DSB formation and repair, in the absence and presence of the DNA hypomethylating agent, 5-azacytidine (5-AzaC), were also investigated using γH2A.X immunofluorescence staining. Here we demonstrate that DNA methylation is generally stable following a single dose of RT; however, a small number of CpG sites are stably altered up to 14 d following exposure. While the radioresistant and radiosensitive cells displayed distinct basal DNA methylation profiles, their susceptibility to DNA damage appeared similar demonstrating that basal DNA methylation has a limited influence on DSB induction at the regions examined. Recovery from DSB induction was also similar between these cells. Treatment with 5-AzaC did not sensitize resistant cells to DNA damage, but rather delayed recruitment of phosphorylated BRCA1 (S1423) and repair of DSBs. These results highlight that stable epigenetic changes are possible following a single dose of RT and may have significant clinical implications for cancer treatment involving recurrent or fractionated dosing regimens.

Novel predictive epigenetic signature for temozolomide in non-G-CIMP glioblastomas.

OBJECTIVE: To identify novel epigenetic signatures that could provide predictive information that is complementary to promoter methylation status of the O-6-methylguanine-DNA methyltransferase (MGMT) gene for predicting temozolomide (TMZ) response, among glioblastomas (GBMs) without glioma-CpGs island methylator phenotype (G-CIMP) METHODS: Different cohorts of primary non-G-CIMP GBMs with genome-wide DNA methylation microarray data were included for discovery and validation of a multimarker signature, combined using a RISK score model. Different statistical analyses and functional experiments were performed for clinical and biological validation. RESULTS: By employing discovery cohorts with radiotherapy (RT) and TMZ versus RT alone and a strict multistep selection strategy, we identified seven CpGs, each of which was significantly correlated with overall survival (OS) of non-G-CIMP GBMs with RT/TMZ, independent of age, MGMT promoter methylation status, and other identified CpGs. A RISK score signature of the 7 CpGs was developed and validated to distinguish non-G-CIMP GBMs with differential survival outcomes to RT/TMZ, but not to RT alone. The interaction analyses also showed differential outcomes to RT/TMZ versus RT alone within the RISK score-based subgroups. The signature could also improve the risk classification by age and MGMT promoter methylation status. Functional experiments showed that HSBP2 appeared to be epigenetically regulated by one identified CpG and was associated with TMZ resistance, but it was not associated with cell proliferation or apoptosis in GBM cell lines. The predictive value of the single CpG methylation of HSBP2 by pyrosequencing was observed in a local cohort of isocitrate dehydrogenase 1 (IDH1) R132H wild-type GBMs. CONCLUSIONS: This novel epigenetic signature might be a promising predictive (but not a general prognostic) biomarker and be helpful for refining the MGMT-based guiding approach to TMZ usage in non-G-CIMP GBMs.

The genome-wide sequence preference of ionising radiation-induced cleavage in human DNA.

For ionising radiation (IR)-induced cellular toxicity, DNA cleavage is thought to be a crucial step. In this paper, the genome-wide DNA sequence preference of gamma radiation-induced cleavage was investigated in purified human DNA. We utilised Illumina short read technology and over 80 million double-strand breaks (DSBs) were analysed in this study. The frequency of occurrence of individual nucleotides at the 50,000 most frequently cleaved sites was calculated and C nucleotides were found to be most prevalent at the cleavage site, followed by G and T, with A being the least prevalent. 5'-C*C and 5'-CC* dinucleotides (where * is the cleavage site) were found to be the present at the highest frequency at the cleavage site; while it was 5'-CC*C for trinucleotides and 5'-GCC*C and 5'-CC*CC for tetranucleotides. The frequency of occurrence of individual nucleotides at the most frequently cleaved sites was determined and the nucleotides in the sequence 5'-GGC*MH (where M is A or C, H is any nucleotide except G) were found to occur most frequently for DNA that was treated with endonuclease IV (to remove blocking 3'-phosphoglycolate termini); and 5'-GSC*MH (where S is G or C) for non-endonuclease IV-treated DNA. It was concluded that GC-rich sequences were preferentially targeted for cleavage by gamma irradiation. This was the first occasion that an extensive examination of the genome-wide DNA sequence preference of IR-induced DSBs has been performed.

Gene hypermethylation in blood leukocytes in humans long term after radiation exposure - Validation set.

Hypermethylation of СpG islands in the promoter regions of several genes with basic protective function in blood leukocytes of individuals exposed to ionizing radiation long time ago (2-46 years), and differential effects of age and radiation exposure on hypermethylation was reported in our previous work. To validate these results, epigenetic modifications were assessed in an independent series of 49 nuclear industry workers from the "Mayak" facility (67-84 years old at sampling) with documented individual accumulated doses from the prolonged external γ-radiation exposure (95.9-409.5 cGy, end of work with radiation:0.3-39 years ago), and in 50 non-exposed persons matched by age. In addition to the genes analyzed before (RASSF1A, p16/INK4A, p14/ARF, GSTP1), four additional loci were analyzed: TP53, ATM, SOD3, ESR1. The frequency of individuals displaying promoter methylation of at least one of the 8 genes (71.4%) was significantly higher in exposed group as compared to the control group (40%), p = .002, OR = 3.75. A significantly elevated frequency of individuals with hypermethylated СpG islands in GSTP1, TP53, SOD3 promoters was revealed among exposed subjects as compared to the control group (p = .012, OR = 8.41; p = .041, OR = 4.02 and p = .009, OR = 3.42, respectively). A similar trend (p = .12, OR = 3.06) was observed for the p16/INK4A gene. As a whole, p16/INK4A and GSTP1 promoter hypermethylation in irradiated subjects from both previously and currently analyzed groups was pronounced. Thus, the direction of the effects was fully confirmed, suggesting the result reproducibility. No statistically significant correlation between promoter methylation and individual radiation dose was found. Further studies are required to create an array of blood epigenetic markers of radiation exposure associating with premature aging and age-related diseases and to accurately evaluate radiation-added effect across the range of doses. SYNTHESIS: The results of studies of epigenetic changes in two independent samples of irradiated subjects indicated the significance of radiation factor in the induction of hypermethylation of CpG islands in gene promoters that is revealed in blood cells years and decades after exposure.

DNA Methylation Patterns in Rat Mammary Carcinomas Induced by Pre- and Post-Pubertal Irradiation.

Several lines of evidence indicate one's age at exposure to radiation strongly modifies the risk of radiation-induced breast cancer. We previously reported that rat mammary carcinomas induced by pre- and post-pubertal irradiation have distinct gene expression patterns, but the changes underlying these differences have not yet been characterized. The aim of this investigation was to see if differences in CpG DNA methylation were responsible for the differences in gene expression between age at exposure groups observed in our previous study. DNA was obtained from the mammary carcinomas arising in female Sprague-Dawley rats that were either untreated or irradiated (γ-rays, 2 Gy) during the pre- or post-pubertal period (3 or 7 weeks old). The DNA methylation was analyzed using CpG island microarrays and the results compared to the gene expression data from the original study. Global DNA hypomethylation in tumors was accompanied by gene-specific hypermethylation, and occasionally, by unique tumor-specific patterns. We identified methylation-regulated gene expression candidates that distinguished the pre- and post-pubertal irradiation tumors, but these represented only 2 percent of the differentially expressed genes, suggesting that methylation is not a major or primary mechanism underlying the phenotypes. Functional analysis revealed that the candidate methylation-regulated genes were enriched for stem cell differentiation roles, which may be important in mammary cancer development and worth further investigation. However, the heterogeneity of human breast cancer means that the interpretation of molecular and phenotypic differences should be cautious, and take into account the co-variates such as hormone receptor status and cell-of-origin that may influence the associations.

Hypermethylation of gene promoters in peripheral blood leukocytes in humans long term after radiation exposure.

Some human genes known to undergo age-related promoter hypermethylation. These epigenetic modifications are similar to those occurring in the course of certain diseases, e.g. some types of cancer, which in turn may also associate with age. Given external genotoxic factors may additionally contribute to hypermethylation, this study was designed to analyzes, using methylation-sensitive polymerase chain reaction (PCR), the CpG island hypermethylation in RASSF1A, CDKN2A (including p16/INK4A and p14/ARF) and GSTP1 promoters in peripheral blood leukocytes of individuals exposed to ionizing radiation long time ago. One hundred and twenty-four irradiated subjects (24-77 years old at sampling: 83 Chernobyl Nuclear Power Plant clean-up workers, 21 nuclear workers, 20 residents of territories with radioactive contamination) and 208 unirradiated volunteers (19-77 years old at sampling) were enrolled. In addition, 74 non-exposed offspring (2-51 years old at sampling) born to irradiated parents were examined. The frequency of individuals displaying promoter methylation of at least one gene in exposed group was significantly higher as compared to the control group (OR=5.44, 95% CI=2.62-11.76, p=3.9×10(-7)). No significant difference was found between the frequency of subjects with the revealed promoter methylation in the group of offspring born to irradiated parents and in the control group. The increase in the number of methylated loci of RASSF1A and p14/ARF was associated with age (ß=0.242; p=1.7×10(-5)). In contrast, hypermethylation of p16/INK4A and GSTP1 genes correlated with the fact of radiation exposure only (ß=0.290; p=1.7×10(-7)). The latter finding demonstrates that methylation changes in blood leukocytes of healthy subjects exposed to radiation resemble those reported in human malignancies. Additional studies are required to identify the dose-response of epigenetic markers specifically associating with radiation-induced premature aging and/or with the development of age-associated cancer and non-cancer diseases.

Fibulin-3 negatively regulates ALDH1 via c-MET suppression and increases γ-radiation-induced sensitivity in some pancreatic cancer cell lines.

Fibulin-3 (FBLN-3) has been postulated to be either a tumor suppressor or promoter depending on the cell type, and hypermethylation of the FBLN-3 promoter is often associated with human disease, especially cancer. We report that the promoter region of the FBLN-3 was significantly methylated (>95%) in some pancreatic cancer cell lines and thus FBLN-3 was poorly expressed in pancreatic cancer cell lines such as AsPC-1 and MiaPaCa-2. FBLN-3 overexpression significantly down-regulated the cellular level of c-MET and inhibited hepatocyte growth factor-induced c-MET activation, which were closely associated with γ-radiation resistance of cancer cells. Moreover, we also showed that c-MET suppression or inactivation decreased the cellular level of ALDH1 isozymes (ALDH1A1 or ALDH1A3), which serve as cancer stem cell markers, and subsequently induced inhibition of cell growth in pancreatic cancer cells. Therefore, forced overexpression of FBLN-3 sensitized cells to cytotoxic agents such as γ-radiation and strongly inhibited the stemness and epithelial to mesenchymal transition (EMT) property of pancreatic cancer cells. On the other hand, if FBLN3 was suppressed in FBLN-3-expressing BxPC3 cells, the results were opposite. This study provides the first demonstration that the FBLN-3/c-MET/ALDH1 axis in pancreatic cancer cells partially modulates stemness and EMT as well as sensitization of cells to the detrimental effects of γ-radiation.

Increased p16 DNA methylation in mouse thymic lymphoma induced by irradiation.

DNA methylation is an important part of epigenetics. In this study, we examined the methylation state of two CpG islands in the promoter of the p16 gene in radiation-induced thymic lymphoma samples. The mRNA and protein levels of P16 were significantly reduced in radiation-induced thymic lymphoma tissue samples. Twenty-three CpG sites of the CpG islands in the p16 promoter region were detected, and the methylation percentages of -71, -63, -239, -29, -38, -40, -23, 46 CpG sites were significantly higher in radiation-induced thymic lymphoma tissue samples than those in matched non-irradiated thymus tissue samples. This study provides new evidence for the methylation state of p16 in the radiation-induced thymic lymphoma samples, which suggests that the methylation of these CpG sites in the p16 promoter may reduce its expression in the thymic lymphoma after irradiation.

Development of TRAIL resistance by radiation-induced hypermethylation of DR4 CpG island in recurrent laryngeal squamous cell carcinoma.

PURPOSE: There are limited therapeutic options for patients with recurrent head and neck cancer after radiation therapy failure. To assess the use of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) as a salvage chemotherapeutic agent for recurrent cancer after radiation failure, we investigated the effect of clinically relevant cumulative irradiation on TRAIL-induced apoptosis. METHODS AND MATERIALS: Using a previously established HN3 cell line from a laryngeal carcinoma patient, we generated a chronically irradiated HN3R isogenic cell line. Viability and apoptosis in HN3 and HN3R cells treated with TRAIL were analyzed with MTS and PI/annexin V-FITC assays. Western blotting and flow cytometry were used to determine the underlying mechanism of TRAIL resistance. DR4 expression was semiquantitatively scored in a tissue microarray with 107 laryngeal cancer specimens. Methylation-specific polymerase chain reaction and bisulfite sequencing for DR4 were performed for genomic DNA isolated from each cell line. RESULTS: HN3R cells were more resistant than HN3 cells to TRAIL-induced apoptosis because of significantly reduced levels of the DR4 receptor. The DR4 staining score in 37 salvage surgical specimens after radiation failure was lower in 70 surgical specimens without radiation treatment (3.03 ± 2.75 vs 5.46 ± 3.30, respectively; P<.001). HN3R cells had a methylated DR4 CpG island that was partially demethylated by the DNA demethylating agent 5-aza-2'-deoxycytidine. CONCLUSION: Epigenetic silencing of the TRAIL receptor by hypermethylation of a DR4 CpG island might be an underlying mechanism for TRAIL resistance in recurrent laryngeal carcinoma treated with radiation.

[Genome-wide screening of CpG island methylation changes induced by low-dose X-ray radiation in mice].

OBJECTIVE: To study the methylation changes in promoter CpG islands induced by low-dose X-ray radiation (LDR). METHODS: Twenty male BALB/c mice were randomly divided into control and fractionated radiation group exposed to 6 MV X-ray for 10 days (0.05 Gy/day). All the mice were sacrificed 2 h after the last radiation on day 10, and blood samples were collected for detecting DNA methylation changes using Roche-NimbleGen mouse DNA methylation 3×720K Promoter Plus CpG Island Array. MeDIP-qPCR was used to further validate the methylation status of specific genes. RESULTS: A total of 811 genes were found to show specific hypermethylation in fractional radiation group as compared with the control group, involving almost all the main biological processes by GO analysis. Eight candidate genes (Rad23b, Tdg, Ccnd1, Ddit3, Llgl1, Rasl11a, Tbx2, and Slc6a15) were confirmed to be hypermethylated in LDR samples by MeDIP-qPCR, consistent with the results of the methylation chip study. CONCLUSION: LDR induces promoter hypermethylation on specific genes, which may contribute to radiation-induced pathogenesis.

[The study of aberrant methylation in blood leukocytes of liquidators of the Chernobyl accident].

The study of aberrant methylation of CpG islands in the promoter regions of genes (P16/CDKN2A, P14/ARF, RASSF1A, GSTP1) in blood leukocytes of liquidators of the Chernobyl accident (n = 83, 38-76 years of age) and control subjects of two groups (n = 48, age ≤ 35 and n = 65, age > 35) was carried out using methylation-sensitive restriction endonuclease analysis followed by PCR. The total number of AciI sites in the analyzed fragments ranged from 2 to 7 for different genes. Only 1 subject (2.1%) from the control group (healthy young individuals, age ≤ 35) has methylation of the studied CpG--dinucleotides of RASSF1A gene. Promoter methylation of at least one of the genes analyzed was observed in 28.92% liquidators and significantly exceeded (p = 0.016) such rate in a one-age (> 35 years of age) control group (12.31%). A significantly elevated frequency (p = 0.023) of individuals with abnormal methylation of GSTP1 gene in the group of liquidators as compared to the control group was revealed. The occurrence of promoter methylation of RASSF1A gene significantly correlated with aging both in the control group (r = 0.214; p = 0.023) and in the liquidators of the Chernobyl accident (r = 0.230; p = 0.036). No similar trend was found for other genes. Multiple regression analysis showed that the growth in the number of methylated loci of a set of genes p16, p14 and GSTP1 is exclusively due to the fact of exposure (OR = 7.32, 95% CI = 2.49-25.83, p-value = 2.7 x 10(-5)). The results obtained demonstrate for the first time the reality of the radiation-induced aberrant methylation of CpG islands in promoters of genes involved in the basic protective, functions of cells in the human body in remote periods after radiation exposure.

[Biologic modulation of ionizing radiation by Toll-like receptors agonists: towards an increase in the therapeutic index of radiotherapy?]. / Modulation biologique des radiations ionisantes par les agonistes des Toll-like receptors : vers une amélioration de l'index thérapeutique de la radiothérapie ?

Toll-like receptors are ubiquitous and very well conserved throughout evolution, with important functions mediating innate and adaptative immunological mechanisms. The importance of these receptors and their agonists has been recently pointed out in immunology and cancerology, although the accurate underlying mechanisms are still under investigation. The association of agonists of these receptors with ionizing radiation has been studied in preclinical experiments with promising results. Part of these compounds is flagellin, which seems to be able to modulate the radiosensitivity of both tumors and healthy tissues.

DNA methylation changes in cells regrowing after fractioned ionizing radiation.

BACKGROUND AND PURPOSE: Repeated exposure to ionizing radiation (IR) can result in adaptive reactions. While DNA methylation changes in adaption to repeated stress exposure are established for a variety of drugs, their role in fractioned ionizing radiation is largely unknown. MATERIAL AND METHODS: MCF7 breast cancer cells were treated 5 times a week with IR in fractions of 2 Gy, resulting in total doses of 10 and 20 Gy. Cells were harvested 48 and 72 h after the last irradiation, as well as after a recovery period of at least 14 d. To identify genes differentially methylated in irradiated versus non-irradiated cells, we used methyl-CpG immunoprecipitation (MCIp) followed by global methylation profiling on CpG island microarrays. RESULTS: MCIp profiling revealed methylation changes in several CpG islands 48 h after FIR with 10 and 20 Gy. Cells receiving a total dose of 10 Gy started regrowing after 14 d and exhibited similar radioresistance as mock-treated cells. Differential methylation of the CpG units associated with FOXC1 (p<0.001) and TRAPPC9 (p<0.001) could be confirmed by time-of-flight mass spectrometry (Sequenom). CONCLUSIONS: In summary, these data indicate that regrowth of MCF7 cells after 10 Gy FIR is associated with locus-specific alterations in DNA methylation.

Detection of changes in DNA methylation induced by low-energy ion implantation in Arabidopsis thaliana.

This study evaluated changes in DNA methylation in Arabidopsis thaliana plants grown from seeds implanted with low-energy N(+) and Ar(+) ions. Methylation-sensitive amplified polymorphism (MSAP) testing revealed altered DNA methylation patterns after ion implantation at doses of 1 × 10(14) to 1 × 10(16) ions/cm(2). Comparison of the MSAP electrophoretic profiles revealed nine types of polymorphisms in ion-implanted seedlings relative to control seedlings, among which four represented methylation events, three represented demethylation events, and the methylation status of two was uncertain. The diversity of plant DNA methylation was increased by low-energy ion implantation. At the same time, total genomic DNA methylation levels at CCGG sites were unchanged by ion implantation. Moreover, a comparison of polymorphisms seen in N(+) ion-implanted, Ar(+) ion-implanted, and control DNA demonstrated that the species of incident ion influenced the resulting DNA methylation pattern. Sequencing of eight isolated fragments that showed different changing patterns in implanted plants allowed their mapping onto variable regions on one or more of the five Arabidopsis chromosomes; these segments included protein-coding genes, transposon and repeat DNA sequence. A further sodium bisulfite sequencing of three fragments also displayed alterations in methylation among either different types or doses of incident ions. Possible causes for the changes in methylation are discussed.

Methyl CpG binding protein 2 (MeCP2) enhances photodimer formation at methyl-CpG sites but suppresses dimer deamination.

Spontaneous deamination of cytosine to uracil in DNA is a ubiquitous source of C→T mutations, but occurs with a half life of ∼50 000 years. In contrast, cytosine within sunlight induced cyclobutane dipyrimidine dimers (CPD's), deaminate within hours to days. Methylation of C increases the frequency of CPD formation at PyCG sites which correlate with C→T mutation hotspots in skin cancers. MeCP2 binds to mCG sites and acts as a transcriptional regulator and chromatin modifier affecting thousands of genes, but its effect on CPD formation and deamination is unknown. We report that the methyl CpG binding domain of MeCP2 (MBD) greatly enhances C=mC CPD formation at a TCmCG site in duplex DNA and binds with equal or better affinity to the CPD-containing duplex compared with the undamaged duplex. In comparison, MBD does not enhance T=mC CPD formation at a TTmCG site, but instead increases CPD formation at the adjacent TT site. MBD was also found to completely suppress deamination of the T=mCG CPD, suggesting that MeCP2 may have the capability to both suppress UV mutagenesis at PymCpG sites as well as enhance it.

Relationship between methylation status of ERCC1 promoter and radiosensitivity in glioma cell lines.

Ionizing radiation represents one of the most important therapies for glioma, a lethal primary brain tumor, while radiotherapy remains a challenge for radiation oncologist because of radioresistance. Radiosensitivity of gliomas determines radiotherapy efficacy. Evidence demonstrated that methylation of CpG Island in the promoter region may result in gene silencing. This study was designed to determine the relationship between methylation status of ERCC1 promoter region and radiosensitivity in glioma cell lines. We investigated the expression levels of ERCC1 transcripts and protein in GBM cell lines. Colony forming experiments was used to measure surviving fraction at 2Gy (SF2) in four human glioma cell lines, MGR1, MGR2, SF767 and T98G. Methylation status in the promoter region of ERCC1 in these glioma cell lines was determined by using bisulphate sequencing and MSP analysis. Radiosensitivity was examined to be heterogeneous in these glioma cell lines. There was a statistical difference in the radiosensitivity between glioma cell lines with and without methylation of ERCC1 gene promoter CpG islands. Furthermore, we promoted ERCC1 expression by 5-azacytidine treatment which resulted in the reduction of radiation-induced cell killing in radiosensitive cell lines. Our data indicate that methylation status of ERCC1 is associated with radiosensitivity in glioma cell lines. It could be used as a new biomarker for predicting the radiosensitivity of human gliomas.

Cyclobutane pyrimidine dimers form preferentially at the major p53 mutational hotspot in UVB-induced mouse skin tumors.

The most prevalent DNA lesion induced by UV irradiation is the cyclobutane pyrimidine dimer (CPD) which forms at positions of neighboring pyrimidines. In mouse skin tumors induced by irradiation with UVB (280-320 nm) lamps or solar UV simulators, a major mutational hotspot occurs at codon 270 (Arg-->Cys) involving a sequence change from 5'-TCGT to 5'-TTGT. We have shown previously that CPD formation by UVB or sunlight is enhanced up to 10-fold at 5'-CCG and 5'-TCG sequences due to the presence of 5-methylcytosine bases. Sequence analysis showed that the CpG at codon 270 is methylated in mouse epidermis at a level of approximately 85%. Irradiation of mouse skin or mouse cells in culture produced the strongest CPD signal within exon 8 at the 5'-TCG sequence which is part of codon 270. Time course experiments showed that CPDs at this particular sequence persist longer than at several neighboring positions. The data suggest that formation of CPDs is responsible for induction of the major p53 mutational hotspot in UV-induced mouse skin tumors.

Formation of 5-formyl-2'-deoxycytidine from 5-methyl-2'-deoxycytidine in duplex DNA by Fenton-type reactions and gamma-irradiation.

5-methyl-2'-deoxycytidine (5-Me-dC) is formed by the enzymatic methylation of dC, primarily in CpG sequences in DNA, and is involved in the regulation of gene expression. In the present study, 5-Me-dC and double-stranded DNA fragments containing 5-Me-dC were either gamma-irradiated or aerobically treated with Fenton-type reagents, Fe(II)-EDTA, Fe(II)-nitrilotriacetic acid, Fe(III)-EDTA-H(2)O(2)-catechol or ascorbic acid-H(2)O(2) under neutral conditions. The formation of 5-formyl-2'-deoxycytidine (5-CHO-dC) was observed upon treatment of both 5-Me-dC and DNA fragments containing 5-Me-dC. The yields of 5-CHO-dC from 5-Me-dC and those of 5-formyl-2'-deoxyuridine from dT were comparable. These results suggest that 5-Me-dC in DNA is as susceptible to oxidation as dT in cells, and raise the possibility that 5-CHO-dC may contribute to the high mutagenic rate observed in CpG sequences in genomic DNA.

p53 mutations in lung cancer following radiation therapy for Hodgkin's disease.

High risks of lung cancer occur after successful treatment of Hodgkin's disease. In addition to tobacco smoking, other risk factors include radiotherapy, chemotherapy, and immunosuppression, although the relative contributions of each are unknown. We conducted p53 mutational spectrum analysis in second lung cancers after radiation therapy for Hodgkin's disease in the Netherlands and in Ontario, Canada. Lung cancer tissues from 11 patients were analyzed by p53 immunohistochemistry and DNA sequence analysis. All were male cigarette smokers, all received radiation therapy, and six also received chemotherapy. The lung cancers occurred 9.8 years (mean) after treatment. Radiation doses to lung lobes that developed the tumors averaged 5.7 Gy (range, 3.7-11.7 Gy). Sequence analysis showed four missense and two silent p53 point mutations in five patients. There were four G:C-->A:T transitions; three of four mutated deoxyguanines occurred on the coding strand, and one was a CpG site. There were two transversions: one G:C-->C:G and one A:T-->C:G. Despite moderate or heavy smoking histories in all patients, the mutational spectrum appears to differ from usual smoking-related lung cancers in which G:C-->T:A transversions predominate. The absence of G:C-->T:A mutations and the prominence of G:C-->A:T transitions, which are characteristic of radiation and oxidative damage, suggest that radiotherapy might have caused some of the p53 mutations. These data illustrate the potential of mutation analysis to determine causes of human cancer. If confirmed in a larger series, these results imply that some radiation-induced cancers can be distinguished from those caused by other factors.