Development of MAPT S305 mutation models exhibiting elevated 4R tau expression, resulting in altered neuronal and astrocytic function.

Due to the importance of 4R tau in the pathogenicity of primary tauopathies, it has been challenging to model these diseases in iPSC-derived neurons, which express very low levels of 4R tau. To address this problem we have developed a panel of isogenic iPSC lines carrying the MAPT splice-site mutations S305S, S305I or S305N, derived from four different donors. All three mutations significantly increased the proportion of 4R tau expression in iPSC-neurons and astrocytes, with up to 80% 4R transcripts in S305N neurons from as early as 4 weeks of differentiation. Transcriptomic and functional analyses of S305 mutant neurons revealed shared disruption in glutamate signaling and synaptic maturity, but divergent effects on mitochondrial bioenergetics. In iPSC-astrocytes, S305 mutations induced lysosomal disruption and inflammation and exacerbated internalization of exogenous tau that may be a precursor to the glial pathologies observed in many tauopathies. In conclusion, we present a novel panel of human iPSC lines that express unprecedented levels of 4R tau in neurons and astrocytes. These lines recapitulate previously characterized tauopathy-relevant phenotypes, but also highlight functional differences between the wild type 4R and mutant 4R proteins. We also highlight the functional importance of MAPT expression in astrocytes. These lines will be highly beneficial to tauopathy researchers enabling a more complete understanding of the pathogenic mechanisms underlying 4R tauopathies across different cell types.

Reporting of serious adverse events during cancer clinical trials to the institutional review board: an evaluation by the research on adverse drug events and reports (RADAR) project.

Global introspection is considered an unreliable method for attribution of causality of serious adverse events (SAEs), yet remains widely used for cancer drug clinical trials. Here, we compare structured case abstraction (SCA) to the routine method for detecting, evaluating, and reporting ADEs during cancer drug clinical trials to an Institutional Review Board (IRB). We obtained all SAE reports (2001-2008) received by one IRB for six clinical trials involving bevacizumab or oxaliplatin for treatment of gastrointestinal cancers. We compared the routine IRB SAE method to SCA for adverse event detection and causality attribution. Of 205 adverse events, 182 events (75%) were not reported; of these, 6 (20%) of 30 SAEs requiring an IRB report were unreported. For the 10 item Naranjo score, the amount of information useful for causality attribution was higher with SCA than the routine method (6.0 vs. 2.4 items, P < .0001). One-fifth of SAEs requiring an IRB report were unreported to the IRB via the routine method. SCA provided more useful information as to whether an SAE was caused by a cancer drug exposure. Our results suggest that SCA may improve SAE detection and the accuracy of attribution of causality during cancer drug clinical trials.

Role of mitochondrial hOGG1 and aconitase in oxidant-induced lung epithelial cell apoptosis.

8-Oxoguanine DNA glycosylase (Ogg1) repairs 8-oxo-7,8-dihydroxyguanine (8-oxoG), one of the most abundant DNA adducts caused by oxidative stress. In the mitochondria, Ogg1 is thought to prevent activation of the intrinsic apoptotic pathway in response to oxidative stress by augmenting DNA repair. However, the predominance of the beta-Ogg1 isoform, which lacks 8-oxoG DNA glycosylase activity, suggests that mitochondrial Ogg1 functions in a role independent of DNA repair. We report here that overexpression of mitochondria-targeted human alpha-hOgg1 (mt-hOgg1) in human lung adenocarcinoma cells with some alveolar epithelial cell characteristics (A549 cells) prevents oxidant-induced mitochondrial dysfunction and apoptosis by preserving mitochondrial aconitase. Importantly, mitochondrial alpha-hOgg1 mutants lacking 8-oxoG DNA repair activity were as effective as wild-type mt-hOgg1 in preventing oxidant-induced caspase-9 activation, reductions in mitochondrial aconitase, and apoptosis, suggesting that the protective effects of mt-hOgg1 occur independent of DNA repair. Notably, wild-type and mutant mt-hOgg1 coprecipitate with mitochondrial aconitase. Furthermore, overexpression of mitochondrial aconitase abolishes oxidant-induced apoptosis whereas hOgg1 silencing using shRNA reduces mitochondrial aconitase and augments apoptosis. These findings suggest a novel mechanism that mt-hOgg1 acts as a mitochondrial aconitase chaperone protein to prevent oxidant-mediated mitochondrial dysfunction and apoptosis that might be important in the molecular events underlying oxidant-induced toxicity.

Kruppel-like factor 4 regulates laminin alpha 3A expression in mammary epithelial cells.

Laminin-5, the major extracellular matrix protein produced by mammary epithelial cells, is composed of three chains (designated alpha3A, beta3, and gamma2), each encoded by a separate gene. Laminin-5 is markedly down-regulated in breast cancer cells. Little is known about the regulation of laminin gene transcription in normal breast cells, nor about the mechanism underlying the down-regulation seen in cancer. In the present study, we cloned the promoter of the gene for the human laminin alpha3A chain (LAMA3A) and investigated its regulation in functionally normal MCF10A breast epithelial cells and several breast cancer cell lines. Using site-directed mutagenesis of promoter-reporter constructs in transient transfection assays in MCF10A cells, we find that two binding sites for Kruppel-like factor 4 (KLF4/GKLF/EZF) are required for expression driven by the LAMA3A promoter. Electrophoretic mobility shift assays reveal absence of KLF4 binding activity in extracts from T47D, MDA-MB 231, ZR75-1, MDA-MB 436, and MCF7 breast cancer cells. Transient transfection of a plasmid expressing KLF4 activates transcription from the LAMA3A promoter in breast cancer cells. A reporter vector containing duplicate KLF4-binding sites in its promoter is expressed at high levels in MCF10A cells but at negligible levels in breast cancer cells. Thus, KLF4 is required for LAMA3A expression and absence of laminin alpha3A in breast cancer cells appears, at least in part, attributable to the lack of KLF4 activity.

Asbestos causes apoptosis in alveolar epithelial cells: role of iron-induced free radicals.

Asbestos causes asbestosis and malignancies by mechanisms that are not fully understood. Alveolar epithelial cell (AEC) injury by iron-induced reactive oxygen species (ROS) is one important mechanism. To determine whether asbestos causes apoptosis in AECs, we exposed WI-26 (human type I-like cells), A549 (human type II-like cells), and rat alveolar type II cells to amosite asbestos and assessed apoptosis by terminal deoxynucleotidyl transferase-mediated deoxyuridine-5'-triphosphate-biotin nick end labeling (TUNEL) staining, nuclear morphology, annexin V staining, DNA nucleosome formation, and caspase 3 activation. In contrast to control medium and TiO2, amosite asbestos and H2O2 each caused AEC apoptosis. A role for iron-catalyzed ROS was suggested by the finding that asbestos-induced AEC apoptosis and caspase 3 activation were each attenuated by either an iron chelator (phytic acid and deferoxamine) or a.OH scavenger (dimethyl-thiourea, salicylate, and sodium benzoate) but not by iron-loaded phytic acid. To determine whether asbestos causes apoptosis in vivo, rats received a single intratracheal instillation of amosite (5 mg) or normal saline solution, and apoptosis in epithelial cells in the bronchoalveolar duct regions was assessed by TUNEL staining. One week after exposure, amosite asbestos caused a 3-fold increase in the percentage of apoptotic cells in the bronchoalveolar duct regions as compared with control (control, 2.1% +/- 0.35%; asbestos, 7.61% +/- 0.15%; n = 3). However, by 4 weeks the number of apoptotic cells was similar to control. We conclude that asbestos-induced pulmonary toxicity may partly be caused by apoptosis in the lung epithelium that is mediated by iron-catalyzed ROS and caspase 3 activation.

Loss of cyclin D2 expression in the majority of breast cancers is associated with promoter hypermethylation.

Cyclin D2 is a member of the D-type cyclins, implicated in cell cycle regulation, differentiation, and malignant transformation. It was noted previously that cyclin D2 is not expressed in the majority of breast cancer cell lines, whereas abundant expression was detected in finite life span human mammary epithelial cells. By reverse transcription-PCR and Western blot analysis, we extended this finding to primary breast carcinomas and show that the majority of these tumors lack expression of cyclin D2 mRNA (18 of 24) and protein (10 of 13). In contrast, both luminal and myoepithelial subpopulations of normal breast tissues expressed cyclin D2. Hypermethylation of the CpG island in the promoter was detected by methylation-specific PCR in nearly half of the breast cancers (49 of 106) and was associated with silencing of cyclin D2 gene expression. Promoter hypermethylation was also detected in ductal carcinoma in situ, suggesting that loss of cyclin D2 expression is an early event in tumorigenesis. Our results suggest that loss of cyclin D2 expression is associated with the evolution of breast cancer.

Cigarette smoke and asbestos activate poly-ADP-ribose polymerase in alveolar epithelial cells.

BACKGROUND: Cigarette smoke augments asbestos-induced bronchogenic carcinoma in a synergistic manner by mechanisms that are not established. One important mechanism may involve alveolar epithelial cell (AEC) injury resulting from oxidant-induced DNA damage that subsequently activates poly (ADP-ribose) polymerase (PARP), an enzyme involved in DNA repair that can deplete cellular energy stores. We previously showed that whole aqueous cigarette smoke extracts (CSE) augment amosite asbestos-induced DNA damage and cytotoxicity to cultured AEC in part by generating iron-induced free radicals. We hypothesized that CSE increase asbestos-induced AEC injury by triggering PARP activation resulting from DNA damage caused by iron-induced free radicals. METHODS: Aqueous CSE were prepared fresh on the day of each experiment. PARP activity in WI-26 (a type I-like cell line) and A549 (a type II-like cell line) cells was assessed by the uptake of labeled NAD over 4 hours and confirmed on the basis of the reduction of PARP levels in the presence of a PARP inhibitor, 3-aminobenzamide (3-ABA). Cell survival was assessed by trypan blue dye exclusion. RESULTS: Hydrogen peroxide (H2O2; 1-250 microM), CSE (0.4-10 vol%), and amosite asbestos (5-250 micrograms/cm2) each caused PARP activation in WI-26 and A549 cells. The combination of asbestos (5 micrograms/cm2) and CSE (0.04-10%) induced WI-26 and A549 cell PARP activation without evidence of synergism. 3-ABA significantly attenuated WI-26 and A549 cell PARP activity and cell death after exposure to H2O2, CSE, and asbestos. Phytic acid, an iron chelator, catalase, and superoxide dismutase each decreased WI-26 cell PARP activation caused by asbestos and CSE. CONCLUSIONS: CSE and asbestos induced PARP activation in cultured AEC in a nonsynergistic manner. These data provide further support that asbestos and cigarette smoke are genotoxic to relevant lung target cells and that iron-induced free radicals in part cause these effects.

Wilms' tumor suppressor gene (WT1) is expressed in primary breast tumors despite tumor-specific promoter methylation.

We analyzed Wilms' tumor suppressor 1 (WT1) expression and its regulation by promoter methylation in a panel of normal breast epithelial samples and primary carcinomas. Contrary to previous reports, WT1 protein was strongly expressed in primary carcinomas (27 of 31 tumors) but not in normal breast epithelium (1 of 20 samples). Additionally, the WT1 promoter was methylated in 6 of 19 (32%) primary tumors, which nevertheless expressed WT1. The promoter is not methylated in normal epithelium. Thus, although tumor-specific methylation of WT1 is established in primary breast cancer at a low frequency, other transcriptional regulatory mechanisms appear to supercede its effects in these tumors. Our results demonstrate expression of WT1 in mammary neoplasia, and that WT1 may not have a tumor suppressor role in breast cancer.

Breast cancer-specific expression of the Candida albicans cytosine deaminase gene using a transcriptional targeting approach.

We constructed a series of adenoviral (Ad) vectors that express the Candida albicans cytosine deaminase (CD) suicide gene under the transcriptional control of either the human alpha-lactalbumin (ALA) or ovine beta-lactoglobulin (BLG) promoter (Ad.ALA.CD and Ad.BLG.CD, respectively). The Ad.ALA.CD and the Ad.BLG.CD vectors converted the prodrug 5-fluorocytosine (5-FC) to the toxic nucleotide analog 5-fluorouracil in a breast cancer cell-specific manner, with a conversion rate of 40% and 52% in T47D cells and 50% and 41% in MCF7 cells, respectively. No significant conversion (< or =3%) was observed in an immortalized nontumorigenic breast epithelial cell line (MCF10A) and a human osteosarcoma cell line (U2OS). Adenovirus vector-based prodrug conversion of the 5-FC in T47D and MCF7 in the presence of 1 mg/mL of 5-FC led to cytotoxicity that resulted in a nearly complete cell death (> or =90%) after 5 days, whereas MCF10A and U2OS cells remained resistant (< or =10%). Nude mice harboring T47D-derived breast tumors that were injected intratumorally (i.t.) with therapeutic adenovirus vectors at a dose of 2 x 10(8) plaque-forming units and treated systemically with 5-FC at a concentration of 500 mg/kg/day showed a marked reduction in tumor mass within 30 days when compared with animals that received vector alone. Animal survival was significantly prolonged after 72 days in mice treated with therapeutic vectors in conjunction with prodrug when compared with control animals. These preclinical data are sufficiently promising to warrant further studies of this transcriptional targeting approach to breast cancer treatment.

Inhibition of laminin-5 production in breast epithelial cells by overexpression of p300.

The transcriptional coactivator p300 is essential for normal embryonic development and cellular differentiation. We have been studying the role of p300 in the transcription of a variety of genes, and we became interested in the role of this coactivator in the transcription of genes important in breast epithelial cell biology. From MCF-10A cells (spontaneously immortalized, nontransformed human breast epithelial cells), we developed cell lines that stably overexpress p300. These p300-overexpressing cells displayed reduced adhesion to culture dishes and were found to secrete an extracellular matrix deficient in laminin-5. Laminin-5 is the major extracellular matrix component produced by breast epithelium. Immunofluorescence studies, as well as experiments using normal matrix, confirmed that the decreased adhesion of p300-overexpressing cells is due to laminin-5-deficient extracellular matrix and not due to loss of laminin-5 receptors. Northern blots revealed markedly decreased levels of expression of two of the genes (designated LAMA3 and LAMC2) encoding the alpha3 and gamma2 chains of the laminin-5 heterotrimer in the cells that overexpress p300, whereas LAMB3 mRNA, encoding the third or beta3 chain of laminin-5, was not markedly reduced. Transient transfection experiments with a vector containing a murine LAMA3 promoter demonstrate that overexpressing p300 down-regulates the LAMA3 promoter. In summary, overexpression of p300 leads to down-regulation of laminin-5 production in breast epithelial cells, resulting in decreased adhesion.

Regulation of expression of N-methylpurine DNA glycosylase in human mammary epithelial cells: role of transcription factor AP-2.

The DNA repair enzyme, N-methylpurine DNA glyclosylase (MPG), is overexpressed in breast cancer as compared with its expression in normal breast epithelial cells. In an effort to determine the mechanism responsible for this difference in expression, we studied rates and regulation of transcription of the MPG gene in normal (HMEC), spontaneously immortalized (MCF10A), and malignant (T47D) mammary epithelial cells. Steady state levels of MPG mRNA are 3-4-fold greater in T47D cells than in MCF10A cells. Nuclear "run-off" transcription measurements revealed MPG transcription rates to be approximately 3-fold greater in the tumor cells than in normal cells. Characterization of the MPG promoter by deletion analysis and transient transfection experiments revealed that all basal promoter activity resided between nucleotides -227 and -81 upstream from the ATG translation start site. Constructs containing this region were expressed at 4-fold greater levels when transfected into malignant T47D cells (56 x baseline) than in MCF10A cells (14 x baseline). Computer database analysis of the region of nucleotides -227 to -81 revealed multiple overlapping Sp1 consensus binding sites and two overlapping consensus AP-2 binding sites located between bases -181 and -169. Electrophoretic mobility shift assays indicated that while Sp1 bound this region of the promoter, nuclear extracts from both cell types contained equal Sp1 binding activity. In contrast, AP-2 binding activity was significantly greater in T47D cells, and Western blots confirmed increased AP-2 protein levels in these cells. Cotransfection into MCF10A cells of the MPG promoter construct and an AP-2 expression plasmid increased MPG promoter activity 2.1-fold. Cotransfection of a dominant negative mutant of AP-2 into T47D cells reduced the extent of MPG promoter-driven transcription by 50%. To investigate the functional significance of the two overlapping AP-2 consensus binding sites, each site was mutated separately. Mutation of the upstream site decreased promoter activity by 15%, but mutation of the downstream site decreased promoter activity by 45% and abolished AP-2 binding to the promoter sequence. These data suggest that AP-2 is important in regulating MPG expression in breast cancer cells, and that the increased amount of AP-2 in these cells plays a major role in directing the increased expression of MPG.

Adenovirus-mediated tissue-targeted expression of the HSVtk gene for the treatment of breast cancer.

In an effort to develop a genetic therapy for the treatment of breast cancer, we constructed adenoviral vectors containing either the beta-galactosidase (beta-gal) reporter gene or the herpes simplex thymidine kinase (HSVtk) suicide gene driven by breast tissue-specific promoters. We utilized upstream regulatory sequences from either the human alpha-lactalbumin (hALA) gene, or the ovine beta-lactoglobulin (oBLG) gene in these vector constructs to target expression of heterologous genes transcriptionally to breast cancer cells both in vitro and in vivo. Data derived from breast tissue-specific reporter vectors in vitro demonstrate that expression from the hALA and oBLG promoters are indeed specific for breast cells (T47D, MCF-7, ZR75-1) when compared with non-breast cells (U2OS, HeLa). Moreover, these vectors displayed tumor cell specificity when compared with the normal MCF-10A breast cell line. These vectors also displayed breast tissue specificity when injected systemically (i.v.) into lactating Balb/c mice, which suggests that these promoters maintain their tissue-specific expression pattern within the context of the adenoviral genome in vivo. Tumors, derived from T47D human breast cancer cells, were established in nude mice and injected with either the tissue-specific reporter or suicide vectors. Results from tumors injected (i.t.) with reporter adenoviruses demonstrate that these promoters are active in T47D cells when grown as established tumors and we observed a marked regression of tumors injected with suicide vectors and treated systemically with gancyclovir (150 mg/kg/day) when compared with control animals. Moreover, mouse survival was prolonged after 35 days in mice undergoing therapy with the suicide vectors in conjunction with gancyclovir when compared with the control animals. These data suggest that the transcriptionally targeted hALA or oBLG driven expression of the HSVtk gene may be a feasible therapy for the treatment of human breast cancer.

Cigarette smoke augments asbestos-induced alveolar epithelial cell injury: role of free radicals.

Cigarette smoke augments asbestos-induced bronchogenic carcinoma by mechanisms that are not established. Alveolar epithelial cell (AEC) injury due to oxidant-induced DNA damage and depletion of glutathione (GSH) and adenosine triphosphate (ATP) may be one important mechanism. We previously showed that amosite asbestos-induces hydroxyl radical production and DNA damage to cultured AEC and that phytic acid, an iron chelator, is protective. We hypothesized that whole cigarette smoke extracts (CSE) augment amosite asbestos-induced AEC injury by generating iron-induced free radicals that damage DNA and reduce cellular GSH and ATP levels. Asbestos or CSE each caused dose-dependent toxicity to AEC (WI-26 and rat alveolar type I-like cells) as assessed by 51chromium release. The combination of asbestos (5 microg/cm2) and CSE (0.O1-0.1%) caused synergistic injury whereas higher doses of each agent primarily had an additive toxic effect. Asbestos (5 microg/cm2) augmented CSE-induced (0.01-1.0%) AEC DNA damage over a 4 h exposure period as assessed by an alkaline unwinding, ethidium bromide fluorometric technique. These effects were synergistic in A549 cells and additive in WI-26 cells. Asbestos (5 microg/cm2) and CSE (0.5-1.0%) reduced A549 and WI-26 cell GSH levels as assessed spectrophotometrically and ATP levels as assessed by luciferin/luciferase chemiluminescence but a synergistic interaction was not detected. Phytic acid (500 microM) and catalase (100 microg/ml) each attenuated A549 cell DNA damage and depletion of ATP caused by asbestos and CSE. However, neither agent attenuated WI-26 cell DNA damage nor the reductions in GSH levels in WI-26 and A549 cells exposed to asbestos and CSE. We conclude that CSE enhance asbestos-induced DNA damage in cultured alveolar epithelial cells. These data provide additional support that asbestos and cigarette smoke are genotoxic to relevant target cells in the lung and that iron-induced free radicals may in part cause these effects.

Altered expression of the DNA repair protein, N-methylpurine-DNA glycosylase (MPG) in breast cancer.

We examined expression of N-methylpurine-DNA glycosylase (MPG), a DNA repair enzyme that removes N-alkylpurine damage, in normal, malignant, and immortalized breast epithelial cells, and breast cancer cell lines (MDA-MB-231, MCF7, T47D). Northern analysis showed increased expression in cancer versus normal breast epithelial cells (2-24-fold). Southern blots revealed no gene amplification or polymorphisms. Immunofluorescence, immunohistochemistry, and Western blot analysis demonstrated increased MPG protein expression in the tumor cells that correlated with elevated glycosylase activity. Since MPG overexpression has been shown to be paradoxically associated with increased susceptibility to DNA damage, up-regulation of this gene may suggest a functional role in breast carcinogenesis.

Mapping of ER gene CpG island methylation-specific polymerase chain reaction.

Southern analysis has shown that DNA from 25% of primary estrogen receptor (ER) alpha-negative breast tumors displays aberrant methylation at one site within the ER gene CpG island. To examine more sites and increase sensitivity, we developed a methylation-specific PCR assay to map methylation of the entire ER CpG island. The island was unmethylated in normal breast tissue and ER-positive breast cancer cell lines, but extensively methylated in all ER-negative cell lines and breast tumors examined. In addition, some of the ER-positive/progesterone receptor-negative and ER-positive/progesterone receptor-positive tumors (about 70% and 35%, respectively) displayed methylation of the ER CpG island, suggesting that this heterogeneity within tumor cell populations could potentially shed light on the etiology of ER-negative recurrent tumors arising from ER-positive tumors.

Methylation of the HIC-1 candidate tumor suppressor gene in human breast cancer.

HIC-1 (hypermethylated in cancer) is a candidate tumor suppressor gene which is located at 17p13.3, a region which frequently undergoes allelic loss in breast and other human cancers. HIC-1 is proposed to be commonly inactivated in human cancers by hypermethylation of a normally unmethylated dense CpG island which encompasses the entire gene. To study whether HIC-1 inactivation may be important to the development of breast cancer, we first measured methylation of the HIC-1 gene in normal breast ductal tissues from microdissected frozen breast tissues and from epithelial cells purified from mammoplasty specimens. Surprisingly, in all normal breast ductal tissues we found approximately equal amounts of densely methylated HIC-1 and completely unmethylated HIC-1. This is in contrast to most normal tissues, in which all copies of HIC-1 are completely unmethylated. We then evaluated 39 primary breast cancer tissues and found virtually complete methylation of the HIC-1 gene in 26 (67%) of the cases. We also found loss of heterozygosity at the telomeric portion of chromosomal arm 17p in 22 of the 26 cases with strongly methylated HIC-1, suggesting that loss of an unmethylated HIC-1 allele may contribute to the inactivation of HIC-1 in cells with a pre-existing methylated allele. Finally, by RNase protection analysis, HIC-1 was found to be expressed in microdissected normal breast ductal tissues and unmethylated tumors but not in tumors with hypermethylation of the HIC-1 gene. These results indicate that hypermethylation of HIC-1 and associated loss of HIC-1 expression is common in primary breast cancer. Furthermore, the HIC-1 gene is densely methylated in approximately one-half of the alleles in normal breast epithelium, which may predispose this tissue to inactivation of this gene by loss of heterozygosity.

Influence of oxygen radical injury on DNA methylation.

One of the most prevalent products of oxygen radical injury in DNA is 8-hydroxyguanosine. Cells must be able to withstand damage by oxygen radicals and possess specific repair mechanisms that correct this oxidative lesion. However, when these defenses are oversaturated, such as under conditions of high oxidative stress, or when repair is inefficient, the miscoding potential of this lesion can result in mutations in the mammalian genome. In addition to causing genetic changes, active oxygen species can lead to epigenetic alterations in DNA methylation, without changing the DNA base sequence. Such changes in DNA methylation patterns can strongly affect the regulation of expression of many genes. Although DNA methylation patterns have been found to be altered during carcinogenesis, little is known about the mechanism(s) that produce this loss of epigenetic controls of gene expression in tumors. Replacement of guanine with the oxygen radical adduct 8-hydroxyguanine profoundly alters methylation of adjacent cytosines, suggesting a role for oxidative injury in the formation of aberrant DNA methylation patterns during carcinogenesis. In this paper, we review both the genetic and epigenetic mechanisms of oxidative DNA damage and its association with the carcinogenic process, with special emphasis on the influence of free radical injury on DNA methylation.