The level of active DNA demethylation compounds in leukocytes and urine samples as potential epigenetic biomarkers in breast cancer patients.

The active DNA demethylation process, which involves TET proteins, can affect DNA methylation pattern. TET dependent demethylation results in DNA hypomethylation by oxidation 5-methylcytosine (5-mC) to 5-hydroxymethylcytosine (5-hmC) and its derivatives. Moreover, TETs' activity may be upregulated by ascorbate. Given that aberrant DNA methylation of genes implicated in breast carcinogenesis may be involved in tumor progression, we wanted to determine whether breast cancer patients exert changes in the active DNA demethylation process. The study included blood samples from breast cancer patients (n = 74) and healthy subjects (n = 71). We analyzed the expression of genes involved in the active demethylation process (qRT-PCR), and 5-mC and its derivatives level (2D-UPLC MS/MS). The ascorbate level was determined using UPLC-MS. Breast cancer patients had significantly higher TET3 expression level, lower 5-mC and 5-hmC DNA levels. TET3 was significantly increased in luminal B breast cancer patients with expression of hormone receptors. Moreover, the ascorbate level in the plasma of breast cancer patients was decreased with the accompanying increase of sodium-dependent vitamin C transporters (SLC23A1 and SLC23A2). The presented study indicates the role of TET3 in DNA demethylation in breast carcinogenesis.

Intracellular ascorbate is a safe-guard and/or reservoir for plasma vitamin C in prostate cancer patients undergoing radiotherapy.

Prostate cancer (PC) represents one of the most common cancer types worldwide and many patients suffering from this kind of cancer are treated with radiotherapy (RTH). Ionizing irradiation is closely associated with reactive oxygen species (ROS) production and oxidative stress. Over the years the role of vitamin C (VC) in cancer prevention has been highlighted as it may be mediated by its ability to neutralize pro-carcinogenic ROS. However, the debate concerning the presence of VC in blood and its beneficial effect on the survival of cancer patients is inconsistent and controversial. To our best knowledge until recently there have been no studies concerning such a role of intracellular VC (iVC). In the present study, blood and intracellular concentrations of vitamin C were analyzed along with the level of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), as an established marker of the stress condition, in leukocytes of PC patients during the course of radiotherapy. The level of intracellular vitamin C significantly decreased in PC patients in comparison with the healthy group, while there were no differences in blood VC. It was observed that a sub-group of the PC patients reacted to RTH decreasing VC in leukocytes (group A), while the other sub-group acted the other way round, significantly increasing its level (group B). Under stressful conditions (RTH) leukocytes react in two different ways. Both ways are in good agreement with two well recognized functions, proposed for iVC; it may serve as a save factor, to protect the cellular DNA, increasing its concentration inside the cell (group B), and as a reservoir decreasing the VC level inside leukocytes and releasing VC into the plasma to rescue its physiological level (group A). It was also demonstrated that there was a relationship between the level of 8-oxodG in leukocytes' DNA and the markers of RTH toxicity.

Unprecedentedly High Level of Intracellular Vitamin C and DNA Epigenetic Marks in Prostate: Relevant for Male Fertility?

BACKGROUND/AIMS: Seminal plasma composition is affected by the physiological state of the prostate, the major male reproductive gland. Semen components, like vitamin C, can modulate sperm function. Vitamin C is an effective scavenger of free radicals and is an essential component of enzymes such as TET proteins involved in the DNA demethylation process. In the present study, a broad range of parameters which may influence the metabolic state of the prostate gland were analysed including blood and prostate tissue vitamin C, epigenetic DNA modifications and 8-oxo-7,8-dihydro-2'-deoxyguanosine in DNA of leukocytes and prostate tissues. METHODS: The experimental material were tissue samples from patients with benign prostatic hyperplasia (BPH), normal/marginal prostate tissues from prostate cancer patients, leukocytes from healthy donors, and blood plasma from BPH patients and healthy donors. We applied ultra-performance liquid chromatography methods with mass spectrometry and/or UV detection. RESULTS: We found an unprecedentedly high level of intracellular vitamin C in all analysed prostatic tissues (benign prostatic hyperplasia and normal, marginal ones), a value much higher than in leukocytes and most human tissues. DNA epigenetic patterns in prostate cells are similar to other soft tissues like the colon, however, its uniqueness is the unprecedentedly high level of 5-(hydroxymethyl)-2'-deoxyuridine and a significant increase in 5-formyl-2'-deoxycytidine value compared to aforementioned tissues. Moreover, the level of 8-oxo-7,8-dihydro-2'-deoxyguanosine, an established marker of oxidative stress, is significantly higher in prostate tissues than in leukocytes and many previously studied soft tissues. CONCLUSION: Our results pointed out that prostatic vitamin C (regarded as the main supplier of the vitamin C to seminal plasma) and the DNA modifications (which may be linked to the regeneration of prostate epithelium) may play important role to maintain the prostate health.

The urinary excretion of epigenetically modified DNA as a marker of pediatric ALL status and chemotherapy response.

The active DNA demethylation process may be linked to aberrant methylation and may be involved in leukemogenesis. We investigated the role of epigenetic DNA modifications in childhood acute lymphoblastic leukemia (ALL) diagnostics and therapy monitoring. We analyzed the levels of 5-methyl-2'-deoxycytidine (5-mdC) oxidation products in the cellular DNA and urine of children with ALL (at diagnosis and during chemotherapy, n = 55) using two-dimensional ultra-performance liquid chromatography with tandem mass spectrometry (2D UPLC-MS/MS). Moreover, the expression of Ten Eleven Translocation enzymes (TETs) at the mRNA and protein levels was determined. Additionally, the ascorbate level in the blood plasma was analyzed. Before treatment, the ALL patients had profoundly higher levels of the analyzed modified DNA in their urine than the controls. After chemotherapy, we observed a statistically significant decrease in active demethylation products in urine, with a final level similar to the level characteristic of healthy children. The level of 5-hmdC in the DNA of the leukocytes in blood of the patient group was significantly lower than that of the control group. Our data suggest that urinary excretion of epigenetic DNA modification may be a marker of pediatric ALL status and a reliable marker of chemotherapy response.

The potential influence of breast cancer estrogen receptors' distribution on active DNA demethylation.

Alterations in DNA methylation may cause disturbances in regulation of gene expression, including drug metabolism and distribution. Moreover, many cancers, including breast cancer, are characterized by DNA hypomethylation and a decreased 5-hydroxymethylcytosine level. The abnormal cell growth found in breast carcinoma might be the result of impaired up-regulation of breast cancer receptors. Receptors' expression in breast cancer determines clinical outcome, and it is possible that they lead to different DNA methylation patterns. Excessive steroid exposure can affect DNA methylation by promoting demethylation of CpG islands in promoter regions of genes, and hence may have an impact on promotion and progression of breast cancer cells. Tamoxifen, as a leading drug in breast cancer hormone therapy, has an ability to act like estrogen or antiestrogen depending on the type and localization of the breast cancer receptor. Further studies are needed to determine whether tamoxifen, similarly to steroids, may evoke changes in methylation pattern.

In vivo evidence of ascorbate involvement in the generation of epigenetic DNA modifications in leukocytes from patients with colorectal carcinoma, benign adenoma and inflammatory bowel disease.

BACKGROUND: A characteristic feature of malignant cells, such as colorectal cancer cells, is a profound decrease in the level of 5-hydroxymethylcytosine, a product of 5-methylcytosine oxidation by TET enzymes. Recent studies showed that ascorbate may upregulate the activity of TET enzymes in cultured cells and enhance formation of their products in genomic DNA. METHODS: The study included four groups of subjects: healthy controls (n = 79), patients with inflammatory bowel disease (IBD, n = 51), adenomatous polyps (n = 67) and colorectal cancer (n = 136). The list of analyzed parameters included (i) leukocyte levels of epigenetic DNA modifications and 8-oxo-7,8-dihydro-2'-deoxyguanosine, a marker of oxidatively modified DNA, determined by means of isotope-dilution automated online two-dimensional ultra-performance liquid chromatography with tandem mass spectrometry, (ii) expression of TET mRNA measured with RT-qPCR, and (iii) chromatographically-determined plasma concentrations of retinol, alpha-tocopherol and ascorbate. RESULTS: Patients from all groups presented with significantly lower levels of 5-methylcytosine and 5-hydroxymethylcytosine in DNA than the controls. A similar tendency was also observed for 5-hydroxymethyluracil level. Patients with IBD showed the highest levels of 5-formylcytosine and 8-oxo-7,8-dihydro-2'-deoxyguanosine of all study subjects, and individuals with colorectal cancer presented with the lowest concentrations of ascorbate and retinol. A positive correlation was observed between plasma concentration of ascorbate and levels of two epigenetic modifications, 5-hydroxymethylcytosine and 5-hydroxymethyluracil in leukocyte DNA. Moreover, a significant difference was found in the levels of these modifications in patients whose plasma concentrations of ascorbate were below the lower and above the upper quartile for the control group. CONCLUSIONS: These findings suggest that deficiency of ascorbate in the blood may be a marker of its shortage in other tissues, which in turn may correspond to deterioration of DNA methylation-demethylation. These observations may provide a rationale for further research on blood biomarkers of colorectal cancer development.

Characteristic profiles of DNA epigenetic modifications in colon cancer and its predisposing conditions-benign adenomas and inflammatory bowel disease.

Background: Active demethylation of 5-methyl-2'-deoxycytidine (5-mdC) in DNA occurs by oxidation to 5-(hydroxymethyl)-2'-deoxycytidine (5-hmdC) and further oxidation to 5-formyl-2'-deoxycytidine (5-fdC) and 5-carboxy-2'-deoxycytidine (5-cadC), and is carried out by enzymes of the ten-eleven translocation family (TETs 1, 2, 3). Decreased level of epigenetic DNA modifications in cancer tissue may be a consequence of reduced activity/expression of TET proteins. To determine the role of epigenetic DNA modifications in colon cancer development, we analyzed their levels in normal colon and various colonic pathologies. Moreover, we determined the expressions of TETs at mRNA and protein level.The study included material from patients with inflammatory bowel disease (IBD), benign polyps (AD), and colorectal cancer (CRC). The levels of epigenetic DNA modifications and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) in examined tissues were determined by means of isotope-dilution automated online two-dimensional ultraperformance liquid chromatography with tandem mass spectrometry (2D-UPLC-MS/MS). The expressions of TET mRNA were measured with RT-qPCR, and the expressions of TET proteins were determined immunohistochemically. Results: IBD was characterized by the highest level of 8-oxodG among all analyzed tissues, as well as by a decrease in 5-hmdC and 5-mdC levels (at a midrange between normal colon and CRC). AD had the lowest levels of 5-hmdC and 5-mdC of all examined tissues and showed an increase in 8-oxodG and 5-(hydroxymethyl)-2'-deoxyuridine (5-hmdU) levels. CRC was characterized by lower levels of 5-hmdC and 5-mdC, the lowest level of 5-fdC among all analyzed tissues, and relatively high content of 5-cadC. The expression of TET1 mRNA in CRC and AD was significantly weaker than in IBD and normal colon. Furthermore, CRC and AD showed significantly lower levels of TET2 and AID mRNA than normal colonic tissue. Conclusions: Our findings suggest that a complex relationship between aberrant pattern of DNA epigenetic modification and cancer development does not depend solely on the transcriptional status of TET proteins, but also on the characteristics of premalignant/malignant cells. This study showed for the first time that the examined colonic pathologies had their unique epigenetic marks, distinguishing them from each other, as well as from normal colonic tissue. A decrease in 5-fdC level may be a characteristic feature of largely undifferentiated cancer cells.

The role of vitamin C in epigenetic regulation.

Vitamin C (L-ascorbic acid) is a micronutrient best known for its anti-scurvy activity in humans. Vitamin C is involved in many biological processes involving enzymatic reactions that are catalyzed by members of dioxygenases which use Fe(II) and 2-oxoglutarate as a co-substrate.The article reviews recent data that suggest the involvement of ascorbate in dioxygenases catalyzed chromatin and DNA modifications which thereby contribute to epigenetic regulation. Concerning chromatin modification, the dioxygenases are involved in distinct demethylation reactions with varying specificity for the position of the lysine on the target histone. TET hydroxylases catalyse the oxidation of methyl groups in the 5 position of cytosine in DNA yielding 5-hydroxymethylcytosine, while further iterative oxidation reactions results in the formation of 5-formylcytosine and 5-carboxylcytosine. A few previous studies demonstrated that ascorbate may enhance generation of 5-hydroxymethylcytosine in cultured cells, probably acting as a cofactor of TETs during hydroxylation of 5-methylcytosine. Physiological concentrations of ascorbate in human serum (10-100 µM) may guarantee stable level of 5-hydroxymethylcytosine, a modification necessary for epigenetic function of the cell. 5-Hydroxymethylcytosine level is substantially decreased in almost all investigated cancers, what may be linked with cancer development. Therefore, it is possible that supplementation with ascorbate could contribute to better management of individual cancer patient. This issue is also discussed in our paper.

Epigenetic modifications and NF-κB pathway activity in Cu,Zn-SOD-deficient mice.

The aim of this study was to examine the possible impact of Cu,Zn-SOD deficiency on the level of epigenetic modifications in different mouse tissues, and the relationship between these modifications and the NF-κB transcription factor activity. Cu,Zn-SOD deficiency did not influence the level of 5mdC or 5hmdC in the analyzed tissues. Statistically significant organ-/tissue-specific differences between the levels of 5mdC and 5hmdC were demonstrated within each genotype. Also correlations between analyzed parameters pointed to wide tissue/genotype variety; we observed a positive correlation between 5mdC and NF-кB proteins, p50 and RelA, in the liver of wild mice, as well as an inverse correlation between 5mdC and p65 in the brain of Cu,Zn-SOD-deficient animals. Moreover, a positive correlation was revealed between 5mdC and 5hmdC in the liver and brain of knockout mice. As the highest levels of both 5mdC and 5hmdC were observed in the brains of analyzed animals regardless of their genotype, and lower, comparable to each other, levels of these modifications were shown in the kidney and liver, active demethylation process seems to be tissue-/organ-specific and does not necessarily rely solely on the redox/oxidation state of cells. According to the most likely scenario, various tissues may differ in terms of their metabolic rates, which has potential influence on cofactors, and consequently on the activity of TET enzymes or activation of TET-independent mechanisms.

Comparison of the absolute level of epigenetic marks 5-methylcytosine, 5-hydroxymethylcytosine, and 5-hydroxymethyluracil between human leukocytes and sperm.

5-Methylcytosine is one of the most important epigenetic modifications and has a profound impact on embryonic development. After gamete fusion, there is a widespread and rapid active demethylation process of sperm DNA, which suggests that the paternal epigenome has an important role during embryonic development. To better understand the epigenome of sperm DNA and its possible involvement in a developing embryo, we determined epigenetic marks in human sperm DNA and in surrogate somatic tissue leukocytes; the analyzed epigenetic modifications included 5-methyl-2'-deoxycytidine, 5-hydroxymethyl-2'-deoxycytidine, and 5-hydroxymethyl-2'-deoxyuridine. For absolute determination of the modification, we used liquid chromatography with UV detection and tandem mass spectrometry techniques with isotopically labeled internal standards. Our analyses demonstrated, for the first time to date, that absolute global values of 5-methyl-2'-deoxycytidine, 5-hydroxymethyl-2'-deoxycytidine, and 5-hydroxymethyl-2'-deoxyuridine in sperm are highly statistically different from those observed for leukocyte DNA, with respective mean values of 3.815% versus 4.307%, 0.797 versus 2.945 per 104 deoxynucleosides, and 5.209 versus 0.492 per 106 deoxynucleosides. We hypothesize that an exceptionally high value of 5-hydroxymethyluracil in sperm (>10-fold higher than in leukocytes) may play a not yet recognized regulatory role in the paternal genome.

Comparison of oxidative stress/DNA damage in semen and blood of fertile and infertile men.

Abnormal spermatozoa frequently display typical features of oxidative stress, i.e. excessive level of reactive oxygen species (ROS) and depleted antioxidant capacity. Moreover, it has been found that a high level of oxidatively damaged DNA is associated with abnormal spermatozoa and male infertility. Therefore, the aim of our study was the comparison of oxidative stress/DNA damage in semen and blood of fertile and infertile men. The broad range of parameters which describe oxidative stress and oxidatively damaged DNA and repair were analyzed in the blood plasma and seminal plasma of groups of fertile and infertile subjects. These parameters include: (i) 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) and 8-oxo-7,8-dihydroguanine (8-oxoGua) levels in urine; (ii) 8-oxodG level in DNA isolated from leukocytes and spermatozoa; (iii) antioxidant vitamins (A, C and E) and uric acid. Urinary excretion of 8-oxodG and 8-oxoGua and the level of oxidatively damaged DNA in leukocytes as well as the level of antioxidant vitamins were analyzed using HPLC and HPLC/GC/MS methods. The results of our study demonstrate that 8-oxodG level significantly correlated with every parameter which describe sperm quality: sperm count, motility and morphology. Moreover, the data indicate a higher level of 8-oxodG in sperm DNA compared with DNA of surrogate tissue (leukocytes) in infertile men as well as in healthy control group. For the whole study population the median values of 8-oxodG/10(6) dG were respectively 7.85 and 5.87 (p=0.000000002). Since 8-oxodG level in sperm DNA is inversely correlated with urinary excretion rate of 8-oxoGua, which is the product of OGG1 activity, we hypothesize that integrity of spermatozoa DNA may be highly dependent on OGG1 activity. No relationship between the whole body oxidative stress and that of sperm plasma was found, which suggests that the redox status of semen may be rather independent on this characteristic for other tissues.

Does morphology of carotid plaque depend on patient's oxidative stress?

OBJECTIVES: This study explored the relationship between oxidative stress biomarkers and stability of carotid plaque. We decided to analyze the broad range of parameters describing oxidative stress in patients with carotid stenosis. DESIGN AND METHODS: 124 consecutive patients undergoing carotid endarterectomy were enrolled in the study group. The control group consisted of 49 patients without symptoms of atherosclerosis. The stability of carotid plaques was assessed using GSM (gray-scale median) scoring system and the study group was divided into three subgroups according to echogenicity of the plaque. The following parameters of oxidative stress/DNA damage were analyzed: i) urinary excretion of the products of oxidative DNA damage repair; ii) the background level of 8-oxo-7,8-dihydro-2'-deoxyguanosine in leukocytes' DNA and in atherosclerotic plaques; and iii) the concentrations of antioxidant vitamins, uric acid and C-reactive protein in plasma. RESULTS: Oxidative stress (described by redox status) was higher in the patient group than in the control group. There is a correlation between oxidative stress of the patients and stability of the plaque, echolucent plaques (GSM<25) being associated with the highest antioxidant level and lowest excretion of DNA repair markers. CONCLUSIONS: The plaque formation/morphology may depend on local environment and is independent of oxidative stress/inflammation observed on the level of the whole body.

[Oxidation and deamination of nucleobases as an epigenetic tool]. / Oksydacyjnie modyfikowane i deaminowane zasady DNA jako czynnik epigenetyczny.

 Recent discoveries have demonstrated that 5-methylcytosine (5mC) may be hydroxymethylated to 5-hydroxymethylcytosine (5hmC) in mammals and that genomic DNA may contain about 0.02-0.7% of 5hmC. The aforementioned modification is the key intermediate of active DNA demethylation and has been named "the sixth base in DNA". Although active DNA demethylation in mammals is still controversial, the most plausible mechanism/s of active 5mC demethylation include involvement of three families of enzymes; i) Tet, which is involved in hydroxylation of 5mC to form 5hmC, which can be further oxidized to 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC); ii) deamination of 5mC (or 5hmC) by AID/APOBEC to form thymine or 5-hydroxymethyluracil (5hmU) mispaired with guanine; iii) the BER pathway induced by involvement of TDG glycosylase to replace the above described base modification (5fC, 5caC, 5hmU) with cytosine to demethylate DNA. A plausible scenario for engagement of TDG glycosylase (or some other G-T glycosylase) is through prior deamination of 5-mC to thymine, which generates a G: T substrate for the enzyme. Here cytidine deaminase of the AID/APOBEC family was implicated in the deamination step. It is possible that TDG may act in concert with these deaminases. It seems that mutations are not the only effect of oxidatively modified DNA bases. These, as yet, understudied aspects of the damage suggest a potential for 8-oxoguanine (8-oxoGua) to affect gene expression via chromatin relaxation. It is possible that 8-oxoGua presence in specific DNA sequences may be widely used for transcription regulation, which suggests the epigenetic nature of 8-oxoGua presence in DNA.

Cu,Zn-superoxide dismutase deficiency in mice leads to organ-specific increase in oxidatively damaged DNA and NF-κB1 protein activity.

Earlier experimental studies have demonstrated that: i) Cu,Zn-superoxide dismutase deficiency leads to oxidative stress and carcinogenesis; ii) dysregulation of NF-κB pathway can mediate a wide variety of diseases, including cancer. Therefore, we decided, for the first time, to examine the level of oxidative DNA damage and the DNA binding activity of NF-κB proteins in SOD1 knockout, heterozygous and wild-type mice. Two kinds of biomarkers of oxidatively damaged DNA: urinary excretion of 8-oxodG and 8-oxoGua, and the level of oxidatively damaged DNA were analysed using HPLC-GC-MS and HPLC-EC. The DNA binding activity of p50 and p65 proteins in a nuclear extracts was assessed using NF-κB p50/p65 EZ-TFA transcription factor assay. These parameters were determined in the brain, liver, kidney and urine of SOD1 knockout, heterozygous and wild-type mice. The level of 8-oxodG in DNA was higher in the liver and kidney of knockout mice than in wild type. No differences were found in urinary excretion of 8-oxoGua and 8-oxodG between wild type and the SOD1-deficient animals. The activity of the p50 protein was higher in the kidneys, but surprisingly not in the livers of SOD1-deficient mice, whereas p65 activity did not show any variability. Our results indicate that in Cu,Zn-SOD-deficient animals the level of oxidative DNA damage and NF-κB1 activity are elevated in certain organs only, which may provide some explanation for organ-specific ROS-induced carcinogenesis.

Oxidatively damaged DNA/oxidative stress in children with celiac disease.

BACKGROUND: Because patients with celiac disease face increased risk of cancer and there is considerable circumstantial evidence that oxidatively damaged DNA may be used as a marker predictive of cancer development, we decided, for the first time, to characterize oxidative stress/oxidative DNA damage in celiac disease patients. METHODS: Two kinds of oxidatively damaged DNA biomarkers, namely, urinary excretion of 8-oxodG and 8-oxoGua, and the level of oxidatively damaged DNA in the leukocytes, as well as the level of antioxidant vitamins were analyzed using high-performance liquid chromatography (HPLC) and HPLC/gas chromatography with isotope dilution mass detection methods. These parameters were determined in three groups: (a) children with untreated celiac disease, (b) patients with celiac disease on a strict gluten-free diet, and (c) healthy children. RESULTS: The mean level of 8-oxodG in DNA isolated from the leukocytes and in the urine samples of the two groups of celiacs was significantly higher than in controls, irrespective of diet. There was no statistically significant difference in these parameters between treated and untreated celiacs. The mean plasma retinol and alpha-tocopherol concentration in the samples of untreated celiacs was significantly lower than in treated celiacs. CONCLUSION: Our results suggest that although diet can be partially responsible for oxidative stress/oxidatively damaged DNA in celiac patients, there is a factor independent of diet. IMPACT: It is possible that celiac disease patients may be helped by dietary supplementation rich in vitamin A (and E) to minimize the risk of cancer development.

Oxidative stress and 8-oxoguanine repair are enhanced in colon adenoma and carcinoma patients.

Oxidative stress is involved in the pathogenesis of colon cancer. We wanted to elucidate at which stage of the disease this phenomenon occurs. In the examined groups of patients with colorectal cancer (CRC, n = 89), benign adenoma (AD, n = 77) and healthy volunteers (controls, n = 99), we measured: vitamins A, C and E in blood plasma, 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) and 8-oxo-7,8-dihydroguanine (8-oxoGua) in leukocytes and urine, leukocyte 8-oxoGua excision activity, mRNA levels of APE1, OGG1, 8-oxo-7,8-dihydrodeoxyguanosine 5'-triphosphate pyrophosphohydrolase (MTH1) and OGG1 polymorphism. The vitamin levels decreased gradually in AD and CRC patients. 8-OxodG increased in leukocytes and urine of CRC and AD patients. 8-OxoGua was higher only in the urine of CRC patients. 8-OxoGua excision was higher in CRC patients than in controls, in spite of higher frequency of the OGG1 Cys326Cys genotype, encoding a glycosylase with decreased activity. mRNA levels of OGG1 and APE1 increased in CRC and AD patients, which could explain increased 8-oxoGua excision rate in CRC patients. MTH1 mRNA was also higher in CRC patients. The results suggest that oxidative stress occurs in CRC and AD individuals. This is accompanied by increased transcription of DNA repair genes, and increased 8-oxoGua excision rate in CRC patients, which is, however, insufficient to counteract the increased DNA damage.

[Mechanism of DNA methylation and demethylation--its role in control of genes expression]. / Mechanizm metylacji i demetylacji DNA--znaczenie w kontroli ekspresji genów.

Cytosine methylation is a post-replicative DNA modification associated with transcriptional repression. This process consists in covalent addition of a methyl group to cytosine within the CpG dinucleotide. DNA methylation is catalyzed by DNA methyltransferases which transfer a methyl group from S-adenosyl-L-methionine to cytosine bases in DNA. Methylation patterns are determined by DNA methyltransferases, but also by the process of DNA demethylation. This review describes biochemical aspects of DNA methylation and demethylation and its role in regulation of genes expression, as well as shows cytosine methylation as promising target for anticancer therapy.

[Global DNA hipomethylation--the meaning in carcinogenesis]. / Globalna hipometylacja DNA--znaczenie w procesie kancerogenezy.

DNA methylation plays an important role in the regulation of gene expression. Tumor cells are characterized by alterations of DNA methylation pattern, namely local CpG island hypermethylation and genome wide hypomethylation. The hypomethylation of the genome affects many repetitive sequences and transposable elements and is believed to results in chromosomal instability and increased mutations events. In this review we summarize the current knowledge concerning epigenetic mechanisms related to cancer, especially the relationship of DNA hypomethylation to carcinogenesis.

Selenium supplementation reduced oxidative DNA damage in adnexectomized BRCA1 mutations carriers.

Some experimental evidence suggests that BRCA1 plays a role in repair of oxidative DNA damage. Selenium has anticancer properties that are linked with protection against oxidative stress. To assess whether supplementation of BRCA1 mutation carriers with selenium have a beneficial effect concerning oxidative stress/DNA damage in the present double-blinded placebo control study, we determined 8-oxodG level in cellular DNA and urinary excretion of 8-oxodG and 8-oxoGua in the mutation carriers. We found that 8-oxodG level in leukocytes DNA is significantly higher in BRCA1 mutation carriers. In the distinct subpopulation of BRCA1 mutation carriers without symptoms of cancer who underwent adnexectomy and were supplemented with selenium, the level of 8-oxodG in DNA decreased significantly in comparison with the subgroup without supplementation. Simultaneously in the same group, an increase of urinary 8-oxoGua, the product of base excision repair (hOGG1 glycosylase), was observed. Therefore, it is likely that the selenium supplementation of the patients is responsible for the increase of BER enzymes activities, which in turn may result in reduction of oxidative DNA damage. Importantly, in a double-blinded placebo control prospective study, it was shown that in the same patient groups, reduction in cancer incidents was observed. Altogether, these results suggest that BRCA1 deficiency contributes to 8-oxodG accumulation in cellular DNA, which in turn may be a factor responsible for cancer development in women with mutations, and that the risk to developed breast cancer in BRCA1 mutation carriers may be reduced in selenium-supplemented patients who underwent adnexectomy.

Elevated level of 8-oxo-7,8-dihydro-2'-deoxyguanosine in leukocytes of BRCA1 mutation carriers compared to healthy controls.

Carriers of BRCA1 mutation face highly increased risk of breast and ovarian cancer and some studies with cell culture suggest that the encoded protein may be involved in oxidatively damaged DNA repair. However, no studies concerning a possible link between oxidatively damaged DNA and BRCA1 deficiency have been conducted with the mutations carriers. Therefore, to assess an involvement of BRCA in oxidative damage to DNA in the present study a broad spectrum of parameters reflecting oxidative stress/DNA damage were analyzed in 3 subject groups; (i) carriers of BRCA1 mutations without symptoms of the disease; (ii) patients with breast or ovarian cancer with the mutations and (iii) the group of healthy subjects recruited from among close relatives of the group of carriers without symptoms of the disease. We found that the endogenous levels of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) in leukocytes DNA and excretion rates of urinary 8-oxodG were significantly higher in the cancer patients than in the healthy carriers. Similarly, to the cancer patient group, 8-oxodG level in leukocytes DNA is significantly higher in the carriers group in comparison with control group. That the control group comprised close relatives of the carriers gives further credit to our finding. Since we did not observe substantial differences in the analyzed markers of oxidative stress between the controls and the carriers, the observed increase in the level may be a result of a deficiency in the repair of 8-oxodG.