Role of O6-methylation in the initiation of GGTase-positive foci

The ability of seven methylating agents to form 7-methylguanine and O6-methylguanine was compared to their ability to initiate carcinogenesis as measured by the initiation of GGTase-positive foci. The seven methylating agents studied were methyl-N-nitroso-p-toluenesulfonamide (diazald), dimethylhydrazine (DMH), dimethylnitrosamine (DMN), dimethylsulfate (DMS), methyl methanesulfonate (MMS), methyl-N-nitro-N-nitrosoguanidine (MNNG) and methyl-N-nitrosourea (MNU). The DNA methylation and initiation of GGTase-positive foci was determined in partial hepatectomized rats. The formation of foci was promoted by 500 ppm sodium phenobarbital in the drinking water. While six of the seven compounds (DMH, DMN, DMS, MMS, MNNG and MNU) produced 7-methylguanine, only the four compounds (DMH, DMN, MNNG and MNU) that produced O6-methylguanine initiated GGTase-positive foci. The extent of O6-methylguanine produced by the methylating agents did not correspond with their potency to initiate GGTase-positive foci. Therefore, the initiation of GGTase-positive foci required the formation of O6-methylguanine. However, some sequential event altered the quantitative relationship of O6-methylguanine formation to the incidence of GGTase-positive foci.     

Comparison of geno- and cytotoxicity of methylnitrosourea on MMR-proficient and MMR-deficient human tumor cell lines

Deficient mismatch repair (MMR) is identified as a mutation of one of four major MMR genes and(or) microsatellite instability. These genomic changes are used as markers of MMR status of the heredity nonpolyposis colorectal cancer (HNPCC) spectrum tumors--familial and sporadic tumors of colon and extracolonic cancers fulfilling Amsterdam clinical criteria II. MMR-deficiency results in mutator phenotype and resistance to geno- and cytotoxicity of alkylating agents. The main cytotoxic damage to DNA in response to chemical methylation is O6-methylguanine (O6-mG). The secondary DNA strand breaks, which are formed during the MMR functioning, are proposed to be required for methylation induced cytotoxicity. We have assumed that the secondary double stand breaks (DSB) upon DNA methylation are able to represent functional efficiency of MMR in cells. The purpose of the paper was to test this assumption on human tumor cells differing in MMR-status and pulse-treated with methylnitrosourea (MNU). We used 3 cell lines: HeLa (MMR-competent endometrial tumor cells), HCT116 (MMR-deficient colorectal carcinoma cells), and Colo320 (sigmoid intestine tumor cells with uncharacterized MMR status). DSBs were evaluated with neutral comet assay. Cytotoxicity/viability was evaluated with MTT-asay and apoptotic index (frequency of morphologically determined apoptotic cells). We show that 1) cytotoxic effect of MNU (250 microM) on HeLa cells was exhibited 3 days after pulse-treatment of cells with MNU; 2) DSBs occurred 48 h after the drug treatment but prior to the onset of apoptosis of HeLa cells; 3) MMR-deficient HCT116 cells were resistant to the drug: no decreased viability, DSBs and apoptosis were observed during 3 days after cell treatment. Both cell lines exhibited high sensitivity to etoposide, classical inductor of unrepairable DSBs and p53. Etoposide has been found to induce DSBs in 6-12 h, which was followed by apoptosis (in 24 h). Colo320 cells exhibited intermediate position between HeLa and HCT116 cell lines in regard to sensitivity to MNU according to MTT-assay and the number of secondary DSBs formed in MNU-treated cells. Nevertheless, in contrast to HeLa cells, these breaks did not induce apoptosis in Colo320 cells. Our data confirm the assumption about case/effect relationship between secondary DNA double strand breaks, induced by monofunctional methylating agent MNU, and functioning of MMR in human tumor cells.     

Genome-wide combination profiling of copy number and methylation offers an approach for deciphering misregulation and development in cancer cells

Copy number changes and DNA methylation alterations are crucial to gene regulation in mammals. Recently, a number of microarray studies have been based on copy number and DNA methylation alterations in order to find clinical biomarkers of carcinogenesis. In this study, we attempted to combine profiles of copy number and methylation patterns in four human cancer cell lines using BAC microarray-based approaches and we detected several clinically important genes which showed genetic and epigenetic relationships. Within the clones analyzed, many contained cancer-related genes involved in cell cycle regulation, cell division, signal transduction, tumor necrosis, cell differentiation, and cell proliferation. One clone included the FHIT gene, a well-known tumor suppressor gene involved in various human cancers. Our combined profiling techniques may provide a method by which to find new clinicopathologic cancer biomarkers, and support the idea that systematic characterization of the genetic and epigenetic events in cancers may rapidly become a reality.     

Role of enzymatic DNA methylation in cell differentiation and carcinogenesis

The enzymatic methylation of specific cytosine residues in DNA plays a part in controlling gene expression. Low methylation levels may be a necessary condition for gene expression. The chemical carcinogens exert their effect on the enzymatic methylation of mammalian DNA and can cause hypomethylation. Demethylated sites do not become remethylated in the subsequent cell cycles. The consequence of DNA hypomethylation may be both stimulation of cell differentiation and initiation of carcinogenesis.     

Epigenetic diagnostics of cancer — the application of DNA methylation markers

In recent years it has become apparent that epigenetic events are potentially equally responsible for cancer initiation and progression as genetic abnormalities. DNA methylation is the main epigenetic modification in humans. Two DNA methylation lesions coexist in human neoplasms: hypermethylation of promoter regions of specific genes within a context of genomic hypomethylation. Aberrant methylation is found at early stages of carcinogenesis and distinct types of cancer exhibit specific patterns of methylation changes. Tumor specific DNA is readily obtainable from different clinical samples and methylation status analysis often permits sensitive disease detection. Methylation markers may also serve for prognostic and predictive purposes as they often reflect the metastatic potential and sensitivity to therapy. As current findings show a great potential of recently characterised methylation markers, more studies in the field are needed in the future. Large clinical studies of newly developed markers are especially needed. The review describes the diagnostic potential of DNA methylation markers.     

GSTP1 DNA methylation and expression status is indicative of 5-aza-2'-deoxycytidine efficacy in human prostate cancer cells

DNA methylation plays an important role in carcinogenesis and the reversibility of this epigenetic modification makes it a potential therapeutic target. To date, DNA methyltransferase inhibitors (DNMTi) have not demonstrated clinical efficacy in prostate cancer, with one of the major obstacles being the inability to monitor drug activity during the trial. Given the high frequency and specificity of GSTP1 DNA methylation in prostate cancer, we investigated whether GSTP1 is a useful marker of DNMTi treatment efficacy. LNCaP prostate cancer cells were treated with 5-aza-2'-deoxycytidine (5-aza-CdR) either with a single high dose (5-20 μM), every alternate day (0.1-10 μM) or daily (0.005-2.5 μM). A daily treatment regimen with 5-aza-CdR was optimal, with significant suppression of cell proliferation achieved with doses of 0.05 μM or greater (p<0.0001) and induction of cell death from 0.5 μM (p<0.0001). In contrast, treatment with a single high dose of 20 μM 5-aza-CdR inhibited cell proliferation but was not able to induce cell death. Demethylation of GSTP1 was observed with doses of 5-aza-CdR that induced significant suppression of cell proliferation (≥ 0.05 μM). Re-expression of the GSTP1 protein was observed only at doses of 5-aza-CdR (≥ 0.5 μM) associated with induction of cell death. Treatment of LNCaP cells with a more stable DNMTi, Zebularine required at least a 100-fold higher dose (≥ 50 μM) to inhibit proliferation and was less potent in inducing cell death, which corresponded to a lack of GSTP1 protein re-expression. We have shown that GSTP1 DNA methylation and protein expression status is correlated with DNMTi treatment response in prostate cancer cells. Since GSTP1 is methylated in nearly all prostate cancers, our results warrant its testing as a marker of epigenetic therapy response in future clinical trials. We conclude that the DNA methylation and protein expression status of GSTP1 are good indicators of DNMTi efficacy.     

The essential role of histone H3 Lys9 di-methylation and MeCP2 binding in MGMT silencing with poor DNA methylation of the promoter CpG island

Silencing of the O (6)-methylguanine-DNA methyltransferase (MGMT) gene, a key to DNA repair, is involved in carcinogenesis. Recent studies have focused on DNA hypermethylation of the promoter CpG island. However, cases showing silencing with DNA hypomethylation certainly exist, and the mechanism involved is not elucidated. To clarify this mechanism, we examined the dynamics of DNA methylation, histone acetylation, histone methylation, and binding of methyl-CpG binding proteins at the MGMT promoter region using four MGMT negative cell lines with various extents of DNA methylation. Histone H3K9 di-methylation (H3me2K9), not tri-methylation, and MeCP2 binding were commonly seen in all MGMT negative cell lines regardless of DNA methylation status. 5Aza-dC, but not TSA, restored gene expression, accompanied by a decrease in H3me2K9 and MeCP2 binding. In SaOS2 cells with the most hypomethylated CpG island, 5Aza-dC decreased H3me2K9 and MeCP2 binding with no effect on DNA methylation or histone acetylation. H3me2K9 and DNA methylation were restricted to in and around the island, indicating that epigenetic modification at the promoter CpG island is critical. We conclude that H3me2K9 and MeCP2 binding are common and more essential for MGMT silencing than DNA hypermethylation or histone deacetylation. The epigenetic mechanism leading to silent heterochromatin at the promoter CpG island may be the same in different types of cancer irrespective of the extent of DNA methylation.     

Epigenetic effects of the continuous exposure to peroxisome proliferator WY-14,643 in mouse liver are dependent upon peroxisome proliferator activated receptor alpha

Peroxisome proliferators are potent rodent liver carcinogens that act via a non-genotoxic mechanism. The mode of action of these agents in rodent liver includes increased cell proliferation, decreased apoptosis, secondary oxidative stress and other events; however, it is not well understood how peroxisome proliferators are triggering the plethora of the molecular signals leading to cancer. Epigenetic changes have been implicated in the mechanism of liver carcinogenesis by a number of environmental agents. Short-term treatment with peroxisome proliferators and other non-genotoxic carcinogens leads to global and locus-specific DNA hypomethylation in mouse liver, events that were suggested to correlate with a burst of cell proliferation. In the current study, we investigated the effects of long-term exposure to a model peroxisome proliferator WY-14,643 on DNA and histone methylation. Male SV129mice were fed a control or WY-14,643-containing (1000ppm) diet for one week, five weeks or five months. Treatment with WY-14,643 led to progressive global hypomethylation of liver DNA as determined by an HpaII-based cytosine extension assay with the maximum effect reaching over 200% at five months. Likewise, trimethylation of histone H4 lysine 20 and H3 lysine 9 was significantly decreased at all time points. The majority of cytosine methylation in mammals resides in repetitive DNA sequences. In view of this, we measured the effect of WY-14,643 on the methylation status of major and minor satellites, as well as in IAP, LINE1 and LINE2 elements in liver DNA. Exposure to WY-14,643 resulted in a gradual loss of cytosine methylation in major and minor satellites, IAP, LINE1 and LINE2 elements. The epigenetic changes correlated with the temporal effects of WY-14,643 on cell proliferation rates in liver, but no sustained effect on c-Myc promoter methylation was observed. Finally, WY-14,643 had no effect on DNA and histone methylation status in Pparalpha-null mice at any of the time points considered in this study. These data indicate the importance of epigenetic alterations in the mechanism of action of peroxisome proliferators and the key role of Pparalpha.     

Genomic patterns of DNA methylation: targets and function of an epigenetic mark

Methylation of cytosines can mediate epigenetic gene silencing and is the only known DNA modification in eukaryotes. Recent efforts to map DNA methylation across mammalian genomes revealed limited DNA methylation at regulatory regions but widespread methylation in intergenic regions and repeats. This is consistent with the idea that hypermethylation is the default epigenetic state and serves in maintaining genome integrity. DNA methylation patterns at regulatory regions are generally stable, but a minor subset of regulatory regions show variable DNA methylation between cell types, suggesting an additional dynamic component. Such promoter de novo methylation might be involved in the maintenance rather than the initiation of silencing of defined genes during development. How frequently such dynamic methylation occurs, its biological relevance and the pathways involved deserve investigation.     

Minichromosome Maintenance Proteins as Markers for Proliferation Zones During Embryogenesis

Regulation of cell proliferation is of fundamental importance for growth andprevention of cancer. An obligatory step in the regulation of cell proliferation is thecontrol of the initiation of DNA synthesis. The minichromosome maintenance (MCM)proteins are essential DNA replication factors crucial for initiating DNA synthesis onceevery cell cycle. Recent studies show that the level of MCM proteins is stringentlyregulated to correlate with cell proliferation and carcinogenesis. Here we discuss ourrecent data, which highlights the usefulness of minichromosome maintenance (MCM)gene expression for detecting proliferating cells as well as zones containingproliferative/stem cells during embryogenesis in a whole organism.     

DNA-repair capacity and lipid peroxidation in chronic alcoholics

The possible impact of long-term overexposure to ethanol was studied in a group of chronic alcoholics in the psychiatric hospital. The level of DNA methylation and unscheduled DNA synthesis (UDS) induced by N-methyl-N-nitrosourea (MNU) in lymphocytes and lipid peroxidation (LPO) in plasma were used as markers of injury caused by alcohol abuse. The data were correlated with plasma levels of some natural antioxidants (vitamins A, C and E) and vitamin B12. The following results were obtained. The degree of DNA methylation by MNU in lymphocytes was the same in the exposed and control groups under our experimental conditions. The DNA excision-repair capacity of lymphocytes measured as UDS was decreased in alcoholics (p less than 0.01) and LPO in plasma was significantly higher (p less than 0.01) as a consequence of alcohol overconsumption. By the simple regression method, a correlation was found between LPO and vitamin C levels (LPO = -0.078 x vit. C + 1.9; p less than 0.05) and between UDS and LPO values (UDS = -0.384 x LPO + 4.1; p less than 0.05). These results support the hypothesis of a connection of cell membrane status and DNA damage and repair and the possible role of active oxygen species in cell damage caused by ethanol.     

The dynamics and prognostic potential of DNA methylation changes at stem cell gene loci in women's cancer

Aberrant DNA methylation is an important cancer hallmark, yet the dynamics of DNA methylation changes in human carcinogenesis remain largely unexplored. Moreover, the role of DNA methylation for prediction of clinical outcome is still uncertain and confined to specific cancers. Here we perform the most comprehensive study of DNA methylation changes throughout human carcinogenesis, analysing 27,578 CpGs in each of 1,475 samples, ranging from normal cells in advance of non-invasive neoplastic transformation to non-invasive and invasive cancers and metastatic tissue. We demonstrate that hypermethylation at stem cell PolyComb Group Target genes (PCGTs) occurs in cytologically normal cells three years in advance of the first morphological neoplastic changes, while hypomethylation occurs preferentially at CpGs which are heavily Methylated in Embryonic Stem Cells (MESCs) and increases significantly with cancer invasion in both the epithelial and stromal tumour compartments. In contrast to PCGT hypermethylation, MESC hypomethylation progresses significantly from primary to metastatic cancer and defines a poor prognostic signature in four different gynaecological cancers. Finally, we associate expression of TET enzymes, which are involved in active DNA demethylation, to MESC hypomethylation in cancer. These findings have major implications for cancer and embryonic stem cell biology and establish the importance of systemic DNA hypomethylation for predicting prognosis in a wide range of different cancers.     

The Controversial Denouement of Vertebrate DNA Methylation Research

The study of the biological role of DNA methylation in vertebrates has involved considerable controversy. Research in this area has proceeded well despite the complexity of the subject and the difficulties in establishing biological roles, some of which are summarized in this review. Now there is justifiably much more interest in DNA methylation than previously, and many more laboratories are engaged in this research. The results of numerous studies indicate that some tissue-specific differences in vertebrate DNA methylation help maintain patterns of gene expression or are involved in fine-tuning or establishing expression patterns. Therefore, vertebrate DNA methylation cannot just be assigned a role in silencing transposable elements and foreign DNA sequences, as has been suggested. DNA methylation is clearly implicated in modulating X chromosome inactivation and in establishing genetic imprinting. Also, hypermethylation of CpG-rich promoters of tumor suppressor genes in cancer has a critical role in downregulating expression of these genes and thus participating in carcinogenesis. The complex nature of DNA methylation patterns extends to carcinogenesis because global DNA hypomethylation is found in the same cancers displaying hypermethylation elsewhere in the genome. A wide variety of cancers display both DNA hypomethylation and hypermethylation, and either of these types of changes can be significantly associated with tumor progression. These findings and the independence of cancer-linked DNA hypomethylation from cancer-linked hypermethylation strongly implicate DNA hypomethylation, as well as hypermethylation, in promoting carcinogenesis. Furthermore, various DNA demethylation methodologies have been shown to increase the formation of certain types of cancers in animals, and paradoxically, DNA hypermethylation can cause carcinogenesis in other model systems. Therefore, there is a need for caution in the current use of demethylating agents as anti-cancer drugs. Nonetheless, DNA demethylation therapy clearly may be very useful in cases where better alternatives do not exist.     

Insight of nitric oxide signaling: A potential biomarker with multifaceted complex mechanism in colorectal carcinogenesis

Colorectal carcinogenesis (CRC) is the most important health concerns throughout the World as the tumour cells rapidly spread and abruptly grow in colon and rectum to further organs. Several etiological factors are associated with colorectal carcinogenesis. During invasion and proliferation of tumour cells, various mechanistic molecular pathways are involved in the cells. Nitric Oxide pathway (NO) is one of the important cellular mechanisms associated with tumour cells initiation, invasion and progression. Epidemiological evidences suggest that NO has potential role in development of cancer. The multidisciplinary action of NO on the initiation of cancer depends on several factors including cell type, metastasis stage, and organs involved. This review emphasizes the biological significance of NO in each step of cancer metastasis, its controversial effects for carcinogenesis including initiation, invasion and progression.     

Protective effects of sodium selenite on killing and mutation by N-methyl-N''-nitro-N-nitrosoguanidine in E. coli.

Sodium selenite was found to protect Escherichia coli cells against killing and mutagenic effects of N-methyl-N′-nitro-N-nitrosoguanidine (MNNG). Such protective effects were not observed when cells were treated with N-methyl-N-nitrosourea (MNU). The protection by sodium selenite was not controlled by the ada gene, which is responsible for the repair of alkylated damage in DNA. A reduction of the amount of glutathione was found when cells were treated with sodium selenite, and glutathione is known to be involved in the methylation of DNA by MNNG, not by MNU. Reduced methylation by MNNG due to the reduction of the amount of glutathione caused by abundant sodium selenite was suggested to be the mechanism of protection.     

Epigenetic changes of CXCR4 and its ligand CXCL12 as prognostic factors for sporadic breast cancer

Chemokines and their receptors are involved in the development and cancer progression. The chemokine CXCL12 interacts with its receptor, CXCR4, to promote cellular adhesion, survival, proliferation and migration. The CXCR4 gene is upregulated in several types of cancers, including skin, lung, pancreas, brain and breast tumors. In pancreatic cancer and melanoma, CXCR4 expression is regulated by DNA methylation within its promoter region. In this study we examined the role of cytosine methylation in the regulation of CXCR4 expression in breast cancer cell lines and also correlated the methylation pattern with the clinicopathological aspects of sixty-nine primary breast tumors from a cohort of Brazilian women. RT-PCR showed that the PMC-42, MCF7 and MDA-MB-436 breast tumor cell lines expressed high levels of CXCR4. Conversely, the MDA-MB-435 cell line only expressed CXCR4 after treatment with 5-Aza-CdR, which suggests that CXCR4 expression is regulated by DNA methylation. To confirm this hypothesis, a 184 bp fragment of the CXCR4 gene promoter region was cloned after sodium bisulfite DNA treatment. Sequencing data showed that cell lines that expressed CXCR4 had only 15% of methylated CpG dinucleotides, while the cell line that not have CXCR4 expression, had a high density of methylation (91%). Loss of DNA methylation in the CXCR4 promoter was detected in 67% of the breast cancer analyzed. The absence of CXCR4 methylation was associated with the tumor stage, size, histological grade, lymph node status, ESR1 methylation and CXCL12 methylation, metastasis and patient death. Kaplan-Meier curves demonstrated that patients with an unmethylated CXCR4 promoter had a poorer overall survival and disease-free survival. Furthermore, patients with both CXCL12 methylation and unmethylated CXCR4 had a shorter overall survival and disease-free survival. These findings suggest that the DNA methylation status of both CXCR4 and CXCL12 genes could be used as a biomarker for prognosis in breast cancer.     

Methylation damage response in hematopoietic progenitor cells

The cellular response to methylation DNA damage was compared in multipotent CD34(+) hematopoietic stem cells and mature CD34(-) cells isolated from cord blood of the same donor. Cytofluorimetric analysis of freshly isolated cord blood cells indicated that both cell types were in the G0/G1 phase of the cell cycle. Quantitative RT-PCR identified a general trend towards high expression of several DNA repair genes in CD34(+) cells compared to their terminally differentiated CD34(-) counterparts. The overexpressed genes included members of the mismatch repair (MMR) (MSH2, MSH6, MLH1, PMS2), base excision repair (AAG, APEX), DNA damage reversal (O(6)-methylguanine DNA methyltransferase) (MGMT), and DNA double strand breaks repair pathways. These differences in gene expression were not apparent in CD34(+) and CD34(-) cells obtained following expansion of CD34(+) cells in a medium containing early acting cytokines. Early progenitor CD34(+) and early precursor CD34(-) cells form the two populations isolated under these experimental conditions, and both contain a significant proportion of cycling cells. The methylating agent N-methyl-N-nitrosourea (MNU) induced similar levels of apoptosis in these cycling CD34(+) and CD34(-) cells. Cytotoxicity required the presence of the MGMT inhibitor O(6)-benzylguanine and the timing of MNU cell death (48 and 72h) was similar in CD34(+) and CD34(-) cells. These data indicate that cycling CD34(+) and CD34(-) cells are equally sensitive to methylation damage. MGMT provides significant protection against MNU toxicity and MGMT and MMR play the expected roles in the MNU sensitivity of these cells.     

Dual actions of the antioxidant chlorophyllin,a glutathione transferase P1-1 inhibitor,in tumorigenesis and tumor progression

Glutathione (GSH) and enzymes related to this antioxidant molecule are often overexpressed in tumor cells and may contribute to drug resistance. Blockade of glutathione transferases (GSTs) has been proposed to potentiate the efficacy of chemotherapeutic drugs in cancer. The aim of this study was to evaluate the effect of chlorophyllin that has antioxidant properties, and also interferes with the activity of GST P1-1, on breast cancers in vitro and in vivo. The in vivo studies were conducted using an N-methyl- N-nitrosourea (MNU)-induced chemical carcinogenesis model in laboratory rats. DNA damage, GST activity, and GSH levels were determined in liver and tumor tissues. Treatment with chlorophyllin increased the GSH levels in the liver and significantly decreased DNA damage in the blood, liver, and tumor tissues. Even though tumorigenesis was delayed in rats receiving chlorophyllin before MNU injections, once the tumors emerged, the progression of tumor appeared to be faster than in the animals that received the carcinogen only. Out of nine breast cell lines, GST P1-1 expression was detected in MCF-12A, MDA-MB-231, and HCC38. Concomitant incubation with chlorophyllin and docetaxel did not significantly affect cell proliferation and viability. Chlorophyllin displayed genoprotective effects that initially delayed tumorigenesis. However, once the tumors were established, it may act as a promoter that facilitates tumor growth, potentially by a mechanism independent of cell proliferation and viability. Our results underline the pros and cons of antioxidant treatment in cancer, even if it has a capacity to inhibit GST P1-1.