The 46359CT polymorphism of DNMT3B is associated with the risk of cervical cancer

Abnormal methylation is related to cancer development. Since DNMT3B is an enzyme that modulates genomic methylation, we hypothesized that genetic variants of the promoter DNMT3B may be associated with an increased risk of developing cervical cancer. Our aim was to investigate the association between ?579GT and 46359CT polymorphisms of DNMT3B and cervical cancer, high-grade squamous intraepithelial lesions (HSIL), and low-grade squamous intraepithelial lesions (LSIL). Samples from 200 healthy women and 130 women with squamous intraepithelial lesions (70 with cervical cancer, 30 with HSIL, and 30 with LSIL) were analyzed. Polymorphism genotyping was performed using PCR and restriction fragment length polymorphism. The ?579GT polymorphism was not associated with cervical cancer, HSIL, or LSIL. The CT genotype of 46359CT polymorphism was significantly associated with cervical cancer risk (OR 8.75, CI 1.27–374.1), whereas the TT genotype was associated with a significantly decreased risk of HSIL (OR 0.66, CI 0.01–0.32) and LSIL (OR 0.11, CI 0.026–0.45). Our results suggest that genotyping the 46359CT polymorphism in DNMT3B may help identify women who are genetically susceptible to cervical cancer development. Additional studies with larger sample sizes are necessary to confirm our findings.     

Polymorphic variants of folate metabolizing genes (C677T and A1298C MTHFR, C1420T SHMT1 and G1958A MTHFD) are not associated with the risk of breast cancer in West Siberian Region of Russia

Breast cancer is the most incident cancer among women. We investigated the role of polymorphisms of folate metabolizing genes MTHFR (C677T and A1298C), SHMT1 (C1420T) and MTHFD (G1258A) in genetic susceptibility to this type of cancer. We determined allele and genotype frequencies in case (850 women with sporadic form of breast cancer) and control (810 women) groups. None of these polymorphisms was significantly associated with breast cancer risk. To increase statistical power of our study, we conducted a meta-analysis which included published genotype data and the results of our work. Meta-analysis also revealed no significant association of studied SNPs with breast cancer.     

Genetic polymorphisms in cytochrome P4501B1 and susceptibility to head and neck cancer

Cytochrome P4501B1 (CYP1B1), a polycyclic aromatic hydrocarbon (PAH) metabolizing CYP, is genetically polymorphic in humans and may be involved in the individual susceptibility to chemical-induced cancer. In the present study, genotype and haplotype frequencies of four single nucleotide polymorphisms (SNPs) in CYP1B1 that cause amino acid changes (Arg-Gly at codon 48, Ala-Ser at codon 119, Leu-Val at codon 432 and Asn-Ser at codon 453) were studied in 150 cases suffering from head and neck squamous cell carcinoma (HNSCC) and in an equal number of controls. A significant difference was observed for the distribution of variant genotypes of Arg48Gly (CYP1B1*2) and Ala119Ser (CYP1B1*2) polymorphisms of CYP1B1 in cases versus controls. No significant differences were observed for the distribution of variant genotypes-Leu432Val (CYP1B1*3) and Asn453Ser (CYP1B1*4), respectively. When the four SNPs were analyzed using a haplotype approach, SNPs at codon 48 (Arg48Gly) and codon 119 (Ala119Ser) exhibited complete linkage disequilibrium (LD) in all the cases and controls. Significant differences in the distribution of the two haplotypes (G-T-C-A and G-T-G-A) were observed both in the cases and in controls. Furthermore, our data indicates a several fold increase in risk in the cases who use tobacco (cigarette smoking or tobacco chewing) or alcohol with the variant genotypes of CYP1B1 (CYP1B1*2 and CYP1B1*3) suggesting the role of gene-environment interaction in the susceptibility to HNSCC.     

DNMT1, DNMT3A and DNMT3B proteins are differently expressed in mouse oocytes and early embryos

DNA methylation is one of the epigenetic mechanisms and plays important roles during oogenesis and early embryo development in mammals. DNA methylation is basically known as adding a methyl group to the fifth carbon atom of cytosine residues within cytosine–phosphate–guanine (CpG) and non-CpG dinucleotide sites. This mechanism is composed of two main processes: de novo methylation and maintenance methylation, both of which are catalyzed by specific DNA methyltransferase (DNMT) enzymes. To date, six different DNMTs have been characterized in mammals defined as DNMT1, DNMT2, DNMT3A, DNMT3B, DNMT3C, and DNMT3L. While DNMT1 primarily functions in maintenance methylation, both DNMT3A and DNMT3B are essentially responsible for de novo methylation. As is known, either maintenance or de novo methylation processes appears during oocyte and early embryo development terms. The aim of the present study is to investigate spatial and temporal expression levels and subcellular localizations of the DNMT1, DNMT3A, and DNMT3B proteins in the mouse germinal vesicle (GV) and metaphase II (MII) oocytes, and early embryos from 1-cell to blastocyst stages. We found that there are remarkable differences in the expressional levels and subcellular localizations of the DNMT1, DNMT3A and DNMT3B proteins in the GV and MII oocytes, and 1-cell, 2-cell, 4-cell, 8-cell, morula, and blastocyst stage embryos. The fluctuations in the expression of DNMT proteins in the analyzed oocytes and early embryos are largely compatible with DNA methylation changes and genomic imprintestablishment appearing during oogenesis and early embryo development. To understand precisemolecular biological meaning of differently expressing DNMTs in the early developmental periods, further studies are required.     

DNMT1, DNMT3A and DNMT3B gene variants in relation to ovarian cancer risk in the Polish population

Studies have demonstrated that changes in DNA methylation of cancer related genes can be an elementary process accounting for ovarian tumorigenesis. Therefore, we evaluated the possible association of single nucleotide polymorphisms (SNPs) of DNA methyltransferases (DNMTs) genes, including DNMT1, DNMT3B, and DNMT3A, with ovarian cancer development in the Polish population. Using PCR–RFLP and HRM analyses, we studied the prevalence of the DNMT1 rs8101626, rs2228611 and rs759920, DNMT3A rs2289195, 7590760, rs13401241, rs749131 and rs1550117, and DNMT3B rs1569686, rs2424913 and rs2424932 SNPs in patients with ovarian cancer (n = 159) and controls (n = 180). The lowest p values of the trend test were observed for the DNMT1 rs2228611 and rs759920 SNPs in patients with ovarian cancer (p _trend = 0.0118 and p _trend = 0.0173, respectively). Moreover, we observed, in the recessive inheritance model, that the DNMT1 rs2228611 and rs759920 SNPs are associated with an increased risk of ovarian cancer development [OR 1.836 (1.143–2.949), p = 0.0114, p _corr = 0.0342, and OR 1.932 (1.185–3.152), p = 0.0078, p _cor=0.0234, respectively]. However, none of other nine studied SNPs displayed significant contribution to the development of ovarian cancer. Furthermore, haplotype and multifactor dimensionality reduction analysis of the studied DNMT1, DNMT3B, and DNMT3A polymorphisms did not reveal either SNP combinations or gene interactions to be associated with the risk of ovarian cancer development. Our results may suggest that DNMT1 variants may be risk factors of ovarian cancer.     

DNMT1 and DNMT3B gene variants and their association with endometriosis in South Indian women

Background

Endometriosis is a multifactorial estrogen dependent gynecological disease characterized by implantation of functional endometrial tissue at ectopic positions. Though this disease is benign, it is associated with an increased risk of malignant transformation. Epigenetic disruptions like aberrant DNA methylation, resulting changes in gene expression capacity, are important in tumor progression and malignant cellular transformation. Therefore, variation in genes involved in DNA methylation might lead to disease susceptibility.

Purpose

To investigate the association between DNA methyl transferases (DNMT1 and DNMT3B) single nucleotide polymorphisms (SNPs) and the risk of endometriosis in South Indian women.

Methods

In the present study, we examined the genotypic and allele distribution of DNMT1 (rs10423341C/A, rs2228611G/Aandrs4804490C/A) and DNMT3B (rs1569686G/T) among the endometriosis patients (n?=?150) and controls (n?=?150). The genotypes were analyzed by polymerase chain reaction (PCR) and sequencing methods. Haplotype frequencies for multiple loci and the standardized disequilibrium coefficient (D?) for pairwise linkage disequilibrium (LD) were surveyed by Haploview Software.

Result

Significant increase in the frequencies of DNMT1 rs10423341 (P?=?0.04601), rs2228611 (P?=?0.00175) and DNMT3B rs1569686 (P?=?0.033) genotypes and alleles was observed in patients compared to controls. In addition, the frequency of A/A/C (P?=?0.0065) haplotype was significantly high in patients. But the DNMT1 (rs4804490) SNP did not show significant association with the disease.

Conclusion

The DNMT1 and DNMT3B polymorphism may constitute an inheritable risk factor for endometriosis in South Indian women. To the best of our knowledge there is no reported study on the association of polymorphisms in DNMT1 and DNMT3B with endometriosis risk.

     

Dynamic subcellular localization of isoforms of the folate pathway enzyme serine hydroxymethyltransferase (SHMT) through the erythrocytic cycle of Plasmodium falciparum

Background

The folate pathway enzyme serine hydroxymethyltransferase (SHMT) converts serine to glycine and 5,10-methylenetetrahydrofolate and is essential for the acquisition of one-carbon units for subsequent transfer reactions. 5,10-methylenetetrahydrofolate is used by thymidylate synthase to convert dUMP to dTMP for DNA synthesis. In Plasmodium falciparum an enzymatically functional SHMT (PfSHMTc) and a related, apparently inactive isoform (PfSHMTm) are found, encoded by different genes. Here, patterns of localization of the two isoforms during the parasite erythrocytic cycle are investigated.

Methods

Polyclonal antibodies were raised to PfSHMTc and PfSHMTm, and, together with specific markers for the mitochondrion and apicoplast, were employed in quantitative confocal fluorescence microscopy of blood-stage parasites.

Results

As well as the expected cytoplasmic occupancy of PfSHMTc during all stages, localization into the mitochondrion and apicoplast occurred in a stage-specific manner. Although early trophozoites lacked visible organellar PfSHMTc, a significant percentage of parasites showed such fluorescence during the mid-to-late trophozoite and schizont stages. In the case of the mitochondrion, the majority of parasites in these stages at any given time showed no marked PfSHMTc fluorescence, suggesting that its occupancy of this organelle is of limited duration. PfSHMTm showed a distinctly more pronounced mitochondrial location through most of the erythrocytic cycle and GFP-tagging of its N-terminal region confirmed the predicted presence of a mitochondrial signal sequence. Within the apicoplast, a majority of mitotic schizonts showed a marked concentration of PfSHMTc, whose localization in this organelle was less restricted than for the mitochondrion and persisted from the late trophozoite to the post-mitotic stages. PfSHMTm showed a broadly similar distribution across the cycle, but with a distinctive punctate accumulation towards the ends of elongating apicoplasts. In very late post-mitotic schizonts, both PfSHMTc and PfSHMTm were concentrated in the central region of the parasite that becomes the residual body on erythrocyte lysis and merozoite release.

Conclusions

Both PfSHMTc and PfSHMTm show dynamic, stage-dependent localization among the different compartments of the parasite and sequence analysis suggests they may also reversibly associate with each other, a factor that may be critical to folate cofactor function, given the apparent lack of enzymic activity of PfSHMTm.     

Splice variants DNMT3B4 and DNMT3B7 overexpression inhibit cell proliferation in 293A cell line

DNA methyltransferase 3B (DNMT3B) is critical in abnormal DNA methylation patterns in cancer cells. Nearly 40 alternatively spliced variants of DNMT3B have been reported. DNMT3B4 and DNMT3B7 are two kinds of splice variants of DNMT3B lacking the conserved methyltransferase motif. In this study, the effect of inactivation of DNMT3B variants, DNMT3B4 and DNMT3B7, on cell proliferation was assessed. pCMV-DNMT3B4 and pCMV-DNMT3B7 recombinant plasmids were developed and stably transfected into 293A cells. 293A cells transfected with plasmid pCMV-DNMT3B4 or pCMV-2B were then treated with G418 to the stable cell lines. After that, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method was used for testing the proliferation level, and flow cytometry was used to test cell cycle distribution of the cell line. The expression of p21 was detected by real-time PCR and Western blot. The methylation status of p21 promoter was detected by methylation-specific PCR (MS-PCR). It was found that DNMT3B4 and DNMT3B7 overexpression could inhibit cell proliferation and increase the expression of p21. Cell cycle analysis demonstrated that inactivation of DNMT3B variants overexpression inhibited cell cycle progression. Inactivation of DNMT3B variants overexpression facilitated p21 expression to delay 293A cell proliferation. These findings indicate that inactivation of DNMT3B variants might play an important role in cell proliferation correlating with the change of p21.     

Complex interaction between serum folate levels and genetic polymorphisms in folate pathway genes: biomarkers of prostate cancer aggressiveness

Little is known about the role of folate and polymorphisms associated with folate metabolism on prostate cancer risk in populations of African origin. We examined the relationship between serum folate and prostate cancer and whether any association was modified by genetic polymorphisms for folate metabolism. The study was case–control in design and consisted of 218 men 40–80 years old with newly diagnosed, histologically confirmed prostate cancer and 236 cancer-free men attending the same urology clinics in Jamaica, March 2005–July 2007. Serum folate was measured by an immunoassay method and genomic DNA evaluated for MTHR (C677T and A1298C), MTRR A66G, and MTR A2756G polymorphisms. Mean serum folate concentration was higher among cases (12.3 ± 4.1 nmol/L) than controls (9.7 ± 4.2 nmol/L). Serum folate concentration showed a positive association with prostate cancer (OR, 4.41; CI, 2.52–7.72 per 10 nmol/L) regardless of grade. No interactions were observed between genotype and folate concentration, but a weak gene effect was observed for MTHFR A1298C and low-grade prostate cancer. Larger studies to investigate the role of gene–gene/gene–diet interactions in Black men are needed.     

VIP receptor antagonists inhibit mammary carcinogenesis in C3(1)SV40T antigen mice

The effects of a vasoactive intestinal peptide (VIP) receptor antagonist on mammary carcinogenesis were investigated using the C3(1)SV40T antigen (ag) mice. Ten microg/day VIPhybrid (VIPhyb) administered daily subcutaneously increased significantly the survival of C3(1)SV40Tag mice. At 5.2 months, VIPhyb significantly reduced the mammary tumor burden in C3(1)SV40Tag mice relative to control animals. 125I-VIP bound with high affinity to mouse mammary tumor homogenate. Because (Lys15, Arg16, Leu27)VIP1-7GRF8-27 (VPAC1 selective) but not Ro25-1553 (VPAC2 selective) inhibited specific 125I-VIP binding to mammary tumor membranes with high affinity, VPAC1 receptors predominate. By RT-PCR, VPAC1 receptor mRNA was detected in mammary tumors. By Western blot, a major 60 Kdalton band was detected in mammary tumor extracts using VPAC1 receptor antisera. By immunocytochemistry, VPAC1-R immunostaining was detected in the cytosol and plasma membrane but not the nucleus of fixed mammary tumor tissue. Using laser capture microdissected tumor cells and surface enhanced laser desorption/ionization (SELDI) techniques on mammary tumor cells, the proteomic profile was altered in mice treated with VIPhyb. Because VPAC1 receptor antagonists increase the survival and reduce the tumor burden in C3(1)SV40Tag mice, they may function as chemopreventive agents in mammary cancer.     

Association between GSTP1, GSTM1 and GSTT1 polymorphisms involved in xenobiotic metabolism and head and neck cancer development

Polymorphisms in the glutathione S-transferase superfamily genes that encodes enzymes involved in the phase II xenobiotic metabolism may lead head and neck cancer development. In this study we investigate the association of A313G and C341T GSTP1 polymorphisms, GSTM1 and GSTT1 null genotypes in the head and neck cancer development, interactions between these polymorphisms,the tumor histopathologic parameters and risk factors (smoking and drinking) were also evaluated in the case–control study. 775 individuals (261 patients/514 controls) were included in the study. Molecular analyzes were performed by PCR and PCR–RFLP; and statistical analyzes by Chi square and multiple logistic regression. Chi square test showed that only the genotype frequencies for GSTM1 and GSTT1 were in Hardy–Weinberg disequilibrium in both groups. Significant results with p ≤ 0.05 showed that age ≥ 48 years (OR = 11.87; 7.55–18.65), smoking (OR = 4.25; 2.70–6.69), drinking (OR = 1.59; 1.02–2.46) were possible predictors for the head and neck cancer development and the presence of A313G GSTP1 polymorphism (OR = 0.62; 0.42–0.92) decreased the risk for this disease. Individuals with the 313AG/GG GSTP1 and age ≥ 48 years (OR = 0.59; 0.38–0.91), male gender (OR = 0.54; 0.35–0.83), smokers (OR = 0.63; 0.40–0.99) and drinkers (OR = 0.57; 0.35–0.95); the GSTM1 null genotype and age < 48 years (OR = 2.46; 1.09–5.55); the GSTT1 null genotype and primary anatomical sites of pharynx (OR = 0.37; 0.17–0.79) and larynx (OR = 3.60; 1.93–6.72), can modulate the risk for the disease development. The variables age ≥ 48 years, smoking and drinking can be predictors for head and neck cancer development; moreover, A313G GSTP1 polymorphism, GSTM1 and GSTT1 null genotypes can modulate the risk for this disease.