study of DNA methyltransferase-3B promoter variant genotype among Iranian sporadic breast cancer patients

Background: DNA methyltransferase-3B [DNMT3B] is an important enzyme responsible for maintaining the DNA methylation pattern in eukaryotic cells. In this study we have investigated the correlation between the 46359C[rightwards arrow]T polymorphism in the DNMT3B gene and the risk of breast cancer incidence among sporadic breast cancer patients in Fars Province, Southern Iran

Methods: In this case-control study, 100 breast cancer patients and 138 healthy control subjects were genotyped for the DNMT3B gene by the polymerase chain reaction-restriction fragment length polymorphism method

Results: The genotype frequency in the case [CC 27%, CT 47%, TT 26%] group significantly [P=0.008] differed from the control [CC 19.56%, CT 67.3%, TT 13%] group. We observed a decreased association between the CT genotype and lymph node involvement in breast cancer patients. Our results have shown that in comparison to the homozygous CC genotype carriers the DNMT3B-CT genotype has a significantly lower risk for breast cancer [OR=0.515, 95% CI=0.267-0.994, P=0.048]

Conclusion: Our case-control study showed that the CT genotype was significantly associated with decreased breast cancer risk. Consistent with these results, a significant decrease of CT genotype among lymph node positive breast cancer patients was observed. However, a larger study population with more clinical data is needed to confirm these results

Quantitative analysis of ErbB1 and ErbB2 genes amplification by a high performance liquid chromatography

Genes for human epidermal growth factor receptors B1 [ErbB1] and B2 [ErbB2] were amplified in breast and ovarian cancers. Both of them were associated with aggressive disease and worse prognosis. The ErbB1 or ErbB2 status of a tumor may provide an indication of the response to ErbB1 and ErbB2 -targeted therapies. For accurate and rapid assessment of amplification of ErbB1 and ErbB2 oncogenes, a High Performance Liquid Chromatography [HPLC] method was developed in this study. DNA was extracted from 30 primary breast tumors and 20 blood samples of healthy donors. ErbB1 and ErbB2 genes along with a reference gene were co-amplificated by Polymerase Chain Reaction [PCR]. The PCR products were separated and quantified using an anion- exchange column within 30 min and in a single step. Optimum resolution was obtained when a sodium chloride gradient and a column temperature of 35°C were used. The results of HPLC analysis of ErbB1 and ErbB2 PCR products were compared with real time PCR method as a gold standard test for 7 tumor samples. The proposed HPLC method was confirmed by real time PCR method. Twenty two and ten of the specimens in our breast cancer cohort showed more than a two-fold amplification of ErbB2 and ErbB1 oncogenes, respectively. Our results were confirmed by real time PCR and showed that HPLC method is a specific, cheap and clinically applicable analytical approach for assessment of ErbB1 and ErbB2 statuses in breast tumors

Effects of L-carnitine and pentoxifylline on the activity of lactate dehydrogenase C[4] isozyme and motility of testicular spermatozoa in mice

Extracted sperm from the testis have poor motility. Moreover, their motility changes during their journey through epidydimis. Meanwhile, they face high concentration of L-carnitine. In addition, lactate dehydrogenase C[4] [LDH-C[4]] gene disorders has been shown to cause impaired sperm motility, leading to infertility in male mice. The aim of this study was to evaluate sperm motility and LDH-C[4] enzyme activity upon L-carnitine [LC] and Pentoxifylline [PTX] administrations in mice. We extracted testicular sperm of 48 mice and divided them into three equal parts. One part was incubated with Ham's F10 medium [control], the other parts were treated with Ham's F10 containing LC and PTX with a final concentration of 1.76 mM, for 30 min at room temperature. Sperm motility was assessed according to the World Health Organization [WHO] criteria. Sperm LDH-C[4] enzyme activity was measured by spectrophotometery method. Statistical analyses were performed using ANOVA and Fisher's LSD test, and a p-value less than 0.05 was considered as a statistically significant difference. Sperm motility increased after 30 min of incubation in LC- and PTX-treated group [p<0.001]. LC and PTX administrations showed a significant increase in the LDHC[4] enzyme activity of sperm compared to that of the controls after 30 min [P=0.04 and 0.01, respectively]. The effects of LC and PTX on motility of sperm can be explained by an increase in LDH-C[4] enzyme activity that may influence male fertility status. We suggest that LC as a non-toxic antioxidant is more suitable for use in assisted reproductive technique protocols than PTX

Production and evaluation of polyclonal rabbit antihuman p53 antibody using bacterially expressed glutathione S-transferase-p53 fusion protein

P53 is a key tumor suppressor gene that is targeted for inactivation during human tumorigenesis. In this study, we produced and characterized polyclonal antihuman p53 antibody. The cDNA encoding the complete human p53 protein was cloned into pGEX-4T-1 and expressed in Escherichia coli as a fusion protein with Schistosoma japonicum glutathione S-transferase [GST]. The rabbits were immunized with the purified p53 recombinant protein. The obtained antisera were purified to increase the specificity of recognition. The sensitivity and specificity of the produced antibody was analyzed by enzyme-linked immunosorbent, immunoblot, immunofluorescence and chromatin immunoprecipitation assays. Enzyme-linked immunosorbent assay showed that immunization with purified GST-p53 produced the high titer [1:10000] polyclonal antibodies with high specificity. Anti-p53 antibody allowed the sensitive detection of native p53 protein in immunoblotting, immunofluorescence and chromatin immunoprecipitation assays. Our results showed that anti-GST-p53 antibody provides a good means for studying the p53 expression pattern and its binding ability to other proteins in tumors

Optimal electroporation condition for small interfering RNA transfection into MDA-MB-468 cell line

Electroporation is a valuable tool for small interfering RNA [siRNA] delivery into cells because it efficiently transforms a wide variety of cell types. Since electroporation condition for each cell type must be determined experimentally, this study presents an optimal electroporation strategy to reproducibly and efficiently transfect MDA-MB 468 human breast cancer cell with siRNA. To identify the best condition, the cells were firstly electroporated without siRNA and cell viability was determined by trypan blue and MTT assays. Then siRNA transfection in the best condition was performed. Western blot analysis was used for monitoring successful siRNA transfection. Results: The best condition for electroporation of this cell line was 220 volt and 975 microF in exponential decay using the Gene Pulser X cell electroporation system. Our data demonstrated that by using proper electroporation condition, DNA methyl transferase mRNA was silenced by 10 nmol DNMT1 siRNA in MDA-MB 468 cells when compared with negative control siRNA electroporation. Analysis of cell viability demonstrated that optimal electroporation condition resulted in 74% and 78% cell viability by trypan blue staining and MTT assay, respectively. Transfection of the MDA-MB-468 breast cancer cell line with siRNA in the obtained electroporation condition was successful and resulted in effective gene silencing and high cellular viability

Association between HIC1 and RASSF1A promoter hypermethylation with MTHFD1 G1958A polymorphism and clinicopathological features of breast cancer in Iranian patients

Ras-associated domain family 1 [RASSF1A] and hypermethylated in cancer [HIC1] genes are methylated more frequently in breast cancer. Genetic factors that alter the DNA methylation levels in normal and tumor tissues could therefore influence the susceptibility to this tumor phenotype. We determined the frequency of aberrant methylation of HIC1 and RASSF1A gene promoters and their association with methylene tetrahydrofolate dehydrogenase [MTHFD1] G1958A polymorphism and major clinical and pathological features of breast cancer in Iranian women. DNA was extracted from 81 primary breast tumors and 100 control blood samples. Gene promoter methylation was analyzed by methylationspecific polymerase chain reaction. Eighty four percent of the breast cancer samples showed total methylation in at least one of two tested loci. We detected HIC1 hypermethylation in 79% of invasive and metastasis tumors and RASSF1A gene hypermethylation in 51% of them. We found no association between HIC1 and RASSF1A gene hypermethylation and MTHFD1 G1958A polymorphism, but a significant correlation between methylation of HIC1 and RASSF1A promoters was indicated [r = 0.24, P = 0.02]. There was a combination between hypermethylation of HIC1 locus and nodal involvement in the studied population [p=0.03]. We found a significant association between total methylation and nodal involvement [P=0.01] as well as tumor size more than 2 cm in all cases [P=0.02]. Methylation of HIC1 and RASSF1A promoters can be used as epigenetic markers to detect the malignant progression of breast carcinoma in Iranian women patients