Mozhgan Rasti

MTHFR C677T and A1298C variant genotypes and the risk of microsatellite instability among Iranian colorectal cancer patients.

Methylenetetrahydrofolate reductase (MTHFR) is a key enzyme in the folate metabolic pathway. We aimed to test the hypothesis that C677T and A1298C variants of MTHFR predispose to microsatellite instable (MSI) colorectal cancer. We determined MTHFR genotypes in 175 sporadic colorectal cancer patients and a total of 231 normal controls in Shiraz, Southern Iran. Among the genotypes found in our samples, MTHFR CT and CT+TT were associated with increased risk for CRC incidence [odds ratio (OR)=2.4, 95% confidence interval (95%CI)=1.8-4.4; OR=2.4, 95%CI=1.6-3.6, respectively]. Double heterozygotes 677CT/1298AC and double homozygote 677TT/1298AA and 677CC/1298CC genotypes also showed a significantly increased risk of developing CRC compared with the wild-type 677CC/1298AA genotypes of the controls. Among the 151 tumors tested, 36 (23.8%) were MSI+. MSI was more common in proximal tumors (OR=10.4; 95%CI=3.9-27.8) and in smokers (OR=2.9; 95%CI=1.3-6.7). In a case-control comparison, the MTHFR 677CT+TT genotype was strongly associated with MSI (OR=2.6; 95%CI=1.3-5.3). Hypermethylation of mismatch repair genes was positively related with MSI incidence in these tumor series (P=0.00). Our data suggest that the MTHFR 677CT+TT variant genotype may be a risk factor for MSI+ cancer.

p53 Binds to Estrogen Receptor 1 Promoter in Human Breast Cancer Cells

Abstractp53 is a tumor suppressor protein that regulates estrogen receptor 1 (ESR1) expression. To investigate the mechanism of ESR1 gene regulation by p53, chromatin immunoprecipitation was applied to assess the binding of p53, DNMT1, HDAC1 and MeCP2 to both silenced ESR1 promoter in MDA-MB-468 cells and active ESR1 promoter in MCF-7 breast cancer cells. The results of chromatin immunoprecipitation experiments showed that p53 protein binds to both unmethylated CpG island of the ESR1 promoter in the ER-positive MCF-7 and the hypermethylated ESR1 promoter in the ER-negative MDA-MB-468 cells. However, repression complex including DNMT1, HDAC1 and MeCP2 is only associated with silenced ESR1 in ER-negative MDA-MB-468 human breast cancer cells. In addition, ectopically expressed wild type p53 failed to reactivate the ESR1 gene in these cells. These results suggest that specific p53 mutations may contribute to loss of estrogen receptor α expression in breast tumors and also support the hypothesis that mutant p53 is likely to impact DNA methylation.

UBE2Q1 in a Human Breast Carcinoma Cell Line: Overexpression and Interaction with p53

The p53 tumor suppressor protein is a principal mediator of growth arrest, senescence, and apoptosis in response to a broad array of cellular damage. p53 is a substrate for the ubiquitin-proteasome system, however, the ubiquitin-conjugating enzymes (E2s) involved in p53 ubiquitination have not been well studied. UBE2Q1 is a novel E2 ubiquitin conjugating enzyme gene. Here, we investigated the effect of UBE2Q1 overexpression on the level of p53 in the MDA-MB-468 breast cancer cell line as well as the interaction between UBE2Q1 and p53. By using a lipofection method, the p53 mutated breast cancer cell line, MDA-MB-468, was transfected with the vector pCMV6-AN-GFP, containing UBE2Q1 ORF. Western blot analysis was employed to verify the overexpression of UBE2Q1 in MDA-MB-468 cells and to evaluate the expression level of p53 before and after cell transfection. Immunoprecipitation and GST pull-down protocols were used to investigate the binding of UBE2Q1 to p53. We established MDA-MB-468 cells that transiently expressed a GFP fusion proteins containing UBE2Q1 (GFP-UBE2Q1). Western blot analysis revealed that levels of p53 were markedly lower in UBE2Q1 transfected MDA-MB-468 cells as compared with control MDA-MB-468 cells. Both in vivo and in vitro data showed that UBE2Q1 co-precipitated with p53 protein. Our data for the first time showed that overexpression of UBE2Q1can lead to the repression of p53 in MDA-MB-468 cells. This repression of p53 may be due to its UBE2Q1 mediated ubiquitination and subsequent proteasome degradation, a process that may involve direct interaction of UBE2Q1with p53.

Mutant p53 binds to estrogen receptor negative promoter via DNMT1 and HDAC1 in MDA-MB-468 breast cancer cells.

DNA methylation and histone deacetylation are two epigenetic mechanisms involved in the lack of estrogen receptor (ER) expression. Our previous studies demonstrated that mutant p53 along with repression complex proteins including DNMT1, HDAC1 and MeCP2 is associated with ER-negative promoter in MDA-MB-468 cells. To elucidate the molecular mechanism of estrogen receptor 1 (ESR1) gene silencing in these cells, we down-regulated DNMT1 and HDAC1 expression using siRNAs and studied the ability of DNMT1, HDAC1, MeCP2 and p53 in binding to ESR1 promoter CpG island. Our results showed that DNMT1 or HDAC1 down-regulation disassembled the repression complex proteins and mutant p53 from ER-negative promoter. The partial demethylation of ESR1 promoter and ER re-expression in down-regulated cells supports these findings. In vivo binding studies demonstrated that mutation of p53 protein in this cell line did not affect its binding capacity to DNMT1, HDAC1 and MeCP2 proteins. Our observations suggest that not only histone deacetylase activity of HDAC1 contributes to inactivation of methylated ESR1 gene but also HDAC1 presence on ESR1 promoter is important for assembly of DNMT1 in repression complex. In addition, our data revealed that mutant p53 protein binds to the promoter of ESR1 through direct interaction with HDAC1 and indirect interaction with DNMT1, MeCP2 proteins in the ER-negative MDA-MB-468 cells.

Additive effect of recombinant Mycobacterium tuberculosis ESAT-6 protein and ESAT-6/CFP-10 fusion protein in adhesion of macrophages through fibronectin receptors

BACKGROUND/PURPOSE Tuberculous granulomas are the sites of interaction between the T cells, macrophages, and extracellular matrix (ECM) to control the infection caused by Mycobacterium tuberculosis (M. tuberculosis). A predominant role of RD-1-encoded secretory proteins, early secreted antigenic target-6 (ESAT-6), and culture filtrate protein-10 (CFP-10) in the formation of granulomas has recently been emphasized. However, the precise molecular events that induce the formation of these granulomatous structures are yet to be elucidated. Macrophages use integrins to adhere to fibronectin (FN) as a major component of the ECM. The major goal of this study was to investigate whether recombinant M. tuberculosis antigens can modulate integrin-mediated macrophage adhesion. METHODS Differentiated THP-1 cell line was stimulated with recombinant ESAT-6, CFP-10, and ESAT-6/CFP-10 proteins and evaluated for alterations in the expression levels of α5β1 and α4β1 by semiquantitative real-time polymerase chain reaction. The role of these recombinant antigens in the cytoskeleton rearrangement was determined by adhesion assay and immunofluorescent microscopy. RESULTS Our data showed that ESAT-6 and ESAT-6/CFP-10 fusion proteins could induce adhesion of macrophages to FN through α4β1 integrin. An increased expression level of α4β1 integrin in comparison with α5β1 integrin in differentiated THP-1 cells was also observed. Results of immunofluorescence studies showed that recombinant proteins-treated THP-1 cells form well-organized stress fibers and focal contacts containing vinculin compared with untreated THP-1 cells. CONCLUSION Increased expression level of α4β1 in differentiated THP-1 cells could suggest the important role of α4β1 integrin in adhesion and focal contact formation of macrophages exposed to M. tuberculosis antigens.