Endocrine Disruptor Regulation of MicroRNA Expression in Breast Carcinoma Cells

Several environmental agents termed "endocrine disrupting compounds" or EDCs have been reported to bind and activate the estrogen receptor-α (ER). The EDCs DDT and BPA are ubiquitously present in the environment, and DDT and BPA levels in human blood and adipose tissue are detectable in most if not all women and men. ER-mediated biological responses can be regulated at numerous levels, including expression of coding RNAs (mRNAs) and more recently non-coding RNAs (ncRNAs). Of the ncRNAs, microRNAs have emerged as a target of estrogen signaling. Given the important implications of EDC-regulated ER function, we sought to define the effects of BPA and DDT on microRNA regulation and expression levels in estrogen-responsive human breast cancer cells.To investigate the cellular effects of DDT and BPA, we used the human MCF-7 breast cancer cell line, which is ER (+) and hormone sensitive. Our results show that DDT and BPA potentiate ER transcriptional activity, resulting in an increased expression of receptor target genes, including progesterone receptor, bcl-2, and trefoil factor 1. Interestingly, a differential increase in expression of Jun and Fas by BPA but not DDT or estrogen was observed. In addition to ER responsive mRNAs, we investigated the ability of DDT and BPA to alter the miRNA profiles in MCF-7 cells. While the EDCs and estrogen similarly altered the expression of multiple microRNAs in MCF-7 cells, including miR-21, differential patterns of microRNA expression were induced by DDT and BPA compared to estrogen.We have shown, for the first time, that BPA and DDT, two well known EDCs, alter the expression profiles of microRNA in MCF-7 breast cancer cells. A better understanding of the molecular mechanisms of these compounds could provide important insight into the role of EDCs in human disease, including breast cancer

Allele-Specific, Age-Dependent and BMI-Associated DNA Methylation of Human MCHR1

Background: Melanin-concentrating hormone receptor 1 (MCHR1) plays a significant role in regulation of energy balance, food intake, physical activity and body weight in humans and rodents. Several association studies for human obesity showed contrary results concerning the SNPs rs133072 (G/A) and rs133073 (T/C), which localize to the first exon of MCHR1. The variations constitute two main haplotypes (GT, AC). Both SNPs affect CpG dinucleotides, whereby each haplotype contains a potential methylation site at one of the two SNP positions. In addition, 15 CpGs in close vicinity of these SNPs constitute a weak CpG island. Here, we studied whether DNA methylation in this sequence context may contribute to population- and age-specific effects of MCHR1 alleles in obesity. \ud Principal Findings: We analyzed DNA methylation of a 315 bp region of MCHR1 encompassing rs133072 and rs133073 and the CpG island in blood samples of 49 individuals by bisulfite sequencing. The AC haplotype shows a significantly higher methylation level than the GT haplotype. This allele-specific methylation is age-dependent. In young individuals (20â\u80\u9330 years) the difference in DNA methylation between haplotypes is significant; whereas in individuals older than 60 years it is not detectable. Interestingly, the GT allele shows a decrease in methylation status with increasing BMI, whereas the methylation of the AC allele is not associated with this phenotype. Heterozygous lymphoblastoid cell lines show the same pattern of allele-specific DNA methylation. The cell line, which exhibits the highest difference in methylation levels between both haplotypes, also shows allele-specific transcription of MCHR1, which can be abolished by treatment with the DNA\ud methylase inhibitor 5-aza-2'-deoxycytidine.\ud Conclusions:We show that DNA methylation at MCHR1 is allele-specific, age-dependent, BMI-associated and affects transcription. Conceivably, this epigenetic regulation contributes to the age- and/or population specific effects reported for MCHR1 in several human obesity studies.\ud \ud doi: 10.1371/journal.pone.0017711\u

Aberrant epigenetic changes and gene expression in cloned cattle dying around birth

<p>Abstract</p> <p>Background</p> <p>Aberrant reprogramming of donor somatic cell nuclei may result in many severe problems in animal cloning. To assess the extent of abnormal epigenetic modifications and gene expression in clones, we simultaneously examined DNA methylation, histone H4 acetylation and expression of six genes (<it>β-actin</it>, <it>VEGF</it>, <it>oct4</it>, <it>TERT</it>, <it>H19 </it>and <it>Igf2</it>) and a repetitive sequence (<it>art2</it>) in five organs (heart, liver, spleen, lung and kidney) from two cloned cattle groups that had died at different stages. In the ED group (early death, n = 3), the cloned cattle died in the perinatal period. The cattle in the LD group (late death, n = 3) died after the perinatal period. Normally reproduced cattle served as a control group (n = 3).</p> <p>Results</p> <p>Aberrant DNA methylation, histone H4 acetylation and gene expression were observed in both cloned groups. The ED group showed relatively fewer severe DNA methylation abnormalities (p < 0.05) but more abnormal histone H4 acetylations (p < 0.05) and more abnormal expression (p < 0.05) of the selected genes compared to the LD group. However, our data also suggest no widespread gene expression abnormalities in the organs of the dead clones.</p> <p>Conclusion</p> <p>Deaths of clones may be ascribed to abnormal expression of a very limited number of genes.</p

Inhibition of focal adhesion kinase suppresses the adverse phenotype of endocrine-resistant breast cancer cells and improves endocrine response in endocrine-sensitive cells

International audienceAcquired resistance to endocrine therapy in breast cancer is a major clinical problem. Previous reports have demonstrated that cell models of acquired endocrine resistance have altered cell–matrix adhesion and a highly migratory phenotype, features which may impact on tumour spread in vivo. Focal adhesion kinase (FAK) is an intracellular kinase that regulates signalling pathways central to cell adhesion, migration and survival and its expression is frequently deregulated in breast cancer. In this study, we have used the novel FAK inhibitor PF573228 to address the role of FAK in the development of endocrine resistance. Whilst total-FAK expression was similar between endocrine-sensitive and endocrine-resistant MCF7 cells, FAK phosphorylation status (Y397 or Y861) was altered in resistance. PF573228 promoted a dose-dependent inhibition of FAK phosphorylation at Y397 but did not affect other FAK activation sites (pY407, pY576 and pY861). Endocrine-resistant cells were more sensitive to these inhibitory effects versus MCF7 (mean IC for FAK pY397 inhibition: 0.43 μM, 0.05 μM and 0.13 μM for MCF7, TamR and FasR cells, respectively). Inhibition of FAK pY397 was associated with a reduction in TamR and FasR adhesion to, and migration over, matrix components. PF573228 as a single agent (0–1 μM) did not affect the growth of MCF7 cells or their endocrine-resistant counterparts. However, treatment of endocrine-sensitive cells with PF573228 and tamoxifen combined resulted in greater suppression of proliferation versus single agent treatment. Together these data suggest the importance of FAK in the process of endocrine resistance, particularly in the development of an aggressive, migratory cell phenotype and demonstrate the potential to improve endocrine response through combination treatment

Structure of the alpha-fetoprotein gene in the mouse.

The mouse alpha-fetoprotein mRNA is the product of a single-copy gene whose mRNA coding sequences are represented discontinuously in the genome. Several EcoRI genomic fragments which contain portions of the alpha-fetoprotein gene have been cloned using the EK2 vector lambda gt WES . lambda B. In addition, a mouse genomic library has been screened to obtain a 15.75-kilobase segment of DNA that includes more than 85% of the alpha-fetoprotein coding sequence. Analyses by restriction endonuclease mapping and electron microscopy showed that the mRNA sequence is interrupted by at least 11 intervening sequences which occupy 90% of the cloned DNA