Ataxia telangiectasia mutated (ATM) inhibition transforms human mammary gland epithelial cells.

Carriers of mutations in the cell cycle checkpoint protein kinase ataxia telangiectasia mutated (ATM), which represent 1-2% of the general population, have an increased risk of breast cancer. However, experimental evidence that ATM deficiency contributes to human breast carcinogenesis is lacking. We report here that in MCF-10A and MCF-12A cells, which are well established normal human mammary gland epithelial cell models, partial or almost complete stable ATM silencing or pharmacological inhibition resulted in cellular transformation, genomic instability, and formation of dysplastic lesions in NOD/SCID mice. These effects did not require the activity of exogenous DNA-damaging agents and were preceded by an unsuspected and striking increase in cell proliferation also observed in primary human mammary gland epithelial cells. Increased proliferation correlated with a dramatic, transient, and proteasome-dependent reduction of p21(WAF1/CIP1) and p27(KIP1) protein levels, whereas little or no effect was observed on p21(WAF1/CIP1) or p27(KIP1) mRNAs. p21(WAF1/CIP1) silencing also increased MCF-10A cell proliferation, thus identifying p21(WAF1/CIP1) down-regulation as a mediator of the proliferative effect of ATM inhibition. Our findings provide the first experimental evidence that ATM is a human breast tumor suppressor. In addition, they mirror the sensitivity of ATM tumor suppressor function and unveil a new mechanism by which ATM might prevent human breast tumorigenesis, namely a direct inhibitory effect on the basal proliferation of normal mammary epithelial cells.

Specific transgenerational imprinting effects of the endocrine disruptor methoxychlor on male gametes.

Endocrine-disrupting chemicals (EDCs), among which methoxychlor (MXC), have been reported to affect the male reproductive system. This study evaluates the possible deleterious effects of MXC on imprinted genes. After administration of the chemical in adult male mice or in pregnant mice we analyzed by pyrosequencing possible methylation defects in two paternally imprinted (H19 and Meg3 (Gtl2)) and three maternally imprinted (Mest (Peg1), Snrpn, and Peg3) genes in the sperm and in the tail, liver, and skeletal muscle DNAs of the adult male mice and of the male offspring. MXC treatment of adult mice decreased the percentages of methylated CpGs of Meg3 and increased those of Mest, Snrpn, and Peg3 in the sperm DNA. MXC treatment of pregnant mice decreased the mean sperm concentrations by 30% and altered the methylation pattern of all the imprinted genes tested in the F1 offspring. In the latter case, MXC effects were transgenerational but disappeared gradually from F1 to F3. MXC did not affect imprinting in the somatic cells, suggesting that it exerts its damaging effects via the process of reprogramming that is unique to gamete development. A systematic analysis at the CpG level showed a heterogeneity in the CpG sensitivity to MXC. This observation suggests that not only DNA methylation but also other epigenetic modifications can explain the transgenerational effects of MXC. The reported effects of EDCs on human male spermatogenesis might be mediated by complex imprinting alterations analogous to those described in this study.

Transgenerational effects of the endocrine disruptor vinclozolin on the methylation pattern of imprinted genes in the mouse sperm.

Endocrine-disrupting chemicals (EDCs), among which is the antiandrogen vinclozolin (VCZ), have been reported to affect the male reproductive system. In this study, VCZ was administered to pregnant mice at the time of embryo sex determination, and its possible effects on the differentially methylated domains (DMDs) of two paternally (H19 and Gtl2) and three maternally (Peg1, Snrpn, and Peg3) imprinted genes were tested in the male offspring. The CpGs methylation status within the five gene DMDs was analyzed in the sperm, tail, liver, and skeletal muscle DNAs by pyrosequencing. In the sperm of controls, the percentages of methylated CpGs were close to the theoretical values of 100 and 0% in paternally or maternally imprinted genes respectively. VCZ decreased the percentages of methylated CpGs of H19 and Gtl2 (respective values 83.1 and 91.5%) and increased those of Peg1, Snrpn, and Peg3 (respective values 11.3, 18.3, and 11.2%). The effects of VCZ were transgenerational, but they disappeared gradually from F1 to F3. The mean sperm concentration of the VCZ-administered female offspring was only 56% of that of the controls in the F1 offspring, and it was back to normal values in the F2 and F3 offspring. In the somatic cells of controls, the percentages of methylated CpGs were close to the theoretical value of 50% and, surprisingly, VCZ altered the methylation of Peg3. We propose that the deleterious effects of VCZ on the male reproductive system are mediated by imprinting defects in the sperm. The reported effects of EDCs on human male spermatogenesis might be mediated by analogous imprinting alterations.

Prenatal Exposure to DEHP Affects Spermatogenesis and Sperm DNA Methylation in a Strain-Dependent Manner.

Di-(2-ethylhexyl)phtalate (DEHP) is a plasticizer with endocrine disrupting properties found ubiquitously in the environment and altering reproduction in rodents. Here we investigated the impact of prenatal exposure to DEHP on spermatogenesis and DNA sperm methylation in two distinct, selected, and sequenced mice strains. FVB/N and C57BL/6J mice were orally exposed to 300 mg/kg/day of DEHP from gestation day 9 to 19. Prenatal DEHP exposure significantly decreased spermatogenesis in C57BL/6J (fold-change = 0.6, p-value = 8.7*10-4), but not in FVB/N (fold-change = 1, p-value = 0.9). The number of differentially methylated regions (DMRs) by DEHP-exposure across the entire genome showed increased hyper- and decreased hypo-methylation in C57BL/6J compared to FVB/N. At the promoter level, three important subsets of genes were massively affected. Promoters of vomeronasal and olfactory receptors coding genes globally followed the same trend, more pronounced in the C57BL/6J strain, of being hyper-methylated in DEHP related conditions. In contrast, a large set of micro-RNAs were hypo-methylated, with a trend more pronounced in the FVB/N strain. We additionally analyze both the presence of functional genetic variations within genes that were associated with the detected DMRs and that could be involved in spermatogenesis, and DMRs related with the DEHP exposure that affected both strains in an opposite manner. The major finding in this study indicates that prenatal exposure to DEHP can decrease spermatogenesis in a strain-dependent manner and affects sperm DNA methylation in promoters of large sets of genes putatively involved in both sperm chemotaxis and post-transcriptional regulatory mechanisms.

Effect of developmental dioxin exposure on methylation and expression of specific imprinted genes in mice.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is an endocrine disruptor affecting the reproductive system in humans. The aim of this study was to evaluate the effects of TCDD administered to pregnant mice at two different doses (2-10 ng/kg/day), on imprinted genes in the male offspring. The degree of methylation and the mRNA expression of Snrpn, Peg3 and Igf2r were analyzed in the sperm, skeletal muscle and liver. TCDD administration (10 ng/kg/day) decreased the sperm count in the male offspring. It did not affect methylation but increased mRNA expression of Snrpn, Peg3, Igf2r and Air ncRNA. In muscle and liver, TCDD (10 ng/kg/day) induced increases in methylation and decreases in mRNA expression of Igf2r. These results show that the robust effects of TCDD on the mRNA expression of Snrpn, Peg3 and Igf2r genes in the sperm and of Igf2r in the muscle and liver are unrelated to changes in methylation in their respective genes.

Prenatal exposure to ethanol: a specific effect on the H19 gene in sperm.

Alcohol exposure during pregnancy induces a range of disorders in the offspring. Methylation changes in imprinted genes may play a role in the teratogenic effects of alcohol. We evaluated the possible effects of alcohol administration in pregnant mice on the methylation pattern of 5 imprinted genes (H19, Gtl2, Peg1, Snrpn and Peg3) in somatic and sperm cell DNAs of the male offspring. The effects observed were a 3% (p < 0.005) decrease in the number of methylated CpGs of H19 in the F1 offspring sperm, a 4% (p < 0.005) decrease in the number of methylated CpGs of H19 in the F2 offspring brain and a 26% (p < 0.05) decrease in the mean sperm concentration. CpGs 1 and 2 of the H19 CTCF-binding site 2 exhibited significant methylation percentage losses. H19 CTCF-binding sites are important for the regulation of Igf2 gene expression. The hypomethylation of H19 may contribute to the decreased spermatogenesis in the offspring.

Superovulation in mice alters the methylation pattern of imprinted genes in the sperm of the offspring.

Some steps of the assisted reproduction techniques, such as superovulation, may interfere with imprinting reprogramming. In the present study, superovulation was induced in the mouse and its possible effects on the differentially methylated domains of 2 paternally (H19 and Gtl2) and 3 maternally (Peg1, Snrpn and Peg3) imprinted genes were tested in the male offspring over 2 generations. The CpGs methylation status was analyzed by pyro- and bisulfite sequencing. In liver, skeletal muscle and tail, no effect of superovulation could be observed. In the sperm, however, a significant 6% decrease in the number of methylated CpGs of H19 and significant 2.8- and 7.0-fold increases in those of Peg1 and Snrpn, respectively were observed following superovulation. The changes were still present in the H19 and Snrpn genes of the second generation offspring. This suggests that superovulation in the mother transgenerationally affects the offspring sperm methylation pattern.