No abstract

This paper addresses the question of whether p-hydroxybenzoic acid, the common metabolite of parabens, possesses oestrogenic activity in human breast cancer cell lines. The alkyl esters of p-hydroxybenzoic acid (parabens) are used widely as preservatives in consumer products to which the human population is exposed and have been shown previously to possess oestrogenic activity and to be present in human breast tumour tissue, which is an oestrogen-responsive tissue. Recent work has shown p-hydroxybenzoic acid to give an oestrogenic response in the rodent uterotrophic assay. We report here that p-hydroxybenzoic acid possesses oestrogenic activity in a panel of assays in human breast cancer cell lines. p-Hydroxybenzoic acid was able to displace [(3)H]oestradiol from cytosolic oestrogen receptor of MCF7 human breast cancer cells by 54% at 5 x 10(6)-fold molar excess and by 99% at 10(7)-fold molar excess. It was able to increase the expression of a stably integrated oestrogen responsive reporter gene (ERE-CAT) at a concentration of 5 x 10(-4) M in MCF7 cells after 24 h and 7 days, which could be inhibited by the anti-oestrogen ICI 182 780 (Faslodex, fulvestrant). Proliferation of two human breast cancer cell lines (MCF7, ZR-75-1) could be increased by 10(-5) M p-hydroxybenzoic acid. Following on from previous studies showing a decrease in oestrogenic activity of parabens with shortening of the linear alkyl chain length, this study has compared the oestrogenic activity of p-hydroxybenzoic acid where the alkyl grouping is no longer present with methylparaben, which has the shortest alkyl group. Intrinsic oestrogenic activity of p-hydroxybenzoic acid was similar to that of methylparaben in terms of relative binding to the oestrogen receptor but its oestrogenic activity on gene expression and cell proliferation was lower than that of methylparaben. It can be concluded that removal of the ester group from parabens does not abrogate its oestrogenic activity and that p-hydroxybenzoic acid can give oestrogenic responses in human breast cancer cells.

SummaryTissue generation and repair requires a stepwise process of cell fate restriction to ensure that adult stem cells differentiate in a timely and appropriate manner. A crucial role has been implicated for Polycomb-group (PcG) proteins and the H3K27me3 repressive histone mark in coordinating the transcriptional programmes necessary for this process, but the targets and developmental timing for this repression remain unclear. To address these questions, we generated novel genome-wide maps of H3K27me3 and H3K4me3 in freshly isolated muscle stem cells. These data, together with the analysis of two conditional Ezh2-null mouse strains, identified a critical proliferation phase in which Ezh2 activity is essential. Mice lacking Ezh2 in satellite cells exhibited decreased muscle growth, severely impaired regeneration and reduced stem cell number, due to a profound failure of the proliferative progenitor population to expand. Surprisingly, deletion of Ezh2 after the onset of terminal differentiation did not impede muscle repair or homeostasis. Using these knockout models and the RNA-Seq and ChIP-Seq datasets, we show that Ezh2 does not regulate the muscle differentiation process in vivo. These results emphasise the lineage and cell-type-specific functions of Ezh2 and Polycomb repressive complex 2.

Since the alkyl esters of p-hydroxybenzoic acid (parabens) can be measured intact in the human breast and possess oestrogenic properties, it has been suggested that they could contribute to an aberrant burden of oestrogen signalling in the human breast and so play a role in the rising incidence of breast cancer. However, although parabens have been shown to regulate a few single genes (reporter genes, pS2, progesterone receptor) in a manner similar to that of 17beta-oestradiol, the question remains as to the full extent of the similarity in the overall gene profile induced in response to parabens compared with 17beta-oestradiol. The GE-Amersham CodeLink 20 K human expression microarray system was used to profile the expression of 19881 genes in MCF7 human breast cancer cells following a 7-day exposure to 5 x 10(-4) M methylparaben, 10(-5) M n-butylparaben and 10(-8) M 17beta-oestradiol. At these concentrations, the parabens gave growth responses in MCF7 cells of similar magnitude to 17beta-oestradiol. The study identified genes which are upregulated or downregulated to a similar extent by methylparaben, n-butylparaben and 17beta-oestradiol. However, the majority of genes were not regulated in the same way by all three treatments. Some genes responded differently to parabens from 17beta-oestradiol, and furthermore, differences in expression of some genes could be detected even between the two individual parabens. Therefore, although parabens possess oestrogenic properties, their mimicry in terms of global gene expression patterns is not perfect and differences in gene expression profiles could result in consequences to the cells that are not identical to those following exposure to 17beta-oestradiol.

Stem cell function is essential for the maintenance of adult tissue homeostasis. Controlling the balance between selfrenewal and differentiation is crucial to maintain a receptive satellite cell pool capable of responding to growth and regeneration cues. The mitogen-activated protein kinase p38a has been implicated in the regulation of these processes but its influence in adult muscle remains unknown. Using conditional satellite cell p38a knockout mice we have demonstrated that p38a restricts excess proliferation in the postnatal growth phase while promoting timely myoblast differentiation. Differentiation was still able to occur in the p38a-null satellite cells, however, but was delayed. An absence of p38a resulted in a postnatal growth defect along with the persistence of an increased reservoir of satellite cells into adulthood. This population was still capable of responding to cardiotoxin-induced injury, resulting in complete, albeit delayed, regeneration, with further enhancement of the satellite cell population. Increased p38c phosphorylation accompanied the absence of p38a, and inhibition of p38c ex vivo substantially decreased the myogenic defect. We have used genomewide transcriptome analysis to characterize the changes in expression that occur between resting and regenerating muscle, and the influence p38a has on these expression profiles. This study provides novel evidence for the fundamental role of p38a in adult muscle homeostasis in vivo.