Differential effects of overexpression of ERalpha and ERbeta in MCF10A immortalised, non-transformed human breast epithelail cells

Background: Cellular effects of oestrogen are mediated by two intracellular receptors ERα and ERβ. However, to compare responses mediated through these two receptors, experimental models are needed where ERα and ERβ are individually stably overexpressed in the same cell type. Methods: We compared the effects of stable overexpression of ERα and ERβ in the MCF10A cell line, which is an immortalised but non-transformed breast epithelial cell line without high endogenous ER expression. Results: Clones of MCF10A cells were characterised which stably overexpressed ERα (10A-ERα2, 10A-ERα13) or which stably overexpressed ERβ (10A-ERβ12, 10A-ERβ15). Overexpression of either ERα or ERβ allowed induction of an oestrogen-regulated ERE-LUC reporter gene by oestradiol which was not found in the untransfected cells. Oestradiol also increased proliferation of 10A-ERα13 and 10A-ERβ12 cells, but not untransfected cells, by 1.3-fold over 7 days. The phytoestrogen, genistein, which is reported to bind more strongly to ERβ than to ERα, could induce luciferase gene expression from an ERE-LUC reporter gene at concentrations of 10−6 M and 10−5 M but only in the clones overexpressing ERβ and not in those overexpressing ERα. Clone 10A-ERβ12 also yielded growth stimulation with 10-6 M genistein. Finally, the overexpression of ERα, but not ERβ, gave rise to increased growth in semi-solid methocel suspension culture in the presence of 70 nM oestradiol, suggesting that overexpression of ERα, but not ERβ, produces characteristics of a transformed phenotype. Conclusions: This provides a model system to compare effects of oestradiol with other oestrogenic ligands in cells stably overexpressing individually ERα or ERβ.

Parabens enable suspension growth of MCF-10A immortalized, non-transformed human breast epithelial cells

Parabens (alkyl esters of p-hydroxybenzoic acid) are used extensively as preservatives in consumer products, and intact esters have been measured in several human tissues. Concerns of a potential link between parabens and breast cancer have been raised, but mechanistic studies have centred on their oestrogenic activity and little attention has been paid to any carcinogenic properties. In the present study, we report that parabens can induce anchorage-independent growth of MCF-10A immortalized but non-transformed human breast epithelial cells, a property closely related to transformation and a predictor of tumour growth in vivo. In semi-solid methocel suspension culture, MCF-10A cells produced very few colonies and only of a small size but the addition of 5 × 10-4 M methylparaben, 10–5 M n-propylparaben or 10–5 M n-butylparaben resulted in a greater number of colonies per dish (P < 0.05 in each case) and an increased average colony size (P < 0.001 in each case). Dose-responses showed that concentrations as low as 10–6 M methylparaben, 10–7 M n-propylparaben and 10–7 M n-butylparaben could increase colony numbers (P = 0.016, P = 0.010, P = 0.008, respectively): comparison with a recent measurement of paraben concentrations in human breast tissue samples from 40 mastectomies (Barr et al., 2012) showed that 22/40 of the patients had at least one of the parabens at the site of the primary tumour at or above these concentrations. To our knowledge, this is the first study to report that parabens can induce a transformed phenotype in human breast epithelial cells in vitro, and further investigation is now justified into a potential link between parabens and breast carcinogenesis.

Cortical hot spots and labyrinths: why cortical neuromodulation for episodic migraine with aura should be personalized

Stimulation protocols for medical devices should be rationally designed. For episodic migraine with aura we outline model-based design strategies toward preventive and acute therapies using stereotactic cortical neuromodulation. To this end, we regard a localized spreading depression (SD) wave segment as a central element in migraine pathophysiology. To describe nucleation and propagation features of the SD wave segment, we define the new concepts of cortical hot spots and labyrinths, respectively. In particular, we firstly focus exclusively on curvature-induced dynamical properties by studying a generic reaction-diffusion model of SD on the folded cortical surface. This surface is described with increasing level of details, including finally personalized simulations using patient's magnetic resonance imaging (MRI) scanner readings. At this stage, the only relevant factor that can modulate nucleation and propagation paths is the Gaussian curvature, which has the advantage of being rather readily accessible by MRI. We conclude with discussing further anatomical factors, such as areal, laminar, and cellular heterogeneity, that in addition to and in relation to Gaussian curvature determine the generalized concept of cortical hot spots and labyrinths as target structures for neuromodulation. Our numerical simulations suggest that these target structures are like fingerprints, they are individual features of each migraine sufferer. The goal in the future will be to provide individualized neural tissue simulations. These simulations should predict the clinical data and therefore can also serve as a test bed for exploring stereotactic cortical neuromodulation.

Effects of aluminium chloride and aluminium chlorohydrate on DNA repair in MCF10A immortalised non-transformed human breast epithelial cells

Use of underarm aluminium (Al)-based antiperspirant salts may be a contributory factor in breast cancer development. At the 10th Keele meeting, Al was reported to cause anchorage-independent growth and double strand DNA breaks in MCF10A immortalised non-transformed human breast epithelial cells. We now report that exposure of MCF10A cells to Al chloride or Al chlorohydrate also compromised DNA repair systems. Longterm (19–21 weeks) exposure to Al chloride or Al chlorohydrate at a 10−4 M concentration resulted in reduced levels of BRCA1 mRNA as determined by real-time RT-PCR and BRCA1 protein as determined by Western immunoblotting. Reduced levels of mRNA for other DNA repair genes (BRCA2, CHK1, CHK2, Rad51, ATR) were also observed using real-time RT-PCR. Loss of BRCA1 or BRCA2 gene function has long been associated with inherited susceptibility to breast cancer but these results suggest that exposure to aluminium-based antiperspirant salts may also reduce levels of these key components of DNA repair in breast epithelial cells. If Al can not only damage DNA but also compromise DNA repair systems, then there is the potential for Al to impact on breast carcinogenesis.