CAPÍTULO IV. SERVICIO AL USUARIO
4.3 Servicios que ofrece la fonoteca
Much of the work published in the literature concerning 3D cultures has focused on the ER negative, non-tumourigenic MCF-10A cell line and has revealed mechanisms and genes involved in the formation and maintenance of the hollow lumen of acini. Limited work concerning the effects of 17β-estradiol on acini formation using the ERα negative, but ERβ positive MCF-10F cell line has demonstrated that E2 treatment results in cells losing their ability to form acini in collagen matrices, but suggests that this effect is ER-independent (Russo et al, 2002; 2003; 2009; 2010). In order to investigate the full spectrum of estrogenic effects on acini formation and the potential involvement of the ERα in these processes, a different cell line was, therefore, required. For that, a non-tumorigenic, ERα and ERβ positive cell line was selected: the MCF-12A cell line.
In Chapter II, we sought to confirm the receptor status of the cell lines to be used to further study the impact of estrogens upon proliferation and apoptosis in a 3D model. In addition to confirming the receptor status of MCF-12A cells, the receptor status of MCF-10A cells was investigated to confirm its ER negative phenotype and to ascertain whether this cell line was GPER-1 competent. Growth curves were also constructed for these cell lines in the presence of E2 and receptor antagonists for both the ER and GPER-1 to investigate the impact of E2 upon proliferation and whether this was mediated by the ER or GPER-1. Finally, the well-studied MCF-7 and MDA-MB-231 cell lines were used as comparison to investigate the effects of E2 upon proliferation in an ER competent and ERα negative tumourigenic cell line.
In Chapter III, we proceeded to optimise an established 3D model for use with MCF-12A cells which has previously been used with MCF-10A cells. After optimising the conditions required to successfully perform 3D culture of MCF-12A cells in reconstituted basement membrane (Matrigel), we
60 established a time-course for the morphogenesis of MCF-12A acini. This was done so that a comparison was available between normal MCF-12A acini formation and acini formation following treatment with estrogenic test compounds.
After establishing the normal morphogenesis of MCF-12A cells in 3D culture and selecting the estrogenic test compounds to perform subsequent 3D studies, a time-course for E2, BPA and n- propylparaben was established in Chapter III. Further to this, in Chapter IV, we decided investigate whether the malformations induced by the estrogenic test compounds were mediated by the ER or GPER-1, using antagonists for these receptors. This work has been submitted for publication (Marchese & Silva, 2012).
After the observation that both of these receptors are implicated in the estrogen-induced disruption of MCF-12A acini, in Chapter V, we proceeded to investigate the non-genomic effects of estrogens using inhibitors of the PI3K and MAPK signalling pathways.
Treatment of 3D cultures with the estrogenic test compounds resulted in deformed acini of increased size and displaying filled lumen. These effects were mediated by both the ER and GPER-1, and were, in part, a consequence of activation of the PI3K and MAPK pathways. We decided then, to investigate how estrogens impacted upon the expression of key apoptotic factors in 3D culture. The first step in doing this was to optimise the primers for future real-time polymerase chain reaction (PCR). This had to be conducted in monolayer due to technical and financial constraints. We then decided in Chapter VI, to investigate first, whether in monolayer culture, E2 affected the gene expression of four key apoptotic factors: BCL-2, BCL-XL, BAX and BAD in four mammary cell lines. After the observation that in monolayer, E2 did not greatly impact upon a single apoptotic factors, but did instead affect the expression of multiple apoptotic factors, we calculated the Bax/Bcl-2 ratio and found that estrogenic treatment did appear to induce an anti-apoptotic influence. Finally, protein analysis of the PI3K and MAPK were performed to investigate whether in monolayer culture, E2 was capable of inducing non- genomic effects, which was previously noticed in 3D cultures of MCF-12A cells in Chapter V.
In order the take the analysis of gene expression one step further, this was then conducted in cells isolated from 3D cultures. Initially, whole MCF-12A acini were extracted from Matrigel to investigate the impact of estrogens upon apoptotic factors and CCND1 (the gene encoding cyclin D1) expression. We observed that E2 does have an impact on gene expression of whole acini, in that CCND1 expression was increased in E2-treated acini. In Chapter VII, we developed a method by which the outer and inner cell populations of MCF-12A acini could be separated based on their integrin β4 expression by
61 fluorescence activated cell sorting (FACS). This enabled us to clearly see that E2 treatment did impact upon gene expression in a way that would promote proliferation of both inner and outer cell populations, and suppress apoptosis, particularly in the case of the centrally located cell population.
Overall, throughout this work we have two main aims. Firstly we aim to investigate whether estrogens can impact upon the 3D architecture of the breast, by using a 3D in vitro model of epithelial cells cultured in reconstituted basement membrane. In the possibility that estrogens, in fact, affect acini formation of MCF-12A cells, our second aim is to dissect the mechanisms through which estrogens act in 3D cultures of this non-tumourigenic epithelial cell line. It is the hope, that the work conducted in this study will enable us to understand the role of both the nuclear ER and transmembrane receptors (GPER- 1) in estrogen-induced malformations of acini.
62