Expression Pattern of the Pro-apoptotic Gene PAR-4 During the Morphogenesis of MCF-10A Human Mammary Epithelial Cells

Abstract

The histological organization of the mammary gland involves a spatial interaction of epithelial and myoepithelial cells with the specialized basement membrane (BM), composed of extra-cellular matrix (ECM) proteins, which is disrupted during the tumorigenic process. The interactions between mammary epithelial cells and ECM components play a major role in mammary gland branching morphogenesis. Critical signals for mammary epithelial cell proliferation, differentiation, and survival are provided by the ECM proteins. Three-dimensional (3D) cell culture was developed to establish a system that simulates several features of the breast epithelium in vivo; 3D cell culture of the spontaneously immortalized cell line, MCF10A, is a well-established model system to study breast epithelial cell biology and morphogenesis. Mammary epithelial cells grown in 3D form spheroids, acquire apicobasal polarization, and form lumens that resemble acini structures, processes that involve cell death. Using this system, we evaluated the expression of the pro-apoptotic gene PAWR (PKC apoptosis WT1 regulator; also named PAR-4, prostate apoptosis response-4) by immunofluorescence and quantitative real time PCR (qPCR). A time-dependent increase in PAR-4 mRNA expression was found during the process of MCF10A acinar morphogenesis. Confocal microscopy analysis also showed that PAR-4 protein was highly expressed in the MCF10A cells inside the acini structure. During the morphogenesis of MCF10A cells in 3D cell culture, the cells within the lumen showed caspase-3 activation, indicating apoptotic activity. PAR-4 was only partially co-expressed with activated caspase-3 on these cells. Our results provide evidence, for the first time, that PAR-4 is differentially expressed during the process of MCF10A acinar morphogenesis.

Keywords: Apoptosis; Breast cancer; Gene expression; MCF10A; PAR-4; Three dimensional (3D) cell culture.

Fig. 1

Expression pattern of PAR-4 in MCF10A cells grown in monolayers and three-dimensional (3D) cell culture. a Immunofluorescence localization of PAR-4. MCF10A cells grown in monolayers were fixed using 4% paraformaldehyde, permeabilized with 0.5% Triton X-100, and then incubated with primary monoclonal antibody anti-PAR-4 (red). b and c, Representative confocal microscopic images of MCF10A cells cultured in growth factor reduced Matrigel for 5 days and examined PAR-4 (red) and laminin V (green) by immunofluorescence. Nuclei were contrastained with Hoestch (blue). Equatorial cross sections of the acini were obtained using a Zeiss LSM Meta 510 laser scanning confocal microscope system (40×)

Fig. 2

PAR-4 mRNA expression pattern during MCF10A morphogenesis. mRNA was extracted from MCF10A cells cultured in growth factor reduced Matrigel for 3, 5, 7, and 10 days and the relative expression of PAR-4 mRNA was determined by quantitative real time PCR using GAPDH as the reference gene. Bars represent the relative gene expression after treatment, compared to control cells (NS). Data are expressed as the mean ± SD of three experiments

Fig. 3

PAR-4 protein expression in different mammary epithelial cell lines. a PAR-4 protein expression was examined by Western blot analysis of protein extracts from the mammary luminal cells MCF10A and from the breast cancer cell lines MCF7 and MDA-MB-231 cultured in media supplemented with 10% serum. Protein expression was analyzed with the mouse monoclonal antibody anti-PHLDA1 (Santa Cruz) and the β-actin antibody (Chemicon) for normalization. b Graphic representation of the quantified results from the experiment

Fig. 4

Expression of PAR-4 and activated caspase 3 in MCF10A cells grown in three-dimensional (3D) cell culture. A to D, Representative confocal microscopic images of MCF10A cells cultured in growth factor reduced Matrigel for 3, 5, 7 and 10 days and examined for PAR-4 (red) and activated caspase 3 (green) by immunofluorescence. Nuclei were contrastained with Hoestch (blue). Equatorial cross sections of the acini were obtained using a Zeiss LSM Meta 510 confocal microscope system (scale bar: 10 μm)