Ction markers along with formation of multi-acinar spheroids (Figure 4). These observations suggest that during acinus morphogenesis, PUMA is involved in the clearance of inner cells while p21 suppresses abnormal cell proliferation in the lumen. This result recapitulates the phenotype of cell polarity altered by knockdown of wild-type p53 or TAp73 [6,7], suggesting that p21 and PUMA function downstream of wild-type p53 and p73 to maintain normal epithelial morphogenesis. In addition, our study is consistent with the recent report, which showed that PUMA/p21 double knockout mice have a phenotype similar to p53 knockout mice upon lethal irradiation, with blocked apoptosis but exacerbated gastro-intestinal epithelial damage [27]. Thus, loss of genes that regulate cell proliferation and apoptosis may lead to tumorigenesis in the mammary gland. Our observations support the postulation that both anti-proliferation and apoptotic activities 11967625 are required for achieving lumen formation in mammary epithelial acini (Figure 7F).EMT plays an important role in embryogenesis and development. During EMT, epithelial cells lose their epithelial features and acquire a fibroblast-like morphology, accompanied with upregulation of mesenchymal markers and enhancement of migratory properties, contributing to pathological processes such as fibrosis and cancer [28,29]. EMT is triggered by diverse signal pathways, including transforming growth factor-b (TGF-b), Wnt, Hedgehog, and Notch [30]. Previous study showed that p21 is responsible for preventing TGF-b from inducing cell proliferation in MCF10A cells [31]. Furthermore, TGF-b confers p21-null cells to mesenchymal transition with increased expression of vimentin and decreased expression of E-cadherin [32]. In addition, loss of p21 enhances, whereas ectopic expression of p21 represses, the features of EMT in transformed human mammary epithelial cell lines [33]. Moreover, p21 prevents Twist transcription factor from repressing E-cadherin expression [33]. Importantly, loss of p21 is correlated with positive vimentin expression in primary human breast cancers [32]. Here, we found that upon knockdown of p21, PUMA and especially both, MCF10A cells undergo EMT and exhibit loss of E-cadherin expression, accumulation of b-catenin in the Epigenetic Reader Domain nucleus, increased expression of laminin V and up-regulated EMT markers (Snail-1, Slug and Twist). In line with this, wePUMA and p21 Regulate Morphogenesis and EMTFigure 5. Knockdown of PUMA and p21 enhances EMT. A-B, Epigenetics Western blots were prepared with extracts from MCF10A cells (lane 1), and MCF10A cells with p21-KD (lane 2), PUMA-KD (lane 3), or PUMA p21-KD (lane 4). MCF10A cells were grown in Matrigel for 20 days. The blots were probed with antibodies against E-cadherin (A), b-catenin (A), laminin V (A), Snail-1 (B), Slug (B), Twist (B), and actin (A ), respectively. C, Top panel: Colony formation assay was performed with MCF10A cells, or MCF10A cells with p21-KD, PUMA-KD or PUMA p21-KD. Cells were cultured for a period of 12 days, then fixed and stained with crystal violet. Bottom panel: The number of colonies was counted and presented as Mean 6 SD from three separate experiments. D, Top panel: Wound healing assay was performed with MCF10A cells and MCF10A cells with p21-KD, PUMA-KD or PUMA p21 -KD. Cell migration was determined by visual assessment of cells migrating into the wound for a period of 24 h using a phase-contrast microscopy. Bottom panel: The time required for wound closure.Ction markers along with formation of multi-acinar spheroids (Figure 4). These observations suggest that during acinus morphogenesis, PUMA is involved in the clearance of inner cells while p21 suppresses abnormal cell proliferation in the lumen. This result recapitulates the phenotype of cell polarity altered by knockdown of wild-type p53 or TAp73 [6,7], suggesting that p21 and PUMA function downstream of wild-type p53 and p73 to maintain normal epithelial morphogenesis. In addition, our study is consistent with the recent report, which showed that PUMA/p21 double knockout mice have a phenotype similar to p53 knockout mice upon lethal irradiation, with blocked apoptosis but exacerbated gastro-intestinal epithelial damage [27]. Thus, loss of genes that regulate cell proliferation and apoptosis may lead to tumorigenesis in the mammary gland. Our observations support the postulation that both anti-proliferation and apoptotic activities 11967625 are required for achieving lumen formation in mammary epithelial acini (Figure 7F).EMT plays an important role in embryogenesis and development. During EMT, epithelial cells lose their epithelial features and acquire a fibroblast-like morphology, accompanied with upregulation of mesenchymal markers and enhancement of migratory properties, contributing to pathological processes such as fibrosis and cancer [28,29]. EMT is triggered by diverse signal pathways, including transforming growth factor-b (TGF-b), Wnt, Hedgehog, and Notch [30]. Previous study showed that p21 is responsible for preventing TGF-b from inducing cell proliferation in MCF10A cells [31]. Furthermore, TGF-b confers p21-null cells to mesenchymal transition with increased expression of vimentin and decreased expression of E-cadherin [32]. In addition, loss of p21 enhances, whereas ectopic expression of p21 represses, the features of EMT in transformed human mammary epithelial cell lines [33]. Moreover, p21 prevents Twist transcription factor from repressing E-cadherin expression [33]. Importantly, loss of p21 is correlated with positive vimentin expression in primary human breast cancers [32]. Here, we found that upon knockdown of p21, PUMA and especially both, MCF10A cells undergo EMT and exhibit loss of E-cadherin expression, accumulation of b-catenin in the nucleus, increased expression of laminin V and up-regulated EMT markers (Snail-1, Slug and Twist). In line with this, wePUMA and p21 Regulate Morphogenesis and EMTFigure 5. Knockdown of PUMA and p21 enhances EMT. A-B, Western blots were prepared with extracts from MCF10A cells (lane 1), and MCF10A cells with p21-KD (lane 2), PUMA-KD (lane 3), or PUMA p21-KD (lane 4). MCF10A cells were grown in Matrigel for 20 days. The blots were probed with antibodies against E-cadherin (A), b-catenin (A), laminin V (A), Snail-1 (B), Slug (B), Twist (B), and actin (A ), respectively. C, Top panel: Colony formation assay was performed with MCF10A cells, or MCF10A cells with p21-KD, PUMA-KD or PUMA p21-KD. Cells were cultured for a period of 12 days, then fixed and stained with crystal violet. Bottom panel: The number of colonies was counted and presented as Mean 6 SD from three separate experiments. D, Top panel: Wound healing assay was performed with MCF10A cells and MCF10A cells with p21-KD, PUMA-KD or PUMA p21 -KD. Cell migration was determined by visual assessment of cells migrating into the wound for a period of 24 h using a phase-contrast microscopy. Bottom panel: The time required for wound closure.
