Cyclin‐dependent kinase inhibitors and basement membrane interact to regulate breast epithelial cell differentiation and acinar morphogenesis

Coppock, Hedley A.; Gilham, David E.; Howell, Anthony; Clarke, Robert B.

doi:10.1111/j.1365-2184.2007.00463.x

Cited by 11 publications

(9 citation statements)

References 55 publications

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“…Karyotype analysis shows mild aneuploidy of 48, XX, 3p-, 9pϩ, 6pϩ, ϩ18, and ϩ16 (45). Previous investigators have identified neither nuclear PR protein in MCF-10A cells by Western blot analysis (34) nor nuclear PR transcript by RT-PCR (8). Our work has shown the same findings, with no evidence of a nuclear PR transcript by RT-PCR or protein by Western blot analysis (unpublished data).…”

Section: Discussionsupporting

confidence: 75%

Progesterone stimulates mitochondrial activity with subsequent inhibition of apoptosis in MCF-10A benign breast epithelial cells

Behera

Dai

Garde

et al. 2009

American Journal of Physiology-Endocrinology and Metabolism

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As an example, progesterone levels correlate with increased epithelial cell proliferation, but there is discordance between the dividing cells and the cells with nuclear progesterone receptor expression. The release of paracrine growth factors from nuclear receptor-positive cells has been postulated as a mechanism, since in vitro studies show a lack of growth effect by progesterone in breast epithelial cells lacking nuclear receptors. This study examined possible nongenomic effects of progesterone in breast epithelia by using MCF-10A cells known to lack nuclear progesterone receptor expression. Treatment for 30 -60 min with progesterone or the progestin, R5020, increased mitochondrial activity as shown by an increase in mitochondrial membrane potential (hyperpolarization) with a concordant increase in total cellular ATP. The reaction was inhibited by a specific progesterone receptor antagonist and not affected by the translation inhibitor cycloheximide. Progestin treatment inhibited apoptosis induced by activation of the FasL pathway, as shown by a decrease in sub-G 1 cell fraction during fluorescence-activated cell sorting and a decrease in caspase 3/7 levels. Progestin treatment did not alter the cell cycle over 48 h. Our study demonstrates a nongenomic action of progesterone on benign breast epithelial cells, resulting in enhanced cellular respiration and protection from apoptosis.progesterone; MCF-10A cells; mitochondria; apoptosis; cellular respiration PREVIOUS STUDIES HAVE SHOWN a discordance between the proliferative action of progesterone in the breast and the content of nuclear progesterone receptors (PR-B and PR-A) in breast epithelial cells. As examples, the mitotic rate of breast epithelial cells is greatest in the progesterone-dominant luteal phase (44). Exogenous administration of estrogen plus progestin results in greater breast epithelial proliferation than estrogen alone (15, 18). The progesterone receptor knockout (PRKO) mouse, an excellent model for the function of nuclear PR in mammary gland development, supports a proliferative role. PR-B is primarily responsible for gland development, with PRKO-B mice showing less extensive ductal development and lack of alveolar terminal end buds (7,29). In contrast to these physiological effects, very few breast epithelial cells appear to express nuclear PR. In immunocytochemical studies of human breast tissue, from 6 to 13% of ductal epithelial cells express PR (6, 37), with the two PR isoforms, B and A, typically localizing in the same cell (14). Immunocytochemical analysis of proliferation by detection of Ki67 antigen in human breast or autoradiographs of [ 3 H]thymidine incorporation in rodent mammary samples show nuclear PR-expressing cells to be nonproliferating. Adjacent cells lacking nuclear PR expression have greater mitotic activity (23). This disassociation between nuclear PR expression and proliferation led to the hypothesis that nuclear PR-expressing cells regulate proliferation of adjacent cells via the control of paracrine factors (4).This ...

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Section: Discussionsupporting

confidence: 75%

Progesterone stimulates mitochondrial activity with subsequent inhibition of apoptosis in MCF-10A benign breast epithelial cells

Behera

Dai

Garde

et al. 2009

American Journal of Physiology-Endocrinology and Metabolism

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“…4 A ), where as in 3D culture only 40% of the cells were positive for pKi-67, which is similar to the in vivo situation (27, 28). The expression of pKi-67 in fewer cells in 3D models compared to monolayer cells has been described before (29, 30) and is most likely the result of differential gene expression induced by differential architectural cues in the two geometries. PDT-mediated cell death triggering in 3D cultures was investigated by incubating the cultures with PICELs containing 20 nM equivalent of FITC for 48 h followed by 488 nm illumination with 5 J/cm 2 .…”

Section: Resultsmentioning

confidence: 60%

Ki-67 as a Molecular Target for Therapy in an In vitro Three-Dimensional Model for Ovarian Cancer

et al. 2010

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Targeting molecular markers and pathways implicated in cancer cell growth is a promising avenue for developing effective therapies. Although the Ki-67 protein (pKi-67) is a key marker associated with aggressively proliferating cancer cells and poor prognosis, its full potential as a therapeutic target has never before been successfully shown. In this regard, its nuclear localization presents a major hurdle because of the need for intracellular and intranuclear delivery of targeting and therapeutic moieties. Using a liposomally encapsulated construct, we show for the first time the specific delivery of a Ki-67-directed antibody and subsequent lighttriggered death in the human ovarian cancer cell line OVCAR-5. Photoimmunoconjugate-encapsulating liposomes (PICEL) were constructed from anti-pKi-67 antibodies conjugated to fluorescein 5(6)-isothiocyanate, as a photoactivatable agent, followed by encapsulation in noncationic liposomes. Nucleolar localization of the PICELs was confirmed by confocal imaging. Photodynamic activation with PICELs specifically killed pKi-67-positive cancer cells both in monolayer and in three-dimensional (3D) cultures of OVCAR-5 cells, with the antibody TuBB-9 targeting a physiologically active form of pKi-67 but not with MIB-1, directed to a different epitope. This is the first demonstration of (a) the exploitation of Ki-67 as a molecular target for therapy and (b) specific delivery of an antibody to the nucleolus in monolayer cancer cells and in an in vitro 3D model system. In view of the ubiquity of pKi-67 in proliferating cells in cancer and the specificity of targeting in 3D multicellular acini, these findings are promising and the approach merits further investigation.

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“…As shown in Figure 2A, the acinar size of MCF10A between egg white and Matrigel was comparable. It is known that when MCF10A cells are cultured in Matrigel, the proliferation marker Ki67 is prevalent during the early phases of acinus formation and decreases at later stages as acini increase in size and slow in their growth (11). Thus we compared the cell proliferation M between egg white and Matrigel cell culture systems.…”

Section: Resultsmentioning

confidence: 99%

Novel Egg White–based 3-D Cell Culture System

et al. 2008

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Although three dimensional (3-D) cell culture systems have numerous advantages over traditional monolayer culture, the currently available 3-D cell culture media are cost-prohibitive for regular use by the majority of research laboratories. Here we show a simple system based on avian egg white that supports growth of cells in 3-D, at a significantly decreased cost. Specifically, we show that growth of immortalized human breast epithelial cells (MCF10A) in egg white-based medium results in formation of acini with hollow lumens, apoptotic clearance of the cells in the lumen, and apicobasal polarization comparable to what has been described using established 3-D culture media such as reconstituted basement membrane preparations (BM). There was no significant difference in MCF10A proliferation and acinar size between egg white and BM. We also cultured different established cell lines, oncogene-transformed MCF10A, and mouse mammary epithelial cells in egg white and BM, and observed similar morphology. In summary, our data convincingly argue that egg white can be used as a suitable alternative model for 3-D cell culture studies. We strongly believe that this simple and inexpensive method should allow researchers to perform 3-D cell culture experiments on a regular basis, and result in a dramatic increase of use of the 3-D cell culture in research. Thus, this finding lays the foundation for significantly increased, cost-effective use of 3-D cultures in cell biology.

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Cyclin‐dependent kinase inhibitors and basement membrane interact to regulate breast epithelial cell differentiation and acinar morphogenesis

Abstract: We conclude that both p21(CIP1) and p27(KIP1) induce partial secretory differentiation of mammary cells in monolayer, but during acinus morphogenesis in 3D culture they have a highly regulated temporal expression pattern.

Cited by 11 publications

References 55 publications

Progesterone stimulates mitochondrial activity with subsequent inhibition of apoptosis in MCF-10A benign breast epithelial cells

Progesterone stimulates mitochondrial activity with subsequent inhibition of apoptosis in MCF-10A benign breast epithelial cells

Ki-67 as a Molecular Target for Therapy in an In vitro Three-Dimensional Model for Ovarian Cancer

Novel Egg White–based 3-D Cell Culture System

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