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dc.contributor.authorCoppock, H A
dc.contributor.authorGilham, David E
dc.contributor.authorHowell, Anthony
dc.contributor.authorClarke, Robert B
dc.date.accessioned2009-06-15T12:03:22Z
dc.date.available2009-06-15T12:03:22Z
dc.date.issued2007-10
dc.identifier.citationCyclin-dependent kinase inhibitors and basement membrane interact to regulate breast epithelial cell differentiation and acinar morphogenesis. 2007, 40 (5):721-40 Cell Prolif.en
dc.identifier.issn0960-7722
dc.identifier.pmid17877612
dc.identifier.doi10.1111/j.1365-2184.2007.00463.x
dc.identifier.urihttp://hdl.handle.net/10541/70473
dc.description.abstractOBJECTIVE: The cyclin-dependent kinase inhibitors (CDKIs), p21(CIP1) and p27(KIP1) regulate growth and differentiation in diverse tissue types. We aimed to determine whether p21(CIP1) or p27(KIP1) could induce a terminally differentiated phenotype in breast cells, and to examine if CDKI expression is regulated by basement membrane interactions. MATERIALS AND METHODS: Effects of increased CDKI expression on the phenotype of MCF-10A breast epithelial cells were examined by retroviral transduction of p21(CIP1) or p27(KIP1) cDNA. RESULTS: Overexpression of p21(CIP1) or p27(KIP1) reduced MCF-10A growth rates in monolayer cultures, altered cellular morphology and stimulated accumulation of neutral lipid droplets, suggesting partial lactational differentiation. However, markers of luminal differentiation (oestrogen and progesterone receptors, alpha-lactalbumin, beta-casein and adipophilin) were absent when examined by reverse transcriptase-polymerase chain reaction and immunohistochemistry. Cell-basement membrane contacts are known to be essential for full mammary epithelial cell differentiation and therefore parental MCF-10A cells were cultured on a basement membrane preparation (Matrigel) in which they form acini. Immunocytochemistry showed that Ki67, the cell proliferation marker, was initially expressed at high levels and as growth decreased p27(KIP1) expression steadily increased. Surprisingly, p21(CIP1) was highest at the early stages of acinus growth and was detected in proliferating cells, as demonstrated by colocalization in dual Ki67/p21(CIP1) immunofluorescence. Overexpression of p21(CIP1) or p27(KIP1) impaired formation of acini, whereas their knockdown, using siRNA, increased acinus formation. CONCLUSION: 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.
dc.language.isoenen
dc.subject.meshBase Sequence
dc.subject.meshBasement Membrane
dc.subject.meshBreast
dc.subject.meshCell Differentiation
dc.subject.meshCell Line
dc.subject.meshCyclin-Dependent Kinase Inhibitor Proteins
dc.subject.meshCyclin-Dependent Kinase Inhibitor p21
dc.subject.meshEpithelial Cells
dc.subject.meshFemale
dc.subject.meshHumans
dc.subject.meshIntracellular Signaling Peptides and Proteins
dc.subject.meshKi-67 Antigen
dc.subject.meshLipid Metabolism
dc.subject.meshMorphogenesis
dc.subject.meshRNA, Small Interfering
dc.subject.meshTransduction, Genetic
dc.titleCyclin-dependent kinase inhibitors and basement membrane interact to regulate breast epithelial cell differentiation and acinar morphogenesis.en
dc.typeArticleen
dc.contributor.departmentCentre for Molecular Medicine, University of Manchester, Manchester, UK.en
dc.identifier.journalCell Proliferationen
html.description.abstractOBJECTIVE: The cyclin-dependent kinase inhibitors (CDKIs), p21(CIP1) and p27(KIP1) regulate growth and differentiation in diverse tissue types. We aimed to determine whether p21(CIP1) or p27(KIP1) could induce a terminally differentiated phenotype in breast cells, and to examine if CDKI expression is regulated by basement membrane interactions. MATERIALS AND METHODS: Effects of increased CDKI expression on the phenotype of MCF-10A breast epithelial cells were examined by retroviral transduction of p21(CIP1) or p27(KIP1) cDNA. RESULTS: Overexpression of p21(CIP1) or p27(KIP1) reduced MCF-10A growth rates in monolayer cultures, altered cellular morphology and stimulated accumulation of neutral lipid droplets, suggesting partial lactational differentiation. However, markers of luminal differentiation (oestrogen and progesterone receptors, alpha-lactalbumin, beta-casein and adipophilin) were absent when examined by reverse transcriptase-polymerase chain reaction and immunohistochemistry. Cell-basement membrane contacts are known to be essential for full mammary epithelial cell differentiation and therefore parental MCF-10A cells were cultured on a basement membrane preparation (Matrigel) in which they form acini. Immunocytochemistry showed that Ki67, the cell proliferation marker, was initially expressed at high levels and as growth decreased p27(KIP1) expression steadily increased. Surprisingly, p21(CIP1) was highest at the early stages of acinus growth and was detected in proliferating cells, as demonstrated by colocalization in dual Ki67/p21(CIP1) immunofluorescence. Overexpression of p21(CIP1) or p27(KIP1) impaired formation of acini, whereas their knockdown, using siRNA, increased acinus formation. CONCLUSION: 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.


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