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


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.


CONCLUSIONS
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.
Coppock
H A
HA
Centre for Molecular Medicine, University of Manchester, Manchester, UK.
Gilham
D E
DE
Howell
A
A
Clarke
R B
RB
D001483:Base Sequence; D001485:Basement Membrane / Q000378:metabolism*; D001940:Breast / Q000166:cytology* / Q000254:growth & development / Q000378:metabolism*; D002454:Cell Differentiation / Q000502:physiology; D002460:Cell Line; D050756:Cyclin-Dependent Kinase Inhibitor Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D050760:Cyclin-Dependent Kinase Inhibitor p27; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism; D005260:Female; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D019394:Ki-67 Antigen / Q000378:metabolism; D050356:Lipid Metabolism; D009024:Morphogenesis; D034741:RNA, Small Interfering / Q000235:genetics; D014161:Transduction, Genetic
C493956:CDKN1A protein, human; C436291:CDKN1B protein, human; D050756:Cyclin-Dependent Kinase Inhibitor Proteins; D050759:Cyclin-Dependent Kinase Inhibitor p21; D047908:Intracellular Signaling Peptides and Proteins; D019394:Ki-67 Antigen; D034741:RNA, Small Interfering; D050760:Cyclin-Dependent Kinase Inhibitor p27