Coppock H A, Gilham D E, Howell A, Clarke R B
Centre for Molecular Medicine, University of Manchester, Manchester, UK.
Cell Prolif. 2007 Oct;40(5):721-40. doi: 10.1111/j.1365-2184.2007.00463.x.
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.
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.
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.
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.
细胞周期蛋白依赖性激酶抑制剂（CDKIs）p21（CIP1）和p27（KIP1）调节多种组织类型的生长和分化。我们旨在确定p21（CIP1）或p27（KIP1）是否能诱导乳腺细胞出现终末分化表型，并研究CDKI表达是否受基底膜相互作用的调控。
通过逆转录病毒转导p21（CIP1）或p27（KIP1）cDNA，检测CDKI表达增加对MCF-10A乳腺上皮细胞表型的影响。
p21（CIP1）或p27（KIP1）的过表达降低了MCF-10A单层培养物中的生长速率，改变了细胞形态，并刺激了中性脂滴的积累，提示部分泌乳分化。然而，通过逆转录酶-聚合酶链反应和免疫组织化学检测时，未发现腔分化标志物（雌激素和孕激素受体、α-乳白蛋白、β-酪蛋白和脂联素）。已知细胞-基底膜接触对于乳腺上皮细胞的完全分化至关重要，因此将亲代MCF-10A细胞培养在基底膜制剂（基质胶）上，它们在其中形成腺泡。免疫细胞化学显示，细胞增殖标志物Ki67最初高水平表达，并随着生长的降低，p27（KIP1）表达稳步增加。令人惊讶的是，p21（CIP1）在腺泡生长早期最高，并在增殖细胞中检测到，这通过双Ki67/p21（CIP1）免疫荧光共定位得以证明。p21（CIP1）或p27（KIP1）的过表达损害了腺泡的形成，而使用小干扰RNA敲低它们则增加了腺泡的形成。
我们得出结论，p21（CIP1）和p27（KIP1）均可在单层培养中诱导乳腺细胞出现部分分泌分化，但在三维培养的腺泡形态发生过程中，它们具有高度调控的时间表达模式。
