The cyclin-dependent kinase inhibitor p27 (Kip1) regulates both DNA synthesis and apoptosis in mammary epithelium but is not required for its functional development during pregnancy

Decreased expression of the cyclin-dependent kinase (CDK) inhibitor p27(Kip1) is common in breast cancer and is associated with poor prognosis. p27 is also an important mediator of steroidal regulation of cell cycle progression. We have therefore investigated the role of p27 in mammary epithelial cell proliferation. Examination of the two major functions of p27, assembly of cyclin D1-Cdk4 complexes and inhibition of Cdk2 activity, revealed that cyclin D1-Cdk4 complex formation was not impaired in p27-/- mammary epithelial cells in primary culture. However, cyclin E-Cdk2 activity was increased approximately 3-fold, indicating that the CDK inhibitory function of p27 is important in mammary epithelial cells. Increased epithelial DNA synthesis was observed during pregnancy in p27-/- mammary gland transplants, but this was paralleled by increased apoptosis. During pregnancy and at parturition, development and differentiation of p27+/+ and p27-/- mammary tissue were indistinguishable. These results demonstrate a role for p27 in both the proliferation and survival of mammary epithelial cells. However, the absence of morphological and cellular defects in p27-/- mammary tissue during pregnancy raises the possibility that loss of p27 in breast cancer may not confer an overall growth advantage unless apoptosis is also impaired.     

Dysregulated expression of cyclin D1 in normal human mammary epithelial cells inhibits all-trans-retinoic acid-mediated G0/G1-phase arrest and differentiation in vitro.

Overexpression of cyclin D1 protein is observed in the majority of breast cancers, suggesting that dysregulated expression of cyclin D1 might be a critical event in breast cancer carcinogenesis. We investigated whether retroviral-mediated expression of cyclin D1 might affect all-trans-retinoic acid (ATRA)-mediated growth inhibition and differentiation of normal cultured human mammary epithelial cells (HMECs). HMECs treated with 1.0 microM ATRA undergo irreversible growth inhibition starting at 24 h and complete G0/G1-phase arrest by Day 3. Cyclin D1 protein levels are observed to decrease in association with the initiation of growth arrest starting at 24 h and then increase by approximately 35% on Day 3. Concomitant with this observed increase in cyclin D1, HMECs undergo morphologic changes consistent with progression to a more differentiated phenotype, including an increase in cell size, increased cell spreading, increased tonofilaments, and accumulation of cytoplasmic vesicles containing lipid. Dysregulated expression of cyclin D1 in HMECs results in inhibition of G0/G1-phase arrest mediated by ATRA. In addition, HMECs expressing exogenous cyclin D1 are resistant to differentiation by ATRA. Our results suggest that coordinated expression of cyclin D1 may be critical for normal mammary epithelial cell homeostasis, and dysregulated expression of cyclin D1 might result in retinoid resistance and promote mammary carcinogenesis.     

Mechanisms of cell cycle arrest in response to TGF-beta in progestin-dependent and -independent growth of mammary tumors

TGF-beta1 modulation of cell cycle components was assessed in an experimental model in which the synthetic progestin medroxyprogesterone acetate (MPA) induced mammary tumors in Balb/c mice. TGF-beta1 inhibited both MPA-induced proliferation of progestin-dependent C4HD epithelial cells and proliferation of the progestin-independent variant cell type C4HI, arresting cells in G(1) phase of the cell cycle. Progestin-independent 60 epithelial cells evidenced reduced response to TGF-beta1 antiproliferative effects. TGF-beta1 inhibition of cyclins D1 and A expression and up-regulation of p21(CIP1) levels were the common findings in all three cell types. In addition, a significant content reduction of cyclin D1/cdk4 and cyclin A/cdk2 complexes was found after TGF-beta1 inhibition of MPA-dependent and -independent proliferation. TGF-beta1 inhibited cyclin D2 expression and up-regulated p27(KIP1) levels only when acting as inhibitor of MPA-induced proliferation of C4HD cells. Regulation of these two cell cycle components resulted in decreased cyclin D2/cdk2 complex and in increased p27(KIP1) association with cdk2 in C4HD cells treated with TGF-beta1. These two molecular mechanisms, unobserved in progestin-independent growth of C4HI or 60 cells, were associated with a significantly higher degree of inhibition of cdk2 kinase activity in C4HD cells compared to that found in TGF-beta-treated C4HI or 60 cells. Reduced sensitivity of 60 cells to the growth-inhibitory effects of TGF-beta1 correlated with significantly lower levels of p15(INK4B), p21(CIP1), and p27(KIP1) expressed in these cells, compared to the levels present in C4HD or C4HI cells, and correlated as well with lack of expression of p16(INK4). Thus, common targets were found to exist in TGF-beta1 inhibitory action on breast cancer cells, but regulation of specific targets was found when TGF-beta1-inhibited proliferation driven by the progesterone receptor.     

Regulatory function of whey acidic protein in the proliferation of mouse mammary epithelial cells in vivo and in vitro

Although possible biological functions of whey acidic protein (WAP) have been suggested, few studies have focused on investigating the function of WAP. This paper describes evidence for WAP function in lobulo-alveolar development in mammary glands in vivo and in the cell cycle progression of mammary epithelial cells in vitro. Ubiquitous overexpression of WAP transgene impaired only lobulo-alveolar development in the mammary glands of transgenic female mice but not other physiological functions, indicating that the inhibitory function of WAP is specific to mammary alveolar cells. The forced expression of WAP significantly inhibited the proliferation of mouse mammary epithelial cells (HC11 cells and EpH4/K6 cells), whereas it did not affect that of NIH3T3 cells. Co-culturing of WAP-clonal cells and control cells using a transwell insert demonstrated that WAP inhibited the proliferation of HC11 cells through a paracrine action but not that of NIH3T3 cells, and that WAP was able to bind to HC11 cells but not to NIH3T3 cells. Apoptosis was not enhanced in the HC11 cells with stable WAP expression (WAP-clonal HC11 cells). BrdU incorporation and FACScan analyses revealed that cell cycle progression from the G0/G1 to the S phase was inhibited in the WAP-clonal HC11 cells. Among G1 cyclins, the expression of cyclin D1 and D3 was significantly decreased in the WAP-clonal HC11 cells. The present results provide the first documented evidence that WAP plays a negative regulatory role in the cell cycle progression of mammary epithelial cells through an autocrine or paracrine mechanism in vivo.     

Pleiotrophin (PTN) Expression and Function and in the Mouse Mammary Gland and Mammary Epithelial Cells

Expression of the heparin-binding growth factor, pleiotrophin (PTN) in the mammary gland has been reported but its function during mammary gland development is not known. We examined the expression of PTN and its receptor ALK (Anaplastic Lymphoma Kinase) at various stages of mouse mammary gland development and found that their expression in epithelial cells is regulated in parallel during pregnancy. A 30-fold downregulation of PTN mRNA expression was observed during mid-pregnancy when the mammary gland undergoes lobular-alveolar differentiation. After weaning of pups, PTN expression was restored although baseline expression of PTN was reduced significantly in mammary glands of mice that had undergone multiple pregnancies. We found PTN expressed in epithelial cells of the mammary gland and thus used a monoclonal anti-PTN blocking antibody to elucidate its function in cultured mammary epithelial cells (MECs) as well as during gland development. Real-time impedance monitoring of MECs growth, migration and invasion during anti-PTN blocking antibody treatment showed that MECs motility and invasion but not proliferation depend on the activity of endogenous PTN. Increased number of mammospheres with laminin deposition after anti-PTN blocking antibody treatment of MECs in 3D culture and expression of progenitor markers suggest that the endogenously expressed PTN inhibits the expansion and differentiation of epithelial progenitor cells by disrupting cell-matrix adhesion. In vivo, PTN activity was found to inhibit ductal outgrowth and branching via the inhibition of phospho ERK1/2 signaling in the mammary epithelial cells. We conclude that PTN delays the maturation of the mammary gland by maintaining mammary epithelial cells in a progenitor phenotype and by inhibiting their differentiation during mammary gland development.     

Neutrophil elastase inhibition of cell cycle progression in airway epithelial cells in vitro is mediated by p27kip1

Neutrophil elastase (NE), a serine protease present in high concentrations in the airways of cystic fibrosis patients, injures the airway epithelium. We examined the epithelial response to NE-mediated proteolytic injury. We have previously reported that NE treatment of airway epithelial cells causes a marked decrease in epithelial DNA synthesis and proliferation. We hypothesized that NE inhibits DNA synthesis by arresting cell cycle progression. Progression through the cell cycle is positively regulated by cyclin complexes and negatively regulated by cyclin-dependent kinase inhibitors (CKI). To test whether NE arrests cell cycle progression, we treated normal human bronchial epithelial (NHBE) cells with NE (50 nM) or control vehicle for 24 h and assessed the effect of treatment on the cell cycle by flow cytometry. NE treatment resulted in G(1) arrest. Arrest in G(1) phase may be the result of CKI inhibition of the cyclin E complex; therefore, we evaluated whether NE upregulated CKI expression and/or affected the interaction of CKIs with the cyclin E complex. Following NE or control vehicle treatment, expression of p27(Kip1), a member of the Cip/Kip family, was evaluated. NE increased p27(Kip1) gene and protein expression. NE increased the coimmunoprecipitation of p27(Kip1) with cyclin E complex, suggesting that p27(Kip1) inhibited cyclin E complex activity. Our results demonstrate that p27 is regulated by NE and is critical for NE-induced cell cycle arrest.     

Cyclin gene expression and growth control in normal and neoplastic human breast epithelium

Recent advances in defining the molecular mechanisms of cell cycle control in eukaryotes provide a basis for beter understanding the hormonal control of cell proliferation in normal and neoplastic breast epithelium. It is now clear that a number of critical steps in cell cycle progression are controlled by families of serine/threonine kinases, the cdks. These kinases are activated by interactions with various cyclin gene products which form the regulatory subunits of the kinase complexes. Several families of cyclins control cell cycle progression in G₁ phase, cyclins C, D and E, or in S, G₂ and mitosis, cyclins A and B. Recent studies have defined the expression and regulation of cyclin genes in normal breast epithelial cells and in breast cancer cell lines. Following growth arrest of T-47D breast cancer cells by serum deprivation restimulation with insulin results in sequential induction of cyclin genes. Cyclin D1 mRNA increases within 1 h of mitogenic stimulation and is followed by increased expression of cyclins D3 and E in G₁ phase, cyclin A in late G₁/early S phase and cyclin B1 in G₂. Similar results were observed following epidermal growth factor stimulation of normal breast epithelial cells. Other hormones—oestrogens and progestins—and growth factors—insulin-like investigated for their effects on G₁ cyclin gene expression. In all cases there was an excellent correlation between the induction of cyclin D1 mRNA and subsequent entry into S phase. Furthermore, growth inhibition by antioestrogens and concurrent G₁ arrest were preceded by an acute decrease in cyclin D1 gene expression. These observations suggest a likely role for cyclin D1 in mediating many of the known hormonal effects on cell proliferation in breast epithelial cells.     

Changes in ErbB2 (her-2/neu), ErbB3, and ErbB4 during growth, differentiation, and apoptosis of normal rat mammary epithelial cells.

Studies were undertaken to examine the natural role of ErbB2, ErbB3, and ErbB4 during the development of normal rat mammary epithelial cells (MECs) in vivo and in vitro. Immunohistochemical analysis demonstrated that mammary gland terminal end buds expressed abundant ErbB2 and ErbB4 but limited ErbB3 in pubescent rats, whereas luminal epithelial cells in nulliparous rats expressed ErbB2, ErbB3, and/or ErbB4. During pregnancy, ductal epithelial cells and stromal cells expressed abundant ErbB3 but limited ErbB2. Although ErbB2 and ErbB3 were downregulated throughout lactation, both receptors were re-expressed during involution. In contrast, ErbB4 was downregulated throughout pregnancy, lactation, and involution. Immunoblotting and immunoprecipitation studies confirmed the developmental expression of ErbB2 and ErbB3 in the mammary gland and the co-localization of distinct ErbB receptors in the mammary gland of nulliparous rats. In agreement with our in vivo findings, primary culture studies demonstrated that ErbB2 and ErbB3 were expressed in functionally immature, terminally differentiated and apoptotic MECs, and downregulated in functionally differentiated MECs. ErbB receptor signaling was required for epithelial cell growth, functional differentiation, and morphogenesis of immature MECs, and the survival of terminally differentiated MECs. Finally, ErbB4 expression did not interfere with functional differentiation and apoptosis of normal MECs.     

Heregulin-dependent delay in mitotic progression requires HER4 and BRCA1

HER4 expression in human breast cancers correlates with a positive prognosis. While heregulin inhibits the growth of HER4-positive breast cancer cells, it does so by undefined mechanisms. We demonstrate that heregulin-induced HER4 activity inhibits cell proliferation and delays G(2)/M progression of breast cancer cells. While investigating pathways of G(2)/M delay, we noted that heregulin increased the expression of BRCA1 in a HER4-dependent, HER2-independent manner. Induction of BRCA1 by HER4 occurred independently of the cell cycle. Moreover, BRCA1 expression was elevated in HER4-postive human breast cancer specimens. Heregulin stimulated c-Jun N-terminal kinase (JNK), and pharmacologic inhibition of JNK impaired heregulin-enhanced expression of BRCA1 and mitotic delay; inhibition of Erk1/2 did not. Knockdown of BRCA1 with small interfering RNA in a human breast cancer cell line interfered with HER4-mediated mitotic delay. Heregulin/HER4-dependent mitotic delay was examined further with an isogenic pair of mouse mammary epithelial cells (MECs) derived from mice harboring homozygous LoxP sites flanking exon 11 of BRCA1, such that one cell line expressed BRCA1 while the other cell line, after Cre-mediated excision, did not. BRCA1-positive MECs displayed heregulin-dependent mitotic delay; however, the isogenic BRCA1-negative MECs did not. These results suggest that heregulin-mediated growth inhibition in HER4-postive breast cancer cells requires BRCA1.     

孕激素诱导cyclin G1在小鼠子宫内膜上皮细胞的表达及其对细胞增殖的影响

本文在体外培养条件下研究卵巢激素诱导小鼠子宫内膜上皮细胞cyclin G1的表达及细胞增殖和细胞周期进程的变化,以探讨孕激素依赖的细胞周期调控因子cyclin G1对子宫内膜上皮细胞增殖的负调控作用.原代培养小鼠子宫内膜上皮细胞,待其生长汇合后分为4组:对照组(C组)、雌激素组(E组)、孕激素组(P组)、雌、孕激素共同作用组(EP组).加入相应激素作用24 h后,用细胞免疫化学方法检测各组细胞cyclin G1的表达水平:四甲基偶氮唑蓝(MTT)比色法检测各组细胞活力,间接观察子宫内膜上皮细胞的增殖情况;用流式细胞仪检测分布在细胞周期各时相的子宫内膜上皮细胞所占百分数.细胞免疫化学结果显示,cyclin G1在C组和E组子宫内膜上皮细胞上无明显表达,而在P组和EP组子宫内膜上皮细胞中表达明显,且定位于细胞核内.MTT法结果显示,与C组相比,E组细胞活力明显增高,而P组和EP组的细胞活力均明显下降,表明雌激素能促进子宫内膜上皮细胞增殖,而孕激素则具有抑制子宫内膜上皮细胞增殖的作用.流式细胞术检测显示,与C组相比,E组中处于S期的子宫内膜上皮细胞百分数增多;P组与EP组中处于S期的子宫内膜上皮细胞百分数明显减少,而处于G1期的细胞百分数和G2/M期的细胞百分数则明显增加.上述结果提示,孕激素依赖的cyclin G1可能通过阻滞细胞周期进程来参与孕激素对子宫内膜上皮细胞增殖的负调控作用.