Lovastatin inhibits mesangial cell proliferation via p27Kip1

Mesangial cell proliferation is a key feature of glomerulonephritis. The hydroxymethylglutaryl-coenzyme A reductase inhibitor lovastatin is known to inhibit cell cycle progression. To determine the inhibitory mechanisms of mesangial cell proliferation by lovastatin, the cyclin-dependent kinase (CDK) activity, and expression of CDK inhibitor (p27Kip1, p21Cip1, and p16INK4) mRNA and protein were measured. Lovastatin inhibited phosphorylation of retinoblastoma protein and mesangial cell proliferation dose dependently. Lovastatin increased the p27Kip1 protein level but produced no changes in the abundance of the p27Kip1 mRNA level both in the presence and absence of mitogens. Treatment with lovastatin revealed the increment of both CDK2- and CDK4-bound-p27Kip1. The experiment using antisense oligonucleotide against p27Kip1 showed significant amelioration of lovastatin-induced cell cycle arrest. Lovastatin reduced both platelet-derived growth factor-stimulated CDK2 and CDK4 kinase activities. In conclusion, lovastatin inhibited mesangial proliferation via translational upregulation or impairment of p27Kip1 protein degradation. Lovastatin serves as a potential therapeutic approach to mesangial proliferative disease.     

Comparative effects of overexpression of p27Kip1 and p21Cip1/Waf1 on growth and differentiation in human colon carcinoma cells

Recent studies have shown that decreased expression of p27Kip1 is associated with high grade tumors and an unfavorable prognosis in several types of human cancer. To clarify the role of p27Kip1 in colon cancer, we have overexpressed this protein in the HT29 colon cancer cell line. The derivatives displayed an increase in the p27Kip1 protein in cyclin E/CDK2 immunoprecipitates and a decrease in cyclin E-associated kinase activity when compared to vector control clones, providing evidence that the overexpressed protein was functional. Clones with a high level of p27Kip1 displayed partial growth inhibition in monolayer culture and a decrease in plating efficiency, even though they expressed increased levels of the cyclin D1 protein. Using alkaline phosphatase expression as a marker, we found that the p27Kip1 overexpressor clones displayed a 2-3-fold increase in sensitivity to induction of differentiation by 2 mM sodium butyrate. In contrast to these results, derivatives of HT29 cells that stably overexpressed p21Cip1/Waf1 displayed decreased sensitivity to the induction of differentiation. These findings may explain why decreased levels of p27Kip1 in certain human cancers is associated with high grade (poorly differentiated) tumors, and suggest that strategies that increase the level of p27Kip1 may be useful in cancer therapy.     

Increased expression of cyclin D1 in a murine mammary epithelial cell line induces p27kip1, inhibits growth, and enhances apoptosis

Cyclin D1 is frequently amplified and/or overexpressed in human breast cancer and several other types of cancer. To examine the role of cyclin D1 in normal mammary epithelial cells, in the present study we have overexpressed human cyclin D1 in the mouse mammary epithelial cell line HC11, using retrovirus-mediated transduction. We found that the cyclin D1 overexpresser clones displayed a decrease in saturation density, a decrease in anchorage-independent growth, an increased fraction of cells in the G(zero)-G1 phase, and increased expression of beta-casein, when compared to the control cells. The latter finding suggested that they were more differentiated. Furthermore, the cyclin D1 overexpressers displayed a marked increase in susceptibility to induction of apoptosis by serum withdrawal or by treatment with hydroxyurea or the protein kinase C inhibitors CGP 41251 and Ro31-8220. Thus, in some mammary epithelial cells, increased expression of cyclin D1 can inhibit growth, induce differentiation, and enhance apoptosis. These effects might be due, at least in part, to the fact that these derivatives displayed increased expression of the p27kip1 inhibitory protein.     

Ectopic expression of p27Kip1 in oligodendrocyte progenitor cells results in cell-cycle growth arrest

Oligodendrocyte differentiation is a complex process believed to be controlled by an intrinsic mechanism associated with cell-cycle arrest. Recently, the cell-cycle inhibitor protein p27 Kip1 has been proposed as a key element in causing growth arrest of oligodendrocyte precursor cells. To investigate the effects of p27 upon oligodendrocyte cell development, we have introduced the p27 cDNA in oligodendrocyte progenitor cells using an adenovirus vector. Progenitor cells normally express low levels of p27. After adenoviral infection and p27 overexpression, progenitor cells were able to undergo cell-cycle arrest, even in the presence of strong mitogens. The effects of p27 were shown to be directly upon cyclin-dependent kinase-2 (CDK2), the protein kinase complex responsible for G1/S transition, as immunodepletion of oligodendrocyte extracts of p27 protein resulted in the activation of CDK2 activity. However, cells that became growth arrested owing to infection with p27 adenovirus did not display conventional oligodendrocyte differentiation markers, such as O4 or O1. Taken together, these data provide mechanistic evidence indicating that p27 is primarily involved in oligodendroglial progenitor proliferation by inhibiting CDK2 activity and inducing oligodendrocyte cell-cycle arrest.     

Interleukin 1alpha and interleukin 6 promote the in vitro growth of both normal and neoplastic human cervical epithelial cells

We report here on a patient who was diagnosed with follicular carcinoma in 1985, and who was treated with total thyroidectomy. Two years later, when metastasis was found in his neck lesion, lung, pelvis and right femur, the patient received 131I treatment. Six years after receiving 131I treatment, the patient presented with hyperthyroidism. Whole-body scan with 131I revealed functioning metastasis in his right femur and pelvis. There was no hot spot in the neck region, confirming that no thyroid tissue remained. Blood panels revealed an increase in both TSH binding inhibitory immunoglobulin (TBII. 36.2%; normal. -10 approximately 10%) and thyroid stimulating antibody (TSAb, 176%; normal, less than 145%). Treatment with antithyroid drugs, dexamethasone and radioisotope therapy rapidly resolved his hyperthyroidism. Thyrotoxicosis and positive TRAb occurred in the absence of thyroid tissue, and many years after the completion of R1 therapy. The overproduction of thyroid hormone can therefore only be attributed to some mechanism of activity in the metastatic tumor tissue.     

Deregulated expression of p27(Kip1) in human breast cancers

Protein complexes composed of cyclins and cyclin-dependent kinases control the orderly progression of mammalian cells through the cell cycle. The p27(Kip1) protein belongs to a family of cyclin-dependent kinase-inhibitory proteins that are negative regulators of cell cycle progression and have been proposed as candidate tumor suppressor genes. However, the p27(Kip1) gene is only rarely mutated in human primary breast carcinomas and breast cancer cell lines. To further address the role of p27(Kip1) in the development of human tumors, we determined by Western blot analysis the levels of expression of the p27(Kip1) protein in a series of human cancer cell lines and found that this protein is expressed at high levels in many of these cell lines, even during exponential growth. The levels of p27(Kip1) were significantly associated with the levels of cyclins D1 and E. In contrast to the high level of p27(Kip1) in breast cancer cell lines, three cell lines established from normal mammary epithelium expressed low levels of this protein. Cell synchronization studies demonstrated deregulation of the expression of p27(Kip1) throughout the cell cycle in two breast cancer cell lines but normal regulation in a normal mammary epithelial cell line. Immunohistochemical studies on p27(Kip1) expression in 52 primary human breast cancers indicated that this protein was also expressed at relatively high levels in 44% of the tumor samples, but it was barely detectable or undetectable in the remaining 56% of the samples. Additional studies are required to determine why some breast cancer cells express relatively high levels of p27(Kip1) despite its known role as an inhibitor of cell cycle progression.     

Inactivation of p27Kip1 by the viral E1A oncoprotein in TGFbeta-treated cells

The adenovirus oncoprotein E1A and the simian virus SV40 large T antigen can both reverse the strong growth-inhibitory effect of transforming growth factor(TGF)-beta on mink lung epithelial cells: exposure of TGF-beta causes these cells to arrest late in the G1 phase of the cell cycle (ref. 3). This arrest correlates with an increase in expression of the protein p15Ink4B (ref. 4), inactivation of the cyclin E/A-cdk2 complex by the inhibitory protein p27Kip1 (refs 5-7), and with the accumulation of unphosphorylated retinoblastoma protein. The rescue by E1A of cells from TGF-beta arrest is partly independent of its binding to retinoblastoma protein. Here we show that E1A directly affects the cyclin-dependent kinase inhibitor p27Kip1 in TGF-beta-treated cells by binding to it and blocking its inhibitory effect, thereby restoring the activity of the cyclin-cdk2 kinase complex. In this way, E1A can overcome the effect of TGF-beta and modulate the cell cycle. To our knowledge, E1A provides the first example of a viral oncoprotein that can disable a cellular protein whose function is to inhibit the activity of cyclin-dependent kinases.     

Reduced levels of the cell-cycle inhibitor p27Kip1 in epithelial dysplasia and carcinoma of the oral cavity

Recent studies have shown that the cyclin-dependent kinase (cdk) inhibitors play important roles in cell cycle progression in normal cells. Alterations in the cdk inhibitors also appear to be important in cancer development in a number of human tumors. p27Kip1 is a member of the CIP/KIP family of cdk inhibitors that negatively regulates cyclin-cdk complexes. Reduced levels of p27Kip1 protein have been identified in a number of human cancers, and in some cases reduced p27Kip1 is associated with an increase in proliferative fraction. In the present study, we examined p27Kip1 protein by immunohistochemistry in 10 normal and 36 dysplastic epithelia and in 8 squamous cell carcinomas from one anatomical site within the oral cavity, the floor of the mouth. Proliferative activity was assessed in serial sections by determining the expression of the cell cycle proteins Ki-67 and cyclin A. p27kip1 protein was significantly reduced in oral dysplasias and carcinomas compared with that in normal epithelial controls. In addition, there was a significant reduction in p27Kip1 protein between low- and high-grade dysplasias, suggesting that changes in p27Kip1 expression may be an early event in oral carcinogenesis. There was increasing expression of Ki-67 and cyclin A proteins with increasingly severe grades of dysplasia compared with normal controls. Although there was a strong correlation between Ki-67 and cyclin A scores (r2= 0.61) for all categories of disease, there was a weak negative correlation between Ki-67 and p27Kip1 levels (r2 = 0.29) and between cyclin A and p27Kip1 levels (r2 = 0.25). In conclusion, this study has found that a reduction in the proportion of cells expressing p27Kip1 protein is frequently associated with oral dysplasia and carcinoma from the floor of the mouth. Furthermore, reductions in p27Kip1 levels are associated with increased cell proliferation, although other changes likely contribute to altered cell kinetics during carcinogenesis at this site.     

Cytoplasmic displacement of cyclin E-cdk2 inhibitors p21Cip1 and p27Kip1 in anchorage-independent cells

Loss of attachment to an extracellular matrix substrate arrests the growth of untransformed cells in the G1 phase. This anchorage-dependent cell cycle arrest is linked to increased expression of the p21Cip1 (p21) and p27Kip1 (p27) cyclin-dependent kinase inhibitors. The result is a loss of cdk2-associated kinase activity, especially that of cyclin E-cdk2. The levels of p21 and p27 are also upregulated in unattached transformed cells, but cyclin E-cdk2 activity remains high, and the cells are able to grow in an anchorage-independent manner. Increased expression of cyclin E and cdk2 appears to be partially responsible for the maintenance of cyclin E-cdk2 activity in transformed cells. To explore further the regulation of cyclin E-cdk2 in transformed cells, we have analysed the subcellular distribution of cyclin-cdk complexes and their inhibitors in normal human fibroblasts, their transformed counterparts, and in various human tumor cell lines. In substrate-attached normal fibroblasts, cyclin E and cdk2 were exclusively in the nuclear fraction, associated with one another. When normal fibroblasts were detached and held in suspension, cyclin E-cdk2 complexes remained nuclear, but were now found associated with the p21 and p27 cdk inhibitors and lacked histone H1 phosphorylating activity. In contrast, the transformed fibroblasts and tumor cells, which are anchorage-independent, had more than half of their cyclin E, cdk2, p21 and p27 in the cytoplasmic fraction, both in attached and suspended cultures. The cytoplasmic p21 and p27 were bound to cyclin E-cdk2, as well as to complexes containing cyclin A and cyclin D. The nuclear cyclin E-cdk2 complexes from the transformed cells grown in suspension contained only low levels of p21 and p27 and had histone H1 kinase activity. Thus, at least three mechanisms contribute to keeping cyclin E-cdk2 complexes active in suspended anchorage-independent cells: cyclin E and cdk2 are upregulated, as reported previously, cdk inhibitors are sequestered away from the nucleus by cytoplasmic cyclin-cdk complexes, and the binding of the inhibitors to nuclear cyclin E-cdk2 complexes is impaired.     

Surface proteins of normal and transformed epithelial liver cells

Surface protein patterns of 12 various tumorigenic and non-tumorigenic liver epithelial cell lines (IAR series) were studied by means of lactoperoxidase catalyzed iodination followed by the gradient sodium dodecyl sulfate polyacrylamide gel electrophoresis. These patterns were found to be very similar except a protein with an apparent mol. wt. about 150 kilodaltons which appeared in cells of tumors derived from in vitro transformed epithelial liver cells. Surface fibronectin was present in all the three non-tumorigenic lines, and in 7 of 9 tumorigenic cell lines examined. The indirect immunofluorescence revealed that in non-tumorigenic lines this protein was localized predominantly in the regions of cell-to-cell contacts, while in tumorigenic cells fibronectin often formed fibrillar networks similar to those of fibroblasts.     

TGF-beta 1 induces the cyclin-dependent kinase inhibitor p27Kip1 mRNA and protein in murine B cells

TGF-beta1 inhibits the cell cycle progression of many types of cells by arresting them in the G1 phase. This cell cycle arrest has been attributed to the regulatory effects of TGF-beta1 on both the levels and the activities of the G1 cyclins and their kinase partners. The activities of these kinases are negatively regulated by a number of proteins, such as p15INK4b, p21WAF1/Cip1, and p27Kip1, that physically associate with cyclins, cyclin-dependent kinases (Cdk), or cyclin-Cdk complexes. In epithelial cell lines, TGF-beta1 was previously shown to inhibit cell cycle progression through down-regulation of Cdk4 and/or up-regulation of p15INK4b and/or p21WAF1/Cip1. However, TGF-beta1 had little or no effect on the p27Kip1 mRNA and protein levels. In this report, we show that, in contrast to observations in epithelial cell lines, TGF-beta1 increased the p27Kip1 mRNA and protein levels in the murine B cell lines CH31 and WEHI231. This TGF-beta1-mediated induction of p27Kip1 also resulted in an increased association of p27Kip1 with Cdk2 and a decreased Cdk2 kinase activity. In contrast to epithelial cells, however, TGF-beta1 had little or no effect on the Cdk4 and p21WAF1/Cip1 protein levels in these B cells. Finally, although several studies suggested a direct role of p53 in TGF-beta1-mediated cell cycle arrest in epithelial cells, TGF-beta1 inhibited cell cycle progression in CH31 even in the absence of wild-type p53. Taken together, these results suggest that TGF-beta1 induces G1 arrest in B cells primarily through a p53-independent up-regulation of p27Kip1 protein.     

p27Kip1: a key mediator of retinoic acid induced growth arrest in the SMS-KCNR human neuroblastoma cell line

Retinoic acid (RA) treatment of SMS-KCNR neuroblastoma (NB) cells leads to G1 growth arrest and neuronal differentiation. To investigate the molecular mechanisms by which RA alters cell growth, we analysed the expression and activity of components of the cell cycle machinery after culture in RA. Within 2 days of RA treatment and prior to the arrest of NB cells in the G1 phase of the cell cycle, there is a complete downregulation of G1 cyclin/Cdk activities. Protein levels for the G1 cyclin/Cdks were essentially unchanged during this time although there was a decrease in the steady-state levels of p67N-Myc and hyperphosphorylated Rb proteins. The Cdk inhibitors, p21Cip1 and p27Kip1 were constitutively expressed in KCNR while p15INK4B and p16INK4A were not detected. RA induced an increase in the expression of p27Kip1 but not p21Cip1. Furthermore, coincident with the decrease in kinase activity there was an increase in G1 cyclin/Cdk bound p27Kip1. These results indicate that changes in the level of p27Kip1 and its binding to G1 cyclin/Cdks may play a key role in RA induced growth arrest of NB cells.     

Critical role for p27Kip1 in cell cycle arrest after androgen depletion in mouse mammary carcinoma cells (SC-3)

The molecular mechanisms underlying androgen-regulated cancer growth and the frequent development of refractoriness to endocrine therapy remain unknown. In this study functional and quantitative alterations in cell cycle regulators after androgen depletion were examined in androgen-dependent mouse mammary carcinoma cells (SC-3) as a model system to clarify the initial response of cancer cells to anti-androgen therapy. FACS analysis of SC-3 cells cultured with or without 10(-7) M testosterone revealed that suppression of cell growth after hormone withdrawal was due to GI arrest. Although cyclin D1/Cdk4 activity decreased along with a reduced level of cyclin D1 protein, this was a later event (48-72 h) than the G1 arrest (24 h). Taken together with the results that constitutive expression of cyclin D1 in SC-3 cells did not overcome the growth suppression following androgen depletion, the existence of an alternative pathway(s) causing G1 arrest was suggested. Cyclin E/Cdk2 and cyclin A/Cdk2 activities decreased significantly at 24 h without apparent changes in the amounts of Cdk2, cyclin E or cyclin A. Among various Cdk inhibitors (CKIs) examined, p27Kip1 was upregulated at both mRNA and protein levels at 24 h after androgen depletion. In addition, immunoprecipitation-Western analysis showed that the amount of p27Kip1 associated with Cdk2 complexes increased as early as 24 h. These results suggest that p27Kip1 CKI is a critical target in the initial response of cancer cells to androgen depletion and plays a key role in Cdk2 inactivation through association with the kinase complex, leading to cell cycle arrest.     

Rapamycin resistance tied to defective regulation of p27Kip1

The potent antiproliferative activity of the macrolide antibiotic rapamycin is known to involve binding of the drug to its cytosolic receptor, FKBP12, and subsequent interaction with targets of rapamycin, resulting in inhibition of p70 S6 kinase (p70S6K). However, the downstream events that lead to inhibition of cell cycle progression remain to be elucidated. The antiproliferative effects of rapamycin are associated with prevention of mitogen-induced downregulation of the cyclin-dependent kinase inhibitor p27Kip1, suggesting that the latter may play an important role in the growth pathway targeted by rapamycin. Murine BC3H1 cells, selected for resistance to growth inhibition by rapamycin, exhibited an intact p70S6K pathway but had abnormally low p27 levels that were no longer responsive to mitogens or rapamycin. Fibroblasts and T lymphocytes from mice with a targeted disruption of the p27Kip1 gene had impaired growth-inhibitory responses to rapamycin. These results suggest that the ability to regulate p27Kip1 levels is important for rapamycin to exert its antiproliferative effects.     

Inactivation of p16 in human mammary epithelial cells by CpG island methylation

Proliferation of human mammary epithelial cells (HMEC) is limited to a few passages in culture due to an arrest in G1 termed selection or mortality stage 0, M0. A small number of cells spontaneously escape M0, continue to proliferate in culture, and then enter a second mortality stage, M1, at which they senesce. Evidence that M0 involves the Rb pathway comes from the observation that expression of human papillomavirus type 16 E7 alleviates the M0 proliferation block, and we further show that the Rb-binding region of E7 is required to allow cells to bypass M0. In contrast, E6 does not prevent HMEC from entering M0 but, rather, is involved in M1 bypass. Here we show that inactivation of the D-type cyclin-dependent kinase inhibitor p16INK4A is associated with escape from the M0 proliferation block. Early-passage HMEC express readily detectable amounts of p16 protein, whereas normal or E6-expressing HMEC that escaped M0 expressed markedly reduced amounts of p16 mRNA and protein. This initial reduction of p16 expression was associated with limited methylation of the p16 promoter region CpG island. At later passages, a further reduction in p16 expression occurred, accompanied by increased CpG island methylation. In contrast, reduction of p16 expression did not occur in E7-expressing HMEC that bypassed M0, due to inactivation of Rb. These observations in the E6-expressing HMEC correlate well with the finding that CpG island methylation is a mechanism of p16 inactivation in the development of human tumors, including breast cancer.     

Cultures of normal human mammary cells

This paper describes a method of obtaining epithelial cells from large quantities of normal human breast tissue and the response of these cells in culture to lactogenic hormones. Suspensions of single cells and clusters of cells resembling normal ductal and alveolar structures were obtained by mechanical disaggregation and subsequent (3h) incubation of tissue fragments in 0.5 mg/ml collagenase. Cells rapidly attached to glass or plastic surfaces within 48 h and grew to form large colonies which maintained their epithelial appearance throughout 2 months of observation. Cell cycling as monitored by DNA synthesis was enhanced by insulin, hydrocortisone, or ovine prolactin (in concentrations of 5.0mug/ml each) at respectively 2,3 and 5 days of incubation. These results were observed in cultures derived from 3 premenopause samples of mammary tissue maintained in medium with 1% fetal calf serum. Prolactin at a concentration of 5 mug/ml induced phosphoprotein synthesis 8-fold over control values. In addition, prolactin induced morphological changes in cells including the development of distended endoplasmic reticulum, large microvilli, and the deposition of glycogen granules. These initial results led to the tentative conclusion that prolactin was sufficient to initiate some of the characteristics in cultured cells normally associated with lactating tissues.     

Cytotoxic effects of adenovirus-mediated wild-type p53 protein expression in normal and tumor mammary epithelial cells

To evaluate the effects of the wild-type p53 expression in normal and tumor cells, we have constructed a recombinant adenovirus vector (E1 minus) expressing human wild-type p53 cDNA (AdWTp53). Infection of normal and tumor cells of lung and mammary epithelial origin with AdWTp53 resulted in high levels of wild-type p53 expression. Production of p53 protein following infection was dependent on the dose of AdWTp53 with maximum amounts of p53 produced following infection with 50 plaque-forming units/cell. AdWTp53 infection inhibited the growth of all human cell lines studied. However, tumor cells that were null for p53 prior to infection (H-358 and MDA-MB-157) and tumor cells that expressed mutant endogenous p53 protein (MDA-MB-231 and MDA-MB-453) were more sensitive to AdWTp53 cytotoxicity than cells that contained the wild-type p53 (MCF-7, MCF-10, 184B5, and normal mammary epithelial cells). All cells exhibited WAF1/Cip1 mRNA and protein induction following AdWTp53 infection. AdWTp53-induced cytotoxicity of human tumor cell lines expressing mutant p53 was mediated by apoptosis as revealed by nucleosomal DNA fragmentation analysis. No detectable nucleosomal DNA fragmentation was observed following AdWTp53 infection of human cells expressing wild-type p53. These data suggest that endogenous p53 status is a determinant of AdWTp53-mediated cell killing of human tumor cells.