Association between human cancer and two polymorphisms occurring together in the p21Waf1/Cip1 cyclin-dependent kinase inhibitor gene

BACKGROUND: The cyclin-dependent kinase inhibitor gene p21Waf1/Cip1 plays a role in signaling cellular growth arrest. In response to DNA damage, p21 is induced by the p53 gene, thereby playing a direct role in mediating p53-induced G1 arrest. Alterations in this gene may adversely affect regulation of cellular proliferation and increase susceptibility for cancer. Two polymorphisms have previously been characterized in the p21 gene: a C-->A transversion at codon 31 (ser-->arg) and a C-->T transition 20 nucleotides downstream from the 3' end of exon 3. METHODS: The codon 31 polymorphism in exon 2 of the p21 gene was identified by restriction digestion (Alw26I) of products amplified by polymerase chain reaction (PCR). The polymorphism downstream from exon 3 of the p21 gene was identified by single strand conformation polymorphism (SSCP) analysis of PCR amplified products and was confirmed by PstI enzyme restriction digestion. DNA variant alleles were confirmed by direct DNA sequencing. The entire coding region and the promoter region (p53 binding domain) of the p21 gene were screened for mutations by SSCP analysis or DNA sequencing. RESULTS: The two polymorphisms were found in 18 of 96 tumor samples lacking p53 alterations (18.8%). Nine of 54 prostate adenocarcinoma samples (16.7%) contained both p21 variants, whereas 9 of 42 squamous cell carcinomas of the head and neck (21.4%) displayed both polymorphisms. Of the 110 controls examined, 10 (9.1%) had both alterations. Both p21 polymorphisms occurred together in all samples examined and there was no indication of mutation in the coding region of the p21 gene or in the p53 binding domain of the promoter region. CONCLUSIONS: These data suggest that p21 gene variants may play a role in increased susceptibility for the development of some types of cancer. In the current study, the authors demonstrated that the occurrence of these two polymorphisms is increased in prostate adenocarcinoma and squamous cell carcinoma of the head and neck. The polymorphic sites may be directly responsible for this apparent increased susceptibility or they may be linked to regulatory region alterations.     

The absence of p27Kip1, an inhibitor of G1 cyclin-dependent kinases, uncouples differentiation and growth arrest during the granulosa->luteal transition

The involvement of cyclin-dependent kinase inhibitors in differentiation remains unclear: are the roles of cyclin-dependent kinase inhibitors restricted to cell cycle arrest; or also required for completion of the differentiation program; or both? Here, we report that differentiation of luteal cells can be uncoupled from growth arrest in p27-deficient mice. In these mice, female-specific infertility correlates with a failure of embryos to implant at embryonic day 4.5. We show by ovarian transplant and hormone reconstitution experiments that failure to regulate luteal cell estradiol is one physiological mechanism for infertility in these mice. This failure is not due to a failure of p27-deficient granulosa cells to differentiate after hormonal stimulation; P450scc, a marker for luteal progesterone biosynthesis, is expressed and granulosa cell-specific cyclin D2 expression is reduced. However, unlike their wild-type counterparts, p27-deficient luteal cells continue to proliferate for up to 3.5 days after hormonal stimulation. By day 5.5, however, these cells withdraw from the cell cycle, suggesting that p27 plays a role in the early events regulating withdrawal of cells from the cell cycle. We have further shown that in the absence of this timely withdrawal, estradiol regulation is perturbed, explaining in part how fertility is compromised at the level of implantation. These data support the interpretation of our previous observations on oligodendrocyte differentiation about a role for p27 in establishing the nonproliferative state, which in some cases (oligodendrocytes) is required for differentiation, whereas in other cases it is required for the proper functioning of a differentiated cell (luteal cell).     

Expression of cyclin-dependent kinase inhibitor p21 in human liver

The p21 protein is a universal inhibitor of cyclin-dependent kinases and of cell-cycle progression and is involved in numerous growth-inhibitory pathways in cell culture systems. Recent studies suggest that p21 regulates hepatocyte cell cycle progression in models of liver regeneration. The present study was designed to investigate the possible involvement of p21 in the control of hepatocyte proliferation in human liver diseases. To examine that, the expression of p21 in clinical liver biopsy specimens was determined by immunohistochemistry. This was correlated with hepatocyte Ki-67 immunostaining (a marker of hepatocyte proliferation in vivo) as well as histologic features. Little p21 or Ki-67 expression was detected in normal human liver or in specimens of nonalcoholic steatohepatitis. In patients with alcoholic hepatitis, increased expression of p21, but not of Ki-67, was observed. In specimens with chronic hepatitis C, hepatocyte p21 expression was significantly correlated with Ki-67 immunostaining, as well as with the degree of inflammation and fibrosis. These results indicate that hepatocyte p21 expression is upregulated in response to hepatic injury and correlates with histologic markers of proliferation and disease activity. This study provides evidence that p21 plays a role in the regulation of hepatocyte proliferation in human liver diseases.     

Interaction with cyclin-dependent kinases and PCNA modulates proteasome-dependent degradation of p21

The cyclin-dependent kinase (CDK) inhibitor p21(Cip1/Waf1) plays an essential role in the control of cell proliferation by modulating the activity of cyclin/CDK complexes in response to various intracellular or extracellular signals. Small variations in p21 expression levels may determine whether it acts as an inhibitor or an assembly factor for cyclin/CDK complexes. It is therefore critical to better characterize the mechanisms regulating p21 abundance. Here, we show, using a tetracycline-regulated system in p53-deficient DLD-1 human colon cancer cells, that p21 protein levels and stability are regulated by the proteasome-dependent degradation pathway and by association with its partners, CDKs and PCNA. A p21 mutant deficient for interaction with CDKs, p21CDK-, displayed an enhanced stability and greatly reduced sensitivity to proteasome-mediated proteolysis, indicating that association with cyclin/CDK complexes may trigger p21 degradation. In contrast, a p21 mutant impaired in the interaction with PCNA, p21PCNA-, exhibited a decreased stability, suggesting that association with PCNA protects p21 from proteasome-dependent degradation. Furthermore, the abundance of p21 itself, in addition to protein-protein interactions, may also modulate p21 stability since we found that high levels of p21 expression overcome proteasome-dependent regulation of p21 accumulation.     

Nitric oxide-induced apoptosis in human pancreatic carcinoma cell lines is associated with a G1-arrest and an increase of the cyclin-dependent kinase inhibitor p21WAF1/CIP1

Nitric oxide (NO) is a messenger molecule with various biological activities including DNA damage. In the present study, we examined the influence of endogenously produced NO on human pancreatic cell lines. In response to cytokine stimulation (tumor necrosis factor alpha, IFN-gamma, and interleukin 1beta), human pancreatic carcinoma cell lines expressed the inducible NO synthase that synthesizes NO, detectable as nitrate and nitrite in the culture supernatants. Endogenously produced NO induced apoptosis in all of the tested pancreatic carcinoma cell lines. In cell cycle analysis, endogenous production of NO revealed a G1-arrest in all of the tested cell lines. This G1-arrest was blockable by addition of NG-monomethyl-L-arginine. These data indicate that NO induces a G1-arrest followed by apoptosis in pancreatic carcinoma cell lines.     

The cyclin-dependent kinase inhibitor p40SIC1 imposes the requirement for Cln G1 cyclin function at Start

In yeast, commitment to cell division (Start) is catalyzed by activation of the Cdc28 protein kinase in late G1 phase by the Cln1, Cln2, and Cln3 G1 cyclins. The Clns are essential, rate-limiting activators of Start because cells lacking Cln function (referred to as cln-) arrest at Start and because CLN dosage modulates the timing of Start. At or shortly after Start, the development of B-type cyclin Clb-Cdc28 kinase activity and initiation of DNA replication requires the destruction of p40SIC1, a specific inhibitor of the Clb-Cdc28 kinases. I report here that cln cells are rendered viable by deletion of SIC1. Conversely, in cln1 cln2 cells, which have low CLN activity, modest increases in SIC1 gene dosage cause inviability. Deletion of SIC1 does not cause a general bypass of Start since (cln-)sic1 cells remain sensitive to mating pheromone-induced arrest. Far1, a pheromone-activated inhibitor of Cln-Cdc28 kinases, is dispensable for arrest of (cln-)sic1 cells by pheromone, implying the existence of an alternate Far1-independent arrest pathway. These observations define a pheromone-sensitive activity able to catalyze Start only in the absence of p40SIC1. The existence of this activity means that the B-type cyclin inhibitor p40SIC1 imposes the requirement for Cln function at Start.     

Cell-free degradation of p27(kip1), a G1 cyclin-dependent kinase inhibitor, is dependent on CDK2 activity and the proteasome

Entry into S phase is dependent on the coordinated activation of CDK4,6 and CDK2 kinases. Once a cell commits to S phase, there must be a mechanism to ensure the irreversibility of this decision. The activity of these kinases is inhibited by their association with p27. In many cells, p27 plays a major role in the withdrawal from the cell cycle in response to environmental cues. Thus, it is likely that p27 is a target of the machinery required to ensure the irreversibility of S-phase entry. We have been interested in understanding the mechanisms regulating p27 at the G1/S transition. In this report, we define a cell-free degradation system which faithfully recapitulates the cell cycle phase-specific degradation of p27. We show that this reaction is dependent on active CDK2 activity, suggesting that CDK2 activity is directly required for p27 degradation. In addition to CDK2, other S-phase-specific factors are required for p27 degradation. At least some of these factors are ubiquitin and proteasome dependent. We discuss the relationships between CDK2 activity, ubiquitin-dependent, and possibly ubiquitin-independent proteasomal activities in S-phase extracts as related to p27.     

p21Cip1/WAF1 is important for differentiation and survival of U937 cells

Vitamin D3 (VD3) induces monocytic differentiation of U937 cells. Induction of p21Cip1/WAF1 (p21) and subsequent G0/G1 cell-cycle arrest are required in this process. Using a system of inducible expression of ectopic p21, we demonstrated the important role of p21 in the induction of monocytic differentiation in U937 cells. Prior induction of antisense-p21 expression significantly suppressed p21 expression, and resulted in inhibition of VD3-induced U937 differentiation. Moreover, induction of expression of antisense-p21 in VD3-differentiated U937 cells resulted in apoptosis of the cells. This was associated with activation of Cdc2 and caspase-3 like protease. Our results suggest that p21 is required for the initiation of the early steps of differentiation as well as survival of differentiated cells.     

Anticarcinogenic effect of a flavonoid antioxidant, silymarin, in human breast cancer cells MDA-MB 468: induction of G1 arrest through an increase in Cip1/p21 concomitant with a decrease in kinase activity of cyclin-dependent kinases and associated cyclins

There is an increasing interest in identifying potent cancer preventive and therapeutic agents against breast cancer. Silymarin, a flavonoid antioxidant isolated from milk thistle, exerts exceptionally high to complete anticarcinogenic effects in tumorigenesis models of epithelial origin. In this study, we investigated the anticarcinogenic effect of silymarin and associated molecular mechanisms, using human breast carcinoma cells MDA-MB 468. Silymarin treatment resulted in a significantly high to complete inhibition of both anchorage-dependent and anchorage-independent cell growth in a dose- and time-dependent manner. The inhibitory effects of silymarin on cell growth and proliferation were associated with a G1 arrest in cell cycle progression concomitant with an induction of up to 19-fold in the protein expression of cyclin-dependent kinase (CDK) inhibitor Cip1/p21. Following silymarin treatment of cells, an incremental binding of Cip1/p21 with CDK2 and CDK6 paralleled a significant decrease in CDK2-, CDK6-, cyclin D1-, and cyclin E-associated kinase activity with no change in CDK2 and CDK6 expression but a decrease in G1 cyclins D1 and E. Taken together, these results suggest that silymarin may exert a strong anticarcinogenic effect against breast cancer and that this effect possibly involves an induction of Cip1/p21 by silymarin, which inhibits the threshold kinase activities of CDKs and associated cyclins, leading to a G1 arrest in cell cycle progression.     

Nuclear accumulation of p21Cip1 at the onset of mitosis: a role at the G2/M-phase transition

Cell cycle arrest in G1 in response to ionizing radiation or senescence is believed to be provoked by inactivation of G1 cyclin-cyclin-dependent kinases (Cdks) by the Cdk inhibitor p21(Cip1/Waf1/Sdi1). We provide evidence that in addition to exerting negative control of the G1/S phase transition, p21 may play a role at the onset of mitosis. In nontransformed fibroblasts, p21 transiently reaccumulates in the nucleus near the G2/M-phase boundary, concomitant with cyclin B1 nuclear translocation, and associates with a fraction of cyclin A-Cdk and cyclin B1-Cdk complexes. Premitotic nuclear accumulation of cyclin B1 is not detectable in cells with low p21 levels, such as fibroblasts expressing the viral human papillomavirus type 16 E6 oncoprotein, which functionally inactivates p53, or in tumor-derived cells. Moreover, synchronized E6-expressing fibroblasts show accelerated entry into mitosis compared to wild-type cells and exhibit higher cyclin A- and cyclin B1-associated kinase activities. Finally, primary embryonic fibroblasts derived from p21-/- mice have significantly reduced numbers of premitotic cells with nuclear cyclin B1. These data suggest that p21 promotes a transient pause late in G2 that may contribute to the implementation of late cell cycle checkpoint controls.     

E-Cadherin-dependent growth suppression is mediated by the cyclin-dependent kinase inhibitor p27(KIP1)

Recent studies have demonstrated the importance of E-cadherin, a homophilic cell-cell adhesion molecule, in contact inhibition of growth of normal epithelial cells. Many tumor cells also maintain strong intercellular adhesion, and are growth-inhibited by cell- cell contact, especially when grown in three-dimensional culture. To determine if E-cadherin could mediate contact-dependent growth inhibition of nonadherent EMT/6 mouse mammary carcinoma cells that lack E-cadherin, we transfected these cells with an exogenous E-cadherin expression vector. E-cadherin expression in EMT/6 cells resulted in tighter adhesion of multicellular spheroids and a reduced proliferative fraction in three-dimensional culture. In addition to increased cell-cell adhesion, E-cadherin expression also resulted in dephosphorylation of the retinoblastoma protein, an increase in the level of the cyclin-dependent kinase inhibitor p27(kip1) and a late reduction in cyclin D1 protein. Tightly adherent spheroids also showed increased levels of p27 bound to the cyclin E-cdk2 complex, and a reduction in cyclin E-cdk2 activity. Exposure to E-cadherin-neutralizing antibodies in three-dimensional culture simultaneously prevented adhesion and stimulated proliferation of E-cadherin transfectants as well as a panel of human colon, breast, and lung carcinoma cell lines that express functional E-cadherin. To test the importance of p27 in E-cadherin-dependent growth inhibition, we engineered E-cadherin-positive cells to express inducible p27. By forcing expression of p27 levels similar to those observed in aggregated cells, the stimulatory effect of E-cadherin-neutralizing antibodies on proliferation could be inhibited. This study demonstrates that E-cadherin, classically described as an invasion suppressor, is also a major growth suppressor, and its ability to inhibit proliferation involves upregulation of the cyclin-dependent kinase inhibitor p27.     

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.     

Potential role of WAF1/Cip1/p21 as a mediator of TGF-beta cytoinhibitory effect

Transforming growth factor-beta (TGF-beta) inhibits cell cycle progression of many types of human cells by arresting them in the G1 phase of the cell cycle. The arrest is mediated through interactions of various cyclin-dependent protein kinases (CDKs) and their inhibitors. We demonstrate that treatment with TGF-beta induces increased levels of WAF1/Cip1/p21, a potent inhibitor of various cyclin-CDK kinase activities, in two colon cancer cell lines (LS1034 and LS513), which are sensitive to TGF-beta-induced growth arrest. The induction in at least one of these cell lines (LS1034,p53-) is p53-independent. No WAF1 induction was observed after TGF-beta treatment in a third cell line (HT-29), which is completely insensitive to the cytoinhibitory effect of TGF-beta. In both LS513 and LS1034, WAF1 induction correlated with reduced cyclin E-associated kinase activity in vitro and suppression of the retinoblastoma susceptibility gene (Rb) protein phosphorylation in vivo. In addition, WAF1 was physically associated with cyclin E in the two sensitive cell lines. These results suggest that WAF1/Cip1/p21 is a mediator of cellular sensitivity to TGF-beta.     

TGF-beta mediated G1 arrest in a human melanoma cell line lacking p15INK4B: evidence for cooperation between p21Cip1/WAF1 and p27Kip1

We have studied TGF-beta mediated G1 arrest in WM35, an early stage human melanoma cell line. These cells have lost p15INK4B expression through loss of one chromosome 9 and rearrangement of the other. In asynchronously growing WM35, TGF-beta caused reductions in cyclin D1, cyclin A and cdk4 proteins and their associated kinase activities and an increase in both p21Cip1/WAF1 and p27Kip1. These findings were confirmed in cells released from quiescence in the presence of TGF-beta, in which TGF-beta inhibited or delayed the reduction in the cdk inhibitors that normally occurs in late G1. In contrast to observations in other cell types, there was an increased association of both p21Cip1/WAF1 and p27Kip1 with cyclin D1/cdk4 and with cyclin E/cdk2 during TGF-beta mediated arrest of asynchronously growing cells. Upregulation of p21Cip1/WAF1 preceded that of p27Kip1. Furthermore, p21Cip1/WAF1 and p27Kip1 were not present in the same cdk complexes but bound distinct populations of target cdk molecules. Both p21Cip1/WAF1 and p27Kip1 immunoprecipitates from asynchronously growing cells contained active kinase complexes. These KIP-associated kinase activities were reduced in TGF-beta arrested cells. It has been proposed that in TGF-beta arrested epithelial cells, up-regulation of p15INK4B and of p15INK4B binding to cdk4 serves to destabilize the association of p27Kip1 with cyclin D1/cdk4, promoting p27Kip1 binding and inhibition of cyclin E/cdk2. Our findings demonstrate that this is not a universal mechanism of G1 arrest by TGF-beta. In TGF-beta arrested WM35, which lack p15INK4B, the increased p21Cip1/WAF1 may serve a similar function to that of p15INK4B: initiating kinase inhibition and providing an additional mechanism to supplement the effect of p27Kip1 on G1 cyclin/cdks.     

Butyrate-induced differentiation of Caco-2 cells is associated with apoptosis and early induction of p21Waf1/Cip1 and p27Kip1

BACKGROUND: Intestinal mucosal turnover is a process of proliferation, differentiation, and apoptosis; the mechanisms remain largely undefined. The purpose of our study was to (1) assess the relationship between apoptosis and enterocyte differentiation and (2) determine whether the cell-cycle inhibitors, p21Waf1/Cip1 and p27Kip1, or the apoptosis inhibitors, Bcl-2 and Bcl-XL, may be involved. METHODS: Gut-derived Caco-2 cells were treated with sodium butyrate. Apoptosis was assessed by Hoechst stain, DNA laddering, and annexin V assay; differentiation was determined by alkaline phosphatase and sucrase activity. RNA and protein were analyzed for expression of p21Waf1/Cip1, p27Kip1, and members of the Bcl-2 family. RESULTS: Treatment of Caco-2 cells with sodium butyrate resulted in the concomitant induction of both differentiation (increased alkaline phosphatase and sucrase activity) and apoptosis. Increased levels of p21Waf1/Cip1 and p27Kip1 mRNA and protein were detected at 24 hours, occurring before apoptosis or differentiation; decreased mRNA levels of Bcl-2 and Bcl-XL were noted at 24 hours. CONCLUSIONS: Differentiation and apoptosis occurred simultaneously in Caco-2 cells, suggesting that apoptosis may be linked to enterocyte differentiation. The induction of p21Waf1/Cip1 and p27Kip1 and the down-regulation of Bcl-2 and Bcl-XL further suggest a link between the cell-cycle mechanisms regulating enterocyte differentiation and apoptosis.