Novel polymorphism in p21(waf1/cip1) cyclin dependent kinase inhibitor gene: association with human esophageal cancer

p21(waf1/cip1), an important regulator of the cell cycle, binds to PCNA and acts as a mediator of the growth suppressing and apoptosis promoting functions of p53. We report a hitherto unobserved polymorphism in the carboxy terminal domain (codon 149) of p21(waf1/cip1) gene, the domain encoding the PCNA binding motif. The codon 149 polymorphism (GAT-->GGT) was observed in 42 of 50 (84%) esophageal squamous cell carcinomas (ESCCs) and eight of 50 (16%) normal individuals. The resultant amino acid substitution from aspartate to glycine may have vital implication in PCNA mediated cell cycle regulation by p21(waf1/cip1). The second polymorphism at codon 31, involving a C-->A transversion at nucleotide 168 (AGC-->AGA) changing the amino acid from serine to arginine, was observed in 2/50 (4%) ESCCs at a relatively lower frequency in the Indian population than that reported in the West. No significant association was observed between p21(wap1/cip1) polymorphism at codon 149 and p21(wap1/cip1) protein expression in ESCC in this cohort of patients. Interestingly, the frequency of p21(wap1/cip1) variants (codon 149) in ESCCs (18 of 19 cases) with wild-type p53 was significantly higher than in tumors with p53 mutations, suggesting that this polymorphism affects the p53 pathway and may play an important role in esophageal tumorigenesis. Analysis of p21(waf1/cip1) expression in relation to p53 gene and protein status revealed its induction by p53-dependent as well as independent pathways in esophageal tumorigenesis.     

The p21(cip1/waf1) cyclin-dependent kinase inhibitor enhances the cytotoxic effect of cisplatin in human ovarian carcinoma cells

The seriousness of ovarian cancer, which is related to the observed link between recurrency and cell cycle control defect, prompted us to explore the effect of ectopic expression of the cdk inhibitor p21(cip1/waf1) on ovarian carcinoma chemosensitivity. The transfection of p21(cip1/waf1) cDNA into SKOV3 and OVCAR3 cells led to reduction of tumor cell growth, enhanced susceptibility to cisplatin-induced apoptosis, and abolition of recurrency after cisplatin exposure. p21(cip1/waf1) gene transfer allowed a marked reduction of the cisplatin concentration needed to erradicate the tumor cell population. These results suggest exploring the possible use of p21(cip1/waf1) as an adjunctive to conventional chemotherapy.     

Prognostic impact of p21/waf1/cip1 in colorectal cancer

In addition to the tumor suppressor gene p53, Cyclin Dependent Kinases (CDK) are well known to influence the cell cycle in normal human tissues and various neoplasias as well. The purpose of our present study was to evaluate the expression of the CDK-inhibitor p21/waf1/cip1 in colorectal cancer with special emphasis on the prognostic impact. Between 1985 and 1991, 294 patients (median age, 65 years) underwent surgical operative therapy for colorectal cancer. Formalin-fixed and paraffin-embedded tumor specimens were investigated. For immunohistochemistry the Catalysed Reporter Deposition (CARD) technique was performed. The survival probability was calculated and possible prognostic risk factors were tested using multivariate analysis. The p21/ waf1/cip1 staining pattern was positive in 197 (67%) specimens and negative in 97 (33%) samples. No significant correlation could been calculated between p21/waf1/cip1 expression and other variables such as age, sex, WHO-Classification, localisation, grading, TNM-classification or UICC-stage. Patients with a positive staining reaction had a significantly better survival (p < 0.0052). Moreover, p21/waf1/cip1 was shown to be an independent prognostic parameter by multivariate analysis (p < 0.022). In contrast with these findings, the p53 tumor status had no impact on survival. P21/ waf1/cip1 appears to be an independent prognostic parameter in colorectal cancer and is associated with a favorable survival. This feature may be related to a cell cycle arrest in the G1 phase induced by p21/waf1/cip1, resulting in lower tumor cell proliferative activity.     

The cyclin-dependent kinase inhibitor p21(cip1/waf1) enhances the cytotoxicity of ganciclovir in HSV-tk transfected ovarian carcinoma cells

Suicide gene therapy could be an attractive addition to the treatment of ovarian carcinomas, for which acquired chemoresistance frequently results in treatment failure. Here we show that transfection of the HSV-tk gene, followed by incubation with up to 1 mM ganciclovir fails to induce cell death in SKOV3 chemoresistant human ovarian carcinoma cells. However, co-transfection of HSV-tk with Cip1/Waf1 encoding the p21(cip1/waf1) inhibitor of cdks, allows 100 microM ganciclovir to eradicate the population of tumor cells. Potentiation of a drug by co-transfer of HSV-tk with Cip1/Waf1could thus represent another therapeutic approach for tumours that are resistant to conventional therapy.     

Paradoxical correlations of cyclin-dependent kinase inhibitors p21waf1/cip1 and p27kip1 in metastatic colorectal carcinoma.

The cyclin-dependent kinase inhibitors (CDIs) p27kip1 and p21waf1/cip1 are key cell cycle-negative regulatory enzymes. The objective of this study was to correlate expression of p27kip1 and p21waf1/cip1 with survival, chemotherapy responsiveness, and expression of the proliferation marker Ki-67 in patients with advanced colorectal cancer. Immunohistochemistry was performed with antibodies to p27kip1, p21waf1/cip1, and Ki-67 on samples from 66 patients with metastatic colorectal carcinoma. Interpretation was performed by visual inspection and automated image analysis. Patients who obtained a response to chemotherapy had greater p21waf1/cip1 tumor staining with a mean of 10.0 positive cells/high-powered field, compared with 4.5 positive cells/high-powered field for nonresponders (P = 0.03). A positive Spearman correlation was seen between Ki-67 and p27kip1 (r = 0.48; P = 0.0001), as well as between Ki-67 and p21waf1/cip1 (r = 0.48; P = 0.0001). A trend toward shorter survival was seen in patients with positive specimens (median survival of 10 months for patients with both p27kip1- and p21waf1/cip1-positive specimens, compared with 22 months for patients with neither p27kip1- nor p21waf1/cip1-positive specimens). In contrast to that previously reported in normal colonic mucosa or early-stage colorectal cancer, we observed positive correlations of Ki-67 with both p27kip1 and p21waf1/cip1, a trend toward greater CDI staining indicating worse prognosis, and greater p21waf1/cip1 staining in tumors that were chemosensitive. These findings suggest that in the metastatic setting, CDIs may show altered function, compared with their role in the normal cell cycle.     

骨肉瘤p21 ras蛋白、p21 wafl/cip1蛋白、PCNA与AgNOR的表达及其临床意义

目的探讨骨肉瘤组织中p21ras蛋白、p21wafl/cip1蛋白、PCNA与AgNOR的表达及其临床意义.方法应用免疫组化方法和组织化学方法检测45例骨肉瘤病人的骨肉瘤组织、20例骨软骨瘤病人的骨软骨瘤组织和20例正常骨组织中p21ras蛋白、p21wafl/cipl蛋白、PCNA与AgNOR的表达情况,对有关临床病理指标综合分析.结果45例骨肉瘤组织中p21ras蛋白、p21wafl/cipl蛋白和PCNA的阳性表达率分别为86.7%、13.3%和100%,AgNOR计数为9.63±2.47,阳性表达率与AgNOR计数均高于骨软骨瘤和正常骨组织;p21ras蛋白的表达与PCNA的表达存在平行关系;AgNOR计数与PCNA的表达呈正相关;四者的表达与骨肉瘤有关临床病理指标之间存在一定的关系.结论检测p21ras蛋白、p21waf1/cip1蛋白、PCNA与AgNOR为骨肉瘤恶性程度和预后的评估提供了重要的辅助参考指标.     

Diallyl disulfide (DADS) increases histone acetylation and p21(waf1/cip1) expression in human colon tumor cell lines

Diallyl disulfide (DADS) is a naturally occurring organosulfur compound, from garlic, which exerts pleiotropic biological effects. In rodents, DADS inhibits colon chemically induced carcinogenesis. DADS anti-promoting effect may partly result from its ability to inhibit tumoral cell proliferation in vivo and in vitro. As far as DADS may modulate the expression of a subset of genes, we investigated DADS effect on histone acetylation, in two human colon tumor cell lines. Our study demonstrates that in Caco-2 and HT-29 cells treated for 6 h, 200 microM DADS increases histone H3 acetylation (x2 and x1.4, respectively). In Caco-2 cells, we also observed histone H4 hyperacetylation, preferentially at the lysine residues 12 and 16. We explored the effects of DADS and one of its metabolites, allyl mercaptan (AM), on histone deacetylase (HDAC) activity: using nuclear extracts of Caco-2 cells, 200 microM DADS decreased HDAC activity by 29% and AM at the same concentration was more efficient (92% inhibition). We also observed that DADS induced an increase in p21(waf1/cip1) expression, at mRNA and protein levels, in both cell lines. This effect was associated with an accumulation of cells in the G2 phase of the cell cycle. Our results suggest that in Caco-2 and HT-29 cells, DADS could inhibit cell proliferation through the inhibition of HDAC activity, histone hyperacetylation and increase in p21(waf1/cip1) expression. The present study provides evidence for cellular and molecular responses triggered by DADS that could be linked to its effect on histone acetylation and play a role in its protective properties on colon carcinogenesis.     

Change in gene expression subsequent to induction of Pnn/DRS/memA: increase in p21(cip1/waf1)

Pnn (PNN) is a nuclear and cell adhesion-related protein. Previous work has suggested that Pnn/DRS/memA is a potential tumor suppressor involved in the regulation of cell adhesion and cell migration. Using the ecdysone-inducible mammalian expression system, a stable inducible GFP-tagged human Pnn gene (PNNGFP) expressing 293 cell line was created (EcR293-PNNGFP). Cells induced to express PNNGFP not only exhibited increased cell-cell adhesion but also exhibited changes in cell growth and cell cycle progression. cDNA array analyses, together with real time PCR, revealed that the effects of exogenously expressed Pnn on cellular behavior may be linked to the regulation of the expression of specific subset genes. This subset includes cell cycle-related genes such as p21(cip1/waf1), CDK4, CPR2; cell migration and invasion regulatory genes such as RhoA, CDK5, TIMP-1, MMP-7, and EMMPRIN; and MIC-1. Concordant with previous observations of Pnn-induced phenotype changes, genes coding for epithelial associated processes and cell division controls were elevated, while those coding for increased cell motility and cellular reorganizations were downregulated. We utilized p21 promoter-luciferase reporter constructs and demonstrated that a marked stimulation of p21 promoter activity in 293 cells correlated with increased Pnn expression. Taken together, these data indicate that Pnn may participate in the regulation of gene expression, thereby, positively promoting cell-cell adhesion, and negatively affecting cell migration and cell proliferation.     

Regulation of iNOS by the p44/42 mitogen-activated protein kinase pathway in human melanoma

Activating mutations of the genes for NRAS and BRAF, components of the p44/42 mitogen-activated protein kinase (MAPK) pathway, are common findings in melanoma. Recent evidence in several nonmelanoma cell systems supports the regulation of the inducible nitric oxide synthase (iNOS) gene by this pathway. On the basis of our data showing that melanoma iNOS expression predicts shortened patient survival, we formulated the hypothesis that activating mutations of NRAS or BRAF, which lead to constitutive activation of the p44/42 MAPK pathway, drive iNOS expression in human melanoma. In the present study, we have shown that inhibition of melanoma iNOS activity by S-methylisothiourea leads to decreased cell proliferation, confirming the importance of iNOS activity for melanoma cell growth. Regulation of melanoma iNOS expression by the p44/42 MAPK pathway was demonstrated by inhibition of the pathway by U0126, and by BRAF RNA interference. To explore this regulatory pathway in human tissue, 20 melanoma tumors were examined for NRAS and BRAF mutations, immunohistochemical evidence of ERK phosphorylation, and iNOS expression. A significant association was found among these three features. We conclude that in human melanoma, activating mutations of NRAS and BRAF drive constitutive iNOS expression and, implicitly, nitric oxide production, contributing to the poor survival of these patients.     

Involvement of p21cip1/waf1 in the anti-proliferative effects of polyethylene glycol in colon carcinogenesis

Polyethylene glycol (PEG) is a safe and effective chemopreventive agent against colorectal carcinogenesis in cell culture, animal models and human subjects. Although the precise molecular mechanism is unclear, we previously reported that PEG suppresses colonic epithelial proliferation. As cellular proliferation is driven by complex G1-S phase transition, we now characterize the role of PEG on cell cycle regulation. We focused our attention on the effect of PEG on the CDK inhibitor p21cip1/waf1, which is implicated in early colon carcinogenesis and is upregulated by non-steroidal anti-inflammatory drugs. These studies were done in the azoxymethane-treated (AOM) rat model as well as in HT-29 colon cancer cells. Immunohistochemical analysis revealed that while AOM decreased the p21 expression (75%, p<0.01) in the premalignant colonic mucosa, PEG induced p21 levels back to normal. These findings paralleled a decreased BrdUrd incorporation (78%, p<0.001) and hypophosphorylated retinoblastoma protein (Rb; by 47%) signifying PEG's antiproliferative activity. Furthermore, in HT-29 cells, PEG decreased proliferation as measured by PCNA (68% reduction), increased p21 expression (2.3-fold), induced cell cycle arrest during G0/G1 phase (45% reduction in S phase cells) and inhibited the phosphorylation of Rb (by 52% compared to untreated). PEG caused greater than a 2-fold induction of protein and mRNA level of p21cip1/waf1 in HT-29 cells. These results demonstrate for the first time that PEG is involved in p21 regulation concomitant with G1S phase cell cycle arrest and it is through these effects that it can exert its anti-proliferative and hence chemopreventive role.     

Early g1 induction of p21/waf1/cip1 in synchronized osteosarcoma cells is independent of p53

Enzymatic activities of cyclin-dependent protein kinases (cdks) are tightly regulated at the level of subunit composition, involving both positive (cyclins) and negative (p21Wafl/Cipl); p16Ink4; p27Kipl) effectors. In the present study, we examined the expression of p21/WAF1/CIP1 in highly synchronized human MG-63 osteosarcoma cells in which the sequential induction of specific cyclins was characterized previously (1). Two distinct peaks of p21/WAF1/CIP1 expression were detected by Northern analysis of serum-stimulated cells: one peak was detectable by 1 hour, reached a maximum at approximately 3 hours, and diminished markedly by 4-6 hours; and a second peak was observed during S phase. The early G1 induction of the 21 kDa gene product was further demonstrated by Western blotting. Both Northern analysis and Western blotting for the p53 tumor suppressor confirmed previous reports that its expression is not detectable in MG-63 cells at any time. The transient induction of p21/WAF1/CIP1 in early G1 was correlated with a transient decrease in p9Ckshs1-agarose precipitable histone H1 kinase activity, as determined by in vitro kinase assays. In contrast, the myelin basic protein kinase activity of anti-Cdk4 immune complexes was not attenuated to a significant extent. Taken together, these studies identify a novel biochemical pathway of p21/WAF1/CIP1 induction operating in p53-deficient osteosarcoma cells; a pathway whose independence from p53 may conceivably be exploited to therapeutic advantage in the treatment of proliferative disorders.     

Anti-sense oligonucleotide of p21(waf1/cip1) prevents interleukin 4-mediated elevation of p27(kip1) in low grade astrocytoma cells

Elevation of the cyclin-dependent kinase (cdk) inhibitor, p27(kip1) is necessary for Interleukin (IL)-4-mediated growth arrest of human low grade astrocytoma (RTLGA) cells and occurs at 24 h of treatment. Pathways involved in IL4 alteration of p27(kip1) are unknown, however. Here we investigated whether other cdk inhibitors contributed to the actions of IL-4 on RTLGA cells. By 12 h of IL-4 treatment, both cdk4 and cdk2 kinase activities against the retinoblastoma protein (pRb) were reduced and nuclear entry of pRb was prohibited. Twelve-hour cdk complexes contained elevated p21(waf1/cip1) but not p27(kip1), p15(ink4B) or p16(ink4A). IL-4 increased p21(waf1/cip1) but not p27(kip1) mRNA levels, and stimulated luciferase activity of a p21(waf1/cip1) promoter-luciferase reporter. In p53-mutant WITG3 cells, IL-4 did not alter p21(waf1/cip1) mRNA and promoter-luciferase activity or p27(kipl) protein, suggesting a need for functional p53. STAT6 phosphorylation by IL-4, however, occurred in both p53-mutant WITG3 and p53-functional RTLGA cells. Pre-treatment of RTLGA with anti-sense but not missense p21(waf1/cip1) oligonucleotide prior to IL-4: (a) restored cdk activities; (b) reduced cdk4-associated p21(waf1/cip1) levels; (c) prevented p27(kipl) elevation; and (d) reversed growth arrest. These results are the first to suggest that p21(waf1/cip1) is essential for IL-4-mediated elevation of p27(kip) and growth arrest of astrocytoma cells.     

A dual role of p21waf1/cip1 gene in apoptosis of HEp-2 treated with cisplatin or methotrexate

We investigated the effects of p21(waf1/cip1) gene overexpression in human laryngeal squamous carcinoma cells HEp-2 lacking p53 protein expression on apoptosis induction upon the treatment with two commonly used chemotherapeutic agents, cisplatin and methotrexate. For that purpose, we employed cDNA arrays and qPCR to monitor gene expression upon treatment with AdCMV-p21 alone or in combination with the chemotherapeutic compounds. We found that p21(waf1/cip1) gene overexpression provoked apoptosis of HEp-2 through the induction of the TNFRSF9 gene and activation of caspase 7. In addition, we have proved that p21(waf1/cip1) can assume a dual role in apoptosis in the same cell system depending on the chemotherapeutic agent: its overexpression enhances apoptosis in cisplatin-treated cells and attenuates apoptotic signals in methotrexate-treated cells. The observed dual role of p21(waf1/cip1) was in direct correlation with the modulation of caspases 3 and 7 activation and changes in the expression of GADD45a gene. The results presented herein encourage future use of targeted p21(waf1/cip1) gene therapy in cancer treatment in a well-defined therapeutic and genetic context.     

Role of p53 and p21waf1/cip1 in senescence-like terminal proliferation arrest induced in human tumor cells by chemotherapeutic drugs.

Exposure of human tumor cell lines to moderate doses of anticancer agents induces terminal proliferation arrest accompanied by morphologic and enzymatic changes that resemble senescence of normal cells. We have investigated the role of p53 and p21waf1/cip1 in the induction of this response in drug-treated tumor cells. Doxorubicin treatment induced the senescence-like phenotype (SLP) and its associated terminal growth arrest in wild-type HCT116 colon carcinoma cells; this response was strongly decreased but not abolished in HCT116 lines with homozygous knockout of p53 or p21. Transduction of HT1080 fibrosarcoma cells with a genetic inhibitor of p53 also decreased the induction of SLP and increased drug-induced mitotic cell death. To determine if drug-stimulated p21 expression was responsible for senescence-like growth arrest, we have expressed different levels of p21 from an inducible promoter. While high-level overexpression of p21 was sufficient to induce SLP in HT1080 cells, the levels of p21 expressed in doxorubicin-treated cells could account for only a fraction of doxorubicin-induced SLP. Our results indicate that p53 and p21 act as positive regulators of senescence-like terminal proliferation arrest, but their function is neither sufficient nor absolutely required for this treatment response in tumor cells.     

Effects of p21cip1/waf1 overexpression on growth, apoptosis and differentiation in human colon carcinoma cells

The cyclin-dependent kinase inhibitor p21cip1/waf1 negatively regulates the progression of cell cycle and the potential usefulness of p21cip1/waf1 gene is proposed in gene therapy. However, studies have demonstrated a protective role of p21cip1/waf1 against apoptosis and little is known about effects of ectopic expression of p21cip1/waf1 on differentiation of colon cancer cells. In the present study, we found diffuse p21cip1/waf1 expression in only a few clinical samples of colorectal cancer with wild-type p53 gene. To explore the role of p21cip1/waf1 in cell growth, apoptosis and differentiation, we constitutively overexpressed p21cip1/waf1 in HT29 colon carcinoma cells. Ectopic overexpression of p21cip1/waf1 was associated with inhibition of CDK2-associated kinase activity, indicating the functionality of the introduced p21cip1/waf1 gene. Overexpression of p21cip1/waf1 caused an appreciable growth inhibition in monolayer and soft agar cultures and it significantly reduced sodium butyrate- but not 5-fluorouracil-induced apoptosis. p21cip1/waf1 overexpressing cells exhibited marked decrease of intestinal differentiation when assayed with intestinal alkaline phosphatase. Our findings suggest that introduction of p21cip1/waf1 gene into colon cancer cells may be useful for inhibiting cell growth but caution should be taken regarding the increased resistance to certain apoptosis-inducing agents and dysregulation of endogenous p21cip1/waf1-mediated differentiation process.     

p21(waf1/cip1)-null human fibroblasts are deficient in nucleotide excision repair downstream the recruitment of PCNA to DNA repair sites

The cyclin-dependent kinase inhibitor p21(waf1/cip1) is known to impair DNA synthesis by binding to PCNA, the co-factor of DNA polymerases delta and epsilon. However, a positive role for p21 in nucleotide excision repair (NER) has been suggested. In this study, the sensitivity to DNA damage and DNA repair efficiency were investigated in p21-null human fibroblasts obtained by targeted homologous recombination. After UV-C irradiation, p21-/- cells showed a threefold reduction in clonogenic survival and an increased susceptibility to apoptosis, as compared with parental p21+/+ cells. Removal of cyclobutane pyrimidine dimers was significantly reduced in p21-/- cells both in the whole genome, and at the level of the rDNA gene cluster, as determined by immunoassay and Southern blot, respectively. After DNA damage, the recruitment of PCNA as detergent-insoluble form associated to DNA repair sites in p21-/- fibroblasts, was comparable to that observed in parental p21+/+ cells. However, PCNA remained associated with DNA for a longer period in p21-/- than in p21+/+ cells. These results suggest that in human cells, p21 is required for NER at a step located downstream the recruitment of PCNA to DNA repair sites.     

Effect of p21waf1/cip1 transgene on radiation induced apoptosis in T cells.

The cyclin kinase inhibitor p21WAF1/Cip1 is upregulated by the tumor suppressor p53. While p21 is central for the G-1 arrest mediated by p53, it is still unclear if p21 also functions as a downstream effector of p53 dependent apoptosis. Apoptosis induced by DNA damage but not dexamethasone is p53 dependent in thymocytes. To investigate the physiological role of p21 in apoptosis, we have generated transgenic mice in which the p21 transgene is targeted for restricted expression in the T cell lineage. Thymocytes from p21 transgenic mice were hypersensitive to cell death induced by DNA damaging agents such as ionizing radiation and UV, but not be dexamethasone. Irradiated p21 transgenic thymocytes had approximately twofold more apoptotic cells as compared to irradiated age matched littermate control mice. Radiation induced death is comparable in thymocytes from p21 + Bcl2 + double transgenic mice and age matched littermate controls, indicating that the Bcl2 transgene rescues the radiation hypersensitivity imposed by p21. However, thymocytes from p53-/- mice even when they expressed the p21 transgene, were resistant to death induced by radiation. Together these results show that thymocytes from p21 transgenic mice are hypersensitive to radiation induced programmed cell death and suggest that the radiation hypersensitivity of p21 transgenic thymocytes involves p53 dependent pathway and signals in addition to p21.