Analysis of p21WAF1/CIP1 in primary bladder tumors

The p21WAF1/CIP1 gene is regulated by p53 and encodes a cyclin-dependent kinase (Cdk)-inhibitor involved in senescence and cell quiescence. The role of p21 as a negative regulator of cell proliferation suggests that it may function as a tumor suppressor gene. However, only a few mutations of the p21WAF1/CIP1 gene have been reported to date. In order to assess potential p21WAF1/CIP1 gene alterations in human bladder cancer, we have examined this gene and its encoded product in a well-characterized cohort of 27 primary bladder tumors. Mobility shifts by single-strand conformation polymorphism in the p21WAF1/CIP1 gene were identified in 2 cases. Sequencing analyses revealed that one of these cases had point mutations in the 3' untranslated region, while the other case had a frame shift mutation at positions 322 (C to A) and a deletion of 8 nucleotides (323-->331; CCG-->ACG, codon 81 Arg-->Thr) that produced a stop signal at codon 83 (Gly--Stop). This tumor had a p21-negative phenotype by immunohistochemistry, but did not lose any allele. We further characterized these cases by the study of TP53 mutations using single-strand conformation polymorphism (PCR-SSCP) and sequencing, as well as immunohistochemical assays. Seven mobility shifts were identified and seven cases showed p53 nuclear accumulation. The two cases displaying mutated p21WAF1/CIP1 had wild-type TP53. It is concluded that p21WAF1/CIP1 gene aberrations are infrequent in bladder carcinoma but may be occasionally identified in primary bladder tumors.     

Altered WAF1 genes do not play a role in abnormal cell cycle regulation in breast cancers lacking p53 mutations

Seventy-five to 80% of breast cancers are negative for p53 gene mutations. We have investigated the possibility that altered WAF1 genes provide an alternative mode of cell cycle disruption in these tumors. DNA from a total of 85 primary breast tumors and cell lines from both the United States and Australia were examined for WAF1 and p53 mutations. With the exception of one primary tumor containing the polymorphic codon 31 (AGC-->AGA), no missense mutations in the WAF1 gene were found in 33 primary tumors or in the 19 cell lines from the United States. By contrast, 2 of 33 tumors from Australia contained tumor-specific missense mutations in the WAF1 gene, while an additional six cases contained the AGC-->AGA polymorphic 31st codon in the WAF1 gene. The p53 mutation frequency in the Australian cohort (18%) was found to be similar to that reported by us (Glebov et al., Cancer Res., 54: 3703-3709, 1994; Runnebaum et al., Proc. Natl. Acad. Sci. USA, 88: 10657-10661, 1991) in the tumors of United States patients (13%) with sporadic breast cancer. Thus, mutations in the WAF1 gene are rare in tumors with or without p53 mutations, suggesting that except in a minor population of breast cancer patients of Caucasian origin, cell cycle dysregulation by mutated p53 or WAF1 genes may not contribute to breast tumor initiation or progression.     

Conversion enzyme inhibitors against the progression of renal and vascular diseases: yes but again

p21WAF1/CIP1 is a potent, tight-binding inhibitor of Cdks which is induced by the wild-type p53 gene. We examined p21WAF1/CIP1 mRNA expression in 16 surgically excised human colorectal tumor and nontumor tissues using Northern blot analysis with reference to the identification of p53 gene mutation. p53 gene mutation was detected in six tumor tissues but not in the other 10 using the PCR-SSCP method. The p21WAF1/CIP1 mRNA level was lower in tumor tissues than in the corresponding nontumor tissues. The mean expression level of p21WAF1/CIP1 mRNA was lower in tissues in which the p53 gene mutation was detected than in those in which it was not, although the difference was not significant. The relative p21WAF1/CIP1 mRNA expression level was significantly higher in the tumors with liver metastases than in those without. These results suggest that the p21WAF1/CIP1 mRNA expression level, which might be a indicator of colorectal cancer malignancy, is suppressed in human colorectal tumor tissues compared with the corresponding nontumor tissues and that the wild-type p53 gene is one of the factors inducing p21WAF1/CIP1 mRNA expression.     

Suppression of p53 activity and p21WAF1/CIP1 expression by vascular cell integrin alphaVbeta3 during angiogenesis

Induction of p53 activity in cells undergoing DNA synthesis represents a molecular conflict that can lead to apoptosis. During angiogenesis, proliferative endothelial cells become apoptotic in response to antagonists of integrin alphavbeta3 and this leads to the regression of angiogenic blood vessels, thereby blocking the growth of various human tumors. Evidence is presented that administration of alphavbeta3 antagonists during angiogenesis in vivo selectively caused activation of endothelial cell p53 and increased expression of the p53-inducible cell cycle inhibitor p21WAF1/CIP1. In vitro studies revealed that the ligation state of human endothelial cell alphavbeta3 directly influenced p53 activity and the bax cell death pathway. Specifically, agonists of endothelial cell alphavbeta3, but not other integrins, suppressed p53 activity, blocked p21WAF1/CIP1 expression, and increased the bcl-2/bax ratio, thereby promoting cell survival. Thus, ligation of vascular cell integrin alphavbeta3 promotes a critical and specific adhesion-dependent cell survival signal during angiogenesis leading to inhibition of p53 activity, decreased expression of p21WAF1/CIP1, and suppression of the bax cell death pathway.     

Effects of ectopic overexpression of p21(WAF1/CIP1) on aneuploidy and the malignant phenotype of human brain tumor cells

p21WAF1/CIP1 is a downstream effector of the p53 tumor suppressor gene and a universal cyclin-dependent kinase (CDK) inhibitor. To determine the ability of p21WAF1/CIP1 to function as a tumor suppressor, we constructed a replication-defective adenovirus vector containing p21WAF1/CIP1 (Adp21WAF1/CIP1) to effect ectopic overexpression in a p53-defective human astrocytoma cell line, U-373MG. We observed a marked decrease in CDC2 and CDK2 kinase activity associated with a corresponding decrease in the amount of CDC2 but not CDK2 protein; a decreased growth potential of Adp21WAF1/CIP1-infected cells demonstrated by diminished [3H]thymidine incorporation, increased cell doubling time and G1-arrested cell cycle; an association between Adp21WAF1/CIP1-infected cells and inhibition of aneuploid cell accumulation; and an alteration of the malignant phenotype of cells was evidenced by the loss of anchorage-independent growth in soft agar and the failure to induce tumorigenesis in both peripheral and intracerebral xenograft models, including the prevention of tumor formation Adp21WAF1/CIP1 infection 2 days post tumor cell implantation. Adp21WAF1/CIP1. Adp21WAF1/CIP1 appears to be a strong candidate for gene therapy studies based on these studies indicating that Adp21WAF1/CIP1 inhibits proliferation, tumorigenicity and aneuploidy in human brain tumor cells.     

Dexamethasone-mediated protection from drug cytotoxicity: association with p21WAF1/CIP1 protein accumulation?

Dexamethasone (DEX)-mediated inhibition of drug-induced, but not CD95 ligand-induced, apoptosis in malignant glioma cells correlates with wild-type p53 status. Here, we examined mechanisms underlying DEX-mediated protection from apoptosis. DEX did not induce p53 expression in two p53 wild-type cell lines (U87MG, LN-229) and did not alter drug-induced p53 accumulation. Forced expression of temperature-sensitive p53val135 in mutant conformation failed to prevent accumulation of endogenous wild-type p53 but acted in a transdominant negative manner to inhibit p53-mediated, camptothecin-induced p21WAF1/CIP1 expression. p53val135-transfected cells retained responsiveness to DEX at restrictive temperature, suggesting that p53 activity is not required for cytoprotection. Forced expression of wild-type p53val135 abrogated the protective effect of DEX, suggesting redundant cytoprotective effects of DEX and p53. Indeed, DEX induced moderate accumulation of p21WAF1/CIP1 in U87MG, LN-229 and p53 mutant LN-18 cells, but not in p53 mutant LN-308 or T98G cells. LN-18 is also the p53 mutant cell line with the best cytoprotective response to DEX. p21WAF1/CIP1 accumulation occurred in the absence of changes in p21WAF1/CIP1 mRNA expression. Wild-type p53 was not required for this DEX effect since DEX induced p21WAF1/CIP1 accumulation in p53val135-transfected LN-229 cells, too. DEX failed to induce p21WAF1/CIP1 expression or cytoprotection in untransformed rat astrocytes. The same lack of modulation of p21WAF1/CIP1 expression and drug toxicity was observed in p21(+/+), p21(+/-) and p21(-/-) human colon carcinoma cells. Paradoxically, while only p21(+/+) and p21(+/-) mouse embryonic fibroblasts showed enhance p21WAF1/CIP1 levels after exposure to DEX, only p21(-/-) fibroblasts were protected from drug toxicity by DEX. The present study links DEX-mediated protection from cancer chemotherapy to a p53-independent pathway of regulating p21WAF1/CIP1 expression in glioma cells but this effect appears to cell type-specific.     

Release of cell cycle constraints in mouse melanocytes by overexpressed mutant E2F1E132, but not by deletion of p16INK4A or p21WAF1/CIP1

Compared to normal melanocytes, melanoma cell lines exhibit overexpression of hyperphosphorylated retinoblastoma tumor suppressor protein (Rb) or a marked decrease in, or lack of, expression of Rb. Hyperphosphorylation of Rb results in increased E2F-mediated transactivation of target genes and cell cycle progression. Using a combination of gene disruption and ectopic expression in growth factor-dependent mouse melanocytes, we studied the roles of E2F1 and the p16INK4A and p21WAF1/CIP1 CKIs (cyclin dependent kinase inhibitors) in the acquisition of TPA (12-O-tetradecanoyl phorbol-13-acetate)-independent growth in culture, a hallmark of melanomas. Surprisingly, melanocytes from p16INK4A- or p21WAF1/CIP1-null mice remained TPA-dependent, and disruption of p21WAF1/CIP1 accelerated cell death in the absence of this mitogen. Disruption of E2F1 had the most profound effect on melanocyte growth, resulting in a fourfold decrease in growth rate in the presence of TPA. Furthermore, enforced overexpression of the DNA-binding-defective E2F1E132 mutant conferred TPA-independence upon melanocytes and was associated with sequestration of Rb and constitutive expression of E2F1 target genes, including p21WAF1/CIP1. We conclude that neutralization of Rb by E2F1E132, but not the disruption of p16INK4A or p21WAF1/CIP1, resulted in the accumulation of free E2F and cell cycle progression. Thus, mechanisms other than the loss of p16INK4A or p21WAF1/CIP1 that activate E2F may play an important role in melanomas.     

Function and conformation of wild-type p53 protein are influenced by mutations in bovine leukemia virus-induced B-cell lymphosarcoma

The mutations of the p53 gene previously represented one of several genetic changes involved in the development of bovine leukemia virus (BLV)-induced lymphosarcoma, while the effects of these mutations on the function of p53 are unknown. We identified four mutations of p53 gene in BLV-infected cattle with lymphosarcoma and demonstrated clearly the existence of two functionally distinct groups of mutants: (i) the mutant forms with substitutions at codons 241 and 242, which were mapped within an evolutionally conserved region and corresponded to the human "hot-spot" mutations, had completely lost the capacities for transactivation and growth suppression and gained transdominant repression activity in p53-null SAOS-2 cells; and (ii) the mutations at codons 206 and 207 were located outside the evolutionally conserved regions. These mutants partially retained the capacity for transactivation and growth suppression and failed to inhibit the transactivation activity of coexpressed wild-type p53, instead showing an enhancement of this activity. In addition, protein analysis using an antibody specific for the mutant form revealed that the mutations at codons 206 and 242 induced a "mutant" conformation of the bovine p53 proteins. Collectively, these results show that mutations of p53 gene in BLV-infected cattle with lymphosarcoma can potentially alter its physiological function and may play an important role in BLV-induced leukemogenesis.     

IFNgamma inhibition of cell growth in glioblastomas correlates with increased levels of the cyclin dependent kinase inhibitor p21WAF1/CIP1

Glioblastoma is a highly aggressive form of brain cancer characterized by uncontrolled cell growth resulting from a loss of cell cycle regulation. In this study we determined the antiproliferative effects of interferon gamma (IFNgamma) on the glioblastoma cell lines T98G, SNB-19 and U-373, focusing on the ability of IFNgamma to increase levels of p21WAF1/CIP1, an important negative regulator of cell cycle events. IFNgamma was found to inhibit the growth of all cell lines, with inhibition ranging from 82.2% to 45.4%. Flow cytometry analysis showed that IFNgamma treatment caused a cell cycle delay in the G1 or S phases. The strength of this delay varied, correlating with the degree by which IFNgamma inhibited proliferation of each cell line. IFNgamma treatment increased the production of the cyclin dependent kinase inhibitor (CKI) p21WAF1/ CIP1 in all cell lines, the level and kinetics of production of which correlated with the degree and stage of inhibition of cellular proliferation. Further, immunoprecipitation of p21WAF1/CIP1 in complexes of p21WAF1/CIP1/cyclin-dependent kinase 2 (cdk2)/cyclin showed that the amount of p21WAF1/CIP1 in the complexes and the inhibition of cdk2-cyclin kinase activity correlated with the level of p21WAF1/CIP1 produced in the cells by IFNgamma. These results show that IFNgamma has significant antiproliferative effects on the glioblastoma cell lines and suggest that p21WAF1/CIP1 plays a role in mediating these effects.     

Cytotoxic effects of recombinant adenovirus p53 and cell cycle regulator genes (p21 WAF1/CIP1 and p16CDKN4) in human prostate cancers

PURPOSE: Overexpressing or restoring the basal levels of tumor suppressor genes in cancer cells can suppress tumorigenicity of cancer cells. In this communication, we compared tumor suppressive activities of three well-defined tumor suppressive genes (p53, p21WAF1/CIP1, and p16CDKN2) delivered individually to prostate cancer cells with adenoviral vector (Ad). MATERIALS AND METHODS: Efficacy of growth inhibition by recombinant adenoviruses bearing p53, p21WAF1/CIP1, or p16CDKN2 (Ad5CMV-p53, Ad5CMV-p21, Ad5CMV-p16) genes were tested in vitro on androgen-dependent (LNCaP) and androgen-independent (C4-2, DU-145, and PC-3) human prostate cancer cells, ex vivo and in vivo on PC-3 tumor. RESULTS: Ad5CMV-p53 was observed to exert the greatest growth inhibitory action on all of the cell lines tested; inhibition appeared to be cytolytic. In comparison to control Ad5CMV-PA added samples, the growth inhibitory action of Ad5CMV-p21 and Ad5CMV-p16 appeared to be cytostatic. Ad5CMV-p53 is more effective than Ad5CMV-p16 and Ad5CMV-p21 in inhibiting the tumor "take" rate. A similar order of antitumor activity was observed when recombinant adenoviruses were injected intratumorily to previously established PC-3 tumors in vivo. CONCLUSION: p53 is the most effective tumor suppressor gene to target human prostate cancer.     

Expression of p21WAF1/CIP1 in adenocarcinoma of the uterine cervix: a possible immunohistochemical marker of a favorable prognosis

BACKGROUND: The purpose of this study was to assess the prognostic significance of the expression of estrogen receptor and cell cycle regulatory gene products in cervical adenocarcinoma. METHODS: In 40 cases of adenocarcinoma of the uterine cervix and 10 normal cervices, expression of estrogen receptor and cell cycle regulatory gene products (cyclin E, p16, p21WAF1/CIP1, p27, p53, and Ki-67) was studied using immunohistochemical techniques. The survival of the patients was analyzed in terms of such variables as the expression of these molecules in the tumor and conventional clinicopathologic features, and the Cox proportional hazards model was used to predict the survival of patients with cervical adenocarcinoma. RESULTS: Expression of estrogen receptor was consistently observed in normal cervical glands, but in cervical adenocarcinoma it was lost (in 28 cases) or significantly diminished (in 12 cases). Normal cervical glandular cells were usually negative for the cell cycle regulatory gene products, whereas 47.5-85% of cervical adenocarcinomas were positive for these molecules. When the expression of these molecules was analyzed, significant positive correlations were found between p16 and p27, cyclin E and p27, and cyclin E and p21WAF1/CIP1. Univariate survival analysis revealed that the presence of parametrial invasion, the presence of lymph node metastasis, negative staining for p21WAF1/CIP1, and a moderately or poorly differentiated tumor all correlated significantly with a poor prognosis. In a stepwise regression analysis, the expression of p21WAF1/CIP1 and negative pelvic lymph nodes were the best predictors of a favorable prognosis. CONCLUSIONS: Expression of p21WAF1/CIP1 correlated with a favorable prognosis for patients with cervical adenocarcinoma and may serve as a useful marker of survival in cases of this disease.     

Platelet-derived growth factor (PDGF)-signaling mediates radiation-induced apoptosis in human prostate cancer cells with loss of p53 function

Platelet-derived growth factor (PDGF) signals a diversity of cellular responses in vitro, including cell proliferation, survival, transformation, and chemotaxis. PDGF functions as a "competence factor" to induce a set of early response genes expressed in G1 including p21WAF1/CIP1, a functional mediator of the tumor suppressor gene p53 in G1/S checkpoint. For PDGF-stimulated cells to progress beyond G1 and transit the cell cycle completely, progression factors in serum such as insulin and IGF-1 are required. We have recently shown a novel role of PDGF in inducing apoptosis in growth-arrested murine fibroblasts. The PDGF-induced apoptosis is rescued by insulin, suggesting that G1/S checkpoint is a critical determinant for PDGF-induced apoptosis. Because recent studies suggest that radiation-induced signal transduction pathways interact with growth factor-mediated signaling pathways, we have investigated whether activation of the PDGF-signaling facilitates the radiation-induced apoptosis in the absence of functional p53. For this study we have used the 125-IL cell line, a mutant p53-containing, highly metastatic, and hormone-unresponsive human prostate carcinoma cell line. PDGF signaling is constitutively activated by transfection with a p28v-sis expression vector, which was previously shown to activate PDGF alpha- and beta- receptors. Although the basal level of p21WAF1/CIP1 expression and radiation-induced apoptosis were not detectable in control 125-IL cells as would be predicted in mutant p53-containing cells, activation of PDGF-signaling induced expression of p21WAF1/CIP1 and radiation-induced apoptosis. Our study suggests that the level of "competence" growth factors including PDGF may be one of the critical determinants for radiation-induced apoptosis, especially in cells with loss of p53 function at the site of radiotherapy in vivo.     

Pulse oximetry at fibre-optic bronchoscopy in local anaesthesia: indication for postbronchoscopy oxygen supplementation?

To investigate the clinical significance of p53 and p21WAF1/CIP1 in the advanced squamous cell carcinoma (SCC) of the pyriform sinus, we performed immunohistochemical staining of p53 and p21WAF1/CIP1 on the biopsy specimens from patients with stage III or stage IV SCC of the pyriform sinus. The results were compared with clinico-pathological features, including age, histological grade, TNM classification, number of neck lymph node metastases on histopathological examination (pLN) and prognosis. Specific staining for p53 and p21WAF1/CIP1 was detected in 36% and 32% of the specimens, respectively. Positive staining of p21WAF1/CIP1 was observed not only in the p53-negative specimens but also in the p53-positive specimens. Age (p < 0.05) and pLN (p < 0.001) were regarded as the significant prognostic factors. The 5-year survival rate of the p53-positive patients (55%) was significantly higher than that of the p53-negative patients (26.5%: p < 0.03). However, there is no significant difference between the p53 groups after controlling pLN. Although it was not statistically significant, the 5-year survival rate of the p21WAF1/CIP1-positive patients (58.8%) was higher than that of the p21WAF1/CIP1-negative patients (26.9%). These results suggest that expressions of p53 and p21WAF1/CIP1 are independent genetic alterations that may play different roles in the SCC of the pyriform sinus. Expression of p53 could not be regarded as an independent prognostic factor at this point. Further studies including the molecular biological analysis should be performed in order to determine the clinical role of p21WAF1/CIP1.     

p21WAF1/CIP1/SDI1 is elevated through a p53-independent pathway by mimosine

Tumor suppressor p53 is a nuclear protein that is induced by DNA damage and is involved in G1 and G2 phase control of the cell cycle. p21WAF1/CIP1/SDI1 (p21), a cyclin-dependent kinase inhibitor, is a downstream target and effector of p53 to induce G1 arrest. Mimosine is a potent reversible late G1 phase blocker of the cell cycle. In this study, we showed that mimosine can increase both p21 mRNA and protein levels, indirectly inhibit cyclin E-associated kinase activity without affecting the cyclin E protein level, block human breast cancer cells (21PT) in the late G1 phase of the cell cycle, and induce a p53-independent p21 pathway in these cells. These results support the possibility of restoring a G1 checkpoint by use of mimosine. They also suggest that the mechanism of the effect of mimosine is complex and may have more than one target in the cell.     

HPV-16 oncogenes E6 and E7 are mutagenic in normal human oral keratinocytes

The mutation frequency of pS189 shuttle vector plasmids is higher in human oral keratinocytes (NHOK) immortalized with cloned human papillomavirus-16 (HPV-16) genome than in primary normal NHOK (NHOK). To determine whether oncoproteins E6 and E7 of HPV-16 are responsible for the higher mutation frequency of the plasmids, we measured the mutation frequency in NHOK and in NHOK expressing the HPV-16 oncogenes (E6, E7, or E6 plus E7). We also measured the mutation frequency in NHOK expressing the E6 or E7 proteins of the non-oncogenic HPV-6b. The mutation frequency, either background or N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced, in NHOK expressing the HPV-16 oncoproteins (E6, E7, or E6 plus E7) was significantly higher than in NHOK. The HPV-16 oncogenes did not alter the nature of the MNNG-induced mutations (G:C-->A:T), but increased the frequency of deletions and insertions with or without MNNG. The background or MNNG-induced mutation frequency in NHOK expressing the HPV-6b E6 or E7 proteins was the same as in NHOK. NHOK and NHOK expressing HPV6b-E6 or E7 were able to arrest the cell cycle and enhance cellular p53, p21(WAF1/CIP1), and Gadd45 levels when exposed to MNNG, whereas NHOK expressing the HPV-16 E6 oncogene did not demonstrate. NHOK expressing HPV-16 E7 were able to enhance cellular p53, p21(WAF1/CIP1), and Gadd45 levels, but failed to arrest cell cycle progression when exposed to MNNG. These data indicate that HPV-16 E6 and E7 oncogenes are mutagenic in human oral keratinocytes and enhance the mutagenic effect of MNNG. However, the E6 and E7 proteins of the 'low risk' HPV-6b did not demonstrate such an ability.