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.     

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.     

FHIT、p21waf1/cip1基因在膀胱移行细胞癌中的表达与意义

目的:通过测定脆性组氨酸三联体基因(FHIT)及p21waf1/cip1基因在膀胱移行细胞癌组织、正常膀胱组织中的表达,探讨FHIT基因以及p21waf1/cip1基因与膀胱癌的关系及其临床意义。方法:采用免疫组织化学SP法对43例膀胱移行细胞癌(BTCC)组织及14例正常膀胱组织中的FHIT基因及p21waf1/cip1基因的蛋白表达进行检测。结果:FHIT蛋白表达与肿瘤的分期、分级无相关性(P>0.05)而p21waf1/cip1蛋白的表达与之有相关性(P<0.05);FHIT蛋白的表达在GI肿瘤、浅表性肿瘤中明显低于在正常膀胱组织中的表达(P<0.05)而p21waf1/cip1蛋白的表达在上述组织比较中无差别(P>0.05);FHIT蛋白的表达在初发肿瘤中与复发肿瘤中无明显差别(P>0.05)而p21waf1/cip1蛋白的表达在上述两种组织比较有明显差别(P<0.05)。FHIT蛋白与p21waf1/cip1蛋白的表达没有相关性(P>0.05)。结论:FHIT基因可能成为早期诊断膀胱移行细胞癌的指标。p21waf1/cip1基因可能成为估计膀胱移行细胞癌的恶性程度及肿瘤侵袭性、预后的指标。FHIT基因在膀胱移行细胞癌中的作用机制可能与p21waf1/cip1基因没有关系。     

Expression of p53 and p21waf1/cip1 in gastric carcinoma: lack of inter-relationship or correlation with prognosis.

AIMS: The cell cycle regulators p53 and p21waf1/cip1 are expressed variably in human cancers. We investigated their expression in gastric carcinoma and determined their inter-relationship and prognostic significance. METHODS: Immunohistochemistry was used to determine their expression in material from 100 resected specimens of gastric carcinoma, and comparison was then made of the degree of expression between each, with conventional clinicopathological indices and with survival. RESULTS: Positivity was found with p53 (40%) and p21 (75%). There was no significant correlation between the expression of each individual marker, nor between each marker and 5-year survival. There appeared to be an association between p53 expression and lymph node metastases, and a higher frequency of p21waf1/cip1 expression in males. CONCLUSIONS: The expression of p53 and p21waf1/cip1 as detected by immunohistochemistry were of no value in predicting the prognosis of patients with gastric carcinoma.     

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.     

Expression of cyclins A and D and p21(waf1/cip1) proteins in renal cell cancer and their relation to clinicopathological variables and patient survival.

We have studied 118 renal cell carcinomas to analyse the expressions of cyclins A and D1 and p21(waf1/cip1), and their relationship to clinical and histopathological parameters as well as to clinical outcome. Cyclins A and D1 and cyclin-dependent kinase inhibitor p21 (waf1/cip1) were not expressed in normal renal tissue. Staining signals of cyclin D1 and p21(waf1/cip1) were always nuclear but cyclin A was also expressed in the cytoplasm of the tumour cells. The mean (range) fractions of cyclin A, cyclin D1 and p21(waf1/cip1)-positive tumour cells were 2.2% (range 0-20%), 23.3% (range 0-90%) and 6.8% (range 0-70%) respectively. The expression of cyclin A was related to venous invasion, high nuclear grade, high mitotic rate, high Ki-67 and high PCNA expressions (P < or = 0.006 for all). The expression of cyclin D1 was linked with age over 65 years, low nuclear grade and high p53 expression (P < or = 0.05 for all). An inverse correlation was present between p21(waf1/cip1) and cyclin D1 (P = 0.011). Cyclin A predicted survival in the entire study group (P = 0.0014), in T1-4/N0-2/M0 (P = 0.0007) and in T1-2/N0/M0 tumours (P = 0.0007). Cyclin A was also a powerful predictor of disease-free survival in T1-4/N0/M0 (P = 0.0027) tumours (P = 0.0007). Cyclin D1 and p21(waf1/cip1) were not significantly related to survival or disease-free survival in any of the groups. In the entire material the independent prognostic factors were the presence of distant metastases (relative risk (RR) 5.16, P < 0.001), T category (RR 2.68, P < 0.001), Ki-67 expression (RR 1.02, P = 0.026) and cyclin A expression (RR 1.12, P = 0.001). The independent predictors in T1-4/N0/M0 tumours were T-category (RR 2.67, P = 0.001) and cyclin A (RR 1.21, P < 0.001), and in T1-2/N0/M0 tumours the only significant predictor was cyclin A (RR 1.19, P = 0.0002). In renal cell carcinoma, cyclin A is a powerful and independent prognostic factor in all clinical stages of the disease, whereas cyclin D1 and p21(waf1/cip1) have no prognostic value.     

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.     

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.     

Abnormal accumulation of copper in LEC rat liver induces expression of p53 and nuclear matrix-bound p21waf 1/cip 1

The LEC rat is an inbred mutant strain which spontaneously developsliver injury and subsequent liver cancer. Liver injury in LECrats has recently been shown to be closely related to abnormalcopper accumulation in the liver. Previously, we reported thatLEC rat hepatocytes lose their growth potential, probably allowingselective growth of preneoplastic cells. In this study, to elucidatethe effects of copper accumulation on the growth activity ofLEC rat hepatocytes, we examined the growth activity and theexpression of p53 and p21^{waf 1/cip 1} in the livers of LEC ratsfed on either a control or a low-copper diet. Potential forcell proliferation of hepatocytes obtained from normal dietfed LEC rats was almost comparable to that of the cells fromage-matched Sprague-Dawley (SD) rats. Northern blot analysisshowed that the expression of p53 and p21^{waf 1/cip 1} was significantlyhigh in the livers of LEC rats fed a control diet, while theexpression of p53 and p21^{waf 1/cip 1} in the LEC rats fed a low-copperdiet was as low as that of SD rat livers. Western blot analysisconsistently showed that the amount of p21^{waf 1/cip 1} boundto the nuclear matrix scaffold of the LEC rat liver was reducedby feeding a low-copper diet. These findings suggest that abnormalaccumulation of copper induced the expression of p53 and p21^{waf1/cip 1}, resulting in the inhibition of cell proliferation ofLEC rat hepatocytes.     

Immunohistochemical study of cell cycle-associated proteins in adenocarcinoma of the uterine cervix treated with radiotherapy alone: P53 status has a strong impact on prognosis

PURPOSE: Because the incidence of adenocarcinoma of the uterine cervix has recently risen, the evaluation of radiotherapy (RT) for this disease has become an increasingly urgent matter. We analyzed the expression of the cell cycle-associated proteins p53, p27, p21/waf1/cip1, and cyclin D1 in cervical adenocarcinomas in correlation with the prognostic significance in tumors treated with RT alone. METHODS AND MATERIALS: The expression of p53, p27, p21/waf1/cip1, and cyclin D1 was studied using an immunohistochemical method in 53 cases of cervical adenocarcinoma treated only with RT. Patients received RT alone between 1965 and 1994. The mean patient age was 61.8 +/- 12.6 years (range, 36-82 years). The number of patients with Stage I, II, III, and IVA disease was 6, 16, 28, and 3, respectively. RESULTS: The number of patients with p53, p27, p21/waf1/cip1, and cyclin D1 positive tumors was 24, 18, 22, and 8, respectively; no statistically significant correlation was noted. The 5-year disease-free survival rate of p53-positive patients was 30%, significantly lower than the 62% for the p53-negative patients (p = 0.02); no statistically significant correlation was noted between disease-free survival and p27, p21/waf1/cip1, and cyclin D1 expression. No statistically significant correlation was observed between local control and expression of any of the proteins. CONCLUSION: Expression of p53 protein has a statistically significant impact on disease-free survival in adenocarcinoma of the uterine cervix treated with RT alone. However, the clinical significance of p27, p21/waf1/cip1, and cyclin D1 protein expression was not obvious.     

P21(WAF1/CIP1) is dispensable for G1 arrest, but indispensable for apoptosis induced by sodium butyrate in MCF-7 breast cancer cells

Sodium butyrate (NaB) has been proposed as a potential anticancer agent. However, its mechanism of action is not totally elucidated. Here, we showed that NaB-induced cell cycle arrest and apoptosis were associated with an increase of P21(waf1/cip1) in MCF-7 breast cancer cells. This increase was more important in the nuclei, as revealed by immunofluorescence analysis. Transient transfections of MCF-7 cells with p21 deficient for interaction with CDK, but not with p21 deficient for interaction with PCNA (p21PCNA-), abrogated NaB-induced cell cycle arrest. This indicated that cell cycle blockage involved the interaction of P21(waf1/cip1) with CDK. However, P21(waf1/cip1) was dispensable, since p21 antisense did not modify cell cycle arrest. On the other hand, NaB-induced apoptosis was abolished by p21 antisense or p21PCNA-. In addition, NaB decreased PCNA levels, but increased the association of PCNA with P21(waf1/cip1). These results suggested that NaB-induced apoptosis required P21(waf1/cip1) and its interaction with PCNA.     

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.     

Protease inhibitor-induced stabilization of p21(waf1/cip1) and cell-cycle arrest in chemical carcinogen-exposed mammary and lung cells.

In previous studies, we have shown that human breast and lung carcinoma cells and mouse nontransformed type II lung cells fail to undergo cell-cycle arrest in G(1) phase in response to treatment with hydrocarbon carcinogens but rather accumulate in the S phase with damaged DNA. This situation may lead to replication of DNA on a damaged template and enhance frequency of mutations. The mechanism of this G(1) arrest failure was examined. Western immunoblot analyses of MCF7 human mammary cancer cells exposed to actinomycin D (used as a positive control for G(1) cell-cycle arrest) or hydrocarbon carcinogens revealed that while all of these chemicals caused an increase in p53, only trace levels of p21(waf1/cip1) protein were observed in the hydrocarbon carcinogen-treated samples. Similarly, in murine lung E10 type II cells, p53 but not p21(waf1/cip1) protein increased in response to benzo[a]pyrene dihydrodiol epoxide. Treatment of either MCF7 mammary or E10 lung cells with the protease inhibitor calpain I resulted in increased levels of p21(waf1/cip1) protein and enhancement of arrest of the cells in early phases of the cell cycle (G(1) and early S phase). The results suggest that failure of cell-cycle arrest in carcinogen-treated mammary and lung cells is related to increased protease-mediated degradation of p21(waf1/cip1) and/or related regulatory proteins.     

Myriocin, a serine palmitoyltransferase inhibitor, suppresses tumor growth in a murine melanoma model by inhibiting de novo sphingolipid synthesis

Advanced melanoma is the most virulent form of cancer and has a poor prognosis. In a previous study, myriocin, an inhibitor of serine palmitoyltransferase, was found to suppress melanoma cell proliferation by cell cycle arrest at the G 2/M phase through decreased sphingolipid levels and increased p53 and p21 (waf1/cip1) expression. ( 1) In the present study, myriocin (1 mg/kg, every other day for 3 weeks) was administered intradermally or intraperitoneally to melanoma mice. Tumor formation was significantly inhibited by intradermal and intraperitoneal administration of myriocin. The expression of Cdc25C, Cdc2 and cyclin B1 was decreased in tumor tissues from myriocin-treated mice, while the expression of p53 and p21 (waf1/cip1) was increased compared with that of the controls. The levels of sphingolipids in serum, liver and tumor tissue from myriocin-treated mice were decreased compared with those of controls. The decreased levels of sphingolipids in serum and liver of melanoma mice treated with myriocin suggests that myriocin may be accessible to tumor tissues of advanced melanoma. Taken together, the suppression of sphingolipid synthesis by myriocin inhibits the expression of Cdc25C or activates the expression of p53 and p21 (waf1/cip1) . This is followed by Cdc2 and cyclin B1 inhibition which results in the suppression of tumor growth.     

Differential expression of p53, p21waf1/cip1 and hdm2 dependent on DNA damage in Bloom's syndrome fibroblasts

The Bloom's syndrome gene, BLM, encodes a protein which bears homology to the RecQ helicases. It is believed to be involved in DNA replication and has been implicated in the maintenance of genomic stability. To investigate whether BLM was involved in cellular responses to DNA damage Bloom's syndrome fibroblasts were treated with either UV or ionizing radiation and the levels of p53 and two of its down stream effectors, p21waf1/cip1 and hdm2, were determined by western blot analysis. Following 20 J/m2 UVC-radiation we observed that the maximal accumulation of p21waf1/cip1 and hdm2 proteins preceded that of p53 in both a normal diploid fibroblast cell strain (GM0038) and in two Bloom's syndrome cell strains. Furthermore, the Bloom's syndrome cells demonstrated a delayed and prolonged accumulation of all three proteins and a delayed recovery of the protein levels back to pre-damage levels compared with the normal cell strain. Conversely, normal and Bloom's syndrome cell response following 2.5 Gy of ionizing radiation was quite similar for p21waf1/cip1 and hdm2, but differed significantly for p53. Maximum accumulation of p53 occurred within 2 h of damage and preceded that of p21waf1/cip1 and hdm2. These results suggest that the BLM protein may play a role in the detection of certain types of DNA damage and in the cellular response to that damage.      

Characterization of cells resistant to the potent histone deacetylase inhibitor spiruchostatin B (SP-B) and effect of overexpressed p21waf1/cip1 on the SP-B resistance or susceptibility of human leukemia cells

We previously showed that the B cell leukemia cell line NALM-6 had the highest susceptibility among a number of leukemia cell lines to spiruchostatin B (SP-B), a potent histone deacetylase (HDAC) inhibitor. We also showed that SP-B-induced cytotoxicity depended on induction of apoptosis that was mediated by p21waf1/cip1 expression. In the present study, we generated and characterized a stable, SP-B-resistant NALM-6 cell line (NALM-6/SP-B) by continuous exposure to SP-B, starting with a low SP-B concentration. NALM-6/SP-B cells were also more resistant to FK228, which has a similar chemical structure to SP-B, and were slightly more resistant to the P-gp substrates doxorubicin and vincristine than parental cells, but displayed similar susceptibility to other HDAC inhibitors and to paclitaxel as the parental cells. There was little change in the basal mRNA expression of HDAC1, p53, Bax, Bcl-2, Fas, caspase-3, c-Myc and MDR1 in NALM-6/SP-B compared to parental cells, but the mRNA expression of p21waf1/cip1 was decreased. The introduction of an exogenous p21waf1/cip1 expression vector restored SP-B induction of NALM-6/SP-B cell apoptosis. Moreover, overexpressed p21waf1/cip1 enhanced SP-B induction of the apoptosis of the human erythroleukemia leukemia cell line K562 which is less susceptible to SP-B than NALM-6 cells. These results suggest that downregulation of p21waf1/cip1, which is a characteristic feature of NALM-6/SP-B cells, was important for their resistance to SP-B, and that this SP-B resistance could be overcome by the introduction of exogenous p21waf1/cip1. Furthermore, introduction of p21waf1/cip1 to other leukemia cells such as K562 may enhance their susceptibility to SP-B. This is the first report of the characterization of SP-B-resistant cells and of the effect of overexpressed p21waf1/cip1 on the resistance or susceptibility of human leukemia cells to SP-B.