Lack of p53-mediated G1 arrest in response to an environmental carcinogen.

The environmental carcinogen, 5-methylchrysene, is a component of cigarette smoke. Its reactive metabolite, anti-5-methylchrysene-1, 2-dihydrodiol-3,4-epoxide (5-MeCDE) mainly reacts with the N(2)-position of guanine residues in the DNA molecule. In this study, we demonstrate that the tumor suppressor protein p53 is stabilized in response to DNA damage by 5-MeCDE but fails to induce the cells' protective mechanism of G1 arrest in the human breast carcinoma cell line, MCF-7. In contrast, actinomycin D treatment of these cells did lead to G1 arrest. Western analyses revealed that, though both actinomycin D and 5-MeCDE treatment stabilized p53, only trace levels of p21(waf1/cip1) were seen in the latter case. This lack of p21(waf1/cip1) expression in 5-MeCDE-treated cells is attributed to a stealth characteristic of this environmental carcinogen that allows it to damage DNA and still escape the p53-mediated cellular defense mechanism of G1 arrest.     

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.     

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.     

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.     

DNA damage-induced cell-cycle phase regulation of p53 and p21waf1 in normal and ATM-defective cells

The ATM-dependent accumulation of p53 and induction of p21waf1 are key events for G1 cell-cycle checkpoint arrest following DNA damage. In ATM-null AT cells, even though the p53 and p21waf1 responses are kinetically delayed and quantitatively reduced, the G1 checkpoint is virtually disrupted, suggesting that these proteins arrive too late in G1 to enforce the arrest. As the precise mechanism remains unclear, we examined the response to DNA double-strand breaks generated by gamma-radiation (IR), to determine if ATM deficiency affects the cell-cycle phase regulation of these molecules. We find that, after irradiation, whereas normal LCL-N cells markedly increase their levels of p53 in all phases of the cell cycle, AT cells fail to show any p53 increase in the G1 phase. In addition, whereas in LCL-N p21waf1 is induced in G1 and G2-M, in AT cells this induction is partly seen in G2-M, but not in G1, indicating a different cell-cycle phase regulation of p53 and p21waf1 as a result of ATM deficiency. The levels and catalytic activity of the p53-targeting kinases ATR and DNA-PK in LCL-N and AT cells are very similar throughout the cell cycle, both before and after IR, thus excluding a phase-specific activity for these kinases. Collectively, our findings demonstrate that, in ATM-deficient cells, the p53-dependent p21waf1 response to DNA damage is not only quantitatively reduced, but also specifically suppressed in the G1 phase, thus providing a mechanistic explanation for the severe disruption of the G1 checkpoint in AT cells.     

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.     

蛋白酶体抑制剂MG132诱导HL-60细胞凋亡前G2/M期阻滞及机制

背景和目的:蛋白酶体(proteasome)抑制剂能够诱导多种肿瘤细胞凋亡,是一种潜在的有应用前景的抗肿瘤剂.本研究旨在探讨蛋白酶体抑制剂MGl32(Z-Leu-Leu-Leu-CHO)诱导白血病细胞HL-60凋亡和C2/M期阻滞的机制.方法:采用荧光显微镜观察、流式细胞术和免疫印迹研究测定MG132诱导HL-60细胞凋亡和周期阻滞及机制.结果:2μmol/L的MG132能够有效地诱导HL-60细胞凋亡,用药后24 h就显现有细胞凋亡;在MG132诱导HL-60细胞凋亡出现之前有一个明显的G2/M期阻滞,加MG132后12 h时G2/M期时相百分比为63.42±2.02;24 h时加MG132组细胞凋亡为16.67±1.48,与对照组G2/M期时相百分比为7.29±3.01及细胞凋亡为0相比,两者之间有显著性差别(P＜0.01);咖啡因CAF能够减少MG132诱导HL-60细胞出现的G2/M期阻滞,同时也减少凋亡细胞的比例;细胞周期检查点的负调控因子p21waf/cip1蛋白在加MG132处理后3 h有明显的表达,但并未能检测到p53和p27蛋白.结论:MG132诱导HL-60细胞凋亡之前有一个明显的G2/M期阻滞,p21蛋白表达明显上调提示:是p21waf/cip1而不是p53或其同源蛋白参与了其中的调控.     

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.      

A p21(waf1)-independent pathway for inhibitory phosphorylation of cyclin-dependent kinase p34(cdc2) and concomitant G(2)/M arrest by the chemopreventive flavonoid apigenin.

Apigenin, a nonmutagenic flavonoid, has been shown to inhibit ultraviolet light-induced skin tumorigenesis when topically applied to mouse skin. Our previous studies have shown that apigenin treatment of cultured mouse keratinocytes induces G(2)/M arrest accompanied by an increase in p53 protein stability and expression of p21(waf1). In this study, we determined whether the G(2)/M arrest induced by apigenin was dependent upon the presence of the cyclin dependent kinase inhibitor p21(waf1). We exposed WWT.8 (p21(waf1) wild-type) and WKO.16 (p21(waf1) null) mouse keratinocytes to various doses of apigenin for 24 h and observed G(2)/M arrest in both cell lines, thereby establishing that the apigenin-induced G(2)/M arrest was p21(waf1) independent. A 4-h treatment with apigenin induced increases in p53 protein level by sixfold and tenfold in the WWT.8 p21(waf1) wild-type cells and WKO.16 p21(waf1) null cells, respectively. After 24 h in WWT.8 cells, p21(waf1) protein also was induced in a dose-dependent manner, but it was not expressed in WKO.16 keratinocytes. We then measured the effect of apigenin treatment on the mammalian homologue of the yeast cdc2 gene (p34(cdc2)) cyclin-dependent kinase and cyclin B1 (cycB1), because these proteins complex to regulate G(2)/M progression. Apigenin treatment decreased the protein level of p34(cdc2), and p34(cdc2) kinase activity was inhibited in both p21(waf1)(+/+) and p21(waf1)(-/-) cell lines by approximately 40%. The inhibition of p34(cdc2) kinase activity by apigenin treatment correlated with increasing levels of p34(cdc2) phosphorylation at Tyr15, a site in the p34(cdc2) kinase that undergoes inhibitory phosphorylation by Wee1 kinase. Apigenin treatment also had no effect on the protein level or activity of the competing phosphatase, cdc25c, which dephosphorylates p34(cdc2) kinase at Tyr15. Apigenin had little effect on the accumulation of cycB1 protein. These results supported the conclusion that G(2)/M arrest induced by apigenin was accompanied by inhibition of the p34(cdc2) cyclin-dependent kinase protein level and activity in a p21(waf1)-independent manner.     

Absence of p53-mediated G1 arrest with induction of MDM2 in sterigmatocystin-treated cells

P53 plays a critical role in G1 checkpoint after DNA damage. MDM2 gene is a p53 target gene and its protein forms a feedback loop with p53 and inhibits p53-mediated G1 arrest. Sterigmatocystin (ST) is a mycotoxin and carcinogen. In this study we show that exposure of cells to ST for 12 or 24 h resulted in failure of G1 arrest at both time points. Accordingly, p53 protein was not increased and p21WAF1 expression was inhibited at 12 h, and both proteins were weakly induced at 24 h after treatment with ST. Meanwhile, MDM2 protein was induced in a p53-dependent fashion by ST at both 12 and 24 h. The induction of MDM2 was coincident with the cellular responses of p53 and p21WAF1, and might contribute to the failure of G1 arrest in ST-treated cells. In addition, ST-treated cells exhibited G2M arrest, regardless of p53 status. Our results indicate that the carcinogenic effects of ST seem to be mediated by failure of p53-mediated G1 checkpoint.     

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.     

Low dose fractionated radiation enhances the radiosensitization effect of paclitaxel in colorectal tumor cells with mutant p53

BACKGROUND: The current study was undertaken to investigate the influence of wild-type or mutant p53 status on the radiosensitizing effect of paclitaxel in colorectal tumor cell lines. METHODS: HCT-116 (contains wild-type p53) and HT-29 (contains mutant p53) established from moderately differentiated colorectal carcinomas were used in this study. Colony-forming assay was performed after exposure to either different radiation doses (0.5-6 gray [Gy]) or paclitaxel (1-10 nM) or in combination. Induction of p53 and p21(waf1/cip1) by these treatments were determined by immunocytochemistry and Western blot analysis. RESULTS: Radiation caused an increase in nuclear p53 and p21(waf1/cip1) proteins in HCT-116 cells, indicating that p53 functionally induced p21(waf1/cip1). However, induction of nuclear p53 and p21(waf1/cip1) protein was not evident in HT-29 cells, suggesting that p53 was not functional in these cells. Survival data showed that the HCT-116 cells (survival fraction of exponentially growing cells that were irradiated at the clinically relevant dose of 2 Gy [SF(2)] = 0.383; dose required to reduce the fraction of cells to 37% [D(0)] = 223 centigray [cGy]) were significantly sensitive to ionizing radiation (P < 0.008) when compared with the HT-29 cells (SF(2) = 0.614; D(0) = 351 cGy). Paclitaxel caused a higher degree of clonogenic inhibition in HCT-116 (D(0) = 0.7 nM) than HT-29 (D(0) = 1.11 nM) cells (P < 0.06). When paclitaxel and radiation were combined, an enhanced radiosensitizing effect (P < 0.05) was observed in HCT-116 cells (SF(2) = 0.138; D(0) = 103 cGy), whereas in HT-29 cells no significant radiosensitization of paclitaxel was observed (SF(2) = 0.608; D(0) = 306 cGy). However, pretreatment with paclitaxel followed by multifractionated low dose radiation (0.5- or 1-Gy fractions for a total dose of 2 Gy) significantly enhanced the radiosensitizing effect in both HCT-116 and HT-29 cells. CONCLUSIONS: The results of the current study suggested that multifractionated radiation given at very low doses after exposure of cells to paclitaxel conferred a potent radiation sensitizing effect irrespective of p53 status.     

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.     

8-60hIPP5m-Induced G2/M Cell Cycle Arrest Involves Activation of ATM/p53/p21cip1/waf1 Pathways and Delayed Cyclin B1 Nuclear Translocation

Protein phosphatase 1 (PP1) is a major serine/threonine phosphatase that controls gene expression andcell cycle progression. The active mutant IPP5 (8-60hIPP5m), the latest member of the inhibitory molecules forPP1, has been shown to inhibit the growth of human cervix carcinoma cells (HeLa). In order to elucidate theunderlying mechanisms, the present study assessed overexpression of 8-60hIPP5m in HeLa cells. Flow cytometricand biochemical analyses showed that overexpression of 8-60hIPP5m induced G2/M-phase arrest, which wasaccompanied by the upregulation of cyclin B1 and phosphorylation of G2/M-phase proteins ATM, p53, p21cip1/waf1and Cdc2, suggesting that 8-60hIPP5m induces G2/M arrest through activation of the ATM/p53/p21cip1/waf1/Cdc2/cyclin B1 pathways. We further showed that overexpression of 8-60hIPP5m led to delayed nuclear translocationof cyclin B1. 8-60hIPP5m also could translocate to the nucleus in G2/M phase and interact with pp1α and Cdc2as demonstrated by co-precipitation assay. Taken together, our data demonstrate a novel role for 8-60hIPP5min regulation of cell cycle in HeLa cells, possibly contributing to the development of new therapeutic strategiesfor cervix carcinoma.