Sensitivity to the non-COX inhibiting celecoxib derivative, OSU03012, is p21(WAF1/CIP1) dependent

OSU03012 is a non-COX inhibiting celecoxib derivative with growth inhibiting and apoptotic activity in many cancer cell lines. To investigate mechanisms related to cell cycle proteins in growth inhibition and apoptosis induced by OSU03012, the primary human oral epithelial cell line, TE1177, was transformed with HPV16 E6 (TE/E6), HPV16 E7 (TE/E7) or empty vector (TE/V). TE/E6 cell lines exhibiting low levels of p53 and undetectable levels of p21(WAF1/CIP1) were sensitized to the growth inhibiting and apoptotic effects of OSU03012. The TE/E7 cell lines expressing low levels of Rb and elevated levels of p53 and p21(WAF1/CIP1) were resistant. OSU03012 reduced the number of cells in the S phase of the TE/E7 and TE/V cell lines with intact p53-p21(WAF1/CIP1) checkpoint, but not in the checkpoint defective TE/E6 cell lines. Treatment with OSU03012 also markedly reduced the levels of cyclin A and Cdk2 in TE/E7 and TE/V, but not in TE/E6 cell lines, which had significantly enhanced basal levels of cyclin A and Cdk2. Consistent with the TE/E6 cell line, p21(WAF1/CIP1)-/- mouse embryo fibroblasts were more sensitive to OSU03012-induced apoptosis as evidenced by PARP and caspase 3 cleavages. These data suggest that p21(WAF1/CIP1) is an important factor in the sensitivity of cells to the growth inhibiting and apoptotic effects of OSU03012.     

F9 embryonal carcinoma cells fail to stop at G1/S boundary of the cell cycle after gamma-irradiation due to p21WAF1/CIP1 degradation

We studied the ability of F9 teratocarcinoma cells to arrest in G1/S and G2/M checkpoints after gamma-irradiation. Wild-type p53 protein was rapidly accumulated in F9 cells after gamma-irradiation, however, this was followed not by a G1/S arrest but by a short and reversible delay of the cell cycle in G2/M. In order to elucidate the reasons of the lack of G1/S arrest in F9 cells, we investigated the expression of p53 downstream target Cdk inhibitor p21WAF1/CIP1. In spite of p53-dependent activation of p21WAF1/CIP1 gene promoter and p21WAF1/CIP1 mRNA accumulation upon irradiation, the p21WAF1/CIP1 protein was not detected by either immunoblot or immunofluorescence techniques. However, the cells treated with a specific proteasome inhibitor lactacystin revealed the p21WAF1/CIP1 protein both in non-irradiated and irradiated cells. Therefore we suggest that p21WAF1/CIP1 protein is degraded by a proteasome-dependent mechanism in F9 cells and the lack of G1/S arrest after gamma-irradiation is due to this degradation. We also examined the expression and activity of cell cycle regulatory proteins: G1- and G2-cyclins and cyclin-dependent kinases. In the absence of functional p21WAF1/CIP1 inhibitor, the activity of G1 cyclin/Cdk complexes was insufficiently inhibited to cause a G1 arrest, whereas a decrease of cdc2 and cyclin B1-associated kinase activities was enough to contribute to a reversible G2 arrest following gamma-irradiation. After gamma-irradiation, the majority of F9 cells undergo apoptosis implying that wt-p53 likely triggers pro-apoptotic gene expression in DNA damaged cells. Elimination of defected cells might ensure maintenance of genome integrity in the remaining cell population.     

Synergistic effects of dexamethasone and genistein on the expression of Cdk inhibitor p21WAF1/CIP1 in human hepatocellular and colorectal carcinoma cells

Previous studies have shown that dexamethasone, a synthetic glucocorticoid, can induce a G1 arrest, however, genistein, a natural isoflavonoid phytoestrogen, induces a G2/M arrest in the cell cycle progression in various cancer cell lines. A block of cell cycle checkpoint by dexamethasone and genistein correlates with a selective induction of cyclin-dependent kinase (Cdk) inhibitor p21WAF1/CIP1 in a tumor suppressor p53-independent manner and abolishment of Cdk2 phosphorylation. In the present study, the effects of dexamethasone and genistein (both singly and combined) on the expression of p21 in human hepatocellular Hep G2 and colorectal Colo320 HSR carcinoma cells were evaluated. Whereas dexamethasone mildly induced the level of p21 protein, genistein strongly increased the expression of p21 protein in our experimental condition. Both compounds also activated p21 promoter reporter constructs. The combined effects of dexamethasone and genistein on the induction of p21 protein and activation of p21 promoter were synergistic in both cell lines. These findings indicate that dexamethasone and genistein act in a synergistic fashion and have potential for combination chemotherapy for the treatment of liver and colon cancer.     

Adriamycin induced G2/M cell cycle arrest in transitional cell cancer cells with wt p53 and p21(WAF1/CIP1) genes

Adriamycin (ADM), widely used for systemic and local treatment of bladder tumors, triggers apoptosis in bladder cancer cells. Here we investigated the effect of ADM on cell cycle progression and expression of cell cycle regulating proteins in bladder cancer cell lines with various p53 and p21(WAF1/CIP1) status. Flowcytometric analysis was used to estimate the cell cycle distribution of T24, HT-1376, RT4, and SCaBER bladder cancer cell lines. Cell cycle regulating proteins were analyzed by Immunoblot. Treatment of RT4 cells, bearing wild type p53 and p21(WAF1/CIP1), with ADM induced expression of both proteins and cell cycle arrest, not in G1, as was anticipated, but in the G2 phase. Simultaneously, Retinoblastoma (Rb) protein expression was decreased. Expression of PCNA, which is a target gene of E2F, was not changed. The results suggest that even if the tumor cells bear wild type (wt) p53 and wt p21(WAF1/CIP1) and both proteins accumulate due to genotoxic stimuli, the cell cycle arrest might happen not in the G1 but in the G2 phase.     

Differential effects of p21(WAF1/CIP1) deficiency on MMTV-ras and MMTV-myc mammary tumor properties

p21(WAF1/CIP1) (p21) functions as a cyclin-dependent kinase (CDK) inhibitor and is a key mediator of p53-dependent growth arrest. However, its role in cell cycle regulation is complex, because it also appears to promote CDK activity in certain experimental contexts. Its potential role in tumor suppression was evaluated in MMTV-ras and MMTV-myc transgenic mice that were interbred to p21(WAF1/CIP1) knockout mice (p21-/-). p21 deficiency had differential effects on tumor incidence and age of onset, proliferation, and apoptosis in the presence of these two oncogenes. Tumors arising in MMTV-ras/p21-/- mice displayed higher S-phase fractions and correspondingly increased cyclin D1 and E/CDK activity than MMTV-ras tumors. In contrast, MMTV-myc/p21-/- tumors had lower S-phase fractions and levels of cyclin D1 and E/CDK activity than MMTV-myc tumors. In both tumor types, changes in cyclin D1 and E/CDK activity were paralleled by changes in the corresponding cyclin protein levels. Tumor cell apoptosis was also differentially influenced by p21 deficiency in the two models. MMTV-ras/p21-/- tumors exhibited a significant increase in spontaneous apoptosis as compared with MMTV-ras tumors, whereas p21 deficiency had minimal effect on apoptosis in MMTV-myc tumors. These results indicate that the effects of p21 expression on cellular proliferation are differentially affected by the expression of different oncogenes, and that p21 may play a role in promoting either growth arrest or proliferation, depending on the specific cellular context.     

Mitomycin C and doxorubicin elicit conflicting signals by causing accumulation of cyclin E prior to p21WAF1/CIP1 elevation in human hepatocellular carcinoma cells

Proteins involved in the G1 phase of the cell cycle are aberrantly expressed, sometimes in mutated forms, in human cancers including human hepatocellular carcinoma. Upon attack by a DNA-damaging anticancer drug, a cell arrests at the G1 phase; this is a safety feature prohibiting entry of DNA-damaged cells into S-phase. p21WAF1/CIP1 prevents damaged cells from progressing to the next cell cycle. Here, we show that, in response to mitomycin C and doxorubicin, human hepatocellular carcinoma cells generate conflicting signals, mediated by cyclin E and p21WAF1/CIP1, which respectively accelerates and represses cell cycle transition. Exposure to these anticancer drugs led to rapid accumulation of cyclin E in both p53-proficient HepG2 and p53-deficient Hep3B cells. Such anticancer drug-induced cyclin?E accumulation influenced the G1-S-phase transition, but not DNA fragmentation-mediated death. In p53-proficient HepG2 cells, accumulation of cyclin E was followed by an increase in the level of p53-dependent p21WAF1/CIP1, thereby inhibiting further the G1-S-phase transition. Sublethal drug concentrations also induced rapid accumulation of cyclin E, but p21WAF1/CIP1 accumulation was delayed, further facilitating the G1-S-phase transition. Eventually, most cells arrested in G2/M. Thus, mitomycin C- or doxorubicin-induced conflicting signals, mediated by cyclin E and p21WAF1/CIP1, are in play in human hepatocellular carcinoma cells. Damaged G1 cells either immediately enter S-phase, or do not do so at all, depending on the extent of DNA damage.     

Induction of p21 by p53 following DNA damage inhibits both Cdk4 and Cdk2 activities

DNA damage often activates the p53-p21 pathway and causes G(1)-phase arrest in mammalian cells. Although there is ample evidence that p21 induction by p53 leads to Cdk2 inhibition, it is unclear whether this checkpoint event also leads to Cdk4 inhibition. Diaminocyclohexane(trans-diacetato)(dichloro) platinum(IV) (DAP), a platinum-based coordination complex, is a DNA-damaging agent that is effective against a variety of tumor cells resistant to the parental drug cisplatin. Our previous studies established that treatment of human cancer cells with low effective concentrations of DAP specifically activates the G(1)-phase checkpoint and simultaneously inhibit Cdk4 and Cdk2 activities. Here we demonstrate that DAP treatment of human cancer cells activates the p53-p21 pathway without activating other known mechanisms that inhibit Cdk4 and Cdk2 activities. The induced p21 binds to both the Cdk4/cyclin D and Cdk2/cyclin E complexes and inhibits both of their kinase activities. Conversely, inhibition of p21 induction by cycloheximide or by p21 gene deletion prevents DAP-induced inhibition of Cdk4 and Cdk2 activities. Attenuated p53 expression and p21 induction also eliminates DAP-induced G(1)-phase arrest and inhibition of Cdk4 and Cdk2 activities. Together, these findings establish that activation of the p53-p21 pathway is responsible for the DAP-induced G(1)-phase checkpoint response and provide the first solid evidence that p21 induction by p53 during a DNA damage-induced G(1)-phase checkpoint response inhibits both Cdk4 and Cdk2 activities.     

Flavopiridol inversely affects p21(WAF1/CIP1) and p53 and protects p21-sensitive cells from paclitaxel

Resting cells are relatively resistant to microtubule-active drugs including paclitaxel (PTX). By causing p53-mediated arrest, pretreatment with low concentrations of doxorubicin (DOX) protected HCT116 cells from the cytotoxicity caused by PTX. Unlike DOX, flavopiridol (FL) did not protect HCT116 cells. Low concentrations of FL (50 nM) induced p21 but not p53. High concentrations of FL (500 nM) decreased levels of p21 and Mdm-2 but dramatically induced p53. Thus, FL reciprocally affects p21 and p53. In LNCaP, a prostate cancer cell line which is highly sensitive to p21-induced growth arrest (p21-sensitive), low concentrations of FL (50 nM) induced p21 (without induction of p53) and caused G1 and G2 arrest. This precluded mitotic arrest, Bcl-2 and Raf-1 phosphorylation, and diminished cell death caused by PTX. In contrast, FL did not protect PC3M, arrest-resitant and highly aggressive prostate cancer cells. Like LNCaP, HL60 and SKBr3 cells are known to be p21-sensitive. As predicted, low concentrations of FL antagonized PTX-mediated cytotoxicity in HL60 and SKBr3 cell lines. In summary, only low concentrations of FL can induce p21, and, in turn, only p21-sensitive cells are protected from PTX.     

Levels of p21(WAF1/CIP1) do not affect radiation-induced cell death in human breast epithelial cells

Loss of the wild-type p53 activity and/or overexpression of the proto-oncogene bcl-2 are frequently detected in breast cancer and suggested to be related to chemotherapy and radiation therapy resistance. To identify the downstream signaling molecules for anti-proliferative and apoptotic activities of p53 and to investigate the interaction of bcl-2 with p53 in human breast epithelial cells, we have used the MCF10A cell line. We previously showed that overexpression of bcl-2 downregulates expression of p21(WAF1/CIP1) (a cyclin dependent kinase inhibitor which mediates p53 dependent G(1) arrest) and suppresses DNA damage-induced apoptosis in MCF10A cells. In the present study, we constitutively overexpressed p21(WAF1/CIP1) in bcl-2 overexpressing MCF10A cells to determine whether downregulation of p21(WAF1/CIP1) is necessary for the anti apoptotic activity of bcl-2, and to investigate the roles of p21(WAF1/CIP1) in p53-mediated cell death upon irradiation. Overexpression of p21(WAF1/CIP1) resulted in growth inhibition, but had no effect on bcl-2 inhibition of apoptosis following irradiation. Also, overexpression of p21(WAF1/CIP1) did not affect the dose-dependent radiation-induced cell lethality as determined by a clonogenic survival assay. These results suggest that bcl-2 downregulation of p21(WAF1/CIP1) is independent of the anti-apoptotic activity of bcl-2, and that p21(WAF/CIP1) is not involved in the p53-mediated cell death pathway.     

p21WAF1/CIP1 protein expression in primary ovarian cancer

p21WAF1/CIP1 protein is a cyclin-dependent kinase inhibitor, able to prevent the CDK2/cyclin E induced retinoblastoma protein (pRB) phosphorylation, thus inhibiting cell cycle progression at G1 phase. p21WAF1/CIP1 protein levels were examined in a series of 102 ovarian tissue samples including normal ovary, primary ovarian tumors, omental metastasis, recurrent disease and residual tumor after chemotherapy exposure, by Western blot analysis. The association of p21WAF1/CIP1 status with clinicopathological parameters and clinical outcome was also investigated. p21WAF1/CIP1 protein was detectable in 76 out of 102 (74%) ovarian tissue samples. We observed a significant trend of p21 levels to gradually increase from normal ovarian tissues (median 0 a.u.) through primary ovarian cancers (median 0.19 a.u.), omental metastases (median 0.33 a.u.) and recurrence of disease (median 0.44 a.u.) (p=0.015). In the group of stage III-IV ovarian cancer patients, p21-positive cases showed a more favourable prognosis with respect to p21-negative cases: the 3-year time to progression (TTP) rate was 58% for p21-positive compared with 33% of p21-negative cases (p=0.036). In conclusion, p21WAF1/CIP1 expression levels seem to be correlated with tumor status at the time of diagnosis and can predict TTP in a selected group of patients.     

The alternative reading frame tumor suppressor inhibits growth through p21-dependent and p21-independent pathways

The alternative reading frame (ARF) tumor suppressor mediates growth arrest or apoptosis through activation of the p53 tumor suppressor. A prevailing concept is that ARF uses p21Cip1/Waf1, a p53-responsive gene and cyclin-dependent kinase (Cdk) inhibitor, to block cell cycle progression. Using p21 nullizygous cells, we demonstrate that p21 is nonessential for the antiproliferative activity of ARF and p53, although it likely governs the arrest through Cdk inactivation when present. ARF overexpression in p21-positive and p21-negative mouse embryo fibroblasts (MEFs), but not in primary cells lacking p53, induced a biphasic (G1 and G2) cell cycle arrest. The ARF-induced growth arrest, regardless of p21 status, coincided with activation of p53 and accumulation of hypophosphorylated retinoblastoma protein (retinoblastoma protein). In ARF-arrested p21-positive cells, the presence of growth-inhibitory retinoblastoma protein correlated with an absence of Cdk2-dependent kinase activity, an increase in p21 association with inactive Cdks, and a lack of cyclin A expression. In contrast, p21-/- mouse embryo fibroblasts were arrested by ARF despite containing elevated levels of cyclin A protein and highly active Cdk2-dependent kinases. These findings provide evidence that ARF can block growth through a p21-independent pathway(s) that overrides Cdk2 activation.     

Expression of cell-cycle regulators, cyclin E and p21WAF1/CIP1, potential prognostic markers for gastric cancer.

AIMS: The deregulation of cyclin, cyclin-dependent kinases (CDKs) and their inhibitors could have a crucial role in the development of diverse human cancers. METHODS: In this study, we analysed the expression of cyclin D1, cyclin E, p21WAF1/CIP1 and p27KIP1 in 84 surgically resected gastric cancers by immunohistochemistry with long-term follow-up (median 38 months). We also evaluated the relation between each cell cycle regulator and various clinicopathological findings, including age, sex, histological grade, tumour location, tumour type and stage and lymph-node metastasis. RESULTS: Overexpression of cyclin D1 and E was detected in 21/84 (25%) and 34/84 (40.5%) patients, respectively. Normal gastric epithelium showed consistently positive immunostain for p21WAF1/CIP1 and p27KIP1 in more than 50% of nuclei. Loss of p21WAF1/CIP1 and p27KIP1 expression was noted in 45/84 (53.6%) and 44/84 (52.4%) patients, respectively. Among the various clinicopathological findings, overexpression of cyclin E was associated with lymph-node metastasis (P=0.003) and recurrence (P=0.043). Loss of p21WAF1/CIP1 expression was more frequent in diffuse type cancers (P=0.005) and was correlated with recurrence (P=0.002) and death (P=0.002). Overexpression of cyclin E and loss of p21WAF1/CIP1 expression were significantly correlated with decreased disease-free (P=0.037; P= 0.001) and overall (P=0.031; P=0.001) survival. CONCLUSIONS: These results suggest that immunohistochemical analysis for cell cycle regulators, especially cyclin E and p21WAF1/CIP1, might be a useful prognostic indicator in gastric cancer.     

Rapamycin disrupts cyclin/cyclin-dependent kinase/p21/proliferating cell nuclear antigen complexes and cyclin D1 reverses rapamycin action by stabilizing these complexes

Rapamycin and its derivatives are promising anticancer agents, but the exact mechanisms by which these drugs induce cell cycle arrest and inhibit tumor growth are unknown. A biochemical analysis of human mammary tumor cell lines indicated that rapamycin-induced antiproliferative effects correlated with down-regulation of cellular p21 levels and the levels of p21 in cyclin-dependent kinase (Cdk) 2 and 4 complexes. Cyclin D1 overexpression reversed rapamycin action and this reversal correlated with increased levels of cellular p21, higher levels of p21 associated with Cdk2, and stabilization of cyclin D1/Cdk2/p21/proliferating cell nuclear antigen (PCNA) complexes. Experiments using a novel cyclin D1-Cdk2 fusion protein or a kinase-dead mutant of the fusion protein indicated that reversal of rapamycin action required not only the formation of complexes with p21 and PCNA but also complex-associated kinase activity. Similar results were observed in vivo. The rapamycin derivative RAD001 (everolimus) inhibited the growth of mouse mammary tumors, which correlated with the disruption of cyclin D1/Cdk2 complexes. The potential implications of these results with respect to the use of rapamycin derivatives in breast cancer therapy are discussed.