Deregulation of p53/p21Cip1/Waf1 pathway contributes to polyploidy and apoptosis of E1A+cHa-ras transformed cells after gamma-irradiation.

The p53/p21Cip1/Waf1-dependent checkpoint control of G1/S and G2/M phases of the cell cycle in response to DNA damage is an important mechanism of genome stability maintenance in normal cells. In many tumor cells, due to frequent point mutations and deletions of p53, the stringent control of the cell cycle and apoptosis is compromised. We have examined the cell cycle control and cell death of the rat embryo fibroblast cells (REF) transformed by E1A+cHa-ras oncogenes and expressing wild type p53. Gamma-irradiation at a dosage of 6 Gy has been used to analyse the p53-dependent trans-activation of the target p21cip1/waf1 gene and the levels of activity of cyclin-dependent kinases. Our results show that the cell cycle inhibitors p21Cip1/Waf1 and p27KIP accumulate in response to irradiation both in REF and E1A+cHa-ras cells. In contrast to normal REF cells, the accumulation of p21Cip1/Waf1 and p27KIP inhibitors, however, does not lead to inhibition of Cdk2 and cyclins E, A-associated kinase activities and to a G1/S block in E1A+cHa-ras cells. It is unlikely that the lack of inhibitory function of p21Cip1/Waf1 can be explained by its inability to bind Cdk2 and Cdk4 kinases or PCNA. Moreover, the p21Cip1/Waf1-associated kinase activity is increased upon gamma-irradiation of E1A+cHa-ras cells. We suggest that inactivation of p21Cip1/Waf1 may be accounted for by its interaction with E1A oncoproducts as the inhibitor is detected in immunoprecipitates using E1A-specific antibodies. During a temporary G2/M delay induced by gamma-irradiation, E1A+cHa-ras transformants continue DNA replication, which leads to accumulation of polyploid cells with lobulated nuclei and micronuclei. Thus, DNA damage of E1A+cHa-ras transformed cells, with a combination of functionally active wild type p53 and inactive p21Cip1/Waf1, contributes to formation of polyploid cells which then die due to apoptosis.     

p21Waf1/Cip1/Sdi1 induces permanent growth arrest with markers of replicative senescence in human tumor cells lacking functional p53.

We have shown previously that wild type p53 can rapidly induce replicative senescence in EJ human bladder carcinoma cells lacking functional p53. A major effector of p53 functions is p21Waf1/Cip1/Sdi1, a potent cyclin-dependent kinase inhibitor. p21Waf1/Cip1/Sdi1 has been shown to be involved in both p53 dependent and independent control of cell proliferation, differentiation and death. To directly investigate the effects of p21Waf1/Cip1/Sdi1 in the p53 response observed in EJ tumor cells, we established p21Waf1/Cip1/Sdi1 inducible lines using the tetracycline-regulatable vector system. p21Waf1/Cip1/Sdi1 induction caused irreversible cell cycle arrest in both G1 and G2/M, and diminished Cdk2 kinase activity. In addition, p21Waf1/Cip1/Sdi1 induction led to morphological alterations characteristic of cells undergoing replicative senescence with morphological, biochemical and ultrastructural markers of the senescent phenotype. Furthermore, sustained p21Waf1/Cip1/Sdi1 induction sensitized EJ cells to apoptotic cell death induced by mitomycin C, a cross-linking DNA damaging agent. These findings support the function of p21Waf1/Cip1/Sdi1 as an inducer of replicative senescence and a major mediator of this phenomenon in response to p53. Moreover, our results imply that therapeutic intervention in human cancers might be aimed at sustained elevation of p21Waf1/Cip1/Sdi1 expression.     

The Aurora kinase inhibitor VX-680 induces endoreduplication and apoptosis preferentially in cells with compromised p53-dependent postmitotic checkpoint function

VX-680 is a potent inhibitor of Aurora kinases that induces the accumulation of cells with > or =4N DNA content, followed by cell death. Here, we define the role of p53 and p21(Waf1/Cip1) in cell cycle perturbations following exposure to VX-680. Endoreduplication and apoptosis in response to VX-680 are limited in A549 and MCF-7 cells expressing wild-type p53, and markedly enhanced in cells lacking p53, including those engineered to express the HPV16-E6 oncoprotein or short interfering RNA pools targeting p53. In contrast, endoreduplication and apoptosis occur in the p53 wild-type cell lines, RKO and U2OS. The difference in response to VX-680 among these cell lines correlates with the timing of induction of p21(Waf1/Cip1) and its ability to inhibit cyclin E-cdk2 activity. In A549 cells, VX-680 induces the expression of p53 and p21(Waf1/Cip1) within 24 hours, with consequent inhibition of cyclin E-cdk2, and reduction of retinoblastoma protein phosphorylation, limiting endoreduplication. In RKO and U2OS cells, the induction of p21(Waf1/Cip1) is delayed and associated with higher residual cyclin E-cdk2 kinase activity and retinoblastoma protein phosphorylation, followed by progressive endoreduplication and apoptosis. Abrogation of p21(Waf1/Cip1) expression by short interfering RNA targeting in A549 cells results in a substantial increase in the degree of endoreduplication, whereas inducible expression of p21(Waf1/Cip1) in p53-negative NCI-H1299 cells inhibits VX-680-induced endoreduplication and cell death. These data suggest that the integrity of the p53-p21(Waf1/Cip1)-dependent postmitotic checkpoint governs the response to Aurora kinase inhibition. Although cells with intact checkpoint function arrest with 4N DNA content, those with compromised checkpoint function are more likely to undergo endoreduplication followed by eventual apoptosis.     

2-Chloroethyl-3-sarcosinamide-1-nitrosourea (SarCNU) inhibits prostate carcinoma cell growth via p53-dependent and p53-independent pathways

BACKGROUND: Prostate carcinoma is the most commonly occurring malignancy in men. Although 2-chloroethyl-3-sarcosinamide-1-nitrosourea (SarCNU), an analog of the chloroethylnitrosoureas, has been used in the treatment of advanced solid tumors, the molecular mechanisms underlying the antineoplastic activity of this agent are not well understood. In the current study, the authors sought to investigate the effects of SarCNU on prostate carcinoma cell growth in vivo and in vitro. METHODS: Male SCID mice underwent subcutaneous implantation (on both flanks) of human CWR-22 and CWR-22R prostate carcinoma xenografts. Mice were treated with either vehicle or 60 or 80 mg SarCNU per kg body weight for 5 days, with tumor growth being assessed every 3 days. Animals were sacrificed 21 days after the final injection, and tumors subsequently were collected, weighed, and processed for analysis. Immunohistochemical analyses were performed to obtain data on the localization of p53 and p21Cip1/Waf1. Cell counting, terminal deoxynucleotidyltransferase-mediated deoxyuridine triphosphate nick end-labeling (TUNEL) assays, cell cycle analyses, Western blotting, and in vitro kinase assays were performed to determine the effects of SarCNU on growth, apoptosis, cell cycle arrest, cell cycle-regulated protein levels, and Cdc-2 activity, respectively. RESULTS: SarCNU reduced tumor incidence and inhibited the growth of CWR-22 and CWR-22R xenografts. In addition, treatment with this agent led to increases in p21Cip1/Waf1 levels and p53 phosphorylation at Ser15. In vitro administration of SarCNU to cells with wild-type p53 (LNCaP and primary CWR-22 cells) and cells with mutant p53 (PC-3 cells) resulted in G2/M arrest and the reduction of cellular Cdc-2 activity. Up-regulation of p53 levels, p53 phosphorylation at Ser15, and p21Cip1/Waf1 levels in primary CWR-22 and LNCaP cells, as well as up-regulation of Cdc-2 phosphorylation at Tyr15 in PC-3 cells, was detected. CONCLUSIONS: SarCNU induced G2/M arrest in prostate carcinoma cells via p53-dependent up-regulation of p21Cip1/Waf1 and p53-independent phosphorylation of Cdc-2 at Tyr15. These findings suggest a potential role for SarCNU in the treatment of prostate malignancies.     

p53-independent inhibition of proliferation and p21(WAF1/Cip1)-modulated induction of cell death by the antioxidants N-acetylcysteine and vitamin E

Epidemiological evidence has suggested an association between diets rich in antioxidants and diminished risks of various types of cancer. Proposed mechanisms for protective effects of antioxidants have involved inhibition of free radical-mediated DNA damage. Recent data suggest that antioxidants may prevent or eliminate cancerous cells through their ability to inhibit proliferation or to induce programmed cell death (PCD). To begin to identify cell cycle and cell death regulatory factors involved in antioxidant-induced growth arrest and PCD, we have studied colorectal carcinoma cells (CRCs) that differ in expression of the tumor suppressor protein p53, and of the cyclin-dependent kinase (CDK) inhibitor p21^Waf1/Cip1. The antioxidants, N-acetylcysteine (NAC) and vitamin E either inhibited proliferation in a p53-independent manner without affecting cell viability or induced cell death. Growth arrest was not associated with upregulation of the CDK inhibitors p21^Waf1/Cip1, p18^ink4c or p16^ink4a, but was associated with a decrease in reactive oxygen species (ROS). In contrast to previous observations, the absence of p21^Waf1/Cip1 increased susceptibility of CRCs to antioxidant-induced PCD. NAC decreased levels of retinoblastoma protein (Rb) phosphorylation in all cells tested, but Rb was cleaved only in cells which underwent NAC-induced death. Although NAC decreased ROS in all cells studied, cell lines in which PCD occurred had higher baseline levels of ROS than cell lines in which proliferation was blocked. These observations suggest that expression of p21^Waf1/Cip1 and basal levels of ROS are important determinants of outcome after antioxidant treatment.     

Association between polymorphism in p21(Waf1/Cip1) cyclin-dependent kinase inhibitor gene and human oral cancer.

The cyclin-dependent kinase inhibitor gene p21(Waf1/Cip1) plays a central role in inducing cellular growth arrest, terminal differentiation, and apoptosis. Alterations in this gene may adversely affect regulation of these processes and increase susceptibility for cancer. We have recently reported a novel polymorphism in the p21(Waf1/Cip1) gene in the Indian population and its association with esophageal cancer. An A-->G transition at codon 149 resulted in amino acid substitution from aspartate to glycine in the proliferating cell nuclear antigen binding COOH-terminal domain of p21(Waf1/Cip1) that may affect PCNA-p21(Waf1/Cip1) interactions, thereby affecting regulation of cellular proliferation, and may increase susceptibility for development of cancer. In a parallel study in our laboratory, we searched for putative p21(Waf1/Cip1) mutations in oral premalignant and malignant lesions. No somatic mutation was detected in exon 2 of p21(Waf1/Cip1). Interestingly, a codon 149 polymorphism variant (A-->G) was identified in 11 of 30 (37%) premalignant lesions (7 of 19 hyperplastic lesions and 4 of 11 dysplastic lesions) and 11 of 30 (37%) squamous cell carcinomas (SCCs). This codon 149 variant was also identified in paired lymphocytes of all of the patients with premalignant lesions and SCCs harboring the variant allele, suggesting the occurrence of a polymorphism. Lymphocyte DNA isolated from 50 unrelated age- and gender-matched healthy subjects was screened for this polymorphism. Seven of 50 (14%) normal controls harbored the A-->G codon 149 variant allele. Immunohistochemical analysis of p21(Waf1/Cip1) protein expression showed immunoreactivity in 19 of these 30 (63%) oral premalignant lesions and 16 of 30 (53%) SCCs. The most intriguing features of the study were: (a) the significant increase in frequency of this polymorphism not only in patients with oral SCCs (P = 0.038), but also in patients with premalignant lesions (P = 0.038), compared with normal controls; and (b) the significantly higher frequency of p21(Waf1/Cip1) variants (codon 149) in oral premalignant lesions (10 of 11 cases) and SCCs (11 of 11 cases) with wild-type p53 (P = 0.045) than in lesions with p53 mutations, suggesting that this polymorphism affects the p53 pathway and may play a vital role in oral tumorigenesis. Furthermore, overexpression of p21 protein in oral lesions harboring missense mutations in the p53 gene suggest a p53-independent role for p21 in the pathogenesis of oral cancer.     

Cyclin D1, p53, and p21Waf1/Cip1 expression is predictive of poor clinical outcome in serous epithelial ovarian cancer.

PURPOSE: Dysregulation of cell cycle control, in particular G(1)-S-phase transition, is implicated in the pathogenesis of most human cancers, including epithelial ovarian cancer (EOC). However, the prognostic significance of aberrant cell cycle gene expression in EOC remains unclear. EXPERIMENTAL DESIGN: The expression of selected genes from the pRb pathway that regulates G(1)-S-phase progression, including cyclin D1, p16(Ink4a), cyclin E, p27(Kip1), p21(Waf1/Cip1), and p53, was examined in a consecutive series of 134 serous EOC using immunohistochemistry and the results correlated to disease outcome. RESULTS: Molecular markers predictive of reduced overall survival in univariate analysis were overexpression of cyclin D1 (P = 0.03) and p53 (P = 0.03) and reduced expression of p27(Kip1) (P = 0.05) and p21(Waf1/Cip1) (P = 0.02), with the latter three also being prognostic for a shorter progression-free interval. In addition, patients displaying overexpression of p53 with concurrent loss of p21(Waf1/Cip1) had a significantly shorter overall (P = 0.0008) and progression-free survival (P = 0.0001). On multivariate analysis, overexpression of cyclin D1 and combined loss of p21(Waf1/Cip1) in the presence of p53 overexpression were independent predictors of overall survival. Similarly, the combination of p21(Waf1/Cip1) loss and p53 overexpression was independently predictive of a shorter progression-free interval. Overexpression of p53 and cyclin E and reduced expression of p27(Kip1) and p21(Waf1/Cip1) were significantly associated with increasing tumor grade. CONCLUSIONS: This study confirms that dysregulation of cell cycle genes is common in EOC, and that aberrant expression of critical cell cycle regulatory proteins can predict patient outcome in serous EOC.     

Loss of the p21(Cip1/Waf1) cyclin kinase inhibitor results in propagation of horizontally transferred DNA.

We have shown previously that phagocytosis of cells dying by apoptosis results in transfer of whole or fragments of chromosomes into the nucleus of the recipient cell. Although DNA transfer was detected in normal cells, stable propagation of the transferred DNA was only observed in cells deficient in p53. Here we show that mouse embryonic fibroblast cells lacking the p21 (Cip1/Waf1) cyclin-kinase inhibitor are able to propagate DNA engulfed by phagocytosis of apoptotic bodies. Feeding mouse embryonic fibroblast p21(-/-) cells with apoptotic bodies derived from a rat fibrosarcoma resulted in focus formation in vitro and tumor formation in vivo. In contrast, cells lacking the p19 alternative reading frame gene did not show any evidence of transformation. These data indicate that p53, via the activation of p21, blocks normal cells from replicating transferred DNA from engulfed apoptotic bodies. This may be one protection level against the propagation of potentially pathological DNA.     

Investigation of the Effect of Zebularine in Comparison to and in Combination with Trichostatin A on p21Cip1/Waf1/ Sdi1, p27Kip1, p57Kip2, DNA Methyltransferases and Histone Deacetylases in Colon Cancer LS 180 Cell Line

Background: The heart of the cell cycle regulatory machine is a group of enzymes named cyclin-dependent kinases (Cdks). The active form of these enzymes includes a kinase and its partner, a cyclin. The regulation of cyclin-Cdk complexes is provided by Cdk inhibitors (CKIs) such as Cip/Kip family comprising p21Cip1/Waf1/Sdi1, p27Kip1, and p57Kip2. The hypermethylation and deacetylation of Cip/Kip gene family seem to be frequent in numerous cancers. It has been indicated that increased expression of DNMTs and HDACs contributes to cancer induction. Previously, we reported the effect of DNA demethylating agents and histone deacetylase inhibitors on histone deacetylase 1, DNA methyltransferase 1, and CIP/KIP family in colon cancer. The current study was designed to evaluate the effect of zebularine in comparison to and in combination with trichostatin A (TSA) on p21Cip1/Waf1/Sdi1, p27Kip1, p57Kip2, DNA methyltransferases (DNMT1, 3a and 3b) and histone deacetylases (HDAC1, 2, and 3) genes expression, cell growth inhibition and apoptosis induction in colon cancer LS 180 cell line. Materials and Methods: The colon cancer LS 180 cell line was cultured and treated with zebularine and TSA. To determine cell viability, apoptosis, and the relative expression level of the genes, MTT assay, cell apoptosis assay, and qRT-PCR were done respectively. Results: Both compounds significantly inhibited cell growth, and induced apoptosis. Furthermore, both compounds increased p21Cip1/Waf1/Sdi1, p27Kip1, and p57Kip2 significantly. Additionally, zebularine and TSA decreased DNMTs and HDACs gene expression respectively. Conclusion: The zebularine and TSA can reactivate the CIP/KIP family through inhibition of DNMTs and HDACs genes activity.     

c-Myc overexpression sensitises colon cancer cells to camptothecin-induced apoptosis

The proto-oncogene c-Myc is overexpressed in 70% of colorectal tumours and can modulate proliferation and apoptosis after cytotoxic insult. Using an isogenic cell system, we demonstrate that c-Myc overexpression in colon carcinoma LoVo cells resulted in sensitisation to camptothecin-induced apoptosis, thus identifying c-Myc as a potential marker predicting response of colorectal tumour cells to camptothecin. Both camptothecin exposure and c-Myc overexpression in LoVo cells resulted in elevation of p53 protein levels, suggesting a role of p53 in the c-Myc-imposed sensitisation to the apoptotic effects of camptothecin. This was confirmed by the ability of PFT-alpha, a specific inhibitor of p53, to attenuate camptothecin-induced apoptosis. p53 can induce the expression of p21(Waf1/Cip1), an antiproliferative protein that can facilitate DNA repair and drug resistance. Importantly, although camptothecin treatment markedly increased p21(Waf1/Cip1) levels in parental LoVo cells, this effect was abrogated in c-Myc-overexpressing derivatives. Targeted inactivation of p21(Waf1/Cip1) in HCT116 colon cancer cells resulted in significantly increased levels of apoptosis following treatment with camptothecin, demonstrating the importance of p21(Waf1/Cip1) in the response to this agent. Finally, cDNA microarray analysis was used to identify genes that are modulated in expression by c-Myc upregulation that could serve as additional markers predicting response to camptothecin. Thirty-four sequences were altered in expression over four-fold in two isogenic c-Myc-overexpressing clones compared to parental LoVo cells. Moreover, the expression of 10 of these genes was confirmed to be significantly correlated with response to camptothecin in a panel of 30 colorectal cancer cell lines.     

p21Waf1/Cip1 acts in synergy with bcl-2 to confer multidrug resistance in a camptothecin-selected human lung-cancer cell line

The realization that chemotherapeutic agents induce apoptosis raises the concern that tumors resistant to chemotherapy are unable to initiate the apoptotic program. In the present study, we examined the apoptosis-resistance mechanism of a multidrug-resistant cell line, A549/CPT, which was established from the human lung-cancer cell line A549 by in vitro selection with gradually increased camptothecin (CPT) concentrations. We found that A549/CPT cells were resistant to anti-cancer drug-induced apoptosis in which caspase-3-like protease activity was attenuated remarkably, compared with parental A549 cells. We observed 2 mechanisms associated with apoptosis resistance in A549/CPT cells: over-expression of anti-apoptotic bcl-2 and elevated expression of p21Waf1/Cip1. Transfection of either bcl-2 or p21Waf1/Cip1 cDNA into parental A549 cells resulted in resistance to apoptosis. Furthermore, the co-treatment of p21Waf1/Cip1 and bcl-2 anti-sense oligodeoxy-nucleotides restored drug susceptibility in A549/CPT cells more effectively than either one of them alone. These results indicate that co-induction of bcl-2 and p21Waf1/Cip1 in A549/CPT cells may be involved in acquired drug resistance by inhibiting caspase-mediated apoptosis. Agents aimed at preventing both bcl-2 and p21Waf1/Cip1 expression may increase the efficiency of chemotherapy.     

Id-1 promotes proliferation of p53-deficient esophageal cancer cells

The helix-loop-helix protein inhibitor of differentiation and DNA binding (Id-1) is known to promote cellular proliferation in several types of human cancer. Although it has been reported that Id-1 is over-expressed in esophageal squamous cell carcinoma (ESCC), its function and signaling pathways in esophageal cancer are unknown. In our study, we investigated the direct effects of Id-1 on esophageal cancer cell growth by transfecting an Id-1 expression vector into an ESCC cell line (HKESC-3), which showed serum-dependent Id-1 expression. Ectopic Id-1 expression resulted in increased serum-independent cell growth and G1-S phase transition, as well as up-regulation of mouse double minute 2 (MDM2) and down-regulation of p21Waf1/Cip1 protein expressions in the transfectant clones in a p53-independent manner. However, overexpression of Id-1 had no effect on the pRB, CDK4 and p16INK4A expressions. Stable transfection of Id-1 antisense expression vector to inhibit the expression of endogenous Id-1 in another ESCC cell line (HKESC-1) reversed the effects on MDM2 and p21Waf1/Cip1. In addition, Id-1 expression protected ESCC cells from Tumor Necrosis Factor (TNF)-alpha-induced apoptosis by up-regulating and activating Bcl-2. In conclusion, our study provides evidence for the first time that Id-1 plays a role in both proliferation and survival of esophageal cancer cells. Our findings also suggest that unlike prostate, hepatocellular and nasopharyngeal carcinomas in which Id-1 induces cell proliferation through inactivation of p16INK4A/RB pathway, the increased cell proliferation observed in ESCC cells may be mediated through a different mechanism.     

Resistance of primary cultured mouse hepatic tumor cells to cellular senescence despite expression of p16(Ink4a), p19(Arf), p53, and p21(Waf1/Cip1)

Primary cultured mouse hepatic cells become senescent within a short period, although rare cells form colonies from which continuously proliferating cell lines can be established. In contrast, hepatic tumor (HT) cells show little senescence and higher colony-forming capacity. To assess this difference, we investigated p16(Ink4a)/p19(Arf)/p53/p21(Waf1/Cip1) expression in primary normal and HT cells, together with cell lines established from both. In primary normal cells, p16(Ink4a)/p19(Arf) were expressed only in association with senescence and disappeared at later stages of colony formation. In contrast, primary HT cells showed sustained p16(Ink4a)/p19(Arf) expression from the beginning. No p16(Ink4a)/p19(Arf) alterations, such as deletion, mutations, or hypermethylation, were detected in the primary HT cells, although most cell lines derived from either normal or HT cell colonies lost p16(Ink4a) or p19(Arf) expression owing to hypermethylation or homozygous deletion of p16(Ink4a)/p19(Arf). On the other hand, primary normal and HT cells and most cell lines showed constitutively elevated expression of p53/p21(Waf1/Cip1), with a further increment after ultraviolet ir-radiation, indicating a functionally normal p53 pathway. These results indicate that primary HT cells are resistant to senescence despite retaining p16(Ink4a)/p19(Arf)/p53/p21(Waf1/Cip1) expression and that loss of p16(Ink4a)/p19(Arf) function is associated only with establishment of the cell lines.     

Novel 1p tumour suppressor Dnmt1-associated protein 1 regulates MYCN/ataxia telangiectasia mutated/p53 pathway

Neuroblastoma (NB) is a paediatric solid tumour which originates from sympathetic nervous tissues. Deletions in chromosome 1p are frequently found in unfavourable NBs and are correlated with v-myc avian myelocytomatosis viral oncogene neuroblastoma derived homolog (MYCN) amplification; however, it remains to be elucidated how the 1p loss contributes to MYCN-related oncogenic processes in NB. In this study, we identified the role of Dnmt1-associated protein 1 (DMAP1), coded on chromosome 1p34, in the processes.We studied the expression and function of DMAP1 in NB and found that low-level expression of DMAP1 related to poor prognosis, unfavourable histology and 1p Loss of heterozygosity (LOH) of primary NB samples. Intriguingly, DMAP1 induced ataxia telangiectasia mutated (ATM) phosphorylation and focus formation in the presence of a DNA damage reagent, doxorubicin. By DMAP1 expression in NB and fibroblasts, p53 was activated in an ATM-dependent manner and p53-downstream pro-apoptotic Bcl-2 family molecules were induced at the mRNA level, resulting in p53-induced apoptotic death. BAX and p21^Cip1/Waf1 promoter activity dependent on p53 was clearly up-regulated by DMAP1. Further, MYCN transduction in MYCN single-copy NB cells accelerated doxorubicin (Doxo)-induced apoptotic cell death; MYCN is implicated in DMAP1 protein stabilisation and ATM phosphorylation in these situations. DMAP1 knockdown attenuated MYCN-dependent ATM phosphorylation and NB cell apoptosis. Together, DMAP1 appears to be a new candidate for a 1p tumour suppressor and its reduction contributes to NB tumourigenesis via inhibition of MYCN-related ATM/p53 pathway activation.