Mutant p53 can delay growth arrest and loss of CDK2 activity in senescing human fibroblasts without reducing p21(WAF1) expression

Functional wild-type p53 is required for human diploid fibroblasts (HDF) to enter an irreversible growth arrest known as replicative senescence. Experimentally, abrogation of p53 function by expression of human papillomavirus type 16 E6 or disruption of a key downstream effector p21 by homologous recombination both extended HDF life span. However, although sufficient to extend life span, p21 down-regulation is not necessary, because expression of a dominant-negative mutant p53 (143(ala)) extends life span without apparently decreasing p21 expression. Given the importance of p53 in cellular senescence and the general assumption that p21 may be the sole mediator of its action in this process, we have investigated how abrogation of p53 function can overcome senescence without lowering expression of p21. We have found up-regulated levels of the cyclin-dependent kinase 2 (cdk2) protein in HDF expressing 143(ala) mutant p53 as compared to senescent controls, together with an increase in p21-free cdk2 which, in conjunction with cyclin E, is able to form an active kinase which can phosphorylate the retinoblastoma protein. However, forced overexpression of cdk2 in near-senescent HDF failed to restore cdk2-associated kinase activity. Our data suggest that p53-mediated senescence depends on factor(s) other than p21 which modulate formation of cyclin E-cdk2 complexes.     

Stat1-dependent, p53-independent expression of p21(waf1) modulates oxysterol-induced apoptosis

7-Ketocholesterol (7kchol) is prominent in atherosclerotic lesions where apoptosis occurs. Using mouse fibroblasts lacking p53, p21(waf1), or Stat1, we found that optimal 7kchol-induced apoptosis requires p21(waf1) and Stat1 but not p53. Findings were analogous in a human cell system. Apoptosis was restored in Stat1-null human cells when wild-type Stat1 was restored. Phosphorylation of Stat1 on Ser(727) but not Tyr(701) was essential for optimum apoptosis. A neutralizing antibody against beta interferon (IFN-beta) blunted Ser(727) phosphorylation and apoptosis after 7kchol treatment; cells deficient in an IFN-beta receptor subunit exhibited blunted apoptosis. IFN-beta alone did not induce apoptosis; thus, 7kchol-induced release of IFN-beta was necessary but not sufficient for optimal apoptosis. In Stat1-null cells, expression of p21(waf1) was much less than in wild-type cells; introducing transient expression of p21(waf1) restored apoptosis. Stat1 and p21(waf1) were essential for downstream apoptotic events, including cytochrome c release from mitochondria and activation of caspases 9 and 3. Our data reveal key elements of the cellular pathway through which an important oxysterol induces apoptosis. Identification of the essential signaling events that may pertain in vivo could suggest targets for therapeutic intervention.     

T-cadherin-mediated cell growth regulation involves G2 phase arrest and requires p21(CIP1/WAF1) expression

Members of the cadherin family have been implicated as growth regulators in multiple tumor types. Based on recent studies from our laboratory implicating T-cadherin expression in mouse brain tumorigenesis, we examined the role of T-cadherin in astrocytoma growth regulation. In this report, we show that T-cadherin expression increased during primary astrocyte physiologic growth arrest in response to contact inhibition and serum starvation in vitro, suggesting a function for T-cadherin in astrocyte growth regulation. We further demonstrate that transient and stable reexpression of T-cadherin in deficient C6 glioma cell lines results in growth suppression. In addition, T-cadherin-expressing C6 cell lines demonstrated increased homophilic cell aggregation, increased cell attachment to fibronectin, and decreased cell motility. Cell cycle flow cytometry demonstrated that T-cadherin reexpression resulted in G2 phase arrest, which was confirmed by mitotic index analysis. This growth arrest was p53 independent, as T-cadherin could still mediate growth suppression in p53(-/-) mouse embryonic fibroblasts. T-cadherin-expressing C6 cell lines exhibited increased p21(CIP1/WAF1), but not p27(Kip1), expression. Lastly, T-cadherin-mediated growth arrest was dependent on p21(CIP1/WAF1) expression and was eliminated in p21(CIP1/WAF1)-deficient fibroblasts. Collectively, these observations suggest a novel mechanism of growth regulation for T-cadherin involving p21(CIP1/WAF1) expression and G2 arrest.     

The p21(WAF1/CIP1) promoter is methylated in Rat-1 cells: stable restoration of p53-dependent p21(WAF1/CIP1) expression after transfection of a genomic clone containing the p21(WAF1/CIP1) gene

Rat-1 cells are used in many studies on transformation, cell cycle, and apoptosis. Whereas UV treatment of Rat-1 cells results in apoptosis, X-ray treatment does not induce either apoptosis or a cell cycle block. X-ray treatment of Rat-1 cells results in both an increase of p53 protein and expression of the p53-inducible gene MDM2 but not the protein or mRNA of the p53-inducible p21(WAF1/CIP1) gene, which in other cells plays an important role in p53-mediated cell cycle block. The lack of p21(WAF1/CIP1) expression appears to be the result of hypermethylation of the p21(WAF1/CIP1) promoter region, as p21(WAF1/CIP1) protein expression could be induced by growth of Rat-1 cells in the presence of 5-aza-2-deoxycytidine. Furthermore, sequence analysis of bisulfite-treated DNA demonstrated extensive methylation of cytosine residues in CpG dinucleotides in a CpG-rich island in the promoter region of the p21(WAF1/CIP1) gene. Stable X-ray-induced p53-dependent p21(WAF1/CIP1) expression and cell cycle block were restored to a Rat-1 clone after transfection with a P1 artificial chromosome (PAC) DNA clone containing a rat genomic copy of the p21(WAF1/CIP1) gene. The absence of expression of the p21(WAF1/CIP1) gene may contribute to the suitability of Rat-1 cells for transformation, cell cycle, and apoptosis studies.     

Mitochondrial ribosomal protein S36 delays cell cycle progression in association with p53 modification and p21(WAF1/CIP1) expression

Ribosomal biogenesis is correlated with cell cycle, cell proliferation, cell growth and tumorigenesis. Some oncogenes and tumor suppressors are involved in regulating the formation of mature ribosome and affecting the ribosomal biogenesis. In previous studies, the mitochondrial ribosomal protein L41 was reported to be involved in cell proliferation regulating through p21(WAF1/CIP1) and p53 pathway. In this report, we have identified a mitochondrial ribosomal protein S36 (mMRPS36), which is localized in the mitochondria, and demonstrated that overexpression of mMRPS36 in cells retards the cell proliferation and delays cell cycle progression. In addition, the mMRPS36 overexpression induces p21(WAF1/CIP1) expression, and regulates the expression and phosphorylation of p53. Our result also indicate that overexpression of mMRPS36 affects the mitochondrial function. These results suggest that mMRPS36 plays an important role in mitochondrial ribosomal biogenesis, which may cause nucleolar stress, thereby leading to cell cycle delay.     

Platelet-derived growth factor (PDGF) receptor-alpha-activated c-Jun NH2-terminal kinase-1 is critical for PDGF-induced p21WAF1/CIP1 promoter activity independent of p53

Platelet-derived growth factor (PDGF) is a potent mitogen for mesenchymal cells. PDGF AA functions as a "competent factor" that stimulates cell cycle entry but requires additional (progression) factors in serum to transit the cell cycle beyond the G1/S checkpoint. Unlike PDGF AA, PDGF B-chain (c-sis) homodimer (PDGF BB) and its viral counterpart v-sis can serve as both competent and progression factors. PDGF BB activates alpha- and beta-receptor subunits (alpha-PDGFR and beta-PDGFR) and induces phenotypic transformation in NIH 3T3 cells, whereas PDGF AA activates alpha-PDGFR only and fails to induce transformation. We showed previously that alpha-PDGFR antagonizes beta-PDGFR-mediated transformation through activation of stress-activated protein kinase-1/c-Jun NH2-terminal kinase-1, whereas both alpha-PDGFR and beta-PDGFR induce mitogenic signals. These studies revealed a striking feature of PDGF signaling; the specificity and the strength of the PDGF growth signal is modulated by alpha-PDGFR-mediated simultaneous activation of growth stimulatory and inhibitory signals, whereas beta-PDGFR mainly induces a growth-promoting signal. Here we demonstrate that PDGF BB activation of beta-PDGFR alone results in more efficient cell cycle transition from G1 to S phase than PDGF BB activation of both alpha-PDGFR and beta-PDGFR. PDGF AA activation of alpha-PDGFR or PDGF BB activation of both alpha- and beta-PDGFRs up-regulates expression of p21WAF1/CIP1, an inhibitor of cell cycle-dependent kinases and a downstream mediator of the tumor suppressor gene product p53. However, beta-PDGFR activation alone fails to induce p21WAF1/CIP1 expression. We also demonstrate that alpha-PDGFR-activated JNK-1 is a critical signaling component for PDGF induction of p21WAF1/CIP1 promoter activity. The ability of PDGF/JNK-1 to induce p21WAF1/CIP1 promoter activity is independent of p53, although the overall p21WAF1/CIP1 promoter activities are greatly reduced in the absence of p53. These results provide a molecular basis for differential regulation of the cell cycle and transformation by alpha- and beta-PDGFRs.     

细胞周期相关蛋白cyclin D1、p53和p21WAF1/Cip1在食管鳞癌中的表达改变及其病理学意义

食管鳞状细胞癌(Esophageal squamous cell carcinoma, ESCC)是我国常见的恶性肿瘤之一, 虽然临床诊治手段正逐步改进, 但中晚期患者5年生存率仍然很低。目前认为细胞周期调控异常与肿瘤发生发展关系密切, 然而相关周期调节蛋白在食管癌患者中的表达改变、临床意义及其应用价值还没有明确结论。文章应用组织微阵列联合免疫组织化学技术(TMA-IHC), 对148例食管鳞癌组织标本中细胞G₁/S期调控蛋白cyclin D1、p53和p21^WAF1/Cip1的表达进行检测, 分析其与临床病理参数之间的相关性。结果显示, cyclin D1与p53蛋白在食管癌细胞中表达升高, p53表达阳性率与区域淋巴结转移显著相关(P = 0.001)。p21^WAF1/Cip1蛋白在肿瘤组织中表达降低, 且p21WAF1/Cip1表达阴性患者的术后生存时间显著短于表达阳性的患者(P = 0.001)。多因素生存分析显示p21WAF1/Cip1是一个独立的预后因素(相对危险度为0.418, P<0.001)。微阵列比较基因组杂交(array-CGH)检测进一步表明45.4%的食管癌患者存在cyclin D1基因扩增。以上结果提示食管鳞癌中存在细胞周期G1/S期调控异常, p21^WAF1/Cip1蛋白可能是一个有应用价值的预后因子。     

Epstein-barr virus latent membrane protein 2B (LMP2B) modulates LMP2A activity

Latent membrane protein 2A (LMP2A) and LMP2B are viral proteins expressed during Epstein-Barr virus (EBV) latency in EBV-infected B cells both in cell culture and in vivo. LMP2A has important roles in modulating B-cell receptor (BCR) signal transduction by associating with the cellular tyrosine kinases Lyn and Syk via specific phosphotyrosine motifs found within the LMP2A N-terminal tail domain. LMP2A has been shown to alter normal BCR signal transduction in B cells by reducing levels of Lyn and by blocking tyrosine phosphorylation and calcium mobilization following BCR cross-linking. Although little is currently known about the function of LMP2B in B cells, the similarity in structure between LMP2A and LMP2B suggests that they may localize to the same cellular compartments. To investigate the function of LMP2B, B-cell lines expressing LMP2A, LMP2B, LMP2A/LMP2B, and the relevant vector controls were analyzed. As was previously shown, cells expressing LMP2A had a dramatic block in normal BCR signal transduction as measured by calcium mobilization and tyrosine phosphorylation. There was no effect on BCR signal transduction in cells expressing LMP2B. Interestingly, when LMP2B was expressed in conjunction with LMP2A, there was a restoration of normal BCR signal transduction upon BCR cross-linking. The expression of LMP2B did not alter the cellular localization of LMP2A but did bind to and prevent the phosphorylation of LMP2A. A restoration of Lyn levels, but not a change in LMP2A levels, was also observed in cells coexpressing LMP2B with LMP2A. From these results, we conclude that LMP2B modulates LMP2A activity.     

p53-, SIRT1-, and PARP-1-independent downregulation of p21WAF1 expression in nicotinamide-treated cells

Nicotinamide at mM concentration is a potent inhibitor of certain key molecules involved in cell survival, such as SIRT1 and PARP-1, and affects cell survival in various conditions in vivo and in vitro. However, the effect of an acute treatment of nicotinamide on gene expression has rarely been closely examined. In our study, the treatment of 10 mM nicotinamide downregulated p21WAF1 expression in various human cells including p53-negative or SIRT1-knockdown cells indicating gene regulation not mediated by p53 or SIRT1. Meanwhile, in the nicotinamide-treated cells, Sp1 activity and protein level was substantially reduced due to increased proteasome-mediated degradation. Our results indicate that nicotinamide treatment attenuates p21WAF1 expression through Sp1 downregulation, and suggest a possible involvement of nicotinamide metabolism in cellular gene expression.     

Reversible phosphorylation at the C-terminal regulatory domain of p21(Waf1/Cip1) modulates proliferating cell nuclear antigen binding

The p53-inducible gene product p21(WAF1/CIP1) plays a critical role in regulating the rate of tumor incidence, and identifying mechanisms of its post-translational regulation will define key pathways that link growth control to p21-dependent tumor suppression. A eukaryotic cell model system has been developed to determine whether protein kinase signaling pathways that phosphorylate human p21 exist in vivo and whether such pathways regulate the binding of p21 to one of its key target proteins, proliferating cell nuclear antigen (PCNA). Although human p21 expressed in Sf9 cells is able to form a complex with human PCNA, the inclusion of cell-permeable phosphatase inhibitors renders p21 protein inactive for PCNA binding. The treatment of this inactive isoform of p21 with alkaline phosphatase restores its binding to PCNA, suggesting that p21 expressed in Sf9 cells is subject to reversible phosphorylation at a key regulatory site(s). A biochemical approach was subsequently used to map the phosphorylation sites within p21, whose modification in vitro can inhibit p21-PCNA complex formation, to the C-terminal domain at residues Thr(145) or Ser(146). A phospho-specific antibody was developed that only bound to full-length p21 protein after phosphorylation in vitro at Ser(146), and this reagent was further used to demonstrate that the inactive isoform of p21 recovered from Sf9 cells treated with phosphatase inhibitors had been phosphorylated in vivo at Ser(146). These data identify the first phosphorylation site within the C-terminal regulatory domain of p21 whose modification in vivo modulates p21-PCNA interactions and define a eukaryotic cell model that can be used to study post-translational signaling pathways that regulate p21.     

p21(CIP1) Controls proliferating cell nuclear antigen level in adult cardiomyocytes

Cell cycle withdrawal associated with terminal differentiation is responsible for the incapability of many organs to regenerate after injury. Here, we employed a cell-free system to analyze the molecular mechanisms underlying cell cycle arrest in cardiomyocytes. In this assay, incubation of S phase nuclei mixed with cytoplasmic extract of S phase cells and adult primary cardiomyocytes results in a dramatic reduction of proliferating cell nuclear antigen (PCNA) protein levels. This effect was blocked by the proteasome inhibitors MG132 and lactacystin, whereas actinomycin D and cycloheximide had no effect. Immunodepletion and addback experiments revealed that the effect of cardiomyocyte extract on PCNA protein levels is maintained by p21 but not p27. In serum-stimulated cardiomyocytes PCNA expression was reconstituted, whereas the protein level of p21 but not that of p27 was reduced. Cytoplasmic extract of serum-stimulated cardiomyocytes did not influence the PCNA protein level in S phase nuclei. Moreover, the hypertrophic effect of serum stimulation was blocked by ectopic expression of p21 and the PCNA protein level was found to be upregulated in adult cardiomyocytes derived from p21 knockout mice. Our data provide evidence that p21 regulates the PCNA protein level in adult cardiomyocytes, which has implications for cardiomyocyte growth control.     

Modulation of antioxidant enzymes p21WAF1 and p53 expression during proliferation and differentiation of human melanoma cell lines

The activities of antioxidant enzymes, and the expression of p21(WAF1) and p53 proteins were studied at different times after subculture during proliferation and differentiation phases. Two human melanoma cell lines were used: IPC182, which is a non-differentiating cell line, and IGR221, which spontaneously differentiates at the end of the exponential growth phase, as evidenced by a marked increase of melanin content and tyrosinase activity. In the two cell lines, the slowing of proliferation coincided with an increase in the activity and amount of immunoreactive superoxide dismutases (SOD1 and SOD2), and a decrease of catalase and glutathione peroxidase activities, and of the glutathione content. The levels of p21WAF1 and p53 proteins were found to be lower in confluent than in proliferative cells. Several parameters were modified only during the differentiation phase of IGR221 cells; in these cells the increase of tyrosinase activity was highly correlated with the increase in SOD2, GST, glutathione reductase, and G6PD activities. The level of glutathione was found to be lower in differentiated IGR221 than in non-differentiated IPC182 cells. These results suggest that p21WAF1 and p53 proteins are not involved in the spontaneous differentiation process of melanoma cells, and that abnormal regulation of the cell cycle inhibition pathway occurred in these cells. The results sustain the hypothesis that alterations of antioxidant enzyme expression are involved in the control of proliferation and differentiation of melanoma cells. Alterations of SOD2 activity may be of particular importance, since variations are observed with both cell growth and cell differentiation.     

Air pollution by carcinogenic PAHs and plasma levels of p53 and p21(WAF1) proteins

We analyzed the effect of exposure to carcinogenic polycyclic aromatic hydrocarbons (c-PAHs) in ambient air on the plasma levels of p53 and p21^WAF1 proteins among city policemen, bus drivers and controls in three European cities: Prague (Czech Republic), Kosice (Slovakia) and Sofia (Bulgaria). p53 and p21^WAF1 proteins are key regulators of the cell cycle and are accepted as universal markers of genotoxic stress and DNA damage. In total 204 exposed subjects (100 smokers, 104 nonsmokers) and 152 controls (54 smokers, 98 nonsmokers) were analyzed. Personal exposure to c-PAHs was evaluated using personal samplers during the working shift. The levels of p53 and p21^WAF1 proteins were assessed by ELISA assay. There were no differences between the levels of either protein between exposed and controls, or smokers and nonsmokers, in any city. However, we observed significant differences in p53 plasma levels in all subjects regardless of the exposure status between the individual cities (median values: 5, 31, 234 pg/ml, p < 0.001, for Prague, Kosice and Sofia, respectively). The levels correspond to the differences in exposure levels to c-PAHs and benzo[a]pyrene (B[a]P) in the individual cities. A multiple linear regression analysis confirmed that c-PAHs exposure is a variable significantly affecting levels of both proteins in all locations. When all subjects were divided into the group exposed to below-median levels of c-PAHs and the group exposed to above-median levels of c-PAHs we found significantly higher p53, as well as p21^WAF1 levels in the above-median exposure group (p53, 167 pg/ml versus 25 pg/ml, p < 0.001; p21^WAF1, 2690 pg/ml versus 2600 pg/ml, p < 0.05). Among all subjects p53 plasma levels were positively correlated with p21^WAF1 levels, exposure to B[a]P, c-PAHs and levels of total DNA adducts; for p21^WAF1 levels we observed the positive correlation with cotinine, c-PAHs exposure, total and B[a]P-like DNA adduct levels. In conclusion our results suggest that p53 and p21^WAF1 proteins plasma levels may be useful biomarkers of c-PAHs environmental exposure.     

MDM2 is a negative regulator of p21WAF1/CIP1, independent of p53

The MDM2 oncogene has both p53-dependent and p53-independent activities. We have previously reported that antisense MDM2 inhibitors have significant anti-tumor activity in multiple human cancer models with various p53 statuses (Zhang, Z., Li, M., Wang, H., Agrawal, S., and Zhang, R. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 11636-11641). We have also provided evidence that MDM2 has a direct role in the regulation of p21, a cyclin-dependent kinase inhibitor. Here we provide evidence supporting functional interaction between MDM2 and p21 in vitro and in vivo. The inhibition of MDM2 with anti-MDM2 antisense oligonucleotide or Short Interference RNA targeting MDM2 significantly elevated p21 protein levels in PC3 cells (p53 null). In contrast, overexpression of MDM2 diminished the p21 level in the same cells by shortening the p21 half-life, an effect reversed by MDM2 antisense inhibition. MDM2 facilitates p21 degradation independent of ubiquitination and the E3 ligase function of MDM2. Instead, MDM2 promotes p21 degradation by facilitating binding of p21 with the proteasomal C8 subunit. The physical interaction between p21 and MDM2 was demonstrated both in vitro and in vivo with the binding region in amino acids 180-298 of the MDM2 protein. In summary, we provide evidence supporting a physical interaction between MDM2 and p21. We also demonstrate that, by reducing p21 protein stability via proteasome-mediated degradation, MDM2 functions as a negative regulator of p21, an effect independent of both p53 and ubiquitination.     

MPP+ inhibits proliferation of PC12 cells by a p21(WAF1/Cip1)-dependent pathway and induces cell death in cells lacking p21(WAF1/Cip1).

The molecular and biochemical mode of cell death of dopaminergic neurons in Parkinson's disease (PD) is uncertain. In an attempt at further clarification we studied the effects of 1-methyl-4-phenylpyridinium (MPP+), the active metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), on dopaminergic PC12 cells. In humans and nonhuman primates MPTP/MPP+ causes a syndrome closely resembling PD. MPP+ toxicity is thought to be mediated by the block of complex I of the mitochondrial electron transport chain. Treatment of undifferentiated PC12 cells with MPP+ primarily inhibited proliferation of PC12 cells and secondarily led to cell death after the depletion of all energy substrates by glycolysis. This cell death showed no morphological characteristics of apoptosis and was not blocked by treatment with caspase inhibitors. The inhibition of cell growth was not dependent on an inhibition of complex I activity since MPP+ also inhibited cell proliferation in SH-SY5Y cells lacking mitochondrial DNA and complex I activity (p0 cells). As shown by flow cytometric analysis, MPP+ induced a block in the G0/G1 to S phase transition that correlated with increased expression of the cyclin-dependent kinase inhibitor p21(WAF1/Cip1) and growth arrest. Since treatment with 1 microM MPP+ caused apoptotic cell death in p21(WAF1/Cip1)-deficient (p21(-/-)) but not in parental (p21(+/+)) mouse embryo fibroblasts, our data suggest that in an early phase MPP+-induced p21(WAF1/Cip1) expression leads to growth arrest and prevents apoptosis until energy depletion finally leads to a nonapoptotic cell death.