Nox2 regulates endothelial cell cycle arrest and apoptosis via p21cip1 and p53

Endothelial cells (EC) express constitutively two major isoforms (Nox2 and Nox4) of the catalytic subunit of NADPH oxidase, which is a major source of endothelial reactive oxygen species. However, the individual roles of these Noxes in endothelial function remain unclear. We have investigated the role of Nox2 in nutrient deprivation-induced cell cycle arrest and apoptosis. In proliferating human dermal microvascular EC, Nox2 mRNA expression was low relative to Nox4 (Nox2:Nox4 approximately 1:13), but was upregulated 24 h after starvation and increased to 8+/-3.5-fold at 36 h of starvation. Accompanying the upregulation of Nox2, there was a 2.28+/-0.18-fold increase in O2.- production, a dramatic induction of p21cip1 and p53, cell cycle arrest, and the onset of apoptosis (all p<0.05). All these changes were inhibited significantly by in vitro deletion of Nox2 expression and in coronary microvascular EC isolated from Nox2 knockout mice. In Nox2 knockout cells, although there was a 3.8+/-0.5-fold increase in Nox4 mRNA expression after 36 h of starvation (p<0.01), neither O2.- production nor the p21cip1 or p53 expression was increased significantly and only 0.46% of cells were apoptotic. In conclusion, Nox2-derived O2.-, through the modulation of p21cip1 and p53 expression, participates in endothelial cell cycle regulation and apoptosis.     

p21cip1 is required for the differentiation of oligodendrocytes independently of cell cycle withdrawal

Differentiation of most cell types requires both establishment of G₁ arrest and the induction of a program related to achieving quiescence. We have chosen to study the differentiation of oligodendrocyte cells to determine the role of p27 and p21 in this process. Here we report that both p27 and p21 are required for the appropriate differentiation of these cells. p27 is required for proper withdrawal from the cell cycle, p21 is not. Instead, p21 is required for the establishment of the differentiation program following growth arrest. Similar observations were made in vivo. We show that p21^–/– cells withdraw from the cell cycle similar to wild-type cells; however, early in animal life, the brain is hypomyelinated, inferring that the loss of p21 delayed myelination in the cerebellum. We found that we could complement or bypass the differentiation failure in p21^–/– cells with either PD98059, an inhibitor of Mek1, or by transducing them with a tat–p16^Ink4a protein. We concluded that the two cdk inhibitors serve non-redundant roles in this program of differentiation, with p27 being responsible for arrest and p21 having a function in differentiation independent of its ability to control exit from the cell cycle.     

Both p53-PUMA/NOXA-Bax-mitochondrion and p53-p21cip1 pathways are involved in the CDglyTK-mediated tumor cell suppression

CDglyTK fusion suicide gene has been well characterized to effectively kill tumor cells. However, the exact mechanism and downstream target genes are not fully understood. In our study, we found that CDglyTK/prodrug treatment works more efficiently in p53 wild-type (HONE1) cells than in p53 mutant (CNE1) cells. We then used adenovirus-mediated gene delivery system to either knockdown or overexpress p53 and its target genes in these cells. Consistent results showed that both p53-PUMA/NOXA/Bcl2-Bax and p53-p21 pathways contribute to the CDglyTK induced tumor cell suppression. Our work for the first time addressed the role of p53 related genes in the CDglyTK/prodrug system.     

The mammalian mid-pachytene checkpoint: meiotic arrest in spermatocytes with a mutation in Atm alone or in combination with a Trp53 (p53) or Cdkn1a (p21/cip1) mutation

ATM, the protein product of the gene mutated in the human autosomal recessive disorder ataxia telangiectasia, is involved in detection of double strand breaks (DSBs) and is a key component of the damage surveillance network of cell cycle proteins. In somatic cells ATM phosphorylates many other proteins including p53, an important regulator of cell cycle control. Mice deficient for Atm are male sterile with arrest and apoptosis occurring at testis epithelial stage IV, which in normal spermatocytes corresponds to mid-pachynema. Unlike the situation in somatic cells, we find no evidence that disruption of the Trp53 (p53) gene, or its down-stream target Cdkn1a (p21/Cip1) results in even a partial rescue of the Atm defect.     

BMP-7-induced cell cycle arrest of anaplastic thyroid carcinoma cells via p21(CIP1) and p27(KIP1).

The aim of the present study was to investigate the effect of bone morphogenetic protein (BMP-7) on thyroid carcinoma cell growth. Addition of BMP-7 inhibited the proliferation of four out of six human anaplastic thyroid carcinoma cell lines, observed as decreased incorporation of (3)H-thymidine and decreased cell number. The growth inhibitory effect was cell density-dependent; sparse cells were inhibited by BMP-7 whereas dense cells were not. Cell cycle analysis by flow cytometry showed an increased fraction of cells in the G1-phase and subsequent decrease in both S- and G2/M-phase after BMP-7 stimulation. Furthermore, BMP-7 caused an upregulation of the cyclin-dependent kinase inhibitors (CDKIs) p21 and p27, decreased activity of Cdk2 and Cdk6, and hypophosphorylation of the retinoblastoma protein (pRb). These findings suggest a growth inhibitory effect of BMP-7 on anaplastic thyroid carcinoma cells by inhibition of Cdk activity shifting the Rb protein to the hypophosphorylated state.     

Caveolin-1 expression negatively regulates cell cycle progression by inducing G(0)/G(1) arrest via a p53/p21(WAF1/Cip1)-dependent mechanism

Caveolin-1 is a principal component of caveolae membranes in vivo. Caveolin-1 mRNA and protein expression are lost or reduced during cell transformation by activated oncogenes. Interestingly, the human caveolin-1 gene is localized to a suspected tumor suppressor locus (7q31.1). However, it remains unknown whether caveolin-1 plays any role in regulating cell cycle progression. Here, we directly demonstrate that caveolin-1 expression arrests cells in the G(0)/G(1) phase of the cell cycle. We show that serum starvation induces up-regulation of endogenous caveolin-1 and arrests cells in the G(0)/G(1) phase of the cell cycle. Moreover, targeted down-regulation of caveolin-1 induces cells to exit the G(0)/G(1) phase. Next, we constructed a green fluorescent protein-tagged caveolin-1 (Cav-1-GFP) to examine the effect of caveolin-1 expression on cell cycle regulation. We directly demonstrate that recombinant expression of Cav-1-GFP induces arrest in the G(0)/G(1) phase of the cell cycle. To examine whether caveolin-1 expression is important for modulating cell cycle progression in vivo, we expressed wild-type caveolin-1 as a transgene in mice. Analysis of primary cultures of mouse embryonic fibroblasts from caveolin-1 transgenic mice reveals that caveolin-1 induces 1) cells to exit the S phase of the cell cycle with a concomitant increase in the G(0)/G(1) population, 2) a reduction in cellular proliferation, and 3) a reduction in the DNA replication rate. Finally, we demonstrate that caveolin-1-mediated cell cycle arrest occurs through a p53/p21-dependent pathway. Taken together, our results provide the first evidence that caveolin-1 expression plays a critical role in the modulation of cell cycle progression in vivo.     

p14ARF induces G2 cell cycle arrest in p53- and p21-deficient cells by down-regulating p34cdc2 kinase activity

The human INK4a gene locus encodes two structurally unrelated tumor suppressor proteins, p16(INK4a) and p14(ARF). Although primarily proposed to require a functional p53.Mdm-2 signaling axis, recently p14(ARF) has been implicated in p53-independent cell cycle regulation. Here we show that p14(ARF) preferentially induces a G(2) arrest in tumor cells lacking functional p53 and/or p21. Expression of p14(ARF) impaired mitotic entry and enforced a primarily cytoplasmic localization of p34(cdc2) that was associated with a decrease in p34(cdc2) kinase activity and reduced p34(cdc2) protein expression. A direct physical interaction between p14(ARF) and p34(cdc2) was, nevertheless, ruled out by lack of co-immunoprecipitation. The p14(ARF)-induced depletion of p34(cdc2) was associated with impaired cdc25C phosphatase expression and a prominent shift to inhibitory Tyr-15-phosphorylation in G(2)-arrested cells lacking either p53, p21, or both. Finally, reconstitution of p34(cdc2) using a constitutively active, phosphorylation-deficient p34(cdc2AF) mutant alleviated this p14(ARF)-induced G(2) arrest, thereby allowing cell cycle progression. Taken together, these data indicate that p14(ARF) arrests cells lacking functional p53/p21 in the G(2) phase of the cell cycle by targeting p34(cdc2) kinase. This may represent an important fail-safe mechanism by which p14(ARF) protects p53/p21-deficient cells from unrestrained proliferation.     

The ability of p53 to activate downstream genes p21(WAF1/cip1) and MDM2, and cell cycle arrest following DNA damage is delayed and attenuated in scid cells deficient in the DNA-dependent protein kinase

scid mouse embryonic fibroblasts are deficient in DNA-dependent protein kinase activity due to a mutation in the C-terminal domain of the catalytic subunit (DNA-PKcs). When exposed to ionizing radiation, the increase in levels of p53 was the same as in normal mouse embryonic fibroblasts. However, the rise in p21(WAF1/cip1) and mdm2 was found to be delayed and attenuated, which correlated in time with delayed onset of G1/S arrest by flow cytometric analysis. The p53-dependent G1 checkpoint was not eliminated: inactivation of p53 by the E6 protein in scid cells resulted in the complete loss of detectable G1/S arrest after DNA damage. Immunofluorescence analysis of normal cells revealed p53 to be localized predominantly within the cytoplasm prior to irradiation and then translocate to the nucleus after irradiation. In contrast, scid cells show abnormal accumulation of p53 in the nucleus independent of irradiation, which was confirmed by immunoblot analysis of nuclear lysates. Taken together, these data suggest that loss of DNA-PK activity appears to attenuate the kinetics of p53 to activate downstream genes, implying that DNA-PK plays a role in post-translational modification of p53, without affecting the increase in levels of p53 in response to DNA damage.     

EDD induces cell cycle arrest by increasing p53 levels

Tight regulation of p53 is essential for its central role in maintaining genome stability and tumor prevention. Here, EDD/ UBR5/hHyd, hereafter called EDD, is identified as a novel regulator of p53. Downregulation of EDD results in elevated p53 protein levels both in transformed and untransformed cells. Concomitant with a rise in p53, the levels of p21, a critical p53 target, are also elevated in these conditions. Surprisingly, EDD knockdown does not affect p53 protein stability, and p53 mRNA levels do not increase significantly upon EDD depletion. Consistent with the function of p53, EDD downregulation triggers a senescent phenotype in fibroblasts at later time points. In addition, the increased p53 levels upon EDD depletion cause a G₁ arrest, as co-depletion of EDD and p53 completely rescues this effect on cell cycle progression.     

p21(Cip1) and p27(Kip1) regulate cell cycle reentry after hypoxic stress but are not necessary for hypoxia-induced arrest

We investigated the role of the cyclin-dependent kinase inhibitors p21(Cip1) and p27(Kip1) in cell cycle regulation during hypoxia and reoxygenation. While moderate hypoxia (1 or 0.1% oxygen) does not significantly impair bromodeoxyuridine incorporation, at very low oxygen tensions (0.01% oxygen) DNA replication is rapidly shut down in immortalized mouse embryo fibroblasts. This S-phase arrest is intact in fibroblasts lacking the cyclin kinase inhibitors p21(Cip1) and p27(Kip1), indicating that these molecules are not essential elements of the arrest pathway. Hypoxia-induced arrest is accompanied by dephosphorylation of pRb and inhibition of cyclin-dependent kinase 2, which results in part from inhibitory phosphorylation. Interestingly, cells lacking the retinoblastoma tumor suppressor protein also display arrest under hypoxia, suggesting that pRb is not an essential mediator of this response. Upon reoxygenation, DNA synthesis resumes by 3.5 h and reaches aerobic levels by 6 h. Cells lacking p21, however, resume DNA synthesis more rapidly upon reoxygenation than wild-type cells, suggesting that this inhibitor may play a role in preventing premature reentry into the cell cycle upon cessation of the hypoxic stress. While p27 null cells did not exhibit rapid reentry into the cell cycle, cells lacking both p21 and p27 entered S phase even more aggressively than those lacking p21 alone, revealing a possible secondary role for p27 in this response. Cdk2 activity is also restored more rapidly in the double-knockout cells when returned to normoxia. These studies reveal that restoration of DNA synthesis after hypoxic stress, but not the S phase arrest itself, is regulated by p21 and p27.     

Human parvovirus B19 nonstructural protein (NS1) induces cell cycle arrest at G(1) phase

Human parvovirus B19 infects predominantly erythroid precursor cells, leading to inhibition of erythropoiesis. This erythroid cell damage is mediated by the viral nonstructural protein 1 (NS1) through an apoptotic mechanism. We previously demonstrated that B19 virus infection induces G(2) arrest in erythroid UT7/Epo-S1 cells; however, the role of NS1 in regulating cell cycle arrest is unknown. In this report, by using paclitaxel, a mitotic inhibitor, we show that B19 virus infection induces not only G(2) arrest but also G(1) arrest. Interestingly, UV-irradiated B19 virus, which has inactivated the expression of NS1, still harbors the ability to induce G(2) arrest but not G(1) arrest. Furthermore, treatment with caffeine, a G(2) checkpoint inhibitor, abrogated the B19 virus-induced G(2) arrest despite expression of NS1. These results suggest that the B19 virus-induced G(2) arrest is not mediated by NS1 expression. We also found that NS1-transfected UT7/Epo-S1 and 293T cells induced cell cycle arrest at the G(1) phase. These results indicate that NS1 expression plays a critical role in G(1) arrest induced by B19 virus. Furthermore, NS1 expression significantly increased p21/WAF1 expression, a cyclin-dependent kinase inhibitor that induces G(1) arrest. Thus, G(1) arrest mediated by NS1 may be a prerequisite for the apoptotic damage of erythroid progenitor cells upon B19 virus infection.     

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。     

High-intensity Raf signal causes cell cycle arrest mediated by p21Cip1.

Activated Raf has been linked to such opposing cellular responses as the induction of DNA synthesis and the inhibition of proliferation. However, it remains unclear how such a switch in signal specificity is regulated. We have addressed this question with a regulatable Raf-androgen receptor fusion protein in murine fibroblasts. We show that Raf can cause a G1-specific cell cycle arrest through induction of p21Cip1. This in turn leads to inhibition of cyclin D- and cyclin E-dependent kinases and an accumulation of hypophosphorylated Rb. Importantly, this behavior can be observed only in response to a strong Raf signal. In contrast, moderate Raf activity induces DNA synthesis and is sufficient to induce cyclin D expression. Therefore, Raf signal specificity can be determined by modulation of signal strength presumably through the induction of distinct protein expression patterns. Similar to induction of Raf, a strong induction of activated Ras via a tetracycline-dependent promoter also causes inhibition of proliferation and p21Cip1 induction at high expression levels. Thus, p21Cip1 plays a key role in determining cellular responses to Ras and Raf signalling. As predicted by this finding we show that Ras and loss of p21 cooperate to confer a proliferative advantage to mouse embryo fibroblasts.