Control of apoptosis in influenza virus-infected cells by up-regulation of Akt and p53 signaling

PI3k-Akt and p53 pathways are known to play anti- and pro-apoptotic roles in cell death, respectively. Whether these pathways are recruited in influenza virus infection in highly productive monkey (CV-1) and canine (MDCK) kidney cells was studied here. Phosphorylation of Akt (Akt-pho) was found to occur only early after infection (5–9 h.p.i). Nuclear accumulation and phosphorylation of p53 (p53-pho), and expression of its natural target p21/waf showed low constitutive levels at this period, whereas all three parameters were markedly elevated at the late apoptotic stage (17–20 h.p.i.). Up-regulation of Akt-pho and p53-pho was not induced by UV-inactivated virus suggesting that it required virus replication. Also, mRNAs of p53 and its natural antagonist mdm2 were not increased throughout infection indicating that p53-pho was up-regulated by posttranslational mechanisms. However, p53 activation did not seem to play a leading role in influenza-induced cell death: (i) infection of CV1 and MDCK cells with recombinant NS1-deficient virus provoked accelerated apoptotic death characterized by the lack of p53 activation; (ii) mixed apoptosis-necrosis death developed in influenza-infected human bronchial H1299 cells carrying a tetracycline-regulated p53 gene did not depend on p53 gene activation by tetracycline. Virus-induced apoptosis and signaling of Akt and p53 developed in IFN-deficient VERO cells with similar kinetics as in IFN-competent CV1-infected cells indicating that these processes were endocrine IFN-independent. Apoptosis in influenza-infected CV-1 and MDCK cells was Akt-dependent and was accelerated by Ly294002, a specific inhibitor of PI3k-Akt signaling, and down-regulated by the viral protein NS1, an inducer of host Akt. The obtained data suggest that influenza virus (i) initiates anti-apoptotic PI3k-Akt signaling at early and middle phases of infection to protect cells from fast apoptotic death and (ii) provokes both p53-dependent and alternative p53-independent apoptotic and/or necrotic (in some host systems) cell death at the late stage of infection. These data have been partially presented at The 3rd Orthomyxovirus Research Conference (sponsored by ESWI and NIH). Abstr. p. 23 entitled: “Influenza virus-specific up-regulation of Akt and Mdm2 in infected cells” by Zhirnov O.P., and Klenk H.D., July 28–21, 2005. Queen’s College, Cambridge, United Kingdom; and at The Annual Meeting of Virology in Munich, March 15–18 (2006)—“Influenza virus-specific up-regulation of Akt, Mdm2, and p53 in infected cells” by O. P. Zhirnov and H. D. Klenk; Book of abstracts, p. 339     

Different cell cycle mechanisms between UV-induced and X-ray-induced apoptosis in WiDr colorectal carcinoma cells

Although DNA-damaging agents such as ultraviolet (UV) and X-ray can induce apoptosis, the difference in the apoptotic mechanism is not clearly understood. In the present study, we investigated the effects of these two genotoxic agents on the induction of DNA damage and subsequent apoptotic cell death from the viewpoint of cell cycle regulation by using WiDr cells. Transient G1 arrest was observed after UV exposure, whereas G2 but not G1 arrest was induced after X-ray irradiation. UV-exposure could induce G1 arrest in both mutant-type (mt-p53) and wild-type p53 (wt-p53) cells, but obvious G1 arrest was not observed in the cells lacking in p53 expression. An increase in the DNA fragmentation was observed at S phase in UV-irradiated cells and at G2 phase in X-irradiated cells, respectively. UV-irradiated cells showed an increase production of p53 protein and accumulation of p21 protein. On the contrary, both p53 and p21 proteins remained at a low level in X-irradiated cells. Treatment with aphidicolin, an S phase blocking agent, prolonged cell cycle arrest and reduced the rate of apoptotic cell death in both UV-irradiated and X-irradiated cells. From these results, it is suggested that UV-induced apoptosis occurs mainly at S phase and is regulated by increased production of p53 and p21 proteins, while X-ray-induced apoptosis occurs after G2 blockade and may be independent of p53.     

Apoptosis induced by adenovirus-mediated p14ARF expression in U2OS osteosarcoma cells is associated with increased Fas expression

The INK4A/ARF locus on chromosome 9 is a tumor suppressor gene frequently mutated in human cancers. In order to study the effects of p14ARF expression in tumor cells, we constructed a recombinant adenovirus containing p14ARF cDNA (Adp14ARF). Adp14ARF infection of U2OS osteosarcoma cells which has wild type p53 and mutant p14ARF revealed high levels of p14 (ARF) expression within 24h. In addition, Adp14ARF-mediated expressing of p14 (ARF) was associated with increased levels of p53, p21, and mdm2 protein. Growth inhibition assays following Adp14ARF infection demonstrated that the growth of U2OS cells was inhibited relative to infection with control virus. Furthermore, TUNEL analysis as well as PARP cleavage assays demonstrated that Adp14ARF infection was associated with increased apoptosis in U2OS cell line and that it was associated with Adp14ARF induced overexpression of Fas and Fas-L. Addition of Fas-L neutralizing antibody NOK-1 decreased Adp14-mediated cell death, indicating that p14 (ARF) induction of the Fas pathway is associated with increased apoptosis. The finding that Adp14ARF infection did not induce Fas expression in U2OS/E6 and MCF/E6 cells suggests that wild type p53 expression may be necessary for Adp14ARF-mediated induction of Fas. The observation that overexpression of p53 by Adp53 infection in MCF-7 does not induce increased Fas protein levels nor apoptotic cell death suggests that p53 overexpression is required but not sufficient enough for apoptosis. These studies suggest there are other mechanisms other than induction of p53 in ARF-mediated apoptosis and gene therapy using Adp14ARF may be a promising treatment option for human cancers containing wild type p53 and mutant or deleted p14 expression.     

Apoptosis induced by doxorubicin in neurotumor cells is divorced from drug effects on ceramide accumulation and may involve cell cycle-dependent caspase activation

Doxorubicin (0.5 microgram/ml) induced caspase-dependent apoptosis in SH-SY5Y neuroblastoma and CHP-100 neuroepithelioma cells. The apoptotic response started to be evident approximately 15 h after drug administration and, as monitored over a 48-h period, was more pronounced in CHP-100 than in SH-SY5Y cells. In both systems, apoptosis was accompanied by elevation of intracellular ceramide levels. Ceramide accumulation was blocked by the ceramide synthase inhibitor fumonisin B(1) (25 microM); this compound, however, did not prevent drug-induced apoptosis. Untreated cells from both lines expressed negligible p53 levels; on the other hand, whereas p53 and p21(Cip1/Waf1) were rapidly up-regulated in doxorubicin-treated SH-SY5Y cells, such a response was not observed in CHP-100 cells. Doxorubicin induced a G(2)/M phase block in both cell lines, but whereas the G(1) phase was markedly depleted in CHP-100 cells, it was substantially retained in SH-SY5Y cells. In the latter system, double G(1) and G(2)/M block largely preceded cell death; however, as apoptosis underwent completion, it selectively targeted late S and G(2)/M cells. Moreover, apoptosis suppression by caspase inhibition did not result in a recovery of the G(1) cell population. These results support the notion that doxorubicin-induced apoptosis and ceramide elevation are divorced events in neuroectodermal tumors and that p53 function is at least dispensable for apoptosis completion. Indeed, as G(1) cells appear to be refractory to doxorubicin-induced apoptosis, p53 up-regulation and p21(Cip1/Waf1) expression may provide an unfavorable setting for the apoptotic action of the drug.     

Upregulation of BNIP3 promotes apoptosis of lung cancer cells that were induced by p53

It has been shown that p53 induces cell apoptosis and the Bcl-2 family plays key roles in this process. However, the molecular mechanism of p53 apoptotic pathway is still unclear. Here, we show that overexpression of exogenous wild-type p53 induced apoptosis in lung cancer cells and high metastasis potential cells had a faster rate of apoptosis than low metastasis potential cells. The expression of pro-apoptotic gene BNIP3 was increased significantly both in Anip973 and 95D cell lines which have high metastasis ability, but not AGZY83-a or little increased in 95C cell lines which possess low metastasis ability. Overexpression of BNIP3 increases apoptotic rate induced by p53 in AGZY83-a cells. Blocking the expression of BNIP3 by siRNA in Anip973 cells decreased apoptotic rate mediated by p53. Taken together, these data suggest that high level expression of BNIP3 mediated rapid apoptosis that was triggered by p53 in lung cancer cells.     

米非司酮对恒河猴母胎界面细胞凋亡和凋亡分子p53表达的影响

胎盘形成过程中发生活跃的细胞增殖、迁移和凋亡等活动。p53蛋白是参与调节细胞周期和凋亡过程的原癌基因。本实验用原位末端标记、蛋白印迹和免疫组织化学方法研究正常和米非司酮(RU486)处理后恒河猴母胎界面绒毛和蜕膜组织细胞凋亡及p53蛋白表达。在正常妊娠的恒河猴母胎界面,凋亡信号主要集中在合体滋养层和细胞柱内的一些滋养层细胞;p53蛋白主要定位于细胞滋养层。在母体蜕膜中,也在部分基质细胞中检测到细胞凋亡和p53蛋白表达。经过RU486处理2d后,胎盘绒毛和母体蜕膜中凋亡细胞数都显著增加,绒毛中增加的凋亡信号集中于细胞滋养层。同时,RU486处理也导致绒毛细胞滋养层和蜕膜基质细胞中p53表达明显增加。以上结果提示,在正常妊娠中,生理性的细胞凋亡和p53表达可能是控制细胞滋养层细胞增殖、保持胎盘组织动态平衡的一个重要机制;RU486终止早孕的可能途径之一是促进母胎界面细胞凋亡,推测p53参与这一过程。     

Mechanism of diepoxybutane‐induced p53 regulation in human cells

Diepoxybutane (DEB) is the most potent active metabolite of the environmental chemical 1,3‐butadiene (BD). BD is a known mutagen and human carcinogen and possesses multisystems organ toxicity. We previously reported the elevation of p53 in human TK6 lymphoblasts undergoing DEB‐induced apoptosis. In this study, we have characterized the DEB‐induced p53 accumulation and investigated the mechanisms by which DEB regulates this p53 accumulation. The elevation of p53 levels in DEB‐exposed TK6 lymphoblasts and human embryonic lung (HEL) human fibroblasts was found to be largely due to the stabilization of the p53 protein. DEB increased the acetylation of p53 at lys‐382, dramatically reduced complex formation between p53 and its regulator protein mdm2 and induced the phosphorylation of p53 at serines 15, 20, 37, 46, and 392 in human lymphoblasts. A dramatic increase in phosphorylation of p53 at serine 15 in correlation to total p53 levels was observed in DEB‐exposed Ataxia Telangiectasia Mutated (ATM) proficient human lymphoblasts as compared to DEB‐exposed ATM‐deficient human lymphoblasts; this implicates the ATM kinase in the elevation of p53 levels in DEB‐exposed cells. Collectively, these findings explain for the first time the mechanism by which p53 accumulates in DEB‐exposed cells and contributes to the understanding of the molecular toxicity of DEB and BD. © 2009 Wiley Periodicals, Inc. J Biochem Mol Toxicol 23:373–386, 2009; Published online in Wiley InterScience ( www.interscience.wiley.com ). DOI 10.1002/jbt.20300     

Characterization of the cell death induced by cadmium in HaCaT and C6 cell lines

Cell death resulting from cadmium (Cd) intoxication has been confirmed to induce both necrosis and apoptosis. The ratio between both types of cell death is dose- and cell-type-dependent. This study used the human keratinocytes HaCaT expressing a mutated p53 and the rat glial cells C6 expressing a wild p53 as models to characterize Cd-induced apoptosis, using sub-lethal and lethal doses. At these concentrations, features of apoptosis were observed 24 h after C6 cell treatment: apoptotic DNA fragmentation and caspase-9 activation, whereas Cd did not induce caspase-3. In HaCaT, Cd did not induce apoptotic DNA fragmentation or caspase-9 and -3 activation. The results also showed that the inhibition of p53 led to a resistance of the C6 cells to 20 µm Cd, decreased the apoptosis and increased the metallothioneins in these cells. p53 restoration increased the sensitivity of HaCaT cells to Cd but did not affect the MT expression. The results suggest that Cd induced apoptosis in C6 cells but a non-apoptotic cellular death in HaCaT cells.     

Characterization of the cell death induced by cadmium in HaCaT and C6 cell lines

Cell death resulting from cadmium (Cd) intoxication has been confirmed to induce both necrosis and apoptosis. The ratio between both types of cell death is dose- and cell-type-dependent. This study used the human keratinocytes HaCaT expressing a mutated p53 and the rat glial cells C6 expressing a wild p53 as models to characterize Cd-induced apoptosis, using sub-lethal and lethal doses. At these concentrations, features of apoptosis were observed 24 h after C6 cell treatment: apoptotic DNA fragmentation and caspase-9 activation, whereas Cd did not induce caspase-3. In HaCaT, Cd did not induce apoptotic DNA fragmentation or caspase-9 and -3 activation. The results also showed that the inhibition of p53 led to a resistance of the C6 cells to 20 µm Cd, decreased the apoptosis and increased the metallothioneins in these cells. p53 restoration increased the sensitivity of HaCaT cells to Cd but did not affect the MT expression. The results suggest that Cd induced apoptosis in C6 cells but a non-apoptotic cellular death in HaCaT cells.     

p53对TGEV感染PK-15细胞4种免疫调节因子mRNA转录水平的影响

为探究p53对IFN-α、MIP-1α、PGK-1、TGF-β1四种免疫调节因子在TGEV感染PK-15细胞中的影响,本研究首先采用CRISPR-Cas9慢病毒系统靶向于PK-15细胞的p53基因构建p53基因敲除(p53-/-)的细胞;再以感染复数为0.1 MOI的TGEV感染p53野生型(p53+/+)和p53-/-PK-15细胞,于不同的感染时间收集细胞并提取细胞总RNA,应用实时荧光定量PCR(qRT-PCR)技术检测四种细胞因子的转录水平。结果表明,构建的靶向于p53基因敲除的PK-15细胞中,p53基因的454碱基位点缺失一个碱基T,细胞的p53蛋白已检测不到;TGEV感染后IFN-αmRNA的相对表达量在两种细胞中均表现为先上升后下降的趋势,但在病毒感染的36 h之前,p53-/-PK-15细胞中的表达量显著低于p53+/+PK-15细胞(p<0.05);MIP-1αmRNA相对表达量随着病毒感染时间的推移而递增,且在p53+/+PK-15细胞中显著高于p53-/-PK-15细胞(p<0.05);TGF-β1 mRNA的相对表达量在p53+/+PK-15细胞中随时间推移总体呈递减趋势,并在病毒感染(post infection,p.i.)12 h之后显著低于p53-/-PK-15细胞(p<0.05);PGK-1 mRNA相对表达量在病毒感染的12 h p53+/+PK-15细胞中虽略有上升,但差异不显著,而在p53-/-PK-15细胞中呈现时间依赖性递增,并显著高于p53+/+PK-15细胞(p<0.05)。以上结果表明:p53对TGEV感染PK-15细胞后的细胞免疫因子起到了关键的调节作用,推测其可能在宿主抗TGEV感染中发挥着重要作用。     

C-terminal peptides of p53 molecules enhance radiation-induced apoptosis in human mutant p53 cancer cells

We propose here a novel p53-targeting radio-cancer therapy using p53 C-terminal peptides for patients having mutated p53. Hoechst 33342 staining showed that X-ray irradiation alone efficiently induced apoptotic bodies in wild-type p53 (wt p53) human head and neck cancer cells transfected with a neo control vector (SAS/neo cells), but hardly induced apoptotic bodies in mutation-type p53 (m p53) cells transfected with a vector carrying the m p53 gene (SAS/m p53). In contrast, transfection of p53 C-terminal peptides (amino acid residues 361-382 or 353-374) via liposomes caused a remarkable increase of apoptotic bodies in X-ray-irradiated SAS/m p53 cells, but did not enhance apoptotic bodies in X-ray-irradiated SAS/neo cells. In immunocytochemical analysis, positively stained cells for active type caspase-3 were observed at high frequency after X-ray irradiation in the SAS/m p53 cells pre-treated with p53 C-terminal peptides. In SAS/neo cells, positively stained cells for active type caspase-3 were observed with X-ray irradiation alone. Furthermore, protein extracts from X-ray-irradiated SAS/m p53 cells showed higher DNA-binding activity of p53 to p53 consensus sequence when supplemented in vitro with p53 C-terminal peptides than extracts from non-irradiated SAS/m p53 cells. These results suggest that radiation treatment in the presence of p53 C-terminal peptides is more effective for inducing p53 -mediated apoptosis than radiation treatment alone or p53 C-terminal peptide treatment alone, especially in m p53 cancer cells. This novel tool for enhancement of apoptosis induction in m p53 cells might be useful for p53-targeted radio-cancer therapy.     

Resveratrol- induced apoptosis is mediated by p53-dependent pathway in Hep G2 cells

Resveratrol, a phytoalexin found in many plants, has been reported to possess a wide range of pharmacological properties and is one of the promising chemopreventive agents for cancer. Here, we examined the antiproliferation effect of resveratrol in two human liver cancer cell lines, Hep G2 and Hep 3B. Our results showed that resveratrol inhibited cell growth in p53-positive Hep G2 cells only. This anticancer effect was a result of cellular apoptotic death induced by resveratrol via the p53-dependent pathway. Here we demonstrated that the resveratrol-treated cells were arrested in G1 phase and were associated with the increase of p21 expression. In addition, we also illustrated that the resveratrol-treated cells had enhanced Bax expression but they were not involved in Fas/APO-1 apoptotic signal pathway. In contrast, the p53-negative Hep 3B cells treated with resveratrol did not show the antiproliferation effect neither did they show significant changes in p21 nor Fas/APO-1 levels. In summary, our study demonstrated that the resveratrol effectively inhibited cell growth and induced programmed cell death in Hepatoma cells on a molecular basis. Furthermore, these results implied that resveratrol might also be a new potent chemopreventive drug candidate for liver cancer as it played an important role to trigger p53-mediated molecules involved in the mechanism of p53-dependent apoptotic signal pathway.     

Two-dimensional gel analysis of apoptosis-specific p53 isoforms induced by 2-methoxyestradiol in human lung cancer cells

The natural metabolic byproduct of estradiol, 2-methoxyestradiol (2-MeOE2), induces apoptosis in human lung cancer cells by a p53-dependent mechanism. The expression of wild-type p53 isoforms was investigated in H1299 non-small cell lung carcinoma cells induced into apoptosis by 2-MeOE2. H1299 cells lack endogenous p53 and undergo predominantly a G1 arrest when infected with a recombinant wild-type p53 adenovirus. However, when H1299 cells transfected with p53 were treated with 2-MeOE2, they underwent rapid and extensive apoptosis. H1299 cells expressing mutant his273 p53 were unaffected by 2-MeOE2, indicating a dependence of 2-MeOE2-mediated apoptosis on the presence of a functional p53. Analysis of wild-type p53 phosphoisoforms in H1299 cells by two-dimensional gel electrophoresis revealed that 2-MeOE2 induced a unique group of acidic p53 isoforms. Although most of the wild-type p53 in untreated H1299 cells migrated as at least five diffuse species with isoelectric points from pH 5.5–6.3, as many as nine additional forms migrating toward the acidic region with pI values from 4.4–5.3 were detected in 2-MeOE2-treated apoptotic cells. Two other agents known to induce apoptosis, vinblastine and actinomycin D, induced a similar pattern of acidic p53 species as that observed for 2-MeOE2. The results indicated that the induction of apoptosis in H1299 cells by 2-MeOE2 is dependent on the upregulation of specific p53 isoforms. Identification of the specific p53 phosphoisoforms induced by MeOE2 will be an important step in understanding the regulation and function of p53 in apoptosis.     

p53 negatively regulates Pin1 expression under ER stress

Accumulating evidence suggests that endoplasmic reticulum (ER) stress plays a major role in the development of many diseases. A previous study indicated that the apoptotic regulator p53 is significantly increased in response to ER stress and participates in ER stress-induced apoptosis. However, the regulators of p53 expression during ER stress are still not fully understood. Here, we investigated whether p53 contributes to the impairment of Pin1 signaling under ER stress. We found that treatment with thapsigargin, a stimulator of p53 expression and an inducer of ER stress, decreased Pin1 expression in HCT116 cells. Also, we identified functional p53 response elements (p53REs) in the Pin1 promoter. Overexpression of p53 significantly decreased Pin1 expression in HCT116 cells while abolition of p53 gene expression induced Pin1 expression. Pin1 expression was significantly increased by treatment with the p53 inhibitor pifithrin-α or down-regulation of p53 expression. Taken together, ER stress decreased Pin1 expression through p53 activation, and this mechanism may be associated with ER stress-induced cell death. These data reported here support the importance of Pin1 as a potential target molecule mediating tumor development.     

Expression of p53 in luminal and glandular epithelium during the growth and regression of rat uterus during the estrous cycle

It has been well recognized that epithelial cells of the rat endometrium cyclically proliferate and die during the estrous cycle. The aim of the present study was to determine p53 expression pattern and correlate it with the the apoptotic pattern of epithelial cells of the rat uterus during the estrous cycle. The p53 mRNA and protein expression pattern was assessed by in situ hybridization and immunohistochemistry. The apoptotic index was determined by using terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) and electron microscopy. The highest p53 mRNA content, detected by in situ hybridization, was observed on the metestrus day both in the luminal and the glandular epithelia. During this period both epithelia presented high proliferation. The content of p53 mRNA markedly decreased in the following days, presenting its minimal values on the estrus day. The highest number of p53 immunopositive nuclei, in both the luminal and the glandular epithelia, was also detected on the metestrus day, while the lowest one was found on estrus day. On the proestrus day, p53 protein was predominantly detected in the glandular epithelium. However, on the estrus day, p53 protein was detected both in the nuclei and in the cytoplasm of luminal epithelial cells, predominantly in the cytoplasm. The highest apoptotic index in both the luminal and the glandular epithelia was observed on the estrus day whereas the lowest one was observed on the proestrus day. The apoptotic index values were higher in the luminal than in the glandular epithelia. The overall results indicate that p53 expression at both mRNA and protein levels is higher on the metestrus day when the apoptotic index is low. This suggests that p53 should play an important physiological role during proliferative phases of the estrous cycle in the rat uterus.