p53 and the hypoxia-induced apoptosis of cultured neonatal rat cardiac myocytes.

Myocyte cell loss is a prominent and important pathogenic feature of cardiac ischemia. We have used cultured neonatal rat cardiac myocytes exposed to prolonged hypoxia as an experimental system to identify critical factors involved in cardiomyocyte death. Exposure of myocytes to hypoxia for 48 h resulted in intranucleosomal cleavage of genomic DNA characteristic of apoptosis and was accompanied by increased p53 transactivating activity and protein accumulation. Expression of p21/WAF-1/CIP-1, a well-characterized target of p53 transactivation, also increased in response to hypoxia. Hypoxia did not cause DNA laddering or cell loss in cardiac fibroblasts. To determine whether the increase in p53 expression in myocytes was sufficient to induce apoptosis, normoxic cultures were infected with a replication-defective adenovirus expressing wild-type human p53 (AdCMV.p53). Infected cells expressed high intracellular levels of p53 protein and exhibited the morphological changes and genomic DNA fragmentation characteristic of apoptosis. In contrast, no genomic DNA fragmentation was observed in myocytes infected with the control virus lacking an insert (AdCMV.null) or in cardiac fibroblasts infected with AdCMV.p53. These results suggest that the intracellular signaling pathways activated by p53 might play a critical role in the regulation of hypoxia-induced apoptosis of cardiomyocytes.     

p53和Bcl-2基因在人和兔心肌细胞中的同源性研究

目的 :探讨兔和人的有核细胞中 p5 3和 Bcl 2基因的同源性。方法 :用人的 p5 3和 Bcl 2基因序列来扩增兔心肌细胞的 DNA,纯化其扩增产物 ,然后进行测序 ,再测定人与兔心肌细胞内 p5 3和 Bcl 2基因的同源性。结果 :兔心肌细胞内 p5 3基因第 6外显子的扩增产物长度为 2 0 8个碱基 ,与人类 p5 3基因第 6外显子的碱基序列进行比较 ,在扩增的 177个碱基片段中完全同源 ,其同源性为 10 0 .0 %;而兔心肌细胞内 Bcl 2基因的扩增产物长度为 2 89个碱基 ,与人类 Bcl 2基因 m RNA的碱基序列进行比较 ,在扩增的 199个碱基片段中有 176个碱基相同 ,其同源性为 88.4%。结论 :兔和人的有核细胞中 p5 3和 Bcl 2基因有很好的同源性 ,在研究兔心肌细胞这两种基因的改变时 ,完全可以用人的 p5 3和 Bcl 2基因序列 (引物 )来扩增。     

黄芩素通过抗氧化及调节细胞内钙离子浓度抑制大鼠缺氧/复氧诱导的心肌细胞凋亡

背景:黄芩素对缺氧复氧损伤的心血管有保护作,但机制至今不清。目的:探讨中药黄芩素对心肌细胞缺氧/复氧损伤导致心肌细胞凋亡的保护作用机制。方法:体外培养大鼠乳鼠心肌细胞培养。实验分3组:正常对照组为正常培养的心肌细胞未做处理;缺氧/复氧组为应用缺氧/复氧方法诱导心肌细胞凋亡损伤;黄芩素预处理组为经黄芩素预处理30min后经缺氧/复氧诱导的心肌细胞。通过检测培养基上清液中乳酸脱氢酶活力检测细胞损伤程度及黄芩苷保护作用;应用原位末端标记细胞法标记细胞后,流式细胞检测心肌细胞凋亡率;应用免疫印迹方法检测心肌细胞凋亡蛋白Bax与抗凋亡蛋白Bcl-2的蛋白表达水平;应用Fura-2-AM负载心肌细胞,实时检测心肌细胞内Ca2+浓度变化。结果与结论:与正常对照组相比,缺氧/复氧组上清液乳酸脱氢酶活性、心肌细胞凋亡率、Bax蛋白含量、心肌细胞Ca2+浓度均增加(P<0.05),Bcl-2蛋白含量降低(P<0.05)。与缺氧/复氧组相比,黄芩素预处理组乳酸脱氢酶含量、心肌细胞凋亡率、Bax蛋白含量及心肌细胞Ca2+浓度均降低(P<0.05),Bcl-2蛋白含量增加(P<0.05)。证实黄芩素能抑制缺氧/复氧导致的心肌细胞凋亡,其作用机制可能与抗氧化与调节心肌细胞内钙离子浓度有关     

Flavopiridol induces apoptosis in glioma cell lines independent of retinoblastoma and p53 tumor suppressor pathway alterations by a caspase-independent pathway

Flavopiridol is a synthetic flavone, which inhibits growth in vitro and in vivo of several solid malignancies such as renal, prostate, and colon cancers. It is a potent cyclin-dependent kinase inhibitor presently in clinical trials. In this study, we examined the effect of flavopiridol on a panel of glioma cell lines having different genetic profiles: five of six have codeletion of p16(INK4a) and p14(ARF); three of six have p53 mutations; and one of six shows overexpression of mouse double minute-2 (MDM2) protein. Independent of retinoblastoma and p53 tumor suppressor pathway alterations, flavopiridol induced apoptosis in all cell lines but through a caspase-independent mechanism. No cleavage products for caspase 3 or its substrate poly(ADP-ribose) polymerase or caspase 8 were detected. The pan-caspase inhibitor Z-VAD-fmk did not inhibit flavopiridol-induced apoptosis. Mitochondrial damage measured by cytochrome c release and transmission electron microscopy was not observed in drug-treated glioma cells. In contrast, flavopiridol treatment induced translocation of apoptosis-inducing factor from the mitochondria to the nucleus. The proteins cyclin D(1) and MDM2 involved in the regulation of retinoblastoma and p53 activity, respectively, were down-regulated early after flavopiridol treatment. Given that MDM2 protein can confer oncogenic properties under certain circumstances, loss of MDM2 expression in tumor cells could promote increased chemosensitivity. After drug treatment, a low Bcl-2/Bax ratio was observed, a condition that may favor apoptosis. Taken together, the data indicate that flavopiridol has activity against glioma cell lines in vitro and should be considered for clinical development in the treatment of glioblastoma multiforme.     

ASPP家族及其与p53相互作用的研究进展

p53是一个肿瘤抑制蛋白,它通过影响编码细胞周期相关蛋白质的基因表达来调控细胞周期的G1停滞,同时还会因为DNA的损伤增多,诱导细胞发生细胞凋亡。p53诱导细胞凋亡的机制多年来一直不太清楚,而最近发现的 p53凋亡刺激蛋白 ASPP蛋白家族对 p53 诱导细胞凋亡的机制的研究有了新的进展。ASPP蛋白家族与 p53 的作用主要是特异性增强 p53 的细胞凋亡功能,而对 p53的细胞生长停滞功能则没有作用。ASPP蛋白家族增强 p53 的细胞凋亡功能是通过增强 p53的促凋亡基因启动子与DNA的结合而促进细胞凋亡,现就此作一综述。     

鼠骨髓间充质干细胞对缺氧/复氧心肌细胞的影响

目的:观察在体外培养条件下骨髓间充质干细胞(mesenchymal stem cell,MSC)对缺氧/复氧新生大鼠心肌细胞的保护作用.方法:无菌条件下分离、培养、传代Wistar大鼠MSC,细胞融合达90%时更换培养基培养24 h,收集细胞培养液作为MSC条件培养基;原代培养新生Wistar大鼠心肌细胞,先用5% CO2、10% H2、85% N2混合气造成细胞缺氧24 h,再给予95% O2 和 5% CO2混合气复氧2 h,建立缺氧/复氧模型.MSC 处理组于复氧时加入MSC 条件培养基,正常细胞作为对照组,分别于复氧后用MTT法、乳酸脱氢酶(lactated dehydrogenase)法测定各组心肌细胞活性的改变;RT-PCR观察细胞凋亡蛋白Bcl-2和Bax的表达.结果: 与对照组比较,缺氧/复氧组细胞活力显著降低(P ＜ 0.01).与缺氧/复氧组比较,MSC处理组细胞活力明显增加(P ＜ 0.01).缺氧/复氧组抗凋亡基因Bcl-2的表达明显降低,促凋亡基因Bax的表达明显增加,而MSC条件培养基可以减少Bax基因的表达,提高Bcl-2/Bax值,与缺氧/复氧组比较差异具有显著性(P ＜ 0.01).结论:MSC对体外缺氧-复氧诱导的心肌细胞的凋亡有保护作用.     

c-Abl-independent p73 stabilization during gemcitabine- or 4'-thio-beta-D-arabinofuranosylcytosine-induced apoptosis in wild-type and p53-null colorectal cancer cells

Nucleoside anticancer drugs like gemcitabine (2'-deoxy-2',2'-difluorocytidine) are potent inducers of p53, and ectopic expression of wild-type p53 sensitizes cells to these agents. However, it is also known that nucleosides are efficient activators of apoptosis in tumor cells that do not express a functional p53. To clarify this issue, we examined the effects of gemcitabine and 4'-thio-beta-d-arabinofuranosylcytosine (T-ara-C) on p73, a structural and functional homologue of p53, whose activation could also account for nucleoside-induced apoptosis because no functionally significant mutations of p73 have been reported in cancers. Acute treatment of HCT 116 colon carcinoma cells with gemcitabine or T-ara-C induced marked cytotoxicity and cleavage of caspase-3 and poly(ADP-ribose) polymerase. T-ara-C and gemcitabine markedly induced p53 accumulation as well as increased levels of phospho-p53 (Ser15/Ser20/Ser46) and induced its binding to a consensus p53 response element. Despite robust activation of p53 by T-ara-C and gemcitabine, we found that wild-type and p53-/- HCT 116 cells exhibited almost equivalent sensitivity towards these nucleosides. Examination of p73 revealed that T-ara-C and gemcitabine markedly increased p73 protein levels and p73 DNA-binding activities in both p53-/- and wild-type cells. Furthermore, T-ara-C- and gemcitabine-induced increases in p73 levels occur due to a decrease in p73 protein turnover. RNA interference studies show that nucleoside-induced p73 increases are independent of c-Abl, a nucleoside-activated kinase recently implicated in p73 stabilization. HCT 116 lines, wherein the downstream p53/p73 targets Bax and PUMA (p53 up-regulated modulator of apoptosis) were deleted, were less sensitive to T-ara-C and gemcitabine. Together, these studies indicate that c-Abl-independent p73 stabilization pathways could account for the p53-independent mechanisms in nucleoside-induced apoptosis.     

一氧化氮对心肌细胞缺氧复氧时脂质过氧化损伤的保护作用

目的:研究一氧化氮对培养鼠心肌细胞缺氧复氧所致脂质过氧化损伤的保护作用,探讨心肌缺血修复机制。 方法:采用细胞缺氧复氧损伤模型,培养细胞随机分为4组:A组正常对照组(培养3 h);B组单纯缺氧/复氧(A/R缺氧2 h复氧1 h);C组缺氧预处理组(缺氧20 min后复氧20 min,然后缺氧复氧);D组一氧化氮预处理组[加入S-亚硝基-已酰青酶胺(s-nitroso-n-acetyl-penicillamine,SNAP)使其终浓度为l mmol/L,预处理40 min后A/R。与复氧后测定培养液中肌酸激酶(creatine kinase,CK)、乳酸脱氢酶(lacfic dehydrogenase,LDH)活性变化及细胞内丙二醛含量和细胞存活率。结果:与正常组比,单纯缺氧/复氧组CK[(941.35±152.53)nkat/L]、 LDH[(7 416.48+984.20)nkat/L]、丙二醛[(1.35±0.26)μmol/g],水平显著井高(P<0.01),细胞存活率(54.68±6.00)％显著降低(P<0.01)。1 mmol/L SNAP预处理组[细胞存活率为(74.55±4.11),CK为(582.45±140.86)nkat/L,LDH为(5 766.15±941.69)nkat/L,丙二醛为(0.89±0.16)μmol/g]和缺氧预处理组[细胞存活率为(74.69±6.14)。CK为(547.94±125.52)nkat/L,LDH为(5 882.34±844.67)nkat/L,丙二醛为(0.85±0.12)μmol/g]上述变化明显减轻(P<0.01)。 结论:一氧化氮可抑制脂质过氧化反应,减轻自由基对心肌     

Recombinant, refolded tetrameric p53 and gonadotropin-releasing hormone-p53 slow proliferation and induce apoptosis in p53-deficient cancer cells

The p53 tumor suppressor is mutated in over 50% of human cancers. Mutations resulting in amino acid changes within p53 result in a loss of activity and consequent changes in expression of genes that regulate DNA repair and cell cycle progression. Replacement of p53 using protein therapy would restore p53 function in p53-deficient tumor cells, with a consequence of tumor cell death and tumor regression. p53 functions in a tetrameric form in vivo. Here, we refolded a wild-type, full-length p53 from inclusion bodies expressed in Escherichia coli as a stable tetramer. The tetrameric p53 binds to p53-specific DNA and, when transformed into a p53-deficient cancer cell line, induced apoptosis of the transformed cells. Next, using the same expression and refolding technology, we produced a stable tetramer of recombinant gonadotropin-releasing hormone-p53 fusion protein (GnRH-p53), which traverses the plasma membrane, slows proliferation, and induces apoptosis in p53-deficient, GnRH-receptor-expressing cancer cell lines. In addition, we showed a time-dependent binding and internalization of GnRH-p53 to a receptor-expressing cell line. We conclude that the GnRH-p53 fusion strategy may provide a basis for constructing an effective cancer therapeutic for patients with tumors in GnRH-receptor-positive tissue types.     

Cycloheximide protects HepG2 cells from serum withdrawal-induced apoptosis by decreasing p53 and phosphorylated p53 levels

Cycloheximide (CHX), an inhibitor of protein synthesis, has been reported to prevent cell death in a wide variety of cell types and produced by different apoptotic stimuli. However, the mechanisms by which CHX protects cells from apoptosis are still unclear. In this study, we investigated whether p53 plays a role in the protection by CHX against serum withdrawal-induced apoptosis. Deprivation of serum from the culture medium causes apoptosis in HepG2 cells, and CHX dramatically protects cells from death. p53, p21, and Bax protein levels were elevated, and cell cycle arrest was produced after serum withdrawal. CHX abolished this elevation of p53, p21, and Bax as well as the cell cycle arrest induced by serum deprivation. The p53 inhibitor pifithrin-alpha protects HepG2 cells against apoptosis induced by serum withdrawal. HepG2 cells expressing a dominant negative form of mutant p53 and Hep3B cells lacking p53 were resistant to serum withdrawal-induced apoptosis. Lowering of p53 by small interfering RNA protects HepG2 cells from serum withdrawal-induced apoptosis. p53 phosphorylation was induced by serum withdrawal and other chemotherapeutic reagents such as actinomycin D, doxorubicin, and etoposide. CHX decreases the levels of phosphorylated p53 (pp53) even in the presence of a proteasome inhibitor, which maintains the total p53 levels, whereas it does not affect the dephosphorylation of pp53. These results suggest the possibility that kinases that phosphorylate p53 might be affected by CHX administration. In summary, CHX protects HepG2 cells from serum withdrawal-induced apoptosis through inhibiting the synthesis of p53 and the phosphorylation of p53.     

Nutlin-3 radiosensitizes hypoxic prostate cancer cells independent of p53

Nutlin-3 is a small-molecule inhibitor that acts to inhibit MDM2 binding to p53 and subsequent p53-dependent DNA damage signaling. Whether Nutlin-3 alters cell toxicity following DNA damage under oxic versus hypoxic conditions has not been studied. The potential radiosensitization (0-10 Gy) properties of Nutlin-3 (dose range, 2-10 micromol/L for up to 24 h) were investigated in vitro using three prostate cancer cell lines, 22RV1 [wild-type p53 (WTp53)], DU145 (mutated p53), and PC-3 (p53-null) under oxic (21% O(2)), hypoxic (0.2% O(2)), and anoxic (0% O(2)) conditions. As a single agent, Nutlin-3 (2-10 micromol/L) stabilized p53 and p21(WAF) levels and was toxic to WTp53-22RV1 cells (IC(50), 4.3 micromol/L) but had minimal toxicity toward p53-deficient cells (IC(50), >10 micromol/L). When combined with radiation under oxic conditions, Nutlin-3 decreased clonogenic survival in all three cell lines: 22RV1 [sensitizing enhancement ratio (SER), 1.24], DU145 (SER, 1.27), and PC-3 (SER, 1.12). Anoxia induced p53 protein expression in 22RV1 cells and this was augmented by Nutlin-3 treatment. Furthermore, Nutlin-3 was more effective as a radiosensitizer under hypoxic conditions particularly in WTp53-expressing cells: 22RV1 (SER, 1.78), DU145 (SER, 1.31), and PC-3 (SER, 1.28). The decrease in clonogenic survival with Nutlin-3 was not correlated to altered levels of radiation-induced apoptosis within the three cell lines. Our results indicate that Nutlin-3 can act as a radiosensitizer via p53-independent mechanisms under low O(2) levels. Nutlin-3 may be a useful adjunct to improve the therapeutic ratio using precision radiotherapy targeted to hypoxic cells and warrants further study in vivo.     

A small-molecule inhibitor of MDMX activates p53 and induces apoptosis

The p53 inactivation caused by aberrant expression of its major regulators (e.g., MDM2 and MDMX) contributes to the genesis of a large number of human cancers. Recent studies have shown that restoration of p53 activity by counteracting p53 repressors is a promising anticancer strategy. Although agents (e.g., nutlin-3a) that disrupt MDM2-p53 interaction can inhibit tumor growth, they are less effective in cancer cells that express high levels of MDMX. MDMX binds to p53 and can repress the tumor suppressor function of p53 through inhibiting its trans-activation activity and/or destabilizing the protein. Here we report the identification of a benzofuroxan derivative [7-(4-methylpiperazin-1-yl)-4-nitro-1-oxido-2,1,3-benzoxadiazol-1-ium, NSC207895] that could inhibit MDMX expression in cancer cells through a reporter-based drug screening. Treatments of MCF-7 cells with this small-molecule MDMX inhibitor activated p53, resulting in elevated expression of proapoptotic genes (e.g., PUMA, BAX, and PIG3). Importantly, this novel small-molecule p53 activator caused MCF-7 cells to undergo apoptosis and acted additively with nutlin-3a to activate p53 and decrease the viability of cancer cells. These results thus show that small molecules targeting MDMX expression would be of therapeutic benefits.     

HIV-induced apoptosis requires the CD4 receptor cytoplasmic tail and is accelerated by interaction of CD4 with p56lck

The roles of the CD4 receptor and the src kinase p56lck were examined in the process of HIV-induced apoptosis of CD4+ T lymphocytes. The presence of the CD4 cytoplasmic tail was found to be essential in delivering an apoptotic signal, and interaction of CD4 with p56lck potentiated HIV-induced apoptosis. Apoptosis, but not HIV replication, was abrogated by deleting the NH2-terminal intracytoplasmic tail of CD4, or by mutating the two critical cysteines in this tail that are responsible for CD4-p56lck interaction. Introduction of p56lck in C8166- 45 or MT-2 cells, CD4+ T cell lines deficient for this protein, greatly increased HIV-induced apoptosis and syncytium formation. The ability of p56lck to deliver an apoptotic signal did not depend on its kinase function, since a kinase-deficient mutant was as effective as its normal counterpart in inducing apoptosis, suggesting that p56lck may act as an adapter to anchor other proteins to transduce the death signal.     

Restoration of p53 function for selective Fas-mediated apoptosis in human and rat glioma cells in vitro and in vivo by a p53 COOH-terminal peptide

We have shown that a COOH-terminal peptide of p53 (amino acids 361-382, p53p), linked to the truncated homeobox domain of Antennapedia (Ant) as a carrier for transduction, induced rapid apoptosis in human premalignant and malignant cell lines. Here, we report that human and rat glioma lines containing endogenous mutant p53 or wild-type (WT) p53 were induced into apoptosis by exposure to this peptide called p53p-Ant. The peptide was comparatively nontoxic to proliferating nonmalignant human and rat glial cell lines containing WT p53 and proliferating normal human peripheral marrow blood stem cells. Degree of sensitivity to the peptide correlated directly with the level of endogenous p53 expression and mutant p53 conformation. Apoptosis induction by p53p-Ant was quantitated by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay and Annexin V staining in human glioma cells in vitro and in a syngeneic orthotopic 9L glioma rat model using convection-enhanced delivery in vivo. The mechanism of cell death by this peptide was solely through the Fas extrinsic apoptotic pathway. p53p-Ant induced a 3-fold increase in extracellular membrane Fas expression in glioma cells but no significant increase in nonmalignant glial cells. These data suggest that p53 function for inducing Fas-mediated apoptosis in gliomas, which express sufficient quantities of endogenous mutant or WT p53, may be restored or activated, respectively, by a cell-permeable peptide derived from the p53 COOH-terminal regulatory domain (p53p-Ant). p53p-Ant may serve as a prototypic model for the development of new anticancer agents with unique selectivity for glioma cancer cells and it can be successfully delivered in vivo into a brain tumor by a convection-enhanced delivery system, which circumvents the blood-brain barrier.     

Exogenous NAD+ supplementation protects H9c2 cardiac myoblasts against hypoxia/reoxygenation injury via Sirt1‐p53 pathway

Nicotinamide adenine dinucleotide (NAD⁺) not only transfers electrons in mitochondrial respiration, but also acts as an indispensable cosubstrate for Sirt1, the class III histone/nonhistone deacetylase. However, NAD⁺ is depleted in myocardial ischemia/reperfusion (IR) injury. The objective of this study was to investigate the role of exogenous NAD⁺ supplementation in hypoxia/reoxygenation (HR)‐stressed H9c2 cardiac myoblasts. Firstly, the effects of distinct treating time points and doses of NAD⁺ supplementation on the viability of HR‐stressed H9c2 cells were detected. Secondly, intracellular NAD⁺ levels in HR‐stressed H9c2 cells at various extracellular NAD⁺ concentrations were determined. Thirdly, the role of NAD⁺ supplementation in HR‐induced cell apoptosis and its relevance to Sirtuin 1‐p53 pathway were investigated. Exogenous NAD⁺ supplementation elevated intracellular NAD⁺ level and reduced HR‐induced cell death in both time‐ and concentration‐dependent manners. It appeared that NAD⁺ supplementation exerted the greatest protection when extracellular concentration ranged from 500 to 1000 μm and when NAD⁺ was added immediately after reoxygenation began. NAD⁺ replenishment restored Sirt1 activity, reduced the acetylation level of p53 (Lys373 & 382), and attenuated cell apoptosis in HR‐stressed H9c2 cells, whereas inhibition of Sirt1 activity alleviated the effects of NAD⁺ replenishment. These results indicated that exogenous NAD⁺ supplementation attenuated HR‐induced cell apoptosis, which was at least partly mediated by restoring Sirt1 activity and subsequently inhibiting p53 activity via deacetylating p53 at lysine 373 and 382.