Preconditioning rabbit cardiomyocytes: role of pH, vacuolar proton ATPase, and apoptosis.

Ischemic preconditioning signals through protein kinase C (PKC) to protect against myocardial infarction. This protection is characterized by diminished intracellular acidification. Acidification is also a feature of apoptosis, and several agents act to prevent apoptosis by preventing acidification through activation of ion channels and pumps to promote cytoplasmic alkalinization. We characterized metabolic inhibition, recovery, and preconditioning through a PKC-dependent pathway in cardiomyocytes isolated from adult rabbit hearts. Preconditioning reduced loss of viability assessed by morphology and reduced DNA nicking. Blockade of the vacuolar proton ATPase (VPATPase) prevented the effect of preconditioning to reduce metabolic inhibition-induced acidosis, loss of viability, and DNA nicking. The beneficial effect of Na+/H+ exchange inhibition, which is thought to be effective through reduced intracellular Na+ and Ca++, was also abrogated by VPATPase blockade, suggesting that acidification even in the absence of Na+/H+ exchange may lead to cell death. We conclude that a target of PKC in mediating preconditioning is activation of the VPATPase with resultant attenuation of intracellular acidification during metabolic inhibition. Inhibition of the "death protease," interleukin-1-beta converting enzyme or related enzymes, also protected against the injury that followed metabolic inhibition. This observation, coupled with the detection of DNA nicking in cells subjected to metabolic inhibition, suggests that apoptotic cell death may be preventable in this model of ischemia/reperfusion injury.     

In vivo treatment with granulocyte colony-stimulating factor does not delay apoptosis in human neutrophils by increasing the expression of the vacuolar proton ATPase.

BACKGROUND: Neutrophils die by apoptosis, and in vivo administration of granulocyte colony-stimulating factor (G-CSF) delays this apoptotic cell death. G-CSF administered in vitro correlates delayed apoptosis with upregulation of the vacuolar proton ATPase (v-ATPase). Because this enzyme requires assembly of membrane and cytosolic domains to function, we hypothesized that in vivo G-CSF would increase synthesis and assembly of v-ATPase components to delay apoptosis. METHODS: Volunteers received G-CSF for 5 days, and each had a paired control. Neutrophils were isolated from subjects before the first and after the fifth injection. Proteins from cytosol or plasma membrane or from whole cell lysates were resolved by SDS-polyacrylamide gel electrophoresis and immunoblotted with antibody to the 33kDa v-ATPase E subunit. Densitometry quantified immunoreactivity. RESULTS: No significant increase on the E subunit occurred between treated and control groups. CONCLUSION: In vivo G-CSF does not increase the amount of v-ATPase in neutrophils. Although G-CSF in vivo delays apoptosis, the mechanism(s) by which this occurs is not known.     

Increased p53 expression induced by APR-246 reprograms tumor-associated macrophages to augment immune checkpoint blockade

In addition to playing a major role in tumor cell biology, p53 generates a microenvironment that promotes antitumor immune surveillance via tumor-associated macrophages. We examined whether increasing p53 signaling in the tumor microenvironment influences antitumor T cell immunity. Our findings indicate that increased p53 signaling induced either pharmacologically with APR-246 (eprenetapopt) or in p53-overexpressing transgenic mice can disinhibit antitumor T cell immunity and augment the efficacy of immune checkpoint blockade. We demonstrated that increased p53 expression in tumor-associated macrophages induces canonical p53-associated functions such as senescence and activation of a p53-dependent senescence-associated secretory phenotype. This was linked with decreased expression of proteins associated with M2 polarization by tumor-associated macrophages. Our preclinical data led to the development of a clinical trial in patients with solid tumors combining APR-246 with pembrolizumab. Biospecimens from select patients participating in this ongoing trial showed that there was a suppression of M2-polarized myeloid cells and increase in T cell proliferation with therapy in those who responded to the therapy. Our findings, based on both genetic and a small molecule–based pharmacological approach, suggest that increasing p53 expression in tumor-associated macrophages reprograms the tumor microenvironment to augment the response to immune checkpoint blockade.     

氧化应激诱导肺泡Ⅱ型上皮细胞凋亡及Bax和p53的表达变化

目的 探讨氧化应激状态下肺泡Ⅱ型上皮细胞(ATⅡ)凋亡的变化规律及Bax和p53表达的变化.方法 用过氧化氢(H2O2,500 μmol/L)处理不同时间模拟机体活性氧攻击损伤ATⅡ细胞的体内情况,建立氧化损伤性细胞模型;用四甲基偶氮唑盐(MTT)比色法检测细胞存活率,流式细胞仪检测细胞凋亡率,荧光显微镜及流式细胞仪检测细胞线粒体膜电位,蛋白质免疫印迹法(Western blotting)检测Bax和p53的蛋白表达变化.结果 与对照组比较,随H2O2刺激时间延长,ATⅡ细胞存活率明显下降(F=85.211,P＜0.05),线粒体膜电位也明显下降(F=72.453,P＜0.05),凋亡率却明显增加(F=54.002,P＜0.05);同时发现Bax蛋白和p53蛋白表达均明显增加(F1=28.118,F2=43.456,P均＜0.05).结论 氧化应激能损伤ATⅡ细胞,使细胞线粒体膜电位下降,细胞凋亡率增加、存活率下降,Bax和p53可能参与了ATⅡ细胞的凋亡.     

整合素连接激酶对阿霉素诱导心肌细胞凋亡的影响

目的探讨高表达整合素连接激酶(ILK)对阿霉素(DOX)诱导心肌细胞凋亡的影响及其机制。方法体外培养乳鼠心肌细胞,分为正常对照组(转染Ad-GFP)、阿霉素损伤组(转染Ad-GFP+DOX)、ILK转染组(转染Ad-ILK+DOX)采用原位末端缺口标记法(TUNEL)检测心肌细胞凋亡率;real-time PCR检测心肌细胞Fas、FasL、Bax、Bel-2 mRNA的表达水平。结果与对照组相比,阿霉素损伤组心肌细胞凋亡率明显升高(P<0.01),Fas、FasL、Bax mRNA的表达水平明显增高(P<0.01),Bcl-2 mRNA的表达明显降低(P<0 01);与阿霉素损伤组相比,ILK转染组心肌细胞凋亡率显著降低(P<0.05),Fas、FasL、Bax mRNA的表达水平明显(均P<0.01),Bcl-2 mRNA的表达明显增加(P<0.05)。结论高表达ILK通过抑制心肌细胞Fas、FasL、Bax mRNA的表达、上调Bcl-2 mRNA的表达而抑制阿霉素诱导的心肌细胞凋亡。     

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。     

Lonidamine induces apoptosis in drug-resistant cells independently of the p53 gene.

Lonidamine, a dichlorinated derivative of indazole-3-carboxylic acid, was shown to play a significant role in reversing or overcoming multidrug resistance. Here, we show that exposure to 50 microg/ml of lonidamine induces apoptosis in adriamycin and nitrosourea-resistant cells (MCF-7 ADR(r) human breast cancer cell line, and LB9 glioblastoma multiform cell line), as demonstrated by sub-G1 peaks in DNA content histograms, condensation of nuclear chromatin, and internucleosomal DNA fragmentation. Moreover, we find that apoptosis is preceded by accumulation of the cells in the G0/G1 phase of the cell cycle. Interestingly, lonidamine fails to activate the apoptotic program in the corresponding sensitive parental cell lines (ADR-sensitive MCF-7 WT, and nitrosourea-sensitive LI cells) even after long exposure times. The evaluation of bcl-2 protein expression suggests that this different effect of lonidamine treatment in drug-resistant and -sensitive cell lines might not simply be due to dissimilar expression levels of bcl-2 protein. To determine whether the lonidamine-induced apoptosis is mediated by p53 protein, we used cells lacking endogenous p53 and overexpressing either wild-type p53 or dominant-negative p53 mutant. We find that apoptosis by lonidamine is independent of the p53 gene.     

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.     

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.     

Bcl-2和p 53蛋白表达在脓毒症大鼠心肌细胞凋亡中的作用研究

目的 探讨Bcl-2和p53基因在脓毒症大鼠心肌细胞凋亡中的作用.方法 将成年雄性SD大鼠随机分为正常对照组(n=6)、假手术组(n=6)、模型组(n=24).以盲肠结扎穿孔术(CLP)复制脓毒症大鼠模型.术后3、9、12和24 h各取6只大鼠的心肌组织备检.以电镜和原位末端缺刻标记法(TUNEL)检测心肌细胞凋亡变化;用免疫组化法检测Bcl-2和p53的蛋白表达.结果 模型组大鼠心肌细胞凋亡指数(AI)随时间延长明显升高,于12 h达峰值C(55.633±2.073)%),24 hC(33.683±2.070)%]较12 h明显下降,但各时间点均明显高于正常对照组[(1.500±0.141)%]和假手术组[(1.567±0.258)%],差异均有统计学意义(P均<0.05).模型组各时间点p53基因的蛋白表达阳性数[3 h最低为(13.817±0.964)%.12 h峰值为(80.567±5.055)%]均较正常对照组C(0.617±0.232)%]和假手术组C(0.600±0.297)%)明显升高(P均<0.05),其变化与TUNEL检测凋亡的结果一致;而Bcl-2基因的蛋白表达阳性数C3 h最高为(31.650±1.799)%,12 h最低为(0.650±0.308)%]均较正常对照组[(47.017±0.691)%]和假手术组[(46.817±0.567)%]明显降低(P均<0.05),其变化与TUNEL检测凋亡的结果趋势相反.结论 细胞凋亡可能是脓毒症心肌损害的机制之一,其调控基因p53和Bcl-2基因的改变或许可以作为脓毒症病情改变的标志,对其进行干预,以改善脓毒症的预后.     

外周型苯二氮(艹卓)受体在缺氧/复氧损伤大鼠心肌细胞凋亡中的作用

外周型苯二氮[艹卓]受体（PBR）是一种由169个氨基酸组成的线粒体外膜蛋白，是线粒体通透性转换孔（PTP）的重要组成部分。PBR基因敲除鼠在发育早期死亡，提示PBR在细胞中扮演了极重要的作用。有关PBR抗缺氧性保护作用正成为国内外的研究热点，本研究以此作为重点，报告如下。     

脓毒症大鼠心肌细胞凋亡与p53和Bcl-2基因表达的关系

目的 探讨脓毒症大鼠心肌细胞凋亡的变化及其与Bcl-2和p53基因mRNA表达的关系.方法 取体重190～220 g的成年雄性SD大鼠42只,随机分为三组,脓毒症组30只,正常对照组6只,假手术组6只;脓毒症组以盲肠结扎穿刺法(CLP)复制脓毒症大鼠模型,于术后3 h、9 h、12h、24 h各处死6只大鼠,假手术组麻醉满意后打开腹腔,正常对照组仅行麻醉术,均取1.0 mm3大小的心肌组织,另外余下的6只脓毒症鼠术后待其自然死亡不取心肌.采用电镜和TUNEL法检测大鼠心肌细胞的凋亡,用RT-PCR方法检测大鼠心肌细胞Bcl-2和p53基因mRNA的表达.结果 电镜下大鼠凋亡的心肌细胞胞膜皱缩,核固缩、边集、碎裂,胞膜突起、凋亡小体形成,TUNEL法检测各时间点的脓毒症大鼠心肌细胞的凋亡率均较正常对照组和假手术对照组明显升高(P＜0.05),术后12h达到峰值;p53 mRNA表达明显升高(P＜0.05),其表达量与心肌细胞凋亡指数呈正相关性(r=0.976,P＜0.05);Bcl-2 mRNA表达明显降低(P＜0.05),其表达量与心肌细胞凋亡指数呈负相关性(r=-0.833,P＜0.05).结论 脓毒症大鼠心肌细胞的凋亡明显增高,p53和Bcl-2 mRNA的表达异常是其可能机制之一.     

Transgenic expression of survivin in keratinocytes counteracts UVB-induced apoptosis and cooperates with loss of p53

The inhibitor of apoptosis protein survivin has been implicated in both cell cycle control and apoptosis resistance. To discriminate between these different roles, we used transgenic expression of survivin in the skin as a model for cell proliferation, differentiation, and apoptosis. Transgenic mice expressing survivin under the control of a keratin-14 promoter developed normally, without histologic abnormalities of the skin or hair, epidermal hyperplasia, or developmental abnormalities of basal or suprabasal epidermis. Keratinocyte proliferation assessed under basal conditions, or after ultraviolet-B (UVB) irradiation, or phorbol ester stimulation was unchanged in survivin transgenic mice. In contrast, survivin expression inhibited UVB-induced apoptosis in vitro and in vivo (i.e., sunburn cell formation), whereas it did not affect Fas-induced cell death. When crossed with p53 knockout mice, transgenic expression of survivin in a p53(+/-) background substituted for the loss of a second p53 allele and further inhibited UVB-induced apoptosis. These data provide the first in vivo evidence that survivin inhibits apoptosis and suggest that this pathway may oppose the elimination of cancerous cells by p53.     

p53凋亡刺激蛋白在非小细胞肺癌中的表达及其临床意义

目的 研究分析p53遗传背景不同的非小细胞肺癌(NSCLC)细胞株中p53凋亡刺激蛋白(ASPP)家族的表达和调节,以及在NSCLC诊断和化疗监测中的临床意义.方法 用实时荧光定量逆转录(FQ-RT)-PCR检测NSCLC细胞株A549(野生型)和NCI-H157(突变型)及37例肺癌患者组织与外周血单个核细胞中ASPP1、ASPP2 mRNA表达,免疫印迹技术检测NSCLC细胞株ASPP1、ASPP2蛋白含量.同时,检测2株细胞对顺铂(CDDP)的敏感性以及化疗药物处理后ASPP1、ASPP2mRNA表达变化.此外,还比较NSCLC患者与37名健康对照者ASPP1、ASPP2 mRNA表达情况,并监测NSCLC患者化疗前后ASPP1、ASPP2 mRNA表达变化.结果 FQ-RT-PCR检测ASPP标准品曲线斜率均值分别为-3.249、-3.358,r＞0.98,标准曲线的扩增效率分别为1.156、1.028;CDDP处理后NCI-H157细胞株的IC_(50)值是3.70μg/ml,A549的IC_(50)是10.48μg/ml.NCI-H157细胞株中ASPP1,ASPP2 mRNA平均表达量比A549分别高2.66倍和6.98倍.肺癌患者组织和外周血单个核细胞中ASPP1 mRNA表达分别为(4.27 ±0.57)×10~3和(2.49±0.32)×10~3,ASPP2 mRNA表达分别为(2.34±0.75)×10~3和(7.00±1.17)×10~3,而健康对照组组织和外周血单个核细胞中ASPP1 mRNA 表达分别为(1.32±0.21)×10~4和(1.46±0.31)×10~4,ASPP2 mRNA表达分别为(1.38±0.19)×10~4和(1.28±0.18)×10~4.肺癌组中ASPP1和ASPP2 mRNA明显低于健康对照组(t值分别为2.58、3.94、3.62、3.76,P均＜0.05).NSCLC组中18例患者术后化疗前ASPP1和ASPP2 mRNA分别为(2.34±0.56)×10~3和(6.64±0.72)×10~3,进行2个周期化疗后分别为(3.66±0.64)×10~3和(9.42±0.44)×10~3,基因表达量均明显升高(t值分别为3.02、4.50,P均＜0.05).结论 NSCLC突变型细胞株ASPP1和ASPP2 mRNA的表达均高于野生型细胞株,ASPP1和ASPP2表达升高有可能增强肿瘤细胞对化疗药物的敏感性.NSCLC患者化疗过程中,ASPP1和ASPP2 mRNA表达的变化可能与化疗药物作用有关,提高ASPP1、ASPP2的表达将有助于肿瘤的治疗.     

结直肠癌p53基因点突变和蛋白表达的研究

目的了解中国人结直肠癌ｐ５３基因的突变谱，探讨聚合酶链反应－单链构象多态（ＰＣＲ－ＳＳＣＰ）银染技术用于研究结直肠癌中ｐ５３基因突变的可行性。方法应用ＰＣＲ－ＳＳＣＰ银染技术检测４１例结直肠癌ｐ５３基因突变，以ＡＢＣ免疫组织化学染色检测结直肠癌Ｐ５３蛋白的表达。结果３４％（１４／４１）的病例显示有ｐ５３基因突变，其中外显子５，６，７和８各有４，１，５和４例突变。２０例有Ｐ５３蛋白异常表达，阳性率为４９％。结论导致Ｐ５３蛋白异常堆积的ｐ５３基因突变是结直肠癌的一种常见的分子结构改变，可能在结直肠癌的发生和发展中起着重要的作用。ＰＣＲ－ＳＳＣＰ银染技术是一简便、快速、有效的检测基因点突变的方法     

异丙酚对离体大鼠心肌缺血/再灌注损伤后细胞凋亡及HSP70表达的影响

目的观察异丙酚对离体大鼠心肌缺血/再灌注损伤的影响并探讨其作用机制。方法应用langendorff离体心脏灌注系统建立心肌缺血/再灌注损伤模型。40只SD大鼠随机分为正常对照组、缺血/再灌注模型(I/R)组、异丙酚15、30、60μmol/L组。除正常对照外,各组分别平衡灌注20min后,常温全心停灌25min,再灌注30min。Powerlab/8s仪记录各组平衡末、缺血前及再灌30min时心率(HR)、左室发展压(LVDP)、左室舒张末压(LVDEP)、左室压力变化速率(±dp/dtmax)、冠脉流量(CF)等心功能指标;测定冠脉流出液中乳酸脱氢酶(LDH)、磷酸肌酸激酶(CK)活性;差速离心法提取心肌线粒体,测定线粒体活力、膜肿胀度、锰超氧化物歧化酶(Mn-SOD)活性和丙二醛(MDA)含量;原位末端转移酶标记法(TUNEL)检测心肌细胞凋亡,免疫组化法测定天冬氨酸特异的半胱氨酸蛋白酶(caspase)-3和热休克蛋白70(HSP70)的表达。结果异丙酚30、60μmol/L能明显改善缺血/再灌注后的心脏机械功能,降低冠脉流出液中LDH、CK的活性(P<0.05);异丙酚在30、60μmol/L浓度情况下心肌线粒体活力有所恢复,膜肿胀度减轻,Mn-SOD活性升高,MDA生成明显减少(P<0.05),心肌HSP70表达增多,心肌细胞凋亡率和caspase-3阳性细胞数明显减少(P<0.05)。结论异丙酚明显减轻缺血/再灌注所致的心肌线粒体的过氧化损伤,上调HSP70的表达,抑制caspase-3表达和心肌细胞凋亡的发生,可能是其心肌保护作用机制之一。     

高温诱导大肠癌细胞程序化死亡与p53基因转录表达改变的实验研究

目的：探讨Ｐ５３基因在高温诱导大肠癌细胞程序优化死亡过程中的转录表达变化,为高温治疗肿瘤提供分子生物学线索。方法选择几种常用高温治疗温度诱导大肠癌细胞呈现ＰＣＤ特征,应用免疫细胞化学和细胞原位杂交技术对含有不同类型Ｐ５３基因的ＨＴ－２９、Ｌｏｖｏ两大肠癌细胞Ｐ５３蛋白表达和Ｐ５３ｍＲＮＡ转录进行了对比分析。结果经４３℃处理３０ｍｉｎ后,两种细胞ｐ５３ｍＲＮＡ转录均有所增强。ＨＴ－２９细胞ｐ５３蛋白