吡格列酮对脂多糖作用下大鼠腹膜间皮细胞增殖、凋亡和TNF-a表达的影响

目的观察吡格列酮(PIO)对脂多糖(LPS)作用下大鼠腹膜间皮细胞(RPMC)细胞增殖、凋亡及其肿瘤坏死因子a(TNF-a)表达的影响。方法胰蛋白酶-EDTA消化法原代培养RPMC;并用MTT,流式细胞仪和ELISA法来分析吡格列酮对LPS影响下RPMC增殖、凋亡及其分泌TNF-α的影响。结果LPS能显著抑制RPMC的增殖(P0.01),促进RPMC的凋亡(P0.01),而5～20μmol/L的吡格列酮能部分逆转LPS对RPMC增殖的抑制作用(P0.01),抑制LPS所致的RPMC的凋亡(P0.01);LPS能显著增加RPMC表达TNF-a,而5～20μmol/L的吡格列酮能减弱LPS上调RPMC表达TNF-α的作用(P0.01)。结论吡格列酮可能通过逆转LPS对RPMC的增殖抑制作用,抑制LPS所致的RPMC的凋亡,减弱LPS上调RPMC表达炎症因子TNF-α的作用,进而促进RPMC的增殖修复,控制腹膜炎症,为改善间皮细胞损伤和防治腹膜透析相关腹膜炎提供实验依据。     

吡格列酮对脂多糖作用下大鼠腹膜间皮细胞增殖、凋亡和TNF-α表达的影响

目的 观察吡格列酮(PIO)对脂多糖(LPS)作用下大鼠腹膜间皮细胞(RPMC)细胞增殖、凋亡及其肿瘤坏死因子α(TNF-α)表达的影响.方法 胰蛋白酶-EDTA消化法原代培养RPMC;并用MTT,流式细胞仪和ELISA法来分析吡格列酮对LPS影响下RPMC增殖、凋亡及其分泌TNF-α的影响.结果 LPS能显著抑制RPMC的增殖(P<0.01),促进RPMC的凋亡(P<0.01),而5～20μmol/L的吡格列酮能部分逆转LPS对RPMC增殖的抑制作用(P<0.01),抑制LPS所致的RPMC的凋亡(P<0.01): LPS能显著增加RPMC表达TNF-α,而5～20μmol/L的吡格列酮能减弱LPS上调RPMC表达TNF-α的作用(P<0.01).结论 吡格列酮可能通过逆转LPS对RPMC的增殖抑制作用,抑制LPS所致的RPMC的凋亡,减弱LPS上调RPMC表达炎症因子TNF-α的作用,进而促进RPMC的增殖修复,控制腹膜炎症,为改善间皮细胞损伤和防治腹膜透析相关腹膜炎提供实验依据.     

普罗布考对脂多糖刺激下大鼠腹膜间皮细胞白细胞介素18的表达和对氧化应激的影响

目的 研究普罗布考对脂多糖(lipopolysaccharide,LPS)刺激下大鼠腹膜间皮细胞(rat peritoneal mesothelial cells,RPMC)白细胞介素18(interleukin-18,IL-18)和氧化应激产物丙二醛(malondialdehyde,MDA)表达的影响.方法原代培养RPMC,第3代腹膜间皮细胞达80%融合时进入试验.用无血清培养液培养24 h使细胞同步化.随机分组:①正常对照组:只加无血清DMEM/F12培养液;②LPS组:不同浓度LPS(1,10,100 mg/L)培养6 h;10 mg/L LPS分别作用于RPMC 0,3,6,12,24 h;③普罗布考组:20,40,80 μmol/L普罗布考预孵育1 h,加入10 mg/L LPS再作用11 h.用实时聚合酶链反应法检测IL-18 mRNA的表达,酶联免疫吸附法检测细胞上清液中IL-18的蛋白水平,硫代巴比妥酸法检测细胞中MDA的含量.结果与正常对照组比较,LPS刺激下RPMC IL-18和MDA的表达明显增加(P＜0.05),且呈浓度依赖性;随着刺激时间的延长,上述指标呈递增趋势,IL-18于12h达高峰(P＜0.01).与10 mg/L LPS组比较,普罗布考可抑制IL-18及MDA的表达(P＜0.05),随普罗布考浓度增加,抑制作用越强.结论 LPS作用下RPMC促炎症因子IL-18及氧化应激产物MDA表达增加,普罗布考能抑制上述指标的表达,修复间皮细胞的损伤,改善腹膜炎症状态.     

高糖对腹膜间皮细胞β-连环蛋白及骨桥蛋白表达的作用

目的 观察高糖对大鼠腹膜间皮细胞(rat peritoneal mesothelial cell RPMC)中β-连环蛋白(β-Catenin)、骨桥蛋白(Osteopontin,OPN)2者mRNA及蛋白表达的影响.方法 胰蛋白酶法行RPMC的原代培养和传代,经鉴定第3代细胞融合至80%时分组.分为:正常对照组;不同浓度葡萄糖(1.5%,2.5%,4.25%) 组作用24h;2.5% 高糖作用不同时间(8,12,24,34,48 h)组,细胞免疫组织化学法检测β-Catenin蛋白在细胞上的表达情况,RealTime-RT-PCR 技术检测β-Catenin、OPN、TGF-β1mRNA的表达.ELISA法检测OPN蛋白表达.结果高糖刺激下RPMC 的β-catenin表达增加,与0 h 组比较,在8h即出现(但不具有统计学意义),12h具有统计学意义,24h达到高峰,48h 仍存在,组间差异均有统计学意义(P＜0.05);高糖可刺激RPMC OPN 的表达显著增加,呈剂量、时间依赖性(P＜0.05);TGF-β表达增加,在8h表达于较高水平,12h降低,24h达到高峰,48h仍存在,与0 h组比较,组间差异均有统计学意义(P＜0.01);高糖刺激下,β-Catenin、TGF-β1 表达显著增加,呈剂量依赖性,组间差异具有统计学意义( P＜0.05).结论 高糖通过上调β-Catenin、OPN的表达,引起腹膜损伤导致腹膜纤维化,β-Catenin、OPN参与了腹膜间皮细胞透析相关性腹膜纤维化的病理过程.     

脂多糖对大鼠腹膜间皮细胞白细胞介素15、6及丙二醛表达的影响

目的研究脂多糖(lipopolysaccharide,LPS)对大鼠腹膜间皮细胞(rat peritoneal me-sothelial cells,RPMC)白细胞介素15(interleukin-15,IL-15)、IL-6及丙二醛(malondialdehyde,MDA)的影响。方法胰蛋白酶法行RPMC的原代培养和传代,经鉴定第3代用于试验。分组:①正常对照组;②LPS组:不同浓度LPS组(1、10、100mg/L)分别作用6h和12h;③不同时间组:10mg/L LPS分别作用于RPMC3、6、12、24h。实时定量PCR法测IL-15mRNA的表达;ELISA法检测细胞培养上清液中IL-15和IL-6的表达;硫代巴比妥法测MDA。结果 LPS可刺激RPMC的IL-15mRNA及蛋白的表达增高且在12h内呈时间剂量依赖性(P<0.05);LPS诱导RPMC IL-6的蛋白表达增高(P<0.05);LPS诱导RPMC MDA的含量表达增高且呈浓度依赖(P<0.05)。结论 IL-15作为炎症因子参与了腹膜透析相关性腹膜炎的病理过程。LPS可上调RPMC IL-15、IL-6和MDA的表达,导致腹膜炎症及氧化应激。     

晚期氧化蛋白产物通过活性氧诱导人腹膜间皮细胞TGF-β1的表达

目的探讨晚期氧化蛋白产物（AOPP）诱导体外培养的人腹膜间皮细胞（HPMCs）对转化生长因子（TGF-β1）表达的影响及内源性活性氧（ROS）在此过程中的调节机制。方法体外制备AOPP-HSA模型，原代培养人腹膜间皮细胞。采用ELISA法检测TGF-β1蛋白水平，采用RT-PCR半定量分析HPYCs中TGF-β1 mRNA的基因表达水平，同时应用流式细胞仪检测细胞内活性氧的水平。结果AOPP-HSA能显著诱导人腹膜间皮细胞TGF-β1的分泌与基因表达，同时增加细胞内ROS的生成，呈时间与剂量依赖关系（P〈0．01），不同浓度的抗氧化剂维生素E和N-乙酰半胱胺酸预处理细胞，可明显地降低细胞内ROS的生成量，同时显著地抑制AOPP-HSA诱导人腹膜间皮细胞TGF-β1的分泌与基因表达，呈剂量依赖关系（P〈0．01），其中维生素E（50μmol／L）组和NAC（10mmol／L）组抑制更加显著。结论体外制备的AOPP可显著诱导人腹膜间皮细胞TGF-β1的分泌与基因表达，部分可能通过内源性ROS介导细胞内信号转导途径调节此过程，抗氧化剂维生素E和N-乙酰半胱胺酸可显著降低细胞ROS的生成量和显著抑制TGF-β1的分泌与基因表达。此研究揭示抗氧化剂在防治腹膜纤维化发生过程也许是一种可行的治疗策略。     

PAMAM纳米载体介导pCTGF-shRNA抑制小鼠腹膜间皮细胞CTGF的表达

目的研究G9PAMAM纳米载体介导的结缔组织生长因子（CTGF）短发夹RNA（shRNA）质粒（pCTGF-shRNA）对小鼠腹膜间皮细胞CTGF表达的影响。方法设计并构建质粒pCTGF1-shRNA、pCTGF2-shRNA和阴性对照pHK-shRNA；应用G9PAMAM将质粒分别转染4．25％葡萄糖刺激下的第3代小鼠腹膜间皮细胞；采用逆转录多聚酶链式反应（RT-PCR）检测细胞CTGFmRNA水平，采用WesternBlot检测CTGF蛋白含量。结果4．25％葡萄糖可刺激小鼠腹膜间皮细胞CTGF的mRNA和蛋白表达明显上调。pcTGF1-shRNA转染组和pCTGF2-shRNA转染组的CTGF表达较高糖组明显下调（P〈0．05），尤其以pCTGF2-shRNA组显著。pHK-shRNA转染组与高糖组比较，差异无显著性（P〉0．05）。结论G9PAMAM纳米载体可介导pCTGF-shRNA转染原代小鼠腹膜间皮细胞，并能抑制高糖诱导的CTGF表达上调，提示PAMAM介导的pCTGF-shRNA可有效地用于腹膜纤维化的基因治疗。     

锌指蛋白A20对脂多糖诱导的大鼠腹膜间皮细胞炎症效应的影响

目的 探讨锌指蛋白A20（zinc finger protein A20）对脂多糖（LPS）诱导的大鼠腹膜间皮细胞（RPMCs）炎症效应的影响及可能机制. 方法 分离及培养RPMCs,常规传代及鉴定.取第2代细胞用于实验研究.将细胞随机分成对照组、LPS组、转染A20组、空载体组.脂质体转染A20质粒（pGEM-T easy A20）至RPMCs 12h后分别在LPS刺激不同时间点收获细胞提取蛋白及细胞上清液.用Western b]otting检测细胞TLR4、p-Iκ Bα、IκBα蛋白的表达;用ELISA法检测培养上清液IL-18蛋白水平. 结果 LPS刺激8h后,转染A20组RPMCs TLR4蛋白表达水平无明显增高,与对照组相比,P=0.223;与LPS组及空载体组相比,差异有显著性（P=0.003,0.002）.LPS刺激1h后,转染A20组RPMCs p-IκB α蛋白无明显降解,p-I κ Bα/I κ Bα比值与对照组相比,P=0.553.与LPS组及空载体组相比,差异有显著性（P=0.001,0.001）.在LPS刺激12h后,转染A20组RPMCs IL-18蛋白分泌水平（479.12±85.79）pg/ml高于对照组（274.34±47.21） pg/ml （P=0.012）,但明显低于LPS组（1049.45±185.01）pg/ml及空载体组（1028.77±192.90） pg/ml （P=0.011,0.015）.结论 A20通过对LPS信号通路中多个相关功能蛋白的负性调控作用,阻抑LPS诱导的RPMCs炎症效应.     

血红素加氧酶-1对高糖诱导大鼠腹膜间皮细胞转化生长因子β1和纤维连接蛋白表达的影响

目的研究血红素加氧酶-1（HO-1）对高糖作用下体外培养的大鼠腹膜间皮细胞（RPMC）转化生长因子β1（TGF-β1）和纤维连接蛋白（FN）表达的影响。方法将原代培养的第3代RPMC随机分为对照组、高糖组、钴原卟啉（CoPP）＋高糖组和CoPP组。同步培养72h后,以反转录聚合酶链反应（RT-PCR）法检测各组细胞TGF-β1和FNmRNA表达情况;酶联免疫吸附法（ELISA）检测细胞上清液中TGF-β1和FN的蛋白质水平。结果高糖组、CoPP＋高糖组和CoPP组的HO-1mRNA及蛋白表达水平均显著高于对照组,其中CoPP＋高糖组显著高于高糖组;高糖组和CoPP＋高糖组的FNmRNA及蛋白表达水平显著高于对照组及CoPP组;高糖组、CoPP＋高糖组TGF-β1mRNA及蛋白表达水平显著高于对照组及CoPP组,其中CoPP＋高糖组显著低于高糖组。结论 HO-1可抑制高糖诱导的RPMCTGF-β1和FN的表达,具有抗腹膜纤维化的作用。     

短发夹结构RNA对人腹膜间皮细胞转化生长因子-β1表达和超微结构的影响

目的 研究靶向转化生长因子-β1（TGF-β1）的短发夹RNA（shRNA）表达载体对腹膜透析液诱导的人腹膜间皮细胞（HPMC）表达TGF-β1的影响,并观察其对HPMC超微结构的影响. 方法 利用pcDU6质粒构建两个靶向TGF-β1的shRNA真核表达载体pcDU6-A1-A2和pcDU6-B1-B2,使用脂质体介导转染腹膜透析液刺激下的HPMC,采用半定量逆转录多聚酶链式反应（RT-PCR）和酶联免疫吸附（ELISA）检测shRNA表达载体对于TGF-β1基因和蛋白质表达水平的抑制效果,使用透射电镜观察HPMC超微结构的改变. 结果 HPMC暴露于4.25%腹膜透析液中48小时后,其上清中TGF-β1的浓度与对照组相比显著增高分别为（39.71±6.60）pg/10^5 cells和（16.32±2.71）pg/10^5 cells,P＜0.01）,1.5%腹膜透析液对于TGF-β1蛋白合成无明显影响;而预先转染了shRNA载体的两组与转染pcDU6载体（空载体）的组相比,其由4.25%腹膜透析液诱导的TGF-β1蛋白增高幅度明显减少,分别下降了39.2%和47.2%（P＜0.01）;两组转染不同的shRNA载体的HPMC之间相比TGF-β1浓度无显著差异,pcDU6载体（空载体）转染组与4.25%腹膜透析液刺激组TGF-β1浓度亦无显著差异.透射电镜显示HPMC暴露于4.25%腹膜透析液可导致细胞扁平、微绒毛减少和线粒体水肿等,而转染shRNA载体组细胞的改变相对较轻. 结论 pcDU6质粒载体介导的短发夹结构RNA（shRNA）能够明显抑制腹膜透析液刺激下的人腹膜间皮细胞TGF-β1的高表达,并减轻其超微结构的异常,提示shRNA表达载体可能有益于腹膜纤维化的防治.     

Cardiac-specific overexpression of insulin-like growth factor I (IGF-1) rescues lipopolysaccharide-induced cardiac dysfunction and activation of stress signaling in murine cardiomyocytes

Lipopolysaccharide (LPS), a component of the outer membrane of Gram-negative bacteria, plays a key role in cardiac dysfunction in sepsis. Low circulating levels of insulin-like growth factor 1 (IGF-1) are found in sepsis, although the influence of IGF-1 on septic cardiac defect is unknown. This study was designed to examine the impact of IGF-1 on LPS-induced cardiac contractile and intracellular Ca2+ dysfunction, activation of stress signal and endoplasmic reticulum (ER) stress. Mechanical and intracellular Ca2+ properties were examined in cardiomyocytes from Fast Violet B and cardiac-specific IGF-1 overexpression mice treated with or without LPS (4 mg kg(-1), 6 h). Reactive oxygen species (ROS), protein carbonyl formation and apoptosis were measured. Activation of mitogen-activated protein kinase pathways (p38, c-jun N-terminal kinase [JNK] and extracellular signal-related kinase [ERK]), ER stress and apoptotic markers were evaluated using Western blot analysis. Our results revealed decreased peak shortening and maximal velocity of shortening/relengthening and prolonged duration of relengthening in LPS-treated Fast Violet B cardiomyocytes associated with reduced intracellular Ca2+ decay. Accumulation of ROS protein carbonyl and apoptosis were elevated after LPS treatment. Western blot analysis revealed activated p38 and JNK, up-regulated Bax, and the ER stress markers GRP78 and Gadd153 in LPS-treated mouse hearts without any change in ERK and Bcl-2. Total protein expression of p38, JNK, and ERK was unaffected by either LPS or IGF-1. Interestingly, these LPS-induced changes in mechanical and intracellular Ca2+ properties, ROS, protein carbonyl, apoptosis, stress signal activation, and ER stress markers were effectively ablated by IGF-1. In vitro LPS exposure (1 microg mL(-1)) produced cardiomyocyte mechanical dysfunction reminiscent of the in vivo setting, which was alleviated by exogenous IGF-1 (50 nM). These data collectively suggested a beneficial of IGF-1 in the management of cardiac dysfunction under sepsis.     

华蟾素对人膀胱肿瘤T24细胞作用机制的实验研究

目的：研究华蟾素对人膀胱肿瘤T24细胞的作用及其机制。方法 MTT法研究华蟾素对细胞的增殖作用;将细胞分为空白对照、Caspase抑制剂、华蟾素、华蟾素＋Caspase抑制剂4组,流式细胞术分析各组细胞凋亡及细胞周期,RT-qPCR法检测各组细胞AIF、Endo G mRNA表达情况。结果华蟾素能抑制T24细胞增殖;与空白组和Caspase抑制剂组相比,华蟾素组与华蟾素＋Caspase抑制剂组细胞凋亡明显（P〈0.05）,细胞周期主要阻断在S期,并且两组细胞内AIF、Endo G mRNA表达明显上调（P〈0.05）。结论华蟾素能抑制T24细胞增殖,通过Caspase依赖途径及Caspase非依赖途径诱导T24细胞凋亡。     

Mesangial cell apoptosis induced by stimulation of the adenosine A3 receptor: signaling and apoptotic events.

Mesangial cell apoptosis has been proposed as a means of resolution of glomerular hypercellularity in proliferative forms of glomerular disease. We previously demonstrated that adenosine causes mesangial cell apoptosis by stimulating the A3-type adenosine receptor. This is a G protein-coupled receptor shown to activate kinases involved in apoptotic signaling. In this work, we assessed changes in phosphorylation of the mitogen-activated protein kinase extracellular signal-regulated kinase (ERK)1/2 and in levels of specific pro- and antiapoptotic proteins following exposure of mesangial cells to the A3 adenosine receptor agonist N(6)-(3-iodobenzyl)-adenosine-5'-N-methyluronamide (IB-MECA). Cultured mesangial cells were incubated with IB-MECA for 30 minutes and 6, 24, and 48 hours. IB-MECA was used at a concentration (30 microM) that induces a reproducible degree of mesangial cell apoptosis. Changes in ERK1/2 phosphorylation and in protein levels of Bcl-2, Bax, and caspase 3 were assessed by Western blot analysis. IB-MECA markedly increased phosphorylation of ERK1/2. This effect peaked at 5 minutes, dissipated by 20 minutes, and was abolished by the inhibitor of ERK phosphorylation, compound U0126, in a dose-dependent manner. This inhibitor had no effect on the extent of IB-MECA-induced apoptosis. Bcl-2 levels progressively declined, whereas those of Bax and activated caspase 3 increased. These observations indicate that stimulation of the A3-type adenosine receptor causes mesangial cell apoptosis via mechanisms independent of ERK activation. The observations also point to an imbalance in the expression of antiapoptotic (Bcl-2) and proapoptotic (Bax, caspase 3) proteins as a potential mechanism underlying adenosine-induced mesangial cell apoptosis.     

Curcumin alleviates lipopolysaccharide induced sepsis and liver failure by suppression of oxidative stress-related inflammation via PI3K/AKT and NF-κB related signaling

In many liver disorders, oxidative stress-related inflammation and apoptosis are important pathogenic components, finally resulting in acute liver failure. Erythropoietin and its analogues are well known to influence the interaction between apoptosis and inflammation in brain and kidney. The study is to clarify the effect of curcumin, a natural plant phenolic food additive, on lipopolysaccharides (LPS)-induced acute liver injury of mice with endotoxemia and associated molecular mechanism from inflammation, apoptosis and oxidative stress levels. And curcumin, lowered serum cytokines, including Interleukin 1beta (IL-1β), Interleukin 6 (IL-6) and tumor necrosis factor (TNF-α), and improved liver apoptosis through suppressing phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) signaling pathway and inhibiting Cyclic AMP-responsive element-binding protein (CREB)/Caspase expression, and decreased oxidative stress-associated protein expression, mainly involving 2E1 isoform of cytochrome P450/nuclear factor E2-related factor 2/reactive oxygen species (CYP2E/Nrf2/ROS) signaling pathway, as well as liver nitric oxide (NO) production in LPS-induced mice. Moreover, curcumin regulated serum alanine transaminase (ALT), aspartate transaminase (AST) and alkaline phosphatase (ALP), accelerated liver antioxidant enzymes, such as superoxide dismutase (SOD), catalase (CAT), glutathione (GSH) and glutathione peroxidase (GSH-px) levels, and inhibited activation of the mitogen-activated protein kinases/c-Jun NH2-terminal kinase (P38/JNK) cascade in the livers of LPS-induced rats. Thus, curcumin treatment attenuates LPS-induced PI3K/AKT and CYP2E/Nrf2/ROS signaling and liver injury. Strategies to inhibit inflammation and apoptosis signaling may provide alternatives to the current clinical approaches to improve oxidative responses of endotoxemia.     

藤黄酸通过MAPK通路诱导Jurkat细胞凋亡的机理

本研究探讨藤黄酸对Jurkat细胞的抑制作用及可能机理。以不同浓度藤黄酸与抑制剂处理Jurkat细胞,AnnexinⅤ/PI双染法检测细胞的凋亡;流式细胞仪检测荧光素激活的caspase 3、caspase 8、caspase 9阳性细胞水平;免疫印迹检测pro-caspase 3、pro-caspase 9、磷酸化JNK及P38蛋白水平。结果表明,藤黄酸呈浓度依赖性诱导Jurkat细胞凋亡;藤黄酸作用Jurkat细胞后caspase 3、caspase 8、caspase 9阳性细胞水平升高,caspase 3、caspase 9活化蛋白及磷酸化JNK及P38蛋白水平升高;ROS、CaMKⅡ、caspase 3、caspase 9、MAPKK、JNK1/2及P38抑制剂减弱藤黄酸诱导Jurkat细胞凋亡的作用;ROS、CaMKⅡ、MAPKK、JNK1/2及P38抑制剂降低藤黄酸作用Jurkat细胞后caspase 3、caspase 9活化蛋白水平;ROS、CaMKⅡ、MAPKK、JNK1/2抑制剂降低藤黄酸作用Jurkat细胞后磷酸化JNK蛋白水平;ROS、CaMKⅡ、MAPKK、P38抑制剂减弱藤黄酸作用Jurkat细胞后磷酸化P38蛋白水平。结论:藤黄酸通过激活caspase途径诱导Jurkat细胞凋亡,该过程可能与ROS-CaMKⅡ-MAPKK-JNK/P38通路有关。     

Rapamycin and UCN-01 synergistically induce apoptosis in human leukemia cells through a process that is regulated by the Raf-1/MEK/ERK, Akt, and JNK signal transduction pathways

Interactions between the protein kinase C and Chk1 inhibitor UCN-01 and rapamycin in human leukemia cells have been investigated in relation to apoptosis induction. Treatment of U937 monocytic leukemia cells with rapamycin (10 nmol/L) in conjunction with a minimally toxic concentration of UCN-01 (100 nmol/L) for 36 hours resulted in marked potentiation of mitochondrial injury (i.e., loss of mitochondrial membrane potential and cytosolic release of cytochrome c, AIF, and Smac/DIABLO), caspase activation, and apoptosis. The release of cytochrome c, AIF, and Smac/DIABLO were inhibited by BOC-D-fmk, indicating that their release was caspase dependent. These events were associated with marked down-regulation of Raf-1, MEK, and ERK phosphorylation, diminished Akt activation, and enhanced phosphorylation of c-Jun NH2-terminal kinase (JNK). Coadministration of UCN-01 and rapamycin reduced the expression levels of the antiapoptotic members of the Bcl-2 family Mcl-1 and Bcl-xL and diminished the expression of cyclin D1 and p34(cdc2). Furthermore, enforced expression of a constitutively active MEK1 or, to a lesser extent, myristoylated Akt construct partially but significantly attenuated UCN-01/rapamycin-mediated lethality in both U937 and Jurkat cell systems. Finally, inhibition of the stress-related JNK by SP600125 or by the expression of a dominant-negative mutant of c-Jun significantly attenuated apoptosis induced by rapamycin/UCN-01. Together, these findings indicate that the mammalian target of rapamycin inhibitor potentiates UCN-01 cytotoxicity in a variety of human leukemia cell types and suggest that inhibition of both Raf-1/MEK/ERK and Akt cytoprotective signaling pathways as well as JNK activation contribute to this phenomenon.