Hoechst 33342 induces apoptosis in HL-60 cells and inhibits topoisomerase I in vivo.

CONTEXT: Bisbenzimides (Hoechst 33342 and Hoechst 33258) are cell-permeable, adenine-thymine-specific dyes that bind to the minor groove of DNA and stain DNA. Hoechst 33342 induces apoptosis in BC3H-1 myocytes and hepatoma cells. OBJECTIVE: To determine if Hoechst 33342 or Hoechst 33258 induces apoptosis in human promyelocytic leukemia cells (HL-60) and inhibits topoisomerase I activity. DESIGN: A variety of methods were used to detect apoptosis: cell viability (trypan blue exclusion), nuclear fluorescence staining (Hoechst 33342 or Hoechst 33258 stained for 10 minutes), flow cytometric quantitation of annexin binding to phosphatidylserine, and DNA fragmentation (agarose gel electrophoresis). Topoisomerase I activity was determined by a plasmid unwinding assay. SETTING: A large teaching hospital and research laboratories. PATIENTS: None. INTERVENTION: None. MAIN OUTCOME MEASUREMENTS: Apoptosis is characterized by decreased cell viability, condensation of nuclear chromatin, increased phosphatidylserine translocation, and DNA fragmentation into oligonucleosomes composed of multiples of 180 to 200 base pairs. Inhibition of endogenous nuclear topoisomerase I is detected by the absence of plasmid unwinding from a tightly coiled to relaxed form. RESULTS: Hoechst 33342, but not Hoechst 33258, induced apoptosis in the HL-60 cells in a time- and dose-dependent manner. Endogenous nuclear topoisomerase I activity in HL-60 cells was inhibited by treatment with Hoechst 33342 but not Hoechst 33258. CONCLUSION: Hoechst 33342-induced HL-60 cell apoptosis may be related to the dye's inhibition of topoisomerase I activity.     

Hoechst 33342-induced apoptosis is associated with decreased immunoreactive topoisomerase I and topoisomerase I-DNA complex formation

Hoechst 33342, but not Hoechst 33258, induces apoptosis and inhibits topoisomerase 1 activity in vivo. Topoisomerase I relaxes superhelical DNA through a single strand breakage/rejoining reaction in which the active site tyrosine links covalently to a 3' phosphate at the break site, forming a transient intermediate called a cleavable complex. The fate of cellular topoisomerase 1 in Hoechst 33342-induced apoptosis is unknown. We analyzed the binding capacity of topoisomerase 1 to 32P-labeled plasmid pCI DNA, the immunoreactive topoisomerase 1 concentration and topoisomerase 1 activity in BC3H-1 myocytes and HL-60 cells treated with Hoechst 33342 and Hoechst 33258 by using covalent transfer of 32P radioactivity from plasmid DNA to topoisomerase 1, Western blotting and topoisomerase 1-mediated plasmid relaxation assay, respectively. Hoechst 33342, but not Hoechst 33258, induced topoisomerase 1 dysfunction in both BC3H-1 myocytes and HL-60 cells measured by (1) a decrease in the topoisomerase 1 to DNA binding capacity or cleavable complex formation; (2) a decrease in intracellular concentration of immunoreactive topoisomerase 1; and (3) an inhibition of nuclear endogenous topoisomerase 1 activity. These results suggest that destruction of immunoreactive topoisomerase 1 and topoisomerase 1-DNA complexes or cleavable complexes results in inhibition of topoisomerase 1 activity, a key step in the Hoechst 33342-induced apoptotic process.     

Fatty acid synthase and its mRNA concentrations are decreased at different times following Hoechst 33342-induced apoptosis in BC3H-1 myocytes

Fatty acid synthase (FAS) regulates the production of fatty acids and plays a role in regulating apoptosis. Hoechst 33342-induced apoptosis in BC3H-1 myocytes was used as a model to explore intracellular changes in FAS protein (Western blot) and FAS mRNA (RT-PCR). Total lipid and individual phospholipid synthesis was inhibited by a lethal dose of Hoechst 33342 (20 microg/ml) while total lipid and phospholipid degradation ([1-14C]-acetate pulse chase method) were not. Hoechst 33342 at 20 microg/ml reduced the concentration of FAS protein, which was followed more than 6 hr later by a reduction in FAS mRNA. In conclusion, the inhibition of fatty acid synthesis induced by 20 microg/ml of Hoechst 33342 is attributed to the degradation of FAS protein by activated caspases rather than by inhibition of FAS enzyme activity or FAS mRNA synthesis.     

Mitochondrial membrane potential change induced by Hoechst 33342 in myelogenous leukemia cell line HL-60

Abstract. Hoechst 33342's effects on apoptosis and mitochondrial membrane potential (delta psi) were investigated in a myelogenous leukemia cell line, HL-60. Delta psi was detected with 2 lipophilic cationic fluorochromes: 3,3'-dihexyloxacarbocyanine iodide [DiOC6(3)] or 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide (JC-1). Mitochondrial mass was measured with nonyl acridine orange (NAO). Protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP) depolarized mitochondria in control experiments. Cell viability was determined by propidium iodide uptake. Hoechst 33342 at 10-20 mg/L decreased fluorescence for DiOC6(3) at 0.5 hr. The fluorescence partially normalized at 3 hr and then progressively decreased at 5-24 hr, resulting in cell shrinkage and death. Mitochondrial mass decreased 40-70% by 1 hr and 70-90% at 24 hr. A lower concentration of Hoechst 33342, 5 mg/L, reduced the delta psi at 0.5 hr, but delta psi returned to control values after 3 hr. Mitochondrial mass decreased 30-40% and then partially normalized, and cell viability was > 92% at 24 hr. Protonophore carbonyl cyanide m-chlorophenylhydrazone lowered delta psi with little cell death. Thus, at high concentration, Hoechst 33342 induces depolarization of delta psi and subsequent apoptosis. Lack of apoptosis at low concentration of Hoechst 33342, despite depolarization of delta psi, indicates that mitochondrial membrane depolarization alone is insufficient to induce apoptosis.     

肽基脯氨酰顺反异构酶抑制剂胡桃醌对宫颈鳞癌SiHa细胞凋亡的作用

目的:探讨肽基脯氨酰顺反异构酶抑制剂胡桃醌对宫颈鳞癌Si Ha细胞增殖和凋亡的影响及其促凋亡的机制。方法:选取处于对数生长期的Si Ha细胞,将其分为空白对照组和不同剂量(10、20、50、80及100μmol/L)胡桃醌给药组,共6组。采用MTT法观察胡桃醌对Si Ha细胞的抑制作用,并计算出半数抑制浓度(IC50=20.4μmol/L),依据IC50确定胡桃醌的有效浓度;选取与IC50接近的浓度(20μmol/L)通过Hoechst 33258及流式细胞术测定胡桃醌对Si Ha细胞凋亡的影响,Western blot检测凋亡相关蛋白caspase-3、8、9以及抑癌基因PTEN的蛋白水平。结果:MTT实验结果显示,与对照组比较,各给药组对细胞活力的抑制作用明显,差异有统计学意义(P0.05);Hoechst 33258检测表明,20μmol/L胡桃醌处理细胞12 h后可出现明显的细胞核典型凋亡细胞形态,流式细胞术检测结果说明胡桃醌可诱导Si Ha细胞出现早期凋亡;Western blot检测显示与正常对照组相比,20μmol/L胡桃醌处理细胞12 h后,cleaved caspase-3、8、9及PTEN的蛋白水平明显增加。结论:胡桃醌可以抑制Si Ha细胞的活力,并诱导细胞凋亡,其机制主要是通过抑制caspase途径并增加抑癌基因的表达。     

幽门螺杆菌对胃上皮细胞SGC-7901凋亡的影响

目的：观察幽门螺杆菌（H. pylori）能否诱导体外培养的胃上皮细胞发生凋亡。方法：采用荧光染色、流式细胞仪和透射电子显微镜技术,观察H. pylori活菌在体外对人胃腺癌细胞系SGC-791凋亡形态与凋亡率的影响。结果：Hoechst 33258荧光染色和流式细胞术两种方法所得结果一致,3.2×10⁷ CFU/L的H. pylori组与对照组的细胞凋亡率无显著差异（P>0.05）,当H. pylori的浓度1.6×10⁸ CFU/L时,随着H. pylori浓度的加大,细胞凋亡率越来越高,均与对照组有显著差异（P<0.01）;细胞110CFU/L的H. pylori共育48 h后,在透射电镜下可见到部分细胞呈现凋亡形态：核中染色质不规则凝集,核膜向多处伸出指状突起,另外还可见胞浆浓缩成均质状,可形成胞浆凋亡小体。结论：H. pylori活菌能以浓度依赖方式诱导体外培养的SGC-791发生凋亡。     

miRNA-7介导Bax和Bcl-2表达对人鼻咽癌CNE-1细胞凋亡的影响

 目的:研究过表达微小RNA-7(microRNA-7,miRNA-7)诱导人鼻咽癌(nasopharyngeal carcinoma,NPC)CNE-1细胞凋亡及其与Bax和Bcl-2表达之间的关联情况。方法:体外利用脂质体Lipofectamine 2000将miRNA-7模拟物转染到人鼻咽癌CNE-1细胞中;采用real-time PCR法检测转染后各实验组细胞中miRNA-7的相对表达情况;CCK-8法检测各组细胞活力的改变;在荧光显微镜下利用Hoechst 33258荧光染色法观察转染后细胞的凋亡;real-time PCR法检测Bax和Bcl-2的mRNA表达水平;Western blot法检测Bax和Bcl-2蛋白表达水平。结果:Real-time PCR结果表明,转染miRNA-7模拟物的CNE-1细胞中其miRNA-7的相对表达水平明显高于无关序列组和空白对照组(P<0.01);转染miRNA-7模拟物后,CNE-1细胞的活力明显下降(P<0.01);Hoechst 33258染色检测可发现典型的凋亡细胞核形态学变化;real-time PCR及Western blot结果显示,转染miRNA-7模拟物的CNE-1细胞中Bax的mRNA及蛋白显著上调(P<0.01),而Bcl-2的mRNA及蛋白则显著下调(P<0.01)。结论:过表达miRNA-7可以通过调节Bax/Bcl-2之间的比例关系来抑制鼻咽癌CNE-1细胞的恶性生长,并促进细胞凋亡。     

靶向COL1A1基因的 shRNA对人乳腺癌MDA-MB-231细胞增殖与凋亡的影响

 目的：探讨抑制Ⅰ型胶原α1链（COL1A1）基因表达对人乳腺癌MDA-MB-231细胞增殖及凋亡的影响。方法：用已构建的pSilencer2.1-U6-COL1A1重组质粒转染乳腺癌细胞MDA-MB-231,采用RT-PCR和Western blotting技术检测重组质粒对COL1A1基因表达的影响,MTT比色法测定细胞增殖的抑制情况,流式细胞术检测细胞周期和细胞凋亡的变化,Hoechst 33258荧光染色观察细胞凋亡的形态学变化。结果：RT-PCR及Western blotting结果证实重组质粒在mRNA和蛋白水平分别显著抑制COL1A1基因表达;pshRNA-COL1A1转染组细胞的增殖速度明显减慢且呈时间依赖关系;与对照组相比,pshRNA-COL1A1组细胞明显阻滞于G₀/G₁期,细胞凋亡率显著升高（P<0.05）,出现细胞质浓缩、核凝聚等凋亡形态学变化。结论：pshRNA-COL1A1能有效抑制乳腺癌MDA-MB-231细胞增殖,并诱导其凋亡,为以COL1A1为靶点的乳腺癌基因治疗提供实验依据。     

噻可拉林协同顺铂促进宫颈癌c4-1及Hela细胞凋亡机制的研究

目的：探讨噻可拉林协同顺铂促进宫颈癌c4-1、Hela细胞凋亡的机制。方法：用不同浓度噻可拉林和顺铂单独或联合作用于体外培养的c4-1、Hela细胞,MTT法检测c4-1、Hela细胞生长抑制率,Hoechst 33342 和AO-EB染色法检测细胞凋亡,Western blot检测细胞Bax、Bcl2、p53、p21蛋白的表达,流式细胞术检测细胞增殖周期。结果：c4-1及Hela细胞经不同浓度的噻可拉林和顺铂单独或联合作用后,细胞的体外增殖活力被明显抑制,呈剂量依赖关系,且在联合使用噻可拉林（10 nmol/L）：顺铂（4 μmol/L）用量时抑制效果最明显（P<0.05）;AO-EB染色法显示,噻可拉林和顺铂联合使用,细胞凋亡更明显。Western blot则显示,Bax、p53、p21表达明显上调,而Bcl2的表达则明显下调。结论：噻可拉林能协同顺铂上调Bax、p53、p21的表达和下调Bcl2的表达,抑制宫颈癌c4-1、Hela细胞增殖同时促进细胞凋亡。     

Actinobacillus actinomycetemcomitans Leukotoxin Induces Apoptosis in HL-60 Cells

Actinobacillus actinomycetemcomitans leukotoxin (Ltx) is a member of the repeats-in-toxin (RTX) family of pore-forming toxins and kills human immune cells. Currently, it remains unclear whether toxin-mediated killing of target cells involves the induction of necrosis or apoptosis. Therefore, the goal of this investigation was to determine whether Ltx is capable of causing apoptotic cell death in toxin-sensitive promyelocytic HL-60 cells. Multiparameter flow cytometric analysis of toxin-treated cells stained with Hoechst 33258 (or 33342) and 7-aminoactinomycin D allowed us to identify four populations: viable cells, early apoptotic cells, late apoptotic and/or secondarily necrotic cells, and a final population that was composed of cellular debris. Compared with control cells, HL-60 cells treated with Ltx exhibited a gradual decrease in forward light scatter with a coincident increase in side light scatter, indicative of a decrease in cell size and organelle condensation, respectively. Additional experiments demonstrated that Ltx-treated cells showed evidence of internucleosomal DNA fragmentation and phosphatidylserine translocation. The results of our studies clearly demonstrate that Ltx can kill HL-60 cells by inducing apoptosis. We hypothesize that elimination of acute inflammatory cells via this mechanism plays a critical role in the pathogenesis of diseases caused by A. actinomycetemeomitans.     

吲哚美辛通过Akt/GSK3β/NAG-1信号通路诱导胃癌细胞的凋亡

目的: 探讨非选择性环氧合酶抑制剂吲哚美辛诱导胃癌细胞凋亡作用与Akt/GSK3β/NAG-1信号通路的关系。方法: MTT法测定胃癌细胞的活力;Hoechst 33258 染色检测细胞形态学改变,流式细胞术检测DNA 含量的变化,Western blotting检测蛋白表达的变化。结果: 吲哚美辛通过caspase通路诱导胃癌细胞MGC-803的凋亡,明显降低Akt和GSK3β的磷酸化水平,同时上调NAG-1的表达。单独抑制PI3K或Akt的活性亦可上调NAG-1的表达,而GSK3β抑制剂预处理后则取消吲哚美辛上调NAG-1表达的作用。结论: 吲哚美辛可通过Akt/GSK3β/NAG-1信号通路诱导胃癌细胞MGC-803的凋亡。     

Expression of p57(KIP2) potently blocks the growth of human astrocytomas and induces cell senescence

Astrocytic tumors frequently exhibit defects in the expression or activity of proteins that control cell-cycle progression. Inhibition of kinase activity associated with cyclin/cyclin-dependent kinase co-complexes by cyclin-dependent kinase inhibitors is an important mechanism by which the effects of growth signals are down-regulated. We undertook the present study to determine the role of p57(KIP2) (p57) in human astrocytomas. We demonstrate here that whereas p57 is expressed in fetal brain tissue, specimens of astrocytomas of varying grade and permanent astrocytoma cell lines do not express p57, and do not contain mutations of the p57 gene by multiplex-heteroduplex analysis. However, the inducible expression of p57 in three well-characterized human astrocytoma cell lines (U343 MG-A, U87 MG, and U373 MG) using the tetracycline repressor system leads to a potent proliferative block in G(1) as determined by growth curve and flow cytometric analyses. After the induction of p57, retinoblastoma protein, p107, and E2F-1 levels diminish, and retinoblastoma protein is shifted to a hypophosphorylated form. Morphologically, p57-induced astrocytoma cells became large and flat with an expanded cytoplasm. The inducible expression of p57 leads to the accumulation of senescence-associated beta-galactosidase marker within all astrocytoma cell lines such that approximately 75% of cells were positive at 1 week after induction. Induction of p57 in U373 astrocytoma cells generated a small population of cells ( approximately 15%) that were nonviable, contained discrete nuclear fragments on Hoechst 33258 staining, and demonstrated ultrastructural features characteristic of apoptosis. Examination of bax and poly-(ADP ribose) polymerase levels showed no change in bax, but decreased expression of poly-(ADP ribose) polymerase after p57 induction in all astrocytoma cell lines. These data demonstrate that the proliferative block imposed by p57 on human astrocytoma cells results in changes in the expression of a number of cell cycle regulatory factors, cell morphology, and a strong stimulus to cell senescence.     

Trx-ASK1在多柔比星诱导的乳鼠心肌细胞凋亡中的作用

目的: 探讨硫氧还蛋白-凋亡信号调节激酶1(Trx-ASK1)在多柔比星(doxorubicin,DOX)诱导的乳鼠心肌细胞凋亡中的作用。方法: 体外分离原代新生SD大鼠心肌细胞,待细胞可见搏动率大于95%、预孵育抗氧化剂ebselen(Ebs)后,给予DOX(1 μmol/L)作用24 h。 MTT检测细胞存活率;活性氧簇(ROS)敏感的荧光探针2',7'-二氯二氢荧光素二乙酯 (H₂DCFDA)检测细胞内ROS含量;细胞凋亡荧光Hoechst 33258试剂盒进行凋亡检测;凋亡试剂盒检测caspase-3的活性;Western blotting法检测聚腺苷酸二磷酸核糖聚合酶1 、ASK1、p-ASK1、p38和p-p38;免疫沉淀法检测Trx-ASK1是否发生分离。结果: 乳鼠心肌细胞给予DOX后细胞内ROS量明显增多,荧光染色可以观察到明显凋亡,给予抗氧化剂Ebs后凋亡得到明显改善。与正常组相比,凋亡组caspase-3活性明显升高(P<0.01);PARP1、ASK1和p38等蛋白活化形式表达增加(P<0.01);Trx-ASK1的分离显著增加(P<0.01)。Ebs保护组与凋亡组相比,caspase-3活性明显降低(P<0.05);PARP1、ASK1和p38等蛋白活化形式表达减少(P<0.01);Trx-ASK1分离明显减少(P<0.05)。结论: 给予DOX后心肌细胞可发生明显的凋亡,Trx与ASK1发生了一定程度的分离,从而使得ASK1介导的凋亡信号通路参与了该凋亡过程,给予Ebs后凋亡得到明显改善。本研究充分说明Trx-ASK1在DOX诱导心肌细胞凋亡过程中发挥着重要的作用,这将有助于进一步研究DOX诱导心肌细胞凋亡的机制。     

肺炎嗜衣原体CPAF诱导THP-1细胞产生前炎症细胞因子和凋亡

目的 研究肺炎嗜衣原体(Chlamydophila pneumoniae,Cpn)衣原体蛋白酶样活性因子(Chlamydial protease-like activity factor,CPAF)能否在体外诱导人单核细胞THP-1产生前炎症细胞因子和凋亡,为进一步探索Cpn感染宿主致病的分子机制提供实验依据.方法 将Cpn CPAF全基因克隆于pGEX6p-2载体上,在大肠杆菌(E coli)中诱导表达重组蛋白,经去内毒素纯化柱和琼脂糖凝胶FF获得纯化且无脂多糖(LPS)污染的重组蛋白GST-CPAF.以不同浓度的该蛋白作用于THP-1,ELISA检测TNF-α吨和IL-6水平.MTT法检测经该蛋白处理后THP-1的增生或抑制作用,用Hoechst33258荧光染色、DNA片段化分析、Armexin V-FITC-PI染色法检测细胞凋亡情况.结果 制备的莺组蛋白GST-CPAF以时间和剂量依赖方式刺激THP-1分泌TNF-α和IL-6,并以剂量依赖方式抑制THP-1增殖;当GST-CPAF刺激THP-1细胞24 h后,能诱导细胞调亡.结论 制备的Cpn重组蛋白GST-CPAF能诱导THP-1产生前炎症细胞因子和凋亡,可能是Cpn致病机制中的一个因素.     

过表达E2F1保护神经元依赖其转录活性

目的探讨E2F1对小脑颗粒神经元死活的影响及机制。方法构建表达质粒pcDNA-E2F1(pE2F1)及突变体pcDNA-E2F1-m132(pm132),转染293细胞验证表达,转染小脑颗粒神经元用6×E2F-luciferase报道基因检测转录活性。转染神经元24 h或48 h后Hoechst 33258核染色,统计转染pE2FI及pm132神经元(GFP阳性细胞)的核固缩率(即凋亡率);或者转染48 h后进行25K、5K处理12hr,统计凋亡率。结果构建质粒表达E2F1及突变体E2F1-m132成功,E2F1具有转录活性而E2F1-m132没有;表达E2F1及E2F1-m132均未诱导神经元凋亡(P>0.05);表达E2F1抑制5K诱导的神经元凋亡(P<0.05),而E2F1-m132没有抑制效应。结论E2F1抑制神经元凋亡依赖其转录活性。     

MDA-7/IL-24对乳腺癌MDA-MB-231细胞生长抑制和凋亡的影响

 摘要：目的 探讨IL-24基因转染乳腺癌MDA-MB-231细胞的增殖抑制和促凋亡作用。方法 利用脂质体将穿梭质粒pDC316-hIL-24-EGFP瞬时转染至乳腺癌MDA-MB-231细胞,通过RT-PCR检测转染后hIL-24基因mRNA的转录,Western blot检测转染后IL-24和Caspase-3蛋白的表达,MTT比色法测定细胞增殖的抑制, 流式细胞仪检测细胞的凋亡和细胞周期的变化,Hoechst 33258染色检测细胞的凋亡情况。结果IL-24基因可在MDA-MB-231细胞中成功转录及表达。IL-24可上调MDA-MB-231细胞中Caspase-3蛋白表达。IL-24基因的表达使得乳腺癌MDA-MB-231细胞出现增殖抑制。流式细胞仪检测显示MDA-MB-231细胞DNA 合成受到抑制,细胞周期主要抑制在G2/M期。Hoechst 33258染色显示IL-24基因转染后MDA-MB-231细胞出现凋亡。结论IL-24基因转染乳腺癌MDA-MB-231细胞后能够抑制细胞增殖并促进其凋亡,其机制之一可能是上调Caspase-3的表达。     

NOD8对H2O2诱导的人肝细胞凋亡的影响

 目的:探讨NOD8对H2O2诱导的人肝细胞L02凋亡的影响。 方法: pEGFP-C2 及pEGFP-NOD8重组质粒经JetPRIME介导转染L02细胞;用H2O2诱导细胞凋亡。实验分为pEGFP-C2组、pEGFP-C2+H2O2组和pEGFP-NOD8+H2O2组。采用MTT法检测细胞活性,Western blotting检测细胞NOD8的蛋白表达,Hoechst 33342染色检测细胞凋亡情况,流式细胞术检测细胞凋亡率,比色法检测细胞caspase-3活性。 结果: 通过MTT检测不同浓度（0.2～2 mmol/L）H2O2刺激6 h后的细胞活性,确定1 mmol/L H2O2为诱导细胞凋亡的剂量。Western blotting检测结果显示,转染pEGFP-NOD8质粒的细胞NOD8蛋白表达明显增加。Hoechst 33342染色法观察发现,pEGFP-C2+H2O2组有较多细胞出现强蓝色荧光细胞核,细胞凋亡较多,而pEGFP-NOD8+H2O2组细胞凋亡明显减少。流式细胞术分析显示,pEGFP-C2+H2O2组的细胞凋亡率明显升高,pEGFP-NOD8+H2O2组的细胞凋亡率则显著下降。pEGFP-C2+H2O2组细胞的caspase-3活性明显升高,而pEGFP-NOD8+H2O2组细胞的caspase-3活性显著下降。 结论: NOD8可抑制H2O2诱导的L02细胞凋亡,其作用机制可能与NOD8抑制细胞的caspase-3活性有关。     

hPOT1基因过表达对HeLa细胞细胞周期和凋亡的影响

目的观察人POT1(protection of telomeres1)基因过表达对HeLa细胞细胞周期和细胞凋亡的影响。方法利用本课题组构建的hPOTl基因真核表达重组质粒pcDNA3-hPOT1,经脂质体介导瞬时转染HeLa细胞;通过RT-PCR和EMSA法(电泳迁移率改变分析)检测外源基因的表达效果,流式细胞术分析细胞周期,Hoechst33342荧光染色检测细胞凋亡。结果pcDNA3-hPOT1重组质粒转染HeLa细胞48h后,mRNA和蛋白质分析表明,外源性hPOT1基因能在HeLa细胞中有效表达,HeLa细胞阻滞于细胞周期s期,而对凋亡无明显影响。结论hPOT1基因可能参与了高等真核细胞细胞周期调控过程,但与细胞凋亡无密切关系。