Inhibition of human telomerase in MKN—45 cell line by antisense hTR expression vector induces cell apoptosis and growth arrest

AIM: To investigate the effects of antisense human telomerase RNA (hTR)on the biologic behavior of human gastric cancer cell line: MKN-45 by gene transfection and its potential role in the gene therapy of gastric cancer. METHODS: The hTR cDNA fragment was cloned from MKN-45 through RT-PCR and subcloned into eukaryotic expression vector (pEF6/V5-His-TOPO) in cis-direction or trans-direction by DNA recombinant methods. The constructed sense, antisense and empty vectors were transfected into MKN-45 cell lines separately by lipofectin-mediated DNA transfection technology. After drug selection, the expression of antisense hTR gene in stable transfectants and normal MKN-45 cells was detected by RT-PCR, the telomerase activity by TRAP, the apoptotic features by PI and Hoechst 33258 staining, the cell cycle distribution by flow cytometry and the population doubling time by cell counting. Comparison among the stable transfectants and normal MKN-45 cells was made. RESULTS: The sense, antisense hTR eukaryotic expression vectors and empty vector were successfully constructed and proved to be the same as original design by restriction endonuclease analysis and sequencing. Then, they were successfully transfected into MKN-45 cell lines separately with lipofectin. The expression of antisense hTR gene was only detected in MKN-45 cells stably transfected with antisense hTR vector (named as MKN-45-ahTR) but not in the control cells. In MKN-45-ahTR, the telomerase activity was inhibited by 75%, the apoptotic rate was increased to 25.3%, the percentage of cells in the G0/G1 phase was increased to 65%, the proliferation index was decreased to 35% and the population doubling time was prolonged to 35.3 hours. However, the telomerase activity, the apoptotic rate, the distribution of cell cycle, the proliferation index and the population doubling time were not different among the control cells. CONCLUSION: Antisense hTR can significantly inhibit telomerase activity and proliferation of MKN-45 cells and induce cell apoptosis. Antisense gene therapy based on telomerase inhibition can be a potential therapeutic approach to the treatment of gastric cancer.     

乙型肝炎病毒X基因对肝细胞系细胞凋亡及端粒酶活性的影响

目的探讨乙型肝炎病毒x基因对肝细胞恶性变的作用机制.方法将带C基因、S基因的载体电转染导入HepG2细胞,筛选表达细胞克隆,复苏带X基因的HepG2细胞.PCR-ELISA检测各株细胞的端粒酶活性.用反义寡核苷酸诱导细胞凋亡,流式细胞仪观测转染了x基因、C基因、S基因细胞的凋亡情况.结果表达细胞克隆经同步化处理,39.50%转染X基因的细胞进入细胞S周期,其端粒酶活性指数395±0.07明显高于其它各组细胞.反义寡核苷酸诱导后,转染X基因细胞凋亡峰明显减小,凋亡率仅1.75%;其细胞活性与反义寡核苷酸浓度成反比.结论乙型肝炎病毒X基因上调肝源细胞端粒酶活性,抑制细胞凋亡,这可能是诱导肝细胞恶性变的又一机制.     

丙型肝炎病毒核心区蛋白对 HepG2细胞周期、细胞凋亡和细胞端粒酶活性的影响

目的　研究丙型肝炎病毒核心区 (HCV C)蛋白对肝癌细胞HepG2细胞周期、细胞凋亡和细胞端粒酶活性的影响。方法　首先运用基因重组技术构建含有HCV C基因的真核表达质粒pcDNA3.1( ) ,然后利用脂质体介导将重组真核表达质粒转染HepG2 ,经G4 18筛选获得稳定转染HepG2细胞 (HCV C转染HepG2细胞 ) ,经逆转录 聚合酶链反应技术 (RT PCR)和间接免疫荧光法证实其中有HCV C蛋白表达。然后进行如下实验 :( 1)利用四甲基偶氮唑蓝比色 (MTT)法检测HCV C转染HepG2细胞、空白质粒转染HepG2细胞和未转染HepG2细胞的生长增殖率 ;经流式细胞术(FACS)检测 3组细胞的细胞周期 ;( 2 )经流式细胞术检测细胞凋亡率 ;( 3)经端粒重复扩增 酶联免疫吸附试验 (TRAP ELISA)法检测上述 3组细胞端粒酶活性表达情况。结果　 ( 1)HCV C转染HepG2细胞增殖率显著高于空白质粒转染HepG2和未转染HepG2细胞增殖率 ;HCV C转染HepG2细胞S期所占百分率高于未转染HepG2细胞S期所占百分率 ;( 2 )HCV C转染HepG2细胞凋亡率显著低于无HCV C转染细胞凋亡率 ;( 3)上述 3组细胞端粒酶活性之间差异无显著性。结论　 ( 1)HCV C蛋白具有抑制细胞凋亡的作用 ;( 2 )HCV C蛋白促进HepG2从G0 /1期进入S期 ,从而可能促进细胞生长增殖 ,抑制细胞凋亡 ;( 3)HCV     

硫代修饰端粒酶反义核酸联合顺铂、阿霉素对胃癌细胞作用的研究

目的：探讨应用硫代修饰人端粒酶RNA（hTR)反义核酸后胃癌细胞对顺铂（DDP）和阿霉素（ADM）敏感性的变化。方法：采用脂质体将针对hTR模版区设计的13相碱基硫代磷酸修饰的反义寡核苷酸CAGTTAGGGTTAG导入胃癌细胞SGC7901，应用四甲基偶氮唑蓝（MTT）法，流式细胞仪和TRAP－PCR－ELISA法测定联合应用化疗药后对细胞增殖，凋亡和端粒酶活性的影响。结果：化疗药物ADM和DDP地端粒酶活性抑制作用不同，而且有明显依赖趋势。hTR反义PS－ODN可增加ADR和DDP抑制胃癌细胞系SGC7901端粒酶活性，诱导细胞凋亡和抑制细胞增殖的作用。结论：hTR反义PS－ODN在体外能增加胃癌细胞系SGC701对化疗药ADR、DDP敏感性，其机制可能与其抑制细胞端粒酶活性，诱导细胞凋亡有关。     

丙型肝炎病毒非结构蛋白4B对肝癌细胞增殖的影响

目的 探讨丙型肝炎病毒非结构蛋白4B(HCV-NS4B)对肝癌细胞增殖及增殖细胞核抗原(PCNA)和周期素D1(cyclinD1)蛋白表达的影响.方法 采用构建好的HCV-NS4B表达载体,以脂质体转染法转染HepG2细胞.RT-PCR、琼脂糖凝胶电泳证实HCV-NS4B在HepG2细胞稳定表达.MTT法绘制生长曲线,观察NS4B对肝细胞生长的影响;流式细胞仪检测细胞周期的情况;免疫细胞化学法检测PCNA和cyclinD1的表达.结果 转染后,生长曲线显示NS4B可促进肝细胞生长;细胞周期检测显示转染NS4B的HepG2细胞进入S期和G2/M期增多(P＜0.05),处于G0/G1期细胞降低(P＜0.05).PCNA和cyclinD1的表达均较空白载体组升高(P＜0.05).结论 HCV-NS4B可干扰肝癌细胞周期,促进肝癌细胞增殖,其作用与上调PCNA、cyclinD1表达有关.     

重组腺病毒载体携带多药耐药基因1反义RNA靶向逆转肝癌细胞多药耐药

目的 探讨携带多药耐药基因1(multidrug resistance gene 1,mdr1)反义RNA的重组腺病毒载体靶向逆转甲胎蛋白阳性(AFP+)的肝癌多药耐药细胞HepG2R的疗效及作用机制.方法 分别构建携带AFP启动子和mdr1基因反义核苷酸片段的重组腺病毒载体Adeno-asmdr及携带AFP启动子和增强绿色荧光蛋白基因的重组腺病毒载体Adeno-EGFP,将Adeno-EGFP转染人正常肝细胞L02(AFP-),人官颈癌细胞HeLa(AFP-)及HepG2(AFP+)细胞,检测增强绿色荧光蛋白基因在各细胞的转录水平;将Adeno-asmdr转染HepG2R细胞,Western blot检测不同时间P-gp170的表达,末端脱氧核苷酸转移酶介导的脱氧三磷酸尿苷缺口末端标记法检测HepG2R细胞凋亡,流式细胞术检测HepG2R细胞在不同药物作用下细胞周期、凋亡率.结果 增强绿色荧光蛋白基因在AFP阳性的HepG2细胞可得到显著转录,而在L02细胞和HeLa细胞,其转录减少,显示了该载体的良好转录活性以及靶向特异性.Adeno-asmdr转染HepG2R细胞后,HepG2R细胞P-gp170表达明显减弱,HepG2R细胞凋亡增加,HepG2R细胞对多种化疗药物的耐受能力明显下降,细胞出现显著的周期阻滞,大量细胞被阻滞于S期和G0/M期,凋亡细胞比例增加.结论 实验构建的Adneo-asmdr重组腺病毒载体可在AFP阳性HepG2R细胞内特异靶向性表达目的 基因,并可有效降低mdrl基因产物P-gp170的表达,从而达到对HepG2R细胞多药耐药的逆转作用.     

携带AFP增强子反义c-fms真核表达载体的构建及其临床意义

目的 构建人肝癌细胞中高效特异表达的反义c-fms真核表达载体,观察其对肝癌细胞生物学行为的影响。方法 采用PCR法扩增人c-fms癌基因第571位酪氨酸为中心的DNA片段,将其反向克隆人pcDNA3载体（命名pAS);将扩增的人甲胎蛋白（AFP）增强子核心区克隆人pAS（命名pAEAS)。磷酸钙法将空载体pcDNA3及反义真核表达载体pAS、pAEAS分别转导入HepG2肝癌细胞及HeLa宫颈癌细胞,观察细胞生长速度及凋亡。结果 人反义c-fms基因片段及AFP增强子核心区片段,测序结果与Genbank中登录的序列一致。导入反义基因的HepG2肝癌细胞生长速度较对照细胞明显减慢（P＜0．05）,pAEAS抑制作用较pAS强（P＜0．05,pcDNA3组、pAS组、pAEAS组HepG2肝癌细胞凋亡率分别为5．25％、14．7％、31．2％（P＜0．01）,pAEAS组细胞DNA出现梯状凋亡带。在HeLa宫颈癌细胞中,pAS及pAEAS组生长速度减慢,但二者差异无显著性（P＞0．05）,pcDNA3组、pAS组、pAEAS组细胞凋亡率分别为3．99％、8．27％、8．86％（P＜0．05）,DNA均未出现梯状凋亡带。结论 携带AFP增强子的人反义c-fms真核表达载体对AFP阳性肝癌细胞生长有选择性抑制作用,可诱导凋亡,是一种新的肝癌基因治疗方法。     

Antisense hTERT inhibits thyroid cancer cell growth

Activation of telomerase represents an early step in carcinogenesis. Increased telomerase expression in malignant thyroid tumors suggests that inactivation of telomerase may represent a potential chemotherapeutic target. The purpose of this study was to inhibit the protein component of telomerase, hTERT, in a human thyroid cancer cell line in vitro and in vivo using an antisense strategy. A 235-bp fragment of hTERT cDNA was subcloned, and sense and antisense hTERT expression vectors were constructed. These vectors were transfected into a human thyroid carcinoma cell line (FRO). Tumorigenic potential was determined by cellular growth assay, rate of apoptosis, anchorage-independent growth, and tumor growth in a nude mouse model. Significant down-regulation of hTERT expression was seen in the antisense transfected cells, compared with control and those transfected with the sense vector. A decrease in telomerase activity by TRAP assay was observed in the antisense hTERT cells but not in cells transfected with the sense hTERT construct. Inhibition of cell growth was observed after approximately 20 population doublings in the antisense-hTERT clones and was associated with an increase in the rate of apoptosis and a change in cellular morphology. Moreover, anchorage-independent growth was reduced in vitro, and tumor growth rate was diminished in vivo in the antisense hTERT clones. Inhibition of telomerase activity with antisense hTERT in human thyroid cancer cells is achievable and may represent a novel target to inhibit tumor growth.     

反义细胞周期素D1基因对肝癌细胞株HepG2的作用

目的通过基因反义封闭技术体外抑制细胞周期素D1(cyclin D1)的表达,研究其对肝癌细胞cyclin D1蛋白表达和细胞增殖的影响。方法以肝癌细胞株HepG2为研究对象,通过转染可表达cyclin D1反义互补脱氧核苷酸(AScDNA)的质粒后,观察cyclin D1反义cDNA对HepG2细胞cyclin D1基因表达及体外增殖活性的影响。结果四甲基偶氮唑盐法检测细胞增殖活性显示转染表达反义cyclin D1的质粒后, HepG2细胞的增殖受到抑制,抑制作用在48 h左右最强;逆转录聚合酶链反应检测显示cyclin D1 mRNA基因的表达明显被抑制;免疫组织化学检测结果显示cyclin D1蛋白表达明显降低; 流式细胞仪检测结果显示G0/G1期的细胞比例增高,G2+M和S期的细胞比例下降,HepG2细胞周期在G1期被阻滞。结论cyclin D1反义cDNA可以特异性的抑制肝癌HepG2细胞株cyclin D1蛋白的表达,从而调控细胞周期,抑制肝癌细胞增殖。利用cyclin D1反义cDNA进行细胞周期调控对于肝细胞癌的生物治疗具有一定的应用前景。     

Inhibition of the p53 tumor suppressor gene results in growth of human aortic vascular smooth muscle cells. Potential role of p53 in regulation of vascular smooth muscle cell growth.

Loss of activity of the p53 tumor suppressor gene product has been postulated in the pathogenesis of human restenosis. Although the antioncogenes p53 and retinoblastoma (Rb) susceptibility gene have been reported to play a pivotal role in cell cycle progression in various cells, the role of p53 and Rb in the growth of human vascular smooth muscle cells (VSMC) has not yet been clarified. We used antisense strategy against p53 and Rb genes by the viral envelope-liposomal method. Transfection of antisense p53 oligodeoxynucleotides (ODN) alone resulted in an increase in DNA synthesis compared with control (P<0.01). Similarly, transfection of antisense Rb ODN alone resulted in a higher DNA synthesis rate than control (P<0.01). Moreover, increase in VSMC number was only induced by transfection of antisense p53 ODN alone or cotransfection of p53/Rb ODN (P<0.01), whereas a single transfection of antisense Rb ODN had little effect on cell number. Therefore, we hypothesized that this discrepancy is due to the induction of apoptosis mediated by p53. Interestingly, apoptotic cells were markedly increased in VSMC transfected with antisense Rb ODN alone, accompanied by the induction of p53 protein. The number of apoptotic cells was attenuated by cotransfection of antisense p53 ODN (P<0.01). We finally examined the molecular mechanisms of apoptosis induced by the absence of Rb. In VSMC transfected with antisense Rb ODN, bax, a promoter of apoptosis, was significantly increased in VSMC transfected with antisense Rb ODN (P<0.01), whereas bcl-2 and Fas did not play a pivotal role in the induction of apoptosis. Overall, these data first demonstrated that the antioncogenes p53 and Rb negatively regulated the cell cycle in VSMC, suggesting that the modulation of their activity may mediate VSMC growth such as that in restenosis and atherosclerosis. The presence of p53 plays a pivotal role in the regulation of apoptosis in human VSMC growth, probably through the bax pathway. These results provide evidence that p53 is a functional link between cell growth and apoptosis in VSMC.     

Apoptosis and its pathway in X gene-transfected HepG2 cells

AIM: To investigate the effect of hepatitis B virus (HBV) X gene on apoptosis and expressions of apoptosis factors in X gene-transfected HepG2 cells. METHODS: The HBV X gene eukaryon expression vector pcDNA3-X was transiently transfected into HepG2 cells by lipid-media transfection. Untransfected HepG2 and HepG2 transfected with pcDNA3 were used as controls. Expression of HBx in HepG2 was identified by RT-PCR. MTT and TUNEL were employed to measure proliferation and apoptosis of cells in-three groups. Semi-quantified RT-PCR was used to evaluate the expression levels of Fas/FasL, Bax/Bcl-xL, and c-myc in each group. RESULTS: HBV X gene was transfected into HepG2 cells successfully. RT-PCR showed that HBx was only expressed in HepG2/pcDNA3-X cells, but not expressed in HepG2 and HepG2/pcDNA3 cells. Analyzed by MTT, cell proliferation capacity was obviously lower in HepG2/pcDNA3-X cells (0.08910±0.003164) than in HepG2(0.14410±0.004927) and HepG2/pcDNA3 cells (0.12150±0.007159) (P<0.05 and P<0.01). Analyzed by TUNEL, cell apoptosis was much more in HepG2/pcDNA3-X cells (980/2 000) than HepG2 (420/2 000), HepG2/pcDNA3 cells (520/2 000) (P<0.05 and P<0.01). Evaluated by semi-quantified RT-PCR, the expression level of Fas/FasL was significantly higher in HepG2 cells transfected with HBx than in HepG2 and HepG2/ pcDNA3 cells (P<0.05 and P<0.01). Bax/Bcl-xL expression level was also elevated in HepG2/pcDNA3-X cells (P<0.05 and P<0.01). Expression of c-myc was markedly higher in HepG2/pcDNA3-X cells than in HepG2 and HepG2/pcDNA3 cells (P<0.05 and P<0.01). CONCLUSION: HBV X gene can impair cell proliferation capacity, improve cell apoptosis, and upregulate expression of apoptosis factors. The intervention of HBV X gene on the expression of apoptosis factors may be a possible mechanism responsible for the change in cell apoptosis and proliferation.     

miR-155在肝细胞癌中的表达及对肝癌细胞增殖的影响

目的:检测微小RNA-155(miR-155)在肝癌组织中的表达并分析其对肝癌细胞增殖和细胞凋亡的影响.方法:采用TagMan MGB探针法荧光定量P C R分析42例原发性肝癌及对应的癌旁组织miR-155的表达;利用miR-155反义寡核苷酸(ASO-miR-155)降低肝癌细胞HepG2和SMMC7721中miR-155的表达;利用MTT比色法检测肝癌细胞增殖的变化,并通过流式细胞技术检测肝癌细胞早期凋亡情况.结果:42例肝癌及癌旁组织标本中,miR-155在52%(22/42)肝癌组织中的表达明显高于癌旁组织(P<0.05);利用脂质体将ASO-miR-155转染肝癌细胞HepG2和SMMC7721后,miR-155的表达明显降低,肝癌细胞HepG2和SMMC7721生长受到明显抑制;并且细胞的早期凋亡明显增加.结论:miR-155在肝癌组织中过表达,降低其表达能明显抑制肝癌细胞的生长并诱导细胞早期凋亡,miR-155有可能成为肝癌治疗的新靶点.     

CEA and AFP expression in human hepatoma cells transfected with antisense IGF-I gene

AIM:To determine whether antisense insulin-like growth factor-I(IGF-I) gene can modulate CEA and AFP expression in human hepatoma cells (HepG2).METHODS: Transfection of HepG2 cells was accomplished using Lipofectin reagent. Northern blot analysis confirmed the antisense IGF-I RNA of the transfected cells. CEA and AFP levels were measured using radioimmunoassay.RESULTS: Human hepatoma cell lines (HepG2) were transfected with antisense IGF-I gene. Northern blot analysis confirmed that antisense IGF-I RNA was expressed in the transfected cells. The effect of antisense IGF-I gene on CEA and AFP expression was demonstrated by the fact that the CEA and AFP levels in the supernatant of transfected cell culture were significantly lower as compared with the parent cells, (CEA 7.0&mgr;g/L plus minus 0.76&mgr;g/L and 3.29&mgr;g/L plus minus 1.80&mgr;g/L (P < 0.05) and AFP 53.63&mgr;g/L plus minus 6.02&mgr;g/L and 9.0&mgr;g/L plus minus 5.26&mgr;g/L (P<0.01), respectively).CONCLUSION: The malignant potentiality of the transfected cells was partially suppressed.Antisense IGF-I gene can modulate the expression of CEA and AFP in human hepatoma cell lines (HepG2)     

Imbalance between cell proliferation and cellular DNA fragmentation in hepatocellular carcinoma

Abstract: Aim/Background: Hepatocellular carcinoma (HCC) is known for its rapid growth. This study was undertaken to determine the expression of proliferative markers, apoptosis (DNA fragmentation) and oncogene products known to regulate apoptosis (p53, bcl-2) in HCC. Methods: 150 Chinese patients with HCC were studied (M:F 128:22, age 14–88 years), Immunohistochemistry was employed to detect cell proliferative markers (PCNA, Ki67), and oncogene products known to regulate apoptosis (p53, bcl-2). DNA fragmentation was determined by terminal dUTP nick end labeling (TUNEL). Results: 98% and 95% of HCC had PCNA (median 2+) and Ki67 (median 2+) detected respectively. TUNEL labeling was detected in only a small number of tumor cells (no labeling in 11%, median 1/1000 cell labeled, range: 0–70/1000 cells). There was no correlation between TUNEL labeling and the clinical parameters (sex, age, cirrhosis, and survival) and the expression of cell proliferative markers. p53 was detected in 53% of the patients (median 1+, range: 0–4+) and bcl-2 was detected in a small proportion of tumor cells in only 13% of the HCCs (range: 0–1+). The expression of p53 and Bcl-2 did not correlate with TUNEL labeling or the natural survival. Conclusions: Cell proliferation in HCC is unmatched by apoptosis, accounting for the rapid growth of this tumor. This lack of apoptosis in HCC is unrelated to the expression of p53 or bcl-2 over-expression.     

Survvin反义寡核苷酸诱导人胃癌细胞凋亡的作用

目的探讨生存素（Survivin）反义寡核苷酸（ASODN）诱导人胃癌细胞SGC7901凋亡的作用。方法设计合成特异性靶向Survivin的ASODN,将胃癌细胞株分为空白对照组（Sham组）、脂质体转染对照组（Lip组）、正义链转染对照组（Lip-SODN组）和ASODN转染组（Lip-ASODN组）。作用48h后,Westemblot法检测各组Survivin表达情况,流式细胞仪检测各组细胞凋亡率,免疫组化SP法检测细胞中PCNA表达情况。结果脂质体介导Survivin ASODN转染后的胃癌细胞Survivin蛋白表达明显下降;ASODN转染组细胞凋亡率明显高于各对照组（P均〈0．05）,各对照组间无统计学差异（P〉0．05）。ASODN转染后胃癌细胞中PCNA表达水平明显降低。结论 Survivin ASODN转染胃癌细胞能下调Survivin蛋白表达,诱导胃癌细胞凋亡,抑制细胞增殖,具有明显的抗癌作用。     

Gene therapy that inhibits NF-κB results in apoptosis of human hepatocarcinoma by recombinant adenovirus

AIM: To investigate whether the recombinant adenovirus induces the TNF-alpha-mediated apoptosis in vivo. METHODS: Human hepatocarcinoma cell line (HepG(2)) cells were transfected into BALB/c nude mice, and the tumor growth curve was drawn. We analyzed apoptosis in HepG(2) cells by TUNEL, HE staining and electron microscopy. RESULTS: AdIkappaBalphaM was expressed stably and efficiently in HepG(2) and could not be degraded by induction of TNF-alpha. Tumor growth in mice could be reduced remarkably if treated by AdIkappaBalphaM plus TNF-alpha. There was apoptosis of > 70% of cells treated with AdIkappaBalphaM plus TNF-alpha and about 50% of cells treated with AdIkappaBalphaM. In contrast, there was few cell apoptosis in HepG(2) cells treated with phosphate buffered saline and AdIkappaBalpha. HepG(2) cells in mice also exhibited a high level of apoptosis after in vivo injection with AdIkappaBalphaM. The tumor growth curve indicated the tumor transfected with AdIkappaBalphaM could be restrained. CONCLUSION: AdIkappaBalphaM gene therapy greatly enhances apoptosis due to inhibition of an NF-kappaB-mediated antiapoptosis signaling pathway.