端粒酶逆转录酶在脐静脉内皮细胞耐受曲古抑菌素A诱导凋亡中的作用

目的观察曲古抑菌素A（TSA）对脐静脉内皮细胞及官颈癌细胞凋亡、端粒酶逆转录酶（hTERT）表达的影响,并探讨hTERT在脐静脉内皮细胞耐受TSA中的作用。方法磺酰罗丹明B （RSB）法检测药物动力学特征;流式细胞仪检测周期改变和凋亡;RT-PCR检测hTERT和p21^Waf1基因表达变化;免疫荧光结合流式细胞术检测hTERT蛋白表达变化;转染hTERT质粒后,PCR-TRAP- ELISA法检测转染细胞端粒酶活性;AnnexinV／PI检测转染细胞在TSA作用下的早期凋亡。结果在大剂量TSA作用脐静脉内皮细胞后,增殖抑制、周期阻滞,但凋亡并不显著;HeLa细胞在相同剂量的TSA作用下凋亡明显。脐静脉内皮细胞经TSA诱导后,hTERT表达上调,p21^Waf1则无明显变化;而HeLa细胞p21^Waf1表达上升,hTERT表达下降。转染显性负突变hTERT的脐静脉内皮细胞,其端粒酶活性显著低于对照组。TSA作用转染不同质粒的脐静脉内皮细胞的凋亡率与对照组差异有统计学意义。结论脐静脉内皮细胞可以耐受大剂量TSA诱导的凋亡,hTERT表达上调可能是脐静脉内皮细胞耐受TSA诱导凋亡的重要机制之一。     

宫颈癌及子宫内膜癌细胞系中hTERT启动子活性的研究

目的 :探讨Hela及HHUC细胞系中hTERT启动子的活性及其与hTERTmRNA表达和端粒酶活性之间的关系。方法 :将hTERT核心启动子基因转入Hela、HHUC细胞系、人正常皮肤表皮细胞及子宫内膜上皮细胞中 ,检测细胞系hTERT启动子的活性、hTERTmRNA表达水平及端粒酶活性。分别以HEK2 93细胞系作为阳性对照 ,HELF细胞系作为阴性对照。结果 :细胞系Hela、HHUC、人正常皮肤表皮细胞及正常子宫内膜上皮细胞中的hTERT启动子活性分别为 2 0 1%、14 9%、0 3%和 0 5 % ;hTERTmRNA相对表达水平分别为 0 6 3、0 5 1、0和 0 ;端粒酶活性分别为 0 393、0 387、0 14 4和 0 15 2。结论 :宫颈癌细胞系及子宫内膜癌细胞系中hTERT启动子活性、hTERTmRNA表达水平和端粒酶活性均特异性增高 ,而在正常角化细胞及子宫内膜上皮细胞中则被抑制或不表达。hTERT启动子活性水平与hTERTmRNA表达水平和端粒酶活性之间有显著相关性     

Antiproliferative effects of the histone deacetylase inhibitor FR901228 on small-cell lung cancer lines and drug-resistant sublines

FR901228 is a novel histone deacetylase (HDAC) inhibitor, and its antiproliferative effects on non-small cell lung cancer cells have been shown in vitro. However, there have been no reports concerning the effects on small-cell lung cancer (SCLC). We have recently demonstrated that the HDAC inhibitors trichostatin A and sodium butyrate inhibit expression of the catalytic subunit of telomerase (hTERT) mRNA and telomerase activity in prostate cancer cells. The present study was designed to evaluate the effects of FR901228 on proliferation and telomerase activity in SCLC cells in vitro. FR901228 at 5 to 10 nM increased the fraction of cells in the G(2)/M and sub-G(1) phases of the cell-cycle, and inhibited the growth of H69, H526 and H82 cell lines. The expression of hTERT mRNA was inhibited 6 hr after treatment, prior to obvious inhibition of cell growth or cell-cycle distribution shifts. The inhibition of hTERT mRNA expression and telomerase activity was not a consequence of cell-growth arrest or apoptosis. Cycloheximide blocked the suppression of hTERT mRNA induced by FR901228, and the inhibition of hTERT mRNA by FR901228 required newly synthesized proteins. FR901228 also effectively inhibited growth of etoposide-resistant UMCC-1/VP-16, irinotecan-resistant PC-6/SN2-5H and cisplatin-resistant H526/CDDP cells having decreased expression of hTERT mRNA and telomerase activity, as well as their parental cells. This implies that SCLC resistant to these key drugs are not cross-resistant to FR901228. The present study suggests that FR901228 may be a promising drug for chemotherapy of cancers including SCLC, even for refractory or relapsing tumors after conventional chemotherapy.     

人端粒酶催化亚基反义核酸对子宫内膜癌细胞的抑制作用

目的 探讨人端粒酶催化亚基(hTERT)基因反义寡核苷酸(AODN)对子宫内膜癌细胞系HEC-1A的生长抑制作用及作用机理。方法 采用四甲基偶氮唑蓝(MTT)法检测AODN对细胞增殖能力及细胞生长的影响;逆转录聚合酶链反应技术(RT-PCR)、定量端粒酶重复扩增法(TRAP)、Westerm blot蛋白免疫印迹法和凋亡相关酶活性测定检测反义核酸对hTERT基因转录、蛋白表达、细胞端粒酶活性以及凋亡相关酶活性的变化。结果 低浓度hTERT基因AODN能够下调HEC-1A细胞HTERT、mRNA含量,抑制细胞hTERT蛋白表达,下调端粒酶活性,并且激活凋亡相关酶Caspase-1和Caspase-3活性;其对细胞的生长抑制作用有明显的时效性和剂量依赖性。结论 hTERT基冈反义核酸能够抑制子宫内膜癌细胞的增殖能力,有望成为内膜癌治疗的新方向。     

Expression of p120 nucleolar proliferating antigen in human gliomas and growth suppression of glioma cells by p120 ribozyme vector

p120 is a nucleolar proliferating antigen which is expressed in tumor cells but not normal resting cells. The expression and localization of p120 in human gliomas were studied by Northern blot analysis, Western blot analysis and immunohistochemistry. All five of the glioma cell lines and all of the glioma specimens we investigated expressed p120 at both the mRNA and protein levels. p120 expression was not detected in adjacent brain tissues. A ribozyme vector was constructed to cleave the first GUC sequence in the coding region of p120 mRNA. This p120 ribozyme vector was transfected into the glioma cell line SF188, which expresses p120. The reduced p120 expression of the transfectant at both the mRNA and protein levels was confirmed. An MTT assay indicated that the transfected cells grew more slowly than control cells. These results indicate that i) p120 has an important role in the proliferation of gliomas, and ii) the ribozyme against p120 mRNA can suppress glioma cell growth.     

腺病毒介导的shRNA沉默hTERT基因表达对鼻咽癌细胞增殖和凋亡的影响

目的探讨靶向人端粒酶反转录酶（hTERT)的短发夹RNA（shRNA)对人鼻咽癌细胞株CNE-2 hTERT表达的影响,及其对鼻咽癌细胞增殖和凋亡的效应。方法构建表达绿色荧光蛋白(EGFP)基因和靶向hTERT基因短发夹RNA的重组腺病毒质粒,观察其对鼻咽癌细胞株(CNE-2)的转染效果,RT-PCR检测hTERT mRNA表达水平,Western blot检测hTERT蛋白表达水平,CCK-8法检测细胞增殖活性,流式细胞仪检测细胞凋亡状况。结果Adv-EGFP-shTERT重组腺病毒质粒转染率可达90%以上,成功转染CNE-2细胞24 h后,hTERT mRNA的表达水平显著下降,转染48 h后,hTERT蛋白表达明显下调,细胞增殖活性受到显著抑制,细胞凋亡率可达23.0%。结论腺病毒载体介导靶向hTERT基因的RNA干扰,能显著抑制端粒酶反转录酶表达,进而抑制端粒酶活性,抑制CNE-2细胞增殖并诱导其凋亡,为鼻咽癌的基因治疗研究提供了理论基础。     

Differentiation and Growth Inhibition of Glioma Cells Induced by Transfer of trk A Proto-oncogene

The induction of growth inhibition and differentiation of a glioma cell line by transfection of trk A cDNA was examined, and production of endogenous nerve growth factor (NGF) also was studied in these cells. When human trk A cDNA was transfected into a human glioma cell line, U-251MG, which lacks expression of both endogenous trk A and low-affinity NGF receptor, the transfectant expressed the exogenous trk A mRNA and a functional high-affinity NGF receptor. Transfection of trk A cDNA caused a partial induction of cell differentiation, G1 arrest, growth inhibition, tyrosine phosphorylation of the trk A proto-oncogene product, and activation of MAP kinase. Exogenous NGF treatment induced further terminal differentiation and growth inhibition. In summary, our data suggest that endogenous NGF secreted by glioma cells has an important role in the induction of glioma-cell differentiation occuring with transfer of exogenous trk A cDNA.