以人端粒酶逆转录酶为靶点的肿瘤治疗

人端粒酶逆转录酶是近几年肿瘤治疗研究的一个热点,包括端粒酶活性抑制剂研究、以人端粒酶逆转录酶为靶点的RNA干扰研究、人端粒酶逆转录酶介导的免疫学研究以及以人端粒酶逆转录酶为靶点的基因治疗研究等,并取得了一定的成果.     

端粒酶逆转录酶小干扰RNA对结肠癌SW480细胞体外抑制作用的观察

目的:探讨RNA干扰(RNAi)对结肠癌SW480细胞人端粒酶逆转录酶(hTERT)基因的抑制效应。方法:化学合成靶向于hTERT基因的4个小分子干扰RNA(siRNA)序列,用阳离子脂质体为载体转染SW480细胞,分为siRNA1～4、siRNA阴性对照和空白对照6组。通过RT-PCR和蛋白质印迹法分别检测结肠癌细胞转染后细胞hTERT mRNA和蛋白水平,并用MTS法检测细胞生长增殖。结果:siRNA1～4组hTERT mRNA相对表达量分别为0.54±0.18、0.56±0.19、0.46±0.10和0.84±0.18,端粒酶相对活性分别为0.57±0.18、0.56±0.19、0.09±0.08和0.70±0.19,siRNA1～4组hTERT蛋白相对表达量分别为0.41±0.18、0.45±0.17、0.08±0.07和0.82±0.16,其中siR-NA1～3组的hTERT mRNA表达量、端粒酶活性和蛋白表达量与空白对照组的差异有统计学意义,P<0.05。SW480细胞在转染后48h时的细胞增长平均抑制率分别为42.14%、39.12%、48.05%和35.85%。提示不同siRNA序列对SW4...     

PAX8 regulates telomerase reverse transcriptase and telomerase RNA component in glioma

Paired box (PAX) developmental genes are frequently expressed in cancers and confer survival advantages on cancer cells. We have previously found that PAX genes are deregulated in glioma. We have now investigated the expression of PAX genes in glioma and their role in telomere maintenance. The mRNA level of PAX8 showed a positive correlation with telomerase activity in glioma biopsies (r(2) = 0.75, P < 0.001) and in established glioma cell lines (r(2) = 0.97, P = 0.0025). We found that PAX8 is able to coordinately transactivate the promoter for both the telomerase catalytic subunit (hTERT) and the telomerase RNA component (hTR) genes. By electrophoretic mobility shift assay, quantitative PCR, and a telomerase activity assay, we show that PAX8 binds directly to the hTERT and hTR promoters, up-regulating hTERT and hTR mRNA, as well as telomerase activity. Additionally, PAX8 small interfering RNA down-regulated hTERT and hTR. Collectively, these results show that PAX8 may have a role in telomerase regulation.     

端粒酶逆转录酶、P53、PCNA在非小细胞肺癌的表达及意义

目的　探讨端粒酶逆转录酶、P5 3、PCNA在非小细胞肺癌组织及癌旁正常组织中的表达及其关系。方法　取自手术切除及肺穿刺经病理证实为肺癌组织标本 6 0例 ,采用免疫组化法检测人端粒酶逆录酶、P5 3、PCNA的表达 ,并取癌旁肺组织 10例作对照。结果　肺癌组织中端粒酶逆转录酶、P5 3、阳性率分别为78 3% ( 4 7/6 0 )和 5 3 3% ( 32 /6 0 ) ,PCNA的指数为 5 8 6 3± 10 6 1。癌旁组织依次为 0 % ( 0 /10 ) ,10 % ( 1/10 )和30 3± 7 3,(P <0 0 1) ;在鳞癌中的表达依次为 72 7% ,5 1 5 % ,5 7 78± 10 6 8;在腺癌中的表达依次为88 2 % ,5 2 9% ,5 9 18± 11 4 7;在腺鳞癌中依次为 80 % ,6 0 % ,6 1 2± 10 6 7,(P >0 0 5 )。临床分期的关系为 :Ⅰ期依次为 71% ,35 5 % ,5 5 74± 10 71;Ⅱ期为 87 5 % ,70 8% ,5 9 79± 9 14 ;Ⅲ期为 80 4 % ,80 1% ,71 6±7 31 (P <0 0 1)。在淋巴结转移阳性病例中表达为 89 7% ,6 0 9% ,6 1 72± 9 74 ;在淋巴结转移阴性病例中为 6 7 7% ,38 7% ,5 5 74± 10 71,(P =0 0 2 8) ;病理分化较高组织中的表达为 70 4 % ,2 9 6 % ,5 5 6 3± 9 72 ,分化中等的为 84 2 % ,6 8 4 % ,5 8 2 6± 10 2 6 ,分化较低的组织中为 85 7% ,78 6 % ,6 4 93± 10 7     

Ret finger protein-like 3 promotes tumor cell growth by activating telomerase reverse transcriptase expression in human lung cancer cells

In this study, we identified ret finger protein-like 3 (RFPL3) as a hTERT promoter binding protein in lung cancer cells. The high hTERT promoter-binding activity of RFPL3 was detected in lung cancer cells compared to normal cells. Chromatin immunoprecipitation confirmed RFPL3 as a tumor-specific hTERT promoter binding protein. Overexpression of RFPL3 activated hTERT promoter and up-regulated hTERT expression and telomerase activity. Inhibition of RFPL3 expression by siRNA suppressed hTERT promoter activation and telomerase activity. Inhibition of RFPL3 by siRNA or shRNA also significantly inhibited tumor cell growth in vitro and in a xenograft mouse model in vivo. Immunohistochemical analysis of 181 human lung adenocarcinomas specimens showed a significant correlation between RFPL3 and hTERT expression. The overexpression of RFPL3 was also associated significantly with lymph node metastasis. Univariate and multivariate Cox model analyses of NSCLC clinical specimens revealed a strong correlation between RFPL3 expression and overall survival. These results demonstrate that RFPL3 is an important cellular factor which promotes lung cancer growth by activating hTERT expression and may be a potential novel therapeutic target for lung cancer.     

Cytotoxic T-cell immunity against telomerase reverse transcriptase in colorectal cancer patients

The catalytic subunit of telomerase (hTERT) has recently been proposed as a potential tumour-associated antigen capable of inducing T-cell mediated immunity in cancer patients. Before any attempts at vaccination with hTERT antigens can be made, one should establish if cancer patients possess cytotoxic T-lymphocytes (CTL) that can recognise hTERT epitopes. The T-cell response against two HLA-A2-specific epitopes of hTERT in 37 colorectal cancer patients and 12 normal controls was analysed using an interferon gamma (IFN-gamma) ELISPOT assay. For comparison the response to HLA-A2-restricted epitopes of CEA and influenza A matrix protein was also measured. CTL that recognised either of the two hTERT epitopes studied were found in 7 (19%) of colorectal cancer patients, with 2 (5%) possessing T-cells that recognised both these peptides. Four (11%) colorectal cancer patients had CTL that reacted to the CEA epitope. No relationship between cancer stage and the presence of specific CTL against hTERT or CEA was observed. None of the normal controls possessed T-cells capable of recognising either the hTERT or the CEA epitopes. However, a similar proportion of patients and normal controls had CTL reactive with the influenza A peptide. The results of this study demonstrate that CTL active against hTERT are present in approximately 20% of colorectal cancer patients irrespective of disease stage. Moreover, these cells are functional, able to secrete IFN-gamma when stimulated with the relevant peptide.     

RNA interference targeting CHFR enhances taxol chemosensitivity in endometrial cancer cells

In recent years, it has been reported that CHFR may be a useful biomarker for chemotherapeutic response to microtubule inhibitors in some tumor cells. The purpose of the present study was to test the hypothesis and to elucidate the underlying mechanism in endometrial cancer cells. First, we effectively inhibited CHFR expression at both the mRNA and protein levels using siRNA targeting the CHFR gene in Ishikawa and Hec-1a cells. We found that inhibition of CHFR expression significantly enhanced the cytotoxicity of taxol to both cell types, which was confirmed again by colony formation assays. Moreover, suppression of CHFR induced a significant increase of the mitotic index and much lower numbers of cells at the G2/M phase in both cells treated with taxol, indicating mitotic checkpoint impairment. On the other hand, the number of apoptotic cells significantly increased in Ishikawa and Hec-1a cells transfected with CHFR siRNA after treatment with taxol, which was associated with cyclin B1 nuclear localization. Our data indicate that RNA interference targeting CHFR can sensitize endometrial cancer cells to taxol and CHFR may be a promising molecular target to enhance the therapeutic effect of taxol for endometrial cancer.     

人端粒酶逆转录酶在宫颈癌及其癌前病变中的研究进展

人端粒酶逆转录酶（hTERT）与癌症的发生发展息息相关。hTERT在宫颈癌及其癌前病变中的作用也已被广泛研究,主要涉及hTERT表达变化与宫颈癌前病变程度的关系以及hTERT表达水平作为宫颈癌诊断和预后的可行性等方面。hTERT在宫颈癌的诊断、预后及治疗中具有极为重要的临床价值。     

短发夹RNA抑制喉癌细胞hTERT基因表达及诱导细胞凋亡

目的:探讨DNA载体途径的RNA干扰技术抑制人端粒酶逆转录酶(hTERT)基因表达及诱导喉癌细胞Hep-2凋亡的作用。方法:构建靶向hTERTmRNA的质粒pshRNA1、pshRNA2及对照质粒pshRNA3、pEGFP,并将其分别转染喉鳞癌Hep-2细胞。共聚焦荧光显微镜观察质粒转染及表达情况;RT-PCR测定hTERTmRNA表达;Western印迹测定hTERT蛋白表达;末端重复片断扩增-酶联免疫吸附(TRAP-ELISA)方法测定细胞端粒酶活性;MTT法研究细胞增殖活性变化;原位细胞凋亡(TUNEL)及透射电子显微镜研究细胞凋亡。结果:①共聚焦荧光显微镜下见大量的细胞呈现绿色荧光,与其他组比较,pshRNA1、2组呈现荧光的死亡细胞显著增加。②pshRNA1、2转染细胞后hTERTmRNA及hTERT蛋白表达显著降低;其端粒酶活性明显受到抑制:转染后2 d pshRNA1组活性为:0.159±0.039、pshRNA2组活性为0.163±0.028,与空白对照组1.523±0.076比较,差异有非常显著性(P<0.005)。③pshRNA1、2转染细胞后可显著抑制细胞增殖、诱导细胞凋亡。这两组细胞透射电镜可见典型细胞凋亡特征。结论:DNA载体途径的RNA干扰技术能有效抑制hTERT基因的表达及端粒酶活性并诱导癌细胞凋亡,此法可能成为抑制肿瘤细胞的新途径。