Inhibition of telomerase with human telomerase reverse transcriptase antisense increases the sensitivity of tumor necrosis factor-α-induced apoptosis in prostate cancer cells

Aim： To investigate the effect of inhibition of telomerase with human telomerase reverse transcriptase （hTERT） antisense on tumor necrosis factor-α （TNF-α-induced apoptosis in prostate cancer cells （PC3）. Methods： Antisense phosphorothioate oligodeoxynucleotide （AS PS-ODN） was synthesized and purified. Telomerase activity was measured using the telomeric repeat amplification protocol （TRAP） and polymerase chain reaction enzyme-linked immunoassay （PCR-ELISA）. hTERT mRNA was measured by reverse transcription PCR （RT-PCR） assay and gel-image system, hTERT protein was detected by immunochemistry and flow cytometry. Cell viability was detected by 3-（4, 5-dimethylthiazol-2-yl）-2,5-Diphenyltetrazolium （MTT） assay. Cell apoptosis was observed by morphological method and determined by flow cytometry. Results： The telomerase activity decreased with time after hTERT AS PS-ODN treatment. The levels of hTERT mRNA decreased with time after hTERT AS PS-ODN treatment, which appeared before the decline of the telomerase activity. The percentage of positive cells of hTERT protein declined with time after hTERT AS PS-ODN treatment, which appeared after the decline of hTERT mRNA. There was no difference in telomerase activity, hTERT mRNA and protein levels between hTERT sense phosphorothioate oligodeoxynucleotide （S PS-ODN） and the control group. The cell viability decreased with time after hTERT AS PS-ODN combined with TNF-α treatment. The percentage of apoptosis increased with time after hTERT AS PS-ODN combined with TNF-α treatment. There was no difference in cell viability and the percentage of apoptosis between hTERT S PS-ODN and the control group. Conclusion： hTERT AS PS-ODN can significantly inhibit telomerase activity by downregulating the hTERT mRNA and protein expression, and inhibition of telomerase with hTERT antisense can enhance TNF-α- induced apoptosis of PC3 cells.

TELOMERASE: A NOVEL TARGET OF ANTITUMOR AGENTS

Telomerase activity was found to be high in various human cancers, but absent in most normal tissues. Its expression pattern made it a novel target for antitumor agents. Several strategies against telomerase were presented in this review. Targeting the telomerase RNA component by oligonucleotide/ribozyme was considered to be one of the most hopeful approaches. Some progresses were made in this area, such as the use of PANs and 2–5A antisense compounds. The relationships among telomerase activity and cell differentiation, signal transduction, oncogene, tumor suppressor gene as well as cell cycle modulation also provided a series of valuable ideas in designing anti-telomerase drugs for cancer therapy. In conclusion, although there is still a long way in understanding the mechanism and regulation of telomerase, the advance of studies on telomerase has allowed the development of numerous strategies for the treatment of cancer.

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.

Comparison of urinary telomerase,CD44,and NMP22 assays for detection of bladder squamous cell carcinoma

Background:Squamous cell carcinoma(SCC)of the bladder is common in many regions around the world.Prognosis is very poor,as most cases are diagnosed at an advanced stage due to a lack of affordable and valid screening markers for this type of cancer.The diagnostic accuracy of urinary nuclear matrix protein-22(NMP22),telomerase activity,and CD44 were evaluated in urine samples of patients with bladder SCC.Materials and methods:We conducted a case-control study comprised of 60 consecutive newly diagnosed bladder SCC patients diagnosed by cystoscopy and histopathological examination,and controls were 60 outpatients with benign urologic conditions and healthy clinic visitors.Urine samples collected from each subject underwent testing for NMP22,telomerase activity,and CD44.Descriptive and correlational statistical analysis of cases and controls were carried out and receiver operating characteristic curve analysis was used to determine optimal cut-off points for the three assays.Results:Area under the curve was calculated at 0.96,0.93,and 0.62 for NMP22,telomerase,and CD44,respectively.Urine levels of NMP22 and telomerase activity were significantly higher in the SCC group compared to controls(p<0.001).Urine CD44 levels were not significantly higher in the SCC group compared to controls(p=0.111).The overall sensitivity of NMP22,telomerase,and CD44 was 96.7%,87%,and 45%,respectively,while the specificity was 85%,88.6%,and 86.7%,respectively.Conclusions:Urinary telomerase activity,followed by NMP22 urine levels,showed high diagnostic yield and could hold potential promise as urinary biomarkers for the diagnosis of bladder SCC.

Prognostic relevance of circulating CK19 mRNA in advanced malignant biliary tract diseases

AIM: To determine the role of circulating tumor cells (CTCs) in prediction of the overall survival of patients with advanced malignant biliary tract obstruction. METHODS: We investigated the prognostic value of CTCs by examining two markers, cytokeratin (CK) 19 and human telomerase reverse transcriptase (hTERT) mRNA, in 40 patients diagnosed with advanced malig- nant biliary tract diseases. Quantitative real-time re- verse transcription polymerase chain reaction was used to detect CK19 and hTERT mRNA in the peripheral blood of these patients. Overall survival was analyzed using the Kaplan-Meier method and Cox regression modeling.RESULTS: Positive CK19 and hTERT mRNA expression was detected in 45% and 60%, respectively, of the 40 patients. Univariable analysis indicated that positive CK19 mRNA expression was significantly associated with worse overall survival (P = 0.009). Multivariable analysis determined that positive CK19 mRNA expres- sion, patient's age and serum bilirubin were each inde- pendently associated with overall survival. CONCLUSION: CK19 mRNA expression levels in pe- ripheral blood appear to provide a valuable marker to predict the overall survival of patients with advanced malignant biliary tract obstruction.

苦参碱对K562细胞端粒酶hTERT-mRNA表达及其酶活性影响作用的研究

目的：观察苦参碱对 K562细胞 hTERT- mRNA表达及端粒酶活性的影响作用 ,并明确两者的相关性。方法：以 0.1～ 2 mg/ml苦参碱处理 K562细胞 48h后， RT- PCR检测其 hTERT- mRNA表达，同时行 TRAP- PCR- ELISA端粒酶活性检测。结果 :K562细胞为一强端粒酶活性细胞株，在浓度分别为 0.1、 0 5、 1 0、 2 0 mg/ml苦参碱作用后， K562细胞 hTERT- mRNA表达明显受抑，同时伴有端粒酶活性下降，抑制率分别为 0 3％、 13 0％、 91 7％和 98 6％。结论：苦参碱可降低 K562细胞 hTERT- mRNA表达 ,同时伴端粒酶活性下降；端粒酶活性强度与 hTERT- mRNA表达有关。

维甲酸和地塞米松对骨肉瘤细胞分化与端粒酶活性的影响

目的 :观察维甲酸 (RA)和地塞米松 (DEX)对骨肉瘤细胞 (HOS)形态和功能分化的诱导作用以及分化后细胞周期与端粒酶活性的变化。方法 :用RA和DEX诱导HOS细胞分化 ,观察分化后瘤细胞的细胞形态和功能的变化。细胞周期用流式细胞仪测定。端粒酶活性用TRAP -银染法检测。结果 :RA和DEX可使体外培养的HOS细胞停留在G2 /M期 ,细胞生长受到明显抑制 ,细胞形态和功能分化成熟 ,碱性磷酸酶 (AP)活性明显升高 ,瘤细胞端粒酶活性随药物作用时间和浓度的增加而明显减弱。结论 :RA和DEX能诱导骨肉瘤细胞形态和功能上的分化 ,端粒酶活性的调节可能发生于细胞周期的S期 ,其活性的下降可作为骨肉瘤细胞分化的指标 。

转基因肿瘤细胞端粒酶活性表达人和小鼠的差异性

目的人和小鼠正常体细胞端粒酶的活性，存在着种属差异性．肿瘤细胞中端粒酶活性的变化是否亦存在种属差异性尚不清楚．因此，我们在人和小鼠的肿瘤细胞系中，分别转导细胞因子基因ＩＬ２和（或）ＴＮＦα，以观察其对肿瘤细胞增殖活性及端粒酶活性的影响．方法首先将目的基因ＩＬ２和（或）ＴＮＦα构建入ｐＬＸＳＮ或ｐＧＣＥＮ逆转录病毒载体，用脂质体法转染ＰＡ３１７细胞进行病毒包装，再以病毒颗粒分别感染ＭＫＮ２８，ＭＫＮ４５，Ｔｃａ８１１３及Ｈ２２细胞，后者经Ｇ４１８筛选、挑克隆及扩增培养，然后用ＰＣＲ，ＲＴＰＣＲ及Ｓｏｕｔｈｅｒｎｂｌｏｔｉｎｇ鉴定其外源基因的整合及表达，并制作细胞生长曲线，以观察肿瘤细胞增殖活性变化，同时用ＴＲＡＰＰＣＲ法观察转基因肿瘤细胞端粒酶活性变化．结果人ＭＫＮ２８，ＭＫＮ４５及Ｔｃａ８１１３转导ＩＬ２或ＴＮＦα后，肿瘤细胞增殖受到不同程度的抑制；相应地，其端粒酶活性也受到抑制．而小鼠Ｈ２２细胞转导ＩＬ２和（或）ＴＮＦα后，增殖未受抑制，端粒酶活性亦未受抑制．结论人和小鼠肿瘤细胞系中转细胞因子基因ＩＬ２和（或）ＴＮＦα后，端粒酶活性及肿瘤细胞增殖发生不同的变化，提示肿瘤细胞中端粒酶的作用机制，在人和小?

PLGA纳米载体介导的端粒酶反义核酸体外转染率及对肝肿瘤细胞的影响

目的通过以PLGA纳米粒作为端粒酶hTERT反义核酸的载体,促进反义核酸对肝肿瘤细胞的抑制作用。方法用绿色荧光蛋白报道基因pEGFP-N1表达质粒DNA转染细胞;观测不同转染方法的转染率,TRAP-ELISA法检测端粒酶活性;噻唑蓝(MTT)法测定端粒酶反义核酸对肝肿瘤细胞HepG2的抑制;过氧化物酶的抗荧光素抗体法检测肿瘤细胞凋亡指数。结果裸DNA和磷酸钙的转染效率均不高,分别为18%和24%;脂质体的转染效率为42%,纳米粒的转染效率最高,达61%;以脂质体或PLGA纳米粒为载体介导端粒酶反义核酸处理的肿瘤细胞,其端粒酶活性和生长与空白对照相比明显下降(P<0.05),两种载体之间的差异也非常显著(P<0.05);处理72h后,纳米粒介导组的肿瘤细胞凋亡率显著高于脂质体介导组,分别为23.1%和16.4%。结论PLGA纳米粒是一种非常有前途的非病毒基因治疗载体。

The prognostic molecular markers in hepatocellular carcinoma

The prognosis of hepatocellular carcinoma (HCC) still remains dismal, although many advances in its clinical study have been made. It is important for tumor control to identify the factors that predispose patients to death. With new discoveries in cancer biology, the pathological and biological prognostic factors of HCC have been studied quite extensively. Analyzing molecular markers (biomarkers) with prognostic significance is a complementary method. A large number of molecular factors have been shown to associate with the invasiveness of HCC, and have potential prognostic significance. One important aspect is the analysis of molecular markers for the cellular malignancy phenotype. These include alterations in DNA ploidy, cellular proliferation markers (PCNA, Ki-67, Mcm2, MIB1, MIA, and CSE1L/CAS protein), nuclear morphology, the p53 gene and its related molecule MD M2, other cell cycle regulators (cyclin A, cyclin D, cyclin E, cdc2, p27, p73), oncogenes and their receptors (such as ras, c-myc, c-fms, HGF, c-met, and erb-B receptor family members), apoptosis related factors (Fas and FasL), as well as telomerase activity. Another important aspect is the analysis of molecular markers involved in the process of cancer invasion and metastasis. Adhesion molecules (E-cadherin, catenins, serum intercellular adhesion molecule-1, CD44 variants), proteinases involved in the degradation of extracellular matrix (MMP-2, MMP-9, uPA, uPAR, PAI), as well as other molecules have been regarded as biomarkers for the malignant phenotype of HCC, and are related to prognosis and therapeutic outcomes. Tumor angiogenesis is critical to both the growth and metastasis of cancers including HCC, and has drawn much attention in recent years. Many angiogenesis-related markers, such as vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), platelet-derived endothelial cell growth factor (PD-ECGF), thrombospondin (TSP), angiogenin, pleiotrophin, and endostatin (ES) levels, as well as intratumor microvessel density (MVD) have been evaluated and found to be of prognostic significance. Body fluid (particularly blood and urinary) testing for biomarkers is easily accessible and useful in clinical patients. The prognostic significance of circulating DNA in plasma or serum, and its genetic alterations in HCC are other important trends. More attention should be paid to these two areas in future. As the progress of the human genome project advances, so does a clearer understanding of tumor biology, and more and more new prognostic markers with high sensitivity and specificity will be found and used in clinical assays. However, the combination of some items, i.e., the pathological features and some biomarkers mentioned above, seems to be more practical for now.

全反式维甲酸和5-Fu对胃癌细胞端粒酶活性和细胞生长的影响

目的观察 ATRA,5-Fu 单独和联合应用对体外培养的胃癌细胞端粒酶活性及细胞生长的影响.方法分别用 ATRA,5-Fu 单独和联合处理胃癌 MGC-803细胞,采用 MTT 法测定细胞活力,采用端粒重复序列扩增法测定端粒酶活性.结果胃癌细胞活力随 ATRA,5-Fu 浓度增高、作用时间的延长其细胞活力逐渐下降,ATRA d1,d3,d5的 IG_(50)分别为>40μmol/L,(20～40)μmol/L,(10～20)μmol/L;5-Fu d1,d3,d 5的 IC_(50)分别为>25/μmol/L,(2～5)/μmol/L,(2～5)μmol/L;40μmol/L ATRA,5μmol/L 5-Fu 及40μmol/LATRA+5μmol/L 5-Fu 处理胃癌细胞3 d 其细胞活力分别为46%,47%,8%(P<0.01),端粒酶活性分别为45.68%(P<0.01),100.00%,46.10%(P<0.01).结论 ATRA,5-Fu 均能抑制胃癌细胞的生长,其抑制作用具有时间和浓度依赖性,且二者合用具有协同抑制作用;ATRA能抑制胃癌细胞端粒酶活性,而5-Fu 对胃癌细胞端粒酶活性无影响,二者合用对胃癌细胞端粒酶活性无协同抑制作用.抑制端粒酶活性可能是 ATRA 抗癌机制之一.

不同抗癌药物对乳癌MCF-7细胞增殖及端粒酶活性的影响

目的 研究乳癌细胞MCF 7在不同抗癌药物存在的情况下端粒酶活性的变化规律。方法 运用细胞记数、台盼蓝染色和MTT法测定细胞增殖能力及其活性 ,同时用TRAP法测定细胞端粒酶活性。观察细胞在不同情况下端粒酶活性变化及其影响因素。结果 在无药物存在时 ,细胞增殖与端粒酶活性增加呈正相关 (r=0 .90 1)。阿霉素、紫杉醇和顺铂都能有效抑制细胞生长 ,以剂量依赖方式和时间依赖方式降低端粒酶活性 ,这种降低与活性细胞减少密切相关。结论 阿霉素、紫杉醇和顺铂抑制MCF 7细胞生长 ,其机制可能与降低端粒酶活性及细胞活性有关。

端粒酶抑制剂对肿瘤细胞及其端粒酶活性的影响

目的 探讨端粒酶抑制剂作为肿瘤治疗药物的可行性。方法 选择人结肠癌细胞株 HC86 93、人肺癌细胞株A5 49以及人肝癌细胞株 L740 2作为靶细胞 ,观察 3′-叠氮 -3′-脱氧胸腺核苷 (AZT)和人端粒酶模板区的硫代反义寡核苷酸 (S- Oligos)对上述几种肿瘤细胞株的作用 ,用 MTT试验测定细胞毒作用 ;3H - Td R掺入试验测定细胞增殖速度 ;用端粒重复序列扩增法 (TRAP)半定量方法测定细胞染毒前后端粒酶活性的变化。结果 反义核苷酸片段和 AZT在一定剂量范围内有抑制肿瘤细胞株繁殖的作用 ,在抑制细胞生长的同时 ,端粒酶活性降低 ,去除抑制剂 2 4h后 ,端粒酶活性仍然维持在较低水平。三种细胞中 ,HC86 93和 L740 2对两种抑制剂较 A5 49细胞株更加敏感 ,两种抑制剂联合应用比单独应用的效果好。结论 AZT和 S- Oligos单独或联合应用在体外有抗肿瘤细胞的作用 ,进一步深入的研究有助于新的抗癌药物的开发。

银染-TRAP法检测端粒酶活性的建立

目的建立检测端粒酶活性的改良银染ＴＲＡＰ法（端粒重复序列扩增法）。方法用银染ＴＲＡＰ法测定人肿瘤细胞株和经加热处理的阴性对照细胞的端粒酶活性。结果肿瘤细胞株的端粒酶活性阳性率为１００％，１０、１００、５００个Ｈｅｌａ细胞均为阳性，加热处理后的细胞端粒酶活性均为阴性。结论提示与核素ＴＲＡＰ法相比较，银染ＴＲＡＰ法是同样特异、敏感，且更快速的端粒酶活性的检测方法，该方法可成为恶性肿瘤的诊断指标之一。

端粒酶在人胚肾上皮细胞转化中的作用

目的:利用原代人胚肾上皮细胞转化模型阐明端粒酶在细胞癌变过程中的作用。方法:用逆转录病毒介导的基因导入法,使人端粒酶催化亚基(humantelomerasecatalyticsubunit,hTERT)、HRas癌基因、猿猴病毒40(simianvirus40,SV40)编码的早期抗原在原代人胚肾上皮细胞中稳定地表达,观察细胞的生长特性、染色体畸变率以及细胞转化特征。结果:端粒酶的激活虽然能延长细胞的寿命,但不能使细胞永生化。如果细胞同时表达端粒酶和SV40编码的大T抗原(largeTantigen,LT),细胞就能获得永生化。在此永生化细胞株中导入HRas癌基因以及SV40编码的小T抗原(smallTantigen,ST),细胞发生恶性转化,表现为在软琼脂上形成克隆并在裸鼠皮下形成肿瘤。与原代细胞相比,永生化细胞株的染色体畸变率无改变,而恶性转化细胞的畸变率明显增加。此外在转化细胞和几种肿瘤细胞中表达阻抑人端粒酶的特异性siRNA,能显著地抑制肿瘤细胞的生长、诱导细胞凋亡并减少软琼脂上形成的克隆数目。结论:细胞永生化是癌变过程的必要阶段,端粒酶的激活不仅是细胞永生化的重要环节,而且在维持肿瘤细胞生长中起重要的作用。

端粒酶反义寡核苷酸及顺铂对恶性脑胶质瘤治疗的协同作用

目的观察端粒酶反义寡核苷酸(ODN)及顺铂对恶性脑胶质瘤治疗的协同作用。方法用15例Ⅲ、Ⅳ级端粒酶活性阳性表达的恶性脑胶质瘤制备单细胞悬液并原代培养,以5μmol／L端粒酶反义ODN抑制恶性胶质瘤细胞端粒酶活性后,用不同浓度顺铂处理细胞。流式细胞仪检测处理前后胶质瘤细胞PCNA、TUNEL阳性百分率变化情况。结果端粒酶反义ODN作用于恶性胶质瘤细胞72h后,TRAP法检测端粒酶活性转为阴性,此时0．3μg／mL顺铂即可对胶质瘤细胞有明显抑制增殖、促进凋亡作用。结论端粒酶反义ODN能抑制端粒酶活性,增加恶性胶质瘤细胞对顺铂的敏感性,与顺铂在治疗恶性脑胶质瘤细胞过程中有协同作用。

端粒酶在细胞永生化及肿瘤诊断中的作用

端粒酶是一种新的肿瘤标记物,细胞永生化是细胞获得持续生长增殖能力的特性,永生化的细胞有无限增殖生长性,可长期传代。综观国内外的研究情况,端粒酶在肿瘤的诊断上尚有可观的价值,已成人类的研究热点,现已确知在85%的人肿瘤组织中发现了端粒酶活性的表达,因此,已把端粒酶作为肿瘤基因诊断的指标和治疗的新靶点。

端粒和端粒酶与衰老、癌症的潜在关系——2009年诺贝尔生理学或医学奖简介

美国科学家伊丽莎白.布莱克本、卡萝尔.格雷德和杰克.绍斯塔克三人同时获得2009年诺贝尔生理学或医学奖,这是由于他们发现"染色体是如何被端粒和端粒酶保护的",这一研究成果揭开了人类衰老和肿瘤发生等生理病理现象的奥秘。本文将就端粒和端粒酶的发现、结构和功能及其与人类衰老、癌症的潜在关系等方面做一简要介绍。

肿瘤患者外周血细胞端粒酶表达及临床意义

目的探讨肿瘤患者外周血单个核细胞端粒酶活性的表达。方法采用端粒重复序列扩增-微孔板杂交法测定恶性肿瘤患者48例,良性患者34例和38例正常对照者外周血单个核细胞端粒酶表达。结果恶性肿瘤和良性肿瘤患者组外周血单个核细胞端粒酶活性比对照组明显增高(P<0.01),且对恶性肿瘤和良性肿瘤患者的检出阳性率分别达77.1%和47.1%。结论外周血单个核细胞端粒酶表达与肿瘤的发生、发展有关,因而检测外周血单个核细胞端粒酶活性有助于肿瘤的早期诊断和治疗。

端粒和端粒酶的结构与功能及其应用

端粒是构成真核生物线状染色体末端重要的 DNA—蛋白质复合结构,DNA 由简单的串联重复序列组成.它的合成由一个特殊的具有反转录活性的核糖核蛋白端粒酶完成.端粒对染色体、整个生物基因组,甚至对细胞的稳定都具有重要意义.端粒酶是由 RNA 模板和蛋白亚基组成的核蛋白颗粒.它解决染色体的末端问题,归属于逆转录酶家族又和逆转录酶有一定的差别.端粒酶的过度表达和细胞的永生化和癌变直接相关.端粒酶的结构和功能决定了它在肿瘤与癌症治疗等方面具有广泛的应用前景.