Reversible downregulation of telomerase activity by hyperthermia in osteosarcoma cells.

Telomerase, a ribonucleoprotein enzyme, maintains telomere length and is expressed by the majority of malignant tumours, but not in normal tissue. Telomerase facilitates the division of tumour cells and its activity has been suggested as a prognostic indicator, but so far the regulation or modulation of telomerase activity has not been described. Hyperthermia has been shown to decrease tumour growth by inhibition of proliferation. Therefore, the effect of hyperthermia on telomerase activity in human osteosarcoma cells was studied. Telomerase activity was measured by the Telomeric Repeat Amplification Protocol (TRAP) assay in three different osteosarcoma cell lines subjected to hyperthermia (42.5 degrees C, 90 min) and in controls cultured under basal conditions (37 degrees C). Telomerase activity was strongly inhibited by hyperthermia and decreased in all cell lines tested after a recovery time of 2 h under basal conditions (37 degrees C) to an activity of approximately 85%, after 12 h approximately 60% and with lowest activity approximately 55% compared to activity of control cells. Telomerase activity then increased and reached the same, i.e. basal, level as before hyperthermia, after 112 h. These results show that hyperthermia results in a reversible downregulation of telomerase activity in osteosarcoma cells. This effect facilities studies on the regulation of telomerase activity and detailed information might lead to new therapeutic strategies.     

Apoptosis induced by hyperthermia in Dunn osteosarcoma cell line in vitro

The effect of hyperthermia at 43.5^oC for 1h on Dunn osteosarcoma cells was studied. With sham-heated cells (37^oC, 1h) as the control, the hyperthermia treated cells were divided into five groups. Time 0 group was the cells that were harvested immediately after heated at 43.5^oC for 1h. Whereas time 3, 6, 12, and 24h groups were the cells that were collected respectively after reincubation at 37^oC for the above different time periods. The appearance of hyperthermiainduced apoptosis of Dunn osteosarcoma cells was demonstrated to be time dependent. With the confocal microscopic study and TUNEL staining, the morphological characteristics of apoptosis, condensed nuclei and fragmented nuclei were obvious when reincubated at 37^oC for 6h after hyperthermic treatment. This hyperthermia-induced apoptosis was further confirmed by flow cytometric analysis on DNA contents. The sub-G₁ region that was proposed as a marker of apoptotic cells was most significantly elevated at 6h after hyperthermic treatment and, thereafter, decreased to the levels of control values by 24h, as the apoptotic cells underwent secondary necrosis and degraded to debris. The DNA strand breaks, considered as the key biochemical event of apoptosis, were detected by the TUNEL assay. This study indicated that hyperthermia (43.5^oC for 1h) can induce apoptotic changes on osteosarcoma cells in vitro very rapidly (within 6h after treatment), and its occurrence might not be detected if the samples are not taken at several early time points after hyperthermia.     

骨肉瘤端粒酶的表达及其与临床的关系

目的 研究骨肉瘤端粒酶的表达,探讨骨肉瘤端粒酶的表达与临床特征的相关性。方法 应用原位杂交方法,检测49例骨肉瘤石蜡标本及10例正常骨组织和10例骨软骨瘤石蜡标本的端粒酶表达活性,并分析骨肉瘤的端粒酶表达活性与临床病理特征的关系。结果 49例骨肉瘤中端粒酶表达阳性39例(阳性率79.6％),而正常骨组织和骨软骨瘤端粒酶阳性表达率分别为0％和10％,与骨肉瘤比较差异极显著(P=0.001);骨肉瘤端粒酶的表达与肿瘤的大小、病理分级、3年存活率等明显相关。结论 端粒酶在骨肉瘤中阳性表达高,有辅助早期诊断的意义;骨肉瘤端粒酶的表达情况可以作为骨肉瘤预后评判的有效指标。     

骨肉瘤端粒酶逆转录酶的定性与定量检测及其意义

目的 对骨肉瘤中人端粒酶逆转录酶(hTERT)的表达进行定性及定量检测,探讨hTERT在骨肉瘤中的表达及意义.方法 用免疫细胞/组织化学法分别定性检测人宫颈癌细胞株HeLa、人骨肉瘤细胞株U2-OS、MG-63、SAOS-2及15例骨肉瘤组织中hTERT的表达情况;用蛋白印迹法(Western-blot,WB)定量检测上述细胞株及7例骨肉瘤组织中hTERT的表达情况.结果 免疫细胞/组织化学显示,HeLa细胞hTERT表达率最高(95.16%);MG-63、SAOS-2、U2-OS细胞系中hTERT阳性率分别为8.75%、5.26%、2.33%;骨肉瘤组织中hTERT表达阳性率26.7% (4/15).蛋白印迹法显示,Hela、MG-63及两个骨肉瘤样本(OS1和OS2)有hTERT表达,U2-OS及SAOS-2中几乎没有hTERT表达.结论 hTERT在骨肉瘤中表达很低,可能无法成为骨肉瘤的临床治疗靶点.     

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）和地塞以（ＤＥＸ）对骨肉瘤细胞（ＨＯＳ）形态和功能分化的诱导作用以及分人化后细胞周期与端粒酶活性变化。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能诱导骨肉瘤细胞分化的形态和     

DETECTION OF HUMAN TELOMERASE ACTIVITY BY TELOMERASE TRAP-ELISA ASSAY

ＤＥＴＥＣＴＩＯＮＯＦＨＵＭＡＮＴＥＬＯＭＥＲＡＳＥＡＣＴＩＶＩＴＹＢＹＴＥＬＯＭＥＲＡＳＥＴＲＡＰ—ＥＬＩＳＡＡＳＳＡＹＷｅｉＬｉｘｉｎ卫立辛ＷｕＭｅｎｇｃｈａｏ吴孟超ＹａｎＺｈｅｎｌｉｎ阎振林ＳｈｅｎＦｅｎｇ沈锋ＸｉｅＴｉａｎｐｅｉ谢天培Ｑｉａ...     

Lysyl oxidase (LOX) mRNA expression and genes of the differentiated osteoblastic phenotype are upregulated in human osteosarcoma cells by suramin

It is well known that suramin influences proliferation and differentiation of tumour cells. To study whether and how suramin effects osteosarcoma (OS) cells, proliferation, differentiation, LOX mRNA expression and telomerase activity (TA) was analysed in the human MG-63 and U-2 OS, and the rat UMR-106 OS cell lines. Data show that suramin inhibited proliferation in the human cell lines and upregulated alkaline phosphatase activity. TA was attenuated in the human cells while in UMR-106 it was not changed. In UMR-106 suramin had no influence on osteocalcin and LOX expression, in the human cells however, both genes were upregulated.     

紫杉醇对卵巢癌细胞端粒酶活性影响的研究

目的观察紫杉醇对卵巢癌HO-8910细胞端粒酶活性的影响,以了解紫杉醇抗癌的可能机理.方法 1～100nmol/L紫杉醇处理HO-8910细胞后,用MTT法测定卵巢癌细胞的生长抑制率,TRAP-银染法测定端粒酶活性变化.结果 1～100nmol/L紫杉醇对卵巢癌细胞有明显抑制作用,呈时间依赖性及剂量依赖性,紫杉醇处理卵巢癌细胞后,对端粒酶活性的检测结果显示,端粒酶活性下调.结论紫杉醇对卵巢癌细胞具有明显抑制作用,其下调端粒酶活性可能是紫杉醇抗癌作用的重要机理之一.     

检测人端粒酶活性的端粒酶TRAP—ELISA法的建立

目的为改进端粒重复序列扩增法（ＴＲＡＰ）存在定量困难、应用同位素及每次检测标本数受限等缺点,研究建立及评价端粒酶ＴＲＡＰＥＬＩＳＡ法。方法端粒酶ＴＲＡＰＥＬＩＳＡ法是将ＴＲＡＰ与ＰＣＲＥＬＩＳＡ系统结合。与常规ＴＲＡＰ法相比较,应用端粒酶ＴＲＡＰＥＬＩＳＡ法检测端粒酶阳性的２９３细胞和阴性对照标本（加热或ＲＮａｓｅ处理和正常人内皮细胞）。结果２９３细胞端粒酶阳性,对１０,１０２,１０３及１０４个细胞检测均为阳性,所测到的吸光度值（Ａ,曾称光密度ＯＤ）依赖于被检２９３细胞数。ＲＮａｓｅ或加热处理标本和正常人内皮细胞均阴性。该方法可在当日观察结果,不需放射性同位素。结论端粒酶ＴＲＡＰＥＬＩＳＡ是一种非放射性同位素、快速及可定量的人端粒酶活性检测方法。     

胆汁脱落细胞端粒酶活性检测在胆道恶性肿瘤诊断中的价值

背景与目的：已有学者对尿液,胰液,肠腔冲洗液,胸水等脱落细胞端粒酶活性进行了检测,探索其对恶性肿瘤的诊断价值。而胆汁脱落细胞端粒酶活性的检测尚少见报道。本文的目的是探讨胆汁脱落细胞端粒酶活性检测及其对胆道恶性梗阻的诊断价值。方法：应用TRAP银染法检测胆汁脱落细胞的端粒酶活性。结果：44例恶性肿瘤胆道梗阻病人获得的胆汁中有33例检测到端粒酶活性阳性,阳性率75．0％,而良性梗阻组19例中只有1例检测到端粒酶弱阳性,阳性率5．3％。胆汁脱落细胞端粒酶活性阳性率与淋巴结转移,远处转移以及分化程度等肿瘤临床病理学特征无关,但胰腺癌和胆管癌病人胆汁中端粒酶活性检测阳性率高。端粒酶活性检测与脱落细胞学检查对比结果显示：44例胆道恶性梗阻的病人中31例进行了胆汁脱落细胞学检查,结果只有3例癌细胞阳性,阳性率9．7％,这3例脱落细胞学检查阳性的胆汁脱落细胞端粒酶活性检测均为阳性。结论：TRAP银染法可有效检测到胆汁脱落癌细胞的端粒酶活性。端粒酶可以作为诊断恶性胆道疾病的分子标志物。胆汁中脱落细胞端粒酶活性检测可作为细胞学检查的补充诊断手段。     

顺铂对人肺癌SPCA-1细胞端粒酶活性的影响

目的 观察顺铂(DDP)对人肺癌SPCA-1细胞端粒酶活性的影响。方法 不同浓度的DDP处理体外培养的人肺癌SPCA-1细胞72h，非放射性标记的改良TRAP法测定端粒酶活性，MTT法测定细胞增殖抑制，透射电镜、流式细胞仪观察细胞凋亡。结果 DDP可抑制SPCA-1细胞端粒酶活性，随DDP浓度增大，端粒酶活性抑制增强。MTT结果显示DDP抑制SPCA-1细胞增殖且抑制率有浓度依赖性。透射电镜可观察到典型的细胞凋亡形态学，流式细胞仪结果表明DDP诱导SPCA-1细胞凋亡在一定浓度范围内呈剂量依赖性。结论 端粒酶活性的抑制可能是DDP发挥抗肺癌活性的作用机制之一，端粒酶活性可作为评价DDP抗肺癌化疗效果的指标之一。     

Arsenic trioxide downregulates telomerase activity in HL-60 cells

Telomeres are the specialized nucleoproteincomplexes at the physical ends of eukaryoticchromosomes[1]. Telomere length decreases along with increasing cycles of cell divisions. Telomere shortening has therefore been proposed to play a role in cellular senescence. Telomerase is a protein-RNA enzyme complex that adds a six-base DNA sequence (TTAGGG) to the ends of chromosomes and thereby prevents their shortening. This enzyme is specifically activated in most malignant tumors but is usuall…     

反义c-myc对MCF-7细胞端粒酶活性的影响

目的研究脂质体LipfectAMINE^TM（LR）介导的c-myc反义寡核苷酸（ASODN）对MCF-7细胞端粒酶活性的影响及其机制。方法端粒重复序列扩增聚丙烯酰胺凝胶电泳（TRAP-PAGE）及端粒重复序列扩增酶联免疫吸附测定（TRAP-ELISA）定量检测不同时间段MCF-7细胞的端粒酶活性,流式细胞仪检测c-myc蛋白表达。结果TRAP-PAGE结果显示,c-myc ASODN对MCF-7细胞的作用,随时间的递增（24h、48h、72h）,端粒酶梯度条带明显地减少,端粒酶活性明显降低。TRAP-ELISA显示,2．5μmol／L的c-myc ASODN作用MCF-7细胞48h吸光度A值降为0．486±0．041,作用72hA值降为0．263±0．034,与未经任何处理的MCF-7细胞A值0．684±0．031相比,端粒酶活性明显受到抑制,分别与正义寡核苷酸组、空白对照组相比差异有统计学意义（P〈0．05）。未经任何处理的MCF-7细胞c-myc蛋白阳性率为（99．684±2．937）％,经c-myc ASODN作用48h c-myc蛋白阳性率低至（61．295±2．825）％,c-myc ASODN作用72h c-myc蛋白阳性率低至（29．482±2．726）％。而c-mye正义寡核苷酸（SODN）无上述作用。结论c-myc ASODN能有效降低MCF-7细胞端粒酶活性和c-myc蛋白表达。     

Modulation of cellular glycosidase activity by hyperthermia

We examined the effect of 45°C hyperthermia on the following glycosidases in CHO cells: β-galactosidase, β-hexosaminidase, β-glucuronidase and α-mannosidase. Among these, lysosomal α-mannosidase exhibited the most dramatic response to hyperthermia with an increase in activity immediately after 45°C hyperthermia. The increase was linearly dose-dependent with a doubling of activity for every 20 min at 45°C. In contrast to α-mannosidase, β-glucuronidase, β-galactosidase, and β-hexosaminidase showed only minor alterations in activity, or none, after hyperthermia of 10 to 60 min at 45°C. Induction of thermotolerance enhanced the heat resistance of β-galactosidase, but caused increased heat sensitivity for α-mannosidase. Intracellular β-galactosidase, measured by histochemical staining, showed a dramatic redistribution in response to mild hyperthermia (10 min, 45°C); the same effect was not observed for β-glucuronidase. The data argue against non-specific activation of lysosomes by hyperthermia, and suggest that cells contain lysosomal subpopulations that are characterized by different heat sensitivities and variable glycosidase contents.     

大肠癌组织中端粒酶活性的研究

目的研究大肠癌组织及相应癌旁组织中的端粒酶活性,探讨其作为肠癌肿瘤标记物的可能性。方法采用ＴＲＡＰ方法研究了４０例大肠组织（包括３７例大肠癌,３例良性大肠疾病）和２０例相应癌旁上皮组织中的端粒酶活性表达。结果２０例癌旁上皮和３例良性疾病组织中,端粒酶活性表达均为阴性;而３７例大肠癌组织中,３５例显示端粒酶活性表达为阳性,阳性率为９４．６％。结论端粒酶是特异性较强的恶性肿瘤基因标志,有可能成为大肠癌早期诊断和治疗的理想标志物     

采用改进方法检测肺癌组织端粒酶活性

用改进的ＴＲＡＰ法检测肺癌的端粒酶活性,探讨端粒酶活性与肺癌的关系。方法：用培养瓶替代液氮瓶收集标本,采用ＴＲＡＰ改进方法,内含３６ｐｂ质控模板预防假阴性,增加强物长度避免二聚体形成,且使反应一步完成,并用银染法显示检测结果,对４５例病理确诊的肺癌手术标本及一株肺癌细胞株的端粒酶活性进行检测。     

丁酸钠诱导MCF-7细胞凋亡过程中端粒酶活性的变化

目的　研究丁酸钠诱导MCF 7细胞凋亡过程中端粒酶活性变化及其机制。方法　采用MTT法和倒置相差显微镜观察不同浓度丁酸钠对MCF 7细胞的抑制作用 ,流式细胞仪和琼脂糖凝胶电泳检测 2 .5mmol/L丁酸钠作用后的细胞凋亡情况 ,TRAP ELISA法检测端粒酶活性变化 ,RT PCR分析端粒酶各组分的mRNA表达情况。结果　丁酸钠对MCF 7细胞的抑制作用具有时间和剂量依赖性 ,AnnexinV/PI双染法显示 ,2 .5mmol/L丁酸钠作用 72h后 ,细胞凋亡率为 84 .3% ,琼脂糖凝胶电泳可见间隔为 180bp的DNA梯状条带。 2 .5mmol/L丁酸钠作用 2 4和 4 8h后 ,端粒酶活性分别下降为1.82± 0 .2 2和 1.6 1± 0 .0 9。RT PCR显示 ,端粒酶逆转录酶 (hTERT)表达下降 ,而端粒酶RNA模板(hTR)和端粒酶相关蛋白 (hTP1)表达无明显改变。结论　丁酸钠诱导MCF 7细胞凋亡过程中端粒酶活性下降 ,其机制可能与丁酸钠下调hTERT转录水平有关。