p53基因对HL-60细胞端粒酶活性和人端粒酶逆转录酶基因表达的影响

为了研究野生型p53基因转染的HL-60细胞端粒酶活性和人端粒酶逆转录酶（hTERT)基因表达的变化，采用脂质体法将野生型p53基因转染HL-60细胞，用TUNEL检测细胞凋亡。用端粒重复扩增法（TRAP）-酶联免疫吸附试验（ELISA）检测端粒酶活性变化，以及用RT-PCR方法检测hTERTmRNA表达的变化。结果显示：转染野生型p53基因的HL-60细胞在32.5℃培养24小时和72小时后，凋亡率分别为8.3%和21.0%,hTERTmRNA和端粒酶活性分别下降至对照组的68.4%和55.8%及27.3%和8.9%。结论：p53基因能下调HL-60细胞hTERTmRNA和端粒酶活性。这可能是野生型p53基因诱导细胞凋亡机制之一。     

Real-time quantitative telomeric repeat amplification protocol assay for the detection of telomerase activity

BACKGROUND: Telomerase is a ribonucleoprotein enzyme associated with immortalization and transformation of human cells. The telomeric repeat amplification protocol (TRAP) is widely used for the detection of telomerase activity. The TRAP method, although highly sensitive and specific because it includes PCR amplification, is laborious and does not provide precise quantitative information. METHODS: We developed a real-time quantitative TRAP (RTQ-TRAP) system by combining a real-time PCR technique with the conventional TRAP method. Telomerase activity in human tumor cell lines and in 13 lymphoma samples was measured using the RTQ-TRAP assay, and the results obtained from the samples using the RTQ-TRAP method were compared with the conventional TRAP method. RESULTS: The RTQ-TRAP method was both accurate and reproducible in measuring telomerase activity in a dilution series of protein extracts from HL60 cells. Telomerase activity in 13 lymphoma samples, as determined by the RTQ-TRAP method, was ninefold lower than that measured by the conventional TRAP method. The half-life of telomerase activity in human tumor cells, as determined using RTQ-TRAP, was much shorter than the half-life reported previously. CONCLUSIONS: Our results suggest that the conventional TRAP assay frequently overestimates telomerase activity in tumor samples. The RTQ-TRAP method is thus a useful tool to rapidly and precisely quantify telomerase activity.     

脑胶质瘤端粒酶活性表达的研究

目的：探讨端粒酶在脑胶质瘤发生、发展中的作用，并研究端粒酶活性是否与其恶性程度相关。方法：应用端粒重复序列扩增技术(TRAP)对48例胶质瘤标本和8例正常脑组织的端粒酶活性进行检测。结果：8例正常脑组织端粒酶活性均为阴性表达，48例胶质瘤标本中23例为表达阳性(47．92％)，低度恶性组与高度恶性组胶质瘤之间端粒酶活性阳性率有明显差异(P=0．001)。结论：端粒酶活性与胶质瘤恶性程度明显相关，端粒酶在胶质瘤发生发展过程中可能具有重要作用。     

As2O3对KM3细胞端粒酶及其逆转录酶作用的研究

为了探讨三氧化二砷(As2O3)对多发性骨髓瘤(MM)细胞株KM3的作用及其可能机制，用台盼蓝拒染法检测细胞生长抑制率，用光镜、透射电镜观察形态变化，用DNA电泳观察As2O3诱导KM3细胞凋亡，用流式细胞仪检测细胞周期的变化，用TRAP-PCR-EUSA法检测As2O3作用KM3细胞后的端粒酶活性变化，并用半定量RT-PCR法检测端粒酶逆转录酶(hTERTmRNA)的表达水平。结果表明：As2O3抑制KM3细胞的生长，具有诱导细胞凋亡的作用；As2O3阻滞细胞于G2期；As2O3可抑制KM3细胞的端粒酶活性和hTERTmRNA的表达，且hTERTmRNA下降趋势与端粒酶活性相一致。结论：端粒酶及其逆转录酶活性的下调可能在As2O3诱导KM3细胞凋亡的过程中起了重要作用。     

Improved TRAP-silver staining versus conventional radioactive TRAP assays: quantification of telomerase activity during immortalization and in pathological human endometrium

OBJECTIVES: To develop a sensitive telomeric repeat amplification protocol (TRAP)-silver staining assay for telomerase activity quantification. DESIGN AND METHODS: TRAP assays were performed by using a TRAPeze telomerase kit with or without [alpha-32P]-dCTP. Amplification products were electrophoresed in polyacrylamide gels and detected by autoradiography or a modified silver staining protocol. Telomerase activity was quantified from radioactive counts or optical density of telomerase products from test extracts and controls. RESULTS: TRAP-silver staining assay was at least as sensitive as radioactive TRAP assay and quantified telomerase activity within linearity from 10 to 3,000 cell equivalents. Both methods quantified a weak telomerase activity in normal endometrial glandular epithelial cells (GEC) and a strong increase in immortalized GEC. In human pathologic endometria (n=24), telomerase activity was correlated with lesion seriousness and distinguished simple hyperplasias from nonhyperplasic or cancerous lesions. CONCLUSIONS: TRAP-silver staining assay is suitable for cell and tissue telomerase activity routine quantification.     

上皮来源恶性肿瘤端粒酶活性检测研究

目的：探讨端粒酶活性检测在上皮来源恶性肿瘤诊断中的作用。方法：采用改良的银染端粒重复序列扩增方法,对上皮来源的120例恶性肿瘤,60例良性肿瘤和40例癌旁组织进行端粒酶活性检测。结果：120例恶性肿瘤中,110例检测到端粒酶活性,阳性率为91．6％;60例良性肿瘤中,3例检测到端粒酶活性,阳性率为5％,40例癌旁组织中,10例检测到端粒酶活性,阳性率为25％,结论：上皮来源的恶性肿瘤发生发展与端粒酶活性密切相关,端粒酶是灵敏的恶性肿瘤标记物。     

人类端粒酶逆转录酶基因反义核苷酸对白血病细胞端粒酶活性的影响

为了探讨人类端粒酶逆转录酶（hTERT)基因反义寡核苷酸（ASODN）对原代培养的急性髓性白血病（AML）和慢性髓性白血病（CML）细胞端粒酶活性的影响。采用间接免疫荧光标记方法，通过流式细胞术检测hTERT基因ASODN作用于AML和CML细胞后hTERT蛋白表达含量的变化。采用端粒酶聚合酶链反应-酶子弟免疫测定（PCR-ELISA0法检测白血病细胞端粒酶活性的改变。结果显示：hTERT ASODN作用于AML和CML细胞24，48和72小时后，hTERT蛋白表达水平不断降低；同时，AML和CML细胞端粒酶活性均逐渐受到抑制。结论：hTERT基因ASODN可以通过特异性地抑制AML和CML细胞hTERT蛋白的表达，抑制AML和CML细胞端粒酶的活性。     

Bioluminescent method for detecting telomerase activity

BACKGROUND: Telomerase is a promising biomarker in cancer diagnosis and therapy. The elongation of telomeric repeats catalyzed by telomerase is accompanied by release of six PP(i) for each TTAGGG repeat (1 pmol PP(i)/310 pg telomeric repeats). We developed a novel method to measure telomerase activity by use of an enzymatic luminometric PP(i) assay (ELIPA). METHODS: Extracts of cell lines and tissues were incubated with primer at 30 degrees C for 30 min. Released PP(i) was converted to ATP by sulfurylase, and ATP was detected by a luciferase bioluminescence system. The ELIPA results were compared with results obtained with the conventional telomeric repeat amplification (TRAP)-ELISA in 42 lung carcinoma tissues and 27 control tissues without malignancy. RESULTS: The lower detection limits of ELIPA and TRAP-ELISA were 5 and 10 cells, respectively. The within-run imprecision (CV) of ELIPA was < or =12%. When compared with TRAP-ELISA, the correlation coefficient (r) was 0.79. When we used the cutoff value from ROC analysis to distinguish malignant and nonmalignant tissues, the sensitivity and specificity of ELIPA were 83% and 96%, respectively, whereas the sensitivity and specificity of TRAP-ELISA were 71% and 96%, respectively. CONCLUSION: ELIPA is a simple and sensitive homogeneous method to quantify telomerase activity.     

端粒酶检测对恶性胸腹水的诊断价值探讨

目的探讨端粒酶活性对良恶性腩腹水的诊断价值。方法用TRAP银染法检测120份脚腹水脱落细胞的端粒酶活性,以瑞氏染色对脱落细胞进行细胞学检杳。结果在37例细胞学阳性的恶性胸腹水中端粒酶全部表达,在48例良性胸腹水中有2例端粒酶表达,在35例细胞学阴性的可疑恶性胸腹水中有29例端粒酶表达,测定的敏感性91.6%,特异性95.8%,阳性预示值97.0%,阴性预示值为88.5%,实验有效率93.3%。结论端粒酶活性表达与恶性胸腹水呈高度相关,检测端粒酶活性是临床鉴别良恶性胸腹水的一种有效方法。     

Telomerase activity in gastrointestinal, bladder and breast carcinomas and their clinical applications

Telomere ends are known to be shortened at every division of the cells. Telomerase is a ribonucleoprotein which compensate for the telomere ends and indispensable for the immortalization of the cells. It is reported that the enzyme is activated in a variety of cancer cells. In the present report, the enzyme was activated in more than 70% of gastrointestinal, bladder and breast cancer by TRAP assay. Moreover, in order to elucidate whether the enzyme activity is useful for the non-invasive detection of exfoliated cancer cells in the colon bowel washings, urine and fine needle aspirate of the breast tumors, TRAP assay was performed. In most of the cases, the positive findings were observed which support that the enzyme activity is useful for the clinical application. The expression of mRNA and protein for hTERT was detected in cancer cells, however, the assay contains the pitfall including the Taq inhibitor or telomerase inhibitor. Moreover, the activity is also detected although it is weak, in the benign or premalignant lesions. Further analysis is required for the clinical application of the method.     

Telomerase activity, and tissue polypeptide specific antigen (TPS) in Egyptian breast cancer patients.

OBJECTIVES: Breast cancer is the most common malignancy among Egyptian women. The aim of this study is to evaluate the role of both telomerase and TPS estimation in assessment of breast cancer. METHODS: The study included 40 patients with breast cancer, and 20 patients with benign breast diseases. Telomerase activity in breast tissues was assessed using TRAP assay. TPS was measured in sera of the patients by ELISA. RESULTS: Telomerase positivity was 15% in benign group vs. 60% in malignant group (p = 0.0009). It was significantly correlated to stage, and lymph node status (p < 0.02). Telomerase positivity showed significant correlation to tumor recurrence (p = 0.0076) in a follow-up period of 36 months. Mean rank of TPS was significantly higher in malignant than benign groups (p < 0.001), and in telomerase positive than telomerase negative patients (p < 0.001). In malignant group, mean rank of TPS was significantly higher in late stages (p < 0.002), in higher grade (p < 0.05), in larger tumor size (p < 0.01), and in lymph node positive patients (p < 0.001). ROC curve was utilized to choose the best cutoff for serum TPS (88 U/L). At this cutoff, the sensitivity was 95%, and the specificity was 75%. At a higher cutoff (109 U/L), TPS positivity was significantly correlated to stage, grade, lymph node status, and telomerase positivity (p < 0.05). CONCLUSION: Telomerase positivity and serum TPS might be used as additional markers for assessment of breast cancer.     

端粒重复扩增生物发光分析法定量检测端粒酶活性

目的：建立端粒酶活性定量检测的TRAP-发光分析法。方法：细胞株、组织样品与引物孵浴后，释放的焦磷酸盐由硫酸化酶作用转变为ATP，用荧光素酶生物发光系统检测发光信号；比较TRAP-发光分析法与TRAP—ELISA的结果。结果：TRAP-发光分析法的线性范围在2-1000个细胞之间。检测结果与TRAP—SYBRGreen染色一致，与TRAP—EHSA法显著相关（r^2=0．992，P〈0.001）。结论：TRAP-发光分析法是一种稳定、快速、实际可行的端粒酶活性的定量分析方法。