Bitisik O, Yavuz S, Yasasever V, Dalay N
Istanbul University, Oncology Institute, Dept. of Basic Oncology.
Res Commun Mol Pathol Pharmacol. 2000;107(1-2):3-12.
Telomeres are repeated DNA sequences, positioned at the ends of chromosomes and are essential for the stable maintenance of chromosomes. The telomere length serves as a mitotic clock determining the remaining replicative capacity of the cell. Telomeric sequences are lost during each cell division, leading to a process thought to contribute to senescence and cell death. The enzyme telomerase adds 5'-TTAGGG-3' repeats to the mammalian telomeres and maintains the telomere length. Telomerase is normally inactive in most somatic cells but telomerase activity is observed in malignancies. In this study telomerase activity was analyzed in patients with chronic myeloid leukemia (CML) and lymphoma by PCR and ELISA. This approach combines highly specific amplification of the telomerase-mediated elongation products with nonradioactive detection in a highly sensitive photometric ELISA. The PCR products were also analyzed by Southern blotting. The telomerase-specific PCR products were seperated by electrophoresis and transferred to a nylon membrane with subsequent detection of the biotinylated amplificates. High activity levels were detected in 17 CML ( 34%) patients. On the other hand, no activity was observed in lymphoma patients. An increase in the shorter telomeric bands was observed in CML patients who displayed a high level of telomerase activity. In contrast to the low enzyme activity, evidence of telomeric repeats were also found in some lymphoma patients by Southern blotting. This may indicate that lymphoma cells may make use of different pathways for maintaining the length of their telomeres.
端粒是位于染色体末端的重复DNA序列，对染色体的稳定维持至关重要。端粒长度充当有丝分裂时钟，决定细胞剩余的复制能力。端粒序列在每次细胞分裂过程中都会丢失，导致一个被认为与衰老和细胞死亡有关的过程。端粒酶将5'-TTAGGG-3'重复序列添加到哺乳动物端粒上，并维持端粒长度。端粒酶在大多数体细胞中通常无活性，但在恶性肿瘤中可观察到端粒酶活性。在本研究中，通过聚合酶链反应（PCR）和酶联免疫吸附测定（ELISA）分析了慢性粒细胞白血病（CML）和淋巴瘤患者的端粒酶活性。这种方法将端粒酶介导的延伸产物的高度特异性扩增与高灵敏度光度ELISA中的非放射性检测相结合。PCR产物也通过Southern印迹法进行分析。端粒酶特异性PCR产物通过电泳分离，转移到尼龙膜上，随后检测生物素化的扩增产物。在17例（34%）CML患者中检测到高活性水平。另一方面，在淋巴瘤患者中未观察到活性。在表现出高水平端粒酶活性的CML患者中，观察到较短端粒条带增加。与低酶活性相反，通过Southern印迹法在一些淋巴瘤患者中也发现了端粒重复序列的证据。这可能表明淋巴瘤细胞可能利用不同途径来维持其端粒长度。
