Izgi Ahu, Gunal Armagan, Yalcin Serap, Gunduz Ufuk
Middle East Technical University, Department of Biological Sciences, 06531 Ankara, Turkey.
Gülhane Millitary Medical Academy, Department of Pathology, 06010 Ankara, Turkey.
Biomed Pharmacother. 2014 Sep;68(7):841-6. doi: 10.1016/j.biopha.2014.08.014. Epub 2014 Aug 20.
The ends of chromosoms, telomeres are bound with a number of proteins which protect and stabilize telomeres against degredation, end to end fusion and aberrant recombinations. Telomeric DNA is bound of two groups of proteins, which are double-stranded telomeric DNA bindings proteins, and single stranded telomeric binding proteins. Among telomere binding proteins, protections of telomere 1 protein is a single stranded telomere binding proteins and suggested to be a significant player for telomere elongation and has an association with an enzyme called as telomerase which is an intrinsic reverse transcriptase. Telomerase synthesizes hexameric telomeric repeats onto the chromosomes thereby compansating telomere loss in immortal cells, such as tumor cells, whereas telomeres are shorthened with each division in normal cells. PCR-based TRAP (telomeric repeat amplification protocol) assay is a very sensitive assay for the detection of enzymatic activity of telomerase even if a few numbers of cancerous cells are available. The association between telomerase activity and hPOT1 expression in colorectal cancer is still unclear. Protein extraction was performed from specimens of matched normal and colorectal cancer specimens. Protein concentrations were determined by Bradford assay. Optimized protein concentrations were used for TRAP Assay. TRAP products were seperated by vertical gel electrophoresis on 12.5% polyacrylamide gels and visualized by silver staining. Gene expression of hPOT1 was determined by qPCR analysis. The results demonstrated that all tumor tissues were telomerase positive whereas all corresponding normal tissue was telomerase negative. Among clinicopathological findings, telomerase activity was found to be associated with stage, histology, localization, distant metastasis and lymph node metastasis of tumor in the current study. Although all of the clinicopathological findings differed in the expression of hPOT1 compared to normal tissues, they did not differ from each other significantly, except side of tumor and lymph node metastasis. Telomerase activity and hPOT1 gene expression may serve as a promising tumor marker for colorectal cancer and there is a close association between the enzymatic activty of telomerase and the expression of human protection of telomere 1 gene.
染色体的末端，即端粒，与多种蛋白质结合，这些蛋白质可保护和稳定端粒，防止其降解、端对端融合及异常重组。端粒DNA与两组蛋白质结合，即双链端粒DNA结合蛋白和单链端粒结合蛋白。在端粒结合蛋白中，端粒保护蛋白1是一种单链端粒结合蛋白，被认为是端粒延长的重要参与者，且与一种名为端粒酶的酶相关联，端粒酶是一种内在的逆转录酶。端粒酶在染色体上合成六聚体端粒重复序列，从而补偿永生细胞（如肿瘤细胞）中端粒的丢失，而在正常细胞中，端粒会随着每次分裂而缩短。基于聚合酶链反应的端粒重复序列扩增法（TRAP）检测是一种非常灵敏的检测端粒酶酶活性的方法，即使只有少量癌细胞也适用。结直肠癌中端粒酶活性与hPOT1表达之间的关联仍不清楚。从配对的正常和结直肠癌标本中进行蛋白质提取。通过Bradford法测定蛋白质浓度。将优化后的蛋白质浓度用于TRAP检测。TRAP产物通过在12.5%聚丙烯酰胺凝胶上进行垂直凝胶电泳分离，并用银染法进行可视化。通过定量聚合酶链反应分析测定hPOT1的基因表达。结果表明，所有肿瘤组织端粒酶均呈阳性，而所有相应的正常组织端粒酶均呈阴性。在本研究的临床病理结果中，发现端粒酶活性与肿瘤的分期、组织学、定位、远处转移和淋巴结转移相关。尽管所有临床病理结果与正常组织相比hPOT1表达存在差异，但除肿瘤部位和淋巴结转移外，它们彼此之间差异不显著。端粒酶活性和hPOT1基因表达可能是结直肠癌有前景的肿瘤标志物，端粒酶的酶活性与人类端粒保护蛋白1基因的表达之间存在密切关联。
