Colitz Carmen Maria Helena, Barden Curtis Andrew, Lu Ping, Chandler Heather Lynn
Department of Veterinary Clinical Science, The Ohio State University, Columbus, OH, USA.
Mol Vis. 2006 Sep 13;12:1067-76.
To determine whether the catalytic subunit of telomerase reverse transcriptase (TERT) is regionally distributed in canine lens epithelial cells (LEC), compare TERT and the RNA subunit of telomerase (TR) mRNA expression and TERT protein expression in normal and cataractous LEC, and to evaluate whether telomerase activity is present in the cytoplasm and nucleus from normal LEC. Finally, the expression of p23 and heat shock protein 90 (hsp90), coactivators of TERT in neoplastic cells, were evaluated in normal and cataractous LEC.
TERT protein was detected by imunohistochemical staining and western immunoblotting in normal and cataractous LEC. Quantitative RT-PCR (qRT-PCR) was used to measure TERT and TR expression. Separated cytoplasmic and nuclear extracts from primary cultures of normal canine LEC were evaluated for TERT protein expression and telomerase activity. Western immunoblotting was performed on normal and cataractous LEC for p23 and hsp90, and coimmunoprecipitation was used to determine whether p23 and hsp90 were interacting with TERT in LEC.
TERT expression in normal lens capsule whole mounts varied by region in normal LEC. All cataractous LEC demonstrated more intense TERT immunostaining in both the nucleus and cytoplasm when compared to normal LEC. Normal LEC expressed less TERT protein and less TERT and TR mRNA than cataractous LEC. Normal LEC expressed hsp90 while cataractous LEC did not; p23 was not significantly expressed in either normal or cataractous LEC. Neither hsp90 nor p23 interacted with TERT.
The localization of TERT in normal LEC corresponded with the LEC's regional functions. There was more cytoplasmic TERT in the central region that corresponds with the need for inhibited apoptosis and for proliferative capabilities; there was more nuclear TERT in the germinal and equatorial regions corresponding with the need for proliferative capabilities. In addition, cataractous LEC demonstrated increased TERT protein and increased TERT and TR mRNA expression than normal LEC corresponding with their increased proliferative potential. However, the telomerase coactivators, p23 and hsp90, are not overexpressed and do not associate with TERT in cataractous LEC, suggesting that telomerase regulation in cataractous LEC, a somatic cell type, differs from that in neoplastic cells.
确定端粒酶逆转录酶（TERT）的催化亚基在犬晶状体上皮细胞（LEC）中是否呈区域分布，比较正常和白内障LEC中端粒酶RNA亚基（TR）的mRNA表达、TERT蛋白表达，并评估正常LEC的细胞质和细胞核中是否存在端粒酶活性。最后，评估肿瘤细胞中端粒酶的共激活因子p23和热休克蛋白90（hsp90）在正常和白内障LEC中的表达情况。
采用免疫组织化学染色和蛋白质免疫印迹法检测正常和白内障LEC中的TERT蛋白。运用定量逆转录聚合酶链反应（qRT-PCR）检测TERT和TR的表达。对原代培养的正常犬LEC分离的细胞质和细胞核提取物进行TERT蛋白表达和端粒酶活性评估。对正常和白内障LEC进行p23和hsp90的蛋白质免疫印迹分析，并采用免疫共沉淀法确定p23和hsp90在LEC中是否与TERT相互作用。
正常晶状体囊膜整装片中TERT在正常LEC中的表达因区域而异。与正常LEC相比，所有白内障LEC的细胞核和细胞质中的TERT免疫染色均更强。正常LEC表达的TERT蛋白、TERT和TR mRNA均少于白内障LEC。正常LEC表达hsp90，而白内障LEC不表达；p23在正常或白内障LEC中均未显著表达。hsp90和p23均未与TERT相互作用。
TERT在正常LEC中的定位与其区域功能相对应。中央区域的细胞质TERT较多，这与抑制细胞凋亡和增殖能力的需求相对应；生发区和赤道区的细胞核TERT较多，这与增殖能力的需求相对应。此外，白内障LEC的TERT蛋白增加，TERT和TR mRNA表达也高于正常LEC，这与其增殖潜力增加相对应。然而，端粒酶共激活因子p23和hsp90在白内障LEC中并未过度表达，也不与TERT相关联，这表明白内障LEC（一种体细胞类型）中的端粒酶调节与肿瘤细胞不同。
