Expression and characterization of the catalytic subunit oftelomerase in normal and cataractous canine lens epithelial cells

[摘要] Purpose: To determine whether the catalytic subunit of telomerasereverse transcriptase (TERT) is regionally distributed in canine lensepithelial cells (LEC), compare TERT and the RNA subunit of telomerase(TR) mRNA expression and TERT protein expression in normal andcataractous LEC, and to evaluate whether telomerase activity is presentin the cytoplasm and nucleus from normal LEC. Finally, the expression ofp23 and heat shock protein 90 (hsp90), coactivators of TERT inneoplastic cells, were evaluated in normal and cataractous LEC.Methods: TERT protein was detected by imunohistochemical stainingand western immunoblotting in normal and cataractous LEC. QuantitativeRT-PCR (qRT-PCR) was used to measure TERT and TR expression. Separatedcytoplasmic and nuclear extracts from primary cultures of normal canineLEC were evaluated for TERT protein expression and telomerase activity.Western immunoblotting was performed on normal and cataractous LEC forp23 and hsp90, and coimmunoprecipitation was used to determine whetherp23 and hsp90 were interacting with TERT in LEC.Results: TERT expression in normal lens capsule whole mounts variedby region in normal LEC. All cataractous LEC demonstrated more intenseTERT immunostaining in both the nucleus and cytoplasm when compared tonormal LEC. Normal LEC expressed less TERT protein and less TERT and TRmRNA than cataractous LEC. Normal LEC expressed hsp90 while cataractousLEC did not; p23 was not significantly expressed in either normal orcataractous LEC. Neither hsp90 nor p23 interacted with TERT.Conclusions: The localization of TERT in normal LEC correspondedwith the LEC's regional functions. There was more cytoplasmic TERT inthe central region that corresponds with the need for inhibitedapoptosis and for proliferative capabilities; there was more nuclearTERT in the germinal and equatorial regions corresponding with the needfor proliferative capabilities. In addition, cataractous LECdemonstrated increased TERT protein and increased TERT and TR mRNAexpression than normal LEC corresponding with their increasedproliferative potential. However, the telomerase coactivators, p23 andhsp90, are not overexpressed and do not associate with TERT incataractous LEC, suggesting that telomerase regulation in cataractousLEC, a somatic cell type, differs from that in neoplastic cells.

[学科分类] 生物化学/生物物理