Purpose: Telomerase is a specialized polymerase that catalyzes synthesis of telomeres in most eukaryotes. When introduced into somatic cells, it extends the proliferative lifespan and prevents replicative senescence. Whether it has similar functions in lens epithelial cells, especially in human lens epithelial cells (HLECs) remains to be determined. In this study, the human telomerase reverse transcriptase (hTERT) catalytic subunit was introduced into HLECs. A stable cell line expressing hTERT was established and the functions of hTERT were studied.
Methods: The telomeric repeat amplification protocol (TRAP) assay was used to analyze the telomerase activity. Western blot analysis was used to examine hTERT expression. Southern blot analysis was used to detect telomere length. HLECs isolated from intact lenses were cultured in DMEM and transfected with hTERT cDNA. The expression of the exogenous hTERT was examined with RT-PCR, Western blot analysis, and TRAP assay. The functions of hTERT were examined with various techniques.
Results: Among the human, bovine, and rabbit lenses examined, only the central epithelium from the 6-month rabbit lens displayed telomerase activity. In both transparent and cataractous human lenses, hTERT activity and expression were not detected. However, the template RNA was present in both types of human lenses. The telomeres in transparent lenses were approximately 1 kb longer than those in cataractous lenses. The primary cultures and later passages of HLECs also displayed no detectable telomerase activity. Introduction of hTERTcDNA into HLECs followed by G418 selection yielded a stable line of HLECs expressing hTERT. In this line, hTERT has supported normal growth after 48 population doublings (PDs) to date and also enhanced antiapoptotic activity against oxidative stress.
Conclusions: Telomere lengths may be associated with cataractogenesis. hTERT introduced into HLECs prevents replicative senescence through telomere synthesis. Furthermore, hTERT displays functions beyond telomere synthesis in normal HLECs.