Telomere dynamics in cells with introduced telomerase: a rapid assay for telomerase activity on telomeres.
Most immortal cell lines derived from human cancers or transformed in vitro maintain telomeres by endogenous expression of telomerase. In the present work, immortal cells that already express endogenous telomerase activity were induced to overexpress an exogenous telomerase (hTERT) and were analyzed for changes in telomere length. Introduction of hTERT into telomerase-positive immortal cell lines results in elevated telomerase activity as measured by the TRAP assay, leading to elongated telomeres in the cell lines tested. We explore possibilities for regulatory differences among the cell lines, including the level of overexpression of the catalytic subunit hTERT and the endogenous levels of telomere binding proteins. Reducing levels of hTERT expression with a construct containing an inefficient translation initiation sequence provided sufficient telomerase expression for maximal rates of telomere elongation. Overexpression of the hTERT alters the telomere length normally maintained in these cells and provides a very useful assay for the rapid analysis of telomerase activity on its native substrate, telomeres.
McChesney
P A
PA
Department of Cell Biology, University of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Boulevard, Dallas, Texas 75390-9039, USA.
Aisner
D L
DL
Frank
B C
BC
Wright
W E
WE
Shay
J W
JW
D015153:Blotting, Western; D002292:Carcinoma, Renal Cell; D020134:Catalytic Domain; D002460:Cell Line; D002461:Cell Line, Transformed; D004268:DNA-Binding Proteins / Q000378:metabolism; D005347:Fibroblasts; D005354:Fibrosarcoma; D006801:Humans; D012313:RNA; D019098:Telomerase / Q000235:genetics* / Q000378:metabolism; D016615:Telomere / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D004268:DNA-Binding Proteins; C422952:telomerase RNA; D012313:RNA; D019098:Telomerase