Enzymatic activity of endogenous telomerase associated with intact nuclei from human leukemia CEM cells.
Telomerase, a telomere-specific DNA polymerase and novel target for chemotherapeutic intervention, is found in many types of cancers. Telomerase activity is typically assayed using an exogenous primer and cellular extracts as the source of enzyme. Since the nuclear organization might affect telomerase function, we developed a system in which telomerase in intact nuclei catalyzes primer extension. Telomerase activity in isotonically isolated nuclei from human CEM cells shows low processivity (addition of up to four TTAGGG repeats). In contrast, telomerase activity which leaks into a 500 g postnuclear supernatant and the activity in a CHAPS extract are highly processive. The nucleotide inhibitor, 7-deaza-dGTP, seems to be more inhibitory against the nuclei-associated enzyme compared to telomerase from cytoplasmic extracts. However, 7-deaza-dATP and ddGTP are less inhibitory against nuclei-associated telomerase. The results suggest that the association of telomerase with the nuclear chromatin affects telomerase activity. Examination of telomerase activity in a more natural nuclear environment may shed new light on the telomerase function and provide a useful system for the evaluation of new telomerase inhibitors.
Fletcher
T M
TM
Institute for Drug Development, 14960 Omicron Drive, San Antonio, Texas, 78245, USA. Fletchet@dce41.nci.nih.gov
Trevino
A
A
Woynarowski
J M
JM
D001483:Base Sequence; D002458:Cell Fractionation / Q000379:methods; D002467:Cell Nucleus / Q000201:enzymology*; D002843:Chromatin / Q000378:metabolism; D004273:DNA, Neoplasm / Q000096:biosynthesis / Q000737:chemistry; D003854:Deoxyribonucleotides / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D007938:Leukemia; D008969:Molecular Sequence Data; D012091:Repetitive Sequences, Nucleic Acid; D013347:Subcellular Fractions / Q000201:enzymology; D013379:Substrate Specificity; D019098:Telomerase / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D002843:Chromatin; D004273:DNA, Neoplasm; D003854:Deoxyribonucleotides; D004791:Enzyme Inhibitors; D019098:Telomerase