Influence of 3'-azido-2',3'-dideoxyguanosine treatment on amounts of TERT and POT1 in human HL60 cells.

In human cells, TERT (telomerase reverse transcriptase) is involved in the synthesis of telomere DNA, and POT1 (protection of telomeres 1) is believed to be a regulator of telomere length. We have reported that long-term treatment of human HL60 cells with 50 microM 3'-azido-2',3'-dideoxyguanosine (AZddG) caused telomeres to shorten significantly during early passages (up to 40-50 days), but that telomere length was then stabilized at approximately 2 kbp during later passages. Additionally, cell growth rates showed no obvious change during culture in the presence of 50 microM AZddG. Western blot analysis of these cells showed that the amounts of TERT and POT1 expressed were increased significantly and slightly, respectively. Furthermore, telomeric 3' G-overhangs (G-tails) of AZddG-treated cells were lengthened. These findings suggest that HL60 cells may develop resistance to telomere erosion induced by AZddG.

Telomere shortening in human HL60 cells by treatment with 3'-azido-2',3'-dideoxynucleosides and telomerase inhibition by their 5'-triphosphates.

Telomerase is thought to play an important role in the mechanism of tumor cell immortalization by maintenance of telomere length. To obtain information on the susceptibility of telomerase to nucleoside analogues, the effects of base-modified 3'-azido-2',3'-dideoxynucleoside triphosphates on the enzyme were investigated. It is suggested that the 2-amino group of the nucleotide purine nucleus is important for the inhibitory activity. Telomere shortening caused by long-term treatment with these nucleosides is also described.

Influence of nucleoside analogue treatment on telomere length and TRF2 amount in human HL60 cells.

Long-term treatment with 3'-azido-2',3'-dideoxy-guanosine (AZddG) results in reproducible telomere shortening in cultured human HL60 cells. TRF2 protein has been implicated in the protection of chromosome ends. It binds to double-strand repeats and may have an indirect role in protecting the G-rich overhang by recruiting other telomere-binding proteins to the G-tail or by mediating the formation of the telomeric t-loop. Western blot analysis demonstrated no change or a slight increase, of the TRF2 protein level in HL60 cells with AZddG-induced telomere shortening. The effects of nucleoside analogues on TRF2 suggest that it is not telomere length per se, but rather the presence or absence of a protective telomere state, which determines whether senescence ensues.

3'-Azido-2',3'-dideoxynucleoside 5'-triphosphates inhibit telomerase activity in vitro, and the corresponding nucleosides cause telomere shortening in human HL60 cells.

Telomerase adds telomeric DNA repeats to the ends of linear chromosomal DNA. 3'-Azido-3'-deoxythymidine 5'-triphosphate (AZTTP) is a known telomerase inhibitor. To obtain more selective and potent inhibitors that can be employed as tools for studying telomerase, we investigated the telomerase-inhibitory effects of purine nucleosides bearing a 3'-down azido group: 3'-azido-2',3'-dideoxyguanosine (AZddG) 5'-triphosphate (AZddGTP), 3'-azido-2',3'-dideoxy-6-thioguanosine (AZddSG) 5'-triphosphate (AZddSGTP), 3'-azido-2',3'-dideoxyadenosine (AZddA) 5'-triphosphate (AZddATP) and 3'-azido-2',3'-dideoxy-2-aminoadenosine (AZddAA) 5'-triphosphate (AZddAATP). Of these, AZddGTP showed the most potent inhibitory activity against HeLa cell telomerase. AZddGTP was significantly incorporated into the 3'-terminus of DNA by partially purified telomerase. However, AZddGTP did not exhibit significant inhibitory activity against DNA polymerases alpha and delta, suggesting that AZddGTP is a selective inhibitor of telomerase. We also investigated whether long-term treatment with these nucleosides could alter telomere length and growth rates of human HL60 cells in culture. Southern hybridization analysis of genomic DNA prepared from cells cultured in the presence of AZddG and AZddAA revealed reproducible telomere shortening.

Telomerase inhibition by 3'-azido-2', 3'-dideoxynucleoside 5'-triphosphates and telomere shortening in human cultured cells by the corresponding nucleosides.

Telomerase is believed to be a good target for the development of antitumor agents. In this study, 3'-azido-2',3'-dideoxy-2-aminoadenosine (AZddAA), 3'-azido-2',3'-dideoxyadenosine (AZddA), 9-(3-azido-2,3-dideoxy-beta-D-ribofuranosyl)-2-aminopurine (AZddAP), 3'-azido-2-chloro-2',3'-dideoxyadenosine (AZddClA) and their triphosphate derivatives were synthesized. Telomerase assay studies showed that the 2-amino group plays an important role in the inhibitory activity of these compounds. In addition, AZddAA was found to cause telomere shortening in of HL60 cells in culture.

Influence of long-term treatment with cytosine arabinoside on telomere length in human HL60 cells.

Immortal tumor cells employ a telomere length maintenance mechanism to escape the normal limits on proliferation. We investigated whether treatment with cytosine arabinoside (AraC), whose triphosphate derivative AraCTP might partially inhibit the synthesis of C-rich telomere strands, is effective for inducing telomere shortening in human HL60 cells. Long-term treatment with 0.01 microM AraC was found to cause significant telomere lengthening, but had no marked effects on cell population doubling rates or morphology.