Influence of 3'-azido-2',3'-dideoxyguanosine treatment on telomere length in human telomerase-immortalized human fibroblast cells.

In order to study telomerase activation in normal cells, a telomerase-immortalized fibroblast cell line, hTERT-BJ1, treated with a telomerase inhibitor, 3'-azido-2',3'-dideoxyguanosine (AZddG), is considered to be a good model. Long-term treatment with AZddG resulted in telomere shortening from 10-20 kbp to 5-6 kbp in cultured hTERT BJ1 cells. However, the telomere length then stabilized. As expected, removal of AZddG from the culture medium induced telomere lengthening in the cells, suggesting that telomerase activity was recovered upon AZddG removal. The effect of recovery of telomerase activity in hTERT-BJ1 cells will be investigated in future studies.

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.

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.