The in Vitro and in Vivo Effects of Telomerase Substrate 6-Thio-2 -Deoxyguanosine

Abstract

Telomerase mediated telomere targeted therapy represents a new approach in cancer therapy. In this study, I report that the nucleoside analogue 6-thio-2’-deoxyguanosine (6-thio-dG) is recognized by the telomerase holoenzyme and incorporated into de novo synthesized telomeres to alter the structure and function of telomeres. This results in structurally and functionally modified telomeres, loss of telomeric end protective complexes, leading to telomere dysfunction. Additionally, 6-thio-dG causes progressive telomere shortening, which is independent from inhibition of telomerase activity in vitro. 6-thio-dG induced telomere dysfunction is observed in hTERT expressing normal human BJ fibroblast cells and cancer cells, but not in telomerase-negative BJ cells. Moreover, one week treatment with 6-thio-dG results in 80-90% cell death for the majority of the cancer cells (H2882, HCC2429, HCC827, HCC15, H2087, HCC4017, HCC515, H2009, H2073), whereas normal BJ fibroblast and human colonic epithelial (HCEC1) cells were largely unaffected. In A549 lung cancer cell based xenograft model studies, intraperitoneally 6-thio-dG treatment (2 mg/kg) caused dramatic tumor reduction as well as telomere dysfunction, superior to that observed for 6-thioguanine (2 mg/kg) treatment. These results indicate that 6-thio-dG may provide a new telomere-addressed telomerase-dependent anti-cancer approach, targeting both genomic and telomeric DNA. It was observed that some of the cancer cell lines (H1819, H1993, H1693) were resistant to 6-thio-dG compared with the sensitive cell lines. The methylation analysis showed that several genes were highly hypermethylated in resistant cell lines. In addition, in gene expression data, there were 3 different genes (FSCN1, TLE2, ALDH1A2) that were differentially expressed between resistant and sensitive cell lines. The results of this study will help in the future directions focusing on 6-thio-dG resistance.