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

Liu X, Takahashi H, Harada Y, Ogawara T, Ogimura Y, Mizushina Y, Saneyoshi M, Yamaguchi T - Nucleic Acids Res. (2007)

Bottom Line: 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).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.

View Article: PubMed Central - PubMed

Affiliation: Biotechnology Research Center, Teikyo University of Science and Technology, Uenohara, Yamanashi, Japan.

ABSTRACT
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.

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Lineweaver–Burk plot analyses of the inhibitory effects of AZTTP, AZddGTP, AZddSGTP, AZddATP and AZddAATP. Relative reaction velocity (relative V) was calculated relative to the highest activity without inhibition, which was taken as 1.
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Figure 3: Lineweaver–Burk plot analyses of the inhibitory effects of AZTTP, AZddGTP, AZddSGTP, AZddATP and AZddAATP. Relative reaction velocity (relative V) was calculated relative to the highest activity without inhibition, which was taken as 1.

Mentions: Next, the inhibitory effects of nucleotide analogs on telomerase were analyzed using Lineweaver–Burk plots (Figure 3). The inhibition modes of AZTTP and AZddGTP were shown to be nearly competitive with dTTP or dGTP, respectively. However, the inhibition mode of AZddGTP, shown in Figure 3, was not competitive in a simple manner with respect to dGTP, but rather in a complex manner, as indicated reproducibly by the concave curves obtained at lower concentrations of dGTP. The inhibition mode of AZddAATP was also not simple. On the other hand, the inhibition mode of AZddSGTP was shown to be due to a mixed-type mechanism. The Ki values of AZTTP, AZddGTP, AZddSGTP, AZddATP and AZddAATP were estimated to be 15, 1.5, 9.0, 4.8 and 0.26 µM, respectively, and the Km values of dTTP, dGTP and dATP were estimated to be 7.9 ± 1.8, 8.2 ± 2.1 and 0.97 ± 0.31 µM, respectively.Figure 3.


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

Liu X, Takahashi H, Harada Y, Ogawara T, Ogimura Y, Mizushina Y, Saneyoshi M, Yamaguchi T - Nucleic Acids Res. (2007)

Lineweaver–Burk plot analyses of the inhibitory effects of AZTTP, AZddGTP, AZddSGTP, AZddATP and AZddAATP. Relative reaction velocity (relative V) was calculated relative to the highest activity without inhibition, which was taken as 1.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2175342&req=5

Figure 3: Lineweaver–Burk plot analyses of the inhibitory effects of AZTTP, AZddGTP, AZddSGTP, AZddATP and AZddAATP. Relative reaction velocity (relative V) was calculated relative to the highest activity without inhibition, which was taken as 1.
Mentions: Next, the inhibitory effects of nucleotide analogs on telomerase were analyzed using Lineweaver–Burk plots (Figure 3). The inhibition modes of AZTTP and AZddGTP were shown to be nearly competitive with dTTP or dGTP, respectively. However, the inhibition mode of AZddGTP, shown in Figure 3, was not competitive in a simple manner with respect to dGTP, but rather in a complex manner, as indicated reproducibly by the concave curves obtained at lower concentrations of dGTP. The inhibition mode of AZddAATP was also not simple. On the other hand, the inhibition mode of AZddSGTP was shown to be due to a mixed-type mechanism. The Ki values of AZTTP, AZddGTP, AZddSGTP, AZddATP and AZddAATP were estimated to be 15, 1.5, 9.0, 4.8 and 0.26 µM, respectively, and the Km values of dTTP, dGTP and dATP were estimated to be 7.9 ± 1.8, 8.2 ± 2.1 and 0.97 ± 0.31 µM, respectively.Figure 3.

Bottom Line: 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).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.

View Article: PubMed Central - PubMed

Affiliation: Biotechnology Research Center, Teikyo University of Science and Technology, Uenohara, Yamanashi, Japan.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus