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Interaction of Several Nucleoside Triphosphate Analogues and 10-Hydroxycamptothecin with Human DNA Topoisomerases¹

Department of Pharmacology, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7365

DNA topoisomerase I (Topo I) can exist in several different molecular weight forms in human leukemic cells. The M_r 98,000 form of Topo I was inhibited by several nucleoside triphosphates and their analogues at a 500 µM concentration in the order: dideoxy-GTP > 2-bromo-dATP > dideoxy-ATP > dideoxy-CTP > 2-fluoro-dATP > 2-chloro-dATP. The same concentration of these nucleoside triphosphates also inhibited the M_r 32,000 and the M_r 35,000 Topo I forms in the order: 2-bromo-dATP > dideoxy-GTP > 2-fluoro-dATP > dideoxy-ATP; however, dideoxy-CTP and 2-chloro-dATP did not inhibit these forms. ATP inhibited both the large and the small molecular weight forms of Topo I at a concentration of 8 mM. DNA topoisomerase II (Topo II) isolated from human leukemic cells requires ATP for its activity. Of the nucleoside triphosphates examined, only dATP could substitute for ATP. In the presence of 500 µM ATP, equimolar concentrations of 2-bromo-dATP, dideoxy-ATP, 2-chloro-dATP, 2-fluoro-dATP, and dideoxy-GTP nucleotide analogues inhibited the unknotting activity of the Topo II enzyme. When the nucleotide analogue concentration was decreased to 250 µM, only 2-bromo-dATP still had a significant inhibitory effect on Topo II. With the exception of 2-bromo-dATP, the analogues studied appeared to inhibit the nicking step of both the Topo I and Topo II enzyme activity. These results differ from previously described mechanisms of inhibition by camptothecin of Topo I and etoposide of Topo II. These enzymatic studies suggest the inhibition of Topo I and Topo II activities could contribute to the cytotoxicity of the respective nucleoside analogues in cell culture, particularly when high concentrations of these nucleoside analogues accumulate as triphosphates inside the cells.

¹ This work was supported by Grant CA44358 from the National Cancer Institute.

² To whom requests for reprints should be addressed, at Department of Pharmacology and Medicine, CB 7365, 917 FLOB, University of North Carolina, Chapel Hill, NC 27599-7365.

Received 7/25/88. Revised 12/ 5/88. Accepted 12/14/88.

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