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Fludarabine Triphosphate Inhibits Nucleotide Excision Repair of Cisplatin-induced DNA Adducts in Vitro¹

Division of Laboratory Medicine [L. L., X-M. L., A. B. G., L-Y. Y.] and Departments of Hematology [M. J. K.] and Clinical Investigation [W. P.], The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030, and Institute of Biophysics, Academy of Sciences of Czech Republic, Brno, Czech Republic [M. S.]

Fludarabine (9-ß-arabinofuranosyl-2-fluoroadenine-5'-monophosphate) is clinically active against chronic lymphocytic leukemia and low-grade lymphomas. We reported previously that fludarabine nucleoside synergistically enhanced cisplatin (CDDP)-induced cytotoxicity in vitro, and that the synergism was concomitant with inhibition of removal of cellular CDDP-induced DNA interstrand cross-links, which are presumably repaired by homologous recombinational repair. To extend our work, we investigated whether fludarabine inhibits nucleotide excision repair (NER) of CDDP-induced DNA intrastrand adducts. The effect of fludarabine on NER was determined using a cell-free system in which a plasmid containing the DNA adducts served as the substrate for repair enzymes in whole-cell extracts from repair-competent cells. To prevent the cell-bound high mobility group box-containing proteins from interfering with repair, cell extracts were depleted with high mobility group box proteins by immunoprecipitation prior to the assay. Repair synthesis, measured by the incorporation of [³²P]dATP or [³²P]dCTP, was inhibited by 50% at 26 or 43 µM fludarabine triphosphate, respectively; the effect was dose dependent and may have resulted from the termination of repair-patch elongation. These results were consistent with those from pulse-chase experiments demonstrating the conversion of nicked circular plasmid to the closed circular form by cell extracts filling the repair gaps. When proliferating cell nuclear antigen-depleted cell extracts were used and aphidicolin was added in the repair assay to arrest NER at the incision/excision stage, 100 µM fludarabine triphosphate inhibited about 55% of the conversion of nicked plasmids from the closed circular damaged plasmid substrate; the inhibition was dose dependent. We conclude that fludarabine triphosphate inhibited NER at the steps of incision and repair synthesis. These results suggest that fludarabine may serve as a potential repair modulator to improve the antitumor efficacies of combination regimens containing agents that induce NER.

¹ This work was supported by National Cancer Institute Grant CA-68137, a Leukemia Society of America Translational research grant, and a Physician Referral Service research grant from The University of Texas M. D. Anderson Cancer Center (all to L-Y. Y.), and by National Cancer Institute Grant CA-28596 (to W. P.).

² To whom requests for reprints should be addressed, at Section of Experimental Laboratory Medicine, Box 54, Division of Laboratory Medicine, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030-4095. Phone: (713) 792-3650; Fax: (713) 745-1627.

Received 8/ 6/96. Accepted 2/17/97.

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