Inhibition of nucleotide excision repair by fludarabine in normal lymphocytes in vitro, measured by the alkaline single cell gel electrophoresis (Comet) assay.
Alkylating agents or platinum analogues initiate several excision repair mechanisms, which involve incision of the DNA strand, excision of the damaged nucleotide, gap filling by DNA resynthesis, and rejoining by ligation. The previous study described that nucleotide excision repair permitted incorporation of fludarabine nucleoside (F-ara-A) into the repair patch, thereby inhibiting the DNA resynthesis. In the present study, to clarify the repair kinetics in view of the inhibition by F-ara-A, normal lymphocytes were stimulated to undergo nucleotide excision repair by ultraviolet C (UV) irradiation in the presence or absence of F-ara-A. The repair kinetics were determined as DNA single strand breaks resulting from the incision and the rejoining using the alkaline single cell gel electrophoresis (comet) assay. DNA resynthesis was evaluated in terms of the uptake of tritiated thymidine into DNA. The lymphocytes initiated the incision step maximally at 1 h, and completed the rejoining process within 4 h after UV exposure. UV also initiated thymidine uptake, which increased time-dependently and reached a plateau at 4 h. A 2-h pre-incubation with F-ara-A inhibited the repair in a concentration-dependent manner, with the maximal inhibition by 5 mM. This inhibitory effect was demonstrated by the reduction of the thymidine uptake and by the inhibition of the rejoining. A DNA polymerase inhibitor, aphidicolin, and a ribonucleotide reductase inhibitor, hydroxyurea, were not so inhibitory to the repair process as F-ara-A at equimolar concentrations. The present findings suggest that inhibition of nucleotide excision repair may represent a novel therapeutic strategy against cancer, especially in the context of resistant cells with an increased repair capacity.
Yamauchi
Takahiro
T
First Department of Internal Medicine, Fukui Medical University, Matsuoka, Fukui 910-1193. tyamauch@fmsrsa.fukui-med.ac.jp
Kawai
Yasukazu
Y
Ueda
Takanori
T
D000970:Antineoplastic Agents / Q000494:pharmacology*; D016590:Aphidicolin / Q000494:pharmacology; D017209:Apoptosis; D020552:Comet Assay / Q000379:methods*; D004260:DNA Repair / Q000187:drug effects*; D004305:Dose-Response Relationship, Drug; D019008:Drug Resistance, Neoplasm; D006801:Humans; D006918:Hydroxyurea / Q000494:pharmacology; D007700:Kinetics; D008214:Lymphocytes / Q000187:drug effects* / Q000378:metabolism*; D013997:Time Factors; D014466:Ultraviolet Rays; D014740:Vidarabine / Q000031:analogs & derivatives* / Q000494:pharmacology*
D000970:Antineoplastic Agents; D016590:Aphidicolin; D014740:Vidarabine; C024352:fludarabine; D006918:Hydroxyurea