Carboplatin has been widely used in patients with solid tumors. However, the clinical experience in the patients with aggressive lymphoma and leukemia have not been satisfactory. Previous in vitro studies including ours have demonstrated that DNA repair induced by DNA damaging agents is an important target for enhancing it′s cytotoxic action of 9-β-D-arabinofuranosyl-2-fluoroadenine (fludarabine) by incorporation into the repair patch. The aim of the present study was to evaluate whether fludarabine inhibited carboplatin-induced DNA repair equally in proliferating cells (CCRF-CEM and K562) and quiescent cells (normal human lymphocytes), and elicited a positive combinational effect in both groups. To address this hypothesis, carboplatin-induced DNA damage and repair was evaluated by the alkaline comet assay. When both human leukemia cells and normal lymphocytes were exposed to carboplatin for 90 min, a rapid increment in the tail moment was observed in a dose-dependent manner, indicating that carboplatin substantially induced DNA damage in both groups. During incubation in fresh media after treatment with carboplatin for 90 min, the tail-moment decreased in proportion to the incubation-period, and recovered to the control level at 4 h in both proliferating and quiescent cells. This steep decrement in the tail-moment indicated that both proliferating and quiescent cells were equally repair-proficient, and have an ability to complete DNA re-joining after removal of damaged DNA. When cells were pulse-treated with fludarabine and carbpolatin for 90 min in combination then transferred to fresh media to allow DNA repair, these repair processes were similarly inhibited by fludarabine in both proliferating and quiescent cells. This inhibitory effect was fludarabine concentration-dependent manner, and was almost complete in the presence of 3 μM fludarabine, indicating that a clinically achievable concentration of fludarabine efficiently inhibited DNA repair in vitro. Under this condition, substantial accumulations of fludarabine triphosphate were observed. The cytotoxic action of these two drugs in combination was evaluated by the trypan blue dye-exclusion method. In lymphocytes, continuous exposure to fludarabine and carboplatin in combination produced an enhancement of cytotoxic effect, in contrast, only additive cytotoxic effect was observed in CCRF-CEM cells. These data suggested that carboplatin provids a new opportunity for incorporation of fludarabine into the repair patch in quiescent cells, leading to an enhancement of cytotoxic action. The mechanistic interaction of F-ara-A with carboplatin may be effective against a dormant population of lymphoma, which may contribute in vivo to drug resistance, as these cells are free from targets of S-phase specific anticancer agents.
[Proc Amer Assoc Cancer Res, Volume 46, 2005]