DNA repair initiated in chronic. lymphocytic leukemia lymphocytes by 4-hydroperoxycyclophosphamide is inhibited by fludarabine and clofarabine

Purpose: Chronic lymphocytic leukemia (CLL) lymphocytes respond to DNA alky lation by excision repair, with the extent of repair increasing as the cell s acquire resistance to alkylating agents. Because incorporation of nucleot ide analogues into the repair patches elicits death signals in quiescent ce lls, the increased capacity for excision repair in alkylator-resistant cell s could facilitate incorporation of nucleotide analogues. We hypothesized t hat the mechanism-based interaction of nucleoside analogues with alkylating agents could elicit greater than additive killing of CLL cells. Experimental Design: Lymphocytes from 50 patients with CLL that were not re fractory to alkylators were treated in vitro with 4-hydroperoxycyclophospha mide (4-HC) with or without prior incubation with fludarabine nucleoside (F -ara-A) or with clofarabine (Cl-F-ara-A). DNA damage repair kinetics were d etermined by the single-cell gel electrophoresis (comet) assay. Cytotoxicit y was assessed by staining with annexin V. Results: CLL lymphocytes promptly initiated and completed excision repair i n response to 4-HC. A 2-h preincubation. with 10 muM F-ara-A or 10 mum Cl-F -ara-A inhibited the repair initiated by 4-HC, with inhibition peaking at t he intracellular concentrations of 50 muM F-ara-ATP or 5 muM Cl-F-ara-ATP. Combining 4-HC with either F-ara-A or Cl-F-ara-A produced more than additiv e apoptotic cell death than the sum of each alone. The increase in cytotoxi city was proportional to the initial magnitude of the DNA incision and, to the extent of repair inhibition by the nucleoside analogues, suggesting clo se correlation between the repair inhibition and induction of cell death. Conclusions: DNA repair, which is active in CLL lymphocytes, may be a biolo gical target for facilitating the incorporation of nucleoside analogues and increasing their, cytotoxicity. Thus, the increased repair capacity associ ated with, resistant disease may be manipulated to therapeutic advantage.