We have established a target-based therapeutic strategy to combine methoxyamine (MX), an inhibitor of base excision repair (BER) with fludarabine, an anticancer drug used in clinic to treat patients with malignant blood diseases. This combined treatment has shown that MX enhances fludarabine induced cell killing in vitro and in xenograft models of colon cancer and HL-60. We hypothesize that MX-initiated potentiation is the consequence of two steps of drug-targeting action: (i) randomly incorporated fludarabine is processed by BER as an abnormal base, resulting in the production of AP sites; and (ii) MX binds to the AP sites, leading to interruption of BER repair. In the present study, we used the Comet Assay to measure the extent of DNA damage induced by MX and fludarabine in malignant lymphocytes from patients with acute lymphocytic leukemia, myeloma or Hodgkin's Lymphoma. Comet assay is a sensitive method to detect DNA lesions, especially AP sites, DNA single and double strand breaks in single cells after exposure to DNA damage agents. Fludarabine alone generated a moderate amount of DNA damage, whereas, MX plus fludarabine, increased DNA strand breaks up to 2 to 5 fold assessed by increases in the tail moment and tail length of the comet. We then correlated DNA damage measured by the Comet Assay with the induction of AP sites formed by fludarabine and modulated by MX. Our data show that the levels of AP sites in both nuclear and mitochondrial DNA (detected using ARP reagent) increased proportionally with the concentration of fludarabine and a significant portion of the AP sites were bound by MX. Since MX bound AP sites persist and are resistant to BER, these sites are lethal lesions. To confirm this process, apoptotic death was measured using Annexin V staining and cell growth assays of these unstimulated primary human lymphoid cells. A 2 to 3-fold increase in Annexin V positive cells and a 3-fold inhibition of cell growth were observed in cells treated with fludarabine plus MX compared to fludarabine alone. These studies not only demonstrate that the combination of MX with fludarabine is a novel and promising therapeutic strategy for malignant lymphoid disorders but also show that the Comet Assay may be a sensitive method to monitor and estimate therapeutic efficacy of MX and fludarabine in clinical use.
[Proc Amer Assoc Cancer Res, Volume 47, 2006]