O-6-benzylguanine potentiates BCNU but not busulfan toxicity in hematopoietic stem cells

Objective. Busulfan (BU) is often used in conditioning regimens prior to bo ne marrow transplantation, but its mechanism of action remains to be resolv ed. We have examined the possibility that BU may exert part of its toxic ef fects via DNA alkylation at the Oh position of guanine as this might provid e an approach to improving the conditioning regimen. Methods. Survival of LAMA-84 and RJKO cells was assessed by colony-forming assay and cell counting, respectively. O-6-alkylguanine-DNA alkyltransferas e (ATase) activity was assayed by transfer of radioactivity from [H-3]-meth ylated DNA, Colony-forming potential of normal human bone marrow cells (BMC ) was measured in the presence of appropriate growth factors as the formati on of both granulocyte-macrophage colony-forming units (CFU-GM) or burst-fo rming unit erythroids (BFU-E) within the same assay. Murine hematopoietic p recursors were grown under a bone marrow stromal cell line to allow measure ment of the frequency of cobblestone area-forming cells (CAFC) that corresp ond to CFU-GM, spleen colony-forming units (CFU-S), and the primitive stem cells with long-term repopulating ability. Results. Inactivation of ATase by O-6-benzylguanine (O-6-BeG) sensitized a human erythromegakaryocytic cell line (LAMA-84) and normal human bone marro w progenitors to 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) but not to BU toxicity, BCNU, but not BU, inactivated ATase in LAMA-84 cells. Overexpress ion of human ATase in cDNA transfected Chinese hamster cells attenuated the toxicity of BCNU but not BU, Finally, the in vivo treatment of mire showed that the depletion of primitive stem cells by BU as measured in the CAFC a ssay was not affected by addition of O-6-BeG. O-6-BeG did, however, dramati cally potentiate BCNU toxicity in all CAFC subsets, leading to depletion of more than 99% stem cells, Conclusion. These data suggest that BU does not elicit toxicity via alkylat ion at the O-6 position of guanine in DNA in a way that can be influenced b y ATase modulation, (C) 2001 International Society for Experimental Hematol ogy. Published by Elsevier Science Inc.