Retrovirus-mediated expression of the base excision repair proteins, formamidopyrimidine DNA glycosylase or human oxoguanine DNA glycosylase, protects hematopoietic cells from N,N ',N ''-triethylenethiophosphoramide (thioTEPA)-induced toxicity in vitro and in vivo

Modulation of DNA damage repair activity could lead to new approaches to re duce cytotoxic side effects of chemotherapy. N,N ' ,N " -Triethylenethiopho sphoramide (thioTEPA) induces the formation of amino-ethyl adducts of guani ne, resulting in imidazole ring opening [formamidopyrimidine (Fapy)] and is associated with significant myelo-suppression in dose-intensive therapies. In Escherichia coil, Fapy lesions are repaired by the Fapy-DNA glycosylase (Fpg) protein. We hypothesized that the expression of the Fpg could increa se resistance of hematopoietic cells to thioTEPA-induced cytotoxicity, Expr ession of Fpg in bone marrow (BM) cells iia a retrovirus vector was associa ted with demonstrable 8-oxodeoxyguanosine DNA glycosylase activity, BM cell s were infected with a recombinant retrovirus, SF91, containing the Fpg gen e and expressing the enhanced green fluorescence protein (EGFP) via an inte rnal ribosomal entry site element. Control mice received BM transduced with the backbone containing IRES-EGFP alone, Fpg-transduced and GFP+ BM hemato poietic cells were resistant in vitro to thioTEPA at multiple concentration s. Mice transplanted with transduced cells were treated. with four doses of thioTEPA (10 mg/kg) given over 7 weeks. Despite low transduction efficienc y; peripheral blood leukocytes, hemoglobin, and platelet counts of thioTEPA -treated Fpg mice were significantly higher than treated control mice (P < 0.05), In addition, after treatment, the BM, spleen, and thymic cellularity as well as the number of GFP+ progenitor cells in the BM of treated mice w ere significantly higher than those of control group. Selection of Fpg-tran sduced cells in vivo was demonstrated by an increase in the mean fluorescen ce intensity of peripheral mononuclear cells of Fpg mice compared with pret reatment value, In addition, a significant increase in the EGFP-bright cell s was demonstrated, suggesting preferential survival of high-expressing hem atopoietic cells. Similar results were demonstrated in vitro with primary B M expressing the human functional counterpart of Fpg, OGG1, These results s how that expression of the Fpg or hOGG1 protein protects hematopoietic cell s from thioTEPA-induced DNA damage and suggest that a high level of express ion of these repair proteins is required to establish resistance to this dr ug. Expression of Fpg and/or OGG1 may provide an novel approach to preventi ng thioTEPA-induced toxicity of primary hematopoietic cells.