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 reduce cytotoxic side effects of chemotherapy. N,N',N"-Triethylenethiophosphoramide (thioTEPA) induces the formation of amino-ethyl adducts of guanine, resulting in imidazole ring opening [formamidopyrimidine (Fapy)] and is associated with significant myelosuppression in dose-intensive therapies. In Escherichia coli, Fapy lesions are repaired by the Fapy-DNA glycosylase (Fpg) protein. We hypothesized that the expression of the Fpg could increase resistance of hematopoietic cells to thioTEPA-induced cytotoxicity. Expression of Fpg in bone marrow (BM) cells via a retrovirus vector was associated with demonstrable 8-oxodeoxyguanosine DNA glycosylase activity. BM cells were infected with a recombinant retrovirus, SF91, containing the Fpg gene and expressing the enhanced green fluorescence protein (EGFP) via an internal ribosomal entry site element. Control mice received BM transduced with the backbone containing IRES-EGFP alone. Fpg-transduced and GFP+ BM hematopoietic cells were resistant in vitro to thioTEPA at multiple concentrations. Mice transplanted with transduced cells were treated with four doses of thioTEPA (10 mg/kg) given over 7 weeks. Despite low transduction efficiency, 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 were significantly higher than those of control group. Selection of Fpg-transduced cells in vivo was demonstrated by an increase in the mean fluorescence intensity of peripheral mononuclear cells of Fpg mice compared with pretreatment value. In addition, a significant increase in the EGFP-bright cells was demonstrated, suggesting preferential survival of high-expressing hematopoietic cells. Similar results were demonstrated in vitro with primary BM expressing the human functional counterpart of Fpg, OGG1. These results show that expression of the Fpg or hOGG1 protein protects hematopoietic cells from thioTEPA-induced DNA damage and suggest that a high level of expression of these repair proteins is required to establish resistance to this drug. Expression of Fpg and/or OGG1 may provide an novel approach to preventing thioTEPA-induced toxicity of primary hematopoietic cells.
Kobune
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Section of Pediatric Hematology/Oncology, Herman B Wells Center for Pediatric Research, Department of Pediatrics, Indiana University School of Medicine, Indianapolis, Indiana 46202, USA.
Xu
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Y
Baum
C
C
Kelley
M R
MR
Williams
D A
DA
D000818:Animals; D018906:Antineoplastic Agents, Alkylating / Q000633:toxicity*; D001854:Bone Marrow Cells / Q000166:cytology / Q000187:drug effects; D004260:DNA Repair; D043382:DNA-Formamidopyrimidine Glycosylase; D029968:Escherichia coli Proteins; D005260:Female; D018014:Gene Transfer Techniques; D005822:Genetic Vectors / Q000235:genetics; D018380:Hematopoietic Stem Cell Transplantation; D006412:Hematopoietic Stem Cells / Q000187:drug effects* / Q000201:enzymology* / Q000502:physiology; D006801:Humans; D016130:Immunophenotyping; D051379:Mice; D008810:Mice, Inbred C57BL; D009699:N-Glycosyl Hydrolases / Q000096:biosynthesis* / Q000097:blood / Q000235:genetics*; D011533:Proviruses / Q000235:genetics; D012190:Retroviridae / Q000235:genetics; D013154:Spleen / Q000166:cytology / Q000187:drug effects; D013852:Thiotepa / Q000633:toxicity*; D013950:Thymus Gland / Q000166:cytology / Q000187:drug effects; D014161:Transduction, Genetic; D016662:Virus Integration
D018906:Antineoplastic Agents, Alkylating; D029968:Escherichia coli Proteins; D013852:Thiotepa; D009699:N-Glycosyl Hydrolases; D043382:DNA-Formamidopyrimidine Glycosylase; C468610:DNA-formamidopyrimidine glycosylase, E coli