Overexpression of glutathione-S-transferase, MGSTII, confers resistance to busulfan and melphalan.
A major obstacle to hematopoietic gene therapy is the lack of appropriate in vivo selection protocols that can raise the presently low numbers of gene-altered stem cells to therapeutically useful levels. Overexpression of glutathione-S-transferases (GST), in combination with busulfan treatment, may provide an exploitable selection mechanism for hematopoietic gene therapy strategies. GST provides a major route of detoxification of a variety of xenobiotics, including alkylating agents used for myeloablative chemotherapy. The only known route of clearance of busulfan is by GST-mediated conjugation. Using a fibroblast cell line as a model, we have tested the effects of overexpression of three human GST (GSTA1, GSTP1, and MGSTII) on cell survival under a busulfan or melphalan challenge. In two separate assay formats using chronic exposure to busulfan, MGSTII conferred a reproducible twofold selective advantage. GSTA1 and GSTP1 had no effect on busulfan resistance, and melphalan resistance was not affected by expression of any of the GSTs in these assays. In an acute (24-hour) melphalan exposure assay, MGSTII conferred about a twofold selective advantage. Busulfan was not toxic in this assay. RTPCR analysis of human bone marrow CD34+ cells showed that MGSTII is not highly expressed in this stem/early progenitor population. These data indicate that MGSTII may be a useful selective agent in hematopoietic gene therapy.
Harkey
Michael A
MA
Division of Clinical Research, Fred Hutchinson Cancer Research Center, Seattle, Washington 98109, USA.
Czerwinski
Maciej
M
Slattery
John
J
Kiem
Hans-Peter
HP
D018952:Antigens, CD34; D018906:Antineoplastic Agents, Alkylating / Q000494:pharmacology*; D001854:Bone Marrow Cells / Q000187:drug effects; D002066:Busulfan / Q000494:pharmacology*; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D004351:Drug Resistance / Q000235:genetics*; D005347:Fibroblasts / Q000187:drug effects*; D005784:Gene Amplification; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008558:Melphalan / Q000494:pharmacology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection; D019076:Transgenes; D015854:Up-Regulation
D018952:Antigens, CD34; D018906:Antineoplastic Agents, Alkylating; D005982:Glutathione Transferase; D002066:Busulfan; D008558:Melphalan