Retroviral-mediated gene transduction of human alkyltransferase complementary DNA confers nitrosourea resistance to human hematopoietic progenitors.
Myelosuppression is the dose-limiting toxicity for nitrosourea chemotherapy due to low levels of the DNA repair protein O6-alkylguanine-DNA alkyltransferase in myeloid precursors. We have shown that high-efficiency myeloproliferative sarcoma virus (vM5MGMT)-mediated transduction of the human MGMT cDNA into murine bone marrow (BM) cells leads to high MGMT expression and increased progenitor resistance to 1,3-bis-(2-chloroethyl) nitrosourea (BCNU) in vitro immediately after infection and after BM transplantation. These experiments were designed to increase MGMT expression in human hematopoietic progenitors. CD34(+) BM cells were isolated over an immunoaffinity column (CEPRATE, CellPro, Inc.), resulting in a mean 66-fold enrichment in clonogenic progenitors (colony-forming unit granulocyte-macrophage + burst-forming unit erythroid + colony-forming unit granulocyte erythroid macrophage = megakaryocyte), with an average progenitor yield of 58 +/- 11.5% and a final population that was 54% CD34(+). Seventy % of progenitors derived from CD34(+) cells were transduced after coculture with AM12-vM5MGMT retroviral producers. vM5MGMT-transduced progenitors were over 2-fold more resistant to concentrations of BCNU between 30 and 50 micrometer than were concurrently LacZ-transduced progenitors (P < 0.003). In vitro selection of transduced, cytokine-stimulated CD34(+) cells with 20 micrometer BCNU resulted in survival of 4.7% of MGMT+ clonogenic progenitors compared to 0.05% of LacZ+ progenitors. These studies indicate that MGMT-transduced human hematopoietic progenitors have increased resistance to nitrosoureas, and in a clinical transplant setting, this strategy may reduce alkylating agent myelosuppression.
Allay
J A
JA
Departments of Medicine and Biology, Molecular Virology Training Program, Case Western Reserve School of Medicine, Cleveland, Ohio 44106, USA.
Koç
O N
ON
Davis
B M
BM
Gerson
S L
SL
D018952:Antigens, CD34 / Q000032:analysis; D002330:Carmustine / Q000009:adverse effects*; D018076:DNA, Complementary / Q000235:genetics*; D019008:Drug Resistance, Neoplasm; D018014:Gene Transfer Techniques; D006412:Hematopoietic Stem Cells / Q000187:drug effects*; D006801:Humans; D019853:O(6)-Methylguanine-DNA Methyltransferase / Q000235:genetics*; D012190:Retroviridae / Q000235:genetics*
D018952:Antigens, CD34; D018076:DNA, Complementary; D019853:O(6)-Methylguanine-DNA Methyltransferase; D002330:Carmustine