Retroviral transduction and expression of the human alkyltransferase cDNA provides nitrosourea resistance to hematopoietic cells.
Myelosuppression is the dose-limiting toxicity for nitrosourea chemotherapy. This toxicity predominantly involves modification of the O6 position of guanine with an alkyl moiety. The enzyme responsible for repair of O6-alkylguanine adducts, O6-alkylguanine-DNA alkyltransferase (alkyltransferase), is expressed at low levels in bone marrow (BM) cells. High alkyltransferase expression prevents the cytotoxicity and carcinogenicity of nitrosoureas in several transgenic and in vitro gene transfer models. We used gene transfer using a novel myeloproliferative sarcoma virus (MPSV) based retrovirus (vM5MGMT) to express the human alkyltransferase cDNA (MGMT) in human and murine hematopoietic cells. Transduced K562 cells had very high levels of alkyltransferase expression and significantly increased resistance to 1,3-bis (2-chloroethyl) nitrosourea (BCNU) as compared with untransduced K562 cells. Primary murine BM progenitors showed a high transduction efficiency with vM5MGMT and have increased BCNU resistance in vitro. After BM transplantation with vM5MGMT-transduced BM cells and BCNU treatment of these mice, BM, spleen and thymus had a 10- to 40-fold increase in alkyltransferase expression that persisted for at least 23 weeks posttransplantation. Progenitor cells procured from mice expressing high levels of alkyltransferase also had increased resistance to BCNU. Thus, an MPSV-based retroviral vector transduces mouse and human hematopoietic cells at high efficiency and results in high levels of gene expression both in vitro and in vivo. Overexpression of the alkyltransferase protein may protect hematopoietic progenitors from nitrosourea-induced myelosuppression.
Allay
J A
JA
Department of Medicine, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106-4937, USA.
Dumenco
L L
LL
Koc
O N
ON
Liu
L
L
Gerson
S L
SL
D000818:Animals; D001483:Base Sequence; D001854:Bone Marrow Cells; D002330:Carmustine / Q000494:pharmacology*; D004351:Drug Resistance / Q000235:genetics; D005822:Genetic Vectors; D018380:Hematopoietic Stem Cell Transplantation; D006412:Hematopoietic Stem Cells / Q000187:drug effects* / Q000201:enzymology; D006801:Humans; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive / Q000473:pathology; D008780:Methyltransferases / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D051379:Mice; D008969:Molecular Sequence Data; D019853:O(6)-Methylguanine-DNA Methyltransferase; D011828:Radiation Chimera; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D012190:Retroviridae / Q000235:genetics*; D013950:Thymus Gland / Q000166:cytology; D014162:Transfection; D014407:Tumor Cells, Cultured
D011993:Recombinant Fusion Proteins; D008780:Methyltransferases; D019853:O(6)-Methylguanine-DNA Methyltransferase; D002330:Carmustine