Protection of hematopoietic cells from O(6)-alkylation damage by O(6)-methylguanine DNA methyltransferase gene transfer: studies with different O(6)-alkylating agents and retroviral backbones.
Overexpression of O(6)-methylguanine DNA methyltransferase (MGMT) can protect hematopoietic cells from O(6)-alkylation damage. To identify possible clinical applications of this technology we compared the effect of MGMT gene transfer on the hematotoxicity induced by different O(6)-alkylating agents in clinical use: the chloroethylnitrosoureas ACNU, BCNU, CCNU and the tetrazine derivative temozolomide. In addition, various retroviral vectors expressing the MGMT-cDNA were investigated to identify optimal viral backbones for hematoprotection by MGMT expression. Protection from ACNU, BCNU, CCNU or temozolomide toxicity was evaluated utilizing a Moloney murine leukemia virus-based retroviral vector (N2/Zip-PGK-MGMT) to transduce primary murine bone marrow cells. Increased resistance in murine colony-forming units (CFU) was demonstrated for all four drugs. In comparison to mock-transduced controls, after transduction with N2/Zip-PGK-MGMT the IC50 for CFU increased on average 4.7-fold for ACNU, 2.5-fold for BCNU, 6.3-fold for CCNU and 1.5-fold for temozolomide. To study the effect of the retroviral backbone on hematoprotection various vectors expressing the human MGMT-cDNA from a murine embryonic sarcoma virus LTR (MSCV-MGMT) or a hybrid spleen focus-forming/murine embryonic sarcoma virus LTR (SF1-MGMT) were compared with the N2/Zip-PGK-MGMT vector. While all vectors increased resistance of transduced human CFU to ACNU, the SF1-MGMT construct was most efficient especially at high ACNU concentrations (8-12 microg/ml). Similar results were obtained for protection of murine high-proliferative-potential colony-forming cells. These data may help to optimize treatment design and retroviral constructs in future clinical studies aiming at hematoprotection by MGMT gene transfer.
Jansen
M
M
Department of Internal Medicine (Cancer Research), West German Cancer Center, University of Essen Medical School, Essen, Germany.
Bardenheuer
W
W
Sorg
U R
UR
Seeber
S
S
Flasshove
M
M
Moritz
T
T
D000477:Alkylating Agents / Q000633:toxicity*; D000478:Alkylation; D000818:Animals; D002330:Carmustine / Q000633:toxicity; D002478:Cells, Cultured; D003114:Colony-Forming Units Assay; D004249:DNA Damage; D018076:DNA, Complementary / Q000235:genetics; D003606:Dacarbazine / Q000031:analogs & derivatives / Q000633:toxicity; D004351:Drug Resistance / Q000235:genetics*; D005822:Genetic Vectors / Q000235:genetics*; D006412:Hematopoietic Stem Cells / Q000187:drug effects* / Q000201:enzymology; D006801:Humans; D008130:Lomustine / Q000633:toxicity; D051379:Mice; D008979:Moloney murine leukemia virus / Q000235:genetics; D015376:Nimustine / Q000633:toxicity; D019853:O(6)-Methylguanine-DNA Methyltransferase / Q000235:genetics / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000502:physiology; D012190:Retroviridae / Q000235:genetics*; D009053:Sarcoma Viruses, Murine / Q000235:genetics; D013155:Spleen Focus-Forming Viruses / Q000235:genetics; D000077204:Temozolomide; D020079:Terminal Repeat Sequences; D014162:Transfection
D000477:Alkylating Agents; D018076:DNA, Complementary; D011993:Recombinant Fusion Proteins; D015376:Nimustine; D008130:Lomustine; D003606:Dacarbazine; D019853:O(6)-Methylguanine-DNA Methyltransferase; D002330:Carmustine; D000077204:Temozolomide