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¹ Division of Hematology/Oncology and Comprehensive Cancer Center, Case Western Reserve University and University Hospitals of Cleveland, Cleveland, Ohio; ² Promega Corporation, Madison, Wisconsin; ³ Regional Blood Center and Faculdade de Medicina de Ribeirão Preto da Universidad de São Paulo, Centro de Terapia Celular, Ribeirão Preto, Brazil; ⁴ The Joseph Gottstein Memorial Cancer Research Laboratory, Department of Pathology, University of Washington School of Medicine, Seattle, Washington; and ⁵ Department of Cellular and Molecular Physiology, Penn State Medical Center, Hershey, Pennsylvania

Requests for reprints: Stanton L. Gerson, Division of Hematology Oncology, BEB-3 Case Western Reserve University, 10900 Euclid Avenue, Cleveland, OH 44106-4955. Phone: 216-368-1177; Fax: 216-368-1166. E-mail: slg5{at}po.cwru.edu

P140K-MGMT and G156A-MGMT genes encode two O⁶-benzylguanine–resistant O⁶-alkylguanine DNA alkyltransferase proteins that confer a high degree of O⁶-benzylguanine and 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) or O⁶-benzylguanine and temozolomide resistance to primary hematopoietic cells. In this study, we directly compared these and three other O⁶-benzylguanine–resistant MGMT genes for their ability to protect the human erythroleukemia cell line, K562, using a direct competitive selection strategy to identify the mutation that conferred the greatest degree of protection from O⁶-benzylguanine and either BCNU or temozolomide. MFG retroviral vector plasmids for each of these mutants [G156A-MGMT (ED₅₀ for O⁶-benzylguanine, 60 µmol/L); and P140K-MGMT, MGMT-2 (S152H, A154G, Y158H, G160S, L162V), MGMT-3 (C150Y, A154G, Y158F, L162P, K165R), and MGMT-5 (N157T, Y158H, A170S; ED₅₀ for benzylguanine, >1,000 µmol/L)] were mixed, and the virus produced from Phoenix cells was transduced into K562 cells. Stringent selection used high doses of O⁶-benzylguanine (800 µmol/L) and temozolomide (1,000 µmol/L) or BCNU (20 µmol/L) administered twice, and following regrowth, surviving clones were isolated, and the MGMT transgene was sequenced. None of the mutants was lost during selection. Using temozolomide, the enrichment factor was greatest for P140K-MGMT (1.7-fold). Using BCNU selection, the greatest enrichment was observed with MGMT-2 (1.5-fold). G156A-MGMT, which is the least O⁶-benzylguanine–resistant MGMT gene of the mutants tested, was not lost during selection but was selected against. The optimal mutant MGMT useful as a drug resistance gene may depend on whether a methylating or chloroethylating agent is used for drug selection. [Mol Cancer Ther 2006;5(1):121–8]

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Note: A.M. Fontes is currently at the Regional Blood Center of Ribeirão Preto, Ribeirão Preto, SP, 14051-140 Brazil. B. Davis is currently at Virexis, Gaithersburg, Maryland. L. Encell is currently at Tanox, Inc., Department of Molecular Biology, 10301 Stella Link, Houston, TX 77025.

⁴ http://www.stanford.edu/group/nolan/protocols/pro_helper_dep.html

Received 7/11/05; revised 10/ 7/05; accepted 10/28/05.