Differential competitive resistance to methylating versus chloroethylating agents among five O6-alkylguanine DNA alkyltransferases in human hematopoietic cells

Aparecida Maria Fontes, Brian M. Davis, Lance P. Encell, Karen Lingas, Dimas Tadeu Covas, Marco Antonio Zago, Lawrence A. Loeb, Anthony E. Pegg, Stanton L. Gerson

Research output: Contribution to journalArticlepeer-review

8 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)121-128
Number of pages8
JournalMolecular cancer therapeutics
Volume5
Issue number1
DOIs
StatePublished - Jan 2006

All Science Journal Classification (ASJC) codes

  • Oncology
  • Cancer Research

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