TY - JOUR
T1 - Differential competitive resistance to methylating versus chloroethylating agents among five O6-alkylguanine DNA alkyltransferases in human hematopoietic cells
AU - Fontes, Aparecida Maria
AU - Davis, Brian M.
AU - Encell, Lance P.
AU - Lingas, Karen
AU - Covas, Dimas Tadeu
AU - Zago, Marco Antonio
AU - Loeb, Lawrence A.
AU - Pegg, Anthony E.
AU - Gerson, Stanton L.
PY - 2006/1
Y1 - 2006/1
N2 - 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.
AB - 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.
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U2 - 10.1158/1535-7163.MCT-05-0236
DO - 10.1158/1535-7163.MCT-05-0236
M3 - Article
C2 - 16432170
AN - SCOPUS:33644856269
SN - 1535-7163
VL - 5
SP - 121
EP - 128
JO - Molecular cancer therapeutics
JF - Molecular cancer therapeutics
IS - 1
ER -