Mechanisms of resistance to 1,3-bis(2-chloroethyl)-1-nitrosourea in human medulloblastoma and rhabdomyosarcoma.
Medulloblastoma (D-341 MED) and rhabdomyosarcoma (TE-671) cell lines, which are resistant to either 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) or the combination of BCNU and O6-benzylguanine (O6-BG), were generated by serial escalation of BCNU. The activities of O6-alkylguanine-DNA alkyltransferase (AGT), glutathione-S-transferase (GST), and total glutathione (GSH) of the parental, BCNU-resistant (BR), and BCNU + O6-BG-resistant (OBR) cells were measured. No significant differences in GST activity or total GSH were seen between the parental, BR, and OBR cells of both TE-671 and D-341 MED. The AGT activities of D-341 MED (BR) and TE-671 (BR) were twice those of D-341 MED and TE-671, respectively, confirming the importance of this enzyme for BCNU resistance. The D-341 MED (OBR) cells did not exhibit any AGT activity, suggesting that another mechanism must play a role in the drug resistance. Fewer DNA interstrand cross-links (ICLs) were observed in D-341 MED (OBR) than in D-341 MED after 8 h BCNU (100-400 microM) treatment. However, the amounts of DNA ICLs observed in D-341 MED and D-341 MED (OBR) were stable after 24 h. Microarray analysis showed the increased expressions of several metallothionein genes and down-regulation of several proapoptotic genes. The AGT activity of TE-671 (OBR) was 223 fmol/mg when the cells were grown in 10 microM O6-BG and decreased to about half this value when the O6-BG concentration was increased 60 microM. The AGT cDNA of TE-671 (OBR) cells was cloned and found to contain a G-to-T transversion at codon 156, resulting in conversion of glycine to cysteine (G156C). In vitro mutagenesis has shown that the G156C AGT mutant is resistant to inactivation by O6-BG. Thus, the selection of a mutant AGT with decreased sensitivity to O6-BG is a significant contributing factor to BCNU + O6-BG resistance.
Bacolod
Manny D
MD
Department of Surgery, Duke University Medical Center, Durham, NC 27710, USA.
Johnson
Stewart P
SP
Ali-Osman
Francis
F
Modrich
Paul
P
Bullock
Nancy S
NS
Colvin
O Michael
OM
Bigner
Darell D
DD
Friedman
Henry S
HS
D000595:Amino Acid Sequence; D017209:Apoptosis; D002330:Carmustine / Q000494:pharmacology*; D003001:Cloning, Molecular; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation; D019008:Drug Resistance, Neoplasm; D015870:Gene Expression; D005978:Glutathione / Q000378:metabolism; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D008527:Medulloblastoma / Q000188:drug therapy*; D008969:Molecular Sequence Data; D019853:O(6)-Methylguanine-DNA Methyltransferase / Q000378:metabolism; D017354:Point Mutation; D012208:Rhabdomyosarcoma / Q000188:drug therapy*; D017386:Sequence Homology, Amino Acid; D013997:Time Factors; D015854:Up-Regulation
D019853:O(6)-Methylguanine-DNA Methyltransferase; D005982:Glutathione Transferase; D005978:Glutathione; D002330:Carmustine