BCNU-sequestration by metallothioneins may contribute to resistance in a medulloblastoma cell line.

PURPOSE: Resistance of neoplastic cells to the alkylating drug BCNU [1,3-bis(2-chloroethyl)-1-nitrosourea] has been correlated with expression of O (6)-methylguanine-DNA methyltransferase, which repairs the O (6)-chloroethylguanine produced by the drug. Other possible mechanisms of resistance include raised levels of glutathione or increased repair of the DNA interstrand cross-links formed by BCNU. Transcriptional profiling revealed the upregulation of several metallothionein (MT) genes in a BCNU-resistant medulloblastoma cell line [D341 MED (OBR)] relative to its parental line. Previous studies have shown that MTs, through their reactive thiol groups can quench nitrogen mustard-derived alkylating drugs. In this report, we evaluate whether MTs can also quench BCNU. METHODS: To demonstrate the binding of BCNU to MT, we used an assay that measured the release of the MT-bound divalent cations (Zn(2+), Cd(2+)) upon their displacement by the drug. We also measured the decomposition rates of BCNU at those reaction conditions. RESULTS: The rate of release of the cations was higher in pH 7.4 than at pH 7.0, which is likely a result of more rapid decomposition of BCNU (thus faster release of MT-binding intermediate) at pH 7.4 than at pH 7.0. CONCLUSION: We demonstrate that resistance to BCNU may be a result of elevated levels of MTs which act by sequestering the drug's decomposition product(s).

Brain tumor cell lines resistant to O6-benzylguanine/1,3-bis(2-chloroethyl)-1-nitrosourea chemotherapy have O6-alkylguanine-DNA alkyltransferase mutations.

The chemotherapeutic activity of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU or carmustine) may be improved by the addition of O6-benzylguanine (O6-BG). The reaction of O6-BG with O6-alkylguanine-DNA alkyltransferase (AGT) prevents the repair of O6-chloroethyl lesions caused by BCNU. In clinics, the combination of O6-BG and BCNU is now being tested for the treatment of brain tumors. However, the effectiveness of this drug regimen may be limited by drug resistance acquired during treatment. To understand the possible mechanisms of resistance of brain tumor cells to the O6-BG/BCNU combination, we generated medulloblastoma cell lines (D283 MED, D341 MED, and Daoy) resistant to the combination of O6-BG and BCNU [O6-BG/BCNU resistant (OBR)]. DNA sequencing showed that all of the parent cell lines express wild-type AGTs, whereas every OBR cell line exhibited mutations that potentially affected the binding of O6-BG to the protein as evidenced previously by in vitro mutagenesis and structural studies of AGT. The D283 MED (OBR), Daoy (OBR), and D341 MED (OBR) cell lines expressed G156C, Y114F, and K165T AGT mutations, respectively. We reported previously that rhabdomyosarcoma TE-671 (OBR) also expresses a G156C mutation. These data suggest that the clonal selection of AGT mutants during treatment with O6-BG plus an alkylator may produce resistance to this intervention in clinical settings.

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.