Treatment for
glioblastoma multiforme includes the
alkylating agent temozolomide combined with ionizing radiation. Persistent O6-guanine methylation by
temozolomide in
O6-methylguanine methyl
transferase negative
tumors causes cytotoxic lesions recognized by DNA mismatch repair, triggering apoptosis. Resistance (intrinsic or acquired) presents obstacles to successful
temozolomide treatment, limiting drug efficacy and life expectancy. Two
glioma cell lines, SNB19 and U373, sensitive to
temozolomide (GI(50) values 36 and 68 microM, respectively) were exposed to increasing
temozolomide concentrations (1-100 microM). Variant cell lines (SNB19VR, U373VR) were generated that displayed acquired
temozolomide resistance (GI(50) values 280 and 289 microM, respectively). Cross-resistance to
mitozolomide was observed in U373VR cells only. In clonogenic and MTT assays, methylguanine
methyltransferase (MGMT) depletion using
O6-benzylguanine sensitized U373VR cells to
temozolomide, indicating the resistance mechanism involves MGMT re-expression. Indeed, Western blot analyses revealed MGMT
protein in cell lysates. In SNB19VR cells, down-regulation of MSH6 message and
protein expression may confer
temozolomide tolerance. Inhibition of
poly(ADP-ribose) polymerase-1 (a key base excision repair (BER)
enzyme) partially restored sensitivity, and DNA repair gene arrays demonstrated up-regulation (>5-fold) of BER gene NTL1 in SNB19VR cells. In conclusion, we have developed two
glioma cell lines whose distinct mechanisms of acquired resistance to
temozolomide, involving expression of MGMT, or inactivation of DNA mismatch repair and recruitment of BER
enzymes, are consistent with clinical observations. These cell lines provide valuable models for the development of strategies to combat
temozolomide resistance.