title: Acquired resistance to temozolomide in glioma cell lines: molecular mechanisms and potential translational applications.
abstract: 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.
authors.affiliation: Centre for Biomolecular Science, School of Pharmacy, University of Nottingham, Nottingham, UK.
authors.forename: Jihong
authors.initials: J
authors.lastname: Zhang
authors.forename: Malcolm F G
authors.initials: MF
authors.lastname: Stevens
authors.forename: Charles A
authors.initials: CA
authors.lastname: Laughton
authors.forename: Srinivasan
authors.initials: S
authors.lastname: Madhusudan
authors.forename: Tracey D
authors.initials: TD
authors.lastname: Bradshaw
mesh_terms: D018906:Antineoplastic Agents, Alkylating / Q000627:therapeutic use*; D002453:Cell Cycle / Q000187:drug effects; D045744:Cell Line, Tumor; D004260:DNA Repair / Q000187:drug effects; D004273:DNA, Neoplasm / Q000235:genetics; D003606:Dacarbazine / Q000031:analogs & derivatives* / Q000627:therapeutic use; D004305:Dose-Response Relationship, Drug; D019008:Drug Resistance, Neoplasm / Q000502:physiology*; D005434:Flow Cytometry; D005909:Glioblastoma / Q000188:drug therapy / Q000201:enzymology / Q000235:genetics; D005910:Glioma / Q000188:drug therapy* / Q000201:enzymology / Q000235:genetics / Q000473:pathology; D006801:Humans; D009588:Nitrogen Mustard Compounds / Q000627:therapeutic use; D019853:O(6)-Methylguanine-DNA Methyltransferase / Q000235:genetics / Q000378:metabolism; D020411:Oligonucleotide Array Sequence Analysis; D011799:Quinazolines / Q000494:pharmacology; D000077204:Temozolomide; D014410:Tumor Stem Cell Assay
chemical_list: D018906:Antineoplastic Agents, Alkylating; D004273:DNA, Neoplasm; C115774:NU 1025; D009588:Nitrogen Mustard Compounds; D011799:Quinazolines; D003606:Dacarbazine; C040369:mitozolomide; D019853:O(6)-Methylguanine-DNA Methyltransferase; D000077204:Temozolomide