title: The L84F polymorphic variant of human O6-methylguanine-DNA methyltransferase alters stability in U87MG glioma cells but not temozolomide sensitivity.
abstract: First-line therapy for patients with glioblastoma multiforme includes treatment with radiation and temozolomide (TMZ), an oral DNA alkylating chemotherapy. Sensitivity of glioma cells to TMZ is dependent on the level of cellular O(6)-methylguanine-DNA methyltransferase (MGMT) repair activity. Several common coding-region polymorphisms in the MGMT gene (L84F and the linked pair I143V/K178R) modify functional characteristics of MGMT and cancer risk. To determine whether these polymorphic changes influence the ability of MGMT to protect glioma cells from TMZ, we stably overexpressed enhanced green fluorescent protein (eGFP)-tagged MGMT constructs in U87MG glioma cells. We confirmed that the wild-type (WT) eGFP-MGMT protein is properly localized within the nucleus and found that L84F, I143V/K178R, and L84F/I143V/K178R eGFP-MGMT variants exhibited nuclear localization patterns indistinguishable from WT. Using MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide] proliferation and clonogenic survival assays, we confirmed that WT cells expressing eGFP-MGMT are resistant to TMZ treatment compared with control U87MG cells, and that each of the polymorphic eGFP-MGMT variants confers similar resistance to TMZ. However, upon exposure to O(6)-benzylguanine (O(6)-BG), a synthetic MGMT inhibitor, the L84F and L84F/I143V/K178R variants were degraded more rapidly than WT or I143V/K178R in a proteasome-dependent manner. Despite the increased O(6)-BG- stimulated protein turnover caused by the L84F alteration, cells expressing L84F eGFP-MGMT did not exhibit altered sensitivity to the combination of O(6)-BG and TMZ compared with WT cells. In conclusion, we demonstrated that the L84F polymorphic variant has altered protein turnover without modifying sensitivity of U87MG cells to TMZ or combined TMZ and O(6)-BG. These findings may provide a clue to determining the clinical significance of MGMT coding-region polymorphisms.
authors.affiliation: Department of Neurology, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095, USA.
authors.forename: Maya
authors.initials: M
authors.lastname: Remington
authors.forename: Jana
authors.initials: J
authors.lastname: Chtchetinin
authors.forename: Karen
authors.initials: K
authors.lastname: Ancheta
authors.forename: Phioanh Leia
authors.initials: PL
authors.lastname: Nghiemphu
authors.forename: Timothy
authors.initials: T
authors.lastname: Cloughesy
authors.forename: Albert
authors.initials: A
authors.lastname: Lai
mesh_terms: D018906:Antineoplastic Agents, Alkylating / Q000494:pharmacology; D015153:Blotting, Western; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D003513:Cycloheximide / Q000494:pharmacology; D003606:Dacarbazine / Q000031:analogs & derivatives / Q000494:pharmacology; D019008:Drug Resistance, Neoplasm / Q000235:genetics*; D004795:Enzyme Stability; D015972:Gene Expression Regulation, Neoplastic / Q000502:physiology*; D005910:Glioma / Q000188:drug therapy / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006147:Guanine / Q000031:analogs & derivatives / Q000494:pharmacology; D006801:Humans; D019853:O(6)-Methylguanine-DNA Methyltransferase / Q000235:genetics* / Q000378:metabolism; D011110:Polymorphism, Genetic / Q000235:genetics*; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D000077204:Temozolomide; D014407:Tumor Cells, Cultured; D014410:Tumor Stem Cell Assay
chemical_list: D018906:Antineoplastic Agents, Alkylating; D011500:Protein Synthesis Inhibitors; D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D006147:Guanine; D003606:Dacarbazine; D003513:Cycloheximide; C008449:O-(6)-methylguanine; D019853:O(6)-Methylguanine-DNA Methyltransferase; D000077204:Temozolomide