Copyright 2009 by the Society for Neuro-Oncology
The L84F polymorphic variant of human O6-methylguanine-DNA methyltransferase alters stability in U87MG glioma cells but not temozolomide sensitivity
Neuro-Oncology Program, Department of Neurology, and Henry E. Singleton Brain Cancer Research Program, David Geffen School of Medicine at UCLA, Los Angeles, CA, USA
Address correspondence to Albert Lai, UCLA Neuro-Oncology Program, Department of Neurology, David Geffen School of Medicine at UCLA, 710 Westwood Plaza, Suite 1-230 RNRC, Los Angeles, CA 90095, USA (albertlai{at}mednet.ucla.edu).
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Abstract |
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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 O6-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 O6-benzylguanine (O6-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 O6-BG– stimulated protein turnover caused by the L84F alteration, cells expressing L84F eGFP-MGMT did not exhibit altered sensitivity to the combination of O6-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 O6-BG. These findings may provide a clue to determining the clinical significance of MGMT coding-region polymorphisms.
Keywords: glioma, O6-benzylguanine (O6-BG), O6-methylguanine-DNA methyltransferase (MGMT), polymorphism, temozolomide, U87MG
Received March 14, 2008; Accepted April 29, 2008