The L84F polymorphic variant of human O6-methylguanine-DNA methyltransferase alters stability in U87MG glioma cells but not temozolomide sensitivity

¹ 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
² Neuro-oncology Program, Department of Neurology and The Henry E. Singleton Brain Cancer Research Program, David Geffen School of Medicine at UCLA, Los Angeles, CA, USA

^* To whom correspondence should be addressed. E-mail: albertlai{at}mednet.ucla.edu.

	Abstract

First-line therapy for patients with glioblastoma multiforme (GBM) 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 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 proliferation and clonogenic survival assays, we confirmed that WT eGFP-MGMT expressing cells are resistant to TMZ treatment compared to control U87MG cells, and found that each of the polymorphic eGFP-MGMT variants confer similar resistance to TMZ. However, upon exposure to 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 to WT expressing cells. In conclusion, we demonstrate that the L84F polymorphic variant has altered protein turnover without modifying sensitivity of U87 cells to TMZ or combined TMZ and O6-BG. These findings may provide a clue to determining the clinical significance of coding region polymorphisms.

Key Words: O6-methylguanine-DNA methyltransferase (MGMT), O6-benzylguanine (O6-BG), temozolomide, glioma, U87MG, polymorphism

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