A radioisotope-nondependent high-sensitivity method for measuring the activity of glioblastoma-related O(6)-methylguanine DNA methyltransferase

Aya Hongo; Ran Gu; Miho Suzuki; Naoto Nemoto; Koichi Nishigaki

doi:10.1016/j.ab.2014.08.012

A radioisotope-nondependent high-sensitivity method for measuring the activity of glioblastoma-related O(6)-methylguanine DNA methyltransferase

Anal Biochem. 2015 Jul 1:480:82-4. doi: 10.1016/j.ab.2014.08.012. Epub 2014 Aug 28.

Authors

Aya Hongo¹, Ran Gu¹, Miho Suzuki², Naoto Nemoto², Koichi Nishigaki³

Affiliations

¹ Department of Functional Materials Science, Graduate School of Science and Engineering, Saitama University, Sakura-ku, Saitama 338-8570, Japan.
² Department of Functional Materials Science, Graduate School of Science and Engineering, Saitama University, Sakura-ku, Saitama 338-8570, Japan; Rational Evolutionary Design of Advanced Biomolecules, Saitama (REDS), Saitama Small Enterprise Promotion Corporation, No. 552, Kawaguchi, Saitama 333-0844, Japan.
³ Department of Functional Materials Science, Graduate School of Science and Engineering, Saitama University, Sakura-ku, Saitama 338-8570, Japan; Rational Evolutionary Design of Advanced Biomolecules, Saitama (REDS), Saitama Small Enterprise Promotion Corporation, No. 552, Kawaguchi, Saitama 333-0844, Japan. Electronic address: koichi@fms.saitama-u.ac.jp.

PMID: 25173514
DOI: 10.1016/j.ab.2014.08.012

Abstract

O(6)-Methylguanine DNA methyltransferase (MGMT) cancels the anticancer effect of temozolomide (drug for glioblastoma), which introduces methylation to DNA. Therefore, developing an MGMT inhibitor is a promising strategy for the treatment of this cancer. For this purpose, a sensitive detection method that does not depend on the conventional radioisotope (RI) method was developed. This was realized by a fluorescence-based method that measured the amount of cleavable restriction sites demethylated by the action of MGMT; this method was enhanced by introducing a polymerase chain reaction (PCR) amplification step. As an assay of enzyme activity, 20-fold higher sensitivity (subnanomolar) was attained compared with our and others' fluorescence-based approaches.

Keywords: Glioblastoma; MGMT; RI-nondependent activity assay; Sensitivity enhancement by PCR; TMZ.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Enzyme Activation
Fluorescence*
Glioblastoma / enzymology*
Glioblastoma / metabolism
Humans
O(6)-Methylguanine-DNA Methyltransferase / analysis*
O(6)-Methylguanine-DNA Methyltransferase / genetics
O(6)-Methylguanine-DNA Methyltransferase / metabolism*
Polymerase Chain Reaction*
Radioisotopes

Substances

Radioisotopes
O(6)-Methylguanine-DNA Methyltransferase