A radioisotope-nondependent high-sensitivity method for measuring the activity of glioblastoma-related O6-methylguanine DNA methyltransferase
| Autor: | Miho Suzuki, Aya Hongo, Naoto Nemoto, Koichi Nishigaki, Ran Gu |
|---|---|
| Rok vydání: | 2015 |
| Předmět: |
Biophysics
Polymerase Chain Reaction Biochemistry DNA methyltransferase Fluorescence law.invention O(6)-Methylguanine-DNA Methyltransferase chemistry.chemical_compound law medicine Humans neoplasms Molecular Biology Polymerase chain reaction Radioisotopes Temozolomide biology Cancer Cell Biology Methylation medicine.disease Molecular biology Enzyme assay Enzyme Activation Restriction site chemistry biology.protein Glioblastoma DNA medicine.drug |
| Zdroj: | Analytical Biochemistry. 480:82-84 |
| ISSN: | 0003-2697 |
| DOI: | 10.1016/j.ab.2014.08.012 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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