Development of an O(6)-alkylguanine-DNA alkyltransferase assay based on covalent transfer of the benzyl moiety from [benzene-3H]O(6)-benzylguanine to the protein

Anal Biochem. 2008 Dec 1;383(1):44-51. doi: 10.1016/j.ab.2008.08.009. Epub 2008 Aug 20.

Abstract

Although it is known that (i) O(6)-alkylguanine-DNA alkyltransferase (AGT) confers tumor cell resistance to guanine O(6)-targeting drugs such as cloretazine, carmustine, and temozolomide and that (ii) AGT levels in tumors are highly variable, measurement of AGT activity in tumors before treatment is not a routine clinical practice. This derives in part from the lack of a reliable clinical AGT assay; therefore, a simple AGT assay was devised based on transfer of radioactive benzyl residues from [benzene-3H]O(6)-benzylguanine ([3H]BG) to AGT. The assay involves incubation of intact cells or cell homogenates with [3H]BG and measurement of radioactivity in a 70% methanol precipitable fraction. Approximately 85% of AGT in intact cells was recovered in cell homogenates. Accuracy of the AGT assay was confirmed by examination of AGT levels by Western blot analysis with the exception of false-positive results in melanin-containing cells due to [3H]BG binding to melanin. Second-order kinetic constants for human and murine AGT were 1100 and 380 M(-1)s(-1), respectively. AGT levels in various human cell lines ranged from less than 500 molecules/cell (detection limit) to 45,000 molecules/cell. Rodent cell lines frequently lacked AGT expression, and AGT levels in rodent cells were much lower than in human cells.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Blotting, Western
  • Cell Line
  • Enzyme Assays / methods*
  • Guanine / analogs & derivatives*
  • Guanine / metabolism
  • Humans
  • O(6)-Methylguanine-DNA Methyltransferase / metabolism*
  • Proteins / metabolism*
  • Reproducibility of Results

Substances

  • Proteins
  • O(6)-benzylguanine
  • Guanine
  • O(6)-Methylguanine-DNA Methyltransferase