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Measurement of O⁶-Alkylguanine-DNA Alkyltransferase Activity in Human Cells and Tumor Tissues by Restriction Endonuclease Inhibition

Laboratory of Molecular Pharmacology [R. S. W., S. H-C, K. W. K.] and Clinical Investigations Branch, Cancer Therapy Evaluation Program [R. S. W.], Division of Cancer Treatment, National Cancer Institute, NIH, Bethesda, Maryland 20892

A sensitive assay for O⁶-alkylguanine-DNA alkyltransferase activity in cell or tumor extracts has been devised. The theoretical basis of the new assay lies in the observation that certain restriction enzymes will not cleave DNA containing methylated bases. Thus, if a synthetic oligodeoxynucleotide with a restriction sequence containing O⁶-methylguanine were incubated with the restriction enzyme, this synthetic oligodeoxynucleotide should remain intact. However, if the guanine-O⁶ methyl group were first removed by O⁶-alkylguanine-DNA alkyltransferase present in certain cell or tissue extracts the synthetic oligodeoxynucleotide would be cleaved by the restriction enzyme.

The parental oligodeoxynucleotide and its restriction products are separated from each other and analyzed on denaturing polyacrylamide gels. The extent of cleavage by the restriction enzyme is a direct assay of the content of O⁶-alkylguanine-DNA alkyltransferase in the cell/tumor extracts. The assay has been tested against cell culture and xenograft tumor systems and has performed in a predictive manner, correctly predicting five Mer^- and three Mer⁺ phenotypes. Furthermore, the assay is quantitative and the number of molecules of the O⁶-alkylguanine-DNA alkyltransferase per cell estimated using this assay agrees with those that have been published.

¹ To whom requests for reprints should be addressed, at CIB/CTEP/NCI/NIH, Landow Bldg., Room 4C37, Bethesda, MD 20892.

Received 6/25/86. Revised 8/20/87. Accepted 8/24/87.

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