QUANTITATIVE IMMUNOHISTOCHEMICAL ESTIMATES OF O-6-ALKYLGUANINE-DNA ALKYLTRANSFERASE EXPRESSION IN NORMAL AND MALIGNANT HUMAN COLON

A major mechanism of resistance to nitrosoureas is O-6-alkylguanine-DN A-alkyltransferase. The alkyltransferase biochemical assay measures me an tissue activity but requires availability of fresh tissue and canno t assess tumor heterogeneity, an important component of tumor resistan ce to alkylating agents. We assessed the levels of alkyltransferase in human colon carcinoma and normal colon by biochemical assay, Western blot, conventional immunohistochemistry, and quantitative immunohistoc hemistry (using 5H7 and mT3.1 monoclonal IgGs) to correlate whole tiss ue levels with cell-specific expression. Alkyltransferase activity was 18.0 +/- 4.6 fmol/mu g DNA in normal colon and 15.0 +/- 6.5 fmol/mu g DNA in tumors. By Western blot estimates, alkyltransferase in normal colon was 14.8 +/- 4.2 fmol/mu g DNA and in tumors was 16.2 +/- 7.8 fm ol/mu g DNA. Alkyltransferase estimates by biochemical and Western blo ts were correlated strongly (P < 0.0001). Conventional immunohistochem istry demonstrated that alkyltransferase was predominantly nuclear and in normal colon was concentrated in glandular epithelial mucosal cell s close to the lumen, whereas in tumors, expression was heterogenous b ut localized to malignant epithelial cells. Two parameters of quantita tive immunohistochemistry, integrated gray and mean gray, were correla ted strongly with each other (P < 0.002) and with biochemical and West ern blot estimates (P = 0.004-0.04). Thus, quantitative immunohistoche mical estimates of alkyltransferase in fixed tissues are a reasonable alternative to biochemical analysis and have an added advantage of ide ntifying heterogeneity of alkyltransferase expression in tumors.