Zaidi N H, Liu L, Gerson S L
Division of Hematology, Department of Medicine, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106-4937, USA.
Clin Cancer Res. 1996 Mar;2(3):577-84.
A major mechanism of resistance to nitrosoureas is O6-alkylguanine-DNA-alkyltransferase. The alkyltransferase biochemical assay measures mean tissue activity but requires availability of fresh tissue and cannot assess tumor heterogeneity, an important component of tumor resistance 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 immunohistochemistry (using 5H7 and mT3.1 monoclonal IgGs) to correlate whole tissue levels with cell-specific expression. Alkyltransferase activity was 18.0 +/- 4.6 fmol/microgram DNA in normal colon and 15.0 +/- 6.5 fmol/microgram DNA in tumors. By Western blot estimates, alkyltransferase in normal colon was 14.8 +/- 4.2 fmol/microgram DNA and in tumors was 16.2 +/- 7.8 fmol/microgram DNA. Alkyltransferase estimates by biochemical and Western blots were correlated strongly (P < 0.0001). Conventional immunohistochemistry demonstrated that alkyltransferase was predominantly nuclear and in normal colon was concentrated in glandular epithelial mucosal cells close to the lumen, whereas in tumors, expression was heterogenous but localized to malignant epithelial cells. Two parameters of quantitative immunohistochemistry, integrated gray and mean gray, were correlated strongly with each other (P < 0.002) and with biochemical and Western blot estimates (P = 0.004-0.04). Thus, quantitative immunohistochemical estimates of alkyltransferase in fixed tissues are a reasonable alternative to biochemical analysis and have an added advantage of identifying heterogeneity of alkyltransferase expression in tumors.
对亚硝基脲产生耐药性的一个主要机制是O6-烷基鸟嘌呤-DNA-烷基转移酶。烷基转移酶生化检测可测量平均组织活性，但需要新鲜组织，且无法评估肿瘤异质性，而肿瘤异质性是肿瘤对烷化剂耐药的一个重要组成部分。我们通过生化检测、蛋白质免疫印迹法、传统免疫组织化学和定量免疫组织化学（使用5H7和mT3.1单克隆IgG）评估人结肠癌和正常结肠中烷基转移酶的水平，以将全组织水平与细胞特异性表达相关联。正常结肠中烷基转移酶活性为18.0±4.6 fmol/μg DNA，肿瘤中为15.0±6.5 fmol/μg DNA。通过蛋白质免疫印迹法估计，正常结肠中烷基转移酶为14.8±4.2 fmol/μg DNA，肿瘤中为16.2±7.8 fmol/μg DNA。生化检测和蛋白质免疫印迹法对烷基转移酶的估计结果高度相关（P<0.0001）。传统免疫组织化学显示，烷基转移酶主要位于细胞核，在正常结肠中集中于靠近管腔的腺上皮黏膜细胞，而在肿瘤中，表达是异质性的，但局限于恶性上皮细胞。定量免疫组织化学的两个参数，积分灰度和平均灰度，彼此高度相关（P<0.002），且与生化检测和蛋白质免疫印迹法的估计结果相关（P=0.004-0.04）。因此，固定组织中烷基转移酶的定量免疫组织化学估计是生化分析的合理替代方法，并且具有识别肿瘤中烷基转移酶表达异质性的额外优势。
