Woodman A C, Sugiyama M, Yoshida K, Sugino T, Borgya A, Goodison S, Matsumura Y, Tarin D
Nuffield Department of Pathology and Bacteriology, University of Oxford, John Radcliffe Hospital, United Kingdom.
Am J Pathol. 1996 Nov;149(5):1519-30.
Many studies have now demonstrated disorganized overexpression of the CD44 gene in various types of human malignant tumors, and this abnormality has emerged as an interesting candidate marker for early cancer diagnosis. The purpose of this work was to analyze and compare the patterns of transcription and translation of this gene in human breast (ZR75-1; MDAMB-435 clone 4A4) and colon (HT29) tumor cell lines and in tumors of the breast, bladder, and colon, with the aim of identifying the most suitable analyte for diagnostic purposes. Transcription was studied by reverse transcription-polymerase chain reaction using CD44-specific primers and probes complementary to exons in the standard (exons 3 to 5 and 16 to 18) and variably expressed regions of this gene (exons 7, 8, 10, 11, and 15). Translation was investigated by Western blot analysis and immunohistochemistry using monoclonal antibodies specific to the standard form of CD44 and to the products of the same variant exons. Southern blot hybridization analysis of the reverse transcription-polymerase chain reaction products showed a large number of CD44 transcripts in tumor cells. Direct comparison of these Southern blots with Western blots on matched tumor-cell-line extracts indicated that most of the diverse mRNA isoforms did not detectably translate into proteins. However, immunohistochemistry of normal and malignant breast (n = 17 and 23, respectively), bladder (n = 5 and 19), and colon (n = 19 and 19) tissue specimens showed increased staining of CD44 standard and CD44 variant proteins in the carcinoma cells. Combination of this information with the data from reverse transcription-polymerase chain reaction and Western blot analysis indicates that the overexpression at the protein level involves only a minority of the aberrant RNA transcripts. We conclude that the development of methods for the accurate quantitation of over-abundant CD44 RNA species in clinical samples offers the most promising approach to improved early diagnosis of malignancy using this new marker.
目前许多研究已证实，CD44基因在各类人类恶性肿瘤中存在无序的过表达，并且这种异常已成为早期癌症诊断中一个有趣的候选标志物。本研究的目的是分析和比较该基因在人乳腺（ZR75 - 1；MDAMB - 435克隆4A4）和结肠（HT29）肿瘤细胞系以及乳腺、膀胱和结肠肿瘤中的转录和翻译模式，以确定最适合用于诊断目的的分析物。使用与该基因标准区域（外显子3至5和16至18）以及可变表达区域（外显子7、8、10、11和15）中的外显子互补的CD44特异性引物和探针，通过逆转录 - 聚合酶链反应研究转录情况。使用针对CD44标准形式以及相同可变外显子产物的单克隆抗体，通过蛋白质印迹分析和免疫组织化学研究翻译情况。对逆转录 - 聚合酶链反应产物的Southern印迹杂交分析显示，肿瘤细胞中有大量的CD44转录本。将这些Southern印迹与匹配的肿瘤细胞系提取物的蛋白质印迹进行直接比较表明，大多数不同的mRNA异构体并未检测到可转化为蛋白质。然而，对正常和恶性乳腺（分别为n = 17和23）、膀胱（n = 5和19）以及结肠（n = 19和19）组织标本进行的免疫组织化学显示，癌细胞中CD44标准蛋白和CD44变体蛋白的染色增加。将这些信息与逆转录 - 聚合酶链反应和蛋白质印迹分析的数据相结合表明，蛋白质水平的过表达仅涉及少数异常RNA转录本。我们得出结论，开发用于准确定量临床样本中过量CD44 RNA种类的方法，为利用这一新标志物改善恶性肿瘤的早期诊断提供了最有前景的途径。
