Lim Y P, Fowler L C, Hixson D C, Wehbe T, Thompson N L
Department of Medical Oncology, Rhode Island Hospital, Brown University School of Medicine, Providence 02903, USA.
Cancer Res. 1996 Sep 1;56(17):3934-40.
TuAg.1 is a tumor-associated membrane glycoprotein first identified in rat hepatocellular carcinoma by monoclonal antibodies (mAbs) 324.5 and 324.9. This oncofetal antigen is also expressed by hepatocytes in cell culture but not normal adult hepatocytes in vivo. Affinity chromatography and preparative continuous elution slab-gel electrophoresis were used to separate TuAg.1 from co-purified actin and immunoglobulin. TuAg.1 was recovered as a series of bands Mr 82,000-90,000, which were pooled and subjected to CNBr digestion for primary amino acid sequence analysis. Computer database analysis of TuAg.1 peptide sequence revealed homology to the rat colon carcinoma-associated antigen pE4, a member of the immunoglobulin gene superfamily. Oligonucleotide primers derived from sequences shared by TuAg.1 and pE4 were used in reverse transcription-PCR to amplify tumor-specific products corresponding to TuAg.1 cDNA. Northern blot analysis with one of these products confirmed the oncofetal expression of transcripts related to TuAg.1/pE4 and indicated an RNA species of different size expressed only in normal liver. Identity between TuAg.1 and pE4 was further confirmed by immunochemical analysis with mAb 324.5 and mAb E4. Both antibodies were reactive with the same protein on transplantable hepatocellular carcinoma AS30D but recognized different epitopes. The reactivity of human tumor cells with mAb 324.5 and 324.9 indicates the presence of a related TuAg.1 molecule expressed in human neoplasia as well.
TuAg.1是一种肿瘤相关膜糖蛋白,最初由单克隆抗体(mAb)324.5和324.9在大鼠肝细胞癌中鉴定出来。这种癌胚抗原在细胞培养中的肝细胞中也有表达,但在体内正常成年肝细胞中不表达。采用亲和层析和制备性连续洗脱平板凝胶电泳从共纯化的肌动蛋白和免疫球蛋白中分离TuAg.1。TuAg.1以一系列分子量为82,000 - 90,000的条带形式回收,将这些条带合并并进行溴化氰消化以进行一级氨基酸序列分析。对TuAg.1肽序列进行计算机数据库分析发现,它与大鼠结肠癌相关抗原pE4具有同源性,pE4是免疫球蛋白基因超家族的成员。源自TuAg.1和pE4共享序列的寡核苷酸引物用于逆转录 - PCR,以扩增与TuAg.1 cDNA相对应的肿瘤特异性产物。用其中一种产物进行Northern印迹分析,证实了与TuAg.1 / pE4相关的转录本的癌胚表达,并表明仅在正常肝脏中表达一种大小不同的RNA种类。用mAb 324.5和mAb E4进行免疫化学分析进一步证实了TuAg.1和pE4之间的同一性。两种抗体都与可移植肝细胞癌AS30D上的同一种蛋白质发生反应,但识别不同的表位。人肿瘤细胞与mAb 324.5和324.9的反应性表明,在人类肿瘤中也存在一种相关的TuAg.1分子。