Ames R S, North S M, Fry K D, Tainsky M A, Nicolson G L, Roth J A
Department of Thoracic Surgery, University of Texas, M. D. Anderson Cancer Center, Houston 77030.
Cancer Res. 1989 May 1;49(9):2312-9.
A Mr 74,000 phosphoglycoprotein (gp74) present on the surface of oncogene-transformed murine cells but not untransformed NIH 3T3 cells was previously identified with mouse monoclonal antibody 45-2D9. The original cell population used as the immunogen was found to consist of two cell populations. The purpose of this study was to characterize these cell populations; determine the distribution of gp74 on normal, transformed, and neoplastic cells; and to characterize the gp74 molecule. Southern hybridization studies of cloned cell populations demonstrated that the immunizing cell population consisted of c-Ha-ras-transfected NIH 3T3 cells and Kirsten sarcoma virus-transformed rat cells (TRF cells). TRF cells showed a high level of gp74 expression. We observed that the expression of gp74 was increased on chemically and spontaneously transformed rat cells compared to untransformed rat cells. No binding of monoclonal antibody 45-2D9 was detected to rat adult and fetal tissue. Immunoperoxidase staining, immunofluorescence flow cytometry, and immunoprecipitation analysis of dimethylbenz[a]anthracene-induced metastatic 13762NF rat mammary adenocarcinoma clonal sublines demonstrated an inverse relationship between gp74 expression and metastatic phenotype. gp74 was immunoprecipitated from two low and medium metastatic clonal sublines (MTC and MTF7), but not from highly metastatic clone MTLn3 cells. Biosynthetic labeling and immunoprecipitation studies demonstrated that gp74 was phosphorylated on serine residues and was not secreted from transformed cells. No detectable protein kinase activity in an immune complex assay was associated with this molecule. We conclude that increased gp74 expression by rat cells is associated with transformed and neoplastic cells.
一种分子量为74,000的磷糖蛋白（gp74）存在于癌基因转化的鼠细胞表面，但未转化的NIH 3T3细胞表面没有，此前已用小鼠单克隆抗体45-2D9鉴定出来。用作免疫原的原始细胞群体被发现由两个细胞群体组成。本研究的目的是对这些细胞群体进行表征；确定gp74在正常、转化和肿瘤细胞上的分布；并对gp74分子进行表征。对克隆细胞群体的Southern杂交研究表明，免疫细胞群体由c-Ha-ras转染的NIH 3T3细胞和 Kirsten肉瘤病毒转化的大鼠细胞（TRF细胞）组成。TRF细胞显示出高水平的gp74表达。我们观察到，与未转化的大鼠细胞相比，化学诱导和自发转化的大鼠细胞上gp74的表达增加。未检测到单克隆抗体45-2D9与大鼠成年和胎儿组织的结合。对二甲基苯并[a]蒽诱导的转移性13762NF大鼠乳腺腺癌克隆亚系进行免疫过氧化物酶染色、免疫荧光流式细胞术和免疫沉淀分析，结果表明gp74表达与转移表型呈负相关。从两个低转移和中转移克隆亚系（MTC和MTF7）中免疫沉淀出了gp74，但未从高转移克隆MTLn3细胞中免疫沉淀出。生物合成标记和免疫沉淀研究表明，gp74在丝氨酸残基上被磷酸化，并且不会从转化细胞中分泌出来。在免疫复合物测定中未检测到与该分子相关的蛋白激酶活性。我们得出结论，大鼠细胞中gp74表达的增加与转化细胞和肿瘤细胞有关。
