Demonstration of a 65 kDa tumor-specific phosphoprotein in urine and serum of rats with N-methyl-N-nitrosourea-induced mammary adenocarcinomas.
Demonstration of a 65 kDa tumor-specific phosphoprotein in urine and serum of rats with N-methyl-N-nitrosourea-induced mammary adenocarcinomas.
复制标题
证实 N-甲基-N-亚硝基脲诱导的乳腺癌大鼠的尿液和血清中存在 65 kDa 肿瘤特异性磷蛋白。
DOI：
10.1093/carcin/14.8.1659
复制
发表时间：
1993
期刊：
影响因子：
4.7
通讯作者：
Hanausek,M
中科院分区：
文献类型：
--
作者：
Mirowski,M;Walaszek,Z;Sherman,U;Adams,AK;Hanausek,M
用抗65 kDa肿瘤相关磷蛋白（p65）的多克隆抗体建立了ELISA法，检测了N-甲基-N-亚硝基脲诱发的大鼠乳腺癌模型大鼠血清和尿液中p65的含量。将高纯度的大鼠p65加入正常尿液和血清中，建立定量标准曲线，平均相关系数分别为0.98和0.99。从不同荷癌大鼠获得的所有尿液和血清样品显示p65浓度高于对照尿液和血清中发现的正常水平。肿瘤负荷与尿和血清中p65浓度的相关系数分别为0.65和0.77。正常人尿和血清中p65平均含量分别为37.0 ± 32.0和48.0 ± 38.0ng/ml。在患乳腺癌大鼠的尿液中，p65平均含量为119.0 ± 35.9ng/ml，血清中p65平均含量为225.4 ± 67.5ng/ml。血清和尿液标志物升高的敏感性、特异性和预测值分别为78.5%、70.0%和78.5%。在体外磷酸化浓缩尿蛋白，等电聚焦，SDS-PAGE和放射自显影，磷酸化形式的65 kDa蛋白质的pI为5.8的荷瘤大鼠的尿液中被确定。该磷蛋白结合到抗磷酸酪氨酸单克隆抗体和抗p65多克隆抗体，如通过Western印迹分析所确定的。在免疫沉淀过程中使用抗p65抗体，主要的放射性和免疫活性带65 kDa和两个较低的摩尔。50和41 kDa的wt带，显然代表p65的降解产物，在体外和体内磷酸化的尿蛋白从乳腺癌荷瘤大鼠获得后，被确定。
Polyclonal antibodies against a 65 kDa tumor-associated phosphoprotein (p65) were used to develop an ELISA to analyze the presence of p65in urine and serum of rats bearingN-methyl-N-nitrosourea-induced mammary gland adeno-carcinomas. Highly purified rat p65was added to normal urine and serum to establish a quantitative standard curve with the average correlation coefficient being 0.98 and 0.99 respectively. All samples of urine and serum obtained from different carcinoma-bearing rats showed p65 concentrations above the normal levels found in the control urine and sera. The correlation coefficient between tumor burden and p65concentration in urine and serum was 0.65 and 0.77 respectively. The average levels of p65in normal urine and normal serum were 37.0 ± 32.0 and 48.0 ± 38.0 ng/ml respectively. In the case of urine obtained from rats bearing mammary adenocarcinomas, the mean p65level was 119.0 ± 35.9 ng/ml and their serum level was 225.4 ± 67.5 ng/ml. Sensitivity, specificity and predictive value for serum and urine marker elevation were 78.5, 70.0 and 78.5% respectively. Followingin vitrophosphorylation of concentrated urinary proteins, isoelectrofocusing, SDS-PAGE and autoradiography, a phosphorylated form of the 65 kDa protein with a pI of 5.8 was identified in the urine of tumor-bearing rats. This phosphoprotein bound to an antiphosphotyrosine monoclonal antibody and an anti-p65polyclonal as determined by Western blot analysis. Using the anti-p65antibodies in an immunoprecipitation procedure, the main radio- and immunoactive band of 65 kDa and two lower mol. wt bands of 50 and 41 kDa, apparently representing degradation products of p65, were identified afterin vitroandin vivophosphorylation of urinary proteins obtained from mammary carcinoma-bearing rats.