DEMONSTRATION OF A 65 KDA TUMOR-SPECIFIC PHOSPHOPROTEIN IN URINE AND SERUM OF RATS WITH N-METHYL-N-NITROSOUREA-INDUCED MAMMARY ADENOCARCINOMAS

Polyclonal antibodies against a 65 kDa tumor-associated phosphoprotein (p65) were used to develop an ELISA to analyze the presence of p65 in urine and serum of rats bearing N-methyl-N-nitrosourea-induced mammary gland adenocarcinomas. Highly purified rat p65 was 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 p65 concentration in urine and serum was 0.65 and 0.77 respectively. The average levels of p65 in 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 p65 level 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. Following in vitro phosphorylation 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-p65 polyclonal as determined by Western blot analysis. Using the anti-p65 antibodies 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 after in vitro and in vivo phosphorylation of urinary proteins obtained from mammary carcinoma-bearing rats.