IMMUNOHISTOCHEMICAL DETECTION OF RIBONUCLEOTIDE REDUCTASE IN HUMAN BREAST-TUMORS

Ribonucleotide reductase (RNR) consists of two non-identical subunits, R1 and R2 and is one of the key enzymes involved in DNA biosynthesis. RNR activity is considerably higher in malignant tumors than in norma l tissues in the rat suggesting that RNR may play an important role in the pathogenesis of human tumors. In order to obtain immunological re agents to study the localization and level of expression of RNR in var ious human tissues, a synthetic peptide containing sequences correspon ding to the COOH-terminal region of the human R2 subunit was used to g enerate rat monoclonal antibodies. The generated rat monoclonal antibo dies (IgG) inhibited RNR enzymatic activity purified from murine P388 leukemia cells. These antibodies were used to immunohistochemically ex amine the distribution of RNR in a small panel of 8 malignant and 4 be nign human breast tumors. Positive immunostaining for RNR was observed in the cytoplasm of human breast carcinoma cells in which a specific 44 kDa specific band of R2 subunit was also detected by Western blot a nalysis. The immunostaining was blocked by preabsorption of the antibo dy with an excess amount of the synthetic peptide immunogen. In 8 of 8 breast carcinomas, positive immunostaining for the R2 subunit was obs erved whereas noninvolved, adjacent breast tissue showed no staining w ith this antibody. In addition, few of the benign breast lesions exhib ited staining with this antibody. These data indicate that these antib odies can immunohistochemically detect RNR in frozen or formalin-fixed , paraffin- embedded tissues and that there is a differential expressi on of RNR between breast tumors and non-involved breast tissue. Immuno histochemical detection of RNR using these antibodies may therefore be useful for the diagnosis of human breast tumors.