No abstract

Nuclear proteins were extracted from purified nuclei of human primary breast tumors (BrT) and bladder tumors and of human normal breast, kidney and lymphocytes by enzymatic treatment. SDS-Polyacrylamide gel electrophoresis of nuclear proteins from breast tumors showed different bands in the molecular weight zones from 25 to 220 kDa which were absent or present only as traces in normal breast tissue. Murine monoclonal antibodies (MAbs) have been produced using nuclear extracts of human primary breast tumors as immunogens. Approximately 2,000 hybridomas were generated from 5 hybridizations. According to their reactivity to BrT nuclear extracts and mammary carcinoma cell line MCF-7, seven hybridomas were selected and cloned. They were further characterized with histological immunoperoxidase assays of formaldehyde-fixed BrT paraffin tissue sections. MAb 6A3 particularly gave strong nuclear staining with all BrT specimens while MAb 1D8 showed both nuclear and cytoplasmic staining with only some of them. Specimens from mammoplasty did not react with these MAbs. Immunoblotting of BrT nuclear extracts as developed with MAbs 6A3 and 1D8 revealed major protein bands with molecular weight of 120 and 130 kDa. The potential use of these MAb-defined BrT-related nuclear proteins as markers for human breast cancer was suggested.

The major antigen of rat kidney (KMA) isolated in an immunochemically purified form was injected into homologous rats at 2-week intervals during 42 weeks. Circulating antibodies against KMA were constantly found in the sera of all immunized animals after 4 months of KMA treatment. Marked proteinuria accompanied by a significant increase in the serum γ-globulin level occurred only several months after the appearance of circulating antibodies. Histological examinations of the KMA-treated rat kidney revealed mild but definite glomerular lesions of the membranous, non-proliferative type. Rat γ-globulin was found as a mixed, linear and granular, deposit along the basement membranes and the mesangial regions. However, rat β_1C-globulin was found as an exclusively granular deposit in the same areas, but more intensively in the mesangium. The acid elution of KMA-treated rat kidney contained γ-globulin which reacted with KMA. As compared with Edgington’s nephritogenic tubular antigen RTE-α₅, KMA was found to be completely different. However, it was also localized exclusively in the proximal tubular cells. Its presence in normal rat serum and liver has been suggested as a source of autologous immunogens in the long-term development of an autoimmune complex nephritis in the rat.

The major antigen of rat kidney (KMA) was isolated by chromatography on DEAE-cellulose and Sephadex G-25 gel filtration. The sedimentation coefficient of the purified antigen was about 1.2 S and its electrophoretic mobility corresponded to that of α2-globulins. Its diffusion coefficient was estimated by the method of Allison and Humphrey as 11.4 × 10−7 cm2 sec−1. On gel filtration through Sephadex G-100, the purified preparation was resolved into two components with identical antigenic properties but of different molecular weights, estimated as 16,600 and 4,100. The concentrations of KMA were determined in different tissues, urine, and serum by immunodiffusion. The probable relationship between physical and immunochemical properties and the possible physio-pathological role of KMA are discussed.

Nuclear proteins were extracted from purified nuclei of 7,12-dimethylbenz(a)anthracene(DMBA)-induced tumors and normal mammary glands of the rat by enzymatic treatment. Of the 34 bands indicated by one-dimensional polyacrylamide gel electrophoresis of nuclear proteins, 6 appeared in high concentration in tumors but were found as traces or undetectable in normal glands; 6 others were clearly shown in the latter but were not detectable or greatly reduced in the former. Two-dimensional electrophoresis identified about 130 and 92 non-histone proteins in normal mammary and tumor cell nuclei respectively. Marked differences in spot density were noted especially in spots (M.W. X 10(-3)/pI) 100/5.7 and 200/5.5 of tumors and 28/7.1, 32/5.4, 36/5.4, 38/6.9, and 68/6.0 of normal tissue. The relationship between these nuclear proteins and the development of mammary tumors is also discussed.