Comparison of nuclear proteins from DMBA-induced mammary tumors and lactating mammary glands by polyacrylamide gel electrophoresis.

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.

Generation of monoclonal antibodies reactive with nuclear proteins of human primary breast tumors.

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.

Immunochemical and enzymatic studies on the soluble antigens of normal human kidney.

The antigenic composition of the soluble extract from normal human kidney was determined with the combined techniques of immunological absorption, immuno-diffusion and immunoelectrophoresis. The kidney extract contained at least eleven non-serum, tissue antigens; some of them were found in the extracts of other organs, but three were kidney-specific. None of the soluble tissue antigens from human kidney was characterized as a lipoprotein; however, three of them stained with the reagent for glycoproteins. Esterase activities were also demonstrated in four non-serum, renal antigens, one of which was present only in the kidney extract and absent in the other organs extracts.