Detection of phosphotyrosine-containing proteins in the nuclear matrix of some tumors

Abstract

Tyrosine-containing proteins were detected by the immunoperoxidase method in the nuclear matrix of the liver and some tumors of mice. Two strips with molecular weights of about 180 and 170 kD are characteristic of hepatoma 22a and Ehrlich's ascitic carcinoma. Immunoelectron study with colloid gold showed that tyrosine-containing proteins and fibronectin are commonly present in the nucleus and cytoplasm. Key Words: nuclear matrix; phosphotyrosine; tumorsOur previous studies showed the predominance of high-molecular proteins in the nuclear matrix of tumors in comparison with normal tissues [1,2]. A microtubule-associated protein, MAP2-1ike protein p260, and fibronectin, which is not found in the nuclei of normal ceils, were detected in this group of proteins in hepatomas [4].In addition, numerous phosphoproteins, among them two strips with molecular weights of about 180 and 170 kD resistant to alkaline hydrolysis, were found in the nuclear matrix of rat hepatoma 27. This i n d i c a t e d the probable presence of phosphotyrosine residue in them [9] (Fig. 1).With this in mind, and considering the importance of tyrosine phosphorylation for proliferation and tumor growth, we investigated the nuclear matrix of hepatoma 22a and Ehrlieh's carcinoma in comparison with normal mouse liver by the immunoehemical reaction with peroxidase-conjugated antibodies. Antibodies conjugated with colloid gold were used to detect the ultrastructural localization of these proteins. In addition, the localization of fibronectin in hepatoma 22a cell nuclei was identified. MATERIALS AND METHODSCell nuclei and nuclear matrix were isolated from mouse liver as described previously [6]. Ehrlich's ascitic carcinoma and hepatoma 22a cells transplanted to (CBAxC57B1/6) F~ mice were destroyed by osmotic shock in distilled water and the nuclei and nuclear matrix were isolated from them as described previously [4].Protein phosphorylation in isolated nuclei (for electrophoresis) or cells (for electron microscopy) was carried out in 10 mM Tris-HC1 buffer, pH 7.5, for 20 min at 30~ [3].The proteins of nuclei and nuclear matrix were separated by polyacrylamide gel electrophoresis in the presence of sodium lauryl sulfate (SDS) [7]. Some of the electrophoregrams were stained with Coomassie blue. Proteins from other electrophoregrams were transferred onto nitrocellulose filters by eleetroblotting [8], and strips of proteins containing phosphotyrosine residue were detected using monoelonal antibodies to phosphotyrosine-containing proteins and antibodies to peroxidase-conjugated murine IgG (Dia-M, Moscow). The transfer of proteins onto the nitrocellulose filters was monitored by staining with 0.1% amido black.