Rat liver nuclear matrix and similar structures derived from isolated Chironomus polytene chromosomes, nuclear envelopes, and intranuclear bodies of frog late oocytes (the karyospheres) were studied by electron microscopy with platinum shadowing and negative staining. We have shown that the treatment of whole nuclei, nuclear envelopes, polytene chromosomes, or karyospheres with nonionic detergent, high salt, and RNase and DNase followed by dilute alkali or hyaluronidase digestion reveals numerous rather uniform granules 25-30 nm in diameter. With omission ofthe nucleases the granules appear to be associated with DNA strands mostly organized in loops. Many granules form clusters and are arranged in linear or arch-like aggregates or cycles resembling the pore complexes. We suppose that these spherical bodies constitute a basic component of the nuclear matrix, chromosome scaffold, and nuclear envelope and are bound together by hyaluronic acid or some similar glycosaminoglycan.When isolated nuclei are extracted with a high-salt solution, the chromatin is removed and a nonhistone residue remains; most ofthe residue is soluble in dilute alkali (1). Nuclei extracted with high-salt solution consist of residual nucleoli, intranuclear ribonucleoprotein network, and remnants of the nuclear enve-
Our previous studies showed a predominance of high molecular weight protein group in tumor nuclear matrices. Contrary to normal cells, proteins of this group are preferentially phosphorylated. Phosphoproteins of hepatoma nuclear matrix are selectively subjected to rapid proteolysis. By alkali treatment and a monoclonal antibody against phosphotyrosyl residue the presence of two high molecular weight bands of phosphotyrosyl-containing proteins was detected in nuclear matrices of tumor but not of normal liver cells. High molecular weight protein group of tumor nuclear matrices revealed also a rapid turnover and preferential incorporation of labeled amino acids selectively inhibited by chloramphenicol.
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.
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