THE CHARACTERIZATION OF THE MONOCLONAL-ANTIBODY TH-10A, SPECIFIC FOR A NUCLEAR-PROTEIN APPEARING IN THE S-PHASE OF THE CELL-CYCLE IN NORMALTHYMOCYTES AND ITS UNREGULATED EXPRESSION IN LYMPHOMA CELL-LINES

A monoclonal antibody (Th-10a) specific for the nuclear protein appear ing in the S phase of the cell cycle in normal mouse thymocytes was de rived by immunizing Wistar rats with a murine thymic lymphoma (TIGN), and its isotype was rat IgG(2a) and had kappa light chain. Immunohisto chemical staining of frozen sections of B10.Thy1.1 newborn thymus and embryonic intestine revealed that this monoclonal antibody reacted str ongly with the nuclear proteins of subcortical thymocytes and the basa l layer of the mucosa? where many cells were dividing, but not with th at of the thymic medullary area. To evaluate the expression of the nuc lear proteins during the cell cycle in detail, the results of an immun ofluorescence analysis of the thymocytes from hydroxyurea-treated B10 mice using Th-10a monoclonal antibody were compared with those of DNA synthesis of these cells with the use of the FITC-conjugated anti-BrdU rd monoclonal antibody. The results indicated that the nuclear protein detected by Th-10a monoclonal antibody was highly expressed in the S phase of normal thymocytes, while the cells in G(1), G(2) and M phases exhibited a low level of the expression. Moreover, the variations in expression of the nuclear proteins in the thymocytes at different time s after hydroxyurea treatment were observed to correspond with the fre quency of DNA synthesizing cells. In contrast, the high level and unre gulated expression of the nuclear protein detected by Th-10a monoclona l antibody was observed throughout the cell cycle of the mouse lymphom a cell lines examined. Since Th-10a monoclonal antibody does not react with the nuclear proteins derived from human, hamster or rat prolifer ating cells, this antibody may recognize a murine specific epitope of the nuclear protein. To further characterize the nuclear proteins, we extracted them from normal thymocytes or thymic lymphomas, and analyse d them by immunoblotting or immunoprecipitation followed by SDS-polyac rylamide gel electrophoresis. The nuclear protein(s) detected by Th-10 a monoclonal antibody was mostly 95 kDa and also 83 kDa polypeptide. T he results also indicated that the 95 kDa nuclear protein was phosphor ylated in vivo.