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
M. Muto et al., 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, Cell proliferation, 28(12), 1995, pp. 645-657
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.