James W. Freeman

Epitope distribution and immunochemical characterization of nucleolar phosphoprotein C23 using ten monoclonal antibodies.

SummaryExtractable nucleolar proteins from HeLa cells were used as a source of antigen to immunize mice for monoclonal antibody (MAb) production. Ten of the resulting MAbs shown to identify nucleolar phosphoprotein (110 kD/pI 5.5) were purified and used in immunochemical studies to further characterize protein C23. All ten MAbs showed nucleolar localization by indirect immunofluorescence; one antibody (FR2) also showed some nucleoplasmic localization that was attributed to a shared epitope between protein C23 and a 72 kD nuclear/nucleolar antigen. Reciprocal antibody cross blocking studies indicated that the ten MAbs identified nine distinct epitopes on protein C23. Interestingly, seven of the nine epitopes were shown by immunofluorescence and competitive ELISA studies to be species related. Immunostained patterns of exponentially growing HeLa cells suggest that protein C23 exists in vivo solely as a 110 kD peptide. However, protein C23 was subject to rapid degradation into a number of proteolytic fragments upon extraction or storage of isolated nucleoli. The failure to find protein C23 related peptides with molecular sizes less than 110 kD in exponentially growing cells and the lack of cytoplasmic localization of any of the ten MAbs suggests that protein C23 is not a prepro-protein processed in vivo to form ribosomal proteins as previously suggested (1).

Discrimination of epitopes identified by monoclonal antibodies by competitive binding to nitrocellulose bound antigens

A new method is described for determining the distribution of epitopes identified by monoclonal antibodies. The method utilizes nitrocellulose membranes as a solid support for antigens which are rapidly adsorbed to nitrocellulose by vacuum-blotting and then used in competitive antibody binding assays. The distribution of epitopes is established by the reciprocal cross-blocking of radiolabeled antibody by increasing concentrations of unlabeled antibody. When unlabeled antibody does not block the binding of labeled antibody to antigen, the 2 antibodies recognize distinct epitopes. When unlabeled antibody blocks the binding of labeled antibody to antigen, the 2 antibodies recognize the same epitope. The method is rapid, sensitive and should be applicable to screening monoclonal antibodies to any epitope.

mRNA levels for human nucleolar protein P120 in tumor and nontumor cells.

A monoclonal antibody to a human tumor nucleolar 120 kD protein was developed by Freeman et al. (Cancer Res. 48: 1244-1251, 1988). Its complementary DNA (cDNA) has been isolated and sequenced (Fonagy et al., submitted). To determine the relative messenger RNA (mRNA) level for protein p120, cellular mRNA was extracted, slot-blotted onto nitrocellulose filters, and hybridized to radioactive p120 cDNA fragments. Human tumor cells contained 15-60 times more p120 mRNA than human term placenta. The rat Novikoff hepatoma ascites cell mRNA hybridized to the p120 cDNA probes, but the p120 monoclonal antibody did not react with the Novikoff hepatoma proteins. Novikoff hepatoma mRNA contained 8 times as much p120 mRNA as normal rat liver. As a control, a cDNA was used for protein B23, an abundant nucleolar protein; there were 3.5, 29, and 14 times more B23 mRNA than p120 mRNA in normal rat liver, Novikoff hepatoma ascites cells, and HeLa cells, respectively. Whereas the increased levels of the mRNA and protein B23 reflect increased activity of the nucleolus for any increment of nucleolar function, the increased levels of p120 mRNA and the p120 protein reflect the activity of the G1 phase of the cell cycle. The elevated level of p120 mRNA in tumors may reflect the heightened G1 cascade in transformed cells.

Biotechnology and Human Tumor Nucleolar Antigens

Cancer cells and most normal resting cells have been readily differentiated for a century by morphologic and biologic criteria but biochemical and immunologic distinctions have not yet withstood the test of time (Busch, 1984).