Alteration of argyrophilic nucleolar organizer region associated (Ag-NOR) proteins in apoptosis-induced human salivary gland cells and human oral squamous carcinoma cells

The level of argyrophilic nucleolar organizer regions (AgNORs) and AgNOR-as sociated proteins (Ag-NOR proteins) varies with cell activity, including ri bosomal biogenesis occurring in proliferating cells. Proteins associated wi th some AgNORs are detected by a specific silver staining. To investigate a possible relationship between apoptosis and the AgNORs or Ag-NOR proteins, we examined the changes of AgNORs and Ag-NOR proteins during apoptosis in a human salivary gland cell line, HSG cells, and a human oral squamous carc inoma cell line, SCC-25 cells. Apoptosis was induced by treatment of HSG an d SCC-25 cells with okadaic acid. Proteins prepared from HSG and SCC-25 cel ls treated with varying concentrations of okadaic acid (OA) were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) fol lowed by transferring to transfer membranes and staining for Ag-NOR protein s by modified Western blot analysis. Four major bands (110 kDa, 43 kDa, 39k Da, and 37 hDa) were detected in the proteins obtained from the control cel ls. The level of the 110-kDa protein decreased in the proteins prepared fro m OA-induced apoptotic cells; however, the reaction intensity of the other three bands was changed in apoptotic cells. An additional band of an 80-kDa Ag-NOR protein appeared and increased in the apoptotic cells. Cellular fra ctionation of HSG cells and SCC-25 cells was done with or without apoptotic induction. An 80-kDa Ag-NOR protein was detected in the nuclear fraction p repared from the apoptotic cells, while the 110-kDa protein decreased in th e nuclear fraction of these cells. The 110-kDa Ag-NOR protein may be nucleo lin (C23) as deduced from its AgNOR staining features, including molecular weight. The 80-kDa protein may be the cleavage product of the 110-kDa prote in, in the cell-free apoptotic system, in which intact nuclei of HSG cells were incubated with the cytosol fraction of apoptotic HSG and SCC-25 cells, the 80-kDa Ag-NOR protein was detected in nuclei incubated with the cytoso l fraction of apoptotic cells, while the level of the 110-kDa protein decre ased. The changes of Ag-NOR proteins in nuclei prepared from SCC-25 cells i ncubated with cytosol fractions prepared from HSG and SCC-25 cells were ide ntical to those of the HSG cells. The alternation of AgNORs in apoptosis-in duced HSG cells was also examined using double staining with Hoechst 33342 and silver nitrate. Hoechst staining revealed typical apoptotic nuclei, whi ch exhibited highly fluorescent condensed chromatin in OA-treated HSG cells . Silver grains representing AgNORs were not detected in the cells undergoi ng apoptosis. The dual-imposition view confirmed that AgNORs, which are vis ible as dots in nucleoli in the control cells, disappeared from the apoptot ic nuclei of HSG cells. Our results indicate that the 110-kDa nucleolar Ag- NOR protein is associated with apoptosis and is cleaved during apoptosis.