An inhibitor of the chymotrypsin-like activity of the multicatalytic proteinase complex (20S proteasome) induces arrest in G2-phase and metaphase in HeLa cells

HeLa cells were treated with different concentrations of an inhibitor of the proteasome chymotrypsin-like activity the peptidyl aldehyde N-benzyloxgcarbonyl-Ile-Glu(O-t-butyl)-Ala-leucinal (PSI). A detailed analysis, which included flow cytometry, cell counting and morphological assessment, was performed. PSI treatment induces a significant reduction of mitotic activity, accompanied by metaphase arrest of the mitotic cells. DNA flow cytometry shows an accumulation of the cells in G₂+ M phases of the cell cycle, which indicates the existence of a proteasome-mediated step in the G₂-phase of the cell cycle. After removal of the inhibitor and supplementation with fresh medium, the cell cycle is resumed, but the mitotic cells show increased misalignment of chromosomes in the metaphase plate. PSI also induces HeLa cells to acquire a fibroblastoid phenotype.