Nuclear scaffold-associated protease: in situ nuclear localization and effects of a protease inhibitor on growth and morphology of a ras-transformed hepatocyte cell line

We have previously identified a multicatalytic protease (MCP) activity associated with the nuclear scaffold (NS) in hepatocytes and fibroblasts. When we used the chloromethylketone protease inhibitor AAPF_cmk, which is targeted to chymotrypsinlike protease activity, we observed a dramatic inhibition of transformation of fibroblasts, with effects that were relatively selective for the NS fraction. Here, we undertook experiments to determine the effects of AAPF_cmk, on Simian Virus 40-immortalized CWSV1 cells compared with a ras-transformed hepatocyte cell line (NR4) derived from CWSV1. We used _biotinAAPF_cmk and fluorescent reagents to demonstrate a nuclear chymotrypsinlike protease activity, which is most prominent at the nuclear envelope. The ras-transformed NR4 cells were highly susceptible to growth inhibition in a dose-dependent manner, showing 85% growth inhibition at 50 μmol/L AAPF_cmk. In contrast, the immortalized CWSV1 cells were not sensitive at the concentrations (10 to 50 μmol/L) of AAPF_cmk tested. In subcellular fractionation studies, the inhibitory effects of AAPF_cmk were confined to the NS fraction. The AAPF_cmk-induced growth inhibition was accompanied by marked morphological changes in ras-transformed cells, without evidence of overt toxicity. No change in DNA content was observed, but a marked increase in organization of actin cytoskeletal elements was seen. These results suggest that a protease activity associated with the nuclear scaffold has important functions in controlling cytoskeletal filament organization and cell replication.