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 fibrobla sts. 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 t hat were relatively selective for the NS fraction. Here, we undertook experiments to determine the effects of AAPF(cmk) on Simian Virus 40-i mmortalized CWSV1 cells compared with a ras-transformed hepatocyte cel l line (NRA) derived ii om CWSV1. We used (biotin)AAPF(cmk) and fluore scent reagents to demonstrate a nuclear chymotrypsinlike protease acti vity, which is most prominent at the nuclear envelope. The ras-transfo rmed NR4 cells were highly susceptible to growth inhibition in a dose- dependent manner, showing 85% growth inhibition at 50 mu mol/L AAPF(cm k). In contrast, the immortalized CWSV1 cells were not sensitive at th e concentrations (10 to 50 mu mol/L) of AAPF(cmk) tested. In subcellul ar fractionation studies, the inhibitory effects of AAPF(cmk) were con fined 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 obser ved, but a marked increase in organization of actin cytoskeletal eleme nts was seen. These results suggest that a protease activity associate d with the nuclear scaffold has important functions in controlling cyt oskeletal filament organization and cell replication.