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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
Author(s) -
Clawson Gary A.,
Ren Ling,
Isom Harriet C.
Publication year - 1995
Publication title -
hepatology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.488
H-Index - 361
eISSN - 1527-3350
pISSN - 0270-9139
DOI - 10.1002/hep.1840220431
Subject(s) - protease inhibitor (pharmacology) , microbiology and biotechnology , protease , in situ , hepatocyte , chemistry , cell culture , morphology (biology) , nuclear localization sequence , biophysics , biology , nucleus , biochemistry , in vitro , virology , genetics , enzyme , virus , viral load , antiretroviral therapy , organic chemistry
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 ros ‐transformed hepatocyte cell line (NR4) derived from CWSV1. We used biotin AAPF 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. (H EPATOLOGY 1995; 22:1230–1235.).