alpha-chymotrypsin and succinyl-alanylalanyl-prolyl-phenylalanine-chloromethylketone

Protease inhibitors have been known to exhibit anticarcinogenic activity in a variety of model systems, although the biological target(s) and mechanism remain enigmatic. Human papillomavirus (HPV) is the primary etiological agent of cervical cancer. Here we show that a nuclear chymotrypsin-like protease activity (NCLPA), which appears to be involved in transformation in several different experimental models, is significantly elevated in keratinocytes infected with high-risk HPV. Further, we demonstrate a marked growth inhibition of organotypic raft cultures, which is specific for cells infected with high-risk HPV types, using a chloromethyl ketone inhibitor previously shown to be relatively selective for the NCLPA. Surprisingly, this HPV-dependent inhibitory effect is independent of any alterations in the NCLPA. This finding has clear implications for the development of novel therapeutics specifically targeted to cervical dysplasias with HPV-infected cells.

Topics: Amino Acid Chloromethyl Ketones; Carcinoma; Cell Culture Techniques; Cell Nucleus; Cell Proliferation; Cells, Cultured; Chymotrypsin; Dimethyl Sulfoxide; Dose-Response Relationship, Drug; Female; Genome, Viral; Humans; Keratinocytes; Male; Oligopeptides; Papillomaviridae; Papillomavirus Infections; Peptide Hydrolases; Protease Inhibitors; Uterine Cervical Neoplasms

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 AAPFcmk, 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 AAPFcmk on Simian Virus 40-immortalized CWSV1 cells compared with a ras-transformed hepatocyte cell line (NR4) derived from CWSV1. We used biotinAAPFcmk 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 mumol/LAAPFcmk. In contrast, the immortalized CWSV1 cells were not sensitive at the concentrations (10 to 50 mumol/L) of AAPFcmk tested. In subcellular fractionation studies, the inhibitory effects of AAPFcmk were confined to the NS fraction. The AAPFcmk-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.

Topics: Amino Acid Chloromethyl Ketones; Animals; Biotin; Cell Division; Cell Line, Transformed; Cell Nucleus; Chymotrypsin; Cytoskeleton; Fluorescent Dyes; Genes, ras; Liver; Microscopy, Confocal; Oligopeptides; Protease Inhibitors; Rats; Serine Endopeptidases