Although chemical carcinogens produce cancer through diverse mechanisms, there are nevertheless commonalities of carcinogen action. These include nuclear enlargement and alterations in RNA transport and nuclear scaffold (NS) NTPase, which controls the nucleocytoplasmic transport process. The NS NTPase is derived from lamins A/C by a novel Ca2+-regulated serine protease which appears to represent a unique multicatalytic protease complex (MPC) associated with the NS. Alterations in this NS protease appear to underlie the NTPase/RNA transport alterations of carcinogenesis, and its chymotrypsin-like activity correlates with anticarcinogenic activity of various protease inhibitors (ie, Bowman-Birk). A potent peptide-chloromethylketone inhibitor (AAPFcmk) was identified, with a K(i)=56 nM, which preferentially inhibited growth of transformed hepatocyte cell lines. Further, AAPFcmk blocked chemical transformation of fibroblasts in vitro even at femtomolar concentrations, selectively affecting the nuclear/NS fractions. We hypothesize that the NS protease may be important in transformation, and that its inhibition may block secondary increases in NS NTPase, thwarting alterations in RNA transport. We have obtained amino acid sequences from components of the NS protease, and using RT/PCR amplifications with serine protease primers have identified an expressed mRNA with homology to a hamster Ca2+-regulated serine protease. Using a similar strategy, we have also obtained a number of novel putative MPC components. This proposal is designed to define the role of the NS protease in carcinogenesis. Specifically, we propose to l) clone and sequence the novel NS serine protease; 2) Determine the nature of the inhibition of cell growth in transformed hepatocyte cell lines; 3) Define the relationship between alterations in NS protease expression and transformation; and 4) Examine whether specific inhibition of the NS protease, using antisense oligonucleotides and targeted ribozymes, blocks transformation of fibroblasts, or blocks growth of transformed hepatocyte cell lines.
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