""""""""DISCONTINUED This project is directed at identifying biochemical mechanisms of tumor cells which may be targets for early detection, intervention and/or therapeutic strategies, with a focus on the enzymatic processes responsible for post-translational processing of inactive precursor prohormones into bioactive peptide hormones."""""""" """"""""Our studies of tumor cell enzymes required for processing of precursor prohormones to active peptide hormones are comprised of three parts: (1) biochemistry of peptidyl amidating enzymes (PAM), (2) biology of PAM enzymes in human cells, (3) growth effect of inhibiting PAM."""""""" """"""""The first part of this study is principally carried out using a variety of protein chemistry and molecular genetic techniques. We previously used a PCR approach to identify novel human- specific forms of human PAM mRNA. We have now repaired point mutations induced by the PCR technique required for cloning of the rare message forms. Each of the four linker region variants is now cloned into an expression vector for production of the individual proteins for biochemical analyses such as activity rate, pH optimum, glycosylation and sulfation status, and post-translational endoproteolytic processing. With Dr. Marquez (who left early 1997) we developed methods for the determination of the active site region of the PAM protein. The method used covalent coupling with a PAM inhibitor, reduction, alkylation, and digestion of the protein to yield smaller fragments, and on-line liquid chromatography electrospray-mass spectrometry to map the peptide digests and determine the identity of modified peptides. We have good evidence for localization of the active site to a region of only 7 amino acids within the 715aa (79kDa) protein."""""""" """"""""The second part of this project involves principally immunochemical techniques. We previously showed that non-neoplastic morphological abnormalities in cigarette- carcinogen-damaged lungs, express PAM. This data shows that abnormal peptide hormone biology is a component of the early stages of carcinogenesis in human lung. We are testing a variety of models of rodent carcinogenesis to identify the most appropriate model for using PAM as a target for inhibition of the peptide-hormone-driven proliferative steps in early carcinogenesis."""""""" """"""""We have previously used growth assays to demonstrate that inhibition of the amidating enzymes results in growth inhibition of lung cancer cell lines. We are testing second- generation PAM inhibitors, developed through a collaboration with GaTech, for use as agents to use in the non-human carcinogenesis models.""""""""
