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 tumors, (3) effect of inhibiting PAM on tumor growth. The first part of this study is principally carried out using a variety of protein chemistry and molecular genetic techniques. We previously reported a pcr approach to identify novel human-specific forms of human PAM mRNA with significant biochemical differences in the activity rate, the pH optimum, glycosylation and sulfation status, and post-translational endoproteolytic processing of the new proteins. We have cloned mRNA for each of the bifunctional PAM forms. These cDNAs have been sequenced and need some minor repair before expression studies can commence. With Dr. Marquez we have been developing methods for the biochemical determination of the active site region of the PAM protein. The method being tried uses 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 identy of modified peptides. Technical details of the method have mostly been finalized using a model disulfide-rich protein, and analyses of the covalently inhibited PAM are commencing. The second part of this project involves immunochemical as well as biochemical techniques. We previously reported that all classes of lung tumor cell lines express high levels of PAM enzymes in vitro and in vivo. We have now extended these findings to individual cells, including non-neoplastic morphological abnormalities in cigarette-carcinogen-damaged lungs, using an immunohistochemical approach on archived human tumor sections. The data shows that abnormal peptide hormone biology is a component of the early stages of carcinogenesis in human lung. We have previously used growth assays to demonstrate that inhibition of the amidating enzymes results in growth inhibition of lung cancer cell lines. Xenograft experiments for pre-clinical evaluation of PAM inhibitors as potential intervention and therapeutic agents have commenced. The first agents tested showed mixed results, and testing of second-generation PAM inhibitors, developed through a collaboration with GaTech, are in progress.
