Amidated peptide hormones are an important class of tumor growth factors in endocrine lung tumor cells and are potentially important in the regulation of tumor progression in lung and other tumor types. Our studies of tumor cell enzymes required for processing of precursor prohormones to active peptide hormones are comprised of three parts: the biochemistry of peptidyl amidating enzymes (PAM), the biology of PAM in human tumors, and the effect on cell growth of inhibiting PAM. The two enzymes responsible for peptide amidation (PHM and PAL) are synthesized from the same gene and mRNA precursor, and we have confirmed that in human tumor cell lines they function both as two separate enzyme activities and as a linked bifunctional enzyme. We developed pcr-based techniques to enable us to differentiate the forms of these enzymes at the mRNA level. We have identified several novel forms of the linker region between the PHM and PAL domains of PAM which involve an exon specific to humans. Alternative mRNA splicing at the transmembrane domain appears to be similar to that reported in other species. We are confirming the presence of these new forms and characterizing the translated enzymes biochemically. We previously reported that non-neuroendocrine non-small cell lung cancer cell lines had unexpectedly high levels of PAM enzymes. This led us to explore PAM expression in several tumor types generally considered to be non-neuroendocrine. We have found high levels of expression of PAM in ovarian tumor cell lines but low expression in colon, prostate, and breast lines. We are expanding these studies via an approved clinical protocol to obtain tissue samples for these tumors to study PAM expression in tumors directly using immunochemical methods and in tissue extracts with our biochemical assay. Several of the small cell lung cancer cell lines which express PAM have been reported to be dependent on self-stimulatory growth loops involving amidated peptides. We previously used growth assays to explore whether inhibition of PAM would result in growth inhibition of these cell lines. Two classes of compounds did inhibit tumor cell growth. To confirm that this inhibition is specific to PAM, we are repeating these studies using transfected cell lines expressing antisense PAM mRNA. The data from these experiments support our original findings. We are continuing these studies using chemical inhibitors in a nude mouse xenograft model.
