Gastrin-releasing peptide (GRP) and neuromedin B (NMB) are the mammalian homologues of the amphibian bombesin-like peptides bombesin and ranatensin, respectively. These two peptides are widely distributed in neurons; but, of greatest clinical importance, GRP occurs commonly in small cell carcinoma of the lung (SCCL) and medullary carcinoma of the thyroid (MCT) and has been shown to be a growth factor for normal and neoplastic pulmonary cells. In neuroendocrine cells of normal adult lung and thyroid, GRP is expressed at low, barely detectable levels. By contrast, in fetal lung and thyroid, and in neoplastic lung and thyroid (MCT and SCCL) GRP gene expression is greatly enhanced. The mechanisms responsible for GRP gene activation in neoplasia and normal development are unknown. Even less is known about NMB gene expression although NMB has growth factor activity similar to GRP, though at 1/10 the potency. During the prior grant period (NIRA) I reported the cloning of the gene and mRNAs which encode human GRP (hGRP) and demonstrated that the GRP gene is activated in human fetal lung and fetal thyroid. My objective in this proposal are (1) to analyze the regulation of GRP gene transcription in SCCL and MCT cell lines and (2) to initiate studies in the analysis of the NMB gene. Specifically we propose to study the following elements of GRP gene transcription: (A) Determine whether the differences in GRP mRNA concentration in different cell lines are caused by changes in GRP gene transcription, (B) Determine the cis-acting elements in the hGRP 5'-flanking region necessary for tissue-specific expression in MCT and SCCL cell lines and to identify putative enhancer and repressor elements, and (C) Determine if certain hormones and growth factors can regulate GRP gene expression in MCT and SCCL cell lines. Gene transcription will be measured by nuclear run-on assay. Regulatory sequences in the 5'-flanking region will be identified by transient expression of hGRP gene/reporter gene constructs (eg., chloramphenicol acetyl transferase, CAT). We will use our recently cloned cDNAs encoding human NMB to: (A) Characterize the tissue-specific expression of the NMB gene by RNA blot analysis and in situ hybridization. (B) Analyze and then compare the human NMB gene to the human GRP gene and determine the chromosomal localization of the NMB gene and (C) Use the NMB cDNA to probe at low stringency for related genes.
