""""""""One of the earlier mandatory steps in the carcinogenesis pathway is the tumor promotion event which allows for the clonal expansion of neoplastic cells. The main focus of this project is to define the potential autocrine/paracrine mechanisms regulating the growth of human tumors and utilize these as rational targets for early detection and intervention of malignant disease. Recently, we have demonstrated that the newly identified hypotensive peptide, adrenomedullin (AM), plays an important role in the proliferative process associated with carcinogenesis, embryogenesis and wound repair. AM has been shown to function as a mitogen, apoptosis survival factor and an angeiogenic factor, all of these characteristics playing a critical part in the in vivo proliferative process. Our prior studies have shown that a variety of human cancer cell lines and tumors have the ability to expressing AM and the AM-receptor (AM-R) which implicates these peptide/receptor partners in a possible autocrine growth mechanism regulating human neoplasm proliferation. We have also found that under hypoxic conditions AM expression in human cancer cell lines is increased 10- to 20-fold over the normoxic conditions. In addition, the 5' and 3' flanking regions of the human AM gene have been shown to express several Hypoxia Response Elements (HRE), DNA motifs which bind the transcription factor Hypoxia Inducible Factor-1 (HIF-1). HIF-1 is a heterodimer composed of HIF-1a/HIF-1b, stabilized during low oxygen tension, and when bound to appropriate HRE sites can drive gene expression. We have shown that embryonic stem cells from HIF-1b knockout mice show greatly reduced expression of AM mRNA (>70%) under hypoxic conditions than compared to their wild-type control counterparts. In addition, HIF-1a null fibroblast cells had the complete absence of AM message expression when exposed to 1% oxygen while the HIF-1a +/+ partner showed a time dependent increase in expression. Given that most human tumors are hypoxic by nature and that AM plays a critical role in growth, our data on HIF-1 driven AM expression suggests the possible involvement of this peptide in the promotional event of carcinogenesis. We have also demonstrated a potential functional role for AM during embryogenesis using a in vitro chicken wing bud model. AM was shown to be highly expressed along the apical ridge of the developing wing bud (ectodermal outcroping which produces morphogens that regulate wing development). Limbless and wingless mutants lacked AM expression at the apical ridge. In bromodeoxyuridine uptake experiments to analyze the proliferative index of endogenous AM, it was shown that exposure to the peptide antagonist AM22-52 caused a marked decrease in the growth of mesenchimal cells (>70% inhibition of label cells). In addition, during prolonged incubations studies, AM22-52 disrupted the normal progression of wing development by causing the early differentiation of cartilage inot bone implicating tha AM not only acts as a mitogen but also in an anti-differentiation factor. We are now evaluating the role of AM in wound repair using a rat skin model with a 8 mm cutaneous punch wound. In our preliminary studies, it was found that topic application of AM (100 nM) once a day caused an increase in tissue granulation and neovascularization ot the wound site compared to untreated controls. In addtion, animal with impaired would healing ability induced by two week prior exposure to intramuscular dexamethasone (0.1mg/kg) showed a greatly improved capacity for wound healing when treated with topic AM which approached that observed in the dexamethasone negative controls. Our collective data has helped to define the important role of AM in carcinogenesis, embryogenesis and wound repair and implicate its potential usefulness as a clinical tool in diverse settings.""""""""
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