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 demonstrated that AM expression in these tumor cell lines is upregulated during oxygen deprevation, an hypoxic state which normally exists in solid human tumors. Our studies have revealed that Hypoxia Inducible Factor-1 (HIF-1) act as the transcription factor which modulates AM expression under reduced oxygen tension by binding to hypoxia respnse elements at the 5? region of the AM gene. This was confirmed using luciferase report constructs transfected into MCF-7 followed by exposer to hypoxic conditions. In addition, cell lines derived from HIF-1 knockout mice which have a greatly reduced or complete absence hypoxia induced AM response. In addition, we have shown that reduced oxygen increased the half-life of the AM message going from 1.6 hours during normoxia to 2.5 hours for hypoxic conditions. Previously, we demonstrated the ability of a neutralizing anti-AM monoclonal antibody (MoAbG6) to block tumor cell growth in vitro and in vivo. In an attempt to find naturally occuring regulators of AM function, we have identified a 120,000 MW serum binding protein in a variety of mammalian species which selectively couples to AM. This interaction was shown to be specific for the intact native molecule and not for fragmentary AM products (AM1-12, AM13-52, AM16-21, AM22-52, AM34-52) or structurally related peptides (amylin or CGRP). Using a labeled ligand western blot technique in combination with preparative HPLC we have purified this 120,000MW AM binding protein, desginated as AMBP-1, to homogeneity and identified it to be human complement factor-H (hCFH) by total amino acid compositions analysis and N-terminal amino acid sequencing via Edman degradation. Commercially available hCFH is now being tested for its ability to regulate AM biology involving carcinogenesis, embyrogenesis and wound repair. The use of naturally occurring AM binding proteins may offer a new approach in suppressing this peptide?s promotional influence on human cancers without invoking a contraindicated host response sometimes seen when using neutralizing murine monoclonal antibodies. - angiogenesis, animal models, binding protein, - Human Tissues, Fluids, Cells, etc.
Showing the most recent 10 out of 13 publications
