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 (factor H) by total amino acid compositions analysis and N-terminal amino acid sequencing via Edman degradation. Factor H is a naturally occurring serum protein which, when complexed with factor I, degrades C3b and blocks the complement cascade pathway of cell lysis. We have shown that commercially available factor H can block AM mediated antimicrobial effects on bacterial pathogens, however, it enhanced AM induced cAMP production in rat and mouse fibroblast cells in a dose dependent manner. We have also demonstrated that AM can augment the proteolytic degradation of C3b generated by the factor H/factor I complex. Factor H expression was identified in several human tumor cell lines (including carcinomas of the lung, breast, colon, ovary, prostate and skin) by northern and western blot analysis. Immunohistochemical examination of pathological specimens of human tumors with commercially available polyclonal antibodies to factor H have demonstated intense staining of neoplastic tissue. Thus the AM/factor H interactive link may be a novel promotion mechanism of carcinogenesis whereby tumor cells can circumvent immune surveillance by suppressing complement mediated lysis.

National Institute of Health (NIH)
Division of Clinical Sciences - NCI (NCI)
Intramural Research (Z01)
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Clinical Sciences
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Martinez, Alfredo; Zudaire, Enrique; Julian, Miguel et al. (2005) Gastrin-releasing peptide (GRP) induces angiogenesis and the specific GRP blocker 77427 inhibits tumor growth in vitro and in vivo. Oncogene 24:4106-13
Julian, Miguel; Cacho, Monica; Garcia, Mario A et al. (2005) Adrenomedullin: a new target for the design of small molecule modulators with promising pharmacological activities. Eur J Med Chem 40:737-50
Cuesta, Natalia; Martinez, Alfredo; Cuttitta, Frank et al. (2005) Identification of adrenomedullin in avian type II pneumocytes: increased expression after exposure to air pollutants. J Histochem Cytochem 53:773-80
Zudaire, Enrique; Cuesta, Natalia; Martinez, Alfredo et al. (2005) Characterization of adrenomedullin in birds. Gen Comp Endocrinol 143:10-20
Martinez, Alfredo; Zudaire, Enrique; Portal-Nunez, Sergio et al. (2004) Proadrenomedullin NH2-terminal 20 peptide is a potent angiogenic factor, and its inhibition results in reduction of tumor growth. Cancer Res 64:6489-94
Martinez, Alfredo; Oh, Hae-Ryong; Unsworth, Edward J et al. (2004) Matrix metalloproteinase-2 cleavage of adrenomedullin produces a vasoconstrictor out of a vasodilator. Biochem J 383:413-8
Martinez, Alfredo; Julian, Miguel; Bregonzio, Claudia et al. (2004) Identification of vasoactive nonpeptidic positive and negative modulators of adrenomedullin using a neutralizing antibody-based screening strategy. Endocrinology 145:3858-65
Zudaire, Enrique; Martinez, Alfredo; Ozbun, Laurent L et al. (2004) Characterization of adrenomedullin in non-human primates. Biochem Biophys Res Commun 321:859-69
Ajona, Daniel; Castano, Zafira; Garayoa, Mercedes et al. (2004) Expression of complement factor H by lung cancer cells: effects on the activation of the alternative pathway of complement. Cancer Res 64:6310-8
Macpherson, Gordon R; Ng, Sylvia S W; Forbes, Siiri L et al. (2003) Anti-angiogenic activity of human endostatin is HIF-1-independent in vitro and sensitive to timing of treatment in a human saphenous vein assay. Mol Cancer Ther 2:845-54

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