The autocrine hypothesis proposes that a cell produces and secretes a hormone-like substance that can interact with specific membrane receptors on its surface to induce effects such as proliferation. Thus, a cancer cell could act to stimulate its own growth. Bombesin and bombesin-like peptides (BLPs) such as gastrin-releasing peptide (GRP) cause various physiological responses in mammals, including stimulation of proliferation of 3T3 mouse fibroblasts and normal human bronchial epithelial cells in vitro and induction of gastrin cell hyperplasia and increased pancreatic DNA content in vivo in rats. Human small-cell lung cancer (SCLC) cell lines produce and secrete BLPs and can express a single class of high-affinity receptors for BLPs. Exogenously added BLPs can also stimulate the clonal growth and DNA synthesis of SCLC in vitro. These findings suggest that BLPs function as autocrine growth factors for this tumour. One way to test this hypothesis is to interrupt the function of the endogenously produced BLPs. Here, we demonstrate that a monoclonal antibody to bombesin binds to the C-terminal region of BLPs, blocks the binding of the hormone to cellular receptors and inhibits the clonal growth of SCLC in vitro and the growth of SCLC xenografts in vivo. These results demonstrate that BLPs can function as autocrine growth factors for human SCLC.

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HIF-1␣ is a normally labile proangiogenic transcription factor that is stabilized and activated in hypoxia. Although the von Hippel Lindau (VHL) gene product, the ubiquitin ligase responsible for regulating HIF-1␣ protein levels, efficiently targets HIF-1␣ for rapid proteasome-dependent degradation under normoxia, HIF-1␣ is resistant to the destabilizing effects of VHL under hypoxia. HIF-1␣ also associates with the molecular chaperone Hsp90. To examine the role of Hsp90 in HIF-1␣ function, we used renal carcinoma cell (RCC) lines that lack functional VHL and express stable HIF-1␣ protein under normoxia. Geldanamycin (GA), an Hsp90 antagonist, promoted efficient ubiquitination and proteasome-mediated degradation of HIF-1␣ in RCC in both normoxia and hypoxia. Furthermore, HIF-1␣ point mutations that block VHL association did not protect HIF-1␣ from GA-induced destabilization. Hsp90 antagonists also inhibited HIF-1␣ transcriptional activity and dramatically reduced both hypoxiainduced accumulation of VEGF mRNA and hypoxiadependent angiogenic activity. These findings demonstrate that disruption of Hsp90 function 1) promotes HIF-1␣ degradation via a novel, oxygen-independent E3 ubiquitin ligase and 2) diminishes HIF-1␣ transcriptional activity. Existence of an Hsp90-dependent pathway for elimination of HIF-1␣ predicts that Hsp90 antagonists may be hypoxic cell sensitizers and possess antiangiogenic activity in vivo, thus extending the utility of these drugs as therapeutic anticancer agents.Hypoxia-inducible factor-1␣ (HIF-1␣) 1 is a component of a transcriptional complex that is extremely labile under normoxia but is stabilized and activated under hypoxia (1). Because HIF-1␣ regulates a variety of processes such as angiogenesis and glucose metabolism, it is acknowledged to be a critically important tumor cell survival factor that is required for tumorigenesis in many cancer models and is expressed in a majority of metastases and late stage tumors. The rapid degradation of HIF-1␣ in normoxic cells is mediated by the tumor suppressor VHL, which, together with a multimeric protein complex, serves as its E3 ubiquitin protein ligase (2-5). Under hypoxic conditions, the stabilization and activation of HIF-1␣ is due to an inability of VHL to associate with and ubiquitinate HIF-1␣. It was recently shown that hypoxic conditions impair the ability of a class of enzymes termed prolyl hydroxylases to modify two separate consensus proline motifs present on HIF-1␣ (6 -10). These modifications are required for VHL to associate with and ubiquitinate HIF-1␣, thereby targeting the protein for proteasome-dependent degradation.HIF-1␣ is constitutively stabilized in normoxic tumors and in cell lines that are VHL null or that express a nonfunctional mutant form of VHL. This occurs in over 50% of sporadic RCCs and clear cell RCCs (11), the most common malignant neoplasm of the kidney, and one of the few human tumors known to depend upon VHL inactivation. The importance of VHL function is further demonstrated in VHL disease, a human cancer ...

Adrenomedullin (AM)1 is a recently identified hypotensive peptide initially isolated from human pheochromocytoma (1). AM and its gene-related peptide, proadrenomedullin N-terminal 20 peptide (PAMP), are the two known bioactive products generated from post-translational enzymatic processing of the 185-amino acid prepro-AM molecule (1-3). Both AM and PAMP are amidated peptides. However, they have been shown to mediate their vasodilatory effects through distinctly different receptor systems (4). AM stimulates adenyl cyclase activity which elevates cAMP levels in smooth muscle cells. It is structurally related to calcitonin gene-related peptide (CGRP), and its vasodilatory effect is inhibited by the CGRP antagonist, CGRP 8 -37 (5-10). Conversely, PAMP has no amino acid sequence homology to AM or CGRP and its biological effects are not blocked by CGRP 8 -37 suggesting the involvement of a separate receptor system (4). Human AM cDNA has been cloned and mRNA expression identified in the adrenal glands, lung, kidney, and heart (2). A high degree of base sequence homology has been found between AM mRNAs isolated from other mammalian species, including rat and pig (11,12). AM has been also implicated as an important regulator of renal function having natriuretic and diuretic action (13, 14), a potent bronchodilator (15), a regulator of certain central brain actions (16,17), and a suppressor of aldosterone, adrenocorticotropin and insulin release (18 -20). The receptor for AM (AM-R) was recently cloned and sequenced (21); it contains seven transmembrane domains and belongs to the G protein-linked receptor superfamily. Finally, we and others have shown that AM is expressed in a variety of human tumors of both pulmonary and neural lineage including small cell lung cancer, adenocarcinoma, bronchoalveolar carcinoma, squamous cell carcinoma, and lung carcinoids; and ganglioblastoma and neuroblastoma (22,23). In an attempt to further study the distribution of AM and its receptor in human tumors and determine their role in these malignant disorders, we have used molecular, biochemical, and in vitro techniques to analyze a variety of human cancer cell lines of lung, breast, brain, ovary, colon, prostate, and hemopoietic lineages. MATERIALS AND METHODS Cell Lines and NormalTissue-Tumor cell lines evaluated in this study were as follows: small cell lung carcinomas (SCLC, H60, H69c, H82, H146, H187, H209, H345, H446, N417, H510, N592, H735, H774, H889, H1092), non-small cell lung carcinomas (NSCLC, H23, H157, H460, H676, H720, H727, H820, H1264, H1385, H1404, H2087, H2228, A549, UMC11), breast (SK-BR-3, ZR75-1, MCF-7, BT-20, MDA-MD231, BT-474, H2380), colon (H630, SNUC-1), nervous system (T98G (glioblastoma), TC106, CHP100, TC17, PNET, Peii, SY5Y, AS, LAN-1, KCNR-C, KCNR-DRA (neuroblastomas of the peripheral nervous system)), ovarian (NIH:OVCAR-3, SKOV3, OVT2, A2780, CP70), prostate (DU-145), adrenal (H295), chondrosarcoma (SW578), and chronic monocytic leukemia (U937). Additional NSCLC cell lines used to evaluate AM-R that are not ...

Every organ in the body requires blood vessels for efficient delivery of oxygen and nutrients, but independent vascular beds are highly specialized to meet the individual needs of specific organs. The vasculature of the brain is tightly sealed, with bloodbrain barrier (BBB) properties developing coincident with neural vascularization. G protein-coupled receptor 124 (GPR124) (tumor endothelial marker 5, TEM5), an orphan member of the adhesion family of G protein-coupled receptors, was previously identified on the basis of its overexpression in tumor vasculature. Here, we show that global deletion or endothelial-specific deletion of GPR124 in mice results in embryonic lethality associated with abnormal angiogenesis of the forebrain and spinal cord. Expression of GPR124 was found to be required for invasion and migration of blood vessels into neuroepithelium, establishment of BBB properties, and expansion of the cerebral cortex. Thus, GPR124 is an important regulator of neurovasculature development and a potential drug target for cerebrovascular diseases.

The aryl hydrocarbon receptor repressor (AHRR) is a bHLH/Per-ARNT-Sim transcription factor located in a region of chromosome 5 (5p15.3) that has been proposed to contain one or more tumor suppressor genes. We report here consistent downregulation of AHRR mRNA in human malignant tissue from different anatomical origins, including colon, breast, lung, stomach, cervix, and ovary, and demonstrate DNA hypermethylation as the regulatory mechanism of AHRR gene silencing. Knockdown of AHRR gene expression in a human lung cancer cell line using siRNA significantly enhanced in vitro anchorage-dependent and -independent cell growth as well as cell growth after transplantation into immunocompromised mice. In addition, knockdown of AHRR in non-clonable normal human mammary epithelial cells enabled them to grow in an anchorage-independent manner. Further, downregulation of AHRR expression in the human lung cancer cell line conferred resistance to apoptotic signals and enhanced motility and invasion in vitro and angiogenic potential in vivo. Ectopic expression of AHRR in tumor cells resulted in diminished anchorage-dependent and -independent cell growth and reduced angiogenic potential. These results therefore demonstrate that AHRR is a putative new tumor suppressor gene in multiple types of human cancers.

We have found markedly deficient expression of the class I major histocompatibility antigens HLA-A,B,C and beta 2m on human small-cell lung cancer (SCLC) lines and fresh tumor samples. The deficit of HLA-A,B,C and beta 2-microglobulin (beta 2m) antigen expression was demonstrated with both radiobinding assays and indirect immunofluorescence assays. Immunoprecipitation of metabolically labeled cells with antibodies to class I antigens showed most SCLC lines to have synthesized almost no beta 2m and HLA-A,B,C proteins. Northern blot analysis, using human HLA-A,B, and beta 2m cDNA probes, showed that almost all SCLC lines tested had markedly decreased amounts of HLA and beta 2m mRNA, but both gene products could be induced with interferon treatment of SCLC lines. We conclude that human SCLC, in contrast to other lung cancer types, is characterized by greatly reduced transcription of HLA-A,B,C and beta 2m genes, which suggests the existence of a mechanism for evading the host immune response to the tumor and of an E1a-like product in this type of tumor cell.

Little is known about the molecular mechanisms that control adrenomedullin (AM) production in human cancers. We demonstrate here that the expression of AM mRNA in a variety of human tumor cell lines is highly induced in a time-dependent manner by reduced oxygen tension (