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 ...