Two distinct bombesin receptor subtypes are expressed and functional in human lung carcinoma cells.

Bombesin-like peptides have been implicated as autocrine growth factors influencing the pathogenesis and progression of some human lung carcinoma cells. To determine the pharmacologic and structural properties of the bombesin receptors expressed in human lung carcinoma cells, cDNA clones encoding a human gastrin-releasing peptide receptor (GRP-R) and a pharmacologically distinct neuromedin-B preferring bombesin-receptor (NMB-R) were isolated from a human small cell lung carcinoma cell line (NCI-H345). After expression in Xenopus oocytes, a GRP-R-specific antagonist was effective in blocking responses elicited from the cloned GRP-R, but not the NMB-R. Both GRP-R and NMB-R mRNA expression was detected at varying levels in a panel of human lung cancer cell lines. These results indicate heterogeneity of bombesin receptor subtypes exists in human lung carcinoma cells and should be considered in the design of bombesin receptor antagonists intended to inhibit tumor cell growth.     

In vitro immunization of human lymphocytes. I. Production of human monoclonal antibodies against bombesin and tetanus toxoid

A procedure for in vitro sensitization of human lymphocytes against bombesin conjugated to tetanus toxoid (BTT) is described. Bombesin is a tetradecapeptide associated with small cell lung carcinoma. We found that antibody responses against bombesin as well as tetanus toxoid could be generated in vitro by culturing nylon-separated human splenic lymphocytes for 6 days with lipopolysaccharide, phytohemagglutinin-activated lymphocyte supernatants, human AB serum, and bombesin conjugated to tetanus toxoid. Cells sensitized by this procedure were fused to murine myeloma cells, NS-1. The specificities of resulting hybrids were analyzed by enzyme-linked immunoassays and competitive inhibition experiments. Hybrids secreting anti-bombesin (IgM) or anti-tetanus toxoid (IgM or IgG) were obtained. The ratio of IgG to IgM antibodies against tetanus toxoid could be increased by using antigen coupled to Sepharose beads. The sensitization procedure described here offers a system for the study of antigenic stimulation of human B lymphocytes in vitro and for the production of human monoclonal antibodies with the desired specificities.     

Cytokeratins in normal lung and lung carcinomas. I. Adenocarcinomas, squamous cell carcinomas and cultured cell lines

The various epithelial cells of the lower respiratory tract and the carcinomas derived from them differ markedly in their differentiation characteristics. Using immunofluorescence microscopy and two-dimensional gel electrophoresis of cytoskeletal proteins from microdissected tissues we have considered whether cytokeratin polypeptides can serve as markers of cell differentiation in epithelia from various parts of the human and bovine lower respiratory tract. In addition , we have compared these protein patterns with those found in the two commonest types of human lung carcinoma and in several cultured lung carcinoma cell lines. By immunofluorescence microscopy, broad spectrum antibodies to cytokeratins stain all epithelial cells of the respiratory tract, including basal, ciliated, goblet, and alveolar cells as well as all tumor cells of adenocarcinomas and squamous cell carcinomas. However, in contrast, selective cytokeratin antibodies reveal cell type-related differences. Basal cells of the bronchial epithelium react with antibodies raised against a specific epidermal keratin polypeptide but not with antibodies derived from cytokeratins characteristic of simple epithelia. When examined by two-dimensional gel electrophoresis, the alveolar cells of human lung show cytokeratin polypeptides typical of simple epithelia (nos. 7, 8, 18 and 19) whereas the bronchial epithelium expresses, in addition, basic cytokeratins (no. 5, small amounts of no. 6) as well as the acidic polypeptides nos. 15 and 17. Bovine alveolar cells also differ from cells of the tracheal epithelium by the absence of a basic cytokeratin polypeptide. All adenocarcinomas of the lung reveal a "simple-epithelium-type" cytokeratin pattern (nos. 7, 8, 18 and 19). In contrast, squamous cell carcinomas of the lung contain an unusual complexity of cytokeratins. We have consistently found polypeptides nos. 5, 6, 8, 13, 17, 18 and 19 and, in some cases, variable amounts of cytokeratins nos. 4, 14 and 15. Several established cell lines derived from human lung carcinomas (SK-LU-1, Calu -1, SK-MES-1 and A-549) show a uniform pattern of cytokeratin polypeptides (nos. 7, 8, 18 and 19), similar to that found in adenocarcinomas. In addition, vimentin filaments are produced in all the cell lines examined, except for SK-LU-1.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effects of bombesin on human small cell lung cancer cells: evidence for a subset of bombesin non-responsive cell lines

The effects of bombesin on three human small cell lung carcinoma cell (SCLC) lines (NCI-H69, NCI-H128, and NCI-H345) have been examined and compared to the effects of the peptide on the mouse fibroblast cell line Swiss 3T3, and the rat pituitary tumor cell line GH3W5. While all three SCLC lines expressed messenger RNA encoding pro-gastrin releasing peptide (GRP), only the NCI-H345 cells expressed detectable membrane receptors for GRP and responded to nanomolar concentrations of bombesin as shown by 125I-GRP binding, total inositol phosphate accumulation, and increased clonal growth in soft agarose. These data show that some SCLC lines are insensitive to bombesin and do not express detectable membrane receptors for GRP.     

Bombesin-like peptides and receptors in human tumor cell lines

Human cancer cell lines were assayed for bombesin-like peptides and receptors. Acid extracts derived from small cell lung cancer, but not other types of cancer had high levels of immunoreactive bombesin. Regardless of patient treatment, site of tumor origin (bone marrow, lymph node, or pleural effusion) or culture conditions, small cell lung cancer cell lines had high levels of bombesin-like peptides. Thus, bombesin levels in small cell lung, but not other types of human cancer, are routinely elevated. Also, small cell lung cancer lines in contrast to other cell lines have a high density of binding sites for a radiolabeled bombesin analogue. The presence of high concentrations of bombesin-like peptides and receptors suggests that bombesin may function as an important regulatory agent in human small cell lung cancer.     

Nicotine, acetylcholine and bombesin are trophic growth factors in neuroendocrine cell lines derived from experimental hamster lung tumors.

Neuroendocrine hamster lung tumors, induced by exposure to 60% hyperoxia and subcutaneous administration of the tobacco-specific nitrosamine 4-(methylnitrosamino)-l-(3-pyridyl)-l-butanone (NNK) for 12 weeks, were placed in cell culture. By subsequent selective transfer of epithelial cells and maintenance in an atmosphere of 8% CO2, cell lines with characteristics of neuroendocrine cells were established. The neuroendocrine markers expressed by these cell lines included electron dense neuroendocrine secretion granules as well as secretion of calcitonin and mammalian bombesin. In keeping with data previously reported for a human neuroendocrine lung tumor cell line, nicotine, acetylcholine, and mammalian bombesin (MB) acted as strong growth factors in these neuroendocrine hamster tumor lines. The mitogenic effect of nicotine and acetylcholine was abolished by nicotinic receptor inhibition while the effects of mammalian bombesin were inhibited by an antagonist of MB receptors. Our data suggest that a receptor-mediated mitogenic effect of nicotine on neuroendocrine lung cells may be instrumental in the induction of smoking-associated small cell lung cancer.     

Bombesin-related peptides induce calcium mobilization in a subset of human small cell lung cancer cell lines

To examine the biochemical basis for growth factor-induced responses in human lung cancer cells, we used the quin2 technique to study the effect of the amphibian peptide bombesin and its congeners including mammalian gastrin-releasing peptide (GRP) on the intracellular free calcium level [Ca2+]i in small cell lung cancer cell lines. In five of eleven cell lines tested, Tyr4-bombesin or GRP elicited a rapid and transient increase in [Ca2+]i. The response was seen with as little as 1 nM ligand, was not affected by membrane depolarization, and derived in part from internal calcium stores. Desensitization to a second addition of active bombesin congeners occurs subsequent to initial addition of Tyr4-bombesin. Structure-activity analysis showed the carboxyl-terminal octapeptide was the active portion of the peptide. Analogs in which the carboxyl terminus was oxidized or deamidated were inactive. Ranatensin, litorin, alytesin, and GRP, but not physalaemin, were as active as Tyr4-bombesin. A monoclonal antibody to the carboxyl terminus of bombesin selectively blocked the increased [Ca2+]i elicited by Tyr4-bombesin. These studies suggest that bombesin congeners can act on some small cell lung cancer cell lines by a pathway utilizing increased [Ca2+]i.     

Bombesin-like peptides elevate cytosolic calcium in small cell lung cancer cells

The ability of bombesin-like peptides to elevate intracellular Ca2+ levels in small cell lung cancer cells was investigated using the fluorescent Ca2+ indicator Fura 2. Nanomolar concentrations of bombesin elevated cytosolic Ca2+ levels in the absence or presence of extracellular Ca2+. Potent bombesin receptor agonists, such as gastrin releasing peptide (GRP) or (GRP)14-27 elevated cytosolic Ca2+ levels whereas inactive compounds such as (D-Trp8)bombesin or (GRP)1-16 did not. Furthermore, the bombesin receptor antagonist (D-Arg1, D-Pro2, D-Trp7,9, Leu11) substance P (30 microM) had no effect on the Ca2+ levels by itself but antagonized the increase in Ca2+ caused by 10 nM or 100 nM bombesin. These data suggest that bombesin receptors may regulate the release of Ca2+ from intracellular organelles in small cell lung cancer cells.     

Bombesin and calcitonin secretion by pulmonary carcinoma is modulated by cholinergic receptors

Three established cell lines derived from human small cell carcinoma of the lung, and known to produce significant amounts of peptide hormones were used to evaluate the regulation of hormone secretion by cholinergic agonists. In two of the cell lines (DMS 53, DMS 153) acetylcholine chloride, bethanechol chloride, and carbamylcholine at the concentrations of 10(-3)M to 10(-5)M stimulated secretion of bombesin and calcitonin as measured by RIA. The third cell line, DMS 406, was not significantly stimulated. Inhibition of induced stimulation by the cholinergic antagonist atropine, but not hexamethonium, indicated the presence of muscarinic rather than the nicotinic type of cholinergic receptors on the stimulatable cells. These receptors appear to mediate hormone secretion comparably to normal endocrine cells.     

Receptor for bombesin with associated tyrosine kinase activity.

The neuropeptide bombesin is known for its potent mitogenic activity on murine 3T3 fibroblasts and other cells. Recently it has been implicated in the pathogenesis of small cell lung carcinoma, in which it acts through an autocrine loop of growth stimulation. Phosphotyrosine (P-Tyr) antibodies have been successfully used to recognize the autophosphorylated receptors for known growth factors. In Swiss 3T3 fibroblasts, phosphotyrosine antibodies identified a 115,000-Mr cell surface protein (p115) that became phosphorylated on tyrosine as a specific response to bombesin stimulation of quiescent cells. The extent of phosphorylation was dose dependent and correlated with the mitogenic effect induced by bombesin, measured by [3H]thymidine incorporation. Tyrosine phosphorylation of p115 was detectable minutes after the addition of bombesin, and its time course paralleled that described for the binding of bombesin to its receptor. Immunocomplexes of phosphorylated p115 and phosphotyrosine antibodies bound 125I-labeled [Tyr4]bombesin in a specific and saturable manner and displayed an associated tyrosine kinase activity enhanced by bombesin. Furthermore, the 125I-labeled bombesin analog gastrin-releasing peptide, bound to intact live cells, was coprecipitated with p115. These data strongly suggest that p115 participates in the structure and function of the surface receptor for bombesin, a new member of the family of growth factor receptors with associated tyrosine kinase activity.     

Bombesin-like peptides in human small cell carcinoma of the lung

The nature of bombesin-like immunoreactive peptides was studied in extracts of small cell carcinoma of the human lung. Three peaks, I, II and III, designated by their increasing retention times, were separated by reversed-phase high performance liquid chromatography (HPLC) with trifluoroacetic acid (TFA) as counter ion. None of the peaks corresponded to bombesin. Peak III was eluted at the same position as porcine gastrin releasing peptide (GRP) but was separated from it in another reversed-phase system using heptafluorobutyric acid (HFBA). Peak II material eluted in the position of bombesin in the HFBA system but not in the TFA system. The elution position of Peak I corresponded to that of the carboxyl terminal fragments of GRP, i.e. GRP18-27 and GRP19-27. This correspondence was observed in each of the reversed-phase and gel filtration systems used. The Peak III peptide was converted to peak I after incubation with trypsin. It was reasoned that this conversion could be one of the steps in the processing of bombesin-like peptides in human small cell carcinoma.     

Bombesin-like peptides in small cell lung cancer: biochemical characterization and secretion from a cell line

High intracellular levels of BN-like peptides are present in tumors and cell lines of small cell carcinoma of the lung (SCCL) as well as the putative precursor cells of this tumor, the pulmonary endocrine cell. In cell line NCI-H209 the density of bombesin-like peptides was 8.9 +/- 1.1 pmol/mg total protein. Gel filtration chromatography of an extract of these cells revealed one major peak of immunoreactivity which coeluted with synthetic bombesin (1620 daltons). Also, high pressure liquid chromatography revealed one major peak of immunoreactivity was present which eluted before synthetic peptide. Therefore, SCCL bombesin-like peptides may be of similar size but are more hydrophilic than synthetic peptide. Cells maintained in culture continuously release bombesin-like peptides into the growth medium. Also, high concentrations of K+ stimulated the secretion of immunoreactive bombesin from cell lines in a Ca++-dependent manner. These SCCL bombesin-like peptides may function as important regulatory agents in the malignant lung.     

Localization of somatostatin-, bombesin-, and serotonin-like immunoreactivity in the lung of the fetal Rhesus monkey

Summary Immunoreactivity of regulatory peptides has been demonstrated in the fetal lung of Macaca mulatta by the peroxidase anti-peroxidase method. Serotonin-immunoreactive neuroepithelial bodies are distributed in the airways from the bronchi to the alveolar ducts. Many neuroepithelial bodies also show bombesin-like immunoreactivity; a very few are immunoreactive to somatostatin antiserum. Four populations of neuroepithelial bodies were identified which contain immunoreactivity for 1) serotonin alone, 2) serotonin and bombesin, 3) serotonin and somatostatin, and 4) serotonin, bombesin, and somatostatin. Since bombesin and somatostatin have been demonstrated to have opposite effects on the release of other peptide hormones, it seems likely that the presence of these same peptides in neuroepithelial bodies may have a similar regulatory role in the lung.     

Bombesin production by human small cell carcinoma of the lung

A series of continuous cell lines of human small cell carcinoma of the lung (SCCL) have been evaluated for the production of bombesin (BN). In early established cultures BN was detected in the medium of 9 out of 11 cell lines and in 6 out of 7 cell homogenates examined. Levels in the medium were frequently higher in cultures of later passages compared to earlier passages of the same line and low levels developed in the two previously negative cell lines. Plasma concentrations were greater than 80 pmol/l in 2 out of 27 (7%) randomly selected patients with SCCL. A culture (DMS 406) established from the tumor of a patient with the highest plasma level (1240 pmol/l) was the highest producer in vitro. The results indicate that BN, which has been demonstrated immunocytochemically to be present in normal bronchial mucosal cells, is frequently produced by SCCL in vitro but elevated plasma levels are infrequently found in patients with this neoplasm.     

Bombesin and substance P analogues differentially regulate G-protein coupling to the bombesin receptor. Direct evidence for biased agonism

Substance P analogues including [d-Arg1,d-Phe5,d-Trp7,9,Leu11]substance P (SpD) act as "broad spectrum neuropeptide antagonists" and are potential anticancer agents that inhibit the growth of small cell lung cancer cells in vitro and in vivo. However, their mechanism of action is controversial and not fully understood. Although these compounds block bombesin-induced mitogenesis and signal transduction, they also have agonist activity. The mechanism underlying this agonist activity was examined. SpD binds to the ligand-binding site of the bombesin/gastrin-releasing peptide receptor and blocks the bombesin-stimulated increase in [Ca2+]i within the same concentration range that causes sustained activation of c-Jun N-terminal kinase and extracellular signal-regulated protein kinase (ERK). The activation of c-Jun N-terminal kinase by SpD and bombesin is blocked by dominant negative inhibition of G(alpha12). The ERK activation by SpD is pertussis toxin-sensitive in contrast to ERK activation by bombesin, which is pertussis toxin-insensitive but dependent on epidermal growth factor receptor phosphorylation. SpD does not simply act as a partial agonist but differentially modulates the activation of the G-proteins G(alpha12), G(i), and G(q) compared with bombesin. This unique ability allows the bombesin receptor to couple to G(i) and at the same time block receptor activation of G(q). Our results provide direct evidence that SpD is acting as a "biased agonist" and that this has physiological relevance in small cell lung cancer cells. This validation of the concept of biased agonism has important implications in the development of novel pharmacological agents to dissect receptor-mediated signal transduction and of highly selective drugs to treat human disease.     

Asbestos exposure induces alveolar epithelial cell plasticity through MAPK/Erk signaling

The inhalation of asbestos fibers is considered to be highly harmful, and lead to fibrotic and/or malignant disease. Epithelial-to-mesenchymal transition (EMT) is a common pathogenic mechanism in asbestos associated fibrotic (asbestosis) and malignant lung diseases. The characterization of molecular pathways contributing to EMT may provide new possibilities for prognostic and therapeutic applications. The role of asbestos as an inducer of EMT has not been previously characterized. We exposed cultured human lung epithelial cells to crocidolite asbestos and analyzed alterations in the expression of epithelial and mesenchymal marker proteins and cell morphology. Asbestos was found to induce downregulation of E-cadherin protein levels in A549 lung carcinoma cells in 2-dimensional (2D) and 3D cultures. Similar findings were made in primary small airway epithelial cells cultured in 3D conditions where the cells retained alveolar type II cell phenotype. A549 cells also exhibited loss of cell-cell contacts, actin reorganization and expression of α-smooth muscle actin (α-SMA) in 2D cultures. These phenotypic changes were not associated with increased transforming growth factor (TGF)-β signaling activity. MAPK/Erk signaling pathway was found to mediate asbestos-induced downregulation of E-cadherin and alterations in cell morphology. Our results suggest that asbestos can induce epithelial plasticity, which can be interfered by blocking the MAPK/Erk kinase activity.     

High affinity receptors for bombesin/GRP-like peptides on human small cell lung cancer

The binding of a radiolabeled bombesin analogue to human small cell lung cancer (SCLC) cell lines was investigated. (125I-Tyr4)bombesin bound with high affinity (Kd = 0.5 nM) to a single class of sites (2,000/cell) using SCLC line NCI-H446. Binding was reversible, saturable and specific. The pharmacology of binding was investigated using NCI-H466 and SCLC line NCI-H345. Bombesin and structurally related peptides, such as gastrin releasing peptide (GRP), but not other peptides, such as substance P or vasopressin, inhibited high affinity (125I-Tyr4)BN binding activity. Finally, the putative receptor, a 78,000 dalton polypeptide, was identified by purifying radiolabeled cell lysates on bombesin or GRP affinity resins and then displaying the bound polypeptides on sodium dodecylsulfate polyacrylamide gels. Because SCLC both produces bombesin/GRP-like peptides and contains high affinity receptors for these peptides, they may function as important autocrine regulatory factors for human SCLC.     

Gut hormones with activity to modulate cellular growth

Recent progress in cancer research revealed that gut hormones have the activity to regulate the cellular growth of cancer cells. Gastrin, cholecystokinin and vasoactive intestinal peptide were demonstrated to stimulate the growth of gastric cancer cells, pancreatic cancer cells and colon cancer cells, respectively. Accordingly, it is possible to assume that these gut hormones may play an important role in the progression of these cancers. Further studies will be required to clarify the role of gut hormones as physiological growth factors in gastrointestinal tissues. The other aspect of gut hormones related with cellular growth is their role as autocrine growth factors. Gastrin-releasing peptide (GRP) is classified as a gut hormone with the structural similarity with amphibian bombesin. Several reported findings indicate that GRP functions as an autocrine growth factor for human small cell lung carcinoma; a monoclonal antibody for GRP is now applied for the therapy of this cancer. It is important to find out other gut hormones functioning as autocrine growth factors.     

Loss of Rab27 function results in abnormal lung epithelium structure in mice

Rab27 small GTPases regulate secretion and movement of lysosome-related organelles such as T cell cytolytic granules and platelet-dense granules. Previous studies indicated that Rab27a and Rab27b are expressed in the murine lung suggesting that they regulate secretory processes in the lung. Consistent with those studies, we found that Rab27a and Rab27b are expressed in cell types that contain secretory granules: alveolar epithelial type II (AEII) and Clara cells. We then used Rab27a/Rab27b double knockout (DKO) mice to examine the functional consequence of loss of Rab27 proteins in the murine lung. Light and electron microscopy revealed a number of morphological changes in lungs from DKO mice when compared with those in control animals. In aged DKO mice we observed atrophy of the bronchiolar and alveolar epithelium with reduction of cells numbers, thinning of the bronchiolar epithelium and alveolar walls, and enlargement of alveolar airspaces. In these samples we also observed increased numbers of activated foamy alveolar macrophages and granulocyte containing infiltrates together with reduction in the numbers of Clara cells and AEII cells compared with control. At the ultrastructural level we observed accumulation of cytoplasmic membranes and vesicles in Clara cells. Meanwhile, AEII cells in DKO accumulated large mature lamellar bodies and lacked immature/precursor lamellar bodies. We hypothesize that the morphological changes observed at the ultrastructural level in DKO samples result from secretory defects in AEII and Clara cells and that over time these defects lead to atrophy of the epithelium.