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Role for membrane and secreted insulin-like growth factor-binding protein-2 in the regulation of insulin-like growth factor action in lung tumors
Abstracts /Lung Cancer 11 (1994) 123-150
Small-cell lung carcinoma cells express different plasma membrane nicotinic acetylcholine receptor subtypes. We have now found that interacting with these receptors (-)-nicotine induces a dose-dependent and stereoselective release of rH]serotonin which is dependent on external calcium and blocked by the specific ganglionic nicotinic antagonist mecam ylamine. Withthesamepotency (-)-nicotinestimulates tumor cell proliferation, an effect also blocked by mecamylamine. Serotonin itself stimulates cell proliferation in a dose-dependent manner, an effect blocked by the selective semtonergic receptor antagonists methiotepine and metergolioe. Tbeae data suggest that nicotine might affect proliferation of small-cell lung carcinoma cells by inducing the release of hormone$ (such as semtonin) with autocrine capabilities and place both the nicotinic and the serotonergic receptors at key positions in the biological and, possibly, pharmacological approach to this human lung cancer.
Transfer and expression of the human interleukin-4 gene in carcinoma and stromal cell lines derived from lung cancer patients Hunt JD, Pippin BA, Landreneau RJ, Jacob WF, Lotze MT, Siegfried JM. Department ofPhartnawlogy,BiomedicalScienceTower,Pittsburgh Univ. School of Medicine, Pittsburgh, PA 15261. J Immunother 1993;14:314-21. Introduction of the interleukin-4 (IL-t) gene into cells derived from human tumor tissue provides a meam for generating a specific tumor vaccine. Such a vaccine could be produced by either transducing tumorderived stromal cells with the IL-4 vector and coinjecting tumor cells, or by transducing the tumor cells themselves. We have developed a protocol for culturing cells from non-small cell lung tumors and routinely produce tumor cultures from 25% of tumors, and stmmal cultures from >80% of specimens. Several of these cultures were transduced with the incompetent retmviral vector GlNaSvi4.25, which encodes the human IL-4 cDNA and the G418-resistance gene. Infection of cells by viral titers of 2-5 x 10’ plaque-forming units/ml, and a multiplicity of infection of 0.1: 1 to I : 1 yielded transfer efficiencies of 3.3-32.0 transfectants per 10’cells in six of eight attempts. Following selection with the neomycin analog 0418, IL-4-producing cells were isolated. IL4 titers ranged from 142 to 593 U/ml/lo6 in a 24-h collection. Successfid transfer of the IL-4 gene was demonstrated by polymerase chain reaction amplification of cDNA derived from reversetranscribed total RNA, by immunohistochemistry, and by enxymelinked immunosorbent assay. The IL-4- producing cells were shown to stimulate the proliferation of autologous peripheral blood lymphocytes in one individual by 7.5-fold over control and by 4. l-fold over non-IL4 producing tumor cells. Gene transfer was performed between 18 and 60 days after acquisition for stmmal cells and within 150 days for tumor cells. Cells from lung cancer patients may have potential for generating tumor vaccines. In addition, use of lung tumor-derived stmmal cells for transfection may have some advantages over dermal fibmblasts for use in gene therapy.
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by a wide range of tumors. In particular, IGFBP-2 is frequently produced by human tumor cells, suggesting that this protein may be an important determinant of IGF action in tumors. In the present study, we investigated IGFBP-2 effects in lung tumor cells by examining the influence of IGFBP-2 on IGF-receptor interaction and the biological actionsofIGF-IandIGF-II. Affinitycms&nkingstudieademonstrated expression of type-1 and type-11 IGF receptors on small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC) cells and the presence of abundant membrane-associated IGFBP in SCLC cells but not in NSCLC cells. An antiserum specific for IGFBP-2 was used in immunoprecipitation and immunoblotting studies which demonstrated that the membrane-associated IGFBP identified by affinity crosslinking in SCLC cells is IGFBP-2. In NSCLC cells, both IGF-I and IGFII bound predominantly to IGF-I receptors, whereas in SCLC cells binding was principally to surface-associated IGFBP-2. SCLC cells failed to respond to IGF-I and -II stimulation in a DNA synthesis assay. For NSCLC cells, IGF-II was a more potent stimulator of DNA synthesis than IGF-I. SolubleIGFBP-2 inhibited the bindiig ofradiolabeled IGF-I and - II to both SCLC and NSCLC cells in a concentrationdependent manner and inhibited IGF-stimulated DNA synthesis in NSCLC cells. These observations indicate that both soluble and membrane-associated IGFBP-2 compete with IGF receptors for ligand binding and, thus, are likely to be important determinants of IGF responsiveness. The findings of the present study also indicate that the type-1 receptor on NSCLC cells contains a high-affinity binding site for IGF-II which presumably mediates the biological effects of IGF-II in these cells, thereby implicating IGF-II in the autocrine/paracrine growth of NSCLC.
Differential expression of platelet-derived endothelial cell growth factorlthymidine phosphorylase in human lung carcinoma eelI lines. Heldii N-E,Usuki K, Bergh I, Westermark B, Heldin C-H. Department of Pathology, University Hospital, S-751 85 Vppsala. Br J Cancer 1993;68:708-I 1. In the present investigation we have studied the expression of plateletderived endothelial cell gmwth factorlthymidine phosphorylase (PD-ECGFfTP) in ten different human lung carcinoma cell lines, four small cell carcinomas and six non-small cell carcinomas. None of the small-cell lung carcinoma cell lines demonstrated expression of PDECGF/TP mRNA. However, four ofsix of thenon-small cell carcinoma cell lines expressed the 1.8 kb PD-ECGF/TP transcript. The cell lines derived from the single squamous cell carcinoma and the two adenocarcinomas expressed the PD-ECGFiTP mRNA, and were found to have the corresponding protein both in cell lysates and conditioned media as determined both by immunoblotting and measurement of thymidine phosphorylase activity. Only one of three studied large cell carcinoma cell lines expressed low levels of PD-ECGF/TP mRNA, but the corresponding PD-ECGF/TP protein was not demonstrated by immunoblotting.
Role for membrane and secreted insulin-like growth factorbinding protein-2 in the regulation of insulin-like growth factor action in lung tumors
High levels of nm23-Hl and m&3-R2 messenger RNA in human squamous-cell lung carcinoma are associated with poor differentiation and advanced tumor stages
Reeve JG, Morgan J, Schwander J, Bleehen NM. MRC Clin. Onwl./ Radiotherap. Unit, Hills Road, Cambridge CB2 2QH. Cancer Res 1993;53:4680-5. The insulin-like growth factors (IGFs) have been implicated in the auto&m and/or paracrine gmwth of a number of tumor typea, including lung tumors. Importantly, insulin-like gmwtb factor-binding proteins (IGFBPs), which both enhance and inhibit tbe physiological and biological actions of the IGFs, have been shown to be secreted in vitro
Engel M, Theisinger B, Seib T, Seitz G, Huwer H, Zang KD et al. Instituteof Human Genetics, Universiry of Saar, Building 68, D-66421 Homburg/Soar. Int J Cancer 1993;55:375-9. Expression of the candidate metastasis-suppressor gene nm23-Hl has been shown to correlate inversely with metastatic potential in some human tumors, but not in all. Until now, few studies have been carried out on the activity of the homologous nm23-H2 gene in human cancer. No nm23 transcription studies exist for human lung cancer so far. To
Small-cell lung carcinoma cells express different plasma membrane nicotinic acetylcholine receptor subtypes. We have now found that interacting with these receptors (-)-nicotine induces a dose-dependent and stereoselective release of rH]serotonin which is dependent on external calcium and blocked by the specific ganglionic nicotinic antagonist mecam ylamine. Withthesamepotency (-)-nicotinestimulates tumor cell proliferation, an effect also blocked by mecamylamine. Serotonin itself stimulates cell proliferation in a dose-dependent manner, an effect blocked by the selective semtonergic receptor antagonists methiotepine and metergolioe. Tbeae data suggest that nicotine might affect proliferation of small-cell lung carcinoma cells by inducing the release of hormone$ (such as semtonin) with autocrine capabilities and place both the nicotinic and the serotonergic receptors at key positions in the biological and, possibly, pharmacological approach to this human lung cancer.
Transfer and expression of the human interleukin-4 gene in carcinoma and stromal cell lines derived from lung cancer patients Hunt JD, Pippin BA, Landreneau RJ, Jacob WF, Lotze MT, Siegfried JM. Department ofPhartnawlogy,BiomedicalScienceTower,Pittsburgh Univ. School of Medicine, Pittsburgh, PA 15261. J Immunother 1993;14:314-21. Introduction of the interleukin-4 (IL-t) gene into cells derived from human tumor tissue provides a meam for generating a specific tumor vaccine. Such a vaccine could be produced by either transducing tumorderived stromal cells with the IL-4 vector and coinjecting tumor cells, or by transducing the tumor cells themselves. We have developed a protocol for culturing cells from non-small cell lung tumors and routinely produce tumor cultures from 25% of tumors, and stmmal cultures from >80% of specimens. Several of these cultures were transduced with the incompetent retmviral vector GlNaSvi4.25, which encodes the human IL-4 cDNA and the G418-resistance gene. Infection of cells by viral titers of 2-5 x 10’ plaque-forming units/ml, and a multiplicity of infection of 0.1: 1 to I : 1 yielded transfer efficiencies of 3.3-32.0 transfectants per 10’cells in six of eight attempts. Following selection with the neomycin analog 0418, IL-4-producing cells were isolated. IL4 titers ranged from 142 to 593 U/ml/lo6 in a 24-h collection. Successfid transfer of the IL-4 gene was demonstrated by polymerase chain reaction amplification of cDNA derived from reversetranscribed total RNA, by immunohistochemistry, and by enxymelinked immunosorbent assay. The IL-4- producing cells were shown to stimulate the proliferation of autologous peripheral blood lymphocytes in one individual by 7.5-fold over control and by 4. l-fold over non-IL4 producing tumor cells. Gene transfer was performed between 18 and 60 days after acquisition for stmmal cells and within 150 days for tumor cells. Cells from lung cancer patients may have potential for generating tumor vaccines. In addition, use of lung tumor-derived stmmal cells for transfection may have some advantages over dermal fibmblasts for use in gene therapy.
129
by a wide range of tumors. In particular, IGFBP-2 is frequently produced by human tumor cells, suggesting that this protein may be an important determinant of IGF action in tumors. In the present study, we investigated IGFBP-2 effects in lung tumor cells by examining the influence of IGFBP-2 on IGF-receptor interaction and the biological actionsofIGF-IandIGF-II. Affinitycms&nkingstudieademonstrated expression of type-1 and type-11 IGF receptors on small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC) cells and the presence of abundant membrane-associated IGFBP in SCLC cells but not in NSCLC cells. An antiserum specific for IGFBP-2 was used in immunoprecipitation and immunoblotting studies which demonstrated that the membrane-associated IGFBP identified by affinity crosslinking in SCLC cells is IGFBP-2. In NSCLC cells, both IGF-I and IGFII bound predominantly to IGF-I receptors, whereas in SCLC cells binding was principally to surface-associated IGFBP-2. SCLC cells failed to respond to IGF-I and -II stimulation in a DNA synthesis assay. For NSCLC cells, IGF-II was a more potent stimulator of DNA synthesis than IGF-I. SolubleIGFBP-2 inhibited the bindiig ofradiolabeled IGF-I and - II to both SCLC and NSCLC cells in a concentrationdependent manner and inhibited IGF-stimulated DNA synthesis in NSCLC cells. These observations indicate that both soluble and membrane-associated IGFBP-2 compete with IGF receptors for ligand binding and, thus, are likely to be important determinants of IGF responsiveness. The findings of the present study also indicate that the type-1 receptor on NSCLC cells contains a high-affinity binding site for IGF-II which presumably mediates the biological effects of IGF-II in these cells, thereby implicating IGF-II in the autocrine/paracrine growth of NSCLC.
Differential expression of platelet-derived endothelial cell growth factorlthymidine phosphorylase in human lung carcinoma eelI lines. Heldii N-E,Usuki K, Bergh I, Westermark B, Heldin C-H. Department of Pathology, University Hospital, S-751 85 Vppsala. Br J Cancer 1993;68:708-I 1. In the present investigation we have studied the expression of plateletderived endothelial cell gmwth factorlthymidine phosphorylase (PD-ECGFfTP) in ten different human lung carcinoma cell lines, four small cell carcinomas and six non-small cell carcinomas. None of the small-cell lung carcinoma cell lines demonstrated expression of PDECGF/TP mRNA. However, four ofsix of thenon-small cell carcinoma cell lines expressed the 1.8 kb PD-ECGF/TP transcript. The cell lines derived from the single squamous cell carcinoma and the two adenocarcinomas expressed the PD-ECGFiTP mRNA, and were found to have the corresponding protein both in cell lysates and conditioned media as determined both by immunoblotting and measurement of thymidine phosphorylase activity. Only one of three studied large cell carcinoma cell lines expressed low levels of PD-ECGF/TP mRNA, but the corresponding PD-ECGF/TP protein was not demonstrated by immunoblotting.
Role for membrane and secreted insulin-like growth factorbinding protein-2 in the regulation of insulin-like growth factor action in lung tumors
High levels of nm23-Hl and m&3-R2 messenger RNA in human squamous-cell lung carcinoma are associated with poor differentiation and advanced tumor stages
Reeve JG, Morgan J, Schwander J, Bleehen NM. MRC Clin. Onwl./ Radiotherap. Unit, Hills Road, Cambridge CB2 2QH. Cancer Res 1993;53:4680-5. The insulin-like growth factors (IGFs) have been implicated in the auto&m and/or paracrine gmwth of a number of tumor typea, including lung tumors. Importantly, insulin-like gmwtb factor-binding proteins (IGFBPs), which both enhance and inhibit tbe physiological and biological actions of the IGFs, have been shown to be secreted in vitro
Engel M, Theisinger B, Seib T, Seitz G, Huwer H, Zang KD et al. Instituteof Human Genetics, Universiry of Saar, Building 68, D-66421 Homburg/Soar. Int J Cancer 1993;55:375-9. Expression of the candidate metastasis-suppressor gene nm23-Hl has been shown to correlate inversely with metastatic potential in some human tumors, but not in all. Until now, few studies have been carried out on the activity of the homologous nm23-H2 gene in human cancer. No nm23 transcription studies exist for human lung cancer so far. To