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106 hormones is most striking at the carboxy-terminus, which is the portion of the molecule necessary and suffi- cient for binding to high affinity cell surface receptors mediating cellular response. GRP is found normally in periphearl nerves of the gut lining and in pulmonary endocrine cells, as well as in lung carcinoid tumors and human small cell lung cancer (SCLC). GRP has been shown to stimulate the growth of normal human bronchial epithelial cells, mouse Swiss 3T3 fibroblast, and some human small cell lung carcinoma cells growing in culture. As GRP has been demonstrated to be synthesized and secreted in some SCLC cells and is also mitogenic for SCLC, it may function as an autocrine growth factor contibuting to the transformed growth properties of this tumor. Molecular analysis of prepro GRP cDNA clones obtained from several human SCLC cell lines demonstrates at least three forms of mature mRNA are synthesized. The trans- lation product of all three forms would make a signal sequence as well as GRP; they differ in the region encoding a GRP associated carboxy-extension pep- tide synthesized in addition to GRP from the prepro GRP mRNA. The dif- ference between the three forms are a consequence of alternative RNA process- ing of the primary transcript from a single human GRP gene. Antisera specific for the differing forms of the GRP associated DeDtide showed sig- nificant quantities in pulmonary en- docrine cells of neonatal and develop- ing lung, as well as in SCLC. These results indicate that the production of multiple peptides from this gene occurs in both normal and malignant tissue. Chromosomal mapping experiments have localized the prepro GRP gene to 18q21. Nucleotide sequence comparison between the human and the rat prepro GRP gene predicts significant conservation be- tween two mammalian species in pre- dicted amino acid sequences for both GRP and the GRP associated peptides, consistent with their serving a biologically significant role. The growth promoting effects of a constitu- tively expressed prepro GRP gene are currently being assessed by transfect- ing GRP gene constructs into a variety of cultured cell hosts. EFFECTS OF PEPTIDE HORMONES ON GROWTH OF SMALL CELL PULMONARY CARCINOMA. Bepler, G., Jaques, G., Rotsch, M., Haeder, M., Heymanns, J., Philipps- University Medical Center, Division of Hematology and Oncology, 3550 Marburg, West Germany. Small cell pulmonary carcinoma (SCPC) expresses a broad range of dif- ferentiation in vitro. Depending on the expression of various markers, SCPC can be subclassified into three types. The classic cell type is defined by high activities of levodopa-decarboxylase, slow growth as tight suspended spheroids, typical cytological and his- tological features of the intermediate cell type according to the WHO class- ification of SCPC, and no c-myc expression. The transitional cell type is defined by low activities of levo- dopa-decarboxylase, moderate growth as loose suspended aggregates, cytological and histological features of the inter- mediate cell type with some large cell- like featuers, and c-myc overexpression. The variant cell type is defined by no dopa-decarboxylase activity, fast growth with sheet and chain formation in suspension, cytological and histological large cell-like features, and c-myc overexpression. We have investigated the production of sixteen different peptide hormones and growth factors in four classic, four transitional, and six variant SCPC cell lines. Bombesin, neurotensin, calcitonin, calcitonin gene related peptide, somatostatin, growth hormone releasing factor, neurokinin A, glucagon, insulin, and ciliary neuronotrophic factor were detectable in some cell lines which predominantly belonged to the classic and transitional cell types of SCPC. We could not detect arginine-vasopressin, growth hormone, substance P, vasoactive intestinal peptide, neuropeptide Y, and nerve growth factor. Insulin binding sites were present on all cell lines investigated, and some cell lines specifically bound bombesin, calcitonin, and epidermal growth factor. Growth effects were detectable for insulin, bombesin related peptides, and tachykinins. Using serum-free conditions, insulin had a peak growth stimulating effect in liquid culture at a concentration of i0 nM in the presence of transferrin. Bombesin and neuromedin A stimulated the clonal growth at a concentration of i0 nM, and a substance P analogue, which acts as a bombesin receptor antagonist, was able to antagonize this stimulation at 0.i - 1.0 uM concentrations. The tachykinins neurokinin A, neurokinin B, physalaemin, and eledoisin inhibited the clonal growth with a peak effect in the range of 0.1 - i0 pM. Peptide in- duced stimulating and inhibiting ef- fects were within a magnitude of two- fold. All other peptide hormones and growth factors tested, including neurotensin, calcitonin, somatostatin, glucagon, ciliary neuronotrophic factor, nerve growth factor, epidermal growth factor, arginine-vasopression, and adrenocorticotropic hormone did not effect the growth of SCPC. We conclude that SCPC growth is partly controlled by such peptides.

Effects of peptide hormones on growth of small cell pulmonary carcinoma

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hormones is most striking at the carboxy-terminus, which is the portion of the molecule necessary and suffi- cient for binding to high affinity cell surface receptors mediating cellular response. GRP is found normally in periphearl nerves of the gut lining and in pulmonary endocrine cells, as well as in lung carcinoid tumors and human small cell lung cancer (SCLC). GRP has been shown to stimulate the growth of normal human bronchial epithelial cells, mouse Swiss 3T3 fibroblast, and some human small cell lung carcinoma cells growing in culture. As GRP has been demonstrated to be synthesized and secreted in some SCLC cells and is also mitogenic for SCLC, it may function as an autocrine growth factor contibuting to the transformed growth properties of this tumor. Molecular analysis of prepro GRP cDNA clones obtained from several human SCLC cell lines demonstrates at least three forms of mature mRNA are synthesized. The trans- lation product of all three forms would make a signal sequence as well as GRP; they differ in the region encoding a GRP associated carboxy-extension pep- tide synthesized in addition to GRP from the prepro GRP mRNA. The dif- ference between the three forms are a consequence of alternative RNA process- ing of the primary transcript from a single human GRP gene. Antisera specific for the differing forms of the GRP associated DeDtide showed sig- nificant quantities in pulmonary en- docrine cells of neonatal and develop- ing lung, as well as in SCLC. These results indicate that the production of multiple peptides from this gene occurs in both normal and malignant tissue. Chromosomal mapping experiments have localized the prepro GRP gene to 18q21. Nucleotide sequence comparison between the human and the rat prepro GRP gene predicts significant conservation be- tween two mammalian species in pre- dicted amino acid sequences for both GRP and the GRP associated peptides, consistent with their serving a biologically significant role. The growth promoting effects of a constitu- tively expressed prepro GRP gene are currently being assessed by transfect- ing GRP gene constructs into a variety of cultured cell hosts.

EFFECTS OF PEPTIDE HORMONES ON GROWTH OF SMALL CELL PULMONARY CARCINOMA. Bepler, G., Jaques, G., Rotsch, M., Haeder, M., Heymanns, J., Philipps- University Medical Center, Division of Hematology and Oncology, 3550 Marburg, West Germany.

Small cell pulmonary carcinoma (SCPC) expresses a broad range of dif- ferentiation in vitro. Depending on the expression of various markers, SCPC can be subclassified into three types. The

classic cell type is defined by high activities of levodopa-decarboxylase, slow growth as tight suspended spheroids, typical cytological and his- tological features of the intermediate cell type according to the WHO class- ification of SCPC, and no c-myc expression. The transitional cell type is defined by low activities of levo- dopa-decarboxylase, moderate growth as loose suspended aggregates, cytological and histological features of the inter- mediate cell type with some large cell- like featuers, and c-myc overexpression. The variant cell type is defined by no dopa-decarboxylase activity, fast growth with sheet and chain formation in suspension, cytological and histological large cell-like features, and c-myc overexpression. We have investigated the production of sixteen different peptide hormones and growth factors in four classic, four transitional, and six variant SCPC cell lines. Bombesin, neurotensin, calcitonin, calcitonin gene related peptide, somatostatin, growth hormone releasing factor, neurokinin A, glucagon, insulin, and ciliary neuronotrophic factor were detectable in some cell lines which predominantly belonged to the classic and transitional cell types of SCPC. We could not detect arginine-vasopressin, growth hormone, substance P, vasoactive intestinal peptide, neuropeptide Y, and nerve growth factor. Insulin binding sites were present on all cell lines investigated, and some cell lines specifically bound bombesin, calcitonin, and epidermal growth factor. Growth effects were detectable for insulin, bombesin related peptides, and tachykinins. Using serum-free conditions, insulin had a peak growth stimulating effect in liquid culture at a concentration of i0 nM in the presence of transferrin. Bombesin and neuromedin A stimulated the clonal growth at a concentration of i0 nM, and a substance P analogue, which acts as a bombesin receptor antagonist, was able to antagonize this stimulation at 0.i - 1.0 uM concentrations. The tachykinins neurokinin A, neurokinin B, physalaemin, and eledoisin inhibited the clonal growth with a peak effect in the range of 0.1 - i0 pM. Peptide in- duced stimulating and inhibiting ef- fects were within a magnitude of two- fold. All other peptide hormones and growth factors tested, including neurotensin, calcitonin, somatostatin, glucagon, ciliary neuronotrophic factor, nerve growth factor, epidermal growth factor, arginine-vasopression, and adrenocorticotropic hormone did not effect the growth of SCPC. We conclude that SCPC growth is partly controlled by such peptides.