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. 1990 Dec;86(6):1904–1912. doi: 10.1172/JCI114923

Cholera toxin inhibits signal transduction by several mitogens and the in vitro growth of human small-cell lung cancer.

J Viallet 1, Y Sharoni 1, H Frucht 1, R T Jensen 1, J D Minna 1, E A Sausville 1
PMCID: PMC329825  PMID: 2174911

Abstract

Cholera toxin (CT) inhibited the in vitro growth of three of four human small-cell lung carcinoma (SCLC) cell lines with a 50% inhibitory concentration of 27-242 ng/ml. Loss of surface membrane ruffling and the capacity of [Tyr4]-bombesin, vasopressin, and fetal calf serum to stimulate increases in intracellular free calcium clearly preceded effects on cellular metabolic activity and cell growth. 125I-[Tyr4]-bombesin binding was unaffected by CT treatment but [Tyr4]-bombesin stimulated phospholipase C activity was decreased in membranes from CT-treated SCLC cells. CT stimulated a rapid but transient increase in intracellular cyclic AMP ([cAMP]i) in SCLC. The effects of CT on susceptible SCLC were not reproduced by elevations of [cAMP]i induced by forskolin or cyclic AMP analogues. GM1 ganglioside, the cellular binding site for CT, was highly expressed in the CT-sensitive but not the CT-resistant SCLC cell lines. In contrast, expression of guanine nucleotide binding protein substrates for ADP-ribosylation by CT was similar. These data demonstrate the existence of a CT-sensitive growth inhibitory pathway in SCLC-bearing GM1 ganglioside. Addition of CT results in decreased responsiveness to several mitogenic stimuli. These results suggest novel therapeutic approaches to human SCLC.

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Selected References

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  1. Aksamit R. R., Backlund P. S., Jr, Cantoni G. L. Cholera toxin inhibits chemotaxis by a cAMP-independent mechanism. Proc Natl Acad Sci U S A. 1985 Nov;82(22):7475–7479. doi: 10.1073/pnas.82.22.7475. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bar-Sagi D., Feramisco J. R. Induction of membrane ruffling and fluid-phase pinocytosis in quiescent fibroblasts by ras proteins. Science. 1986 Sep 5;233(4768):1061–1068. doi: 10.1126/science.3090687. [DOI] [PubMed] [Google Scholar]
  3. Berridge M. J., Downes C. P., Hanley M. R. Lithium amplifies agonist-dependent phosphatidylinositol responses in brain and salivary glands. Biochem J. 1982 Sep 15;206(3):587–595. doi: 10.1042/bj2060587. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Biffen M., Martin B. R. Polyphosphoinositide labeling in rat liver plasma membranes is reduced by preincubation with cholera toxin. J Biol Chem. 1987 Jun 5;262(16):7744–7750. [PubMed] [Google Scholar]
  5. Bray P., Carter A., Simons C., Guo V., Puckett C., Kamholz J., Spiegel A., Nirenberg M. Human cDNA clones for four species of G alpha s signal transduction protein. Proc Natl Acad Sci U S A. 1986 Dec;83(23):8893–8897. doi: 10.1073/pnas.83.23.8893. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Brunk U., Schellens J., Westermark B. Influence of epidermal growth factor (EGF) on ruffling activity, pinocytosis and proliferation of cultivated human glia cells. Exp Cell Res. 1976 Dec;103(2):295–302. doi: 10.1016/0014-4827(76)90266-4. [DOI] [PubMed] [Google Scholar]
  7. Carmichael J., DeGraff W. G., Gazdar A. F., Minna J. D., Mitchell J. B. Evaluation of a tetrazolium-based semiautomated colorimetric assay: assessment of chemosensitivity testing. Cancer Res. 1987 Feb 15;47(4):936–942. [PubMed] [Google Scholar]
  8. Carney D. N., Cuttitta F., Moody T. W., Minna J. D. Selective stimulation of small cell lung cancer clonal growth by bombesin and gastrin-releasing peptide. Cancer Res. 1987 Feb 1;47(3):821–825. [PubMed] [Google Scholar]
  9. Carney D. N., Gazdar A. F., Bepler G., Guccion J. G., Marangos P. J., Moody T. W., Zweig M. H., Minna J. D. Establishment and identification of small cell lung cancer cell lines having classic and variant features. Cancer Res. 1985 Jun;45(6):2913–2923. [PubMed] [Google Scholar]
  10. Cho-Chung Y. S., Clair T., Shepheard C., Berghoffer B. Arrest of hormone-dependent mammary cancer growth in vivo and in vitro by cholera toxin. Cancer Res. 1983 Apr;43(4):1473–1476. [PubMed] [Google Scholar]
  11. Chopra D. P., Siddiqui K. M., Cooney R. A. Effects of insulin, transferrin, cholera toxin, and epidermal growth factor on growth and morphology of human fetal normal colon epithelial cells. Gastroenterology. 1987 Apr;92(4):891–904. doi: 10.1016/0016-5085(87)90962-0. [DOI] [PubMed] [Google Scholar]
  12. Cuttitta F., Carney D. N., Mulshine J., Moody T. W., Fedorko J., Fischler A., Minna J. D. Bombesin-like peptides can function as autocrine growth factors in human small-cell lung cancer. 1985 Aug 29-Sep 4Nature. 316(6031):823–826. doi: 10.1038/316823a0. [DOI] [PubMed] [Google Scholar]
  13. Dixon S. J., Stewart D., Grinstein S., Spiegel S. Transmembrane signaling by the B subunit of cholera toxin: increased cytoplasmic free calcium in rat lymphocytes. J Cell Biol. 1987 Sep;105(3):1153–1161. doi: 10.1083/jcb.105.3.1153. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Fain J. N., Wallace M. A., Wojcikiewicz R. J. Evidence for involvement of guanine nucleotide-binding regulatory proteins in the activation of phospholipases by hormones. FASEB J. 1988 Jul;2(10):2569–2574. doi: 10.1096/fasebj.2.10.2838362. [DOI] [PubMed] [Google Scholar]
  15. Fischer J. B., Schonbrunn A. The bombesin receptor is coupled to a guanine nucleotide-binding protein which is insensitive to pertussis and cholera toxins. J Biol Chem. 1988 Feb 25;263(6):2808–2816. [PubMed] [Google Scholar]
  16. Gerlier D., Thomasset N. Use of MTT colorimetric assay to measure cell activation. J Immunol Methods. 1986 Nov 20;94(1-2):57–63. doi: 10.1016/0022-1759(86)90215-2. [DOI] [PubMed] [Google Scholar]
  17. Goshima K., Masuda A., Owaribe K. Insulin-induced formation of ruffling membranes of KB cells and its correlation with enhancement of amino acid transport. J Cell Biol. 1984 Mar;98(3):801–809. doi: 10.1083/jcb.98.3.801. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Green H. Cyclic AMP in relation to proliferation of the epidermal cell: a new view. Cell. 1978 Nov;15(3):801–811. doi: 10.1016/0092-8674(78)90265-9. [DOI] [PubMed] [Google Scholar]
  19. Heikkila R., Trepel J. B., Cuttitta F., Neckers L. M., Sausville E. A. Bombesin-related peptides induce calcium mobilization in a subset of human small cell lung cancer cell lines. J Biol Chem. 1987 Dec 5;262(34):16456–16460. [PubMed] [Google Scholar]
  20. Holmgren J., Lange S., Lindholm L., Lönnroth C., Lönnroth I. In vivo modulation of intracellular cAMP and cell growth of a lymphatic tumour in mice by cholera toxin. Exp Cell Res. 1977 Aug;108(1):31–39. doi: 10.1016/s0014-4827(77)80006-2. [DOI] [PubMed] [Google Scholar]
  21. Imboden J. B., Shoback D. M., Pattison G., Stobo J. D. Cholera toxin inhibits the T-cell antigen receptor-mediated increases in inositol trisphosphate and cytoplasmic free calcium. Proc Natl Acad Sci U S A. 1986 Aug;83(15):5673–5677. doi: 10.1073/pnas.83.15.5673. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  23. Lechner J. F., Haugen A., Autrup H., McClendon I. A., Trump B. F., Harris C. C. Clonal growth of epithelial cells from normal adult human bronchus. Cancer Res. 1981 Jun;41(6):2294–2304. [PubMed] [Google Scholar]
  24. Magnani J. L., Smith D. F., Ginsburg V. Detection of gangliosides that bind cholera toxin: direct binding of 125I-labeled toxin to thin-layer chromatograms. Anal Biochem. 1980 Dec;109(2):399–402. doi: 10.1016/0003-2697(80)90667-3. [DOI] [PubMed] [Google Scholar]
  25. Milligan G. Guanine nucleotide regulation of the pertussis and cholera toxin substrates of rat glioma C6 BU1 cells. Biochim Biophys Acta. 1987 Jul 6;929(2):197–202. doi: 10.1016/0167-4889(87)90176-5. [DOI] [PubMed] [Google Scholar]
  26. Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods. 1983 Dec 16;65(1-2):55–63. doi: 10.1016/0022-1759(83)90303-4. [DOI] [PubMed] [Google Scholar]
  27. Moss J., Fishman P. H., Manganiello V. C., Vaughan M., Brady R. O. Functional incorporation of ganglioside into intact cells: induction of choleragen responsiveness. Proc Natl Acad Sci U S A. 1976 Apr;73(4):1034–1037. doi: 10.1073/pnas.73.4.1034. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Moss J., Vaughan M. ADP-ribosylation of guanyl nucleotide-binding regulatory proteins by bacterial toxins. Adv Enzymol Relat Areas Mol Biol. 1988;61:303–379. doi: 10.1002/9780470123072.ch6. [DOI] [PubMed] [Google Scholar]
  29. Nånberg E., Rozengurt E. Temporal relationship between inositol polyphosphate formation and increases in cytosolic Ca2+ in quiescent 3T3 cells stimulated by platelet-derived growth factor, bombesin and vasopressin. EMBO J. 1988 Sep;7(9):2741–2747. doi: 10.1002/j.1460-2075.1988.tb03128.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Rozengurt E. Early signals in the mitogenic response. Science. 1986 Oct 10;234(4773):161–166. doi: 10.1126/science.3018928. [DOI] [PubMed] [Google Scholar]
  31. Sharoni Y., Viallet J., Trepel J. B., Sausville E. A. Effect of guanine and adenine nucleotides on bombesin-stimulated phospholipase C activity in membranes from Swiss 3T3 and small cell lung carcinoma cells. Cancer Res. 1990 Sep 1;50(17):5257–5262. [PubMed] [Google Scholar]
  32. Simonds W. F., Goldsmith P. K., Codina J., Unson C. G., Spiegel A. M. Gi2 mediates alpha 2-adrenergic inhibition of adenylyl cyclase in platelet membranes: in situ identification with G alpha C-terminal antibodies. Proc Natl Acad Sci U S A. 1989 Oct;86(20):7809–7813. doi: 10.1073/pnas.86.20.7809. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Smith C. D., Chang K. J. Regulation of brain phosphatidylinositol-4-phosphate kinase by GTP analogues. A potential role for guanine nucleotide regulatory proteins. J Biol Chem. 1989 Feb 25;264(6):3206–3210. [PubMed] [Google Scholar]
  34. Spiegel S., Fishman P. H. Gangliosides as bimodal regulators of cell growth. Proc Natl Acad Sci U S A. 1987 Jan;84(1):141–145. doi: 10.1073/pnas.84.1.141. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Stewart S. J., Prpic V., Johns J. A., Powers F. S., Graber S. E., Forbes J. T., Exton J. H. Bacterial toxins affect early events of T lymphocyte activation. J Clin Invest. 1989 Jan;83(1):234–242. doi: 10.1172/JCI113865. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Straub R. E., Gershengorn M. C. Thyrotropin-releasing hormone and GTP activate inositol trisphosphate formation in membranes isolated from rat pituitary cells. J Biol Chem. 1986 Feb 25;261(6):2712–2717. [PubMed] [Google Scholar]
  37. Swope S. L., Schonbrunn A. The biphasic stimulation of insulin secretion by bombesin involves both cytosolic free calcium and protein kinase C. Biochem J. 1988 Jul 1;253(1):193–202. doi: 10.1042/bj2530193. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Taylor-Papadimitriou J., Purkis P., Fentiman I. S. Cholera toxin and analogues of cyclic AMP stimulate the growth of cultured human mammary epithelial cells. J Cell Physiol. 1980 Mar;102(3):317–321. doi: 10.1002/jcp.1041020306. [DOI] [PubMed] [Google Scholar]
  39. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Trepel J. B., Moyer J. D., Cuttitta F., Frucht H., Coy D. H., Natale R. B., Mulshine J. L., Jensen R. T., Sausville E. A. A novel bombesin receptor antagonist inhibits autocrine signals in a small cell lung carcinoma cell line. Biochem Biophys Res Commun. 1988 Nov 15;156(3):1383–1389. doi: 10.1016/s0006-291x(88)80785-x. [DOI] [PubMed] [Google Scholar]
  41. Trepel J. B., Moyer J. D., Heikkila R., Sausville E. A. Modulation of bombesin-induced phosphatidylinositol hydrolysis in a small-cell lung-cancer cell line. Biochem J. 1988 Oct 15;255(2):403–410. doi: 10.1042/bj2550403. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Urumow T., Wieland O. H. Evidence for a cholera-toxin-sensitive G-protein involved in the regulation of phosphatidylinositol 4-phosphate kinase of rat liver membranes. Biochim Biophys Acta. 1988 Nov 18;972(2):232–238. doi: 10.1016/0167-4889(88)90121-8. [DOI] [PubMed] [Google Scholar]
  43. Viallet J., Minna J. D. Gastrin-releasing peptide (GRP, mammalian bombesin) in the pathogenesis of lung cancer. Prog Growth Factor Res. 1989;1(2):89–97. doi: 10.1016/0955-2235(89)90003-3. [DOI] [PubMed] [Google Scholar]
  44. Woll P. J., Rozengurt E. [D-Arg1,D-Phe5,D-Trp7,9,Leu11]substance P, a potent bombesin antagonist in murine Swiss 3T3 cells, inhibits the growth of human small cell lung cancer cells in vitro. Proc Natl Acad Sci U S A. 1988 Mar;85(6):1859–1863. doi: 10.1073/pnas.85.6.1859. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Yatani A., Imoto Y., Codina J., Hamilton S. L., Brown A. M., Birnbaumer L. The stimulatory G protein of adenylyl cyclase, Gs, also stimulates dihydropyridine-sensitive Ca2+ channels. Evidence for direct regulation independent of phosphorylation by cAMP-dependent protein kinase or stimulation by a dihydropyridine agonist. J Biol Chem. 1988 Jul 15;263(20):9887–9895. [PubMed] [Google Scholar]

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