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The morphological response of Kc-H cells to ecdysteroids: Hormonal specificity

  • Lucy Cherbas
  • Christopher D. Yonge
  • Peter Cherbas
  • Carroll M. Williams
Article

Summary

Cells of the line Kc, derived fromDrosophila melanogaster embryos, extend long processes when exposed to ecdysteroid hormones. We have devised a quantitative assay for this morphological response, using the subline Kc-H. The assay was used to characterize the conditions required for the response. A halfmaximal response is elicited by approximately 10−8M 20-hydroxyecdysone; the response is saturated by 10−7M 20-hydroxyecdysone, which causes detectable elongation within a few hours, and a maximal response after 2–3 days. The response occurs substantially normally in the absence of serum, during growth in suspension, and in over-crowded cultures. It is not elicited by cyclic nucleotides, vertebrate growth factors, or a variety of other non-ecdysteroid reagents. Of 60 ecdysteroid compounds tested, only those which were active in other insect test systems elicited the response, and the concentrations required were approximately proportional to the concentrations active in other in vitro systems. We conclude that the response of Kc cells to 20-hydroxyecdysone retains basic features of the ecdysteroid response of intact tissues and therefore that Kc cells are a useful model system for studying ecdysteroid action.

Key words

Cell line Drosophila Ecdysone Ecdysterone Hormones 

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References

  1. Agui N (1973) Quantitative bioassay of moulting hormone in vitro. Appl Entomol Zool 8:236–239Google Scholar
  2. Agui N (1976) Attempts to establish insect endocrine system in vitro. In: Maramorosch K (ed) Invertebrate tissue culture. Academic Press, New York, pp 133–160Google Scholar
  3. Agui N, Fukaya M (1973) Effects of moulting hormones and prothoracic glands on the development of wing discs of the cabbage armyworm (Mamestra brassicae L.) in vitro (Lepidoptera: Noctuidae). Appl Entomol Zool 8:73–82Google Scholar
  4. Ashburner M (1971) Induction of puffs in polytene chromosomes of in vitro cultured salivary glands ofDrosophila melanogaster. Nature New Biol 230:222–223Google Scholar
  5. Ashburner M (1973) Sequential gene activation by ecdysone in polytene chromosomes ofDrosophila melanogaster. I. Dependence upon ecdysone concentration. Dev Biol 35:47–61Google Scholar
  6. Ashburner M, Richards G (1976) The role of ecdysone in the control of gene activity in the polytene chromosomes ofDrosophila. In: Lawrence PA (ed) Insect development. Wiley, New York, pp 203–225Google Scholar
  7. Berger E, Frank M, Abell MC (1980) Ecdysone-induced changes in protein synthesis in embryonicDrosophila cells in culture. In: Kurstak E, Maramorosch K, Dübendorfer A (eds) Invertebrate systems in vitro. Elsevier/North Holland, Amsterdam, pp 195–208Google Scholar
  8. Berger E, Ringler R, Alahiotis S, Frank M (1978) Ecdysone-induced changes in morphology and protein synthesis inDrosophila cell cultures. Dev Biol 62:498–511Google Scholar
  9. Best-Belpomme M, Courgeon A-M (1977) Ecdysterone and acetylcholinesterase activity in culturedDrosophila cells. FEBS Lett 82:345–347Google Scholar
  10. Best-Belpomme M, Courgeon A-M, Echalier G (1980) Development of a model for the study of ecdysteroid action:Drosophila melanogaster cells established in vitro. In: Hoffmann J (ed), Progress in ecdysone research. Elsevier/North Holland, Amsterdam, in pressGoogle Scholar
  11. Best-Belpomme M, Courgeon A-M, Rambach A (1978) β-Galactosidase is induced by hormone inDrosophila melanogaster cell cultures. Proc Natl Acad Sci USA 75:6102–6106Google Scholar
  12. Bliss CI (1970) Statistics in biology, vol 2. McGraw-Hill, New York, p 172Google Scholar
  13. Bollenbacher WE, Galbraith MN, Gilbert LI, Horn DHS (1977) In vitro metabolism of 3β-hydroxy-, and 3β, 14α-dihydroxy-[3α-3H]-5β-cholest-7-en-6-one by the prothoracic glands ofManduca sexta. Steroids 29:47–63Google Scholar
  14. Borst DW, Bollenbacher WE, O'Connor JD, King DS, Fristrom JW (1974) Ecdysone levels during metamorphosis ofDrosophila melanogaster. Dev Biol 39:308–316Google Scholar
  15. Boyd M, Ashburner M (1977) The hormonal control of salivary gland secretion inDrosophila melanogaster: Studies in vitro. J Insect Physiol 23:517–523Google Scholar
  16. Bullière F, Bullière D (1977) Régénération, différenciation et hormones de mues chez l'embryon de blatte en culture in vitro. Wilhelm Roux's Archives 182:255–275Google Scholar
  17. Canonica L, Danieli B, Weisza-Vincze I, Ferrari G (1972) Structure of muristerone A, a new phytoecdysone. Chem Commun 1972:1060–1061Google Scholar
  18. Caruelle JP, Cassier P, Joulie-Delorme C (1978) Dose-effect relationships of α- and β-ecdysone on cuticular deposition and cellular alterations of larval epidermis ofSchistocerca gregaria in vitro. Ann Sci Nat Zool Biol Anim 20:53–78Google Scholar
  19. Cherbas L, Cherbas P (1970) Distribution and metabolism of α-ecdysone in pupae of the silkworm,Antheraea polyphemus. Biol Bull 138:115–128Google Scholar
  20. Cherbas L, Cherbas P, Savakis C, Demetri G, Manteuffel-Cymborowska M, Yonge CD, Williams CM (1980) Studies of ecdysteroid action on aDrosophila cell line. In: Kurstak E, Maramorosch K, Dübendorfer A (eds) Invertebrate systems in vitro. Elsevier/North Holland, Amsterdam, pp 217–228Google Scholar
  21. Cherbas P, Cherbas L, Williams CM (1977) Induction of acetylcholinesterase activity by β-ecdysone in aDrosophila cell line. Science 197:275–277Google Scholar
  22. Cherbas P, Cherbas L, Demetri G, Manteuffel-Cymborowska M, Savakis C, Yonge CD, Williams CM (1980) Ecdysteroid hormone effects on aDrosophila cell line. In: Roy AK, Clark JH (eds), Gene regulation by steroid hormones. Springer, Berlin Heidelberg New York, pp 278–308Google Scholar
  23. Chihara CJ, Petri WH, Fristrom JW, King DS (1972) The assay of ecdysones and juvenile hormones onDrosophila imaginal disks in vitro. J Insect Physiol 18:1115–1123Google Scholar
  24. Chong YK, Galbraith MN, Horn DHS (1970) Isolation of deoxycrustecdysone, deoxyecdysone, and α-ecdysone from the fernBlechnum minus. Chem Commun 1970:1217–1218Google Scholar
  25. Courgeon A-M (1972a) Action of insect hormones at the cellular level. Morphological changes of a diploid cell line ofDrosophila melanogaster treated with ecdysone and several analogues in vitro. Exp Cell Res 74:327–336Google Scholar
  26. Courgeon A-M (1972b) Effects of α- and β-ecdysone on in vitro diploid cell multiplication inDrosophila melanogaster. Nature New Biol 238:250–251Google Scholar
  27. Courgeon A-M (1975) Action conjuguée de l'hormone juvénile et de l'ecdysterone sur des lignées cellulaires de Drosophile in vitro. CR Acad Sci Paris Ser D 280:2563–2566Google Scholar
  28. Doane WW (1973) Role of hormones in insect development. In: Counce SJ, Waddington CH (eds), Developmental systems: Insects, vol 2. Academic Press, New York, pp 291–497Google Scholar
  29. Dübendorfer A, Shields G, Sang JH (1975) Development and differentiation in vitro ofDrosophila imaginal disc cells from dissociated early embryos. J Embryol Exp Morphol 33:487–498Google Scholar
  30. Fain MJ, Riddiford LM (1977) Requirements for molting of the crochet epidermis of the tobacco hornworm larva in vivo and in vitro. Wilhelm Roux's Archives 181:285–307Google Scholar
  31. Fristrom JH, Yund MA (1976) Characteristics of the action of ecdysones onDrosophila imaginal discs cultured in vitro. In: Maramorosch K (ed) Invertebrate tissue culture, Academic Press, New York, pp 161–178Google Scholar
  32. Fukushima T, Yagi S (1975) Hormonal effect on cultivated insect tissues. III. Effects of α- and β-ecdysones or prothoracic glands on spermiogenesis in two noctuid moths in vitro (Lepidoptera: Noctuidae). Appl Entomol Zool 10:220–225Google Scholar
  33. Galbraith MN, Horn DHS, Middleton EJ, Thomson JA (1973) Biological activity of synthetic molting hormone analogues in the blowflyCalliphora stygia. Experientia 29:19Google Scholar
  34. Galbraith MN, Horn DHS, Porter QN, Hackney RJ (1968) Structure of podecdysone A, a steroid with moulting hormone activity from the bark ofPodocarpus elatus R. Br. Chem Commun 1968:971–972Google Scholar
  35. Goodwin TW, Horn DHS, Karlson P, Koolman J, Nakanishi K, Robbins WE, Siddall JB, Takemoto T (1978) Ecdysteroids: A new generic term. Nature 272:122Google Scholar
  36. Gvozdev VA, Kakpakov VT, Mukhovatova LM, Polukarova LG, Tarantul VZ (1974) Effect of ecdysterone on cell growth and macromolecular synthesis in established embryonic cell lines ofDrosophila melanogaster. Ontogenez 5:33–42Google Scholar
  37. Hetru C, Lagueux M, Bang L, Hoffmann JA (1978) Adult ovaries ofLocusta migratoria contain the sequence of biosynthetic intermediates for ecdysone. Life Sci. 22:2141–2154Google Scholar
  38. Hikino H, Hikino Y (1970) Arthropod molting hormones. Fortschr Chem Org Naturst 28:256–312Google Scholar
  39. Hikino H, Hikino Y, Nomoto K, Takemoto T (1968) Cyasterone, an insect metamorphosing substance fromCyathula capitata: Structure. Tetrahedron 24:4895–4906Google Scholar
  40. Hodgetts RB, Sage B, O'Connor JD (1977) Ecdysone titers during postembryonic development ofDrosophila melanogaster. Dev Biol 60:310–317Google Scholar
  41. Hoffmeister F, Nakanishi K, Koreeda M, Hsu HY (1968) The moulting hormone activity of ponasterones in theCalliphora test. J Insect Physiol 14:53–54Google Scholar
  42. Horn DHS (1971) The ecdysones. In: Jacobson M, Crosby DG (eds) Naturally occurring insecticides. Dekker, New York, pp 333–459Google Scholar
  43. Imai S, Hori M, Fujioka S, Murata E, Goto M, Nakanishi K (1968a) Isolation of four new phytoecdysones, makisterone A, B, C, D, and the structure of makisterone A, a C28 steroid. Tetrahedron Lett 1968:3883–3886Google Scholar
  44. Imai S, Fujioka S, Murata E, Sasakawa Y, Nakanishi K (1968b) The structures of three additional phytoecdysones fromPodocarpus macrophyllus, makisterone B, C, and D. Tetrahedron Lett 1968:3887–3890Google Scholar
  45. Imai S, Toyosato T, Sakai M, Sato Y, Fujioka S, Murata E, Goto M (1969) Isolation of cyasterone and ecdysterone from plant materials. Chem Pharm Bull Jpn 17:340–342Google Scholar
  46. Jizba J, Herout V, Šorm F (1967) Polypodine B—a novel ecdysonelike substance from plant material. Tetrahedron Lett 1967:5139–5143Google Scholar
  47. Kimura S, Kobayashi M (1975) Prothoracicotropic action of ecdysone analogues inBombyx mori. J Insect Physiol 21:417–421Google Scholar
  48. King DS, Siddall JB (1969) Conversion of α-ecdysone to β-ecdysone by crustaceans and insects. Nature 221:955–956Google Scholar
  49. Kuroda Y (1969) Growth and differentiation of embryonic cells ofDrosophila melanogaster in vitro. Jpn J Genet [Suppl 1] 44:42–50Google Scholar
  50. Landureau JC (1976) Insect cell and tissue culture as a tool for developmental biology. In: Kurstak E, Maramorosch K (eds), Invertebrate tissue culture. Academic Press, New York, pp 101–130Google Scholar
  51. Laverdure A-M (1976) Recherche des conditions nécéssaires à l'évolution de l'ovaire de la nymphe deTenebrio molitor. Etude en culture in vitro. Bull Soc Zool Fr [Suppl 2] 101:8–11Google Scholar
  52. Lee E, Liu Y-T, Solomon PH, Nakanishi K (1976) Stereospecific conversion of diosgenin to α-ecdysone. J Am Chem Soc 83:1634–1635Google Scholar
  53. Marks EP (1973) Deposition of insect cuticle in vitro: Differential responses to α- and β-ecdysone. Gen Comp Endocrinol 21:472–477Google Scholar
  54. Maroy P, Dennis R, Beckers C, Sage BA, O'Connor JD (1978) Demonstration of an ecdysteroid receptor in a cultured cell line ofDrosophila melanogaster. Proc Natl Acad Sci USA 75:6035–6038Google Scholar
  55. Milner MJ, Haynie JL (1979) Fusion ofDrosophila eye-antennal imaginal discs during differentiation in vitro. Wilhelm Roux's Archives 185:363–370Google Scholar
  56. Milner MJ, Sang JH (1974) Relative activities of α-ecdysone and β-ecdysone for the differentiation in vitro ofDrosophila melanogaster imaginal discs. Cell 3:141–143Google Scholar
  57. Mitsuhashi J, Grace TDC (1970) The effects of insect hormones on the multiplication rates of cultured insect cells in vitro. Appl Entomol Zool 5:182–188Google Scholar
  58. Mitsui T, Riddiford LM (1976) Pupal cuticle formation byManduca sexta epidermis in vitro: Patterns of ecdysone sensitivity. Dev Biol 54:172–186Google Scholar
  59. Morisaki M, Awata N, Fujimoto Y, Ikekawa N (1975) Steroidal allenes as inhibitors of sterol biosynthesis. Chem Commun 1975:362–363Google Scholar
  60. Moriyama H, Nakanishi K, King DS, Okauchi T, Siddall JB, Hafferl W (1970) On the origin and metabolic fate of α-ecdysone in insects. Gen Comp Endocrinol 15:80–87Google Scholar
  61. Mukhovatova LM, Kakpakov VT (1975) Effect of α- and β-ecdysone on the differentiation ofDrosophila melanogaster imaginal discs cultivated in vitro. Ontogenez 6:80–87Google Scholar
  62. Oberlander H (1969) Effects of ecdysone, ecdysterone, and inokosterone on the in vitro initiation of metamorphosis of wing disks ofGalleria mellonella. J Insect Physiol 15:297–304Google Scholar
  63. Oberlander H (1976) Dissociation and reaggregation of fat body cells during insect metamorphosis. In: Kurstak E, Maramorosch K (eds) Invertebrate tissue culture. Academic Press, New York, pp 241–246Google Scholar
  64. Oberlander H, Ferkovich SM, Van Essen F, Leach CE (1978) Chitin biosynthesis in imaginal discs cultured in vitro. Wilhelm Roux's Archives 185:95–98Google Scholar
  65. Oberlander H, Leach CE (1975) Uptake and incorporation of D-glucosamine-6-3H into imaginal disks: Effects of alpha-ecdysone and beta-ecdysone in vitro. Insect Biochem 5:235–242Google Scholar
  66. Oberlander H, Leach CE, Tomblin C (1973) Cuticle deposition in imaginal disks of three species of Lepidoptera: Effects of ecdysones in vitro. J Insect Physiol 19:993–998Google Scholar
  67. O'Connor JD, Maroy P, Beckers C, Dennis R, Alvarez CM, Sage BA (1980) Ecdysteroid receptors in culturedDrosophila cells. In: Roy AK, Clark JH (eds) Gene regulation by steroid hormones. Springer, Berlin Heidelberg New York, pp 263–277Google Scholar
  68. Ohmori K (1974) Effect of ecdysterone contact period on the development of wing disks of the fleshfly,Sarcophaga peregrina, in vitro. J Insect Physiol 20:1697–1706Google Scholar
  69. Ohmori K, Ohtaki T (1973) Effects of ecdysone analogues on development and metabolic activity of wing disks of the fleshfly,Sarcophaga peregrina, in vitro. J Insect Physiol 19:1199–1210Google Scholar
  70. Ohtaki T, Milkman RD, Williams CM (1967) Ecdysone and ecdysone analogues: Their assay on the fleshflySarcophaga peregrina. Proc Natl Acad Sci USA 58:981–984Google Scholar
  71. Richards G (1978) Sequential gene activation by ecdysone in polytene chromosomes ofDrosophila melanogaster. VI. Inhibition by juvenile hormone. Dev Biol 66:32–42Google Scholar
  72. Riddiford LM (1976) In vitro action of ecdysone and juvenile hormone on epidermal commitment in the tobacco hornworm. In: Kurstak E, Maramorosch K (eds) Invertebrate tissue culture. Academic Press, New York, pp 213–222Google Scholar
  73. Riddiford LM (1978) Ecdysone-induced change in cellular commitment of the epidermis of the tobacco hornworm,Manduca sexta, at the initiation of metamorphosis. Gen Comp. Endocrinol 34:438–446Google Scholar
  74. Rimpler H (1969) Pterosteron, Polypodin B, und ein neues ecdysonartiges Steroid (Viticosteron E) ausVitex megapotamica (Verbenaceae). Tetrahedron Lett 1969:329–333Google Scholar
  75. Robb JA (1969) Maintenance of imaginal discs ofDrosophila melanogaster in chemically defined media. J Cell Biol 41:876–885Google Scholar
  76. Robbins WE, Kaplanis JN, Thompson MJ, Shortino TJ, Cohen CF, Joyner SC (1968) Ecdysones and analogs: Effects on development and reproduction in insects. Science 161:1158–1160Google Scholar
  77. Robbins WE, Kaplanis JN, Thompson MJ, Shortino TJ, Joyner SC (1970) Ecdysones and synthetic analogs: Molting hormone activity and inhibitive effects on insect growth, metamorphosis, and reproduction. Steroids 16:105–125Google Scholar
  78. Robertson J, Pipa R (1973) Metamorphic shortening of interganglionic connectives ofGalleria mellonella (Lepidoptera) in vitro: Stimulation by ecdysone analogs. J Insect Physiol 19:673–679Google Scholar
  79. Rosset R (1978) Effects of ecdysone on aDrosophila cell line. Exp Cell Res 111:31–36Google Scholar
  80. Sláma K, Romanuk M, Šorm F (1974) Insect hormones and bioanalogues. Springer, Berlin Heidelberg New YorkGoogle Scholar
  81. Solomon F (1979) Detailed neurite morphologies of sister neuroblastoma cells are related. Cell 16:165–169Google Scholar
  82. Svoboda JA, Thompson MJ, Robbins WE, Kaplanis JN (1978) Insect steroid metabolism. Lipids 13:742–753Google Scholar
  83. Szöllösi A, Landureau J-C (1977) Imaginal cell differentiation in the spermiduct ofSamia cynthia (Lepidoptera): Responses in vitro to ecdysone and ecdysterone. Biol Cell 28:23–36Google Scholar
  84. Takeda N (1972) Effect of some phytoecdysones on the spermatogenesis of the slug moth prepupa,Monema flavescens Walker (Lepidoptera: Heterogeneidae). Appl Entomol Zool 7:37–39Google Scholar
  85. Takemoto T, Ogawa S, Nishimoto N, Mue K (1967) Studies on the consituents ofAchyranthis radix. V. Insect hormone activity of ecdysterone and inokosterone on the flies. Yakagaku Zasshi 87:1481–1483Google Scholar
  86. Thompson MJ, Robbins WE, Cohen CF, Kaplanis JN, Dutky SR, Hutchins RFN (1971) Synthesis and biological activity of 5β-hydroxy analogs of α-ecdysone. Steroids 17:399–409Google Scholar
  87. Wyatt GR (1972) Insect hormones. In: Litwack G (ed) Biochemical actions of hormones, vol 2. Academic Press, New York, pp 385–490Google Scholar
  88. Wyatt SS, Wyatt GR (1971) Stimulation of RNA and protein synthesis in silkmoth pupal wing tissue by ecdysone in vitro. Gen Comp Endocrinol 16:369–374Google Scholar
  89. Wyss C (1976) Juvenile hormone analogue counteracts growth stimulation and inhibition by ecdysones in clonalDrosophila cell line. Experientia 32:1272–1274Google Scholar
  90. Wyss C (1980) Cell hybrid analysis of ecdysone sensitivity and resistance inDrosophila cell lines. In: Kurstak E, Maramorosch K, Dübendorfer (eds) Invertebrate systems in vitro. Elsevier/North Holland, Amsterdam, in pressGoogle Scholar
  91. Yund MA, King DS, Fristrom JW (1978) Ecdysteroid receptors in imaginal discs ofDrosophila melanogaster. Proc Natl Acad Sci USA 75:6039–6043Google Scholar
  92. Zdarek J, Denlinger DL (1975) Action of ecdysoids, juvenoids, and non-hormonal agents on termination of pupal diapause in the fleshfly. J Insect Physiol 21:1193–1202Google Scholar

Copyright information

© Springer-Verlag 1980

Authors and Affiliations

  • Lucy Cherbas
    • 1
  • Christopher D. Yonge
    • 1
  • Peter Cherbas
    • 1
  • Carroll M. Williams
    • 1
  1. 1.Cellular and Developmental Biology, The Biological LaboratoriesHarvard UniversityCambridgeUSA

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