Skip to main content
NCBI home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1994 May 2;125(4):743–753. doi: 10.1083/jcb.125.4.743

Retinoic acid disrupts the Golgi apparatus and increases the cytosolic routing of specific protein toxins

PMCID: PMC2120076  PMID: 8188744

Abstract

All-trans retinoic acid can specifically increase receptor mediated intoxication of ricin A chain immunotoxins more than 10,000 times, whereas fluid phase endocytosis of ricin A chain alone or ricin A chain immunotoxins was not influenced by retinoic acid. The immunotoxin activation by retinoic acid does not require RNA or protein synthesis and is not a consequence of increased receptor binding of the immunotoxin. Vitamin D3 and thyroid hormone T3, that activate retinoic acid receptor (RAR) cognates, forming heterodimers with retinoid X receptor (RXR), do not affect the potency of immunotoxins. Among other retinoids tested, 13-cis retinoic acid, which binds neither RAR nor RXR, also increases the potency of the ricin A chain immunotoxin. Therefore, retinoic acid receptor activation does not appear to be necessary for immunotoxin activity. Retinoic acid potentiation of immunotoxins is prevented by brefeldin A (BFA) indicating that in the presence of retinoic acid, the immunotoxin is efficiently routed through the Golgi apparatus en route to the cytoplasm. Directly examining cells with a monoclonal antibody (Mab) against mannosidase II, a Golgi apparatus marker enzyme, demonstrates that the Golgi apparatus changes upon treatment with retinoic acid from a perinuclear network to a diffuse aggregate. Within 60 min after removal of retinoic acid the cell reassembles the perinuclear Golgi network indistinguishable with that of normal control cells. C6-NBD-ceramide, a vital stain for the Golgi apparatus, shows that retinoic acid prevents the fluorescent staining of the Golgi apparatus and eliminates fluorescence of C6-NBD-ceramide prestained Golgi apparatus. Electron microscopy of retinoic acid-treated cells demonstrates the specific absence of any normal looking Golgi apparatus and a perinuclear vacuolar structure very similar to that seen in monensin-treated cells. This vacuolization disappears after removal of the retinoic acid and a perinuclear Golgi stacking reappears. These results indicate that retinoic acid alters intracellular routing, probably through the Golgi apparatus, potentiating immunotoxin activity indepedently of new gene expression. Retinoic acid appears to be a new reagent to manipulate the Golgi apparatus and intracellular traffic. As retinoic acid and immunotoxins are both in clinical trials for cancer therapy, their combined activity in vivo would be interesting to examine.

Full Text

The Full Text of this article is available as a PDF (3.1 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Allenby G., Bocquel M. T., Saunders M., Kazmer S., Speck J., Rosenberger M., Lovey A., Kastner P., Grippo J. F., Chambon P. Retinoic acid receptors and retinoid X receptors: interactions with endogenous retinoic acids. Proc Natl Acad Sci U S A. 1993 Jan 1;90(1):30–34. doi: 10.1073/pnas.90.1.30. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Casellas P., Bourrie B. J., Gros P., Jansen F. K. Kinetics of cytotoxicity induced by immunotoxins. Enhancement by lysosomotropic amines and carboxylic ionophores. J Biol Chem. 1984 Aug 10;259(15):9359–9364. [PubMed] [Google Scholar]
  3. Chaudhary V. K., Jinno Y., FitzGerald D., Pastan I. Pseudomonas exotoxin contains a specific sequence at the carboxyl terminus that is required for cytotoxicity. Proc Natl Acad Sci U S A. 1990 Jan;87(1):308–312. doi: 10.1073/pnas.87.1.308. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chen C. H., Kuwazuru Y., Yoshida T., Nambiar M., Wu H. C. Isolation and characterization of a brefeldin A-resistant mutant of monkey kidney Vero cells. Exp Cell Res. 1992 Dec;203(2):321–328. doi: 10.1016/0014-4827(92)90005-s. [DOI] [PubMed] [Google Scholar]
  5. Doms R. W., Russ G., Yewdell J. W. Brefeldin A redistributes resident and itinerant Golgi proteins to the endoplasmic reticulum. J Cell Biol. 1989 Jul;109(1):61–72. doi: 10.1083/jcb.109.1.61. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Draper R. K., Simon M. I. The entry of diphtheria toxin into the mammalian cell cytoplasm: evidence for lysosomal involvement. J Cell Biol. 1980 Dec;87(3 Pt 1):849–854. doi: 10.1083/jcb.87.3.849. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Endo Y., Mitsui K., Motizuki M., Tsurugi K. The mechanism of action of ricin and related toxic lectins on eukaryotic ribosomes. The site and the characteristics of the modification in 28 S ribosomal RNA caused by the toxins. J Biol Chem. 1987 Apr 25;262(12):5908–5912. [PubMed] [Google Scholar]
  8. Frankel A. E., Ring D. B., Tringale F., Hsieh-Ma S. T. Tissue distribution of breast cancer-associated antigens defined by monoclonal antibodies. J Biol Response Mod. 1985 Jun;4(3):273–286. [PubMed] [Google Scholar]
  9. Gonatas N. K., Steiber A., Kim S. U., Graham D. I., Avrameas S. Internalization of neuronal plasma membrane ricin receptors into the Golgi apparatus. Exp Cell Res. 1975 Sep;94(2):426–431. doi: 10.1016/0014-4827(75)90508-x. [DOI] [PubMed] [Google Scholar]
  10. Gregg E. O., Bridges S. H., Youle R. J., Longo D. L., Houston L. L., Glennie M. J., Stevenson F. K., Green I. Whole ricin and recombinant ricin A chain idiotype-specific immunotoxins for therapy of the guinea pig L2C B cell leukemia. J Immunol. 1987 Jun 15;138(12):4502–4508. [PubMed] [Google Scholar]
  11. Grossbard M. L., Freedman A. S., Ritz J., Coral F., Goldmacher V. S., Eliseo L., Spector N., Dear K., Lambert J. M., Blättler W. A. Serotherapy of B-cell neoplasms with anti-B4-blocked ricin: a phase I trial of daily bolus infusion. Blood. 1992 Feb 1;79(3):576–585. [PubMed] [Google Scholar]
  12. Heyman R. A., Mangelsdorf D. J., Dyck J. A., Stein R. B., Eichele G., Evans R. M., Thaller C. 9-cis retinoic acid is a high affinity ligand for the retinoid X receptor. Cell. 1992 Jan 24;68(2):397–406. doi: 10.1016/0092-8674(92)90479-v. [DOI] [PubMed] [Google Scholar]
  13. Hudson T. H., Grillo F. G. Brefeldin-A enhancement of ricin A-chain immunotoxins and blockade of intact ricin, modeccin, and abrin. J Biol Chem. 1991 Oct 5;266(28):18586–18592. [PubMed] [Google Scholar]
  14. Johnson V. G., Wilson D., Greenfield L., Youle R. J. The role of the diphtheria toxin receptor in cytosol translocation. J Biol Chem. 1988 Jan 25;263(3):1295–1300. [PubMed] [Google Scholar]
  15. Kliewer S. A., Umesono K., Mangelsdorf D. J., Evans R. M. Retinoid X receptor interacts with nuclear receptors in retinoic acid, thyroid hormone and vitamin D3 signalling. Nature. 1992 Jan 30;355(6359):446–449. doi: 10.1038/355446a0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Ktistakis N. T., Roth M. G., Bloom G. S. PtK1 cells contain a nondiffusible, dominant factor that makes the Golgi apparatus resistant to brefeldin A. J Cell Biol. 1991 Jun;113(5):1009–1023. doi: 10.1083/jcb.113.5.1009. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Laurent G., Pris J., Farcet J. P., Carayon P., Blythman H., Casellas P., Poncelet P., Jansen F. K. Effects of therapy with T101 ricin A-chain immunotoxin in two leukemia patients. Blood. 1986 Jun;67(6):1680–1687. [PubMed] [Google Scholar]
  18. Ledger P. W., Uchida N., Tanzer M. L. Immunocytochemical localization of procollagen and fibronectin in human fibroblasts: effects of the monovalent ionophore, monensin. J Cell Biol. 1980 Dec;87(3 Pt 1):663–671. doi: 10.1083/jcb.87.3.663. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Levin A. A., Sturzenbecker L. J., Kazmer S., Bosakowski T., Huselton C., Allenby G., Speck J., Kratzeisen C., Rosenberger M., Lovey A. 9-cis retinoic acid stereoisomer binds and activates the nuclear receptor RXR alpha. Nature. 1992 Jan 23;355(6358):359–361. doi: 10.1038/355359a0. [DOI] [PubMed] [Google Scholar]
  20. Lippincott-Schwartz J., Glickman J., Donaldson J. G., Robbins J., Kreis T. E., Seamon K. B., Sheetz M. P., Klausner R. D. Forskolin inhibits and reverses the effects of brefeldin A on Golgi morphology by a cAMP-independent mechanism. J Cell Biol. 1991 Feb;112(4):567–577. doi: 10.1083/jcb.112.4.567. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Lippincott-Schwartz J., Yuan L. C., Bonifacino J. S., Klausner R. D. Rapid redistribution of Golgi proteins into the ER in cells treated with brefeldin A: evidence for membrane cycling from Golgi to ER. Cell. 1989 Mar 10;56(5):801–813. doi: 10.1016/0092-8674(89)90685-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Lippincott-Schwartz J., Yuan L., Tipper C., Amherdt M., Orci L., Klausner R. D. Brefeldin A's effects on endosomes, lysosomes, and the TGN suggest a general mechanism for regulating organelle structure and membrane traffic. Cell. 1991 Nov 1;67(3):601–616. doi: 10.1016/0092-8674(91)90534-6. [DOI] [PubMed] [Google Scholar]
  23. Lipsky N. G., Pagano R. E. A vital stain for the Golgi apparatus. Science. 1985 May 10;228(4700):745–747. doi: 10.1126/science.2581316. [DOI] [PubMed] [Google Scholar]
  24. Lipsky N. G., Pagano R. E. Intracellular translocation of fluorescent sphingolipids in cultured fibroblasts: endogenously synthesized sphingomyelin and glucocerebroside analogues pass through the Golgi apparatus en route to the plasma membrane. J Cell Biol. 1985 Jan;100(1):27–34. doi: 10.1083/jcb.100.1.27. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Mangelsdorf D. J., Ong E. S., Dyck J. A., Evans R. M. Nuclear receptor that identifies a novel retinoic acid response pathway. Nature. 1990 May 17;345(6272):224–229. doi: 10.1038/345224a0. [DOI] [PubMed] [Google Scholar]
  26. Olsnes S., Pihl A. Different biological properties of the two constituent peptide chains of ricin, a toxic protein inhibiting protein synthesis. Biochemistry. 1973 Jul 31;12(16):3121–3126. doi: 10.1021/bi00740a028. [DOI] [PubMed] [Google Scholar]
  27. Olsnes S., Sandvig K., Refsnes K., Pihl A. Rates of different steps involved in the inhibition of protein synthesis by the toxic lectins abrin and ricin. J Biol Chem. 1976 Jul 10;251(13):3985–3992. [PubMed] [Google Scholar]
  28. Pelham H. R. Control of protein exit from the endoplasmic reticulum. Annu Rev Cell Biol. 1989;5:1–23. doi: 10.1146/annurev.cb.05.110189.000245. [DOI] [PubMed] [Google Scholar]
  29. Pelham H. R. Multiple targets for brefeldin A. Cell. 1991 Nov 1;67(3):449–451. doi: 10.1016/0092-8674(91)90517-3. [DOI] [PubMed] [Google Scholar]
  30. Petkovich M. Regulation of gene expression by vitamin A: the role of nuclear retinoic acid receptors. Annu Rev Nutr. 1992;12:443–471. doi: 10.1146/annurev.nu.12.070192.002303. [DOI] [PubMed] [Google Scholar]
  31. Repa J. J., Hanson K. K., Clagett-Dame M. All-trans-retinol is a ligand for the retinoic acid receptors. Proc Natl Acad Sci U S A. 1993 Aug 1;90(15):7293–7297. doi: 10.1073/pnas.90.15.7293. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Sandvig K., Olsnes S. Effects of retinoids and phorbol esters on the sensitivity of different cell lines to the polypeptide toxins modeccin, abrin, ricin and diphtheria toxin. Biochem J. 1981 Mar 15;194(3):821–827. doi: 10.1042/bj1940821. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Sandvig K., Prydz K., Hansen S. H., van Deurs B. Ricin transport in brefeldin A-treated cells: correlation between Golgi structure and toxic effect. J Cell Biol. 1991 Nov;115(4):971–981. doi: 10.1083/jcb.115.4.971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Wales R., Roberts L. M., Lord J. M. Addition of an endoplasmic reticulum retrieval sequence to ricin A chain significantly increases its cytotoxicity to mammalian cells. J Biol Chem. 1993 Nov 15;268(32):23986–23990. [PubMed] [Google Scholar]
  35. Wu Y., Mikulski S. M., Ardelt W., Rybak S. M., Youle R. J. A cytotoxic ribonuclease. Study of the mechanism of onconase cytotoxicity. J Biol Chem. 1993 May 15;268(14):10686–10693. [PubMed] [Google Scholar]
  36. Yoshida T., Chen C. C., Zhang M. S., Wu H. C. Disruption of the Golgi apparatus by brefeldin A inhibits the cytotoxicity of ricin, modeccin, and Pseudomonas toxin. Exp Cell Res. 1991 Feb;192(2):389–395. doi: 10.1016/0014-4827(91)90056-z. [DOI] [PubMed] [Google Scholar]
  37. Youle R. J., Colombatti M. Hybridoma cells containing intracellular anti-ricin antibodies show ricin meets secretory antibody before entering the cytosol. J Biol Chem. 1987 Apr 5;262(10):4676–4682. [PubMed] [Google Scholar]
  38. Youle R. J., Neville D. M., Jr Kinetics of protein synthesis inactivation by ricin-anti-Thy 1.1 monoclonal antibody hybrids. Role of the ricin B subunit demonstrated by reconstitution. J Biol Chem. 1982 Feb 25;257(4):1598–1601. [PubMed] [Google Scholar]
  39. Zhang X. K., Hoffmann B., Tran P. B., Graupner G., Pfahl M. Retinoid X receptor is an auxiliary protein for thyroid hormone and retinoic acid receptors. Nature. 1992 Jan 30;355(6359):441–446. doi: 10.1038/355441a0. [DOI] [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES