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. 1993 Jul;92(1):431–440. doi: 10.1172/JCI116585

In vitro modulation of cisplatin accumulation in human ovarian carcinoma cells by pharmacologic alteration of microtubules.

R D Christen 1, A P Jekunen 1, J A Jones 1, F Thiebaut 1, D R Shalinsky 1, S B Howell 1
PMCID: PMC293629  PMID: 8100837

Abstract

We have previously shown that forskolin and 3-isobutyl-1-methylxanthine (IBMX) increased accumulation of cisplatin (DDP) in DDP-sensitive 2008 human ovarian carcinoma cells in proportion to their ability to increase cAMP. Since the major function of cAMP is to activate protein kinase A, it was conjectured that the stimulation of DDP accumulation was mediated by a protein kinase A substrate. We now show that exposure of 2008 cells to forskolin resulted in phosphorylation of a prominent 52-kD membrane protein. Microsequencing of the band demonstrated it to be human beta-tubulin. Similarly, pretreatment of 2008 cells with the microtubule stabilizing drug taxol increased platinum accumulation in a dose-dependent manner. In 11-fold DDP-resistant 2008/C13*5.25 cells, decreased DDP accumulation was associated with enhanced spontaneous formation of microtubule bundles and decreased expression of beta-tubulin and the tubulin-associated p53 antioncogene relative to 2008 cells. 2008/C13*5.25 cells had altered sensitivity to tubulin-binding drugs, being hypersensitive to taxol and cross-resistant to colchicine. We conclude that pharmacologic alterations of tubulin enhance accumulation of DDP, and that the DDP-resistant phenotype in 2008/C13*5.25 cells is associated with tubulin abnormalities.

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Selected References

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

  1. Andrews P. A., Howell S. B. Cellular pharmacology of cisplatin: perspectives on mechanisms of acquired resistance. Cancer Cells. 1990 Feb;2(2):35–43. [PubMed] [Google Scholar]
  2. Andrews P. A., Jones J. A., Varki N. M., Howell S. B. Rapid emergence of acquired cis-diamminedichloroplatinum(II) resistance in an in vivo model of human ovarian carcinoma. Cancer Commun. 1990;2(2):93–100. doi: 10.3727/095535490820874641. [DOI] [PubMed] [Google Scholar]
  3. Andrews P. A., Murphy M. P., Howell S. B. Differential potentiation of alkylating and platinating agent cytotoxicity in human ovarian carcinoma cells by glutathione depletion. Cancer Res. 1985 Dec;45(12 Pt 1):6250–6253. [PubMed] [Google Scholar]
  4. Andrews P. A., Velury S., Mann S. C., Howell S. B. cis-Diamminedichloroplatinum(II) accumulation in sensitive and resistant human ovarian carcinoma cells. Cancer Res. 1988 Jan 1;48(1):68–73. [PubMed] [Google Scholar]
  5. Banks L., Matlashewski G., Crawford L. Isolation of human-p53-specific monoclonal antibodies and their use in the studies of human p53 expression. Eur J Biochem. 1986 Sep 15;159(3):529–534. doi: 10.1111/j.1432-1033.1986.tb09919.x. [DOI] [PubMed] [Google Scholar]
  6. Blose S. H., Meltzer D. I., Feramisco J. R. 10-nm filaments are induced to collapse in living cells microinjected with monoclonal and polyclonal antibodies against tubulin. J Cell Biol. 1984 Mar;98(3):847–858. doi: 10.1083/jcb.98.3.847. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Boekelheide K., Arcila M. E., Eveleth J. cis-diamminedichloroplatinum (II) (cisplatin) alters microtubule assembly dynamics. Toxicol Appl Pharmacol. 1992 Sep;116(1):146–151. doi: 10.1016/0041-008x(92)90156-m. [DOI] [PubMed] [Google Scholar]
  8. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  9. Burnouf D., Gauthier C., Chottard J. C., Fuchs R. P. Single d(ApG)/cis-diamminedichloroplatinum(II) adduct-induced mutagenesis in Escherichia coli. Proc Natl Acad Sci U S A. 1990 Aug;87(16):6087–6091. doi: 10.1073/pnas.87.16.6087. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Cabanillas F., Pathak S., Grant G., Hagemeister F. B., McLaughlin P., Swan F., Rodriguez M. A., Trujillo J., Cork A., Butler J. J. Refractoriness to chemotherapy and poor survival related to abnormalities of chromosomes 17 and 7 in lymphoma. Am J Med. 1989 Aug;87(2):167–172. doi: 10.1016/s0002-9343(89)80692-8. [DOI] [PubMed] [Google Scholar]
  11. Cabral F., Barlow S. B. Mechanisms by which mammalian cells acquire resistance to drugs that affect microtubule assembly. FASEB J. 1989 Mar;3(5):1593–1599. doi: 10.1096/fasebj.3.5.2646163. [DOI] [PubMed] [Google Scholar]
  12. Chibber R., Ord M. J. The mutagenic and carcinogenic properties of three second generation antitumour platinum compounds: a comparison with cisplatin. Eur J Cancer Clin Oncol. 1989 Jan;25(1):27–33. doi: 10.1016/0277-5379(89)90047-3. [DOI] [PubMed] [Google Scholar]
  13. DiSaia P. J., Sinkovics J. G., Rutledge F. N., Smith J. P. Cell-mediated immunity to human malignant cells. A brief review and further studies with two gynecologic tumors. Am J Obstet Gynecol. 1972 Dec 1;114(7):979–989. doi: 10.1016/0002-9378(72)90109-3. [DOI] [PubMed] [Google Scholar]
  14. Eastman A. Characterization of the adducts produced in DNA by cis-diamminedichloroplatinum(II) and cis-dichloro(ethylenediamine)platinum(II). Biochemistry. 1983 Aug 2;22(16):3927–3933. doi: 10.1021/bi00285a031. [DOI] [PubMed] [Google Scholar]
  15. Garrison J. C. Measurement of hormone-stimulated protein phosphorylation in intact cells. Methods Enzymol. 1983;99:20–36. doi: 10.1016/0076-6879(83)99037-7. [DOI] [PubMed] [Google Scholar]
  16. Gozes I., Barnstable C. J. Monoclonal antibodies that recognize discrete forms of tubulin. Proc Natl Acad Sci U S A. 1982 Apr;79(8):2579–2583. doi: 10.1073/pnas.79.8.2579. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hewick R. M., Hunkapiller M. W., Hood L. E., Dreyer W. J. A gas-liquid solid phase peptide and protein sequenator. J Biol Chem. 1981 Aug 10;256(15):7990–7997. [PubMed] [Google Scholar]
  18. Huber G., Matus A. Differences in the cellular distributions of two microtubule-associated proteins, MAP1 and MAP2, in rat brain. J Neurosci. 1984 Jan;4(1):151–160. doi: 10.1523/JNEUROSCI.04-01-00151.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Janski A. M., Cornell N. W. Subcellular distribution of enzymes determined by rapid digitonin fractionation of isolated hepatocytes. Biochem J. 1980 Feb 15;186(2):423–429. doi: 10.1042/bj1860423. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Joost H. G., Habberfield A. D., Simpson I. A., Laurenza A., Seamon K. B. Activation of adenylate cyclase and inhibition of glucose transport in rat adipocytes by forskolin analogues: structural determinants for distinct sites of action. Mol Pharmacol. 1988 Apr;33(4):449–453. [PubMed] [Google Scholar]
  21. Köpf-Maier P., Mühlhausen S. K. Changes in the cytoskeleton pattern of tumor cells by cisplatin in vitro. Chem Biol Interact. 1992 May;82(3):295–316. doi: 10.1016/0009-2797(92)90002-3. [DOI] [PubMed] [Google Scholar]
  22. Mann S. C., Andrews P. A., Howell S. B. Modulation of cis-diamminedichloroplatinum(II) accumulation and sensitivity by forskolin and 3-isobutyl-1-methylxanthine in sensitive and resistant human ovarian carcinoma cells. Int J Cancer. 1991 Jul 30;48(6):866–872. doi: 10.1002/ijc.2910480613. [DOI] [PubMed] [Google Scholar]
  23. Manso-Martínez R., Palomares R., Pariente F. Mammalian brain microtubules are sensitive to cyclic AMP in vitro. Arch Biochem Biophys. 1984 Nov 15;235(1):196–203. doi: 10.1016/0003-9861(84)90268-6. [DOI] [PubMed] [Google Scholar]
  24. Massart C., Le Tellier C., Lucas C., Gibassier J., Leclech G., Nicol M. Effects of cisplatin on human thyrocytes in monolayer or follicle culture. J Mol Endocrinol. 1992 Jun;8(3):243–248. doi: 10.1677/jme.0.0080243. [DOI] [PubMed] [Google Scholar]
  25. Matthes T., Wolff A., Soubiran P., Gros F., Dighiero G. Antitubulin antibodies. II. Natural autoantibodies and induced antibodies recognize different epitopes on the tubulin molecule. J Immunol. 1988 Nov 1;141(9):3135–3141. [PubMed] [Google Scholar]
  26. Maxwell S. A., Ames S. K., Sawai E. T., Decker G. L., Cook R. G., Butel J. S. Simian virus 40 large T antigen and p53 are microtubule-associated proteins in transformed cells. Cell Growth Differ. 1991 Feb;2(2):115–127. [PubMed] [Google Scholar]
  27. McGuire W. P., Rowinsky E. K., Rosenshein N. B., Grumbine F. C., Ettinger D. S., Armstrong D. K., Donehower R. C. Taxol: a unique antineoplastic agent with significant activity in advanced ovarian epithelial neoplasms. Ann Intern Med. 1989 Aug 15;111(4):273–279. doi: 10.7326/0003-4819-111-4-273. [DOI] [PubMed] [Google Scholar]
  28. Minotti A. M., Barlow S. B., Cabral F. Resistance to antimitotic drugs in Chinese hamster ovary cells correlates with changes in the level of polymerized tubulin. J Biol Chem. 1991 Feb 25;266(6):3987–3994. [PubMed] [Google Scholar]
  29. Papasozomenos S. C., Binder L. I. Phosphorylation determines two distinct species of Tau in the central nervous system. Cell Motil Cytoskeleton. 1987;8(3):210–226. doi: 10.1002/cm.970080303. [DOI] [PubMed] [Google Scholar]
  30. Peyrot V., Briand C., Momburg R., Sari J. C. In vitro mechanism study of microtubule assembly inhibition by cis-dichlorodiammine-platinum(II). Biochem Pharmacol. 1986 Feb 1;35(3):371–375. doi: 10.1016/0006-2952(86)90207-8. [DOI] [PubMed] [Google Scholar]
  31. Rowinsky E. K., Burke P. J., Karp J. E., Tucker R. W., Ettinger D. S., Donehower R. C. Phase I and pharmacodynamic study of taxol in refractory acute leukemias. Cancer Res. 1989 Aug 15;49(16):4640–4647. [PubMed] [Google Scholar]
  32. Rowinsky E. K., Donehower R. C., Jones R. J., Tucker R. W. Microtubule changes and cytotoxicity in leukemic cell lines treated with taxol. Cancer Res. 1988 Jul 15;48(14):4093–4100. [PubMed] [Google Scholar]
  33. Sullivan K. F., Cleveland D. W. Identification of conserved isotype-defining variable region sequences for four vertebrate beta tubulin polypeptide classes. Proc Natl Acad Sci U S A. 1986 Jun;83(12):4327–4331. doi: 10.1073/pnas.83.12.4327. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Weinstein B., Solomon F. Phenotypic consequences of tubulin overproduction in Saccharomyces cerevisiae: differences between alpha-tubulin and beta-tubulin. Mol Cell Biol. 1990 Oct;10(10):5295–5304. doi: 10.1128/mcb.10.10.5295. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Zhen W., Link C. J., Jr, O'Connor P. M., Reed E., Parker R., Howell S. B., Bohr V. A. Increased gene-specific repair of cisplatin interstrand cross-links in cisplatin-resistant human ovarian cancer cell lines. Mol Cell Biol. 1992 Sep;12(9):3689–3698. doi: 10.1128/mcb.12.9.3689. [DOI] [PMC free article] [PubMed] [Google Scholar]

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