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Dictyostelium discoideum Protocols pp 185–204Cite as

Pharmacogenetics of Resistance to Cisplatin and Other Anticancer Drugs and the Role of Sphingolipid Metabolism

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 983))

Abstract

Dictyostelium discoideum has proven to be a useful lead genetic system for identifying novel genes and pathways responsible for the regulation of sensitivity to the widely used anticancer drug cisplatin. Resistance to cisplatin is a major factor limiting the efficacy of the drug in treating many types of cancer. Studies using unbiased insertional mutagenesis in D. discoideum have identified the pathway of sphingolipid metabolism as a key regulator in controlling sensitivity to cisplatin. Using the genetic tools including directed homologous recombination and ectopic gene expression available with D. discoideum has shown how pharmacological modulation of this pathway can increase sensitivity to cisplatin, and these results have been extensively translated to, and validated in, human cells. Strategies, experimental conditions, and methods are presented to enable further study of resistance to cisplatin as well as other important drugs.

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References

  1. AmericanCancerSociety (2010) Cancer facts and figures (2010) http://www.cancer.org/Research/CancerFactsFigures/index

  2. Siddik ZH (2003) Cisplatin: mode of cytotoxic action and molecular basis of resistance. Oncogene 22:7265–7279

    Article  PubMed  CAS  Google Scholar 

  3. Rabik CA, Dolan ME (2007) Molecular mechanisms of resistance and toxicity associated with platinating agents. Cancer Treat Rev 33:9–23

    Article  PubMed  CAS  Google Scholar 

  4. Wernyj RP, Morin PJ (2004) Molecular mechanisms of platinum resistance: still searching for the Achilles’ heel. Drug Resist Updat 7:227–232

    Article  PubMed  CAS  Google Scholar 

  5. Williams JG (2010) Dictyostelium finds new roles to model. Genetics 185:717–726

    Article  PubMed  CAS  Google Scholar 

  6. Williams RS, Boeckeler K, Gräf R, Müller-Taubenberger A, Li Z, Isberg RR, Wessels D, Soll DR, Alexander H, Alexander S (2006) Towards a molecular understanding of human diseases using Dictyostelium discoideum. Trends Mol Med 12:415–424

    Article  PubMed  CAS  Google Scholar 

  7. Glöckner G, Eichinger L, Szafranski K, Pachebat JA, Bankier AT, Dear PH, Lehmann D, Baumgart C, Parra G, Abril JF, Guigo R, Kumpf K, Tunggal B, Cox E, Quail MA, Platzer M, Rosenthal A, Noegel AA (2002) Sequence and analysis of chromosome 2 of Dictyostelium discoideum. Nature 418:79–85

    Article  PubMed  Google Scholar 

  8. Eichinger L, Pachebat JA, Glöckner G, Rajandream MA, Sucgang R, Berriman M, Song J, Olsen R, Szafranski K, Xu Q, Tunggal B, Kummerfeld S, Madera M, Konfortov BA, Rivero F, Bankier AT, Lehmann R, Hamlin N, Davies R, Gaudet P, Fey P, Pilcher K, Chen G, Saunders D, Sodergren E, Davis P, Kerhornou A, Nie X, Hall N, Anjard C, Hemphill L, Bason N, Farbrother P, Desany B, Just E, Morio T, Rost R, Churcher C, Cooper J, Haydock S, van Driessche N, Cronin A, Goodhead I, Muzny D, Mourier T, Pain A, Lu M, Harper D, Lindsay R, Hauser H, James K, Quiles M, Madan Babu M, Saito T, Buchrieser C, Wardroper A, Felder M, Thangavelu M, Johnson D, Knights A, Loulseged H, Mungall K, Oliver K, Price C, Quail MA, Urushihara H, Hernandez J, Rabbinowitsch E, Steffen D, Sanders M, Ma J, Kohara Y, Sharp S, Simmonds M, Spiegler S, Tivey A, Sugano S, White B, Walker D, Woodward J, Winckler T, Tanaka Y, Shaulsky G, Schleicher M, Weinstock G, Rosenthal A, Cox EC, Chisholm RL, Gibbs R, Loomis WF, Platzer M, Kay RR, Williams J, Dear PH, Noegel AA, Barrell B, Kuspa A (2005) The genome of the social amoeba Dictyostelium discoideum. Nature 435:43–57

    Article  PubMed  CAS  Google Scholar 

  9. Kessin RH (2001) Dictyostelium—evolution, cell biology, and the development of multicellularity. Cambridge Univ. Press, Cambridge

    Book  Google Scholar 

  10. Kuspa A, Loomis WF (1992) Tagging developmental genes in Dictyostelium by restriction enzyme-mediated integration of plasmid DNA. Proc Natl Acad Sci U S A 89:8803–8807

    Article  PubMed  CAS  Google Scholar 

  11. Loomis WF (1987) Genetic tools for Dictyostelium discoideum. Methods Cell Biol 28:31–65

    Article  PubMed  CAS  Google Scholar 

  12. Newell PC (1982) Genetics. In: Loomis WF (ed) The development of Dictyostelium discoideum. Academic, New York

    Google Scholar 

  13. Garcia MXU, Roberts C, Alexander H, Stewart AM, Harwood A, Alexander S, Insall RH (2002) Methanol and acriflavine resistance in Dictyostelium are caused by loss of catalase. Microbiology 148:333–340

    PubMed  CAS  Google Scholar 

  14. Li GC, Alexander H, Schneider N, Alexander S (2000) Molecular basis for resistance to the anticancer drug cisplatin in Dictyostelium. Microbiology 146:2219–2227

    PubMed  CAS  Google Scholar 

  15. Alexander S, Min J, Alexander H (2006) Dictyostelium discoideum to human cells: pharmacogenetic studies demonstrate a role for sphingolipids in chemoresistance. Biochim Biophys Acta 1760:301–309

    Article  PubMed  CAS  Google Scholar 

  16. Min J, Stegner A, Alexander H, Alexander S (2004) Overexpression of sphingosine-1-phosphate lyase or inhibition of sphingosine kinase in Dictyostelium discoideum results in a selective increase in sensitivity to platinum based chemotherapy drugs. Eukaryot Cell 3:795–805

    Article  PubMed  CAS  Google Scholar 

  17. Min J, Traynor D, Stegner AL, Zhang L, Hanigan MH, Alexander H, Alexander S (2005) Sphingosine kinase regulates the sensitivity of Dictyostelium discoideum cells to the anticancer drug cisplatin. Eukaryot Cell 4:178–189

    Article  PubMed  CAS  Google Scholar 

  18. Min J, Van Veldhoven PP, Zhang L, Hanigan MH, Alexander H, Alexander S (2005) Sphingosine-1-phosphate lyase regulates sensitivity of human cells to select ­chemotherapy drugs in a p38-dependent manner. Mol Cancer Res 3:287–296

    Article  PubMed  CAS  Google Scholar 

  19. Min J, Mesika A, Sivaguru M, Van Veldhoven PP, Alexander H, Futerman AH, Alexander S (2007) (Dihydro)ceramide synthase 1 regulated sensitivity to cisplatin is associated with the activation of p38 mitogen-activated protein kinase and is abrogated by sphingosine kinase 1. Mol Cancer Res 5:801–812

    Article  PubMed  CAS  Google Scholar 

  20. Sridevi P, Alexander H, Laviad EL, Min J, Mesika A, Hannink M, Futerman AH, Alexander S (2010) Stress-induced ER to Golgi translocation of ceramide synthase 1 is dependent on proteasomal processing. Exp Cell Res 316:78–91

    Article  PubMed  CAS  Google Scholar 

  21. Sridevi P, Alexander H, Laviad EL, Pewzner-Jung Y, Hannink M, Futerman AH, Alexander S (2009) Ceramide synthase 1 is regulated by proteasomal mediated turnover. Biochim Biophys Acta 1793:1218–1227

    Article  PubMed  CAS  Google Scholar 

  22. Oskouian B, Sooriyakumaran P, Borowsky AD, Crans A, Dillard-Telm L, Tam YY, Bandhuvula P, Saba JD (2006) Sphingosine-1-phosphate lyase potentiates apoptosis via p53- and p38-dependent pathways and is down-regulated in colon cancer. Proc Natl Acad Sci U S A 103:17384–17389

    Article  PubMed  CAS  Google Scholar 

  23. Reiss U, Oskouian B, Zhou J, Gupta V, Sooriyakumaran P, Kelly S, Wang E, Merrill AH Jr, Saba JD (2004) Sphingosine-phosphate lyase enhances stress-induced ceramide generation and apoptosis. J Biol Chem 279:1281–1290

    Article  PubMed  CAS  Google Scholar 

  24. Alexander S, Alexander H (2011) Lead genetic studies in Dictyostelium discoideum and translational studies in human cells demonstrate that sphingolipids are key regulators of sensitivity to cisplatin and other anticancer drugs. Semin Cell Dev Biol 22:97–104

    Article  PubMed  CAS  Google Scholar 

  25. Van Driessche N, Alexander H, Min J, Kuspa A, Alexander S, Shaulsky G (2007) Global transcriptional responses to cisplatin in Dictyostelium discoideum identify potential drug targets. Proc Natl Acad Sci U S A 104:15406–15411

    Article  PubMed  Google Scholar 

  26. Sussman M (1987) Cultivation and synchronous morphogenesis of Dictyostelium under controlled experimental conditions. Methods Cell Biol 28:9–29

    Article  PubMed  CAS  Google Scholar 

  27. Manstein DJ, Schuster HP, Morandini P, Hunt DM (1995) Cloning vectors for the production of proteins in Dictyostelium discoideum. Gene 162:129–134

    Article  PubMed  CAS  Google Scholar 

  28. Loomis WF, Kuspa A (eds) (2005) Dictyostelium genomics. Horizon Bioscience, Norfolk

    Google Scholar 

  29. Adley KE, Keim M, Williams RS (2006) Pharmacogenetics: defining the genetic basis of drug action and inositol trisphosphate analysis. Methods Mol Biol 346:517–534

    PubMed  CAS  Google Scholar 

  30. Keim M, Williams RS, Harwood AJ (2004) An inverse PCR technique to rapidly isolate the flanking DNA of Dictyostelium insertion mutants. Mol Biotechnol 26:221–224

    Article  PubMed  CAS  Google Scholar 

  31. Faix J, Kreppel L, Shaulsky G, Schleicher M, Kimmel AR (2004) A rapid and efficient method to generate multiple gene disruptions in Dictyostelium discoideum using a single selectable marker and the Cre-loxP system. Nucleic Acids Res 32:e143

    Article  PubMed  Google Scholar 

  32. Dubin M, Nellen W (2010) A versatile set of tagged expression vectors to monitor protein localisation and function in Dictyostelium. Gene 465:1–8

    Article  PubMed  CAS  Google Scholar 

  33. Li G, Foote C, Alexander S, Alexander H (2001) Sphingosine-1-phosphate lyase has a central role in the development of Dictyostelium discoideum. Development 128:3473–3483

    PubMed  CAS  Google Scholar 

  34. Williams RS (2005) Pharmacogenetics in model systems: defining a common mechanism of action for mood stabilisers. Prog Neuropsychopharmacol Biol Psychiatry 29:1029–1037

    Article  PubMed  CAS  Google Scholar 

  35. Alexander H, Vomund AN, Alexander S (2003) Viability assay for Dictyostelium for use in drug studies. Biotechniques 35:464–470

    PubMed  CAS  Google Scholar 

  36. Min J, Sridevi P, Alexander S, Alexander H (2006) Sensitive cell viability assay for use in drug screens and for studying the mechanism of action of drugs in Dictyostelium discoideum. Biotechniques 41:591–595

    Article  PubMed  CAS  Google Scholar 

  37. Veldhoven V (1999) Sphingosine-1-phosphate lyase. Methods Enzymol 311:244–254

    Article  Google Scholar 

  38. Pewzner-Jung Y, Brenner O, Braun S, Laviad EL, Ben-Dor S, Feldmesser E, Horn-Saban S, Amann-Zalcenstein D, Raanan C, Berkutzki T, Erez-Roman R, Ben-David O, Levy M, Holzman D, Park H, Nyska A, Merrill AH Jr, Futerman AH (2010) A critical role for ceramide synthase 2 in liver homeostasis: II. insights into molecular changes leading to hepatopathy. J Biol Chem 285:10911–10923

    Article  PubMed  CAS  Google Scholar 

  39. Pewzner-Jung Y, Park H, Laviad EL, Silva LC, Lahiri S, Stiban J, Erez-Roman R, Brugger B, Sachsenheimer T, Wieland F, Prieto M, Merrill AH Jr, Futerman AH (2010) A critical role for ceramide synthase 2 in liver homeostasis: I. alterations in lipid metabolic pathways. J Biol Chem 285:10902–10910

    Article  PubMed  CAS  Google Scholar 

  40. King J, Insall R (2006) Parasexual genetics using axenic cells. Methods Mol Biol 346:125–135

    PubMed  CAS  Google Scholar 

  41. King J, Insall RH (2003) Parasexual genetics of Dictyostelium gene disruptions: identification of a ras pathway using diploids. BMC Genet 4:12

    Article  PubMed  Google Scholar 

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Acknowledgments

Work done in the authors’ laboratory was supported by the National Institute of General Medical Sciences (GM53929) and the University of Missouri Research Board (CB000359).

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Authors and Affiliations

  1. Division of Biological Sciences, University of Missouri, Columbia, MO, USA

    Stephen Alexander, William S. Swatson & Hannah Alexander

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  1. Stephen Alexander
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  2. William S. Swatson
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  3. Hannah Alexander
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Correspondence to Stephen Alexander .

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Editors and Affiliations

  1. , Centre for Biochemistry, University of Cologne, Joseph-Stelzmann-Str. 52, Köln, 50931, Nordrhein-Westfalen, Germany

    Ludwig Eichinger

  2. , Centre for Biomedical Research, University of Hull, Hull, HU6 7RX, Montserrat

    Francisco Rivero

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Alexander, S., Swatson, W.S., Alexander, H. (2013). Pharmacogenetics of Resistance to Cisplatin and Other Anticancer Drugs and the Role of Sphingolipid Metabolism. In: Eichinger, L., Rivero, F. (eds) Dictyostelium discoideum Protocols. Methods in Molecular Biology, vol 983. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-302-2_10

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  • DOI: https://doi.org/10.1007/978-1-62703-302-2_10

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-301-5

  • Online ISBN: 978-1-62703-302-2

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