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

  • Stephen AlexanderEmail author
  • William S. Swatson
  • Hannah Alexander
Part of the Methods in Molecular Biology book series (MIMB, volume 983)


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.

Key words

Sphingosine-1-phosphate Sphingosine kinase Sphingosine-1-phosphate lyase Ceramide Chemotherapy 



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).


  1. 1.
    AmericanCancerSociety (2010) Cancer facts and figures (2010)
  2. 2.
    Siddik ZH (2003) Cisplatin: mode of cytotoxic action and molecular basis of resistance. Oncogene 22:7265–7279PubMedCrossRefGoogle Scholar
  3. 3.
    Rabik CA, Dolan ME (2007) Molecular mechanisms of resistance and toxicity associated with platinating agents. Cancer Treat Rev 33:9–23PubMedCrossRefGoogle Scholar
  4. 4.
    Wernyj RP, Morin PJ (2004) Molecular mechanisms of platinum resistance: still searching for the Achilles’ heel. Drug Resist Updat 7:227–232PubMedCrossRefGoogle Scholar
  5. 5.
    Williams JG (2010) Dictyostelium finds new roles to model. Genetics 185:717–726PubMedCrossRefGoogle Scholar
  6. 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 12:415–424PubMedCrossRefGoogle Scholar
  7. 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 418:79–85PubMedCrossRefGoogle Scholar
  8. 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 435:43–57PubMedCrossRefGoogle Scholar
  9. 9.
    Kessin RH (2001) Dictyostelium—evolution, cell biology, and the development of multicellularity. Cambridge Univ. Press, CambridgeCrossRefGoogle Scholar
  10. 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 89:8803–8807PubMedCrossRefGoogle Scholar
  11. 11.
    Loomis WF (1987) Genetic tools for Dictyostelium discoideum. Methods Cell Biol 28:31–65PubMedCrossRefGoogle Scholar
  12. 12.
    Newell PC (1982) Genetics. In: Loomis WF (ed) The development of Dictyostelium discoideum. Academic, New YorkGoogle Scholar
  13. 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 148:333–340PubMedGoogle Scholar
  14. 14.
    Li GC, Alexander H, Schneider N, Alexander S (2000) Molecular basis for resistance to the anticancer drug cisplatin in Dictyostelium. Microbiology 146:2219–2227PubMedGoogle Scholar
  15. 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 1760:301–309PubMedCrossRefGoogle Scholar
  16. 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 3:795–805PubMedCrossRefGoogle Scholar
  17. 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 4:178–189PubMedCrossRefGoogle Scholar
  18. 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 3:287–296PubMedCrossRefGoogle Scholar
  19. 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 5:801–812PubMedCrossRefGoogle Scholar
  20. 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 316:78–91PubMedCrossRefGoogle Scholar
  21. 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 1793:1218–1227PubMedCrossRefGoogle Scholar
  22. 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 103:17384–17389PubMedCrossRefGoogle Scholar
  23. 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 279:1281–1290PubMedCrossRefGoogle Scholar
  24. 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 22:97–104PubMedCrossRefGoogle Scholar
  25. 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 104:15406–15411PubMedCrossRefGoogle Scholar
  26. 26.
    Sussman M (1987) Cultivation and synchronous morphogenesis of Dictyostelium under controlled experimental conditions. Methods Cell Biol 28:9–29PubMedCrossRefGoogle Scholar
  27. 27.
    Manstein DJ, Schuster HP, Morandini P, Hunt DM (1995) Cloning vectors for the production of proteins in Dictyostelium discoideum. Gene 162:129–134PubMedCrossRefGoogle Scholar
  28. 28.
    Loomis WF, Kuspa A (eds) (2005) Dictyostelium genomics. Horizon Bioscience, NorfolkGoogle Scholar
  29. 29.
    Adley KE, Keim M, Williams RS (2006) Pharmacogenetics: defining the genetic basis of drug action and inositol trisphosphate analysis. Methods Mol Biol 346:517–534PubMedGoogle Scholar
  30. 30.
    Keim M, Williams RS, Harwood AJ (2004) An inverse PCR technique to rapidly isolate the flanking DNA of Dictyostelium insertion mutants. Mol Biotechnol 26:221–224PubMedCrossRefGoogle Scholar
  31. 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:e143PubMedCrossRefGoogle Scholar
  32. 32.
    Dubin M, Nellen W (2010) A versatile set of tagged expression vectors to monitor protein localisation and function in Dictyostelium. Gene 465:1–8PubMedCrossRefGoogle Scholar
  33. 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 128:3473–3483PubMedGoogle Scholar
  34. 34.
    Williams RS (2005) Pharmacogenetics in model systems: defining a common mechanism of action for mood stabilisers. Prog Neuropsychopharmacol Biol Psychiatry 29:1029–1037PubMedCrossRefGoogle Scholar
  35. 35.
    Alexander H, Vomund AN, Alexander S (2003) Viability assay for Dictyostelium for use in drug studies. Biotechniques 35:464–470PubMedGoogle Scholar
  36. 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 41:591–595PubMedCrossRefGoogle Scholar
  37. 37.
    Veldhoven V (1999) Sphingosine-1-phosphate lyase. Methods Enzymol 311:244–254CrossRefGoogle Scholar
  38. 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 285:10911–10923PubMedCrossRefGoogle Scholar
  39. 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 285:10902–10910PubMedCrossRefGoogle Scholar
  40. 40.
    King J, Insall R (2006) Parasexual genetics using axenic cells. Methods Mol Biol 346:125–135PubMedGoogle Scholar
  41. 41.
    King J, Insall RH (2003) Parasexual genetics of Dictyostelium gene disruptions: identification of a ras pathway using diploids. BMC Genet 4:12PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2013

Authors and Affiliations

  • Stephen Alexander
    • 1
    Email author
  • William S. Swatson
    • 1
  • Hannah Alexander
    • 1
  1. 1.Division of Biological SciencesUniversity of MissouriColumbiaUSA

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