Advertisement

Strahlentherapie und Onkologie

, Volume 185, Issue 5, pp 331–338 | Cite as

Additive Effects of 5-Aza-2’-deoxycytidine and Irradiation on Clonogenic Survival of Human Medulloblastoma Cell Lines

  • Ina Patties
  • Jutta Jahns
  • Guido Hildebrandt
  • Rolf-Dieter Kortmann
  • Annegret Glasow
Original Article

Background and Purpose:

In recent years, epigenetic modulators were introduced into tumor therapy. Here, the authors investigated the antitumor effect of 5-aza-2’-deoxycytidine-(5-aza-dC-)induced demethylation combined with irradiation on human medulloblastoma (MB) cells, which form the most common malignant brain tumor in children.

Material and Methods:

Three MB cell lines were treated with 5-aza-dC in a low-dose (0.1 μM, 6 days) or high-dose (3/5 μM, 3 days) setting and irradiated with 2, 4, 6, or 8 Gy single dose on an X-ray unit. Methylation status and mRNA expression of three candidate genes were analyzed by methylation-specific PCR (polymerase chain reaction) and quantitative real-time RT-PCR. Cell survival and mortality were determined by trypan blue exclusion test. Proliferation was analyzed by BrdU incorporation assay, and long-term cell survival was assessed by clonogenic assay.

Results:

5-aza-dC treatment resulted in partial promoter demethylation and increased expression of hypermethylated candidate genes. A significant decrease of vital cell count, proliferation inhibition and increase of mortality was observed in 5-aza-dC-treated as well as in irradiated MB cells, whereby combination of both treatments led to additive effects. Although high-dose 5-aza-dC treatment was more effective in terms of demethylation, clonogenic assay revealed no differences between high- and low-dose settings indicating no relevance of 5-aza-dC-induced demethylation for decreased cell survival. MB cells pretreated with 5-aza-dC showed significantly lower plating efficiencies than untreated cells at all irradiation doses investigated. Analysis of surviving curves in irradiated MB cells, however, revealed no significant differences of α-, β-values and 2-Gy surviving fraction with or without 5-aza-dC treatment.

Conclusion:

5-aza-dC did not enhance radiation sensitivity of MB cells but significantly reduced the clonogenicity versus irradiation alone, which merits further investigation of its potential clinical application in MB possibly by combination with other chemotherapeutic agents.

Key Words:

Irradiation Epigenetic Medulloblastoma Clonogenic assay 5-aza-2’-deoxycytidine Radiosensitivity 

Additive Effekte von 5-Aza-2’-deoxycytidin und Bestrahlung auf das klonogene Überleben von humanen Medulloblastomzelllinien

Hintergrund und Ziel:

In den letzten Jahren wurden epigenetische Modulatoren in die Tumortherapie eingeführt. In dieser Arbeit untersuchten die Autoren den Antitumoreffekt der 5-Aza-2’-deoxycytidin-(5-aza-dC-)induzierten Demethylierung in Kombination mit Bestrahlung auf humane Medulloblastom-(MB-)Zellen, welche die häufigsten malignen Hirntumoren im Kindesalter bilden.

Material und Methodik:

Drei MB-Zell-Linien wurden mit 5-aza-dC in Niedrigdosis (0,1 μM, 6 Tage) oder Hochdosis (3/5 μM, 3 Tage) behandelt und mit 2, 4, 6 oder 8 Gy Einzeldosis bestrahlt. Die Untersuchung des Methylierungsstatus und der mRNA-Expression von drei Kandidatengenen erfolgte durch methylierungsspezifische PCR (Polymerase-Kettenreaktion) und quantitative Real-Time-RT-PCR. Lebendzellraten und Mortalität wurden durch Trypanblau-Ausschlusstest und die Proliferationsrate im BrdU-Assay bestimmt. Das Langzeitüberleben wurde durch einen klonogenen Assay ermittelt.

Ergebnisse:

Die 5-aza-dC-Behandlung führte zur partiellen Promotordemethylierung und zu einem Anstieg der Expression hypermethylierter Kandidatengene. Eine signifikante Verminderung der Lebendzellzahl und Proliferation bei Zunahme der Mortalität wurde sowohl in 5-aza-dC-behandelten als auch in bestrahlten MB-Zellen beobachtet. Bei kombinierter Behandlung summierten sich die Effekte der Einzelbehandlungen. Während die Inkubation mit 5-aza-dC in Hochdosis hinsichtlich der Demethylierung effektiver war als in Niedrigdosis, ergaben sich im klonogenen Assay keine Unterschiede zwischen beiden Behandlungsschemata, was darauf hinweist, dass die 5-aza-dC-induzierte Demethylierung nicht relevant für die Verminderung des Zellüberlebens ist. Mit 5-aza-dC vorbehandelte Zellen zeigten im untersuchten Bestrahlungsdosisbereich eine signifikant niedrigere „plating efficiency“ als unbehandelte Zellen. Die Überlebenskurven bestrahlter MB-Zellen wiesen jedoch keine signifikanten Unterschiede der α/β-Werte und der Überlebensfraktion nach 2 Gy in 5-aza-dC-behandelten gegenüber unbehandelten Zellen auf.

Schlussfolgerung:

5-aza-dC erhöhte die Strahlensensitivität von MB-Zellen nicht, reduzierte jedoch die Klonogenität signifikant gegenüber alleiniger Bestrahlung, was weitere Untersuchungen zur potentiellen klinischen Anwendung von 5-aza-dC bei MB, möglicherweise in Kombination mit anderen Chemotherapeutika, rechtfertigt.

Schlüsselwörter:

Bestrahlung Epigenetik Medulloblastom Klonogener Assay 5-Aza-2’-deoxycytidin Radiosensitivität 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. 1.
    Aparicio A, Eads CA, Leong LA, et al. I trial of continuous infusion 5-aza-2′-deoxycytidine. Cancer Chemother Pharmacol 2003;51:231–9.PubMedGoogle Scholar
  2. 2.
    Appleton K, Mackay HJ, Judson I, et al. Phase I and pharmacodynamic trial of the DNA methyltransferase inhibitor decitabine and carboplatin in solid tumors. J Clin Oncol 2007;25:4603–9.PubMedCrossRefGoogle Scholar
  3. 3.
    Atallah E, Kantarjian H, Garcia-Manero G. The role of decitabine in the treatment of myelodysplastic syndromes. Expert Opin Pharmacother 2007;8: 65–73.PubMedCrossRefGoogle Scholar
  4. 4.
    Chabot GG, Rivard GE, Momparler RL. Plasma and cerebrospinal fluid pharmacokinetics of 5-aza-2′-deoxycytidine in rabbits and dogs. Cancer Res 1983;43:592–7.PubMedGoogle Scholar
  5. 5.
    Chai G, Li L, Zhou W, et al. HDAC inhibitors act with 5-aza-2′-deoxycytidine to inhibit cell proliferation by suppressing removal of incorporated abases in lung cancer cells. PLoS ONE 2008;3:e2445.PubMedCrossRefGoogle Scholar
  6. 6.
    Clark SJ, Harrison J, Paul CL, et al. High sensitivity mapping of methylated cytosines. Nucleic Acids Res 1994;22:2990–7.PubMedCrossRefGoogle Scholar
  7. 7.
    Crawford JR, MacDonald TJ, Packer RJ. Medulloblastoma in childhood: new biological advances. Lancet Neurol 2007;6:1073–85.PubMedCrossRefGoogle Scholar
  8. 8.
    Egger G, Liang G, Aparicio A, et al. Epigenetics in human disease and prospects for epigenetic therapy. Nature 2004;429:457–63.PubMedCrossRefGoogle Scholar
  9. 9.
    Fang X, Zheng C, Liu Z, et al. Enhanced sensitivity of prostate cancer DU145 cells to cisplatinum by 5-aza-2′-deoxycytidine. Oncol Rep 2004;:523–6.PubMedGoogle Scholar
  10. 10.
    Glasow A, Prodromou N, Xu K, et al. Retinoids and myelomonocytic growth factors cooperatively activate RARA and induce human myeloid leukemia cell differentiation via MAP kinase pathways. Blood 2005;105:341–9.PubMedCrossRefGoogle Scholar
  11. 11.
    Gollob JA, Sciambi CJ, Peterson BL, et al. Phase I trial of sequential low-dose 5-aza-2′-deoxycytidine plus high-dose intravenous bolus interleukin-2 in patients with melanoma or renal cell carcinoma. Clin Cancer Res 2006;12:4619–27.PubMedCrossRefGoogle Scholar
  12. 12.
    Gonzalez-Gomez P, Bello MJ, Alonso ME, et al. Promoter methylation status of multiple genes in brain metastases of solid tumors. Int J Mol Med 2004;13:93–8.PubMedGoogle Scholar
  13. 13.
    Hansen MB, Skov L, Menne T, et al. Gene transcripts as potential diagnostic markers for allergic contact dermatitis. Contact Dermatitis 2005;53:100–6.PubMedCrossRefGoogle Scholar
  14. 14.
    Harada K, Toyooka S, Shivapurkar N, et al. Deregulation of caspase 8 and 10 expression in pediatric tumors and cell lines. Cancer Res 2002;62:5897–901.PubMedGoogle Scholar
  15. 15.
    Hou P, Ji M, Yang B, et al. Quantitative analysis of promoter hypermethylation in multiple genes in osteosarcoma. Cancer 2006;106:1602–9.PubMedCrossRefGoogle Scholar
  16. 16.
    Jozwiak J, Grajkowska W, Wlodarski P. Pathogenesis of medulloblastoma and current treatment outlook. Med Res Rev 2007;27:869–90.PubMedCrossRefGoogle Scholar
  17. 17.
    Kanai Y, Hui AM, Sun L, et al. DNA hypermethylation at the D17S5 locus and reduced HIC-1 mRNA expression are associated with hepatocarcinogenesis. Hepatology 1999;29:703–9.PubMedCrossRefGoogle Scholar
  18. 18.
    Karagiannis TC, El-Osta A. Modulation of cellular radiation responses by histone deacetylase inhibitors. Oncogene 2006;25:3885–93.PubMedCrossRefGoogle Scholar
  19. 19.
    Khan R, Aggerholm A, Hokland P, et al. A pharmacodynamic study of 5-azacytidine in the P39 cell line. Exp Hematol 2006;34:35–43.PubMedCrossRefGoogle Scholar
  20. 20.
    Kortmann RD, Kuhl J, Timmermann B, et al. Current and future strategies in interdisciplinary treatment of medulloblastomas, supratentorial PNET (primitive neuroectodermal tumors) and intracranial germ cell tumors in childhood. Strahlenther Onkol 2001;177:447–61.PubMedCrossRefGoogle Scholar
  21. 21.
    Kuzmin I, Liu L, Dammann R, et al. Inactivation of RAS association domain family 1A gene in cervical carcinomas and the role of human papillomavirus infection. Cancer Res 2003;63:1888–93.PubMedGoogle Scholar
  22. 22.
    Li LC, Dahiya R. MethPrimer: designing primers for methylation PCRs. Bioinformatics 2002;18:1427–31.PubMedCrossRefGoogle Scholar
  23. 23.
    Lindsey JC, Lusher ME, Anderton JA, et al. Identification of tumor-specific epigenetic events in medulloblastoma development by hypermethylation profiling. Carcinogenesis 2004;25:661–8.PubMedCrossRefGoogle Scholar
  24. 24.
    Lindsey JC, Lusher ME, Anderton JA, et al. Epigenetic deregulation of multiple S100 gene family members by differential hypomethylation and hypermethylation events in medulloblastoma. Br J Cancer 2007;97:267–74.PubMedCrossRefGoogle Scholar
  25. 25.
    Lusher ME, Lindsey JC, Latif F, et al. Biallelic epigenetic inactivation of the RASSF1A tumor suppressor gene in medulloblastoma development. Cancer Res 2002;62:5906–11.PubMedGoogle Scholar
  26. 26.
    Momparler RL, Bouffard DY, Momparler LF, et al. Pilot phase I–II study on 5-aza-2′-deoxycytidine (decitabine) in patients with metastatic lung cancer. Anticancer Drugs 1997;8:358–68.PubMedCrossRefGoogle Scholar
  27. 27.
    Mund C, Hackanson B, Stresemann C, et al. Characterization of DNA demethylation effects induced by 5-aza-2′-deoxycytidine in patients with myelodysplastic syndrome. Cancer Res 2005;65:7086–90.PubMedCrossRefGoogle Scholar
  28. 28.
    Munshi A, Kurland JF, Nishikawa T, et al. Histone deacetylase inhibitors radiosensitize human melanoma cells by suppressing DNA repair activity. Clin Cancer Res 2005;11:4912–22.PubMedCrossRefGoogle Scholar
  29. 29.
    Palii SS, Van Emburgh BO, Sankpal UT, et al. DNA methylation inhibitor 5-aza-2′-deoxycytidine induces reversible genome-wide DNA damage that is distinctly influenced by DNA methyltransferases 1 and 3B. Mol Cell Biol 2008;28:752–71.PubMedCrossRefGoogle Scholar
  30. 30.
    Patel R, Shervington L, Lea R, et al. Epigenetic silencing of telomerase and a non-alkylating agent as a novel therapeutic approach for glioma. Brain Res 2008;1188:173–81.PubMedCrossRefGoogle Scholar
  31. 31.
    Samlowski WE, Leachman SA, Wade M, et al. Evaluation of a 7-day continuous intravenous infusion of decitabine: inhibition of promoter-specific and global genomic DNA methylation. J Clin Oncol 2005;23:3897–905.PubMedCrossRefGoogle Scholar
  32. 32.
    Schmelz K, Sattler N, Wagner M, et al. Induction of gene expression by 5-aza-2′-deoxycytidine in acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) but not epithelial cells by DNA-methylation-dependent and -independent mechanisms. Leukemia 2005;19:103–11.PubMedGoogle Scholar
  33. 33.
    Shang D, Ito N, Kamoto T, et al. Demethylating agent 5-aza-2′-deoxycytidine enhances susceptibility of renal cell carcinoma to paclitaxel. Urology 2007;69:1007–12.PubMedCrossRefGoogle Scholar
  34. 34.
    Shang D, Liu Y, Matsui Y, et al. Demethylating agent 5-aza-2′-deoxycytidine enhances susceptibility of bladder transitional cell carcinoma to cisplatin. Urology 2008;71:1220–5.PubMedCrossRefGoogle Scholar
  35. 35.
    Sterzing F, Schubert K, Sroka-Perez G, et al. Helical tomotherapy. Experiences of the first 150 patients in Heidelberg. Strahlenther Onkol 2008;184: 8–14.PubMedCrossRefGoogle Scholar
  36. 36.
    Strenger V, Sovinz P, Lackner H, et al. Intracerebral cavernous hemangioma after cranial irradiation in childhood. Incidence and risk factors. Strahlenther Onkol 2008;184:276–80.PubMedCrossRefGoogle Scholar
  37. 37.
    Timmermann B, Kortmann RD, Kuhl J, et al. Combined postoperative irradiation and chemotherapy for anaplastic ependymomas in childhood: results of the German prospective trials HIT 88/89 and HIT 91. Int J Radiat Oncol Biol Phys 2000;46:287–95.PubMedCrossRefGoogle Scholar
  38. 38.
    Timmermann B, Lomax AJ, Nobile L, et al. Novel technique of craniospinal axis proton therapy with the spot-scanning system. Avoidance of patching multiple fields and optimized ventral dose distribution. Strahlenther Onkol 2007;183:685–8.PubMedCrossRefGoogle Scholar
  39. 39.
    Vibhakar R, Foltz G, Yoon JG, et al. Dickkopf-1 is an epigenetically silenced candidate tumor suppressor gene in medulloblastoma. Neurooncology 2007;9:135–44.Google Scholar
  40. 40.
    Welzel G, Fleckenstein K, Mai SK, et al. Acute neurocognitive impairment during cranial radiation therapy in patients with intracranial tumors. Strahlenther Onkol 2008;184:647–54.PubMedCrossRefGoogle Scholar
  41. 41.
    Zhang Y, Jung M, Dritschilo A, et al. Enhancement of radiation sensitivity of human squamous carcinoma cells by histone deacetylase inhibitors. Radiat Res 2004;161:667–74.PubMedCrossRefGoogle Scholar

Copyright information

© Urban & Vogel, Muenchen 2009

Authors and Affiliations

  • Ina Patties
    • 1
  • Jutta Jahns
    • 1
  • Guido Hildebrandt
    • 1
    • 2
  • Rolf-Dieter Kortmann
    • 1
  • Annegret Glasow
    • 1
    • 3
  1. 1.Department of Radiotherapy and RadiooncologyUniversitätsklinikum Leipzig AöRLeipzigGermany
  2. 2.Department of RadiotherapyUniversity of RostockRostockGermany
  3. 3.Klinik und Poliklinik für Strahlentherapie und RadioonkologieUniversitätsklinikum Leipzig AöRLeipzigGermany

Personalised recommendations