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Alteration of apoptotic signaling molecules as a function of time after radiation in human neuroblastoma cells

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Abstract

Ascertaining the time-dependent regulation of induced apoptosis and radioresistance is important to understand the relationship between the level of spontaneous apoptosis in cells and their radiosensitivity. Accordingly, we investigated the time-dependent expression of apoptosis related genes and radioresistance in neuroblastoma cells. Serum-starved human SK-N-MC cells were exposed to low linear energy transfer (LET) radiation (2 Gy) and incubated for 15, 30, 45 min, and 48 h. Radioresistance was investigated by examining the NFκB DNA-binding activity, cellular toxicity, DNA fragmentation, and expression of apoptotic signal transduction molecules. NFκB DNA binding activity was analyzed using electrophoretic mobility shift assay (EMSA). Cellular toxicity was measured using MTT assay. DNA fragmentation was quantified by labeling with fluorescein-conjugated deoxynucleotides. Microarray analysis was performed using cDNA microarray and relative gene expression was measured as % GAPDH and, subsequently validated using Q-PCR. Induction of NFκB analyzed using EMSA showed an increased DNA-binding activity at all time points investigated. Induced DNA fragmentation was observed after 15, 30, and 45 min post-radiation. Relatively, induced fragmentation was reduced after 48 h. Compared to the untreated controls cellular toxicity was induced with low LET radiation after 15, 30, and 45 min. Conversely, cytotoxicity was relatively less at 48 h after low LET radiation. Microarray analysis after low LET radiation revealed time-dependent modulation of apoptosis-related genes that are involved in radio-adaptation, spontaneous apoptosis-related early-responsive genes and late response genes. These results suggest that the time-dependent regulation of apoptotic response may determine the relationship between the level of spontaneous apoptosis in cells and their radiosensitivity.

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References

  1. Bernstein ML, Leclerc JM, Bunin G, Brisson L et al (1992) A population-based study of neuroblastoma incidence, survival, and mortality in North America. J Clin Oncol 10:323–329

    PubMed  CAS  Google Scholar 

  2. Haas-Kogan DA, Swift PS, Selch M et al (2003) Impact of radiotherapy for high-risk neuroblastoma: a Children’s Cancer Group study. Int J Radiat Oncol Biol Phys 56:28–39

    Article  PubMed  Google Scholar 

  3. Ikeda H, August CS, Goldwein JW et al (1992) Sites of relapse in patients with neuroblastoma following bone marrow transplantation in relation to preparatory “debulking” treatments. J Pediatr Surg 27:1438–1441

    Article  PubMed  CAS  Google Scholar 

  4. Lonergan GJ, Schwab CM, Suarez ES, Carlson CL (2002) Neuroblastoma, ganglio neuroblastoma, and ganglioneuroma: radiologic-pathologic correlation. Radiographics 22:911–934

    PubMed  Google Scholar 

  5. Ladenstein R, Lasset C, Hartmann O et al (1993) Impact of megatherapy on survival after relapse from stage 4 neuroblastoma in patients over 1 year of age at diagnosis: a report from the European Group for Bone Marrow Transplantation. J Clin Oncol 11:2330–2341

    PubMed  CAS  Google Scholar 

  6. Kushner BH, Wolden S, LaQuaglia MP et al (2001) Hyperfractionated low-dose radiotherapy for high-risk neuroblastoma after intensive chemotherapy and surgery. J Clin Oncol 19:2821–2828

    PubMed  CAS  Google Scholar 

  7. Matthay KK, Villablanca JG, Seeger RC et al (1999) Treatment of high-risk neuroblastoma with intensive chemotherapy, radiotherapy, autologous bone marrow transplantation, and 13-cis-retinoic acid. Children’s Cancer Group. N Engl J Med 341:1165–1173

    Article  PubMed  CAS  Google Scholar 

  8. McGinn CJ, Kinsella TJ (1992) The experimental and clinical rationale for the use of S-phase-specific radiosensitizers to overcome tumor cell repopulation. Semin Oncol 19:21–28

    PubMed  CAS  Google Scholar 

  9. Chen X, Shen B, Xia L et al (2002) Activation of nuclear factor kappa B in radioresistance of TP53-inactive human keratinocytes. Cancer Res 62:1213–1221

    PubMed  CAS  Google Scholar 

  10. Li Z, Xia L, Lee LM et al (2001) Effector genes altered in MCF-7 human breast cancer cells after exposure to fractionated ionizing radiation. Radiat Res 155:543–553

    Article  PubMed  CAS  Google Scholar 

  11. Guo G, Yan-Sanders Y, Lyn-Cook BD et al (2003) Manganese superoxide dismutase-mediated gene expression in radiation-induced adaptive responses. Mol Cell Biol 23:2362–2378

    Article  PubMed  CAS  Google Scholar 

  12. Joki T, Carroll RS, Dunn IF et al (2001) Assessment of alterations in gene expression in recurrent malignant glioma after radiotherapy using complementary deoxyribonucleic acid microarrays. Neurosurgery 48:195–201

    Article  PubMed  CAS  Google Scholar 

  13. Lehnert S (2000) Prediction of tumor response to therapy: molecular markers and the microenvironment. Apoptosis and chips: an overview of the proceedings. Radiat Res 154:121–124

    Article  PubMed  CAS  Google Scholar 

  14. Ishigami T, Uzawa K, Higo M et al (2007) Genes and molecular pathways related to radioresistance of oral squamous cell carcinoma cells. Int J Cancer 120:2262–2270

    Article  PubMed  CAS  Google Scholar 

  15. Fukuda K, Sakakura C, Miyagawa K et al (2004) Differential gene expression profiles of radioresistant oesophageal cancer cell lines established by continuous fractionated irradiation. Br J Cancer 91:1543–1550

    Article  PubMed  CAS  Google Scholar 

  16. Rosen EM, Fan S, Rockwell S, Goldberg ID (1999) The molecular and cellular basis of radiosensitivity: implications for understanding how normal tissues and tumors respond to therapeutic radiation. Cancer Invest 17:56–72

    Article  PubMed  CAS  Google Scholar 

  17. Guo WF, Lin RX, Huang J et al (2005) Identification of differentially expressed genes contributing to radioresistance in lung cancer cells using microarray analysis. Radiat Res 164:27–35

    Article  PubMed  CAS  Google Scholar 

  18. Ogawa K, Utsunomiya T, Mimori K et al (2006) Differential gene expression profiles of radioresistant pancreatic cancer cell lines established by fractionated irradiation. Int J Oncol 28:705–713

    PubMed  CAS  Google Scholar 

  19. Fornace AJ Jr, Amundson SA, Bittner M et al (1999) The complexity of radiation stress responses:analysis by informatics and functional genomics approaches. Gene Expr 7:387–400

    PubMed  CAS  Google Scholar 

  20. Maity A, McKenna WG, Muschel RJ (1994) The molecular basis for cell cycle delays following ionizing radiation: a review. Radiother Oncol 31:1–13

    Article  PubMed  CAS  Google Scholar 

  21. Schmidt-Ullrich RK, Contessa JN, Dent P et al (1999) Molecular mechanisms of radiation-induced accelerated repopulation. Radiat Oncol Investig 7:321–330

    Article  PubMed  CAS  Google Scholar 

  22. Dewey WC, Ling CC, Meyn RE (1995) Radiation-induced apoptosis: relevance to radiotherapy. Int J Radiat Oncol Biol Phys 33:781–796

    PubMed  CAS  Google Scholar 

  23. Meyn RE, Stephens LC, Milas L (1996) Programmed cell death and radioresistance. Cancer Metastasis Rev 15:119–131

    Article  PubMed  CAS  Google Scholar 

  24. Sirzen F, Zhivotovsky B, Nilsson A et al (1998) Spontaneous and radiation-induced apoptosis in lung carcinoma cells with different intrinsic radiosensitivities. Anticancer Res 18:695–699

    PubMed  CAS  Google Scholar 

  25. Wheeler JA, Stephens LC, Tornos C et al (1995) ASTRO Research Fellowship: apoptosis as a predictor of tumor response to radiation in stage IB cervical carcinoma. Int J Radiat Oncol Biol Phys 32:1487–1493

    PubMed  CAS  Google Scholar 

  26. Dubray B, Breton C, Delic J et al (1997) In vitro radiation-induced apoptosis and tumour response to radiotherapy: a prospective study in patients with non-Hodgkin lymphomas treated by low-dose irradiation. Int J Radiat Biol 72:759–760

    Article  PubMed  CAS  Google Scholar 

  27. Levine EL, Renehan A, Gossiel R et al (1995) Apoptosis, intrinsic radiosensitivity and prediction of radiotherapy response in cervical carcinoma. Radiother Oncol 37:1–9

    Article  PubMed  CAS  Google Scholar 

  28. Aravindan N, Natarajan M, Shaw AD (2006) Fenoldopam inhibits nuclear translocation of nuclear factor kappa B in a rat model of surgical ischemic acute renal failure. J Cardiothorac Vasc Anesth 20:179–186

    Article  PubMed  CAS  Google Scholar 

  29. Aravindan N, Cata JP, Dougherty PM, Shaw AD (2006): Effect of fenoldopam on ischemia/reperfusion-induced apoptosis. Ren Fail 28:337–344

    Article  PubMed  CAS  Google Scholar 

  30. Aravindan N, Cata JP, Hoffman L et al (2006) Effects of isoflurane, pentobarbital, and urethane on apoptosis and apoptotic signal transduction in rat kidney. Acta Anaesthesiol Scand 50:1229–1237

    Article  PubMed  CAS  Google Scholar 

  31. Tang Y, Lu A, Aronow BJ et al (2002) Genomic responses of the brain to ischemic stroke, intracerebral haemorrhage, kainate seizures, hypoglycemia, and hypoxia. Eur J Neurosci 15:1937–1952

    Article  PubMed  Google Scholar 

  32. Vidair CA, Chen CH, Ling CC, Dewey WC (1996) Apoptosis induced by X-irradiation of rec-myc cells is postmitotic and not predicted by the time after irradiation or behavior of sister cells. Cancer Res 56:4116–4118

    PubMed  CAS  Google Scholar 

  33. Fanidi A, Harrington EA, Evan GI (1992) Cooperative interaction between c-myc and bcl-2 proto-oncogenes. Nature 359:554–556

    Article  PubMed  CAS  Google Scholar 

  34. Chen CH, Zhang J, Ling CC (1994) Transfected c-myc and c-Ha-ras modulate radiation-induced apoptosis in rat embryo cells. Radiat Res 139:307–315

    Article  PubMed  CAS  Google Scholar 

  35. Hallahan DE, Spriggs DR, Beckett MA et al (1989) Increased tumor necrosis factor alpha mRNA after cellular exposure to ionizing radiation. Proc Natl Acad Sci USA 86:10104–10107

    Article  PubMed  CAS  Google Scholar 

  36. Van Antwerp DJ, Martin SJ, Kafri T et al (1996) Suppression of TNF-alpha-induced apoptosis by NF-kappaB. Science 274:787–789

    Article  PubMed  Google Scholar 

  37. Voboril R, Weberova-Voborilova J (2006) Constitutive NF-kappaB activity in colorectal cancer cells: impact on radiation-induced NF-kappaB activity, radiosensitivity, and apoptosis. Neoplasma 53:518–523

    PubMed  CAS  Google Scholar 

  38. Nam SY, Chung HY (2005): The suppression of radiation-induced NF-kappaB activity by dexamethasone correlates with increased cell death in vivo. Biochem Biophys Res Commun 336:603–608

    Article  PubMed  CAS  Google Scholar 

  39. Kim BY, Kim KA, Kwon O (2005) NF-kappaB inhibition radiosensitizes Ki-Ras-transformed cells to ionizing radiation. Carcinogenesis 26:1395–1403

    Article  PubMed  CAS  Google Scholar 

  40. Nakanishi C, Toi M (2005) Nuclear factor-kappa B inhibitors as sensitizers to anticancer drugs. Nat Rev Cancer 5:297–309

    Article  PubMed  CAS  Google Scholar 

  41. Ravi R, Bedi A (2004) NF-kappaB in cancer—a friend turned foe. Drug Resist Updat 7:53–67

    Article  PubMed  CAS  Google Scholar 

  42. Boothman DA, Majmudar G, Johnson T (1994) Immediate X-ray-inducible responses from mammalian cells. Radiat Res 138:S44–S46

    Article  PubMed  CAS  Google Scholar 

  43. Prasad AV, Mohan N, Chandrasekar B, Meltz ML (1994) Activation of nuclear factor kappa B in human lymphoblastoid cells by low-dose ionizing radiation. Radiat Res 138:367–372

    Article  PubMed  CAS  Google Scholar 

  44. Matsubara J, Turcanu V, Poindron P, Ina Y (2000): Immune effects of low-dose radiation: short-term induction of thymocyte apoptosis and long-term augmentation of T-cell-dependent immune responses. Radiat Res 153:332–338

    Article  PubMed  CAS  Google Scholar 

  45. Skov KA (1994) Molecular, cellular, and genetic basis of radiosensitivity at low doses: a case of inducible repair? Radiat Res 138:S1–S4

    Article  PubMed  CAS  Google Scholar 

  46. Coleman MA, Yin E, Peterson LE et al (2005) Low-dose irradiation alters the transcript profiles of human lymphoblastoid cells including genes associated with cytogenetic radioadaptive response. Radiat Res 164:369–382

    Article  PubMed  CAS  Google Scholar 

  47. Koterov AN, Filippovich IV (2002) Radioadaptive response in vitro of unstimulated rat lymphocytes according to metallothionein test. Radiats Biol Radioecol 42:130–135

    PubMed  CAS  Google Scholar 

  48. Lanza V, Pretazzoli V, Olivieri G et al (2005) Transcriptional response of human umbilical vein endothelial cells to low doses of ionizing radiation. J Radiat Res (Tokyo) 46:265–276

    Article  CAS  Google Scholar 

  49. Hang H, Lieberman HB (2000) Physical interactions among human checkpoint control proteins HUS1p, RAD1p, and RAD9p, and implications for the regulation of cell cycle progression. Genomics 65:24–33

    Article  PubMed  CAS  Google Scholar 

  50. Eichholtz-Wirth H, Sagan D (2002) Altered signaling of TNFalpha-TNFR1 and SODD/BAG4 is responsible for radioresistance in human HT-R15 cells. Anticancer Res 22:235–240

    PubMed  CAS  Google Scholar 

  51. Neta R, Oppenheim JJ, Schreiber RD et al (1991) Role of cytokines (interleukin 1, tumor necrosis factor, and transforming growth factor beta) in natural and lipopolysaccharide-enhanced radioresistance. J Exp Med 173:1177–1182

    Article  PubMed  CAS  Google Scholar 

  52. Sandur SK, Ichikawa H, Sethi G et al (2006) Plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone) suppresses NF-kappaB activation and NF-kappaB-regulated gene products through modulation of p65 and IkappaBalpha kinase activation, leading to potentiation of apoptosis induced by cytokine and chemotherapeutic agents. J Biol Chem 281:17023–17033

    Article  PubMed  CAS  Google Scholar 

  53. Yi MJ, Park SH, Cho HN et al (2002) Heat-shock protein 25 (Hspb1) regulates manganese superoxide dismutase through activation of Nfkb (NF-kappaB). Radiat Res 158:641–649

    Article  PubMed  CAS  Google Scholar 

  54. Skvara H, Thallinger C, Wacheck V et al (2005) Mcl-1 blocks radiation-induced apoptosis and inhibits clonogenic cell death. Anticancer Res 25:2697–2703

    PubMed  CAS  Google Scholar 

  55. Chin C, Bae JH, Kim MJ et al (2005) Radiosensitization by targeting radioresistance-related genes with protein kinase A inhibitor in radioresistant cancer cells. Exp Mol Med 37:608–618

    PubMed  CAS  Google Scholar 

  56. Nix P, Cawkwell L, Patmore H et al (2005) Bcl-2 expression predicts radiotherapy failure in laryngeal cancer. Br J Cancer 92:2185–2189

    Article  PubMed  CAS  Google Scholar 

  57. Reed JC (1996) Mechanisms of Bcl-2 family protein function and dysfunction in health and disease. Behring Inst Mitt 97:72–100

    PubMed  CAS  Google Scholar 

  58. Schwartz JL, Jordan R, Slovic J et al (2007) Induction and loss of a TP53-dependent radioadaptive response in the human lymphoblastoid cell model TK6 and its abrogation by BCL2 over-expression. Int J Radiat Biol 83:153–159

    Article  PubMed  CAS  Google Scholar 

  59. Ferrer I (1999) Role of caspases in ionizing radiation-induced apoptosis in the developing cerebellum. J Neurobiol 41:549–558

    Article  PubMed  CAS  Google Scholar 

  60. Ramaswamy M, Efimova EV, Martinez O et al (2004) IG20 (MADD splice variant-5), a proapoptotic protein, interacts with DR4/DR5 and enhances TRAIL-induced apoptosis by increasing recruitment of FADD and caspase-8 to the DISC. Oncogene 23:6083–6094

    Article  PubMed  CAS  Google Scholar 

  61. Heminger K, Jain V, Kadakia M et al (2006) Altered gene expression induced by ionizing radiation and glycolytic inhibitor 2-deoxy-glucose in a human glioma cell line: implications for radio sensitization. Cancer Biol Ther 5:815–823

    PubMed  CAS  Google Scholar 

  62. Hildesheim J, Bulavin DV, Anver MR (2002) Gadd45a protects against UV irradiation-induced skin tumors, and promotes apoptosis and stress signaling via MAPK and p53. Cancer Res 62:7305–7315

    PubMed  CAS  Google Scholar 

  63. Morgan MJ, Thorburn J, Thomas L et al (2001) An apoptosis signaling pathway induced by the death domain of FADD selectively kills normal but not cancerous prostate epithelial cells. Cell Death Differ 8:696–705

    Article  PubMed  CAS  Google Scholar 

  64. Chazal M, Marionnet C, Michel L et al (2002) P16(INK4A) is implicated in both the immediate and adaptative response of human keratinocytes to UVB irradiation. Oncogene 21:2652–2661

    Article  PubMed  CAS  Google Scholar 

  65. Kataoka T, Schroter M, Hahne M et al (1998) FLIP prevents apoptosis induced by death receptors but not by perforin/granzyme B, chemotherapeutic drugs, and gamma irradiation. J Immunol 161:3936–3942

    PubMed  CAS  Google Scholar 

  66. Chaudhry MA (2006) Radiation-induced gene expression profile of human cells deficient in 8-hydroxy-2′-deoxyguanine glycosylase. Int J Cancer 118:633–642

    Article  PubMed  CAS  Google Scholar 

  67. Padanilam BJ (2003) Cell death induced by acute renal injury: a perspective on the contributions of apoptosis and necrosis. Am J Physiol Renal Physiol 284:F608–F627

    PubMed  CAS  Google Scholar 

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Authors and Affiliations

  1. OUPB 1430, Department of Radiation Oncology, College of Medicine, University of Oklahoma Health sciences Center, 825 North East 10th Street, Oklahoma City, OK, 73104, USA

    Natarajan Aravindan, Rakhesh Madhusoodhanan & Terence S. Herman

  2. Department of Radiation Oncology, University of Texas Health Science Center at San Antonio, San Antonio, TX, USA

    Mohan Natarajan

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  1. Natarajan Aravindan
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  2. Rakhesh Madhusoodhanan
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  3. Mohan Natarajan
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Correspondence to Natarajan Aravindan.

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Aravindan, N., Madhusoodhanan, R., Natarajan, M. et al. Alteration of apoptotic signaling molecules as a function of time after radiation in human neuroblastoma cells. Mol Cell Biochem 310, 167–179 (2008). https://doi.org/10.1007/s11010-007-9678-0

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