Skip to main content

Advertisement

SpringerLink
Log in

Differentiation Decreased Telomerase Activity in Rat Glioblastoma C6 Cells and Increased Sensitivity to IFN-γ and Taxol for Apoptosis

  • Original Paper
  • Published:
Neurochemical Research Aims and scope Submit manuscript

Abstract

Glioblastoma is the deadliest and most prevalent brain tumor, which is not yet amenable to any treatments. Therefore, new and innovative therapeutic strategies need to be developed for treating this deadly disease. We found that all-trans retinoic acid (ATRA) or 13-cis retinoic acid (13-CRA) induced astrocytic differentiation with down regulation of telomerase activity in rat glioblastoma C6 cells and enhanced sensitivity of the cells to interferon-gamma (IFN-γ) or taxol (TXL) for apoptosis. Sensitivity of differentiated cells to IFN-γ or TXL was greatly increased for apoptosis with increases in calcineurin expression, Bax:Bcl-2 ratio, mitochondrial release of cytochrome c, and expression and activity of calpain and caspases. Treatment with IFN-γ activated caspase-8 indicating induction of apoptosis via the receptor-mediated pathway. Notably, IFN-γ activated the signal transducer and activator of transcription-1 (STAT-1) for signaling via binding to gamma activator sequence (GAS), whereas TXL activated Raf-1 kinase for inactivation of Bcl-2 by its phosphorylation. We confirmed involvement of different proteolytic mechanisms in cell death by pretreating the cells with caspase-8 inhibitor II, calpeptin (calpain inhibitor), and caspase-9 inhibitor I, and caspase-3 inhibitor IV. Results demonstrated that retinoids induced astrocytic differentiation with down regulation of telomerase activity and worked synergistically to enhance sensitivity of cells to the cytotoxic agent IFN-γ and the cytostatic agent TXL for apoptosis. This combination therapy for differentiation and apoptosis could be highly effective for controlling the malignant growth of glioblastoma.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8
Fig. 9
Fig. 10
Fig. 11
Fig. 12
Fig. 13
Fig. 14
Fig. 15

Similar content being viewed by others

References

  1. Rasheed BK, Wiltshire RN, Bigner SH, Bigner DD (1999) Molecular pathogenesis of malignant gliomas. Curr Opin Oncol 11:162–167

    Article  PubMed  CAS  Google Scholar 

  2. Breitman TR, Collins SJ, Keene BR (1981) Terminal differentiation of human promyelocytic leukemic cells in primary culture in response to retinoic acid. Blood 57:1000–1004

    PubMed  CAS  Google Scholar 

  3. Reynolds CP, Kane DJ, Einhorn PA, Matthay KK, Crouse VL, Wilbur JR, Shurin SB, Seeger RC (1991) Response of neuroblastoma to retinoic acid in vitro and in vivo. Prog Clin Biol Res 366:203–211

    PubMed  CAS  Google Scholar 

  4. Bunn PA Jr, Hoffman SJ, Norris D, Golitz LE, Aeling JL (1994) Systemic therapy of cutaneous T-cell lymphomas (mycosis fungoides and the Sezary syndrome). Ann Intern Med 121:592–602

    PubMed  Google Scholar 

  5. Aebi S, Kroning R, Cenni B, Sharma A, Fink D, Los G, Weisman R, Howell SB, Christen RD (1997) All-trans retinoic acid enhances cisplatin-induced apoptosis in human ovarian adenocarcinoma and in squamous head and neck cancer cells. Clin Cancer Res 3:2033–2038

    PubMed  CAS  Google Scholar 

  6. Todesco A, Carli M, Iacona I, Frascella E, Ninfo V, Rosolen A (2000) All-trans retinoic acid and interferon-alpha in the treatment of a patient with esistant metastatic osteosarcoma. Cancer 89:2661–2666

    Article  PubMed  CAS  Google Scholar 

  7. Marley SB, Davidson RJ, Goldman JM, Gordon MY (2002) Effects of combinations of therapeutic agents on the proliferation of progenitor cells in chronic myeloid leukaemia. Br J Haematol 116:162–165

    Article  PubMed  CAS  Google Scholar 

  8. Pili R, Kruszewski MP, Hager BW, LantzJ, Carducci MA (2001) Combination of phenylbutyrate and 13-cis retinoic acid inhibits prostate tumor growth and angiogenesis. Cancer Res 61:1477–1485

    PubMed  CAS  Google Scholar 

  9. Thalasila A, Poplin E, Shih J, Dvorzhinski D, Capanna T, Doyle-Lindrud S, Beers S, Goodin S, Rubin E, DiPaola RS (2003) A phase I trial of weekly paclitaxel, 13-cis retinoic acid, and interferon-α in patients with prostate cancer and other advanced malignancies. Cancer Chemother Pharmacol 52:119–124

    Article  PubMed  CAS  Google Scholar 

  10. Vaishampayan U, Flaherty L, Du W, Hussain M (2001) Phase II evaluation of paclitaxel, α-interferon, and cis-retinoic acid in advanced renal cell carcinoma. Cancer 92:519–523

    Article  PubMed  CAS  Google Scholar 

  11. Allenby G, Bocquel M T, Saunders M, Kazmer S, Speck J, Rosenberger M, Lovey A, Kastner P, Grippo JF, Chambon P, et al (1993) Retinoic acid receptors and retinoid X receptors: interactions with endogenous retinoic acids. Proc Natl Acad Sci USA 90:30–34

    Article  PubMed  CAS  Google Scholar 

  12. Veal GJ, Errington J, Redfern CP, Pearson AD, Boddy AV (2002) Influence of isomerisation on the growth inhibitory effects and cellular activity of 13-cis and all-trans retinoic acid in neuroblastoma cells. Biochem Pharmacol 63:207–215

    Article  PubMed  CAS  Google Scholar 

  13. Fujiwara-Akita H, Maesawa C, Honda T, Kobayashi S, Masuda T (2005) Expression of human telomerase reverse transcriptase splice variants is well correlated with low telomerase activity in osteosarcoma cell lines. Int J Oncol 26:1009–1016

    PubMed  CAS  Google Scholar 

  14. Tchirkov A, Rolhion C, Kemeny JL, Irthum B, Puget S, Khalil T, Chinot O, Kwiatkowski F, Perissel B, Vago P, Verrelle P (2003) Clinical implications of quantitative real-time RT-PCR analysis of hTERT gene expression in human gliomas. Br J Cancer 88:516–520

    Article  PubMed  CAS  Google Scholar 

  15. Liu WJ, Zhang YW, Zhang ZX, Ding J (2004) Alternative splicing of human telomerase reverse transcriptase may not be involved in telomerase regulation during all-trans retinoic acid-induced HL-60 cell differentiation. J Pharmacol Sci 96:106–114

    Article  PubMed  CAS  Google Scholar 

  16. Kominsky S, Johnson HM, Bryan G, Tanabe T, Hobeika AC, Subramaniam PS, Torres B (1998) IFN-γ inhibition of cell growth in glioblastomas correlates with increased levels of the cyclin dependent kinase inhibitor p21WAF1/CIP1. Oncogene 17:2973–2979

    Article  PubMed  CAS  Google Scholar 

  17. Tada M, Diserens AC, Desbaillets I, de Tribolet N (1994) Analysis of cytokine receptor messenger RNA expression in human glioblastoma cells and normal astrocytes by reverse-transcription polymerase chain reaction. J Neurosurg 80:1063–1073

    Article  PubMed  CAS  Google Scholar 

  18. Knupfer MM, Poppenborg H, Van Gool S, Domula M, Wolff JE (1997) IFN-γ inhibits proliferation and adhesion of T98G human malignant glioma cells in vitro. Klin Padiatr 209:271–274

    PubMed  CAS  Google Scholar 

  19. Bach EA, Aguet M, Schreiber RD (1997) The IFN-γ receptor: a paradigm for cytokine receptor signaling. Annu Rev Immunol 15: 563–591

    Article  PubMed  CAS  Google Scholar 

  20. Decker T, Kovarik P, Meinke A (1997) GAS elements: a few nucleotides with a major impact on cytokine-induced gene expression. J Interferon Cytokine Res 17:121–134

    PubMed  CAS  Google Scholar 

  21. Kano A, Watanabe Y, Takeda N, Aizawa S, Akaike T (1997) Interferon-gamma inhibits proliferation and adhesion of T98G human malignant glioma cells in vitro. J Biochem (Tokyo) 121:677–683

    CAS  Google Scholar 

  22. Ossina NK, Cannas A, Powers VC, Fitzpatrick PA, Knight JD, Gilbert JR, Shekhtman EM, Tomei LD, Umansky SR, Kiefer MC (1997) IFN-γ modulates a p53-independent apoptotic pathway and apoptosis-related gene expression. J Biol Chem 272:16351–16357

    Article  PubMed  CAS  Google Scholar 

  23. Ray SK, Wilford GG, Crosby CV, Hogan EL, Banik NL (1999) Diverse stimuli induce calpain overexpression and apoptosis in C6 glioma cells. Brain Res 829:18–27

    Article  PubMed  CAS  Google Scholar 

  24. Danesi R, Figg WD, Reed E, Myers CE (1995) Paclitaxel (taxol) inhibits protein isoprenylation and induces apoptosis in PC-3 human prostate cancer cells. Mol Pharmacol 47:1106–1111

    PubMed  CAS  Google Scholar 

  25. Jordan MA, Wendell K, Gardiner S, Derry WB, Copp H, Wilson L (1996) Mitotic block induced in HeLa cells by low concentrations of paclitaxel (Taxol) results in abnormal mitotic exit and apoptotic cell death. Cancer Res 56:816–825

    PubMed  CAS  Google Scholar 

  26. Horwitz SB (1994) Taxol (paclitaxel): mechanisms of action. Ann Oncol 6:S3-S6

    Google Scholar 

  27. Haldar S, Jena N, Croce CM (1995) Inactivation of Bcl-2 by phosphorylation. Proc Natl Acad Sci USA 10:4507–4511

    Article  Google Scholar 

  28. Salah-Eldin AE, Inoue S, Tsukamoto S, Aoi H, Tsuda M (2003) An association of Bcl-2 phosphorylation and Bax localization with their functions after hyperthermia and paclitaxel treatment. Int J Cancer 103:53–60

    Article  PubMed  CAS  Google Scholar 

  29. Das A, Banik NL, Patel NJ, Ray SK (2004) Dexamethasone protected human glioblastoma U87MG cells from temozolomide induced apoptosis by maintaining Bax:Bcl-2 ratio and preventing proteolytic activities. Mol Cancer 3:36 (1–10)

    Google Scholar 

  30. Kim NW, Piatyszek MA, Prowse KR, Harley CB, West MD, Ho PL, Coviello GM, Wright WE, Weinrich SL, Shay JW (1994) Specific association of human telomerase activity with immortal cells and cancer. Science 66:2011–2015

    Article  Google Scholar 

  31. Grynkiewicz G, Poenie M, Tsien RY (1995) A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem 260:3440–3450

    Google Scholar 

  32. Hansen CA, Monck JR, Williamson JR (1990) Measurement of intracellular free calcium to investigate receptor-mediated calcium signaling. Methods Enzymol 191:691–706

    Article  PubMed  CAS  Google Scholar 

  33. Kang D, Nishida J, Iyama A, Nakabeppu Y, Furuichi M, Fujiwara T, Sekiguchi M, Takeshige K (1995) Intracellular localization of 8-oxo-dGTPase in human cells, with special reference to the role of the enzyme in mitochondria. J Biol Chem 270:14659–14665

    Article  PubMed  CAS  Google Scholar 

  34. Ray SK, Neuberger TJ, Deadwyler G, Wilford G, DeVries GH, Banik NL (2002) Calpain and calpastatin expression in primary oligodendrocyte culture: preferential localization of membrane calpain in cell processes. J Neurosci Res 70:561–569

    Article  PubMed  CAS  Google Scholar 

  35. Nudson WA, Rovnak J, Buechner M, Quackenbush SL (2003) Walleye dermal sarcoma virus Orf C is targeted to the mitochondria. J Gen Virol 84:375–381

    Article  PubMed  CAS  Google Scholar 

  36. Das A, Sribnick EA, Wingrave JM, Del Re AM, Woodward JJ, Appel SH, Banik NL, Ray SK (2005) Calpain activation in apoptosis of ventral spinal cord 4.1 (VSC4.1) motoneurons exposed to glutamate: calpain inhibition provides functional neuroprotection. J Neurosci Res 81:551–562

    Article  PubMed  CAS  Google Scholar 

  37. Pique M, Barragan M, Dalmau M, Bellosillo B, Pons G, Gil J (2000) Aspirin induces apoptosis through mitochondrial cytochrome c release. FEBS Lett 480:193–196

    Article  PubMed  CAS  Google Scholar 

  38. Butt AM (1991) Macroglial cell types, lineage, and morphology in the CNS. Ann N Y Acad Sci 633:90–95

    Article  PubMed  CAS  Google Scholar 

  39. Tascos NA, Parr J, Gonatas NK (1982) Immunocytochemical study of the glial fibrillary acidic protein in human neoplasms of the central nervous system. Hum Pathol 13:454–458

    Article  PubMed  CAS  Google Scholar 

  40. Chiu FC, Goldman JE (1985) Regulation of glial fibrillary acidic protein (GFAP) expression in CNS development and in pathological states. J Neuroimmunol 8:283–292

    Article  PubMed  CAS  Google Scholar 

  41. Yamada O, Takanashi M, Ujihara M, Mizoguchi H (1998) Down-regulation of telomerase activity is an early event of cellular differentiation without apparent telomeric DNA change. Leuk Res 22:711–717

    Article  PubMed  CAS  Google Scholar 

  42. Neradil J, Veselska R, Svoboda A (2005) The role of actin in the apoptotic cell death of P19 embryonal carcinoma cells. Int J Oncol 27:1013–1021

    PubMed  CAS  Google Scholar 

  43. Kass GE, Orrenius S (1999) Calcium signaling and cytotoxicity. Environ Health Perspect 107:25–35

    Article  PubMed  CAS  Google Scholar 

  44. Roy M, Chakrabarty S, Sinha D, Bhattacharya RK, Siddiqi M (2003) Anticlastogenic, antigenotoxic and apoptotic activity of epigallocatechin gallate: a green tea polyphenol. Mutat Res 523–524:33–41

    PubMed  Google Scholar 

  45. Krammer PH (2000) CD95’s deadly mission in the immune system. Nature 407:789–95

    Article  PubMed  CAS  Google Scholar 

  46. Desagher S, Osen-Sand A, Nichols A, Eskes R, Montessuit S, Lauper S, Maundrell K, Antonsson B, Martinou JC (1999) Bid-induced conformational change of Bax is responsible for mitochondrial cytochrome c release during apoptosis. J Cell Biol 144:891–901

    Article  PubMed  CAS  Google Scholar 

  47. Chen M, He H, Zhan S, Krajewski S, Reed JC, Gottlieb RA (2001) Bid is cleaved by calpain to an active fragment in vitro and during myocardial ischemia/reperfusion. J Biol Chem 276:30724–30728

    Article  PubMed  CAS  Google Scholar 

  48. Piskurich JF, Linhoff MW, Wang Y, Ting JP (1999) Two distinct gamma interferon-inducible promoters of the major histocompatibility complex class II transactivator gene are differentially regulated by STAT1, interferon regulatory factor 1, and transforming growth factor beta. Mol Cell Biol 19:431–440

    PubMed  CAS  Google Scholar 

  49. Kazuhito Y, Hidenori I. Stanley JK (1999) Bcl-2 is phosphorylated and inactivated by an Ask1/Jun N-terminal protein kinase pathway normally activated at G2/M. Mol Cell Biol 19:8469–8478

    Google Scholar 

  50. Wang HG, Pathan N, Ethell IM, Krajewski S, Yamaguchi Y, Shibasaki F, McKeon F, Bobo T, Franke TF, Reed JC (1999) Ca2+-induced apoptosis through calcineurin dephosphorylation of Bad. Science 284:339–343

    Article  PubMed  CAS  Google Scholar 

  51. Nath R, Raser KJ, Stafford D, Hajimohammadreza I, Posner A, Allen H, Talanian RV, Yuen P, Gilbertsen RB, Wang KK (1996) Non-erythroid α-spectrin breakdown by calpain and interleukin-1ß-converting-enzyme-like protease(s) in apoptotic cells: contributory roles of both protease families in neuronal apoptosis. Biochem J 319:683–690

    PubMed  CAS  Google Scholar 

  52. Wang KK, Posmantur R, Nath R, McGinnis K, Whitton M, Talanian RV, Glantz SB, Morrow JS (1998) Simultaneous degradation of αII- and ßII-spectrin by caspase 3 (CPP32) in apoptotic cells. J Biol Chem 273:22490–22497

    Article  PubMed  CAS  Google Scholar 

  53. Wang KK, Posmantur R, Nadimpalli R, Nath R, Mohan P, Nixon RA, Talanian RV, Keegan M, Herzog L, Allen H (1998) Caspase-mediated fragmentation of calpain inhibitor protein calpastatin during apoptosis. Arch Biochem Biophys 356:187–196

    Article  PubMed  CAS  Google Scholar 

  54. Sakahira H, Enari M, Nagata S (1998) Cleavage of CAD inhibitor in CAD activation and DNA degradation during apoptosis. Nature 391:96–99

    Article  PubMed  CAS  Google Scholar 

  55. Hietakangas V, Poukkula M, Heiskanen KM, Karvinen JT, Sistonen L, Eriksson JE (2003) Erythroid differentiation sensitizes K562 leukemia cells to TRAIL-induced apoptosis by downregulation of c-FLIP. Mol Cell Biol 23:1278–1291

    Article  PubMed  CAS  Google Scholar 

  56. Toda M, Miura M, Asou H, Toya S, Uyemura K (1994) Cell growth suppression of astrocytoma C6 cells by glial fibrillary acidic protein cDNA transfection. J Neurochem 63:1975–1978

    Article  PubMed  CAS  Google Scholar 

  57. Linskey ME, Gilbert MR (1995) Glial differentiation: a review with implications for new directions inneuro-oncology. Neurosurgery 36:1–21

    Article  PubMed  CAS  Google Scholar 

  58. Rothwell NJ (1997) Neuroimmune interactions: the role of cytokines. Br J Pharmacol 121:841–847

    Article  PubMed  CAS  Google Scholar 

  59. Sawada M, Itoh Y, Suzumura A, Marunouchi T (1993) Expression of cytokine receptors in cultured neuronal and glial cells. Neurosci Lett 160:131–134

    Article  PubMed  CAS  Google Scholar 

  60. Deshpande RV, Goust JM, Chakrabarti AK, Barbosa E, Hogan EL, Banik NL (1995) Calpain expression in lymphoid cells. Increased mRNA and protein levels after cell activation. J Biol Chem 270:2497–2505

    Article  PubMed  CAS  Google Scholar 

  61. Wang Q, Yang W, Uytingco MS, Christakos S, Wieder R (2000) 1,25-Dihydroxy vitamin D3 and all-trans retinoic acid sensitize breast cancer cells to chemotherapy-induced cell death. Cancer Res 60:2040–2048

    PubMed  CAS  Google Scholar 

  62. Figueroa-Masot XA, Hetman M, Higgins MJ, Kokot N, Xia Z (2001) Taxol induces apoptosis in cortical neurons by a mechanism independent of Bcl-2 phosphorylation. J Neurosci 21:4657–4667

    PubMed  CAS  Google Scholar 

  63. Blagosklonny MV, Giannakakou P, Deiry WS, Kingston DG, Higgs PI, Neckers L, Fojo T (1997) Raf-1/Bcl-2 phosphorylation: a step from microtubule damage to cell death. Cancer Res 57:130–135

    PubMed  CAS  Google Scholar 

  64. Ray SK, Matzelle DD, Wilford GG, Hogan EL, Banik NL (2001) Cell death in spinal cord injury (SCI) requires de novo protein synthesis: Calpain inhibitor E-64-d provides neuroprotection in SCI lesion and penumbra. Ann N Y Acad Sci 939:436–449

    Article  PubMed  CAS  Google Scholar 

  65. Waters SL, Sarang SS, Wang KK, Schnellmann RG (1997) Calpains mediate calcium and chloride influx during the late phase of cell injury. J Pharmacol Exp Ther 283:1177–1184

    PubMed  CAS  Google Scholar 

  66. Gil-Parrado S, Fernandez-Montalvan A, Assfalg-Machleidt I, Popp O, Bestvater F, Holloschi A, Knoch TA, Auerswald EA, Welsh K, Reed JC, Fritz H, Fuentes-Prior P, Spiess E, Salvesen GS, Machleidt W (2002) Ionomycin-activated calpain triggers apoptosis: A probable role for Bcl-2 family members. J Biol Chem 277:27217–27226

    Article  PubMed  CAS  Google Scholar 

  67. Scarlett JL, Sheard PW, Hughes G, Ledgerwood EC, Ku HH, Murphy MP (2000) Changes in mitochondrial membrane potential during staurosporine-induced apoptosis in Jurkat cells. FEBS Lett 475:267–272

    Article  PubMed  CAS  Google Scholar 

  68. Adams JM, Cory S (2001) Life-or-death decisions by the Bcl-2 protein family. Trends Biochem Sci 26:61–66

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

This study was supported in part by the R01 grants from the NCI (CA-91460) and NINDS (NS-57811) and a Spinal Cord Injury Research Foundation grant from the State of SC (SCIRF-0803) to S.K.R.

Author information

Authors and Affiliations

  1. Department of Neurosciences, Medical University of South Carolina, 96 Jonathan Lucas Street, Suite 325E, P.O. Box 250606, Charleston, SC, 29425, USA

    Arabinda Das, Naren L. Banik & Swapan K. Ray

Authors
  1. Arabinda Das
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Naren L. Banik
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Swapan K. Ray
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Swapan K. Ray.

Additional information

Special issue in honor of Naren Banik.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Das, A., Banik, N.L. & Ray, S.K. Differentiation Decreased Telomerase Activity in Rat Glioblastoma C6 Cells and Increased Sensitivity to IFN-γ and Taxol for Apoptosis. Neurochem Res 32, 2167–2183 (2007). https://doi.org/10.1007/s11064-007-9413-y

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11064-007-9413-y

Keywords

Search

Navigation