Hostname: page-component-78c5997874-lj6df Total loading time: 0 Render date: 2024-11-05T06:52:29.940Z Has data issue: false hasContentIssue false
  • English
  • Français

Cell-Cycle Gene Expression in Lovastatin-Induced Medulloblastoma Apoptosis

Published online by Cambridge University Press:  02 December 2014

Wei Wang  and
Robert J.B. Macaulay
Wei Wang
Affiliation:
Laboratory of Cellular Oncology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA
Robert J.B. Macaulay
Affiliation:
Department of Pathology and Laboratory Medicine, QEII Health Sciences Center and Dalhousie University, Halifax, Nova Scotia B3H 1V8, Canada
Rights & Permissions [Opens in a new window]

Abstract

Core share and HTML view are not available for this content. However, as you have access to this content, a full PDF is available via the ‘Save PDF’ action button.
Background:

3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase is a key rate-limiting enzyme in the mevalonate pathway, which generates precursors both for cholesterol biosynthesis and for the production of nonsteroidal mevalonate derivatives that are involved in a number of growth-regulatory processes. We have reported that lovastatin, a competitive inhibitor of HMG-CoA reductase, not only inhibits medulloblastoma proliferation in vitro, but also induces near-complete cell death via apoptosis. The mechanism of this phenomenon is unclear. Possible involvement of changes in expression of certain cell-cycle related genes led us to study some of them in more detail.

Methods:

Medulloblastoma cell lines were exposed in vitro to lovastatin, and the effects of gene expression changes were studied using RT-PCR, antisense oligonucleotide, DNA electrophoresis and Western blotting analysis.

Results:

1) Levels of total Ras gene mRNA and individual Ras gene mRNA are stable in lovastatin treatment in all examined medulloblastoma cell lines. 2) Blocking c-myc gene over-expression does not enhance medulloblastoma cell sensitivity to lovastatin. 3) Following lovastatin treatment, p16 expression exhibits no change, but pronounced increases of p27 KIP1 protein are observed in all examined cell lines. Lovastatin induces pronounced increases of p21WAF1 protein only in Daoy and UW228, but not in D283 Med and D341 Med. 4) Following lovastatin treatment, increased p53 protein is detected only in D341 Med, and bax protein is unchanged in all cell lines.

Conclusion:

Lovastatin-induced growth inhibition and apoptosis in medulloblastoma are not dependent on the regulation of Ras and c-myc gene expression, but may be mediated by p27KIP1 gene expression. Lovastatin-induced apoptosis in medulloblastoma is probably p53 independent, but p53 and p21WAF1 gene expression may also mediate anti-proliferative effects of lovastatin on specific medulloblastoma cell lines.

Résumé:

RÉSUMÉ: Introduction:

La 3-hydroxy-3-méthylglutaryl-coenzyme A (HGM-CoA) réductase est un enzyme limitant clé de la voie du mévalonate qui génère des précurseurs tant pour la biosynthèse du cholestérol que pour la production de dérivés non stéroïdiens du mévalonate qui sont impliqués dans certains processus régulateurs de la croissance. Nous avons rapporté que la lovastatine, un inhibiteur non compétitif de l'HMG-CoA réductase, inhibe la prolifération du médulloblastome in vitro et induit également la mort cellulaire presque complète via l'apoptose. Le mécanisme sous-jacent à ce phénomène n'est pas clair. La possibilité que des changements dans l'expression de certains gènes du cycle cellulaire soient impliqués nous a incités à en étudier quelques-uns de plus près.

Méthodes:

Des lignées cellulaires de médulloblastome ont été exposées in vitro à la lovastatine et les effets des changements dans l'expression génique ont été étudiés au moyen de RT-PCR, d'oligonucléotides antisenses, de l'électrophorèse de l'ADN et du buvardage western.

Résultats:

Les niveaux d'ARNm de tous les gènes Ras et des gènes Ras pris individuellement sont stables dans toutes les lignées cellulaires de médulloblastome étudiées après traitement par la lovastatine. 2) Le fait de bloquer la surexpression du gène c-myc n'augmente pas la sensibilité des cellules de médulloblastome à la lovastatine. 3) Suite au traitement par la lovastatine, l'expression de p16 ne change pas, mais on observe des augmentations considérables de la protéine p27KIP1 dans toutes les lignées cellulaires examinées. La lovastatine provoque des augmentations marquées de la protéine p21WAF1 dans le lignées Daoy et UW228, mais pas dans D283 Med et D341 Med. 4) Suite au traitement par la lovastatine, une augmentation de la protéine p53 est détectée seulement dans D341 Med et la protéine Box demeure inchangée dans toutes les lignées cellulaires.

Conclusions:

L'inhibition de la croissance et l'apoptose induites par la lovastatine dans le médulloblastome ne sont pas dépendantes de la régulation de l'expression des gènes Ras et c-myc, mais elles pourraient être médiées par l'expression du gène p27KIP1. L'apoptose induite par la lovastatine dans le médulloblastome est probablement indépendante de p53, mais l'expression des gènes p53 et p21WAF1 peut également médier des effets antiprolifératifs de la lovastatine dans des lignées cellulaires spécifiques de médulloblastome.

Type
Research Article
Information
Canadian Journal of Neurological Sciences , Volume 30 , Issue 4 , November 2003 , pp. 349 - 357
Copyright
Copyright © The Canadian Journal of Neurological 2003

References

1. Jay, V, Becker, LE. Brain tumors. Curr Opin Neurol Neurosurg 1990;3:934–942.Google Scholar
2. Packer, RJ, Sutton, LN, Elterman, R, et al. Outcome for children with medulloblastoma treated with radiation and cisplatin, CCNU, and vincristine chemotherapy. J Neurosurg 1994;81:690–698.CrossRefGoogle ScholarPubMed
3. Rorke, LB, Trojanowski, JQ, Lee, VM, et al. Primitive neuroectodermal tumors of the central nervous system. Brain Pathol 1997;7:765–784.CrossRefGoogle ScholarPubMed
4. Tomlinson, FH, Scheithauer, BW, Meyer, FB, et al. Medulloblastoma: I. Clinical, diagnostic, and therapeutic overview. J Child Neurol 1992;7:142–155.CrossRefGoogle ScholarPubMed
5. Nishiyama, K, Funakoshi, S, Izumoto, S, et al. Long-term effects of radiation for medulloblastoma on intellectual and physical development. A case report of monozygotic twins. Cancer 1994;73:2450–2455.Google Scholar
6. Packer, RJ. Chemotherapy for medulloblastoma/primitive neuroectodermal tumors of the posterior fossa. Ann Neurol 1990;28:823–828.Google Scholar
7. Tishler, DM, Weinberg, KI, Sender, LS, et al. Multidrug resistance gene expression in pediatric primitive neuroectodermal tumors of the central nervous system. J Neurosurg 1992;76:507–512.Google Scholar
8. Cohen, BH, Zweidler, P, Goldwein, JW, et al. Ototoxic effect of cisplatin in children with brain tumors. Pediatr Neurosurg 1990;16:292–296.Google Scholar
9. Torres, CF, Rebsamen, S, Silber, JH, et al. Surveillance scanning of children with medulloblastoma [see comments]. N Engl J Med 1994;330:892–895.CrossRefGoogle ScholarPubMed
10. Tobert, JA, Hitzenberger, G, Kukovetz, WR, et al. Rapid and substantial lowering of human serum cholesterol by mevinolin (MK-803), an inhibitor of hydroxymethylglutaryl-coenzyme A reductase. Atherosclerosis 1982;41:61–65.Google Scholar
11. Keyomarsi, K, Sandoval, L, Band, V, et al. Synchronization of tumor and normal cells from G1 to multiple cell cycles by lovastatin. Cancer Res 1991;51:3602–3609.Google ScholarPubMed
12. Sumi, S, Beauchamp, RD, Townsend, CJ, et al. Inhibition of pancreatic adenocarcinoma cell growth by lovastatin. Gastroenterology 1992;103:982–989.CrossRefGoogle ScholarPubMed
13. Macaulay, RJB, Wang, W, Dimitroulakos, J, et al. lovastatin-induced apoptosis of human medulloblastoma cell lines in vitro . J Neuro-oncol 1999;42:1–11.Google Scholar
14. Wang, W, Macaulay, RJB. Apoptosis of medulloblastoma cells in vitro follows inhibition of farnesylation using manumycin A. Int J Cancer 1999;82:430–434.3.0.CO;2-9>CrossRefGoogle ScholarPubMed
15. Bansal, N, Houle, AG, Melnykovych, G. Comparison of dexamethasone and lovastatin (mevinolin) as growth inhibitors in cultures of T-cell derived human acute leukemia lines (CEM). Leuk Res 1989;13:875–882.Google Scholar
16. Perez, SD, Mollinedo, F. Inhibition of isoprenoid biosynthesis induces apoptosis in human promyelocytic HL-60 cells. Biochem Biophys Res Commun 1994;199:1209–1215.Google Scholar
17. Jones, KD, Couldwell, WT, Hinton, DR, et al. Lovastatin induces growth inhibition and apoptosis in human malignant glioma cells. Biochem Biophys Res Commun 1994;205:1681–1687.CrossRefGoogle ScholarPubMed
18. Di Matola, T, D’Ascoli, F, Luongo, C, et al. Lovastatin-induced apoptosis in thyroid cells: involvement of cytochrome c and lamin B. Eur J Endocrinol 2001;145:645–650.CrossRefGoogle ScholarPubMed
19. Feleszko, W, Mlynarczuk, I, Olszewska, D, et al. Lovastatin potentiates antitumor activity of doxorubicin in murine melanoma via an apoptosis-dependent mechanism. Int J Cancer 2002;100:111–118.Google Scholar
20. Larner, J, Jane, J, Laws, E, et al. A phase I-II trial of lovastatin for anaplastic astrocytoma and glioblastoma multiforme. Am J Clin Oncol 1998;21:579–583.CrossRefGoogle ScholarPubMed
21. Kim, WS, Kim, MM, Choi, HJ, et al. Phase II study of high-dose lovastatin in patients with advanced gastric adenocarcinoma. Invest New Drugs 2001;19:81–83.Google Scholar
22. Wang, W, Macaulay, RJB. Mevalonate prevents lovastatin-induced apoptosis in medulloblastoma cell lines. Can J Neurol Sciences 1999;26:305–310.Google Scholar
23. Kohl, NE, Conner, MW, Gibbs, JB, et al. Development of inhibitors of protein farnesylation as potential chemotherapeutic agents. J Cell Biochem (Suppl) 1995;22:145–150.Google Scholar
24. Dimster, DD, Schafer, WR, Rine, J. Control of RAS mRNA level by the mevalonate pathway. Mol Biol Cell 1995;6:59–70.CrossRefGoogle Scholar
25. Holstein, SA, Wohlford-Lenane, CL, Hohl, RJ. Consequences of mevalonate depletion. Differential transcriptional, translational, and post-translational up-regulation of Ras, Rap1a, RhoA, and RhoB. J Biol Chem 2002;277:10678–10682.Google Scholar
26. Barbacid, M. ras genes. Annu Rev Biochem 1987;56:779–827.Google Scholar
27. Bigner, SH, Friedman, HS, Vogelstein, B, et al. Amplification of the c-myc gene in human medulloblastoma cell lines and xenografts [published erratum appears in Cancer Res 1990 Jun 15;50(12):3809]. Cancer Res 1990;50:2347–2350.Google Scholar
28. Friedman, HS, Burger, PC, Bigner, SH, et al. Phenotypic and genotypic analysis of a human medulloblastoma cell line and transplantable xenograft (D341 Med) demonstrating amplification of c-myc . Am J Pathol 1988;130:472–484.Google ScholarPubMed
29. Askew, DS, Ashmun, RA, Simmons, BC, et al. Constitutive c-myc expression in an IL-3-dependent myeloid cell line suppresses cell cycle arrest and accelerates apoptosis. Oncogene 1991;6:1915–1922.Google Scholar
30. Evan, GI, Wyllie, AH, Gilbert, CS, et al. Induction of apoptosis in fibroblasts by c-myc protein. Cell 1992;69:119–128.Google Scholar
31. Hermeking, H, Eick, D. Mediation of c-myc-induced apoptosis by p53. Science 1994;265:2091–2093.Google Scholar
32. Park, C, Lee, I, Kang, WK. Lovastatin-induced E2F-1 modulation and its effect on prostate cancer cell death. Carcinogenesis 2001;22:1727–1731.Google Scholar
33. Grana, X, Reddy, EP Cell cycle control in mammalian cells: role of cyclins, cyclin dependent kinases (CDKs), growth suppressor genes and cyclin-dependent kinase inhibitors (CKIs). Oncogene 1995;11:211–219.Google ScholarPubMed
34. Hall, M, Peters, G. Genetic alterations of cyclins, cxyclin-dependent kinase, and Cdk inhibitors in human cancer. Adv Cancer Res 1996;68:67–108.Google Scholar
35. Sherr, CJ. Cancer cell cycles. Science 1996;274:1672–1677.CrossRefGoogle ScholarPubMed
36. Sherr, CJ, Roberts, JM. Inhibitors of mammalian G1 cyclin-dependent kinases. Genes Dev 1995;9:1149–1163.CrossRefGoogle ScholarPubMed
37. Hengst, L, Reed, SI. Translational control of p27Kip1 accumulation during the cell cycle. Science 1996;271:1861–1864.CrossRefGoogle ScholarPubMed
38. Poon, RY, Toyoshima, H, Hunter, T. Redistribution of the CDK inhibitor p27 between different cyclin. CDK complexes in the mouse fibroblast cell cycle and in cells arrested with lovastatin or ultraviolet irradiation. Mol Biol Cell 1995;6:1197–1213.Google Scholar
39. Rao, S, Lowe, M, Herliczek, TW, et al. Lovastatin mediated G1 arrest in normal and tumor breast cells is through inhibition of CDK2 activity and redistribution of p21 and p27, independent of p53. Oncogene 1998;17:2393–2402.Google Scholar
40. Gray-Bablin, J, Rao, S, Keyomarsi, K. Lovastatin induction of cyclin-dependent kinase inhibitors in human breast cells occurs in a cell cycle-independent fashion. Cancer Res 1997;57:604–609.Google Scholar
41. Giermasz, A, Makowski, M, Kozlowska, E, et al. Potentiating antitumor effects of a combination therapy with lovastatin and butyrate in the Lewis lung carcinoma model in mice. Int J Cancer 2002;97:746–750.Google Scholar
42. Lee, SJ, Ha, MJ, Lee, J, et al. Inhibition of the 3-hydroxy-3-methylglutaryl-coenzyme A reductase pathway induces p53-independent transcriptional regulation of p21(mFi,CIP1) in human prostate carcinoma cells. J Biol Chem 1998;273:10618–10623.Google Scholar
43. Vidal, A, Millard, SS, Miller, JP, et al. Rho activity can alter the translation of p27 mRNA and is important for RasV12-induced transformation in a manner dependent on p27 status. J Biol Chem 2002;277:16433–16440.Google Scholar
44. Cohen, JJ, Duke, RC. Glucocorticoid activation of a calcium-dependent endonuclease in thymocyte nuclei leads to cell death. J Immunol 1984;132:38–42.Google Scholar
45. Levine, AJ, Momand, J, Finlay, CA. The p53 tumour suppressor gene. Nature 1991;351:453–456.Google Scholar
46. Yonish, RE, Resnitzky, D, Lotem, J, et al. Wild-type p53 induces apoptosis of myeloid leukaemic cells that is inhibited by interleukin-6. Nature 1991;352:345–347.Google Scholar
47. Kastan, MB, Onyekwere, O, Sidransky, D, et al. Participation of p53 protein in the cellular response to DNA damage. Cancer Res 1991;51(23 Pt 1):6304–6311.Google Scholar
48. Oltvai, ZN, Milliman, CL, Korsmeyer, SJ. Bcl-2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programmed cell death. Cell 1993;74:609–619.CrossRefGoogle ScholarPubMed
49. Miyashita, T, Reed, JC. Tumor suppressor p53 is a direct transcriptional activator of the human bax gene. Cell 1995;80:293–299.Google Scholar
50. Yin, C, Knudson, CM, Korsmeyer, SJ, et al. Bax suppresses tumorigenesis and stimulates apoptosis in vivo . Nature 1997;385:637–640.Google Scholar
51. Grasso, L, Mercer, WE. Pathways of p53-dependent apoptosis. Vitam Horm 1997;53:139–173.CrossRefGoogle ScholarPubMed
52. Liebermann, DA, Hoffman, B, Steinman, RA. Molecular controls of growth arrest and apoptosis: p53-dependent and independent pathways. Oncogene 1995;11:199–210.Google Scholar
53. Keles, GE, Berger, MS, Srinivasan, J, et al. Establishment and characterization of four human medulloblastoma-derived cell lines. Oncol Res 1995;7:493–503.Google ScholarPubMed
54. Heikkila, R, Schwab, G, Wickstrom, E, et al. A c-myc antisense oligodeoxynucleotide inhibits entry into S phase but not progress from G0 to G1. Nature 1987;328:445–449.Google Scholar
55. Wickstrom, EL, Bacon, TA, Gonzalez, A, et al. Human promyelocytic leukemia HL-60 cell proliferation and c-myc protein expression are inhibited by an antisense pentadecadeoxynucleotide target against c-myc mRNA. Proc Natl Acad Sci USA 1988;85:1028–1032.CrossRefGoogle ScholarPubMed
56. Holt, JT, Redner, RL, Nienhuis, AW. An oligomer complementary to c-myc mRNA inhibits proliferation of HL-60 promyelocytic cells and induces differentiation. Mol Cell Biol 1988;8:963–973.Google Scholar
57. Koster, R, Blatt, LM, Streubert, M, et al. Consensus-interferon and platelet-derived growth factor adversely regulate proliferation and migration of kaposi’s sarcoma cells by control of c-myc expression. Am J Pathol 1996;149:1871–1885.Google ScholarPubMed
58. Kaptein, JS, Lin, CKE, Wang, CL, et al. Anti-IgM-mediated regulation of c-myc and its possible relationship to apoptosis. J Biol Chem 1996;271:18875–18884.Google Scholar
59. Magee, AI, Gutierrez, L, McKay, IA, et al. Dynamic fatty acylation of p21N-ras. EMBO J 1987;6:3353–3357.Google Scholar
60. Schafer, WR, Kim, R, Sterne, R, et al. Genetic and pharmacologicalsuppression of oncogenic mutations in ras genes of yeast and humans. Science 1989;245:379–385.Google Scholar
61. Leonard, S, Beck, L, Sinensky, M. Inhibition of isoprenoid biosynthesis and the post-translational modification of pro-p21. J Biol Chem 1990;265:5157–5160.Google Scholar
62. Mangues, R, Corral, T, Kohl, NE, et al. Antitumor effect of a farnesyl protein transferase inhibitor in mammary and lymphoid tumors overexpressing N-ras in transgenic mice. Cancer Res 1998;58:1253–1259.Google ScholarPubMed
63. Whyte, DB, Kirschmeier, P Hockenberry, TN, et al. K- and N-Ras are geranylgeranylated in cells treated with farnesyl protein transferase inhibitors. J Biol Chem 1997;272:14459–14464.Google Scholar
64. Green, DR, Mahboubi, A, Nishioka, W, et al. Promotion and inhibition of activation-induced apoptosis in T-cell hybridomas by oncogenes and related signals. Immunol Rev 1994;142:321–342.CrossRefGoogle ScholarPubMed
65. Duffy, MJ. Cellular oncogenes and suppressor genes as prognostic markers in cancer. Clin Biochem 1993;26:439–447.Google Scholar
66. Dimitroulakos, J, Yeger, H. HMG-CoA reductase mediates the biological effects of retinoic acid on human neuroblastoma cells: lovastatin specifically targets P-glycoprotein-expressing cells. Nat Med 1996;2:326–333.Google Scholar
67. Agarwal, B, Halmos, B, Feoktistov, AS, et al. Mechanism of lovastatin-induced apoptosis in intestinal epithelial cells. Carcinogenesis 2002;23:521–528.Google Scholar
68. Vitale, M, Di Matola, T, Rossi, G, et al. Prenyltransferase inhibitors induce apoptosis in proliferating thyroid cells through a p53-independent CrmA-sensitive, and caspase-3-like protease-dependent mechanism. Endocrinology 1999;140:698–704.CrossRefGoogle ScholarPubMed
69. Laufs, U, Marra, D, Node, K, et al. 3-Hydroxy-3-methylglutaryl-CoA reductase inhibitors attenuate vascular smooth muscle proliferation by preventing rho GTPase-induced down-regulation of p27(Kip1). J Biol Chem 1999;274:21926–21931.Google Scholar
70. Tanaka, T, Tatsuno, I, Uchida, D, et al. Geranylgeranyl-pyrophosphate, an isoprenoid of mevalonate cascade, is a critical compound for rat primary cultured cortical neurons to protect the cell death induced by 3-hydroxy-3-methylglutaryl-CoA reductase inhibition. J Neurosci 2000;20:2852–2859.Google Scholar
71. Borner, MM, Myers, CE, Sartor, O, et al. Drug-induced apoptosis is not necessarily dependent on macromolecular synthesis or proliferation in the p53-negative human prostate cancer cell line PC-3. Cancer Res 1995;55:2122–2128.Google Scholar
72. Caelles, C, Helmberg, A, Karin, M. p53-dependent apoptosis in the absence of transcriptional activation of p53-target genes [see comments]. Nature 1994;370:220–223.Google Scholar
73. Wagner, AJ, Kokontis, JM, Hay, N. Myc-mediated apoptosis requires wild-type p53 in a manner independent of cell cycle arrest and the ability of p53 to induce p21waf1,cip1. Genes Dev 1994;8:2817–2830.Google Scholar
74. Guillouf, C, Rosselli, F, Sjin, RT, et al. Role of a mutant p53 protein in apoptosis: characterization of a function independent of transcriptional trans-activation. Int J Oncol 1998;13:107–114.Google Scholar
75. Guillouf, C, Rosselli, F, Krishnaraju, K, et al. p53 involvement i control of G2 exit of the cell cycle: role in DNA damage-induced apoptosis. Oncogene 1995;10:2263–2270.Google Scholar
76. Jin, Z, Dicker, DT, El-Deiry WS. Enhanced sensitivity of G1 arrested human cancer cells suggests a novel therapeutic strategy using a combination of simvastatin and TRAIL. Cell Cycle 2002;1:82–89.Google Scholar