Brain Tumor Pathology

, Volume 18, Issue 2, pp 73–81 | Cite as

Phenotypic changes associated with exogenous expression of p16INK4a in human glioma cells

  • Akio Noguchi
  • Nobuyuki Ito
  • Hiroki Sawa
  • Motoo Nagane
  • Mitsuhiro Hara
  • Isamu Saito
Original Article


The tumor suppressorp16/CDKN2A/INK4a gene is frequently mutated, mostly by homozygous deletions in high-grade gliomas. Although the p16 protein suppresses cell proliferation primarily through inhibition of cell-cycle progression at the G1 phase, other phenotypic changes in glioma cells associated with p16INK4a alterations have not been fully described. To determine the roles of p16 alterations in glioma formation, we have established ecdysonedriven inducible p16 expression in the human glioblastoma cell line CL-4, which were derived from p16-null U87MG cells. Here we show that exogenous p16 expression in CL-4 cells results in morphological changes, with large and flattened cytoplasm, which are associated with increased formation of cytoplasmic actin-stress fibers and vinculin accumulation in the focal adhesion contacts. Adhesion of CL-4 cells to extracellular matrix proteins, such as laminin, fibronectin, and type IV collagen, significantly increased upon exogenous p16 expression, which correlated with increased expression of integrin α5 and αv. Expression of a small GTP-binding protein, Rac, also decreased. Following epidermal growth factor stimulation, phosphorylation of MAP kinases ERK1 and 2 and induction of an early immediate gene product, c-Fos, were significantly reduced in CL-4 cells with p16 expression. These results suggest that the tumor suppressor p16 may exert its antitumor effects through modulation of multiple aspects of glioblastoma phenotypes, including proliferation, invasiveness, and responsiveness to extracellular growth stimuli.

Key words

p16 Glioma Integrin Rac Adhesion Growth factor 


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  1. 1.
    Fults D, Brockmeyer D, Tullous MW, et al (1992) p53 mutation and loss of heterozygosity on chromosome 17 and 10 during human astrocytoma progression. Cancer Res 52:674–679PubMedGoogle Scholar
  2. 2.
    Nobori T, Miura K, Wu DJ, et al (1994) Deletion of the cyclin-dependent kinase-4 inhibitor gene in multiple human cancer. Nature (Lond) 368:753–756CrossRefGoogle Scholar
  3. 3.
    Kamb A, Gruis NA, Weaver-Feldhaus J, et al (1994) A cell cycle regulator potentially involved in genesis of many tumor types. Science 264:436–440PubMedGoogle Scholar
  4. 4.
    Fujimoto M, Fults DW, Thomas GA, et al (1989) Loss of heterozygosity on chromosome 10 in human glioblastoma. Genomics 4:210–214PubMedCrossRefGoogle Scholar
  5. 5.
    Henson JW, Schnitker BL, Correa KM, et al (1994) The retinoblastoma gene is involved in malignant progression of astrocytomas. Ann Neurol 36:714–721PubMedCrossRefGoogle Scholar
  6. 6.
    Hamel W, Westphal M, Shepard HM (1993) Loss in expression of the retinoblastoma gene product in human gliomas is associated with advanced disease. J Neurooncol 16:159–165PubMedCrossRefGoogle Scholar
  7. 7.
    Li J, Yen C, Liaw D, et al (1997) PTEN, a putative protein tyrosine phosphatase gene mutated in human brain, breast, and prostate cancer. Science 275:1943–1947PubMedCrossRefGoogle Scholar
  8. 8.
    Steck PA, Pershouse MA, Jasser SA, et al (1997) Identification of a candidate tumor suppressor gene, MMAC1, at chromosome 10q23. 3 that is mutated in multiple advanced cancers. Nat Genet 15:356–362PubMedCrossRefGoogle Scholar
  9. 9.
    Agosti RM, Leuthhold M, Gullick WJ, et al (1992) Expression of the epidermal growth factor receptor in astrocytic tumors is specifically associated with glioblastoma multiforme. Virchows Arch (A) Pathol Anat 420:321–325CrossRefGoogle Scholar
  10. 10.
    Venter DJ, Bevan KL, Ludwig RL, et al (1991) Retino-blastoma gene deletions in human glioblastomas. Oncogene 6:445–448PubMedGoogle Scholar
  11. 11.
    Schmidt EE, Ichimura K, Reifenberger G, et al (1994) CDKN2(p16/MTS1) gene deletion or CDK4 amplification occurs in majority of glioblastomas. Cancer Res 54:6321–6324PubMedGoogle Scholar
  12. 12.
    Giani C, Finocchiaro G (1994) Mutation rate of the CDKN2 gene in malignant gliomas. Cancer Res 54:6338–6339PubMedGoogle Scholar
  13. 13.
    Costello JF, Berger MS, Huang HS, et al (1996) Silencing of p16/ CDKN2 expression in human gliomas by methylation and chromatin condensation. Cancer Res 56:2405–2410PubMedGoogle Scholar
  14. 14.
    Fujita M, Enomoto T, Haba T, et al (1997) Alteration of p16 and p15 genes in common epithelial ovarian tumors. Int J Cancer 74:148–155PubMedCrossRefGoogle Scholar
  15. 15.
    Ng MH, Chung YF, Lo KW, et al (1997) Frequent hypermethylation of p16 and p15 in multiple myeloma. Blood 89:2500–2506PubMedGoogle Scholar
  16. 16.
    Martinez-Delgado B, Fernandez-Piqueras J, Garcia MJ, et al (1997) Hypermethylation of a 5′ CpG island of p16 is a frequent event in non-Hodgkin's lymphoma. Leukemia 11:425–428PubMedCrossRefGoogle Scholar
  17. 17.
    Schmidt EE, Ichimura K, Reifenberger G, et al (1994) CDKN2 (p16/MTS1) gene deletion or CDK4 amplification occurs in the majority of glioblastomas. Cancer Res 54:6321–6324PubMedGoogle Scholar
  18. 18.
    Arap W, Nishikawa R, Furunari FB, et al (1995) Replacement of the p16/CDKN2 gene suppresses, human glioma cell growth. Cancer Res 55:1351–1354PubMedGoogle Scholar
  19. 19.
    Mao L, Merlo A, Bedi G, et al (1995) A novel p16INK4A transcript. Cancer Res 55:2995–2997PubMedGoogle Scholar
  20. 20.
    Quelle DE, Zindy F, Ashmun RA, et al (1995) Alternative reading frames of the INK4a tumor suppressor gene encode two unrelated proteins capable of inducing cell cycle arrest. Cell 83: 993–1000PubMedCrossRefGoogle Scholar
  21. 21.
    Arap W, Knudsen E, Sewell DA, et al (1997) Functional analysis of wild-type and malignant glioma derived CDKN2A β alleles: evidence for an RB-independent growth suppressive pathway. Oncogene 15:2013–2020PubMedCrossRefGoogle Scholar
  22. 22.
    Zhang Y, Xiong Y, Yarbrough WG (1998) ARF promotes MDM2 degradation and stabilized p53: ARF-INK4a locus deletion impairs both the Rb and p53 tumor suppression pathway. Cell 92:725–734PubMedCrossRefGoogle Scholar
  23. 23.
    Fueyo J, Gomez-Manzano C, Yung WK, et al (1996) Adenovirus-mediated p16/CDKN2 gene transfer induces growth arrest and modifies the transformed phenotype of glioma cells. Oncogene 12:103–110PubMedGoogle Scholar
  24. 24.
    Sawa H, Kamada H, Arato-Ohshima T, et al (1999) Exogenous expression of p16INK4a is associated with decrease in telomerase activity. J Neurooncol 42:47–57CrossRefGoogle Scholar
  25. 25.
    Craig SW, Johnson RP (1996) Assembly of focal adhesion: progress, paradigms, and portents. Curr Opin Cell Biol 8:74–85PubMedCrossRefGoogle Scholar
  26. 26.
    Hall A (1998) Rho GTPases and the actin cytoskeleton. Science 279:509–514PubMedCrossRefGoogle Scholar
  27. 27.
    Ridley AJ, Paterson HF, Johnston CL, et al (1992) The small-GTP-binding protein rac regulates growth factor-induced membrane ruffling. Cell 70:401–410PubMedCrossRefGoogle Scholar
  28. 28.
    Kozma R, Ahmed S, Best A, et al (1995) The Ras-related protein CdC42Hs and bradykinin promote formation of peripheral actin microspikes and filopodia in Swiss 3T3 fibroblasts. Mol Cell Biol 15:1942–1952PubMedGoogle Scholar
  29. 29.
    Schmitz AA, Govek EE, Botter B, et al. Rho GTPases: signaling, migration, and invasion. Exp Cell Res 261:1–12Google Scholar
  30. 30.
    Saruta K (1998) Immunohistochemical analysis of human glioblastoma and exogenous expression of the wild-type p16 gene in human glioblastoma U87MG cells. J Kyorin Med Soc 29:187–199Google Scholar
  31. 31.
    Uhrborn L, Nister M, Westermark B (1997) Induction of senescence in human malignant glioma cells by p16 INK4A. Oncogene 15:505–514CrossRefGoogle Scholar
  32. 32.
    Dirks PB, Patel K, Hubbard SL, et al (1997) Retinoic acid and the cyclin dependent kinase inhibitors synergistically alter proliferation and morphology of U343 astrocytoma cells. Oncogene 15: 2037–2048PubMedCrossRefGoogle Scholar
  33. 33.
    Higashi H, Suzuki-Takahashi I, Yoshida E, et al (1997) Expression of p16INK4a suppresses the unbounded and anchorage-independent growth of a glioblastoma cell line that lacks p16INK4a. Biochem Biophys Res Commun 231:743–750PubMedCrossRefGoogle Scholar
  34. 34.
    Weinel RJ, Rosendahl A, Neuman K, et al (1992) Expression and function of VLA α2, α3, α5 and α6 integrin receptors in pancreatic carcinoma. Int J Cancer 52:827–833PubMedGoogle Scholar
  35. 35.
    Tennenbaum T, Yuspa SH, Grover A, et al (1992) Extracellular matrix receptors and mouse skin carcinogenesis: altered expression linked to appearance of early markers of tumor progression. Cancer Res 52:2966–2976PubMedGoogle Scholar
  36. 36.
    Varner JA, Emerson DA, Juliano RL (1995) Integrin α5β1 expression negatively regulates cell growth: reversal by attachment to fibronectin. Mol Cell Biol 6:725–740Google Scholar
  37. 37.
    Boulton TG, Nye SH, Robbins DJ, et al (1991) ERKs: a family of protein-serine/threonine kinases that are activated and tyrosine phosphorylated in response to insulin and NGF. Cell 65:663–675PubMedCrossRefGoogle Scholar
  38. 38.
    Crews CM, Erikson RL (1992) Purification of a murine protein-tyrosine/ threonine kinases that phosphorylates and activates theErk-1 gene product: relationship to fission yeastbyr1 gene product. Proc Natl Acad Sci (USA) 89:8205–8209CrossRefGoogle Scholar
  39. 39.
    Crews CM, Alessandrini A, Erikson RL (1992) The primary structure of MEK, a protein kinase that phosphorylates the ERK gene product. Science 258:478–480PubMedGoogle Scholar
  40. 40.
    Lallemand D, Spyrou G, Yaniv M, et al (1997) Variations in Jun and Fos protein expression and AP-1 activity in cycling, resting and stimulated fibroblasts. Oncogene 14:819–830PubMedCrossRefGoogle Scholar

Copyright information

© The Japan Society of Brain Tumor Pathology 2001

Authors and Affiliations

  • Akio Noguchi
    • 1
  • Nobuyuki Ito
    • 1
  • Hiroki Sawa
    • 1
    • 2
  • Motoo Nagane
    • 1
  • Mitsuhiro Hara
    • 3
  • Isamu Saito
    • 1
  1. 1.Department of NeurosurgeryKyorin University School of MedicineTokyoJapan
  2. 2.Hokuto Hospital, ObihiroHokkaidoJapan
  3. 3.Department of NeurosurgeryOsaka Municipal UniversityOsakaJapan

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