Skip to main content
PMC home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1996 Mar;16(3):1126–1137. doi: 10.1128/mcb.16.3.1126

Cytoplasmic sequestration of wild-type p53 protein impairs the G1 checkpoint after DNA damage.

U M Moll 1, A G Ostermeyer 1, R Haladay 1, B Winkfield 1, M Frazier 1, G Zambetti 1
PMCID: PMC231095  PMID: 8622657

Abstract

Wild-type p53 protein is abnormally sequestered in the cytoplasm of a subset of primary human tumors including neuroblastomas (NB) (U. M. Moll, M. LaQuaglia, J. Benard, and G. Riou, Proc. Natl. Acad. Sci. USA 92:4407-4411, 1995; U. M. Moll, G. Riou, and A. J. Levine, Proc. Natl. Acad. Sci.USA 89:7262-7266, 1992). This may represent a nonmutational mechanism for abrogating p53 tumor suppressor function. To test this hypothesis, we established the first available in vitro model that accurately reflects the wild-type p53 sequestration found in NB tumors. We characterized a series of human NB cell lines that overexpress wild-type p53 and show that p53 is preferentially localized to discrete cytoplasmic structures, with no detectable nuclear p53. These cell lines, when challenged with a variety of DNA strand-breaking agents, all exhibit impaired p53-mediated G1 arrest. Induction analysis of p53 and p53-responsive genes show that this impairment is due to suppression of nuclear p53 accumulation. Thus, this naturally occurring translocation defect compromises the suppressor function of p53 and likely plays a role in the tumorigenesis of these tumors previously thought to be unaffected by p53 alterations.

Full Text

The Full Text of this article is available as a PDF (5.0 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bueso-Ramos C. E., Manshouri T., Haidar M. A., Huh Y. O., Keating M. J., Albitar M. Multiple patterns of MDM-2 deregulation in human leukemias: implications in leukemogenesis and prognosis. Leuk Lymphoma. 1995 Mar;17(1-2):13–18. doi: 10.3109/10428199509051698. [DOI] [PubMed] [Google Scholar]
  2. Chen J. W., Cha Y., Yuksel K. U., Gracy R. W., August J. T. Isolation and sequencing of a cDNA clone encoding lysosomal membrane glycoprotein mouse LAMP-1. Sequence similarity to proteins bearing onco-differentiation antigens. J Biol Chem. 1988 Jun 25;263(18):8754–8758. [PubMed] [Google Scholar]
  3. Chen Y., Chen C. F., Riley D. J., Allred D. C., Chen P. L., Von Hoff D., Osborne C. K., Lee W. H. Aberrant subcellular localization of BRCA1 in breast cancer. Science. 1995 Nov 3;270(5237):789–791. doi: 10.1126/science.270.5237.789. [DOI] [PubMed] [Google Scholar]
  4. Davidoff A. M., Iglehart J. D., Marks J. R. Immune response to p53 is dependent upon p53/HSP70 complexes in breast cancers. Proc Natl Acad Sci U S A. 1992 Apr 15;89(8):3439–3442. doi: 10.1073/pnas.89.8.3439. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Davidoff A. M., Pence J. C., Shorter N. A., Iglehart J. D., Marks J. R. Expression of p53 in human neuroblastoma- and neuroepithelioma-derived cell lines. Oncogene. 1992 Jan;7(1):127–133. [PubMed] [Google Scholar]
  6. Debbas M., White E. Wild-type p53 mediates apoptosis by E1A, which is inhibited by E1B. Genes Dev. 1993 Apr;7(4):546–554. doi: 10.1101/gad.7.4.546. [DOI] [PubMed] [Google Scholar]
  7. Demers G. W., Foster S. A., Halbert C. L., Galloway D. A. Growth arrest by induction of p53 in DNA damaged keratinocytes is bypassed by human papillomavirus 16 E7. Proc Natl Acad Sci U S A. 1994 May 10;91(10):4382–4386. doi: 10.1073/pnas.91.10.4382. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Eady J. J., Peacock J. H., McMillan T. J. Host cell reactivation of gamma-irradiated adenovirus 5 in human cell lines of varying radiosensitivity. Br J Cancer. 1992 Jul;66(1):113–118. doi: 10.1038/bjc.1992.226. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Feitelson M. A., Zhu M., Duan L. X., London W. T. Hepatitis B x antigen and p53 are associated in vitro and in liver tissues from patients with primary hepatocellular carcinoma. Oncogene. 1993 May;8(5):1109–1117. [PubMed] [Google Scholar]
  10. Fontoura B. M., Sorokina E. A., David E., Carroll R. B. p53 is covalently linked to 5.8S rRNA. Mol Cell Biol. 1992 Nov;12(11):5145–5151. doi: 10.1128/mcb.12.11.5145. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Funk W. D., Pak D. T., Karas R. H., Wright W. E., Shay J. W. A transcriptionally active DNA-binding site for human p53 protein complexes. Mol Cell Biol. 1992 Jun;12(6):2866–2871. doi: 10.1128/mcb.12.6.2866. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Gannon J. V., Lane D. P. Protein synthesis required to anchor a mutant p53 protein which is temperature-sensitive for nuclear transport. Nature. 1991 Feb 28;349(6312):802–806. doi: 10.1038/349802a0. [DOI] [PubMed] [Google Scholar]
  13. Ginsberg D., Michael-Michalovitz D., Ginsberg D., Oren M. Induction of growth arrest by a temperature-sensitive p53 mutant is correlated with increased nuclear localization and decreased stability of the protein. Mol Cell Biol. 1991 Jan;11(1):582–585. doi: 10.1128/mcb.11.1.582. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Green J., Griffiths G., Louvard D., Quinn P., Warren G. Passage of viral membrane proteins through the Golgi complex. J Mol Biol. 1981 Nov 15;152(4):663–698. doi: 10.1016/0022-2836(81)90122-4. [DOI] [PubMed] [Google Scholar]
  15. Gusterson B. A., Anbazhagan R., Warren W., Midgely C., Lane D. P., O'Hare M., Stamps A., Carter R., Jayatilake H. Expression of p53 in premalignant and malignant squamous epithelium. Oncogene. 1991 Oct;6(10):1785–1789. [PubMed] [Google Scholar]
  16. Halperin E. C., Cox E. B. Radiation therapy in the management of neuroblastoma: the Duke University Medical Center experience 1967-1984. Int J Radiat Oncol Biol Phys. 1986 Oct;12(10):1829–1837. doi: 10.1016/0360-3016(86)90326-3. [DOI] [PubMed] [Google Scholar]
  17. Harper J. W., Adami G. R., Wei N., Keyomarsi K., Elledge S. J. The p21 Cdk-interacting protein Cip1 is a potent inhibitor of G1 cyclin-dependent kinases. Cell. 1993 Nov 19;75(4):805–816. doi: 10.1016/0092-8674(93)90499-g. [DOI] [PubMed] [Google Scholar]
  18. Harris C. C., Hollstein M. Clinical implications of the p53 tumor-suppressor gene. N Engl J Med. 1993 Oct 28;329(18):1318–1327. doi: 10.1056/NEJM199310283291807. [DOI] [PubMed] [Google Scholar]
  19. Hinds P. W., Finlay C. A., Frey A. B., Levine A. J. Immunological evidence for the association of p53 with a heat shock protein, hsc70, in p53-plus-ras-transformed cell lines. Mol Cell Biol. 1987 Aug;7(8):2863–2869. doi: 10.1128/mcb.7.8.2863. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Hollstein M., Rice K., Greenblatt M. S., Soussi T., Fuchs R., Sørlie T., Hovig E., Smith-Sørensen B., Montesano R., Harris C. C. Database of p53 gene somatic mutations in human tumors and cell lines. Nucleic Acids Res. 1994 Sep;22(17):3551–3555. [PMC free article] [PubMed] [Google Scholar]
  21. James M., Mansbridge J., Kidson C. Ultraviolet radiation sensitivity of proliferating and differentiated human neuroblastoma cells. Int J Radiat Biol Relat Stud Phys Chem Med. 1982 May;41(5):547–556. doi: 10.1080/09553008214550621. [DOI] [PubMed] [Google Scholar]
  22. Juven T., Barak Y., Zauberman A., George D. L., Oren M. Wild type p53 can mediate sequence-specific transactivation of an internal promoter within the mdm2 gene. Oncogene. 1993 Dec;8(12):3411–3416. [PubMed] [Google Scholar]
  23. Kastan M. B., Onyekwere O., Sidransky D., Vogelstein B., Craig R. W. Participation of p53 protein in the cellular response to DNA damage. Cancer Res. 1991 Dec 1;51(23 Pt 1):6304–6311. [PubMed] [Google Scholar]
  24. Kastan M. B., Zhan Q., el-Deiry W. S., Carrier F., Jacks T., Walsh W. V., Plunkett B. S., Vogelstein B., Fornace A. J., Jr A mammalian cell cycle checkpoint pathway utilizing p53 and GADD45 is defective in ataxia-telangiectasia. Cell. 1992 Nov 13;71(4):587–597. doi: 10.1016/0092-8674(92)90593-2. [DOI] [PubMed] [Google Scholar]
  25. Koop C. E., Schnaufer L. The management of abdominal neuroblastoma. Cancer. 1975 Mar;35(3 Suppl):905–909. doi: 10.1002/1097-0142(197503)35:3+<905::aid-cncr2820350708>3.0.co;2-6. [DOI] [PubMed] [Google Scholar]
  26. Kruman I. I., Kostenko M. A., Gordon RYa, Popov V. I., Umansky S. R. Differentiation and apoptosis of murine neuroblastoma cells N1E115. Biochem Biophys Res Commun. 1993 Mar 31;191(3):1309–1318. doi: 10.1006/bbrc.1993.1360. [DOI] [PubMed] [Google Scholar]
  27. Kulesz-Martin M. F., Lisafeld B., Huang H., Kisiel N. D., Lee L. Endogenous p53 protein generated from wild-type alternatively spliced p53 RNA in mouse epidermal cells. Mol Cell Biol. 1994 Mar;14(3):1698–1708. doi: 10.1128/mcb.14.3.1698. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Lane D. P., Benchimol S. p53: oncogene or anti-oncogene? Genes Dev. 1990 Jan;4(1):1–8. doi: 10.1101/gad.4.1.1. [DOI] [PubMed] [Google Scholar]
  29. Lane D. P. Cancer. p53, guardian of the genome. Nature. 1992 Jul 2;358(6381):15–16. doi: 10.1038/358015a0. [DOI] [PubMed] [Google Scholar]
  30. Lin D., Fiscella M., O'Connor P. M., Jackman J., Chen M., Luo L. L., Sala A., Travali S., Appella E., Mercer W. E. Constitutive expression of B-myb can bypass p53-induced Waf1/Cip1-mediated G1 arrest. Proc Natl Acad Sci U S A. 1994 Oct 11;91(21):10079–10083. doi: 10.1073/pnas.91.21.10079. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Livingstone A., Mairs R. J., Russell J., O'Donoghue J., Gaze M. N., Wheldon T. E. N-myc gene copy number in neuroblastoma cell lines and resistance to experimental treatment. Eur J Cancer. 1994;30A(3):382–389. doi: 10.1016/0959-8049(94)90260-7. [DOI] [PubMed] [Google Scholar]
  32. Livingstone L. R., White A., Sprouse J., Livanos E., Jacks T., Tlsty T. D. Altered cell cycle arrest and gene amplification potential accompany loss of wild-type p53. Cell. 1992 Sep 18;70(6):923–935. doi: 10.1016/0092-8674(92)90243-6. [DOI] [PubMed] [Google Scholar]
  33. Lu X., Lane D. P. Differential induction of transcriptionally active p53 following UV or ionizing radiation: defects in chromosome instability syndromes? Cell. 1993 Nov 19;75(4):765–778. doi: 10.1016/0092-8674(93)90496-d. [DOI] [PubMed] [Google Scholar]
  34. Martinez J., Georgoff I., Martinez J., Levine A. J. Cellular localization and cell cycle regulation by a temperature-sensitive p53 protein. Genes Dev. 1991 Feb;5(2):151–159. doi: 10.1101/gad.5.2.151. [DOI] [PubMed] [Google Scholar]
  35. McMillan T. J., Cassoni A. M., Edwards S., Holmes A., Peacock J. H. The relationship of DNA double-strand break induction to radiosensitivity in human tumour cell lines. Int J Radiat Biol. 1990 Sep;58(3):427–438. doi: 10.1080/09553009014551781. [DOI] [PubMed] [Google Scholar]
  36. Melino G., Annicchiarico-Petruzzelli M., Piredda L., Candi E., Gentile V., Davies P. J., Piacentini M. Tissue transglutaminase and apoptosis: sense and antisense transfection studies with human neuroblastoma cells. Mol Cell Biol. 1994 Oct;14(10):6584–6596. doi: 10.1128/mcb.14.10.6584. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Michieli P., Chedid M., Lin D., Pierce J. H., Mercer W. E., Givol D. Induction of WAF1/CIP1 by a p53-independent pathway. Cancer Res. 1994 Jul 1;54(13):3391–3395. [PubMed] [Google Scholar]
  38. Mietz J. A., Unger T., Huibregtse J. M., Howley P. M. The transcriptional transactivation function of wild-type p53 is inhibited by SV40 large T-antigen and by HPV-16 E6 oncoprotein. EMBO J. 1992 Dec;11(13):5013–5020. doi: 10.1002/j.1460-2075.1992.tb05608.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Moll U. M., LaQuaglia M., Bénard J., Riou G. Wild-type p53 protein undergoes cytoplasmic sequestration in undifferentiated neuroblastomas but not in differentiated tumors. Proc Natl Acad Sci U S A. 1995 May 9;92(10):4407–4411. doi: 10.1073/pnas.92.10.4407. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Moll U. M., Riou G., Levine A. J. Two distinct mechanisms alter p53 in breast cancer: mutation and nuclear exclusion. Proc Natl Acad Sci U S A. 1992 Aug 1;89(15):7262–7266. doi: 10.1073/pnas.89.15.7262. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Momand J., Zambetti G. P., Olson D. C., George D., Levine A. J. The mdm-2 oncogene product forms a complex with the p53 protein and inhibits p53-mediated transactivation. Cell. 1992 Jun 26;69(7):1237–1245. doi: 10.1016/0092-8674(92)90644-r. [DOI] [PubMed] [Google Scholar]
  42. Negrini M., Sabbioni S., Haldar S., Possati L., Castagnoli A., Corallini A., Barbanti-Brodano G., Croce C. M. Tumor and growth suppression of breast cancer cells by chromosome 17-associated functions. Cancer Res. 1994 Apr 1;54(7):1818–1824. [PubMed] [Google Scholar]
  43. Nelson W. G., Kastan M. B. DNA strand breaks: the DNA template alterations that trigger p53-dependent DNA damage response pathways. Mol Cell Biol. 1994 Mar;14(3):1815–1823. doi: 10.1128/mcb.14.3.1815. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Noda A., Ning Y., Venable S. F., Pereira-Smith O. M., Smith J. R. Cloning of senescent cell-derived inhibitors of DNA synthesis using an expression screen. Exp Cell Res. 1994 Mar;211(1):90–98. doi: 10.1006/excr.1994.1063. [DOI] [PubMed] [Google Scholar]
  45. Oliner J. D., Kinzler K. W., Meltzer P. S., George D. L., Vogelstein B. Amplification of a gene encoding a p53-associated protein in human sarcomas. Nature. 1992 Jul 2;358(6381):80–83. doi: 10.1038/358080a0. [DOI] [PubMed] [Google Scholar]
  46. Oren M., Maltzman W., Levine A. J. Post-translational regulation of the 54K cellular tumor antigen in normal and transformed cells. Mol Cell Biol. 1981 Feb;1(2):101–110. doi: 10.1128/mcb.1.2.101. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Prives C., Bargonetti J., Farmer G., Ferrari E., Friedlander P., Wang Y., Jayaraman L., Pavletich N., Hubscher U. DNA-binding properties of the p53 tumor suppressor protein. Cold Spring Harb Symp Quant Biol. 1994;59:207–213. doi: 10.1101/sqb.1994.059.01.025. [DOI] [PubMed] [Google Scholar]
  48. Scheffner M., Werness B. A., Huibregtse J. M., Levine A. J., Howley P. M. The E6 oncoprotein encoded by human papillomavirus types 16 and 18 promotes the degradation of p53. Cell. 1990 Dec 21;63(6):1129–1136. doi: 10.1016/0092-8674(90)90409-8. [DOI] [PubMed] [Google Scholar]
  49. Shaulsky G., Ben-Ze'ev A., Rotter V. Subcellular distribution of the p53 protein during the cell cycle of Balb/c 3T3 cells. Oncogene. 1990 Nov;5(11):1707–1711. [PubMed] [Google Scholar]
  50. Shaulsky G., Goldfinger N., Ben-Ze'ev A., Rotter V. Nuclear accumulation of p53 protein is mediated by several nuclear localization signals and plays a role in tumorigenesis. Mol Cell Biol. 1990 Dec;10(12):6565–6577. doi: 10.1128/mcb.10.12.6565. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Shaulsky G., Goldfinger N., Peled A., Rotter V. Involvement of wild-type p53 protein in the cell cycle requires nuclear localization. Cell Growth Differ. 1991 Dec;2(12):661–667. [PubMed] [Google Scholar]
  52. Shaulsky G., Goldfinger N., Tosky M. S., Levine A. J., Rotter V. Nuclear localization is essential for the activity of p53 protein. Oncogene. 1991 Nov;6(11):2055–2065. [PubMed] [Google Scholar]
  53. Sidell N., Koeffler H. P. Modulation of Mr 53,000 protein with induction of differentiation of human neuroblastoma cells. Cancer Res. 1988 Apr 15;48(8):2226–2230. [PubMed] [Google Scholar]
  54. Speir E., Modali R., Huang E. S., Leon M. B., Shawl F., Finkel T., Epstein S. E. Potential role of human cytomegalovirus and p53 interaction in coronary restenosis. Science. 1994 Jul 15;265(5170):391–394. doi: 10.1126/science.8023160. [DOI] [PubMed] [Google Scholar]
  55. Takahashi K., Sumimoto H., Suzuki K., Ono T. Protein synthesis-dependent cytoplasmic translocation of p53 protein after serum stimulation of growth-arrested MCF-7 cells. Mol Carcinog. 1993;8(1):58–66. doi: 10.1002/mc.2940080112. [DOI] [PubMed] [Google Scholar]
  56. Takahashi T., Nau M. M., Chiba I., Birrer M. J., Rosenberg R. K., Vinocour M., Levitt M., Pass H., Gazdar A. F., Minna J. D. p53: a frequent target for genetic abnormalities in lung cancer. Science. 1989 Oct 27;246(4929):491–494. doi: 10.1126/science.2554494. [DOI] [PubMed] [Google Scholar]
  57. Ueda H., Ullrich S. J., Gangemi J. D., Kappel C. A., Ngo L., Feitelson M. A., Jay G. Functional inactivation but not structural mutation of p53 causes liver cancer. Nat Genet. 1995 Jan;9(1):41–47. doi: 10.1038/ng0195-41. [DOI] [PubMed] [Google Scholar]
  58. Vindeløv L. L., Christensen I. J., Nissen N. I. A detergent-trypsin method for the preparation of nuclei for flow cytometric DNA analysis. Cytometry. 1983 Mar;3(5):323–327. doi: 10.1002/cyto.990030503. [DOI] [PubMed] [Google Scholar]
  59. Vojtesek B., Lane D. P. Regulation of p53 protein expression in human breast cancer cell lines. J Cell Sci. 1993 Jul;105(Pt 3):607–612. doi: 10.1242/jcs.105.3.607. [DOI] [PubMed] [Google Scholar]
  60. Wagner I., Arlt H., van Dyck L., Langer T., Neupert W. Molecular chaperones cooperate with PIM1 protease in the degradation of misfolded proteins in mitochondria. EMBO J. 1994 Nov 1;13(21):5135–5145. doi: 10.1002/j.1460-2075.1994.tb06843.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Wang X. W., Forrester K., Yeh H., Feitelson M. A., Gu J. R., Harris C. C. Hepatitis B virus X protein inhibits p53 sequence-specific DNA binding, transcriptional activity, and association with transcription factor ERCC3. Proc Natl Acad Sci U S A. 1994 Mar 15;91(6):2230–2234. doi: 10.1073/pnas.91.6.2230. [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. White E. Tumour biology. p53, guardian of Rb. Nature. 1994 Sep 1;371(6492):21–22. doi: 10.1038/371021a0. [DOI] [PubMed] [Google Scholar]
  63. Wu X., Bayle J. H., Olson D., Levine A. J. The p53-mdm-2 autoregulatory feedback loop. Genes Dev. 1993 Jul;7(7A):1126–1132. doi: 10.1101/gad.7.7a.1126. [DOI] [PubMed] [Google Scholar]
  64. Xiong Y., Hannon G. J., Zhang H., Casso D., Kobayashi R., Beach D. p21 is a universal inhibitor of cyclin kinases. Nature. 1993 Dec 16;366(6456):701–704. doi: 10.1038/366701a0. [DOI] [PubMed] [Google Scholar]
  65. Yew P. R., Berk A. J. Inhibition of p53 transactivation required for transformation by adenovirus early 1B protein. Nature. 1992 May 7;357(6373):82–85. doi: 10.1038/357082a0. [DOI] [PubMed] [Google Scholar]
  66. Yewdell J. W., Gannon J. V., Lane D. P. Monoclonal antibody analysis of p53 expression in normal and transformed cells. J Virol. 1986 Aug;59(2):444–452. doi: 10.1128/jvi.59.2.444-452.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  67. Yin Y., Tainsky M. A., Bischoff F. Z., Strong L. C., Wahl G. M. Wild-type p53 restores cell cycle control and inhibits gene amplification in cells with mutant p53 alleles. Cell. 1992 Sep 18;70(6):937–948. doi: 10.1016/0092-8674(92)90244-7. [DOI] [PubMed] [Google Scholar]
  68. el-Deiry W. S., Kern S. E., Pietenpol J. A., Kinzler K. W., Vogelstein B. Definition of a consensus binding site for p53. Nat Genet. 1992 Apr;1(1):45–49. doi: 10.1038/ng0492-45. [DOI] [PubMed] [Google Scholar]
  69. el-Deiry W. S., Tokino T., Velculescu V. E., Levy D. B., Parsons R., Trent J. M., Lin D., Mercer W. E., Kinzler K. W., Vogelstein B. WAF1, a potential mediator of p53 tumor suppression. Cell. 1993 Nov 19;75(4):817–825. doi: 10.1016/0092-8674(93)90500-p. [DOI] [PubMed] [Google Scholar]

Articles from Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES