Asian Pacific Journal of Cancer Prevention

Asian Pacific Journal of Cancer Prevention (아시아태평양암예방학회)
권호 표지
DOI QR코드

DOI QR Code

UBE2Q1 in a Human Breast Carcinoma Cell Line: Overexpression and Interaction with p53

  • Shafiee, Sayed Mohammad (Departments of Biochemistry- Recombinant Protein Laboratory, School of Medicine, Shiraz University of Medical Sciences) ;
  • Rasti, Mozhgan (Departments of Biochemistry- Recombinant Protein Laboratory, School of Medicine, Shiraz University of Medical Sciences) ;
  • Seghatoleslam, Atefeh (Departments of Biochemistry- Recombinant Protein Laboratory, School of Medicine, Shiraz University of Medical Sciences) ;
  • Azimi, Tayebeh (Departments of Biochemistry- Recombinant Protein Laboratory, School of Medicine, Shiraz University of Medical Sciences) ;
  • Owji, Ali Akbar (Departments of Biochemistry- Recombinant Protein Laboratory, School of Medicine, Shiraz University of Medical Sciences)
  • Published : 2015.05.18
Download PDF
⟨ Previous Next ⟩

Abstract

The p53 tumor suppressor protein is a principal mediator of growth arrest, senescence, and apoptosis in response to a broad array of cellular damage. p53 is a substrate for the ubiquitin-proteasome system, however, the ubiquitin-conjugating enzymes (E2s) involved in p53 ubiquitination have not been well studied. UBE2Q1 is a novel E2 ubiquitin conjugating enzyme gene. Here, we investigated the effect of UBE2Q1 overexpression on the level of p53 in the MDA-MB-468 breast cancer cell line as well as the interaction between UBE2Q1 and p53. By using a lipofection method, the p53 mutated breast cancer cell line, MDA-MB-468, was transfected with the vector pCMV6-AN-GFP, containing UBE2Q1 ORF. Western blot analysis was employed to verify the overexpression of UBE2Q1 in MDA-MB-468 cells and to evaluate the expression level of p53 before and after cell transfection. Immunoprecipitation and GST pull-down protocols were used to investigate the binding of UBE2Q1 to p53. We established MDA-MB-468 cells that transiently expressed a GFP fusion proteins containing UBE2Q1 (GFP-UBE2Q1). Western blot analysis revealed that levels of p53 were markedly lower in UBE2Q1 transfected MDA-MB-468 cells as compared with control MDA-MB-468 cells. Both in vivo and in vitro data showed that UBE2Q1 co-precipitated with p53 protein. Our data for the first time showed that overexpression of UBE2Q1can lead to the repression of p53 in MDA-MB-468 cells. This repression of p53 may be due to its UBE2Q1 mediated ubiquitination and subsequent proteasome degradation, a process that may involve direct interaction of UBE2Q1with p53.

Keywords

References

  1. Bremm A, Komander D (2011). Emerging roles for Lys11-linked polyubiquitin in cellular regulation. Trends Biochem Sci, 36, 355-63.
  2. Brooks CL, Gu W (2003). Ubiquitination, phosphorylation and acetylation: the molecular basis for p53 regulation. Curr Opin Cell Biol, 15, 164-71. https://doi.org/10.1016/S0955-0674(03)00003-6
  3. Chang R, Wei L, Lu Y, et al (2015). Upregulated expression of ubiquitin-conjugating enzyme E2Q1 (UBE2Q1) is associated with enhanced cell proliferation and poor prognosis in human hapatocellular carcinoma. J Mol Histol, 46, 45-56. https://doi.org/10.1007/s10735-014-9596-x
  4. Chen S, Wang DL, Liu Y, et al (2012). RAD6 regulates the dosage of p53 by a combination of transcriptional and posttranscriptional mechanisms. Mol Cell Biol, 32, 576-87. https://doi.org/10.1128/MCB.05966-11
  5. Dahlmann B (2007). Role of proteasomes in disease. BMC Biochem, 8, 3. https://doi.org/10.1186/1471-2091-8-3
  6. Devine T, Dai MS (2013). Targeting the ubiquitin-mediated proteasome degradation of p53 for cancer therapy. Curr Pharm Des 19, 3248-62. https://doi.org/10.2174/1381612811319180009
  7. Gerard B, Sanders MA, Visscher DW, et al (2012). Lysine 394 is a novel Rad6B-induced ubiquitination site on beta-catenin. Biochim Biophys Acta, 1823, 1686-96. https://doi.org/10.1016/j.bbamcr.2012.05.032
  8. Goldstein G, Scheid M, Hammerling U, et al (1975). Isolation of a polypeptide that has lymphocyte-differentiating properties and is probably represented universally in living cells. Proc Natl Acad Sci U S A, 72, 11-5. https://doi.org/10.1073/pnas.72.1.11
  9. Hoeller D, Hecker CM, Wagner S, et al (2007). E3-independent monoubiquitination of ubiquitin-binding proteins. Mol Cell, 26, 891-8. https://doi.org/10.1016/j.molcel.2007.05.014
  10. Hohenstein P, Giles RH (2003). BRCA1: a scaffold for p53 response? Trends Genet, 19, 489-94. https://doi.org/10.1016/S0168-9525(03)00193-8
  11. Karami K, Cheraghi M, Amori N, et al (2014). Common cancers in Khuzestan province, south west of Iran, during 2005-2011. Asian Pac J Cancer Prev, 15, 9475-8. https://doi.org/10.7314/APJCP.2014.15.21.9475
  12. Lacroix M, Toillon RA, Leclercq G (2006). p53 and breast cancer, an update. Endocr Relat Cancer, 13, 293-325. https://doi.org/10.1677/erc.1.01172
  13. Lai Z, Yang T, Kim YB, et al (2002). Differentiation of Hdm2-mediated p53 ubiquitination and Hdm2 autoubiquitination activity by small molecular weight inhibitors. Proc Natl Acad Sci USA, 99, 14734-9. https://doi.org/10.1073/pnas.212428599
  14. Lee JT, Gu W (2010). The multiple levels of regulation by p53 ubiquitination. Cell Death Differ, 17, 86-92. https://doi.org/10.1038/cdd.2009.77
  15. Maeda H, Miyajima N, Kano S, et al (2009). Ubiquitin-conjugating enzyme UBE2Q2 suppresses cell proliferation and is down-regulated in recurrent head and neck cancer. Mol Cancer Res, 7, 1553-62. https://doi.org/10.1158/1541-7786.MCR-08-0543
  16. Mani A, Gelmann EP (2005). The ubiquitin-proteasome pathway and its role in cancer. J Clin Oncol, 23, 4776-89. https://doi.org/10.1200/JCO.2005.05.081
  17. Moll UM, Petrenko O (2003). The MDM2-p53 interaction. Mol Cancer Res, 1, 1001-8.
  18. Muller J, Eilers M (2008). Ubiquitination of myc: proteasomal degradation and beyond. Ernst Schering Found Symp Proc, 2008, 99-113.
  19. Nikseresht M, Seghatoleslam A, Monabati A, et al (2010). Overexpression of the novel human gene, UBE2Q2, in breast cancer. Cancer Genet Cytogenet, 197, 101-6. https://doi.org/10.1016/j.cancergencyto.2009.11.020
  20. Ohta T, Fukuda M (2004). Ubiquitin and breast cancer. Oncogene, 23, 2079-88. https://doi.org/10.1038/sj.onc.1207371
  21. Rasti M, Arabsolghar R, Khatooni Z, et al (2012). p53 Binds to estrogen receptor 1 promoter in human breast cancer cells. Pathol Oncol Res, 18, 169-75. https://doi.org/10.1007/s12253-011-9423-6
  22. Rodriguez MS, Desterro JM, Lain S, et al (2000). Multiple C-terminal lysine residues target p53 for ubiquitin-proteasome-mediated degradation. Mol Cell Biol, 20, 8458-67. https://doi.org/10.1128/MCB.20.22.8458-8467.2000
  23. Saville MK, Sparks A, Xirodimas DP, et al (2004). Regulation of p53 by the ubiquitin-conjugating enzymes UbcH5B/C in vivo. J Biol Chem, 279, 42169-81. https://doi.org/10.1074/jbc.M403362200
  24. Seghatoleslam A, Bozorg-Ghalati F, Monabati A, et al (2014). UBE2Q1, as a down regulated gene in pediatric acute lymphoblastic leukemia. Int J Mol Cell Med, 3, 95-101.
  25. Seghatoleslam A, Monabati A, Bozorg-Ghalati F, et al (2012a). Expression of UBE2Q2, a putative member of the ubiquitin-conjugating enzyme family in pediatric acute lymphoblastic leukemia. Arch Iran Med, 15, 352-5.
  26. Seghatoleslam A, Nikseresht M, Shafiee SM, et al (2012b). Expression of the novel human gene, UBE2Q1, in breast tumors. Mol Biol Rep, 39, 5135-41. https://doi.org/10.1007/s11033-011-1309-x
  27. Seghatoleslam A, Zambrano A (2009). Effects of over-expression of LOC92912 gene on cell cycle progression. Iran J Med Sci, 34, 227-84.
  28. Seghatoleslam A, Zambrano A, Millon R, et al (2006). Analysis of a novel human gene, LOC92912, over-expressed in hypopharyngeal tumours. Biochem Biophys Res Commun, 339, 422-9. https://doi.org/10.1016/j.bbrc.2005.11.026
  29. Shafiee SM, Seghatoleslam A, Nikseresht M, et al (2013). UBE2Q1 expression in human colorectal tumors and cell lines. Mol Biol Rep, 40, 7045-51. https://doi.org/10.1007/s11033-013-2824-8
  30. Shafiee SM, Seghatoleslam A, Nikseresht M, et al (2014). Expression status of UBE2Q2 in colorectal primary tumors and cell Lines. Iran J Med Sci, 39, 196-202.
  31. Tang XK, Wang KJ, Tang YK, et al (2014). Effects of ubiquitin-conjugating enzyme 2C on invasion, proliferation and cell cycling of lung cancer cells. Asian Pac J Cancer Prev, 15, 3005-9. https://doi.org/10.7314/APJCP.2014.15.7.3005
  32. Voutsadakis IA (2013). Ubiquitin- and ubiquitin-like proteins-conjugating enzymes (E2s) in breast cancer. Mol Biol Rep, 40, 2019-34. https://doi.org/10.1007/s11033-012-2261-0
  33. Waite KA, Eng C (2003). BMP2 exposure results in decreased PTEN protein degradation and increased PTEN levels. Hum Mol Genet, 12, 679-84. https://doi.org/10.1093/hmg/ddg069
  34. Wan C, Chen J, Hu B, et al (2014). Downregulation of UBE2Q1 is associated with neuronal apoptosis in rat brain cortex following traumatic brain injury. J Neurosci Res, 92, 1-12. https://doi.org/10.1002/jnr.23305
  35. Wei W, Jin J, Schlisio S, et al (2005). The v-Jun point mutation allows c-Jun to escape GSK3-dependent recognition and destruction by the Fbw7 ubiquitin ligase. Cancer Cell, 8, 25-33. https://doi.org/10.1016/j.ccr.2005.06.005
  36. Yang Y, Li CC, Weissman AM (2004). Regulating the p53 system through ubiquitination. Oncogene, 23, 2096-106. https://doi.org/10.1038/sj.onc.1207411
  37. Zhu S, Yao F, Li WH, et al (2013). PKC-dependent activation of the ubiquitin proteasome system is responsible for high glucose-induced human breast cancer MCF-7 cell proliferation, migration and invasion. Asian Pac J Cancer Prev, 14, 5687-92. https://doi.org/10.7314/APJCP.2013.14.10.5687

Cited by

  1. Differences in Prognostic Factors between Early and Late Recurrence Breast Cancers vol.16, pp.15, 2015, https://doi.org/10.7314/APJCP.2015.16.15.6575
  2. Induction of cell proliferation, clonogenicity and cell accumulation in S phase as a consequence of human UBE2Q1 overexpression vol.12, pp.3, 2016, https://doi.org/10.3892/ol.2016.4860