Volume 11, Issue 4 (Vol.11 No.4 Jan 2023)                   rbmb.net 2023, 11(4): 672-683 | Back to browse issues page


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Department of Bacteriology and Virology, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran & Gastroenterohepatology Research Center, Shiraz University of Medical Sciences, Shiraz, Iran.
Abstract:   (1463 Views)
Background: Suppression of p53 is an important mechanism in Epstein-Barr virus associate-tumors and described as EBNA1-USP7 which is a key axis in p53 suppression. Thus, in this study, we aimed to evaluate the function of EBNA1 on the expression of p53-inhibiting genes including HDAC-1, MDM2, MDM4, Sirt-3, and PSMD10 and the influence of USP7 inhibition using GNE-6776 on p53 at protein/mRNA level.

Methods:  The electroporation method was used to transfect the BL28 cell line with EBNA1. Cells with stable EBNA1 expression were selected by Hygromycin B treatment. The expression of seven genes, including PSMD10, HDAC-1, USP7, MDM2, P53, Sirt-3, and MDM4, was evaluated using a real-time PCR assay. For evaluating the effects of USP7 inhibition, the cells were treated with GNE-6776; after 24 hours and 4 days, the cells were collected and again expression of interest genes was evaluated.

Results: MDM2 (P=0.028), MDM4 (P=0.028), USP7 (P=0.028), and HDAC1 (P=0.015) all showed significantly higher expression in EBNA1-harboring cells compared to control plasmid transfected cells, while p53 mRNA expression was only marginally downregulated in EBNA1 harboring cells (P=0.685). Four-day after treatment, none of the studied genes was significantly changed. Also, in the first 24-hour after treatment, mRNA expression of p53 was downregulated (P=0.685), but after 4 days it was upregulated (P=0.7) insignificantly.

Conclusions: It seems that EBNA1 could strongly upregulate p53-inhibiting genes including HDAC1, MDM2, MDM4, and USP7. Moreover, it appears that the effects of USP7 suppression on p53 at protein/mRNA level depend on the cell nature; however, further research is needed.
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Type of Article: Original Article | Subject: Microbiology
Received: 2022/07/29 | Accepted: 2022/08/14 | Published: 2023/04/3

References
1. Grywalska E, Rolinski J. Epstein-Barr virus-associated lymphomas. Semin Oncol. 2015;42(2):291-303. [DOI:10.1053/j.seminoncol.2014.12.030] [PMID]
2. Zanella L, Riquelme I, Buchegger K, Abanto M, Ili C, Brebi P. A reliable Epstein-Barr Virus classification based on phylogenomic and population analyses. Sci Rep. 2019;9(1):9829. [DOI:10.1038/s41598-019-45986-3] [PMID] [PMCID]
3. Zanelli M, Sanguedolce F, Palicelli A, Zizzo M, Martino G, Caprera C, et al. EBV-Driven Lymphoproliferative Disorders and Lymphomas of the Gastrointestinal Tract: A Spectrum of Entities with a Common Denominator (Part 2). Cancers (Basel). 2021;13(18):4527. https://doi.org/10.3390/cancers13184578 [DOI:10.3390/cancers13184527] [PMCID]
4. Fields Virology: DNA Viruses. Howley PM, Knipe DM, Cohen JL, Damania BA, editors. Wolters Kluwer Health/Lippincott Williams & Wilkins; 2021.
5. Stuhlmann-Laeisz C, Oschlies I, Klapper W. Detection of EBV in reactive and neoplastic lymphoproliferations in adults-when and how? J Hematop. 2014;7(4):165-170. [DOI:10.1007/s12308-014-0209-0] [PMID] [PMCID]
6. Wilson JB, Manet E, Gruffat H, Busson P, Blondel M, Fahraeus R. EBNA1: Oncogenic Activity, Immune Evasion and Biochemical Functions Provide Targets for Novel Therapeutic Strategies against Epstein-Barr Virus- Associated Cancers. Cancers (Basel). 2018;10(4):109. [DOI:10.3390/cancers10040109] [PMID] [PMCID]
7. Reisman D, Yates J, Sugden B. A putative origin of replication of plasmids derived from Epstein-Barr virus is composed of two cis-acting components. Mol Cell Biol. 1985;5(8):1822-32. https://doi.org/10.1128/mcb.5.8.1822-1832.1985 [DOI:10.1128/MCB.5.8.1822] [PMID] [PMCID]
8. Kessler BM, Fortunati E, Melis M, Pals CE, Clevers H, Maurice MM. Proteome changes induced by knock-down of the deubiquitylating enzyme HAUSP/USP7. J Proteome Res. 2007;6(11):4163-72. [DOI:10.1021/pr0702161] [PMID]
9. Pozhidaeva A, Bezsonova I. USP7: Structure, substrate specificity, and inhibition. DNA repair. 2019;76:30-9. [DOI:10.1016/j.dnarep.2019.02.005] [PMID] [PMCID]
10. Wang Z, Kang W, You Y, Pang J, Ren H, Suo Z, et al. USP7: novel drug target in cancer therapy. Front Pharmacol. 2019;10:427. [DOI:10.3389/fphar.2019.00427] [PMID] [PMCID]
11. Qi SM, Cheng G, Cheng XD, Xu Z, Xu B, Zhang WD, et al. Targeting USP7-mediated deubiquitination of MDM2/MDMX-p53 pathway for cancer therapy: are we there yet? Front Cell Dev Biol. 2020;8:233. [DOI:10.3389/fcell.2020.00233] [PMID] [PMCID]
12. Schauer NJ, Liu X, Magin RS, Doherty LM, Chan WC, Ficarro SB, et al. Selective USP7 inhibition elicits cancer cell killing through a p53-dependent mechanism. Sci Rep. 2020; 10(1):5324. [DOI:10.1038/s41598-020-62076-x] [PMID] [PMCID]
13. Saridakis V, Sheng Y, Sarkari F, Holowaty MN, Shire K, Nguyen T, et al. Structure of the p53 binding domain of HAUSP/USP7 bound to Epstein-Barr nuclear antigen 1: implications for EBV-mediated immortalization. Mol Cell. 2005;18(1):25-36. [DOI:10.1016/j.molcel.2005.02.029] [PMID]
14. AlQarni S, Al-Sheikh Y, Campbell D, Drotar M, Hannigan A, Boyle S, et al. Lymphomas driven by Epstein-Barr virus nuclear antigen-1 (EBNA1) are dependant upon Mdm2. Oncogene. 2018;37(29):3998-4012. [DOI:10.1038/s41388-018-0147-x] [PMID] [PMCID]
15. Renouf B, Hollville E, Pujals A, Tetaud C, Garibal J, Wiels J. Activation of p53 by MDM2 antagonists has differential apoptotic effects on Epstein-Barr virus (EBV)-positive and EBV-negative Burkitt's lymphoma cells. Leukemia. 2009;23(9):1557-63. [DOI:10.1038/leu.2009.92] [PMID]
16. Ghotaslou A, Samii A, Boustani H, Ghalesardi OK, Shahidi M. AMG-232, a New Inhibitor of MDM-2, Enhance Doxorubicin Efficiency in Pre-B Acute Lymphoblastic Leukemia Cells. Rep Biochem Mol Biol 2022;11(1):111-124. [DOI:10.52547/rbmb.11.1.111] [PMID] [PMCID]
17. Zheng X, Wang J, Wei L, Peng Q, Gao Y, Fu Y, et al. Epstein-Barr virus microRNA miR-BART5-3p inhibits p53 expression. Virol J. 2018;92(23):e01022-18. [DOI:10.1128/JVI.01022-18] [PMID] [PMCID]
18. Luo J, Su F, Chen D, Shiloh A, Gu W. Deacetylation of p53 modulates its effect on cell growth and apoptosis. Nature. 2000;408(6810):377-81. [DOI:10.1038/35042612] [PMID]
19. Shadfan M, Lopez-Pajares V, Yuan ZM. MDM2 and MDMX: Alone and together in regulation of p53. Transl Cancer Res. 2012;1(2):88-89.
20. Shvarts A, Steegenga WT, Riteco N, Van Laar T, Dekker P, Bazuine M, et al. MDMX: a novel p53‐binding protein with some functional properties of MDM2. The EMBO J. 1996;15(19):5349-57. [DOI:10.1002/j.1460-2075.1996.tb00919.x] [PMID]
21. Danovi D, Meulmeester E, Pasini D, Migliorini D, Capra M, Frenk R, et al. Amplification of Mdmx (or Mdm4) directly contributes to tumor formation by inhibiting p53 tumor suppressor activity. Mol Cell Biol. 2004;24(13):5835-43. [DOI:10.1128/MCB.24.13.5835-5843.2004] [PMID] [PMCID]
22. Chen Y, Fu L, Wen X, Wang XY, Liu J, Cheng Y, et al. Sirtuin-3 (SIRT3), a therapeutic target with oncogenic and tumor-suppressive function in cancer. Cell Death Dis. 2014;5(2):e1047.. [DOI:10.1038/cddis.2014.14] [PMID] [PMCID]
23. Chen J, Wang A, Chen Q. SirT3 and p53 deacetylation in aging and cancer. J Cell Physiol. 2017;232(9):2308-11. [DOI:10.1002/jcp.25669] [PMID]
24. Kashyap D, Varshney N, Parmar HS, Jha HC. Gankyrin: At the crossroads of cancer diagnosis, disease prognosis, and development of efficient cancer therapeutics. Adv Cancer Biol Metastasis. 2021;4:100023. [DOI:10.1016/j.adcanc.2021.100023]
25. Dowran R, Sarvari J, Moattari A, Fattahi MR, Ramezani A, Hosseini SY. Analysis of TLR7, SOCS1 and ISG15 immune genes expression in the peripheral blood of responder and non-responder patients with chronic Hepatitis C. Gastroenterol Hepatol Bed Bench. 2017;10(4):272-277.
26. Hussain H, Raj S, Ahmad S, Razak MFA, Wan Mohamud WN, Bakar J, Ghazali HM. Determination of cell viability using acridine orange/propidium iodide dual-spectrofluorometry assay. Cogent Food & Agriculture. 2019;5(1):158239. [DOI:10.1080/23311932.2019.1582398]
27. Hassig CA, Schreiber SL. Nuclear histone acetylases and deacetylases and transcriptional regulation: HATs off to HDACs. Curr Opin Chem Biol. 1997;1(3):300-8. [DOI:10.1016/S1367-5931(97)80066-X] [PMID]
28. Yamaguchi H, Woods NT, Piluso LG, Lee HH, Chen J, Bhalla KN, et al. p53 acetylation is crucial for its transcription-independent proapoptotic functions. J. Biol. Chem. 2009;284(17):11171-83. [DOI:10.1074/jbc.M809268200] [PMID] [PMCID]
29. Edwards RH, Dekroon R, Raab-Traub N. Alterations in cellular expression in EBV infected epithelial cell lines and tumors. PLoS Pathog. 2019;15(10):e1008071. [DOI:10.1371/journal.ppat.1008071] [PMID] [PMCID]
30. Momand J, Zambetti GP, Olson DC, George D, Levine AJ. The mdm-2 oncogene product forms a complex with the p53 protein and inhibits p53-mediated transactivation. cell. 1992;69(7):1237-45. [DOI:10.1016/0092-8674(92)90644-R] [PMID]
31. Oliner JD, Pietenpol JA, Thiagalingam S, Gyuris J, Kinzler KW, Vogelstein B. Oncoprotein MDM2 conceals the activation domain of tumour suppressor p53. Nature. 1993;362(6423):857-60. [DOI:10.1038/362857a0] [PMID]
32. Lotfi Garavand A, Mohammadi M, Mohammadzadeh S. Evaluation of TP53 Codon 72, P21 Codon 31, and MDM2 SNP309 Polymorphisms in Iranian Patients with Acute Lymphocytic Leukemia. Rep Biochem Mol Biol. 2020;9(1):26-32. [DOI:10.29252/rbmb.9.1.26] [PMID] [PMCID]
33. Marine JC, Jochemsen AG. MDMX (MDM4), a Promising Target for p53 Reactivation Therapy and Beyond. Cold Spring Harb Perspect Med. 2016;6(7):a026237. [DOI:10.1101/cshperspect.a026237] [PMID] [PMCID]
34. Ma Y, Walsh MJ, Bernhardt K, Ashbaugh CW, Trudeau SJ, Ashbaugh IY, et al. CRISPR/Cas9 screens reveal Epstein-Barr virus-transformed B cell host dependency factors. Cell Host Microbe. 2017;21(5):580-91.e7. [DOI:10.1016/j.chom.2017.04.005] [PMID] [PMCID]
35. Zhou L, Ouyang T, Li M, Hong T, Mhs A, Meng W, et al. Ubiquitin-Specific Peptidase 7: A Novel Deubiquitinase That Regulates Protein Homeostasis and Cancers. Front Oncol. 2021;11:784672-. [DOI:10.3389/fonc.2021.784672] [PMID] [PMCID]
36. Bhattacharya S, Chakraborty D, Basu M, Ghosh MK. Emerging insights into HAUSP (USP7) in physiology, cancer and other diseases. Signal Transduct Target Ther. 2018;3(1):1-12. [DOI:10.1038/s41392-018-0012-y] [PMID] [PMCID]
37. He Y, Li W, Lv D, Zhang X, Zhang X, Ortiz YT, et al. Inhibition of USP7 activity selectively eliminates senescent cells in part via restoration of p53 activity. Aging Cell. 2020;19(3):e13117. [DOI:10.1111/acel.13117]
38. Agathanggelou A, Smith E, Davies NJ, Kwok M, Zlatanou A, Oldreive CE, et al. USP7 inhibition alters homologous recombination repair and targets CLL cells independently of ATM/p53 functional status. Blood. 2017;130(2):156-66. [DOI:10.1182/blood-2016-12-758219] [PMID]
39. Li S, Banck M, Mujtaba S, Zhou M-M, Sugrue MM, Walsh MJ. p53-induced growth arrest is regulated by the mitochondrial SirT3 deacetylase. PloS one. 2010;5(5):e10486. [DOI:10.1371/journal.pone.0010486] [PMID] [PMCID]
40. Jing H, Hu J, He B, YL NA, Stupinski J, Weiser K, et al. A SIRT2-selective inhibitor promotes c-Myc oncoprotein degradation and exhibits broad anticancer activity. Cancer cell. 2016;29(3):297-310. [DOI:10.1016/j.ccell.2016.02.007] [PMID] [PMCID]
41. Higashitsuji H, Higashitsuji H, Itoh K, Sakurai T, Nagao T, Sumitomo Y, et al. The oncoprotein gankyrin binds to MDM2/HDM2, enhancing ubiquitylation and degradation of p53. Cancer Cell. 2005;8(1):75-87. [DOI:10.1016/j.ccr.2005.06.006] [PMID]
42. Wang C, Li Y, Chu CM, Zhang XM, Ma J, Huang H, et al. Gankyrin is a novel biomarker for disease progression and prognosis of patients with renal cell carcinoma. EBioMedicine. 2019;39:255-64. [DOI:10.1016/j.ebiom.2018.12.011] [PMID] [PMCID]
43. Kashyap D, Baral B, Jakhmola S, Singh AK, Jha HC. Helicobacter pylori and Epstein-Barr Virus Coinfection Stimulates Aggressiveness in Gastric Cancer through the Regulation of Gankyrin. mSphere. 2021;6(5):e0075121. [DOI:10.1128/mSphere.00751-21] [PMID] [PMCID]
44. Ribeiro J, Malta M, Galaghar A, Silva F, Afonso LP, Medeiros R, Sousa H. P53 deregulation in Epstein-Barr virus-associated gastric cancer. Cancer Lett. 2017;404:37-43. [DOI:10.1016/j.canlet.2017.07.010] [PMID]

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