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HEK293 Hücreleri Üzerine Endoplazmik Retikulum Stresi Uyaranı Tunikamisinin Etkisi

Year 2018, Volume: 8 Issue: 3, 71 - 78, 02.12.2018

Abstract

DOI:
10.26650/experimed.2018. 462982


Amaç: Endoplazmik Retikulum (ER), protein
katlanması ve işlenmesi yanı sıra Ca+2 ve glikojen deposu ve hücre membran
lipidlerinin biyogenezi gibi fonksiyonlara sahiptir. Hipoksi, viral
enfeksiyonlar, yanlış protein katlanmaları, protein birikimleri ve bazı
kimyasallar hücrede, ER stresine sebep olarak katlanmamış protein yanıtı (UPR)
sistemlerini devreye sokmaktadır. Bu kimyasallardan biri olan Tunikamisin,
asparajin-bağlı glikoproteinlerin sentezinin ilk adımında görev alan GPT
(GlcNAc fosfotransferaz) enzimini inhibe ederek hücrede ER stresine sebep olan
bir antibiyotiktir. Bu çalışmada, ER stresini in vitro araştırmak için en uygun
deney şartlarının belirlenmesi amaçlanmıştır.

Gereç ve Yöntem: HEK293 hücre hattı uygun
kültür koşullarında çoğaltılarak 5 farklı dozda (0,5-1-2-5 ve 10 µg/mL), 12
saatlik Tunikamisin uygulanmıştır. 1-2-4-8 ve 12 saatlik sürelerle ışık
mikroskobunda morfolojik değerlendirilmeleri yapılmıştır. Normal HEK293
hücreleri ile 0,5 ve 1 µg/mL Tunikamisin uygulanan hücrelerden total RNA
izolasyonu ve cDNA sentezi yapılmıştır. Tunikamisin uygulamasının ER stres
açısından değerlendirilmesi için UPR sisteminden seçilen CHOP, BiP, GADD34,
ATF4, EDEM1, ASK1, XBP1 (spliced, unspliced ve total), TRAF2, HERP, PDIA4
genlerinin qRT-PCR yöntemi ile gen ekspresyonları normal hücrelere göre
kıyaslanmıştır.

Bulgular: Tunikamisin uygulamasından
1-2-4-8 ve 12 saatlik ışık mikroskobunda HEK293 hücrelerinin morfolojilerinin
sadece 0,5 ve 1 µg/mL konsantrasyonlarda korunduğu gözlenmiştir. Diğer dozlarda
ise daha ilk saatten hücrelerin morfolojik yapılarının bozulduğu gözlenmiştir.
qRT-PCR yöntemi ile UPR-ilişkili genlerden BiP, ASK1, PDIA4, s-xBP1, us-XBP1,
t-XBP1, HERP ve CHOP rölatif kantitasyon değerlerinde Tunikamisin uygulanmış
HEK293 hücrelerin normal hücrelere göre anlamlı bir artış saptanmıştır. ASK1
ile aynı yolaktan olmasına rağmen TRAF2 geni rölatif kantitasyon değerinde
azalma gözlenmiştir.







Sonuç: HEK293 hücrelerinde ER stresinin
oluşturulabilmesinde 0.5 ve 1 µg/mL
lik  Tunikamisin uygulaması uygun bulunmuştur.
Daha yüksek dozların HEK293 hücrelerini apoptoza yönlendirdiği düşünülmektedir. 

References

  • Schwarz DS, Blower MD. The endoplasmic reticulum: structure, function and response to cellular signaling. Cell Mol Life Sci 2016; 73(1): 79-94.
  • Oslowski CM, Urano F. Measuring ER stress and the unfolded protein response using mammalian tissue culture system. Methods Enzymol 2011; 490: 71-92.
  • Martins AS, Alves I, Helguero L, Domingues MR, Neves BM. The Unfolded Protein Response in Homeostasis and Modulation of Mammalian Immune Cells. Int Rev Immunol 2016; 35(6): 457-476.
  • Marciniak SJ, Yun CY, Oyadomari S, Novoa I, Zhang Y, Jungreis R, ve ark. CHOP induces death by promoting protein synthesis and oxidation in the stressed endoplasmic reticulum. Genes Dev 2004; 18(24): 3066-3077.
  • Takatsuki A, Arima K, Tamura G. Tunicamycin, a new antibiotic. I. Isolation and characterization of tunicamycin. J Antibiot (Tokyo) 1971; 24(4): 215-223.
  • Yoshida H. ER stress and diseases. FEBS J 2007; 274(3): 630-658.
  • Hetz C. The unfolded protein response: controlling cell fate decisions under ER stress and beyond. Nat Rev Mol Cell Biol 2012; 13(2): 89-102.
  • Tong Q, Wu L, Jiang T, Ou Z, Zhang Y, Zhu D. Inhibition of endoplasmic reticulum stress-activated IRE1alpha-TRAF2-caspase-12 apoptotic pathway is involved in the neuroprotective effects of telmisartan in the rotenone rat model of Parkinson's disease. Eur J Pharmacol 2016; 776: 106-115.
  • Malhotra JD, Kaufman RJ. The endoplasmic reticulum and the unfolded protein response. Semin Cell Dev Biol 2007; 18(6): 716-731.
  • Schroder M, Kaufman RJ. The mammalian unfolded protein response. Annu Rev Biochem 2005; 74: 739-789.
  • Wakabayashi S, Yoshida H. The essential biology of the endoplasmic reticulum stress response for structural and computational biologists. Comput Struct Biotechnol J 2013; 6: e201303010.
  • Yirrell DL, Roome AP, Darville JM, Ashley CR, Harbour J. Comparison of the continuous cell line 293 with human embryo kidney cells and human embryo fibroblast cells for the cultivation of ocular viruses. J Clin Pathol 1983; 36(9): 996-999.
  • Roche Molecular Systems. Universal ProbeLibrary Assay Design Center 09.07.2018; Erişim 18.07.2018, https://lifescience.roche.com/en_tr/brands/universal-probe-library.html#assay-design-center.
  • IntegratedDNATechnologies. OligoAnalyzer 3.1. 01.05.2018; Erişim 18.07.2018, https://eu.idtdna.com/calc/analyzer.
  • Szegezdi E, Logue SE, Gorman AM, Samali A. Mediators of endoplasmic reticulum stress-induced apoptosis. EMBO Rep. 2006 Sep;7(9):880-885.
  • Shinjo S, Mizotani Y, Tashiro E, Imoto M. Comparative analysis of the expression patterns of UPR-target genes caused by UPR-inducing compounds. Biosci Biotechnol Biochem 2013; 77(4): 729-735.
  • Neuber C, Uebeler J, Schulze T, Sotoud H, El-Armouche A, Eschenhagen T. Guanabenz interferes with ER stress and exerts protective effects in cardiac myocytes. PLoS One 2014; 9(6): e98893.
  • Weichert N, Kaltenborn E, Hector A, Woischnik M, Schams A, Holzinger A, ve ark. Some ABCA3 mutations elevate ER stress and initiate apoptosis of lung epithelial cells. Respir Res 2011; 12: 4.
  • Güleş Ö, Eren Ü. Apoptozun Belirlenmesinde Kullanılan Yöntemler. Y.Y.Ü. Veteriner Fakültesi Dergisi 2008; 2: 73-78.

The Effect of Endoplasmic Reticulum Stress Activator-Tunicamycin on HEK293 Cells

Year 2018, Volume: 8 Issue: 3, 71 - 78, 02.12.2018

Abstract

DOI:
10.26650/experimed.2018. 462982


Objective: Among the functions of the
Endoplasmic Reticulum (ER) are many different reactions that include the
regulation of protein folding and modifications in the lumen, as well as the
use of Ca+2 and glycogen storage, the biogenesis of cell membrane lipids. ER
homeostasis becomes unbalanced and is recognized as ER stress by the cell. It
triggers Unfolded Protein Responce (UPR) systems. Hypoxia, viral infections,
unfolded protein accumulation, and some chemicals cause ER stress. Among the
chemicals, tunicamycin is an antibiotic known to induce ER stress in the cell
by inhibiting the enzyme GlcNAc phosphotransferase (GPT), which is involved in
the first step of synthesis of asparagine-linked glycoproteins. The aim of this
study is to determine the optimal experimental conditions for investigating ER
stress in vitro.

Material and Method: The HEK293 cells were
cultured at the appropriate culture conditions and treated with five different
doses (0.5, 1, 2, 5, and 10
μg/mL) of tunicamycin for 12 hours. The
morphology of the cells was evaluated with a light microscope for 1, 2, 4, 8,
and 12 hours. The total RNA isolation and cDNA synthesis were performed from
normal and treated HEK293 cells with 0.5 and 1
μg/mL
of tunicamycin. Expressions of CHOP, BiP, GADD34, ATF4, EDEM1, ASK1, XBP1
(spliced, unspliced and total), TRAF2, HERP, and PDIA4 selected from the UPR
system were compared to normal cells for the evaluation of ER stress via
tunicamycin application by qRT-PCR

Results: After 1, 2, 4, 8, and 12 hour
observations using light microscopy after treatment of tunicamycin, the
morphology of HEK293 cells were preserved at concentrations of only 0.5 and 1
μg/mL. In all other doses, the morphological structures of the cells
were observed to be impaired within the first hour. Relative quantitation
values of BiP, ASK1, PDIA4, s-xBP1, us-XBP1, t-XBP1, HERP, and CHOP were
increased significantly on HEK293 cells treated with tunicamycin as commpared
to normal cells. Despite ASK1 being in the same pathway, a decrease of TRAF2
relative quantitation was observed.







Conclusion: It was found that the
appropriate doses to induce ER stress with tunicamycin utilizing an HEK293 cell
culture were 0.5 and 1
μg/mL. Higher doses are thought to lead
to cell apoptosis.

References

  • Schwarz DS, Blower MD. The endoplasmic reticulum: structure, function and response to cellular signaling. Cell Mol Life Sci 2016; 73(1): 79-94.
  • Oslowski CM, Urano F. Measuring ER stress and the unfolded protein response using mammalian tissue culture system. Methods Enzymol 2011; 490: 71-92.
  • Martins AS, Alves I, Helguero L, Domingues MR, Neves BM. The Unfolded Protein Response in Homeostasis and Modulation of Mammalian Immune Cells. Int Rev Immunol 2016; 35(6): 457-476.
  • Marciniak SJ, Yun CY, Oyadomari S, Novoa I, Zhang Y, Jungreis R, ve ark. CHOP induces death by promoting protein synthesis and oxidation in the stressed endoplasmic reticulum. Genes Dev 2004; 18(24): 3066-3077.
  • Takatsuki A, Arima K, Tamura G. Tunicamycin, a new antibiotic. I. Isolation and characterization of tunicamycin. J Antibiot (Tokyo) 1971; 24(4): 215-223.
  • Yoshida H. ER stress and diseases. FEBS J 2007; 274(3): 630-658.
  • Hetz C. The unfolded protein response: controlling cell fate decisions under ER stress and beyond. Nat Rev Mol Cell Biol 2012; 13(2): 89-102.
  • Tong Q, Wu L, Jiang T, Ou Z, Zhang Y, Zhu D. Inhibition of endoplasmic reticulum stress-activated IRE1alpha-TRAF2-caspase-12 apoptotic pathway is involved in the neuroprotective effects of telmisartan in the rotenone rat model of Parkinson's disease. Eur J Pharmacol 2016; 776: 106-115.
  • Malhotra JD, Kaufman RJ. The endoplasmic reticulum and the unfolded protein response. Semin Cell Dev Biol 2007; 18(6): 716-731.
  • Schroder M, Kaufman RJ. The mammalian unfolded protein response. Annu Rev Biochem 2005; 74: 739-789.
  • Wakabayashi S, Yoshida H. The essential biology of the endoplasmic reticulum stress response for structural and computational biologists. Comput Struct Biotechnol J 2013; 6: e201303010.
  • Yirrell DL, Roome AP, Darville JM, Ashley CR, Harbour J. Comparison of the continuous cell line 293 with human embryo kidney cells and human embryo fibroblast cells for the cultivation of ocular viruses. J Clin Pathol 1983; 36(9): 996-999.
  • Roche Molecular Systems. Universal ProbeLibrary Assay Design Center 09.07.2018; Erişim 18.07.2018, https://lifescience.roche.com/en_tr/brands/universal-probe-library.html#assay-design-center.
  • IntegratedDNATechnologies. OligoAnalyzer 3.1. 01.05.2018; Erişim 18.07.2018, https://eu.idtdna.com/calc/analyzer.
  • Szegezdi E, Logue SE, Gorman AM, Samali A. Mediators of endoplasmic reticulum stress-induced apoptosis. EMBO Rep. 2006 Sep;7(9):880-885.
  • Shinjo S, Mizotani Y, Tashiro E, Imoto M. Comparative analysis of the expression patterns of UPR-target genes caused by UPR-inducing compounds. Biosci Biotechnol Biochem 2013; 77(4): 729-735.
  • Neuber C, Uebeler J, Schulze T, Sotoud H, El-Armouche A, Eschenhagen T. Guanabenz interferes with ER stress and exerts protective effects in cardiac myocytes. PLoS One 2014; 9(6): e98893.
  • Weichert N, Kaltenborn E, Hector A, Woischnik M, Schams A, Holzinger A, ve ark. Some ABCA3 mutations elevate ER stress and initiate apoptosis of lung epithelial cells. Respir Res 2011; 12: 4.
  • Güleş Ö, Eren Ü. Apoptozun Belirlenmesinde Kullanılan Yöntemler. Y.Y.Ü. Veteriner Fakültesi Dergisi 2008; 2: 73-78.
There are 19 citations in total.

Details

Primary Language English
Subjects Clinical Sciences
Journal Section Research Article
Authors

Muhammed Abdulvahid Kalkan 0000-0002-3443-9260

Burçak Vural 0000-0001-6392-7645

Evrim Kömürcü Bayrak 0000-0003-1271-1208

Publication Date December 2, 2018
Submission Date October 1, 2018
Published in Issue Year 2018 Volume: 8 Issue: 3

Cite

Vancouver Kalkan MA, Vural B, Kömürcü Bayrak E. The Effect of Endoplasmic Reticulum Stress Activator-Tunicamycin on HEK293 Cells. Experimed. 2018;8(3):71-8.