The effect of UV irradiation on Bcl-2 and Bax proteins in mouse embryonic fibroblast culture

Džinić, Tamara (2012) The effect of UV irradiation on Bcl-2 and Bax proteins in mouse embryonic fibroblast culture. Diploma thesis, Faculty of Science > Department of Biology.

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Abstract

UV irradiation is a potential carcinogen which induces apoptosis trough membrane death receptors or by activation of intrinsic apoptotic pathway. Bcl-2 acts as anti-apoptotic and Bax acts as pro-apoptotic protein in intrinsic apoptotic pathway. FADD (Fas- associated death domain) protein as a positive regulator of apoptosis and negative regulator of necroptosis acts in signal conduction from cell death receptors to caspase 8, respectively to RIP1 kinase. The effect of UVB irradiation on wild type and knock-out mouse embryonic fibroblasts for FADD and involvement of intrinsic apoptotic pathway in UVB induced apoptosis by Western blotting of Bcl-2 and Bax proteins were investigated. Experiments included exposure of cells to 600 J/m2 of UVB irradiation and treatment with necrostatin-1, inhibitor of RIP1 kinase. Apoptosis on cellular level was investigating using TUNEL method for fluorescence microscopy in adherent cells and the percentage of cells in apoptosis was quantitatively determined using TUNEL method for flow citometry. Researches showed that wild type of mouse embryonic fibroblasts die by processes of apoptosis and necroptosis after exposure to UVB irradiation. In FADD-/- fibroblasts, no matter of irradiation and inhibition of RIP1 kinase, no DNA fragmentation was detected. UVB irradiation causes decrease of anti-apoptotic Bcl-2 protein level in irradiated cells no matter to the absence of FADD or inhibition of RIP1 kinase. UVB irradiation causes increase of Bax protein level in wild type of fibroblasts. The amount of Bax protein depends on the presence of FADD and the activity of RIP1 kinase. Defect of only one component, FADD or RIP1, leads to degradation of Bax after irradiation, whereas defect of both components leads to increase of Bax level after irradiation.

Item Type: Thesis (Diploma thesis)
Keywords: UVB, apoptosis, necroptosis, Bcl-2, Bax
Supervisor: Marijanović, Inga
Co-supervisor: Josipović, Pavle
Date: 2012
Number of Pages: 32
Subjects: NATURAL SCIENCES > Biology
Divisions: Faculty of Science > Department of Biology
Depositing User: Grozdana Sirotic
Date Deposited: 03 Jun 2014 12:10
Last Modified: 03 Jun 2014 12:10
URI: http://digre.pmf.unizg.hr/id/eprint/2161

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