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Novel role of the human kinase VRK1 in response to DNA Damage induced by oxidative stress

AutorCampillo-Marcos, Ignacio ; Salzano, Marcella ; Lazo, Pedro A.
Fecha de publicación2015
Citación15th ASEICA International Congress (2015)
Resumen[Introduction]: In basal conditions, cells are exposed to exogenous and endogenous agents, such as ionizing radiation (IR) or oxidative stress, among others, which are responsible for double- and single-strand breaks (DSBs and SSBs, respectively) in the DNA. As a consequence of this, cells can activate different DNA repair mechanisms based on the type of DNA lesions. All these mechanisms take part in a global process called DNA Damage Response (DDR), essential to maintain the integrity and stability of genetic information. It is widely known that oxidative stress induces both SSBs and DSBs, particularly in cells with high metabolic rates, like cancer or neural cells. For this reason, it is particularly relevant to know the proteins which detect and repair the damage, returning to homeostatic equilibrium afterwards. Recently, VRK1 has been described to be required for the assembly of 53BP1 foci and participates in the recruitment and formation of γ H2AX foci in response to ionizing radiation. Furthermore, VRK1 interacts with p53 and is activated by UV-induced DNA damage. Overall, these processes could be an indicator of the crucial role this kinase may play in several steps of DDR. [Objectives]: Our aim is to determine whether VRK1 is activated and participates in the assembly of 53BP1 foci after inducing oxidative stress in human cancer cell lines.
[Methods]: To study VRK1 activation by oxidative stress, cell lines were starved and treated with 10 mM of hydrogen peroxide (H2O2). Next, endogenous p53 was immunoprecipitated at different points in time and the level of threonine 18 phosphorylation, which depends on VRK1 activation, was determined with a phosphospecific antibody. Moreover, in vitro assays were performed, using purified protein GST-p53 (1-85) as substrate and analyzing its phosphorylation in threonine 18. Along this same line, the assembly of 53BP1 foci was assessed by immunofluorescence in the presence or absence of VRK1. [Results]: In this work analysis, we can observe that VRK1 is activated by oxidative stress in the absence of serum, which reduces VRK1 activation mediated by growth factors. Exclusively under H2O2 exposition, this kinase is able to activate and phosphorylate p53. In turn, what it is shown is that VRK1 plays an essential role in DNA repair after oxidative stress induction. When cell lines were treated with H2O2, the number of 53BP1 foci increased considerably for the following 30-60 minutes, beginning to decrease at that point. On the contrary, VRK1 depletion was accompanied by a huge reduction of the number of 53BP1 foci, despite the fact that cells had been previously treated with hydrogen peroxide. [Conclusions]: Based on these results, we conclude that VRK1 participates in DNA repair process after H2O2 exposure, because it is activated and required for the assembly of 53BP1 foci under these conditions. Furthermore, it could also be possible that this kinase plays a crucial role in the early steps of DDR, mainly indicated by its hypothetical involvement in the recruitment and formation of γ H2AX foc.
DescripciónResumen del trabajo presentado al 15th ASEICA International Congress, celebrado en Sevilla (España) del 21 al 23 de octubre de 2015.
Aparece en las colecciones: (IBMCC) Comunicaciones congresos
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