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Evaluation of the DNA damage in response to radiation-induced bystander effects on mesenchymal stem cells

Grant number: 15/18366-7
Support Opportunities:Scholarships abroad - Research Internship - Post-doctor
Effective date (Start): December 01, 2015
Effective date (End): January 31, 2016
Field of knowledge:Biological Sciences - Biology
Principal Investigator:Ricardo Ambrósio Fock
Grantee:Amanda Nogueira Pedro
Supervisor: Kathryn D. Held
Host Institution: Faculdade de Ciências Farmacêuticas (FCF). Universidade de São Paulo (USP). São Paulo , SP, Brazil
Research place: Harvard University, Boston, United States  
Associated to the scholarship:13/23285-0 - Evaluation of the bystander effects in both mesenchymal stem cells and immunomodulation, BP.PD


Accidental and occupational exposure to ionizing radiation (IR) can induces cellular damage in many living organisms at sub-lethal doses. The energy deposited on the cells leads to the breakage of chemical bonds, damaging both basic structures and the DNA, resulting in a range of lesions in which DNA double strand breaks (DSB) have a pivotal role in determining whether cells survive radiation exposure. Cells possess several mechanisms to repair the damaged DNA, but once it is not correctly repaired or even unrepaired, chromosomal aberrations, loss of genetic material and cell death can occur as a direct effect. One consequence of chromosomal aberrations is the formation of micronuclei (MN), once chromosomes did not attach properly to the spindle during the segregation process in anaphase. Mammalian cells possess mechanisms for DSB repair and prevention of chromosomal rearrangements that involves a range of molecules, such as histone H2AX. Its activation is one of the earliest events in cells following exposure to DNA damaging agents. Phosphorylation of H2AX occurs in the chromatin surrounding a DSB site such that hundreds to of ³-H2AX molecules surround one DSB to form a focus which may function both to open the chromatin structure and to serve as a platform for the accumulation of many factors involved in the DNA damage response. DNA damage induction is not an exclusive feature of the irradiated cells. Cells surrounding those that were directly exposed to IR can exhibit a similar behavior: they die or show chromosomal instability and other abnormalities. This phenomenon is called bystander effect. Studies in vitro point to 2 main pathways involved in radiation-induced bystander effects (RIBE): gap junction communications and soluble factors secreted by the cells. In vivo, these indirect responses of the cells to IR are referred to as abscopal effect, in which the immune system seems to play a key role in promoting tumour cell killing or its regression, while genomic instability, cell death and oncogenic transformation of normal tissue also occurs as an off-target effect, which remains an issue to be solved. A deeper knowledge of the mechanisms involved in RIBE may allow the development of suitable strategies to enhance tumour cell killing and / or protect normal tissue cells from the damaging consequences of IR exposure.Regarding this issue, this work aims to evaluate the DNA damage related to the RIBE in normal mesenchymal stem cells (MSC), using the following strategies: analysis of the formation of MN and quantification of ³-H2AX molecules. C3H10T1/2 immortalized lineage of MSC from mouse embryos will be used. For the bystander system, MSC will be cultured in transwell inserts (1 ¼m) and transferred to the wells containing irradiated MSC immediately after irradiation. The irradiation will be performed in a conventional, broad-field 250 kVp X-ray machine in a dose rate of 300cGy/min, at single doses ranging from 0.5 to 6 Gy. Chromosome damage will be evaluated using the standard cytokinesis-block MN technique using cytochalasin-B; at least 500 binucleated cells in 10 view fields were examined under a fluorescence microscope. Thus, MN will be evaluated by flow cytometry using a commercial kit (In Vitro MicroFlow®); validation of this technique for MSC is advantageous, once FACS-based approach allows the simultaneously collection of multiple parameters, giving clues about the genotoxic mode of action (clastogenicity or aneugenicity). DNA damage will be also evaluated by flow cytometry, given by the geometric mean of fluorescence intensity of ³ -H2AX. Altogether these assays significantly contribute to address RIBE on MSC due their specificity in the analysis of DNA damage. (AU)

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