Human cell response to ionizing radiation in ground gravity and microgravity condition

In eukaryotic cells, ionizing radiation (IR) induces damages to proteins, lipids and DNA, directly or indirectly, as a result of free radical formation. Among the numerous types of DNA lesions, the double-strand breaks (DSBs) are particularly important, since an inefficient or inaccurate repair may lead to cell death or genomic instability. The presence of DSBs leads to a complex DNA damage response, consisting in a cascade of cellular events, which involve sensing the damage, signal transduction to the effectors of DNA repair, cell cycle arrest and apoptosis induction. In mammals, a very early step in the cellular response to DSBs is the phosphorylation of the histone H2AX (γ-H2AX) at the sites of DNA damage by members of the phosphatidylinositol-3-OH kinase (ATM, DNA-PK and ATR). This event plays a critical role in the recruitment of signaling–repair proteins (i.e 53BP1, Mre11, Rad50, Nbs1) to the sites of damage to form the ionizing radiation-induced foci (IRIF), which contain hundreds to thousands of proteins. DNA damage and repair can be quantified in individual cells by monitoring the kinetics of formation and disappearance of IRIF that accumulate at sites of DSBs; in particular, the rate of loss of γ-H2AX foci correlates with the progression of DSB repair. Cell signaling events in response to ionising radiation depend on environmental conditions occurring during DNA repair, besides genetic and physiological features of the biological systems. For this reason we have studied and compared the human cell response to IR in different gravity conditions, normal gravity as on Earth and reduced gravity as in space environment where exposure to cosmic radiation during space missions is associated to the reduction of gravitational force, which is approximately 10-4-10-6g. Indeed, space environment is characterized by the presence of ionizing radiation in the form of charged atomic particles travelling at close to the speed of light, which represents the most significant factor limiting humans’ ability to participate in long-duration space missions, and also by a condition of weightlessness called microgravity. As reported in literature, microgravity effects on astronauts include: immune cell function suppression, skeletal muscle atrophy, cardiovascular problems and loss of calcium and minerals from bone. In-flight cell cultures and ground models of microgravity showed inhibition of lymphocyte proliferation, suppression or alteration in cytokine secretion, modifications of cytoskeleton, and also increase of chromosome aberrations and apoptosis. The question whether radiation effects are influenced by microgravity is still open and it is an important point in the risk estimation of space missions. In our experiments, microgravity condition was obtained in laboratory using of the bioreactor “Rotating Wall Vessel” (Synthecon, Inc., Houston, Texas), which simulates the weightlessness, an aspect of spaceflights. This condition is called “modeled microgravity” (MMG). In the first part of this project, DSB rejoining was investigated in human PBL irradiated with γ-rays and incubated in 1g or MMG during repair time. Formation and disappearance of γ-H2AX foci were monitored at various times after irradiation by in situ immunofluorescence; in the same samples the apoptotic index and the DNA fragmentation were determined, the last one was measured by pulsed-field gel electrophoresis (PFGE) and the fraction of DNA released in the gel (FR) was considered as a measure of DSBs. Results obtained provided evidences that MMG incubation during repair time affected cell survival, apoptosis and delayed DSB rejoining, increasing the genotoxic effects of ionising radiation. On the base of these evidences, we investigated if ionizing radiation and modeled microgravity could have an effect on cells focusing on the expression profile of microRNAs: negative regulators of gene expression. MicroRNAs (miRNAs) are a recently discovered class of small (~22nt) endogenously expressed translational-repressor RNAs that play a key role in many cellular pathways. In animal cells, these molecules bind to complementary sequences in the 3’-untraslated region (3’UTR) of the target messenger RNAs (mRNAs) of protein-coding genes, to direct their translational repression. For this reason, miRNAs have been implicated in numerous biological processes including developmental timing, cell fate decisions, cell death and proliferation, stem cell function, tumorigenesis and disease. Aim: The aims of this project were: i) to analyze the efficiency of DNA repair occurring in modeled microgravity culture conditions focusing on DSBs repair kinetics; ii) to investigate if ionizing radiation and modeled microgravity could have a synergistic action on human cells by comparing radio-responsive miRNA in ground gravity and MMG. Activity carried out: The presence of nuclear γ-H2AX foci and the apoptotic index were monitored by in situ immunofluorescence and western blot in not- and γ-irradiated Peripheral Blood Lymphocytes (PBL), at 0.5, 2, 6 and 24h from irradiation, in 1g and MMG. In the same samples was studied the DSBs repair analyzing the fraction of DNA released (FR) after Pulsed-field gel electrophoresis (PFGE), this value was calculated either by measuring optical density of DNA migrating in the gel and by quantity of DNA (ng) retained in the plug/wells. MiRNA expression profile of human PBL, irradiated with γ-rays (0.2-2Gy) and incubated in MMG and in parallel 1g condition, was examined through “Human miRNA microarray Kit V2” (Agilent) and quantitative real-time PCR (RT-qPCR). In addition, we used “Whole Human Genome Oligo Microarray” (Agilent) to determine the gene expression profile, in the same PBL analysed for miRNA profiling, with the aim to identify the most likely miRNA targets by integration of miRNA and mRNA expression data in an anti-correlation analysis. Finally, to identify biological pathways most involved in radiation cell response we performed a Gene Ontology (GO) analysis, on significant anti-correlated target genes identifying biological pathways significantly enriched (P