Increased Mutagenic Joining Of Enzymatically-induced DNA Double-strand Breaks In High Charge And Energy Particle-irradiated Human Cells

Exposure to ionizing radiation creates DNA double-strand breaks (DSBs), which can be repaired by three possible pathways:classical (Ku-dependent) non-homologous end-joining (C-NHEJ), alternative NHEJ (Alt-NHEJ), or homologous recombination repair (HRR). It has been proposed that exposure to HZE particle radiation influences pathwaychoice, and in particular that it may create an unfavorable intracellular and intercellular environment for C-NHEJ by releasing small DNA fragments that interfere with the function of Ku protein, an essential participant in C-NHEJ.To furtherinvestigate the effect of HZE exposure on DSBs non-homologous end-joining, we examined the effect of exposure on repair of a subsequent I-SceI-induced DSB. We used genetically engineered cell line reporter designed to report end-joiningevents. Mutagenic joining of nearby I-SceI sites on the same chromosome results in eGFP expression, whereasjoining of unlinked I-SceI sites results in DsRed expression.Cells were exposed to0Gy,0.3Gy and1.0Gy of600MeV/u Fe and same dose of320kv X-Ray or about equivalent dose of y-rays (0Gy,1Gy and3Gy). Subsequent end-joining repair were tested at intervals following recovery. One way ANOVA was used to evaluate the differences between exposed and unexposed groups. Significant differences in overall frequency of mutagenic NHEJ have been observed at day1and day7post HZE exposure (P<0.05), while there is no change in the X-Ray or γ-rays groups. More cells at S/G2phase were observed till14days post HZE irradiation and only observed cell cycle arrest at day1post X-Ray exposure. Clonogenic survival assay showed a dose-dependent survival rate of cells post HZE radition. Genome-wide expression profiling was performed to explore changes in transcription level in HZE samples after7days of recovery. A striking list of inflammatory factors were significantly up-regulated comparing to mock-irradiated controls, which has been confirmed by quantative real time RT-PCR. This finding indicates a novel cellular response pattern evokes by HZE radiation which leads to significantly more error prone DNA damage repair and will last at least7days.We also established a pharmacological model which was able mimic the mutagenic repair phenotype by inhibition of DNA-PK by a biomolecule called NU7441. By inhibiting another factor Mrel1which is one of the important factor in alt-NHEJ, rescued the mutagenic phenotype in some extent, suggesting alt-NHEJ played an role in this process. We used a transwell co-culture system to investigate the indirect effect of DNA-PK inhibiton, surprisingly, the mutagenic repair phenotype is transferrable to the naive cells from the target cells, indicating the function of DNA-PK inhibiton induced secretom or exosome.Part One:The effect of HZE particle radiation on cell survival and growthObjectives:To investigate the impact of HZE particle radiation on the clonogenic cell survival and overall cell growth rate.Methods:Culture cells to exponetial phase, for clonogenic cell survival assay, expose to0Gy,0.3Gy or1.0Gy of600MeV/u Fe,1000MeV/u Ti or320kV X-rays, each group with three biological relicates, culture till cell colonies are visible, fix and stain cells, plot according to the survival fraction in each group. Analyze cell cycel phase distribution and population doubling rate of the600MeV/u Fe exposed samples after different recovery time.Results:①Of the three type of radiation, the600MeV/u Fe particle exposure generates the lowest surviving fraction at each dose comparing with1000MeV/u Ti and320kV X-rays.②For the cell cycle and cell growth, high dose HZE exposed culture has a slight G2arrest, which appear from1day post exposure and attenuated during recovery time, the cell population doubling rate after high dose HZE exposure showed a less than one doulbing delay.Conclusion:The colonogenic cell survival rate is ralated the LET, indicating the relative biological effectiveness is radiation quality dependent in the tested cell line within the range challenged. HZE exposure induced a transient cell G2phase arrest, and cell growth delay, indicating an continuous repair process.Part Two:The effect of HZE particle radiation on mutagenic repair phenotypeObjectives:To investigate the effct of HZE exposure on subsequent DNA double-strand repair, explore the end-joining pattern in both intra-chromosome and inter-chromosome. Also to investigate the rate cells with residual unresolved y-H2AX foci and fraction of cells with micronuclei as biomarkers of genome instability.Methods:The reporter cell line contains two cassettes, which are able to detect either intra-chromsomal ligation or inter-chromosomal translocation by activating different fluorescent proteins. Samples were exposed to0Gy,0.3Gy or1.0Gy of600MeV/u Fe particles, allow to recover for1,7,14,21or28days, induce DSB by lentiviral I-SceI vector, mutagenic repair rate were analyzed by FACS. To test the factor of radiaton quality an extra group of samples were exposed to equal dose of320kV X-rays, or approxamate equivalent dose of y-rays, allowed to recover for1or7days as a control, mutagenic repair rate were investigated the same as HZE exposed groups.Expose reporter cells to0Gy,0.3Gy or1.0Gy600MeV/u Fe particle radiation, fix and stain cells at1,7,14or21days post exposure, score y-H2AX foci number and cell with micronuclei in100cells at each time point.Results:A substantial and significant increase of mutagenic repair, both intra-chromosomal distal ligation or inter-chromosomal translocaton, lasted for up to two weeks, was observed in the high dose HZE exposed group, which were not present in either low dose HZE group, X-rays or y-rays treated cultures.Number of cells with residual y-H2AX foci (>3per cell) decreases over time. Exposure to1.0Gy of HZE particle radiation increased the frequency of cells with excess y-H2AX foci by about seven-fold at1day post exposure. As with micronuclei, the frequency declined at later times, although it still exceeded background at21days post irradiation. Exposure to0.3Gy HZE radiation or to X-rays increased the frequency of repair foci, although the effect was smaller than with HZE particle radiation and in the case of X-rays more transient.Conclusion:The mutagenic repair is dose dependent and radiation quality dependent, residual effect could last for up to two weeks. HZE exposure caused genome instability is dose dependent and radiation quality dependent, the effect might be attenuated by overgrowth of healthy cells.Part Three:Effct of HZE exposure on whole genome expression profileObjectives:To investigate the overall change of continuous cellular response to HZE exposure at transcription level.Methods:Reporter cells were exposed to0Gy,0.3Gy or1.0Gy of600MeV/u, allow the cells to recover for7days, extract total RNA, generate cDNA by reverse transcriptase PCR, perform whole genome profiling by microarray. Verify a set of genes by real-time qPCR.Results:Principal component analysis (PCA) revealed a good segeragation of diffent dose groups. A small subset (less than250) of genes showed significant alteration, of which, A distinct subset of secretory factors were up-regulated. The results were verified by real-time qPCR on selected8genes.Conclusion:HZE exposure alters gene expression profile in the reporter cells, especially on the secretory factors include cytokines or chemokines, which might indicate a more active cell-cell communication.Part Four:A pharmacological model to mimic the mutagenic reapirObjectives:To set up an model to mimic and magnify the effect caused by HZE exposure, based on a hypothesis of the HZE particle radiation will compromise classical non-homologous end-joining (C-NHEJ) and activate a more error-prone alternative non-homologous end-joining (alt-NHEJ). Hence to further explore the machenism. Methods:Infect reporter cells with I-SceI lentiviral vector, after infection, change medium with DNA-PK inhibitor to block C-NHEJ, apply samples to FACS analysis3days post infection to exam the change of intra-chromosomal distal end-joining and inter-chromosomal translocation. Set up another group inhibiting to to explore the role of alt-NHEJ.To exam the secretary factor mediated non-targeted effect, co-culture cells after DNA-PK inhibition with non-treated cells, test the mutagenic repair phenotype in the naive cells.Results:DNA-PK inhibition showed an～4fold increase in intra-chromosomal distal ligation, a more than10fold increase in inter-chromosomal translocation. Inhibition of Mre11decreasd intra-chromsomal distal ligation at～25%and decreased inter-chromosomal translocation at～50%.The naive cells co-cultured with DNA-PK inhibitor treated cells showed an significant increase at～3fold in intra-chromosomal distal ligation, also an similar increase in inter-chromosomal translocation.Conclusion:The pharmacological model mimics the mutagenic repair phenotype in the reporter cell line. The results provided evidences that this specific mutagenice repair is due to compromise of C-NHEJ, in the same time, activation of alt-NHEJ. Transwell co-culture experiment showed this phenotype is transmeasable through the secretome in the medium via increase cell-cell communication.