The Spatial Function And The Mechanism Of Damage Signal Transduction In Caenorhabditis Elegans

For a long time,radiation induced damage effects have been thought to occur only in irradiated cells,whereas with the discovery of RIBE(radiation induced bystander effects),non-irradiated cells have been shown to present the biological effects similar to that in irradiated cells.RIBE poses a challenge to the traditional linear model of radiological carcinogenicity and radiation protection measures.Then,RIBE has become one of the most focus fields in radiation biology research worldwide,which involves the generation,transduction and response of the radiation damage signal,as well as the cascade pathway.At present,although the phenomenon of RIBE has been confirmed,the differential activation of the radiation damage signal indifferent tissues is not clear,the related signal pathways involved in the RIBE process have not yet been elucidated neither.Therefore,developing new radiation technologies,from in vitro cells to in vivo organisms,analyzing the biological responses and mechanisms in more accurate conditions,are of great significance for radiation protection and tumor therapy.This research was based on the single particle microbeam facility in the key laboratory of ion beam of the Chinese Academy of Sciences and the animal model Caenorhabditis elegans.The digestive organ posterior pharynx bulb and the reproductive organ vulva of C.elegans were separately employed as the irradiated sites to explore the spatial function and molecular mechanisms in radiation induced bystander effects in vivo.The main results of this study are as follows:1.After quantitative irradiation of the posterior pharynx bulb and the vulva of C.elegans with 3.2 MeV proton by the CAS-LIBB single particle microbeam,the results presented radiation induced bystander effects both intra-and inter-system.Compared with pharynx irradiation,irradiation to vulva could induce higher apoptotic germ cell corpses,even at tow doses.Different tissues exhibited different sensitivities to radiation,provided the evidence of system-specific radiation sensitivity in the induction of bystander responses.Both two kinds of RIBE decreased the progeny of irradiated worms in a dose-dependent fashior,and the adverse effect on fecundity caused by pharynx irradiation was less significant than by vulva irradiation.Radiation signals could induce more severe genetic damage in intra-system RIBE and initiate genomic instability in the bystander proliferative germ line of worms.2.Using the fluorescence reporter strain HUS-1::GFP,we observed both intra-and inter-system irradiation significantly caused a dramatic increase in foci in the germline and compared with pharynx irradiation,irradiation to vulva induced more severe damage to the germline,demonstrated that both two non-targeted radiation stimulated the cellular DNA damage in the distant germ line of worms and more severe DNA damage was in intra-system RIBE.After the employment of single gene knockout mutant hus-1(op241),cep-1(w40),egl-1(n487),ced-4(n1162),ced-3(n717)and systemic RNA interference of mrt-1,hus-1,cep-1,ced-4,the results revealed that these DNA damage response genes were indispensable for both two kinds of RIBE and the core apoptotic pathway was required for radiation induced bystander germ cell death.3.In order to further determine the spatial function of the DDR pathway,two recprocal tissue-specific RNAi mutants rrf-1(lg)and ppw-1(lg)have been adopted,and the genes in the bystander signalling pathways were knocked down separately in germ cells or in somatic cells.Mrt-2/hus-1/cep-1/ced-4 were found to mainly function as bystander effectors in the germline.Compared to DDR networks in somatic tissue are of no use for RIBE in intra-system,DNA damage checkpoint protein MRT-2 and HUS-1 are partially required in somatic tissue to regulate germ cell death for inter-system RIBE.4.To study systemic expression level of ROS,two transgenetic mutants CF1553 and SJ4100,which specifically indicated the expression of GFP-labeled mitochondrial manganese superoxide dismutase SOD-3 and the unfolded protein response of the mitochondria were used.Despite the increased incidence of DNA damage observed in bystander germline,the enhanced level of ROS production was observed systemically for both two site irradiation,and could be recovered by the free radical quencher DMSO,suggesting that oxidative damage played a pivotal role in the transduction of RIBE.Moreover,by comparing these two kinds of RIBE,we discovered that irradiation at vulva produced a higher level of ROS production than that at pharynx.5.It has been shown that the involvement of ceramide has a relationship with DDR pathway.The absence of ceramide synthase could effectively inhibit the apoptosis induced by localized radiation using either the lagr-1(gk327)and hyl-1(ok976)gene single mutants,or lagr-1(gk327);hyl-1(ok976)gene double mutants.However,sphk-1(ok 1097)gene mutant did not lead a significant change in germ cell apoptosis.The results indicated that the ceramide biosynthetic pathway was involved in the induction of radiation induced bystander effects of C.elegans.Since mRNA expression of egl-1/ced-13 gene after deletion of ceramide metabolic synthase gene had no significant change compared to the wild type,ceramide and CEP-1 might play a synergistic effect on radiation-induced germ cell apoptosis.