Gene Expression Profile Of Bystander Effect In Human Lymphoblast Cells

With more and more widely adoption of nuclear energy in economic and military,theunderlying damage of ionization radiation to human being increases. Ionizing radiation hasunique characteristics as a genotoxic agent in terms of the DNA damage it produces.Thisdamage can cause mutation including activation of proto-oncogene ,inactivation ormutation of anti-oncogene ,DNA mismatch repair ,loss contol of cell proliferation ,signal transduction change in specific gene or chromatosome. The study on themechanisums has been a hotspot of biology and radiobiology. Since radiation-inducedbiologic bystander effects (RIBBEs) derive from the cellular processing of the physicalradiation insult,a large body of evidence pose great challenges to the conventional ideasuch as"only nucleus damage cause death","only directly- irratiated cause insult".Radiation induces genic instability,cytoplasm irradiated mutation and bystander effect hasthe same important role in radiation carcinogenesis. Bystander effects induced by low doseof ionizing radiation have been shown to be widely existed in many cell types and mayhave a significant impact on radiation risk assessment. Though the mechanisms underlyingthis process are not clear,the discovery and study of bystander effect play a significant rolein studying the essence of radiobiological effect.Radiation—induced bystander effect means indirectly-irradiated cells have theanalogical biological reaction with directly-irradiated cells. It has been suggested thatirradiated cells are capable of providing signals to the neighboring unirradiated cellsresulting in damage to these cells. Nowadays,the study of bystander effect mainlyconcentrates on two directions. First,biological endpoints of the bystander effect: theendpoints of the bystander effect include cell killing,increaseing intracellular reactiveoxygen species,enhancing cell growth,induction of apoptosis,chromosome aberrationsand mutations,gene expression change,induction of genomic instability and neoplastictransformation; Secondly, research of bystander effect mechanism: although muchevidence for the existence of bystander responses to ionizing radiation has beenaccumulated recently,the mechanisms underlying the observed effects remain obscure. Ithas been suggested that the underlying mechanisms involve in the production of reactive oxygen species ,cell production and direct cell-to-cell signal transduction via gapjunctional intercellular communication .The nature of the bystander effect signal and how itimpacts the unirradiated cells remains to be elucidated. Examination of the changes in geneexpression could provides clues to understanding the bystander effect and could define thesignal pathways involved in sustaining damage to these cells. The microarray technologyserves as a tool to gain insight into the molecular pathways leading to bystander effect.Objects1. To identify the genes involved in bystander effect by comparing the gene expressionprofiles of co-cultured non-irradiated cells with normal cells,irradiated cells.2. To identify the gene and protain changes of FHIT gene in direct- irradiated cells andbysander cells by different dosage of gamma-ray.3. To identify the gene and protain changes of PTEN gene in direct- irradiated cells andbysander cells by different dosage of gamma-ray.MethodsHuman lymphoblast cells were irradiated with 0.5 or 2.0 Gy gamma-ray and wereco-cultured with non-irradiated cells in transwell for 8 hours. Then,the total RNA of theco-cultured non-irradiated cells (bystander cells),and normal cells were isolated foranalysis. Gene expression profiles were investigated by cDNA microarrays and analyzedby gene onthology method,including biological process,molecular function,cellularcomponent. In this foundation,PTEN gene is carried on with the real-time fluorescencequota PCR for confirmation,FHIT gene with the real-time fluorescence quota PCR,Western Blot for confirmation.Results1.It is found that direct- irradiated cells and its bystander cells have differentexpression. After difference dosage of gamma-ray,direct- irradiated cells and its bystandercells have their specific gene expression.There were 171 and 165 different gene expression levels between 0.5 Gy bystandercells and normal cells,and 2.0 Gy bystander cells and normal cells,respectively. Therewere 226 and 365 different gene expression levels between 0.5 Gy irradiated cells and 0.5Gy bystander cells,and 2.0 Gy irradiated cells and 2.0 Gy bystander cells,respectively.There were 397 and 222 different gene expression levels between 2.0 Gy irradiated cellsand 0.5 Gy irradiated cells,and 2.0 Gy bystander cells and 0.5 Gy bystander cells, respectively.After the gene onthology analysis,20 out of 171 ,17out of 165 ,12 out of 226 ,19 out of 365 genes showed significant difference. Most of these genes belong to signaltransduction pathway,regulation of cell cycle,immunoglobulin gene superfamily,andDNA damage response.Simultaneously,we also find three genes have consistent changes in each sample.Moreover,heat shock 60kDa protein 1 gene is down-regulate in directed irradiated 2.0Gycells,but up-regulate in other samples.2. FHIT gene is down-regulated in both direct-irradiated cells and bystander cells,but the extent is not predominant.It is also down-regulated at protain level in bothdirect-irradiated cells and bystander cells,and the extent is prodominent when the dosageof gamma ray increase.The decline extent in direct- irradiated cells is larger than that inbystander cells.3. PTEN gene expression has relation to the dose of gamma ray.Its decline extent islarger in 0.5Gy than 2.0Gy cells. PTEN gene expression is also relation to radiationmodality.Its decline extent is larger in bystander cells than that in direct irradiated cells.Conclusion1. The directly-irradiated cell may cause bystander cell presant different expressiongenes;The different dosage of gamma can cause difference expression gene. It is true thatradiation can cause bystander effect and involve in multitudinous genes expression.2. In direct radiation cells and bystander cells,FHIT is down-regulation inmolecular level and protein level.And there is dose-dependent between gamma ray andprotain decline extent.3. In the filted genes,PTEN which in the bystander cell 0.5Gy is down-regulationmore than normal cells.PTEN has close relation to the low dose biological effects ofionizing radiation and bystander effect.