The Genomic Instability Induced By Ionizing Irradiation In Human Liver Cells

Objective: Radiation-induced genomic instability(RIGI) in human liver cells was detected at the cellular, molecular and proteomic level. cDNA chip and proteomic analysis was conducted upon progeny of irradiated human liver cells to provide experimental data for exploring the molecular mechanism of RIGI.Merhods: (1) The cloning efficiency, micronucleus frequency and apoptosis efficiency of human liver cells 7702(HL-7702) irradiated by 60Co-γrays were detected, and the method of single cell gel electrophoresis(SCGE) was used to measure DNA chains damage. (2) The progeny of HL-7702 cells were irradiated by 0, 2, 4, 6, 8, and 10Gy of 60Coγ-irradiation, and the effects mentioned above were detected in the progeny of the irradiated cells. (3) The progeny were secondly irradiated with 2Gy of 60Coγ- irradiation, and the delayed effects were detected. (4) cDNA gene chip was used to measure the transcriptional profile in progeny of HL-7702 cells exposed to 0, 2, 4, and 6Gy 60Coγ-irradiation, and the differentially expressed genes were further identified by Quantitative real-time PCR. A pathway-based network was constructed using a software (Genespring GX10) to analyze the functional relations among the differential genes. (5) Two-dimensional electrophoresis(2-DE) was used to screen the proteins differentially expressed in the progeny of human liver cells surviving from ionizing radiation, and mass spectrometry was used to identify the protein-spots significantly altered in expression. (6) The differential expression proteins of GATA-1, HSP60 were verified by Western blot. (7) Laser confocal scanning microscopy(LGSM) was used to detect the differential expression proteins of EIF5Aand HSP60 to confirm the 2-DE result.Results: (1) The cloning efficiency decreased with the increase of doses after the initial irradiation, while micronucleus frequency, percentage of apoptosis and comet rate increased with the increase of doses. (2) Damage of the survival cells secondly irradiated was correlated with the original irradiation doses. (3) A total of 71 differentially expressed genes were screened, most of which associated with transduction, cell cycle regulation, cellular immunity, cytoskeleton and movement, cell replication and repair mechanism. A pathway-based network was constructed to reveal the biological functions of RAN,CDT1,IER3,V-FOS.(4) A total of 42 differentially expressed proteins from the progeny of irradiated cells were screened, of which 17 were identified by MALDI-TOF-MS analysis, including 4 up-rugulated and 13 down-regulated proteins. The up-regulated expression of two proteins, mitochondrial heat shock 60kD protein (HSP60) and globin transcription factor 1 (GATA-1), were further confirmed by immunoblotting. (5) The differentially expressed proteins of HSP60 and EIF5A were localized in the cytoplasm, and the expression of HSP60 was up-regulated in the progeny of irradiated cells in a dose-dependent manner, which was consistent with the result of 2-DE.Conclusions: (1) Radiation-induced genomic instability may sustain in the progeny of surviving cells. The delayed damage after a second irradiation was correlated to the original irradiation dose. (2) A second irradiation plays an important role in transforming the genomic instability to the progeny, to make the damage more easily to be detected. (3) Irradiation can induce differentially expressed genes and proteins in the progeny of irradiated cells, which may be applied as potential biomarkers of radiation damage in future studies.