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The standard paradigm for Ionizing radiation (IR) effects involved DNA damage with dsDNA breaks as triggers for mutation, cell death and transformation. However, a growing body of evidence reported non-targeted effects, like bystander responses, genomic instability, gene induction, adaptive responses and low dose hypersensitivity which may be dependent on the genotype of cell targets. We found that the EGR1 is efficient in radiosensitive (SF2:0.41 and D0: 176cGy) A549 lung carcinoma cells but not in radioresistant (SF2: 0.69 and D0: 192 cGy) H460 cells suggestive of a possible role for EGR-1 in IR resistance. Induction of TNFα was observed in A549 cells but not H-460 with direct IR (10Gy) or when indirect IR (medium from the cells directly exposed to 10 Gy IR) was combined with 2 Gy direct IR indicating that the induction of TNFα, (EGR1 target), in these cells is a function of EGR1. Interestingly, both the cells showed significant clonogenic inhibition and increased apoptosis in response to 10 Gy indirect IR with a proportionate increase when 2 Gy direct IR was given 24 hrs after 10 Gy indirect IR exposure. A reversal of these effects was seen with neutralizing antibody against TNFα in A549 but not H460 cells. Lack of TNFα induction in H460 cells, led us to focus on genes that are inducible by IR and involved in p53-independent apoptosis. One such gene that met these criteria was TNF-related apoptosis-inducing ligand (TRAIL). Significant induction of TRAIL was observed in H-460 cells but not in A-549 in response to 10 Gy direct IR or a combination of indirect 10Gy with a direct 2Gy IR. These effects were negated by neutralizing antibody against TRAIL suggesting a role for TRAIL in apoptosis and clonogenic inhibition in H460 but not A549 cells. Direct or indirect exposure of A549 but not H460 cells to IR, showed increased activation of NFκB as demonstrated by increased co-localization in the nucleus possibly due to activation of NFκB by TNFα which is exclusively upregulated in A549 cells in response to IR. In response to IR, A549 cells showed increased Bax induction with no changes in bcl-2. Reciprocal changes were observed when H460 cells were treated with IR. Chromatin Immunoprecipitation (ChIP) analysis using A549 cells showed the enrichment of TNFα promoter but not that of Egr-1, TRAIL, Bax or internal control HG6PDH with EGR-1 antibody, suggesting a role or EGR1 in regulation of TNFα. Further knock out of EGR-1 expression with SiRNA, resulted in decreased expression of TNFα in A549 cells with 10Gy indirect IR or 10Gy indirect IR supplemented with 2 Gy. Thus differential responses of A549 and H460 with IR may be dependent on the genotype of the cells.

[Proc Amer Assoc Cancer Res, Volume 46, 2005]