PMC

HPRT^– mutation of bystander cells in mixed cultures of ρ⁺ and ρ⁰ cells using the Columbia microbeam. A. ρ⁺ cells were used as the bystander cells when 10% of ρ⁰ or ρ⁺ cells were irradiated with 20 alpha particles each. B. ρ⁰ cells were used as the bystander cells when 10% of ρ⁺ or ρ⁰ cells were irradiated with 20 alpha particles each. C.HPRT^– mutation of bystander and directly irradiated ρ⁺ cells exposed to a 0.5Gy dose of alpha particles using the special designed strip dishes. D. Same as C with ρ⁰ cells. Data are pooled from 3–5 independent experiments. Bars represent ± SD.

Effects of TNFα in the bystander effects. A. Combined treatment of NHLF with TNFα (20 ng/ml) and IL1β (2 ng/ml) induced COX-2 expression as determined by Western blot analysis. B. EMSA of NF-κB DNA-binding activity in control and bystander cells, with or without anti-TNF mAb (5 μg/ml) in the medium. C. Effects of treatment with the inhibitory anti-TNF mAb (5 μg/ml) on COX-2 levels in control, α-irradiated and bystander cells. D. Clonogenic survival assay of NHLF after indicated treatment with and without inhibitory anti-TNF mAb (5 μg/ml). Data are pooled from three independent experiments. Bar represents ± SD.

A unifying model of the signaling pathways involved in radiation-induced bystander effects. Secreted or membrane forms of cytokines such as TNFα activate IKK-mediated phosphorylation of IκB, which releases NF-κB, that enters the nucleus and acts as a transcription factor for COX-2 and iNOS. TNFα also activates MAPK pathways (ERK, JNK and p38) that via AP-1 transcription factor additionally up-regulates expression of COX-2 () and iNOS, which stimulate the production of nitric oxide. Mitochondrial damage facilities the production of hydrogen peroxide that migrates freely across plasma membrane and is subjected to antioxidant removal. Activation of COX-2 provides a continuous supply of reactive radicals and cytokines for the propagation of the bystander signals either through gap junction or medium.

A. Effect of the nitric oxide scavenger, c-PTIO (20μM, 2 hr before and maintained overnight after irradiation) on HPRT^– mutant fractions of ρ⁺ and ρ⁰ cells. B. Effect of the NF-κB inhibitor, Bay 11-7082 (1μM, 2 hr before and maintained overnight after irradiation) on HPRT^– mutant fractions of ρ⁺ and ρ⁰ cells. Data are from 3–4 independent experiments. Error bars represent SD. C. Characterization of NF-κB DNA binding activities of control, bystander cells and directly irradiated (0.5 Gy dose of alpha particles) ρ⁺ and ρ⁰ cells using EMSA. FP: free labeled oligonucleotide probe. D. Western blot analyse of COX-2 and iNOS protein levels in bystander and directly irradiated (0.5Gy dose of alpha particles) ρ⁺ and ρ⁰ cells. β-Actin was used as loading controls.

Effects of NF-κB inhibition on NF-κB-dependent COX-2 and iNOS protein levels, and micronuclei formation in bystander and directly irradiated normal human lung fibroblasts (NHLF). Cells were treated by 0.5Gy alpha particles in special designed strip dishes with or without Bay 11-7082 (1 μM). A. EMSA was performed with nuclear extracts of directly irradiated and bystander NHLF 16 h after irradiation. Two NF-κB DNA-binding complexes, p65-p50 and p50-p50, are indicated. FP: free labeled oligonucleotide probe. B. Western blot analysis of COX-2, iNOS, BAX and β-actin levels in NHLF after indicated treatment. C and D. Effect of Bay 11-7082 and c-PTIO (1 μM and 20 μM, respectively) on micronuclei formation in irradiated and bystander cells. Data are pooled from three independent experiments. Bar represents ± SD.

Characterization of mitochondrial DNA depleted human skin fibroblasts (ρ⁰) and their parental cells (ρ⁺). A. PCR amplification of genomic DNA from ρ⁰ and ρ⁺ cells using two different segments of mitochondrial DNA as primers. PCR products for the two primer sets: primer A (251 bp) and primer B (261 bp). B. Assessment of mitochondrial membrane potential (ΔΨm) in ρ⁺ and ρ⁰ cells using DiOC6 staining (20 nM) and flow cytometry. ρ⁰ cells show lower membrane potential compared with ρ⁺ cells. C. Assessment of superoxide production in ρ⁰ and ρ⁺ cells using hydroethidine (2μM) and flow cytometry. ρ⁰ cells show lower superoxide production compared with ρ⁺ cells. D. Rates of oxygen consumption for ρ⁺ and ρ⁰ cells using an oxygen electrode unit. Average of duplicate determinants from two or three independent clones from each line; bars represent ± SD.