It has generally been considered that important biological effects of ionizing radiation arise as a direct consequence of DNA damage occurring in irradiated cells. We have examined this hypothesis by exposing cells to very low fluences of ␣-particles, similar to those emitted by radon gas, such that as few as 1% of the cells in a population are traversed by a particle and thus receive any radiation exposure. By using the endpoints of changes in gene expression and induction of DNA damage, we show that nonirradiated ''bystander'' cells participate in the overall response of confluent density-inhibited populations of cultured fibroblast and epithelial cells. I t has long been thought that the important biological effects of radiation in a cell population are a direct consequence of DNA damage occurring in the irradiated cells: unrepaired or misrepaired DNA damage in these cells is responsible for the genetic effects of radiation. Presumably, no effect would be expected in cells in the population that received no direct radiation exposure. Recently, however, evidence has been presented indicating that genetic changes such as increased levels of sister chromatid exchanges (1, 2), mutations (3, 4), micronuclei (5), and DNA damage-inducible proteins (6, 7) occur in a greater-thanexpected number of cells in cultures exposed to very low fluences of ␣-particles, fluences in which only a small fraction of the cells are actually traversed by a particle track and thus directly exposed to radiation. Finally, it has been shown that when some cells were labeled with tritiated thymidine in a three-dimensional multicellular cluster model, a cytotoxic effect was transmitted to adjoining nonlabeled cells present in the same cluster (8).Overall, these studies indicate that radiation traversal through the nucleus of a cell is not a necessary prerequisite to producing genetic damage or a biological response; cells in a population that are in the vicinity of directly hit cells can also respond to the radiation exposure. These nonirradiated cells that express genetic damage or changes in the expression of stress-induced genes have been termed ''bystander cells.'' The present investigation was designed to determine the mechanisms by which damage signals may be transmitted from irradiated to nonirradiated bystander cells.We previously presented preliminary evidence for the involvement of gap junction-mediated intercellular communication (GJIC) in the molecular events leading to the modulation of gene expression in bystander cells (7). In these studies, confluent density-inhibited cultures of normal human fibroblasts were exposed to low fluences of ␣-particles in the presence or absence of lindane, a chemical inhibitor of GJIC. Changes in gene expression were measured by Western blotting. The participation of bystander cells in the overall cellular response to the radiation stress was inferred from the observations that, the effect was significantly greater than expected based on the fraction of directly irradiated cells in the population...

We demonstrate by western analysis that the expression levels of TP53 (formerly known as p53), CDKN1A (formerly known as p21Waf1), CDC2 (formerly known as p34cdc2), CCNB1 (cyclin B1) and RAD51 are significantly modulated in confluent, density-inhibited human diploid cell populations exposed to doses where only a small fraction of the nuclei are actually traversed by an alpha-particle track. The extent of modulation of TP53 and CDKN1A is significantly reduced in the presence of the gap junction inhibitor lindane and in irradiated low-density cell populations. In situ immunofluorescence studies show that at doses where about 2% of the nuclei would be traversed by an alpha particle, induction of CDKN1A occurs in more cells than predicted. Furthermore, the induced cells are present in isolated aggregates of neighboring cells. Therefore, our studies at the gene expression level indicate that similar signaling pathways are induced in bystander cells that are not traversed by an alpha particle as in traversed cells, and that biological effects in cell populations are not restricted to the response of individual cells to the DNA damage they receive.

We have previously shown that chronic exposure of plateau-phase C3H 10 T1/2 cells to (60)Co gamma radiation at doses as low as 10 cGy protected the cells against neoplastic transformation by a subsequent large acute radiation exposure. We have also shown that this induced resistance to neoplastic transformation correlated with an increased ability to repair radiation-induced chromosome breaks. We now show that a single exposure of quiescent cells to doses as low as 0.1 cGy also reduces the risk of neoplastic transformation, from the spontaneous level to a rate three- to fourfold below that level. Higher doses, up to 10 cGy at the same dose rate (0.24 cGy/min), did not reduce the neoplastic transformation frequency further. This protective effect was seen only in irradiated cells that were allowed to incubate at 37 degrees C before release from contact inhibition. Cells released into low-density subcultures immediately after irradiation had unchanged neoplastic transformation frequencies. These results demonstrate that low or chronic exposure to radiation can induce processes which protect the cell against naturally occurring as well as radiation-induced alterations that lead to cell transformation. If similar processes are induced in human cells, the results also suggest that a single low dose, at background or occupational exposure levels, may in some circumstances reduce rather than increase cancer risk, a conclusion inconsistent with the linear no-threshold model of cancer risk from radiation.

Evidence accumulated over the past two decades has indicated that exposure of cell populations to ionizing radiation results in significant biological effects occurring in both the irradiated and nonirradiated cells in the population. This phenomenon, termed the 'bystander response', has been shown to occur both in vitro and in vivo and has been postulated to impact both the estimation of risks of exposure to low doses/low fluences of ionizing radiation and radiotherapy. Several mechanisms involving secreted soluble factors, oxidative metabolism and gapjunction intercellular communication have been proposed to regulate the radiation-induced bystander effect. Our current knowledge of the biochemical and molecular events involved in the latter two processes is reviewed in this article.

Widespread evidence indicates that exposure of cell populations to ionizing radiation results in significant biological changes in both the irradiated and nonirradiated bystander cells in the population. We investigated the role of radiation quality, or linear energy transfer (LET), and radiation dose in the propagation of stressful effects in the progeny of bystander cells. Confluent normal human cell cultures were exposed to low or high doses of 1GeV/u iron ions (LET ~ 151 keV/μm), 600 MeV/u silicon ions (LET ~ 51 keV/μm), or 1 GeV protons (LET ~ 0.2 keV/μm). Within minutes after irradiation, the cells were trypsinized and co-cultured with nonirradiated cells for 5 h. During this time, irradiated and nonirradiated cells were grown on either side of an insert with 3-μm pores. Nonirradiated cells were then harvested and allowed to grow for 20 generations. Relative to controls, the progeny of bystander cells that were co-cultured with cells irradiated with iron or silicon ions, but not protons, exhibited reduced cloning efficiency and harbored higher levels of chromosomal damage, protein oxidation and lipid peroxidation. This correlated with decreased activity of antioxidant enzymes, inactivation of the redox-sensitive metabolic enzyme aconitase, and altered translation of proteins encoded by mitochondrial DNA. Together, the results demonstrate that the long-term consequences of the induced nontargeted effects greatly depend on the quality and dose of the radiation and involve persistent oxidative stress due to induced perturbations in oxidative metabolism. They are relevant to estimates of health risks from exposures to space radiation and the emergence of second malignancies after radiotherapy.

The translationally controlled tumor protein (TCTP) is essential for survival by mechanisms that as yet are incompletely defined. Here we describe an important role of TCTP in response to DNA damage. Upon exposure of normal human cells to low-dose γ rays, the TCTP protein level was greatly increased, with a significant enrichment in nuclei. TCTP up-regulation occurred in a manner dependent on ataxia-telangiectasia mutated (ATM) kinase and the DNA-dependent protein kinase and was associated with protective effects against DNA damage. In chromatin of irradiated cells, coimmunoprecipitation experiments showed that TCTP forms a complex with ATM and γH2A.X, in agreement with its distinct localization with the foci of the DNA damage-marker proteins γH2A.X, 53BP1, and P-ATM. In cells lacking TCTP, repair of chromosomal damage induced by γ rays was compromised significantly. TCTP also was shown to interact with p53 and the DNA-binding subunits, Ku70 and Ku80, of DNA-dependent protein kinase. TCTP knockdown led to decreased levels of Ku70 and Ku80 in nuclei of irradiated cells and attenuated their DNA-binding activity. It also attenuated the radiation-induced G 1 delay but prolonged the G 2 delay. TCTP therefore may play a critical role in maintaining genomic integrity in response to DNA-damaging agents.low dose ionizing radiation | adaptive responses | DNA repair | cell cycle checkpoints | genomic stability

Evidence accumulated over the past two decades has indicated that exposure of cell populations to ionizing radiation results in significant biological effects occurring in both the irradiated and non-irradiated cells in the population. This phenomenon, termed the "bystander response", has been shown to occur both in vitro and in vivo. Experiments have indicated that genetic alterations, changes in gene expression and lethality occur in bystander cells that neighbor directly irradiated cells. Furthermore, cells recipient of growth medium harvested from irradiated cultures exhibit responses similar to those of the irradiated cells. Several mechanisms involving secreted soluble factors, gap-junction intercellular communication and oxidative metabolism have been proposed to regulate the radiation-induced bystander effect. In this review, our current knowledge of this phenomenon and its potential impact both on the estimation of risks of exposure to low doses/low fluences of ionizing radiation and on radiotherapy is discussed.

It has generally been considered that important biological effects of ionizing radiation arise as a direct consequence of DNA damage occurring in irradiated cells. We have examined this hypothesis by exposing cells to very low fluences of ␣-particles, similar to those emitted by radon gas, such that as few as 1% of the cells in a population are traversed by a particle and thus receive any radiation exposure. By using the endpoints of changes in gene expression and induction of DNA damage, we show that nonirradiated ''bystander'' cells participate in the overall response of confluent density-inhibited populations of cultured fibroblast and epithelial cells. It has long been thought that the important biological effects of radiation in a cell population are a direct consequence of DNA damage occurring in the irradiated cells: unrepaired or misrepaired DNA damage in these cells is responsible for the genetic effects of radiation. Presumably, no effect would be expected in cells in the population that received no direct radiation exposure. Recently, however, evidence has been presented indicating that genetic changes such as increased levels of sister chromatid exchanges (1, 2), mutations (3, 4), micronuclei (5), and DNA damage-inducible proteins (6, 7) occur in a greater-thanexpected number of cells in cultures exposed to very low fluences of ␣-particles, fluences in which only a small fraction of the cells are actually traversed by a particle track and thus directly exposed to radiation. Finally, it has been shown that when some cells were labeled with tritiated thymidine in a three-dimensional multicellular cluster model, a cytotoxic effect was transmitted to adjoining nonlabeled cells present in the same cluster (8).Overall, these studies indicate that radiation traversal through the nucleus of a cell is not a necessary prerequisite to producing genetic damage or a biological response; cells in a population that are in the vicinity of directly hit cells can also respond to the radiation exposure. These nonirradiated cells that express genetic damage or changes in the expression of stress-induced genes have been termed ''bystander cells.'' The present investigation was designed to determine the mechanisms by which damage signals may be transmitted from irradiated to nonirradiated bystander cells.We previously presented preliminary evidence for the involvement of gap junction-mediated intercellular communication (GJIC) in the molecular events leading to the modulation of gene expression in bystander cells (7). In these studies, confluent density-inhibited cultures of normal human fibroblasts were exposed to low fluences of ␣-particles in the presence or absence of lindane, a chemical inhibitor of GJIC. Changes in gene expression were measured by Western blotting. The participation of bystander cells in the overall cellular response to the radiation stress was inferred from the observations that, the effect was significantly greater than expected based on the fraction of directly irradiated cells in the population...