Direct evidence for the participation of gap junction-mediated intercellular communication in the transmission of damage signals from alpha -particle irradiated to nonirradiated cells

Abstract

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...

A proliferation-dependent bystander effect in primary porcine and human urothelial explants in response to targeted irradiation

“…Those studies were performed with cells at low densities where cell-to-cell contact was minimal and therefore a media-related factor was involved. However, other studies in confluent monolayers of fibroblasts showed that GJIC also played a role in propagation of the bystander effect (Azzam et al, 1998(Azzam et al, , 2001). Urothelium has highly developed connexin-mediated intercellular communication (Grossman et al, 1994;Lyng et al, 1996) and this could explain the more pronounced bystander effect observed in the urothelial outgrowth in comparison with fibroblast cultures.…”

“…Several studies (Azzam et al, 1998(Azzam et al, , 2001) have demonstrated that the bystander effect is dependent on gap junction intercellular communication (GJIC) in confluent cultures of primary human diploid fibroblasts exposed to low fluences of a-particles. These showed that p53 and p21 mediated pathways are activated (Azzam et al, 2000).…”

ATR-dependent radiation-induced γH2AX foci in bystander primary human astrocytes and glioma cells

“…However, the mechanisms involved in bystander signalling have only partially been elucidated and are probably dependent on cell type and end points investigated. The involvement of soluble factors like reactive oxygen species (ROS) (Azzam et al, 2002;Shao et al, 2003aShao et al, , 2005, nitric oxide (NO) (Shao et al, 2003b(Shao et al, , 2005Sokolov et al, 2005) and cytokines released from irradiated cells as well as gap junction intercellular communication (Azzam et al, 2001;Shao et al, 2003a;Mitchell et al, 2004;Sokolov et al, 2005) have recently been reported. Most interestingly, several cytokines can be induced by ROS and NO/NOS (Ayache et al, 2002;Hsu and Wen, 2002;Kosmidou et al, 2002;Hwang et al, 2004;Ryan et al, 2004) providing a possible link between different factors potentially involved in bystander signalling.…”

Constitutive nitric oxide acting as a possible intercellular signaling molecule in the initiation of radiation-induced DNA double strand breaks in non-irradiated bystander cells

“…The culture medium was replaced every 2 days and the cells were irradiated at day 3 under full confluent condition. At that time, about 92% of the cells were in G0-G1 as determined by flow cytometry (Azzam et al, 2001). …”