Cell proximity is a prerequisite for the proliferative response of bystander cells co‐cultured with cells irradiated with γ‐rays

Abstract

Background In a recent study, we showed that unirradiated cells, when they are in the presence of cells irradiated with γ‐rays, are characterized by enhanced cell growth (Cytometry 2003;54A:1–7). However, the mechanisms and factors involved in the proliferative response of bystander cells are largely unknown. The aim of the current work was to investigate the possible role of spatial proximity of cells, including gap junctional intercellular communication (GJIC), in transmitting proliferation signals from cells irradiated with γ‐rays to unirradiated cells (bystander cells). Methods Confluent monolayers of rat liver epithelial cells (WB‐F344) were irradiated with 137Cs γ‐rays at doses ranging between 0.5 and 10 Gy. The impact of cell proximity on the proliferative response of bystander cells was determined by co‐culturing different densities of irradiated and unirradiated cells (ratio 1:1) followed 24 h later by two‐scheme flow cytometric analysis of their proliferation. To determine whether soluble extracellular factors play a role in this response, irradiated cells were plated together with unirradiated cells at the same ratio (1:1), but they were not allowed to directly contact unirradiated cells by using porous low‐protein binding polyethylene terephthalate membranes. After 24 h, the numbers of unirradiated cells in the co‐culture with irradiated cells were compared with the numbers of unirradiated cells in the control (unirradiated cells co‐cultured with unirradiated cells). To investigate the possible involvement of GJIC in mediating the proliferative response in the bystander cells, the proliferation status of unirradiated GJIC‐incompetent WB‐aB1 cells was compared with that of unirradiated WB‐F344 cells that were GJIC competent. In all of the protocols, the two populations of co‐cultured cells were distinguished by labeling one population with fluorescent cell tracers, such as membrane‐resident or CFDA SE (carboxy) fluorescein diacetate, succinimidyl ester that metabolizes intracellularly. Results Unirradiated cells that were co‐cultured with irradiated cells, but were not allowed to directly contact them, did not show any changes in the proliferation rate compared with that of unirradiated cells in the control. Unirradiated cells that were co‐cultured as a mixture with irradiated cells showed enhanced and statistically significant proliferation, particularly when they were plated together more densely. The proliferation rate of bystander WB‐aB1 cells was apparently higher than that of bystander WB‐F344 cells; however, the difference was statistically insignificant. Conclusions In the present experimental model, the spatial proximity of cells is a crucial element for transmitting growth stimulation signals from irradiated cells to neighboring unirradiated cells. Direct cell‐to‐cell contact appears to be required for transmitting these signals. Neither functional GJIC nor soluble extracellular factors released by irradiated cells into the culture medium appear to play significant roles in this proc...

Biological effects in unirradiated human tissue induced by radiation damage up to 1 mm away

“…The range of the bystander signal in tissue, up to 1 mm, corresponds to Ϸ50-75 cell diameters. This surprisingly long range implies either that directly damaged cells produce long-range, diffusible bystander signals, perhaps through autocrine͞paracrine mechanisms (34)(35)(36)(37), or that a cell relay system is active, in which cells signal only their immediate neighbors (juxtacrine signaling), the signal being relayed by spatially intermediate, unirradiated bystander cells (38)(39)(40).…”

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

“…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. End points used for the study of bystander effects in vitro have included micronuclei formation (Azzam et al, 2002;Kashino et al, 2004;Shao et al, 2004), gene mutations and genomic instability (Zhou et al, 2000), gene expression changes (Azzam et al, 2002;Yang et al, 2005), transformation (Sawant et al, 2001), proliferation (Gerashchenko and Howell, 2003), cell survival, apoptosis (Belyakov et al, 2002;Lyng et al, 2002), cell cycle arrest (Azzam et al, 2000) and most recently the induction of gH2AX foci in bystander cells (Hu et al, 2005;Sokolov et al, 2005;Yang et al, 2005).…”

Characterization of cell‐cycle progression and growth of WB‐F344 normal rat liver epithelial cells following gamma‐ray exposure

“…Specifically, Azzam et al (7) used these cells to provide direct evidence for the participation of GJIC in the transmission of damage signals from ␣-particles irradiated to bystander cells. Using WB-F344 cells, radiation-induced bystander effects have also been observed by Gerashchenko and Howell (8,9). It was found that WB-F344 cells that were irradiated with ␥-rays stimulated proliferation in unirradiated bystander cells (8).…”

“…It was found that WB-F344 cells that were irradiated with ␥-rays stimulated proliferation in unirradiated bystander cells (8). Close proximity between the irradiated and bystander cells was a prerequisite for the proliferative response of the bystanders (9).…”