Gap Junction Communication and the Propagation of Bystander Effects Induced by Microbeam Irradiation in Human Fibroblast Cultures: The Impact of Radiation Quality

Abstract

Understanding the mechanisms underlying the bystander effects of low doses/low fluences of low- or high-linear energy transfer (LET) radiation is relevant to radiotherapy and radiation protection. Here, we investigated the role of gap-junction intercellular communication (GJIC) in the propagation of stressful effects in confluent normal human fibroblast cultures wherein only 0.036–0.144% of cells in the population were traversed by primary radiation tracks. Confluent cells were exposed to graded doses from monochromatic 5.35 keV X ray (LET ~6 keV/μm), 18.3 MeV/u carbon ion (LET ~103 keV/μm), 13 MeV/u neon ion (LET ~380 keV/μm) or 11.5 MeV/u argon ion (LET ~1,260 keV/μm) microbeams in the presence or absence of 18-α-glycyrrhetinic acid (AGA), an inhibitor of GJIC. After 4 h incubation at 37°C, the cells were subcultured and assayed for micronucleus (MN) formation. Micronuclei were induced in a greater fraction of cells than expected based on the fraction of cells targeted by primary radiation, and the effect occurred in a dose-dependent manner with any of the radiation sources. Interestingly, MN formation for the heavy-ion microbeam irradiation in the absence of AGA was higher than in its presence at high mean absorbed doses. In contrast, there were no significant differences in cell cultures exposed to X-ray microbeam irradiation in presence or absence of AGA. This showed that the inhibition of GJIC depressed the enhancement of MN formation in bystander cells from cultures exposed to high-LET radiation but not low-LET radiation. Bystander cells recipient of growth medium harvested from 5.35 keV X-irradiated cultures experienced stress manifested in the form of excess micronucleus formation. Together, the results support the involvement of both junctional communication and secreted factor(s) in the propagation of radiation-induced stress to bystander cells. They highlight the important role of radiation quality and dose in the observed effects.

A correlation of long term effects and radiation quality in the progeny of bystander cells after microbeam radiations: The experimental study of radiotherapy for cancer risk mitigation

“…At the time of irradiation, the cells were confluent, thus allowing direct communication through gap junctions; approximately 94% of the cells were in the G 1 /G 0 phase as analyzed by a flow cytometer (data not shown). For all irradiation experiments carried out at the two different facilities, the same passage of cell, the same cell culture medium and the same experimental protocol were used [10,12].…”

“…The beam characteristics, biological irradiation procedures, and dosimetry have been described previously [10,12,18]. Cells (5  10 6 cell/dish) were cultured on microbeam dishes with 8 µm-thick kapton polyimide film on the bottom, and cultured for 1 day before irradiation to form confluent monolayers.…”

“…Irradiation was carried out by using the 256 (16 × 16)-cross-strip method in the center of the microbeam dishes (containing MEM growth medium). At each spot, a dose of 0.4 Gy was delivered within a beam area of 20 µm × 20 µm, so that around 0.036-0.144% of the total cells attached on the bottom of the microbeam dishes were hit by IR [10,12]. The irradiated and control samples were carried out at room temperature under ambient conditions, and the exposure time was less than 10 min.…”

“…However, the underlying bystander responses and genomic instability mechanisms after high-LET carbon ions are still poorly understood and investigated. In this context, previous studies have suggested that the bystander effects dependence of LET and radiation dose [10]. Although, a little known about the late effects in the progeny of bystander cells for many generations after exposed to high-LET carbon ions.…”

“…However, there is a significant evidence suggesting that similar effects can also be seen in non-irradiated bystander cells, a phenomenon known as the non-targeted effects which include bystander effects, genomic instability and others [1][2][3][4][5][6][7][8][9]. Bystander effect refers to the phenomenon that the induction of biological effects in non-irradiated bystander cells whose nucleus has not been directly traversed by IR [10]. Genomic instability is found when a cell population surviving after IR, shows an increased rate of chromosome aberrations, gene mutations, the delayed cell death in the progeny of cells surviving irradiation and a hallmark of cancer cells [11,12].…”