Genomic instability induced in distant progeny of bystander cells depends on the connexins expressed in the irradiated cells

Abstract

Purpose: To examine the time window during which intercellular signaling though gap junctions mediates non-targeted (bystander) effects induced by moderate doses of ionizing radiation; and to investigate the impact of gap junction communication on genomic instability in distant progeny of bystander cells.

Materials and methods: A layered cell culture system was developed to investigate the propagation of harmful effects from irradiated normal or tumor cells that express specific connexins to contiguous bystander normal human fibroblasts. Irradiated cells were exposed to moderate mean absorbed doses from 3.7 MeV α particle, 1000 MeV/u iron ions, 600 MeV/u silicon ions, or ¹³⁷Cs γ rays. Following 5 h of co-culture, pure populations of bystander cells, unexposed to secondary radiation, were isolated and DNA damage and oxidative stress was assessed in them and in their distant progeny (20-25 population doublings).

Results: Increased frequency of micronucleus formation and enhanced oxidative changes were observed in bystander cells co-cultured with confluent cells exposed to either sparsely ionizing (¹³⁷Cs γ rays) or densely ionizing (α particles, energetic iron or silicon ions) radiations. The irradiated cells propagated signals leading to biological changes in bystander cells within 1 h of irradiation, and the effect required cellular coupling by gap junctions. Notably, the distant progeny of isolated bystander cells also exhibited increased levels of spontaneous micronuclei. This effect was dependent on the type of junctional channels that coupled the irradiated donor cells with the bystander cells. Previous work showed that gap junctions composed of connexin26 (Cx26) or connexin43 (Cx43) mediate toxic bystander effects within 5 h of co-culture, whereas gap junctions composed of connexin32 (Cx32) mediate protective effects. In contrast, the long-term progeny of bystander cells expressing Cx26 or Cx43 did not display elevated DNA damage, whereas those coupled by Cx32 had enhanced DNA damage.

Conclusions: In response to moderate doses from either sparsely or densely ionizing radiations, toxic and protective effects are rapidly communicated to bystander cells through gap junctions. We infer that bystander cells damaged by the initial co-culture (expressing Cx26 or Cx43) die or undergo proliferative arrest, but that the bystander cells that were initially protected (expressing Cx32) express DNA damage upon sequential passaging. Together, the results inform the roles that intercellular communication play under stress conditions, and aid assessment of the health risks of exposure to ionizing radiation. Identification of the communicated molecules may enhance the efficacy of radiotherapy and help attenuate its debilitating side-effects.

Keywords: Bystander effects; channel permeability; gap junctions; genomic instability; non-targeted effects; radiation quality.

Figure 1.. AG1522 normal human fibroblasts establish gap-junction intercellular communication in a layered cell co-culture system.

(A) Schematic representation of the layered cell co-culture system. (B) AG1522 cells labeled with CellTracker Orange and Calcein were seeded on the top side of permeable microporous membrane inserts with 1 m-pores. By 2 h, Calcein permeated to bystander cells grown on the bottom side of the insert.

Figure 2.. Transfer of ¹⁴C-labeled metabolites from irradiated to bystander cells through gap-junctions.

AG1522 cells labeled with D[¹⁴C-U]-glucose for 24 h were chased in non-radioactive medium for 4 h. They were then exposed to 4 Gy from ¹³⁷Cs γ rays, harvested by trypsinization, and seeded on the top side of permeable microporous membrane inserts containing a monolayer of bystander cells growing on the bottom side of the insert. Following 4 h of co-culture in the absence or presence of 50 μM AGA or 100 μM La³⁺, bystander cells were harvested and incorporated radioactivity was measured as cpm/ug of protein.

Figure 3.. Biological changes in AG1522 bystander cells when co-cultured with irradiated cells.

Donor control or irradiated AG1522 cells exposed to mean absorbed dose of 80 cGy from 3.7 MeV α particles were seeded on the top side of permeable microporous membrane inserts with bystander AG1522 cells growing on their underside. After 5 h of co-culture, the bystander cells were collected. [A] Effect of elapsed time between irradiation of donor cells and co-culture with bystander cells on formation of micronuclei in bystander cells. [B] Flow cytometric analyses of non-dyed bystander cells co-cultured with dyed donor cells. AG1522 donor cells labeled with CellTracker Orange and Calcein were seeded on the top side of the inserts containing non-dyed bystander cells on the bottom side. After 5 h of co-culture, the cells were collected and submitted to Flow Cytometry to determine purity. [C] Effect of inhibitors of connexin channels and hemi channels (50 μM AGA) and hemi channels (100 μM La³⁺) during 5 h co-culture of bystander cells with irradiated donor cells on the induction of micronuclei in bystander cells.

Figure 4.. Micronucleus formation in AG1522 bystander cells co-cultured with AG1522 cells exposed to different types of radiation that differ in their LET.

[A] Donor control or irradiated cells exposed to 3.7 MeV α particles (80 cGy), 1000 MeV/u iron ions (2 Gy) or ¹³⁷Cs γ rays (4 Gy) were seeded, immediately after irradiation, on the top side of permeable microporous membrane inserts with bystander AG1522 cells growing on their underside. Following 5 h of co-culture, the bystander cells were harvested and assayed for micronuclei formation. [B] Effect of inhibitors of connexin channels and hemi channels (50 μM AGA) and hemi channels (100 μM La³⁺) during 5 h co-culture of bystander cells with irradiated (2 Gy of 1 GeV/u ⁵⁶Fe ions) donor cells on induction of micronuclei in bystander cells.

Figure 5.. Oxidative stress in AG1522 bystander cells.

[A] Protein carbonylation, and [B] lipid peroxidation (i.e. 4-HNE protein adduct accumulation) as revealed by immunoblotting in confluent bystander cells that had been in co-culture for 5 h with cell populations exposed to mean doses of 0, 5 or 10 cGy of 3.7 MeV α particles using the layered cell culture strategy. Relative to controls, bystander cells exhibited different levels of protein cabonylation and lipid peroxidation.

Figure 6.. Micronucleus formation in distant progeny of bystander cells.

Control or irradiated donor AG1522 cells were seeded on the top side of permeable microporous membrane inserts with bystander AG1522 cells growing on the bottom side of the inserts. Following 5 h of co-culture, bystander cells were harvested, sub-cultured for 25 population doublings and assayed for micronuclei formation. [A] Effect of elapsed time between irradiation of donor cells (80 cGy from 3.7 MeV α particles) and co-culture with bystander cells. [B] Genomic instability induced in distant progeny of bystander cells whose parental cells were co-cultured with cells from cultures exposed to 3.7 MeV α particles (80 cGy), 1000 MeV/u Fe ions (200 cGy), 600 MeV/u Si ions (50 cGy), or ¹³⁷Cs γ rays (400 cGy). [C] Effect of inhibitors of intercellular communication: 50 μ M AGA or 100 μ M La³⁺.

Figure 7.. The effect of the connexin expressed in irradiated cells on genomic instability in distant progeny of bystander cells.

Micronucleus formation in the progeny of bystander AG1522 cells that were co-cultured with either AG1522 cells, HeLa cells devoid of functional connexins, or HeLa cells expressing either Cx26, Cx32 or Cx43 within minutes following exposure to mean absorbed doses of 0 or 50 cGy of 3.7 MeV α particles. Micronuclei formation was assayed in progeny cells that underwent 24 population doublings.