Characterizing biological effects in cells exposed to different types of ionizing radiation and understanding the underlying mechanisms is relevant not only to issues in radiotherapy and radiation protection but also to basic knowledge of the cellular responses to stress, particularly oxidizing and clastogenic stresses. Extensive data have shown that the deposition of radiation energy into cells can cause damage to all cellular macromolecules and, depending on dose, could result in serious injury to the traversed cells (58
). However, cells employ various strategies for detecting damage and repairing it (59
). Holding cells in the confluent density-inhibited state after irradiation or maintaining them in growth factor-depleted medium was shown to influence the fraction of cells that survive the irradiation because of the repair of PLD (2
). Although PLD repair has been studied extensively for decades, the molecular and biochemical events mediating its expression remain incompletely understood, particularly for cells exposed to high-LET radiations. Such studies would have important implications for radiotherapy, because α particles and high-charge/high-energy particles, another type of high-LET radiation, are being used increasingly in cancer treatment (60
). Understanding the biological effects that occur shortly or a few hours after exposure to such particles may help potentiate their therapeutic efficacy and clarify the associated risks to irradiated, or bystander, normal tissues adjacent to the tumor target. Furthermore, the results of this study, although for high doses of radiation, are pertinent to our understanding of signaling events mediating low-dose effects that are relevant in radiation protection, because humans may be exposed to significant doses of α particles or high-charge and high-energy particles during specialized activities such as mining and or prolonged space travel, respectively.
Using human fibroblasts exposed to γ rays, a low-LET radiation, or α particles, a high-LET radiation, we have shown that holding α-particle-exposed cells in the confluent state for several hours after irradiation results in decreased viability () rather than the increased cell viability that occurs in γ-irradiated cells (). After 3 h of confluent holding, α-particle irradiation was over 12 times more effective than γ irradiation at inducing cell killing (); in contrast, when survival is measured shortly after irradiation, an RBE of 5 is deduced at the 10% survival level. Significantly, our data indicate that gap junction communication mediates the propagation of events that lead to the increased toxic effects seen with α-particle radiation. Treatment of cells with a gap junction inhibitor () attenuated the enhanced lethal effect: When cells were irradiated and held in confluence in the presence of 18-α-glycyrrhetinic acid, a sparing of the enhanced toxicity was observed, and survival was similar to that measured shortly after irradiation (). However, clonogenic survival was not increased as it was in γ-irradiated cells that were held in confluence after irradiation. The sparing effect was associated with a decrease in micronucleus formation (). The decrease in the fraction of micronucleated cells was observed in cell populations that were subcultured for the assay shortly after exposure, suggesting that the gap junction-mediated propagation of events leading to increased lethality in α-particle-irradiated cell cultures occurs rapidly after exposure. In contrast, treatment of γ-irradiated cells with AGA did not result in a remarkable effect.
Because chemical inhibitors may not be necessarily specific in their effect, we investigated the role of GJIC in the propagation of lethal effects among α-particle-irradiated cells more directly. When cells transfected with Cx43-siRNA were exposed to an 80-cGy lethal dose of α particles and held in confluence for 3 h, clonogenic survival was increased (22 ± 1%, P
< 0.0001) when compared with scrambled siRNA-transfected cells () and was associated with a decrease in micronucleus formation (). It is likely that the signaling molecules propagated through gap junctions act to induce lethality in cells in the exposed population that are traversed by a small number of tracks that fail to kill the cell when survival is measured shortly after irradiation. The deposition of energy from particulate radiation is known to occur in a nonuniform pattern [reviewed in ref. (62
)], and in AG1522 fibroblast cultures exposed to 80 cGy, ~1.6, 4.8, 9.5 and 14% of the cells would be traversed on average by 1, 2, 3 or 4 particle tracks, respectively (42
). The communicated molecules may have induced processes that led to greater killing in these cells. In this context, it would be of interest to know how many α-particle traversals would kill an AG1522 cell. Together, our data are consistent with those of Jensen and Glazer (63
) that showed greater cell killing by cisplatin in high-density cell cultures of gap junction-proficient cells. They extend our previous findings and those of others showing that GJIC is an important mechanism that mediates the propagation of stressful effects from irradiated to nonirradiated cells in low-fluence α-particle-irradiated cultures (64
). Relative to cells assayed shortly after irradiation, the data in show that a significant increase in micronucleus formation after a 3-h holding period occurred in cells from cultures exposed to an α-particle dose of 10 cGy in which 50% of the cells in the exposed population are bystanders.
The propagation of toxic effects among high-dose α-particle-irradiated cells would be of significance in radioimmunotherapy with antibodies conjugated to α-particle emitters (67
). Although loss of GJIC is widely regarded to correlate with tumorigenic phenotypes, there are exceptions. Specifically, substantial evidence indicates that increased levels of connexin expression and of GJIC are correlated with invasiveness, extravasation and metastasis in a variety of cancer cells. It has also been noted that primary tumors that are initially GJIC impaired become GJIC competent at the metastatic stage (68
). Thus, in those situations in which tumors are treated by radioimmunotherapy with α-particle emitters, GJIC may potentiate killing of both targeted and nontargeted cells in the tumor. Although the potentiating effect on cell killing observed in this study is small (), the cumulative effect in therapeutic regimens involving repeated administration of α-particle emitters would become significant. For tumor cells with reduced GJIC, development of drugs and methods that recover or increase GJIC may provide a new and potent way to enhance treatment of these tumors with high-LET radiations. Thus enhancement of GJIC by chemotherapeutic agents in tumor cells, coupled with radiotherapy using α particles, and the associated transmission of toxic compounds between cells in the irradiated tumor would offer a therapeutic gain. By corollary, transmission of toxic effects from irradiated to neighboring normal bystander cells would pose a health risk if affected normal bystander cells undergo genetic changes but yet survive and become prone to neoplastic transformation.
In addition to the role of GJIC in enhancing the toxic effects of high-fluence α particles, we investigated whether the increase in oxidative stress detected 3 h after irradiation () contributes to the observed increase in cell killing (). To this end, we measured clonogenic survival in α-particle-irradiated cells in which the antioxidant GPX was ectopically overexpressed. Similar to the enhanced toxicity described in , holding empty vector-transduced cells in the confluent state for 3 h after exposure to a mean dose of 80 cGy resulted in a significant decrease in survival (). Ectopic overexpression of GPX significantly attenuated cell killing measured shortly after irradiation, indicating that oxidative stress contributes to cell killing in α-particle-irradiated cells. It is of interest to note that the yield of H2
in irradiated cells is thought to increase with increasing LET (70
). Thus, by more efficiently scavenging H2
in α-particle-irradiated cells, overexpressed GPX would protect against chemical changes to cellular macromolecules caused by H2
or by hydroxyl and superoxide radicals that result from its dissociation by the Haber-Weiss reaction (71
). However, holding GPX-transduced cells for 3 h after α-particle irradiation did not increase survival or decrease micronucleus formation over what was observed when cells were assayed shortly after irradiation (). The latter results suggest that death-inducing or clastogenic factors other than or in addition to oxidizing species may be directly communicated through gap junctions to enhance killing of irradiated cells that would otherwise survive. Signaling events that lead to activation of nucleases may be involved.
Although the increase in lipid peroxidation and protein carbonylation observed in our studies during confluent holding of α-particle-irradiated cells () may be caused by excess ROS generated from an effect of the radiation on oxidative metabolism, ROS generated at the time of irradiation may have contributed to the effect. Whereas ~60 ROS per nanogram of tissue were estimated to be generated from a hit caused by 137
Cs γ rays (67
) (i.e., ~10.4 ROS per cell nucleus, using a nuclear mass of ~173 pg, thus corresponding to a yield of about 1 ROS/100 eV), we estimate that over 2000 ROS are generated from an α-particle traversal, corresponding to a concentration of ~19 nM
ROS in the nucleus. Such a concentration can obviously cause extensive oxidative damage. The data in show an increase in 4-HNE adducts in proteins occurring within minutes after irradiation. Regardless, the net result is enhancement of cell killing that may be due to an effect of protein carbonylation and lipid peroxidation on organelle structure and function (e.g. plasma membrane) (72
) as well as DNA repair proteins and their accessories (73
). Oxidative damage to proteins may render them prone to segregation and degradation. It is noteworthy that carbonylation is unrepairable (74