In this study, we investigated the potential influence of p53 on ultraviolet (UV) signal generation and response of bystander cells to the UV signals generated by beta-irradiated cells. Five cell lines of various p53 status (HaCaT, mutated; SW48, wild-type; HT29, mutated; HCT116 +/+ , wild-type; HCT116 –/– , null) were irradiated with beta particles from tritium. Signal generation (photon emission at 340 ± 5 nm) was quantified from irradiated cells using a photomultiplier tube. Bystander response (clonogenic survival) was assessed by placing reporter cell flasks directly superior to irradiated signal-emitting cells. All cell lines emitted significant quantities of UV after tritium exposure. The magnitudes of HaCaT and HT29 photon emission at 340 nm were similar to each other while they were significantly different from the stronger signals emitted from SW48, HCT116 +/+ and HCT116 –/– cells. In regard to the bystander responses, HaCaT, HCT116 +/+ and SW48 cells demonstrated significant reductions in survival as a result of exposure to emission signals. HCT116 –/– and HT29 cells did not exhibit any changes in survival and thus were considered to be lacking the mechanisms or functions required to elicit a response. The survival response was found not to correlate with the observed signal strength for all experimental permutations; this may be attributed to varying emission spectra from cell line to cell line or differences in response sensitivity. Overall, these results suggest that the UV-mediated bystander response is influenced by the p53 status of the cell line. Wild-type p53 cells (HCT116 +/+ and SW48) demonstrated significant responses to UV signals whereas the p53-null cell line (HCT116 –/– ) lacked any response. The two mutated p53 cell lines exhibited contrasting responses, which may be explained by unique modulation of functions by different point mutations. The reduced response (cell death) exhibited by p53-mutated cells compared to p53 wild-type cells suggests a possible role of the assessed p53 mutations in radiation-induced cancer susceptibility and reduced efficacy of radiation-directed therapy.

We examined bystander cell death produced in T98G cells by exposure to irradiated cell conditioned medium (ICCM) produced by high-energy 20 MeV electrons at a dose rate of 10 Gy min −1 and doses up to 20 Gy. ICCM induced a bystander response in T98G glioma cells, reducing recipient cell survival by more than 25% below controls at 5 and 10 Gy. Higher doses increased survival to near control levels. ICCM was analyzed for the presence of transforming growth factor α (TGF-α) and transforming growth factor β1 (TGF-β1). Monoclonal antibodies for TGF-α (mAb TGF-α) and TGF-β1 (mAb TGF-β1) were added to the ICCM to neutralize any potential effect of the cytokines. The results indicate that TGF-α was not present in the ICCM and addition of mAb TGF-α to the ICCM had no effect on bystander cell survival. No active TGF-β1 was present in the ICCM; however, addition of mAb TGF-β1 completely abolished bystander death of reporter cells at all doses. These results indicate that bystander cell death can be induced in T98G glioma if a large enough radiation stress is applied and that TGF-β1 plays a downstream role in this response.

Mothersill, C., Seymour, R. J. and Seymour, C. B. Increased Radiosensitivity in Cells of Two Human Cell Lines Treated with Bystander Medium from Irradiated Repair-Deficient Cells. Radiat. Res. 165, 26–34 (2006). Radiation-induced bystander factors have been shown to be more toxic if they are from medium harvested from irradiated repair-deficient cells. The aim of this study was to test the hypothesis that the radiosensitivity of repair-proficient cells can be increased by exposing them to medium-borne factors harvested from sensitive cells and vice versa. Cells from a mismatch repair (MMR)-deficient cell line (Raji 10) with a sensitive response to radiation or the wild-type parent cell line were irradiated to 0.5 Gy γ rays and then monitored for growth rate in their own medium or in the alternative conditioned medium. In other experiments, cells or conditioned medium were added to reporter cells (HPV-G, which are relatively sensitive keratinocytes, or highly radioresistant HT29 cells). The subsequent responses of the two cell lines to a 0.5-Gy dose of 60 Co γ rays were measured. The results show that prior exposure of resistant cells to medium from irradiated sensitive cells reduced the clonogenic survival of the subsequently irradiated resistant cells. The reverse is also true. Measurement of the apoptosis index and BCL2 expression confirmed that the harvested medium was capable of modulating apoptosis after irradiation. This may have important applications in tumor therapy and also in the understanding of mechanisms involved in induction of adaptive responses.

Mothersill, C., Seymour, R. J. and Seymour, C. B. Bystander Effects in Repair-Deficient Cells. Radiat. Res. 161, 256–263 (2004). One of the current hypotheses concerning the role of bystander effects in biological systems is that they are protective because they terminate division in cells with collateral or possibly pre-existing DNA damage that is not properly repaired. Following the logic of this hypothesis led us to consider that cell lines that are repair deficient should have larger than usual bystander effects. To test this, several different “repair- deficient” cell lines were used for bystander experiments. Response was monitored by determining the cloning efficiency or, in the case of non-adherent cell lines, the cell number. The results show that the repair-deficient human cell lines and surviving progeny produced moderate to severe bystander- induced death effects in either autologous cells or a reporter cell line. Normal “repair-proficient” lines, which were matched as far as possible, have very much less severe or absent bystander-inducible effects on cloning efficiency. Cells of hamster cell lines derived from CHO-K1 cells did not produce similar severe effects. The results suggest that repair- deficient human cell lines, irrespective of the actual repair defect, may respond to the occurrence of DNA damage in the population by removing large numbers of cells from the proliferating pool.

Mothersill, C. and Seymour, C. B. Bystander and Delayed Effects after Fractionated Radiation Exposure. Radiat. Res. 158, 626–633 (2002). Human immortalized keratinocytes were exposed to a range of single or fractionated doses of γ rays from 60 Co, to medium harvested from donor cells exposed to these protocols, or to a combination of radiation and irradiated cell conditioned medium (ICCM). The surviving fractions after direct irradiation or exposure to ICCM were determined using a clonogenic assay. The results show that medium harvested from cultures receiving fractionated irradiation gave lower “recovery factors” than direct fractionated irradiation, where normal split-dose recovery occurred. The recovery factor is defined here as the surviving fraction of the cells receiving two doses (direct or ICCM) separated by an interval of 2 h divided by the surviving fraction of cells receiving the same dose in one exposure. After treatment with ICCM, the recovery factors were less than 1 over a range of total doses from 5 mGy–5 Gy. Varying the time between doses from 10 min to 180 min did not alter the effect of ICCM, suggesting that two exposures to ICCM are more toxic than one irrespective of the dose used to generate the response. In certain protocols using mixtures of direct irradiation and ICCM, it was possible to eliminate the bystander effect. If bystander factors are produced in vivo , then they may reduce the sparing effect of the dose fractionation.

Mothersill, C., Seymour, C. B. and Joiner, M. C. Relationship between Radiation-Induced Low-Dose Hypersensitivity and the Bystander Effect. Radiat. Res. 157, 526–532 (2002). Recent advances in our knowledge of the biological effects of low doses of ionizing radiation have shown two unexpected phenomena: a “bystander effect” that can be demonstrated at low doses as a transferable factor(s) causing radiobiological effects in unexposed cells, and low-dose hyper-radiosensitivity and increased radioresistance that can be demonstrated collectively as a change in the dose–effect relationship, occurring around 0.5–1 Gy of low-LET radiation. In both cases, the effect of very low doses is greater than would be predicted by conventional DNA strand break/repair-based radiobiology. This paper addresses the question of whether the two phenomena have similar or exclusive mechanisms. Cells of 13 cell lines were tested using established protocols for expression of both hyper-radiosensitivity/increased radioresistance and a bystander response. Both were measured using clonogenicity as an end point. The results showed considerable variation in the expression of both phenomena and suggested that cell lines with a large bystander effect do not show hyper-radiosensitivity. The reverse was also true. This inverse relationship was not clearly related to the TP53 status or malignancy of the cell line. There was an indication that cell lines that have a radiation dose–response curve with a wide shoulder show hyper-radiosensitivity/increased radioresistance and no bystander effect. The results may suggest new approaches to understanding the factors that control cell death or the sectoring of survival at low radiation doses.

Lyng, F. M., Seymour, C. B. and Mothersill, C. Initiation of Apoptosis in Cells Exposed to Medium from the Progeny of Irradiated Cells: A Possible Mechanism for Bystander-Induced Genomic Instability? Radiat. Res. 157, 365–370 (2002). Genomic instability and bystander effects have recently been linked experimentally both in vivo and in vitro. The aim of the present study was to determine if medium from irradiated cells several passages distant from the original exposure could initiate apoptosis in unirradiated cells. Human keratinocytes (from the HPV-G cell line) were irradiated with 0.5 Gy or 5 Gy γ rays. Medium was harvested at each passage up to the 7th passage (approximately 35 population doublings) postirradiation and transferred to unirradiated keratinocytes. Intracellular calcium levels, mitochondrial membrane potential, and the level of reactive oxygen species were all monitored for 24 h after medium transfer. Rapid calcium fluxes (within 30 s), loss of mitochondrial membrane potential, and increases in reactive oxygen species (from 6 h after medium transfer) were observed in the recipient cells. There was no significant difference between medium conditioned by cells irradiated with 0.5 or 5 Gy. The effect of medium from progeny was the same as the initial effect reported previously and did not diminish with increasing passage number. The data suggest that initiating events in the cascade that leads to apoptosis are induced in unirradiated cells by a signal produced by irradiated cells and that this signal can still be produced by the progeny of irradiated cells for several generations.

Killing of unirradiated cells by medium from cultures of irradiated cells implies the release of a cytotoxic substance by the irradiated cells. The finding of the γ-ray-induced cytotoxic effect exclusively in epithelial cells and not in fibroblasts suggested that tissue architecture or cell communication might be important. Normal human keratinocytes and fibroblasts and radiosensitive carcinoma cells were irradiated as single cells, microcolonies of three or four cells, or confluent monolayers. The medium was removed and filtered, and cultures which had never been irradiated were seeded at cloning densities and treated with the medium from the irradiated cells. It was found that the degree of cell-cell contact had no effect on the ability of medium from irradiated epithelial cell cultures to reduce the clonogenic survival of unirradiated cells. Cell density was the only important factor. Inhibition of gap junction intercellular communication using the tumor promoter phorbol myristate acid (PMA), which closes gap junctions, increased killing by the bystander effect when the PMA was added to epithelial cells prior to irradiation. Rescue of epithelial cells exposed to the medium from the irradiated cells was not possible even after only 30 min exposure. This suggests that a signal transduction mechanism may control death or survival by the bystander effect rather than by release of a factor which is directly cytotoxic.

Explant cultures of normal human uroepithelium were established, exposed to a range of 60 Co γ-ray doses from 0.1-5 Gy and grown for 14 days. Expression of Myc, p53 and Bcl2 proteins in the epithelial cells which grew from irradiated explants was measured in situ using immunocytochemistry. The results show that overexpression of Bcl2 with low Myc expression correlated with resistance to radiation as shown by the extent of growth detected on day 14. Strong staining for Myc coupled with low or absent Bcl2 expression generally correlated with radiosensitivity, although the level of p53 of the culture was critical in these cases. None of the proteins on their own correlated with radiation response. What appeared to be critical was the balance of cells expressing Bcl2 and Myc proteins. Building on the results presented in a previous paper which showed a division of cultures from patients into those showing monotonic and non-monotonic responses, this study presents results for explant cultures from a greater number of patients and attempts to characterize the profile of expression of the above proteins in the uroepithelium of these patients. It shows that high Bcl2/Myc ratios were found in cultures which showed a non-monotonic and resistant dose response. Where Myc was the dominant protein in the culture postirradiation, a radiosensitive and monotonic response tended to occur. Since the proteins are being detected in the distant progeny of irradiated cells, it is likely that changes induced by radiation in the cell population are stable. The measurement of these two proteins can be made in cultured biopsy material and may therefore have predictive value in radiotherapy and radiation protection. Both normal and tumor biopsies from bladder mucosa showed similar correlations between Bcl2/Myc ratios and growth postirradiation.

Recent evidence from our laboratory suggests that the fraction of cells with lethal mutations is lost from the population by apoptosis. The relationship of this process to genetic instability and carcinogenesis is unclear. To examine this, tumorigenic cell populations derived from spontaneously occurring, neoplastically transformed C3H 10T1/2 foci and from radiation-induced foci were compared with wild-type C3H 10T1/2 cell populations to determine the frequency of induction of lethal mutations postirradiation. Lethal mutations did not occur in the progeny of cells from type 3 foci derived from cultures of spontaneously occurring or radiation-induced neoplastically transformed cells but were very frequent in the progeny of irradiated wild-type cells. Normal human cells (HPV-immortalized human keratinocytes and primary human normal uroepithelium) were then treated with carcinogens or transfected with the Ha-ras oncogene to see if these carcinogenic events affected the yield of lethal mutations postirradiation. In each case, cells which were exposed to a carcinogenic agent had reduced numbers of lethal mutations, elevated levels of stable p53 and Bcl-2 proteins and reduced evidence of apoptosis. It is suggested that lethal mutations may represent an active safety mechanism which may deal with radiation-induced genomic instability and which is disabled early in carcinogenesis.

Recent results using very low doses of radiation have suggested that there is a hypersensitive region where cultures show an enhanced level of cell killing leading to a non-monotonic survival curve. This effect has been observed at doses below 2 Gy in mammalian systems and at much higher doses in insect cells. In this paper we report observation of the effect in primary human uroepithelial cell cultures. The effect was measured using a postirradiation proliferation assay where irradiated explants of standard size were allowed to proliferate for 14 days after exposure to 60 Co γ irradiation. By 14 days the majority of cultures derived from explants irradiated with 2-5 Gy showed little evidence of growth inhibition and cell numbers approached or even exceeded those obtained in the controls. There was, however, a significant reduction in cell number and growth rate in all cultures exposed to doses lower than 1 Gy. Oncoprotein (p53, c-myc, bcl-2, p21 ras) and EGFR expression were also measured in these cultures and were significantly increased. Morphological evidence of apoptosis was present in all irradiated cultures at 4 h after exposure, but this persisted for longer periods in cultures exposed to low doses.

Uroepithelium cultured from normal patients without cancer (60 individuals) was found to segregate into four subtypes based on the level of carcinogen treatment needed to induce abnormal p53 and c-myc. Twenty-two percent of patient cultures never showed abnormal p53 expression, even after chronic exposure to nitrosamines, in addition to irradiation. In these cultures, c-myc expression was confined to viable, normal-appearing cells at the growing edge of the culture and to apoptotic bodies. Twenty-eight percent of cultures were negative for abnormal p53 unless challenged with both radiation and chronic administration of nitrosamines, while a further 26% required only a single dose of radiation to induce the abnormal protein. The remaining patients had tissue which, while initially negative for stable p53, became positive when put into culture and stimulated to grow. The c-myc protein was overexpressed in all cultures with abnormal p53. It would appear that elevated expression of conformationally inactive p53 and of high levels of c-myc represents an early response of normal uroepithelial cells to carcinogen challenge. It also appears that a relatively high number of patients without cancer express these proteins when their cells are challenged to grow; a pre-exposure to environmental carcinogens such as nitrosamines in cigarette smoke is likely to be involved.

When explants of human uroepithelium or esophageal epithelium are exposed to acute doses of radiation (cobalt-60), the cells which grow out to form the primary cultures show a number of abnormal features. These include the development of characteristic nonsenescent foci. These foci have previously been shown to be c-myc positive and to have an abnormal, tumor-like ultrastructure. Expression of c-myc and the level of stable p53 proteins have now been examined in these cultures 2 weeks after irradiation. Both proteins occurred in dividing cells at the growing edge of the explant and in the foci. The expression of c-myc appeared to be correlated with growth. As expected, variation between individual cultures of normal human cells was noted in the expression of stable p53 protein. Most control uroepithelial cell cultures were negative, but a small cohort showed a wide range of values. The control cultures from the esophageal tissues had high expression of p53, and this decreased marginally after irradiation. Cells positive for p53 were always in cycle and were usually positive for c-myc as well. It would appear from these results that the expression of c-myc and the stable form of the p53 protein occur in irradiated primary cultures of normal human cells both in foci which also express a number of abnormalities and in "edge" cells which are dividing. Cultures of unirradiated cells from esophagus and a small number of uroepithelial samples had high levels of p53. Possible reasons for this are discussed.