Individual variation in the production of a `bystander signal' following irradiation of primary cultures of normal human urothelium
Carmel Mothersill1,4,
David Rea3,
Eric G. Wright2,
Sally A. Lorimore2,
Dennis Murphy3,
Colin B. Seymour1 and
Kiaran O'Malley3
1 Radiation and Environmental Science Centre, Dublin Institute of Technology, Kevin Street, Dublin 8, Republic of Ireland,
2 Department of Cell Pathology, Ninewells Hospital Medical School, Dundee, Scotland, UK and
3 Department of Urology, Beaumont Hospital, Dublin 9, Republic of Ireland
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Abstract
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The existence of a bystander effect following both alpha and gamma irradiation of many cell lines is not now in dispute. The significance of this effect for cancer risk assessment and radiotherapy treatment planning requires demonstration of its relevance in vivo. The problem in demonstrating the existence of the effect in vivo is that other systemic effects may mask or confound the effect being investigated and it is practically impossible to attribute an effect in a particular cell to a signal produced in another irradiated cell. To approach this problem, we have developed an assay where fragments of human tissue can be irradiated ex vivo and the media harvested and added to unirradiated, allogenic explants or to a clonogenic cell line which has a well characterized and stable response to the bystander signal. The variation in production of the signal from patient to patient can thus be assessed using molecular and cellular endpoints. A study using tissue from over 100 patients and from mouse strains with well characterized responses to low level radiation exposure shows that there is variation in the effect of the signal produced by irradiated tissue from different patients. Gender, smoking status and the existence of a bladder malignancy influence the expression of the signal by normal urothelium. The effects of exposure to medium containing the signal are transmitted to distant progeny of the exposed cell population. The results may be important not only for understanding radiation risk mechanisms for protection but also for radiotherapy treatment planning where they may open new avenues for development of drugs for combined therapy.
Abbreviations: ICCM, irradiated cell conditioned medium; MRC, Medical Research Council; PBS, phosphate buffered saline.
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Introduction
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There is considerable evidence going back to 1968 that cells exposed to low and very low doses of radiation can produce a factor which affects the survival and function of unexposed cells (see for example refs 15). The effect has been detected in numerous cell lines, and after both densely ionizing and sparsely ionizing exposures. A number of experimental approaches have been used including very low dose alpha particle fluences where not every cell is traversed by a track (3,4,6,7), microbeam irradiation of single cells or parts of a cell in a field (810) or transfer of medium harvested from irradiated cells to cultures of unirradiated cells (5,11). While the effect has become known in the radiobiology literature as the `bystander effect' it is very similar to a cytokine-mediated effect and clearly may, but does not always, require gap junction-mediated transfer of the factor from cell to cell (6,11). The response of cells to the bystander signal can include induction of apoptosis, induction of genomic instability or delayed death, or induction of mutations (7,10,12). Elevated levels of proteins associated with the above effects and with a generalized stress response have also been detected. While the effect has been widely detected, it is not always present in a cell line. Medium transfer between responding and non-responding cell lines has clearly shown that signal production by an irradiated cell and response to that signal by a recipient cell can be distinguished as separate processes (5,13). Both processes appear to be p53 independent, although there is evidence that the response in fibroblast cells may require a functional p53 pathway (14).
One of the major effects of exposure of cell populations to the bystander signal is the induction of delayed effects in surviving progeny. Seymour and Mothersill (12) showed lethal mutations (delayed cell death) in the progeny of irradiated human keratinocyte cells. Lorimore et al. (7) showed that if primary bone marrow cultures were partially shielded then irradiated with alpha particles, the shielded parts of the culture gave rise to high levels of non-clonal chromosome and chromatid aberrations in the progeny. Nagasawa and Little (15) found high levels of de novo mutations in the progeny of cells from populations exposed to very low fluences of alpha particles.
The association between exposure to the bystander signal and production of delayed effects in the population is not limited to qualitative observations. The dose response curve for both bystander effects and delayed effects has been found to be already saturated at a dose of 0.05 Gy gamma radiation, or following single track microbeam irradiation or single track alpha particles in systems where dose response work has been carried out (3,7,9,16). Quantitatively, the number of cells affected in a population exposed to the bystander signal is relatively high and of the same order of magnitude to the numbers which show delayed effects in their progeny. This has led to speculation that the bystander factor or signal may be involved in the mechanism of induction of delayed effects.
Our group has shown previously a clear genetic basis for the production of delayed effects following irradiation. This has been shown in bone marrow and urothelium from pure inbred mouse strains and also as individual variation in response in primary cultures of human urothelium from a large number of patients (17,18). In view of the possible link between delayed effects and bystander signal production/response, it was decided to look for evidence of a genetic variation in the production of the bystander factor following exposure of cultured urothelium to low doses of gamma radiation.
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Methods
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Source of tissue
Human urothelial specimens
Surgically excised pieces of ureter (34 cm), surgically excised pieces of bladder and bladder biopsies (2x0.5 cm cold cut) were obtained from over 100 patients attending the urology clinic for cystoscopy at Beaumont Hospital, Dublin. All patients had given informed consent and the hospital's ethics committee approved the study. The age, sex, cancer history and smoking history were obtained for all patients and noted along with the reason for admission. All specimens were collected in cold sterile phosphate buffered saline (PBS) and were processed immediately on receipt from the hospital (within 3 h of removal).
Mouse urothelial specimens
Bladders were dissected from CBA/H and C57/BL6 mice maintained in the Medical Research Council's (MRC) facility at Harwell, Didcot, UK. Purebred mice of both sexes were used. The bladders were opened, rinsed in Earle's balanced salt solution and treated exactly as human specimens. The animal studies were carried out in compliance with the guidance issued by the MRC (19). The samples were coded at the MRC and the cultures were analysed by operators in Dublin, blinded with respect to the sex and genetic background of the animals.
Explant culture
The methods have been published in detail previously (20). Any modifications are incorporated in the following brief description: human and mouse tissues were cleaned of fat and cut into small fragments ~1 mm2 using sterile scalpels. The tissue pieces were placed in 10 ml of digest solution containing 10 mg/ml collagenase type IV (Sigma chemicals, Poole, Dorset, UK) in 0.25% trypsin solution and incubated at 37°C for 30 min. The tissue fragments were plated as individual explants in 25 cm2, 40 ml flasks (Nunc, Rochester, NY) containing 2 ml of start-up medium. This medium is RPMI-1640 containing 10% fetal calf serum, 5 ml penicillin streptomycin solution, 5 ml L-glutamine, 1 µg/ml hydrocortisone and 100 mIU insulin. All the above reagents were obtained from Gibco-Biocult Ltd. (Irvine, Scotland, UK). Cultures were maintained in an incubator at 37°C in an atmosphere of 5% CO2 in air.
Bystander experiments
Two approaches were used to assess the ability of human explants to produce or respond to a radiation induced bystander factor.
Method 1
Explants were divided into two groups, group one was directly exposed to irradiation, and the explants received a medium change after 1 h to Clonetics KGM serum (Clonetics, San Diego, California) free formulation and were then left undisturbed in the incubator for 14 days. The second group was not directly irradiated but explants were exposed to the medium harvested from the first group for 1 h. They were then transferred to Clonetics KGM and allowed to grow undisturbed for 14 days. The harvested medium was always filtered through 0.22 µ Nalgene filters (Nalgene, Rochester, NY) to remove debris or cells. Controls, which received medium from unirradiated cultures, were carried with each experiment. The response of the directly irradiated cells and the recipients of the medium (termed irradiated cell conditioned medium or ICCM) was assessed after the 14 day period, in formalin fixed cultures using the following endpoints: growth, morphological evidence of apoptosis or necrosis, and expression of p53, bcl-2 and BAX proteins were detected using immunocytochemistry.
The first method required large amounts of tissue to produce adequate controls and replicates. It is also rather qualitative; therefore, the following method was developed.
Method 2
Urothelial explants are set up and irradiated as before. The medium was harvested after 1 h, filtered and added to human keratinocyte cells immortalized using the HPV-16 virus (HPV-G line obtained as a gift from J.DiPaolo, NIH, Bethesda, MD, USA; see ref. 21 for details). This line is clonogenic with a very stable cloning efficiency of 2530% under the conditions used. It has a well characterized and stable bystander response to ICCM generated by irradiation of the cell line as shown by a reduction in cloning efficiency of ~40% over a wide range of radiation doses (16). This makes it ideal as a reporter system, which can be used to compare, using a quantitative endpoint, the individual human and mouse urothelial cultures for ability to produce the bystander signal.
Irradiation
Where indicated, cultures were irradiated at room temperature using a cobalt 60 teletherapy unit at flask to source distance of 80 cm. The dose rate during these experiments was ~2 Gy/min.
Immunochemistry
Expression of proteins involved in the apoptotic pathway (bcl-2, BAX and p53) was examined in the primary cultures following direct irradiation or exposure to ICCM. These methods have been described in detail previously (22). The reasons for selection of the antibodies and the experimental and statistical controls are also to be found in the above paper. Briefly, cultures were fixed in 10% unbuffered formalin and stored at 4°C until processed. Processing always took place within 7 days of fixation. The explant outgrowth was processed in situ on the flask bottom to enable spatial distribution to be related to the type of cell and degree of differentiation. Cultures were stained for p53 (p53-240 Novocastra Ltd, Newcastle, UK), bcl-2 (Dako Ltd, Uden, The Netherlands), and BAX (Santa Cruz Labs Ltd, California, USA). Immunochemistry was performed using an appropriate Vectastain ABC kit (Burlingame, California, USA) for mouse monoclonal or rabbit polyclonal antibodies. Diaminobendizine was used to express the positive reaction and cultures were lightly counter-stained with Harris Haematoxylin. Two explants were stained for each antibody and over 200 cells were scored per explant over five fields using a Leica image analysis system. The detection threshold for positivity was set using positive control sections from a positive block, obtained from the cell pathology service. Positive and negative control sections were run with every immunocytochemistry run to correct for run variability. All cultures were coded and random samples were re-scored by a second person in the laboratory. As a result of the small amounts of material (~1 mg total cells), and the desirability of using in situ approaches, it was not possible to check expression levels using western analysis.
Quantification of growth, apoptosis and necrosis in explants
All explants from the direct dose or ICCM treated group were used to assess the extent of growth inhibition relative to the appropriate control. Numbers of apoptotic and necrotic cells were scored by pooling data from all explants per dose group during the immunocytochemical scoring. Morphological criteria under the light microscope were used in cultures stained for p53, bcl-2 or BAX proteins, because of the necessity to conserve the very limited amounts of material. Electron microscopy was used to validate the light microscopic observations in selected cultures. Cells were scored as `apoptotic' if the nucleus was fragmented or shrunken and if the cell was pycnotic. `Necrotic' cells had a swollen or burst nucleus.
Statistical analysis
Cultures from over 100 human patients were assessed in the study. These were used to provide medium for the keratinocyte reporter system experiments (method 2 above), and where the quantity of material permitted, to assess the effect of transferring ICCM to explants not exposed to direct irradiation (method 1 above). Biopsies could not be used to set up large experiments; therefore, most patient cultures were only exposed to 0, 0.5 and 5 Gy cobalt 60, as this was sufficient to allow the response to be detected. Data are presented with a breakdown into smokers/non-smokers and male/female groups. Patients with tumours were analysed separately. The experiments with the CBA/H and C57/BL6 mouse strains were repeated twice with eight animals per group per experiment. Cultures were exposed to 0, 0.5 or 5 Gy irradiation. Data are presented as mean ± SE and significance was determined using the MannWhitney U test. Culture success rate was high (>95% of explants attached and grew) and sufficient spare explants were set up to ensure that measurements could be made on eight explants per experimental point. This gives SE between similarly treated explants from the same patient of <5%.
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Results
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Table Ia
shows the results for method 1 where ICCM was harvested from irradiated urothelial explants and transferred to unirradiated explants from the same patient. Biopsies from 20 patients were used for this protocol. A large piece of tissue from a signal patient was used to set up 20 replicate cultures to test for intra-individual variation as a result of the technique. The variation was ±4.1% between explants. The data are grouped using retrospective analysis of growth response post-direct irradiation, into those with a type 1 or radioresistant response and those with a type 2 or radiosensitive response. The basis for this division is presented and discussed extensively by Mothersill et al. (18). The data show that there is considerable variation in both the direct response and in the response to ICCM within the group. Overall, patient cultures from the type 2 category, also show more reduction in explant outgrowth post-exposure to direct irradiation or ICCM These data are summarized in Table II
and are all extremely significant (P < 0.0001). It is interesting that while the direct irradiation causes increased growth inhibition after 5 Gy compared with 0.5 Gy, the effect of ICCM shows a flat dose response, whereby the effect after exposure to ICCM harvested from 5 Gy irradiated cultures is the same as that seen after 0.5 Gy. In Table Ib
, the corresponding scores for apoptotic bodies and necrotic cells in the cultures are shown. Again type 2 responding cultures have higher levels of dead cells in the explant outgrowth, than patients with a type 1 response. All necrotic and apoptotic cells are included under the `dead cells' heading in Table Ib
because scoring apoptosis 14 days post-treatment is problematic because of the possibility of secondary necrosis. The ultimate extent of cell growth and overall numbers of dead cells in the explant are not significantly different at the 0.5 Gy dose whether direct irradiation or ICCM are used. At the 5 Gy dose level, there are more dead cells in the direct irradiation group than in the ICM treated cultures. The results for immunocytochemical expression of p53, bcl-2 and BAX are shown in Figure 1ac
, which shows cumulative frequency plots for each protein. The individual variation is again apparent and the effect of ICCM exposure is very similar to the effect of direct irradiation. As a result of the small number of specimens suitable for this series of experiments it was not possible to analyse the data with respect to patient history.
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Table II. Summary of statistical analysis (MannWhitney U test for non-parametric populations) of the data presented in Figures 27     and Table I
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Table Ib. Percentage of cells in the explant outgrowth that were dead because of apoptosis or necrosis measured 14 days after irradiation/exposure to ICM
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In Figure 2
, data are shown for method 2. Here the endpoint is clonogenic survival of the HPV transfected human keratinocyte line (HPV-G). This method has the considerable advantage that fewer explants are needed to demonstrate the bystander effect for a given patient and this method is the only possible one for use with biopsies. Where adequate supplies of tissue permitted, correlations were carried out between both methods. These gave qualitatively similar results, although explant outgrowth and clonogenic survival are such different endpoints that quantitative correlations are meaningless. The pattern of bystander response for the cell line when exposed to ICCM from its own cells is shown in Figure 3
and can be seen to be very stable over the dose range used in these experiments. Clearly the cloning efficiency of the HPV-G cells is affected by the ICCM from the urothelial explants. The degree of reduction in cloning efficiency is variable ranging from no effect to 80% reduction. The values found for the CBA/H and C57/BL6 mice are also shown on the frequency plots. These show less effect on cloning efficiency than the human samples but the trend is for the CBA/H mouse strain which is more prone to radiogenic malignancies and chromosomal instability, to show a smaller reduction in cloning efficiency than the C57/BL6 strain. While the latter is susceptible to lymphoma, epithelial cancers are rare and chromosomal instability is less pronounced. In Figures 4 and 5
the data are broken down into smokers and non-smokers (Figure 4
) and male or female (Figure 5
). The figures show that ICCM harvested from irradiated cultures from smokers or males induce less reduction in cloning efficiency than that from non-smokers or females. When the effect of smoking was analysed separately for males and females, the ICCM from male smokers caused significantly less reduction in the clonogenic survival of HPV-G cells (P = 0.001) than male non-smoker ICCM but the female statistic was not quite significant (P = 0.074). The summary statistics are presented in Table II
. The gender trend is also seen in both mouse strains. The cytotioxic bystander effect was reduced or absent when ICCM was harvested from cultures of tissue from tumours or normal adjacent tissue from tumour bearing individuals (Figure 6
). In fact the effect of medium from the tumours in these patients increased the clonogenic survival of reporter cells although the increase was not significant. This suggests that the signal production may be down regulated or absent in this group as well as in male smokers.

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Fig. 2. Individual variation in the cytotioxic properties of ICCM derived from tissue cultures from human and mouse urothelium (PE, plating efficiency).
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Fig. 4. Individual variation in the cytotioxic properties of ICCM derived from tissue cultures from smokers versus non-smokers (PE, plating efficiency).
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Fig. 5. Individual variation in the cytotioxic properties of ICCM derived from tissue cultures from males versus females (PE, plating efficiency).
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Fig. 6. Mean plating efficiency of HPV-G cells following exposure to ICCM in tissue cultures derived from normal urothelium or associated transitional cell carcinoma.
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There is considerable evidence that the effects of exposure to the bystander signal are long term (7,12); therefore, the ICCM exposed keratinocytes were kept in culture and grown on from colonies to confluent monolayers. These were subcultured into ordinary medium and re-tested for cloning efficiency. The results for keratinocyte progeny of human and mouse progenitors originally exposed to ICCM are shown in Figure 7ac
. Figure 7a
shows the overall variation, in Figure 7b
the data are broken down into male and female responses and in Figure 7c
the effect of smoking is shown. The statistical parameters for these data are also shown on Table II
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Three points are clear. First, the effect of ICCM exposure is detectable in progeny of exposed cells some 30 cell generations after removal of the signal. Although there is significantly less effect overall in the progeny the result represents a reduction of ~35% in the cloning efficiency of progeny whose progenitor cells were treated once with ICCM. This confirms previous results for cell lines showing that delayed effects persist at high frequencies for many generations.
Second, the inter-patient variation seen in the progenitor cells is also seen in the progeny. Patient tissues and the mouse strain, which produced large initial bystander effects gave rise to reporter cell progeny with similarly large delayed effects. An attempt was made to statistically correlate the initial and delayed response for the entire data set. While there is a clear trend for the two to agree, the correlation coefficients are not significant because of the number of patient cultures that showed no delayed response.
Third, the gender and smoking related differences are still apparent. Among the subgroups that gave significant correlations in the statistical analysis are total non-smokers initial versus delayed response (Spearman correlation = 5.77, considered highly significant), and male smokers initial versus delayed response (Spearman correlation = 0.41 considered significant).
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Discussion
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The aims of this paper were twofold. First, to see if there was individual variation in the expression of a bystander signal and/or response in irradiated human urothelial tissue explants and, if so, whether the response to direct irradiation correlated with the response to ICCM. The second aim was to test a new method of quantifying the bystander effect using a clonogenic cell line known to respond to the signal. This allows signal production to be dissociated from response and also allows a more precise quantification of the early and delayed effects of ICCM, which is not possible in the primary culture system. The results show clearly that there is individual variation in the growth post-ICCM exposure of urothelial explants. Much of the reason for the reduced growth in some cultures is death of cells by apoptosis or necrosis. The levels of dead cells on day 14 are clearly different. Previous work by this group (18) identified two major subtypes of response to irradiation in human urothelial explant cultures. One is sensitive to irradiation and the cultures show evidence of both apoptosis and necrosis. The other is resistant to the cytotoxic effects of irradiation and shows little or no cell death in the outgrowths by day 14 post-exposure. When validated against mouse strains with known differences in response to radiation (17), the results suggested that the death of cells post-irradiation in the explants was protective against delayed genomic instability, as the cytocidal effects were very apparent in the stable mouse strain and not in the unstable strain. It is interesting, therefore, to note that the effect of ICCM follows a similar trend. Type 1 resistant human urothelial explants show only small effects when exposed to ICCM while the sensitive type 2 explants show larger effects. The mouse strains also follow the trend.
While the data shown in the tables is interesting, it is not very quantitative and because the explants cannot be propagated for longer than ~21 days, it is not possible to get long-term information on fate of the cells. A further problem is that signal production and response are probably two separate processes and they cannot be independently analysed using this approach. For this reason it was decided to try to develop an assay for the bystander factor that could be standardized and quantified. The results presented here using the clonogenic human keratinocyte cell line as recipient for the signal, suggest that this approach works. The signal production and response are obviously very stable when ICCM from the cell line itself is used. This is very important as it means that differences in the response to donor ICCM from the patient cultures can be attributed to the donated medium and not to a variable response in the cell line. The results using the reporter system show high variability in the extent of reduction of cloning efficiency. Using this system it is possible to work with biopsy material and to accumulate large numbers of tissue samples relatively quickly. This permits more detailed analysis of the data to determine the impact of gender and smoking history, ICCM from smoker urothelial cultures causes a smaller reduction in cloning efficiency than ICCM generated from non-smoker tissue. Similarly, the trend for ICCM generated from male tissues to have a smaller effect than that from female tissue is confirmed using the clonogenic assay. It is interesting that the tissue with the type 2 response generates a larger bystander effect in the reporter system than the type 1 samples. This is all consistent with a hypothesis that cell death induced by low doses of direct irradiation is protective in that it eliminates cells from the population that are carrying damage. The smoker data then reflects a deregulated response to the damage signal so that damaged cells are not destroyed but persist possibly carrying dangerous mutations or genomic instability (23,24). There are, however, some interesting implications for extending this hypothesis to cover the bystander factor data because a protective mechanism implies a benefit to the population. One implication is that it is difficult to see why it is protective for the tissue/organism that un-hit cells in a population are destroyed or undergo mutation. The release of a signal may perhaps `alert' cells that a cytotoxic insult has happened and may lead to widespread induction of repair or other protection systems. Some of these may act to rid the tissue culture of pre-existing damage. There is some evidence for this from experiments where ICCM was used in lieu of a conditioning dose of radiation. Cells receiving the ICCM pre-treatment had a much greater survival following a challenge dose than those just receiving the dose (11). There is also evidence from the `adaptive response' or radiation hormesis field (25), where it is known that pre-exposure of cells to extremely low doses (<0.01 Gy) can make them more resistant to subsequent high dose exposure. Other evidence comes from the HRS/IRR (Hyper Radio Sensitivity/Induced Radio Resistance) field (26) where some cell lines show a transition from a very sensitive low dose response to a more resistant response at high doses. As mechanisms underlying damage sensing and response in cells become clearer, it is probable that an explanation for the biological importance of bystander effects will become apparent.
In conclusion, media harvested from irradiated cultures of tissue from human and mouse bladder, contains a signal or factor that can induce protein expression and cell death in unirradiated, allogenic explants or reduce the cloning efficiency of a human epithelial cell line. The induced effects are transmissible to progeny. The magnitude of the effect varies from patient to patient and is less in biopsies from males, male smokers and tumour bearing individuals. The data are consistent with the hypothesis that the bystander signal can induce effective radiation damage response pathways in normal cells but that signal production at least is affected by smoking and in tumour bearing tissues
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Notes
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4 To whom correspondence should be addressed Email: cmothersill{at}rsc.iol.ie 
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Acknowledgments
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We acknowledge the support of the Irish Cancer Society and the EU Radiation Protection Programme who partly funded this work. We also want to thank Saint Luke's Hospital Dublin, for access to their Cobalt Unit.
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References
|
---|
-
Hollowell,J.G. and Littlefield,L.G. (1968) Chromosome damage induced by plasma of X-rayed patients: an indirect effect of X-ray. Proc. Soc. Exp. Biol. Med., 129, 240244.[Medline]
-
Lloyd,D.C. and Moquet,J.E. (1985) The clastogenic effect of irradiated plasma. Int. J. Radiat. Biol., 47, 433444.
-
Nagasawa,H. and Little,J.B. (1992) Induction of sister-chromatid exchanges by extremely low doses of alpha particles. Cancer Res., 52, 63946396.[Abstract]
-
Deshpande,A., Goodwin,E.H., Bailey,S.M., Marrone,B.L. and Lehnert,B.E. (1996) Alpha-particle-induced sister chromatid exchange in normal human lung fibroblasts: evidence for an extranuclear target. Radiat. Res., 145, 260267.[Medline]
-
Mothersill,C. and Seymour,C.B. (1997) Medium from irradiated human epithelial cells but not human fibroblasts reduces the clonogenic survival of unirradiated cells. Int. J. Radiat. Biol., 71, 421427.[Medline]
-
Assam,E.I., deToledo,S.M., Gooding,T. and Little,J.B. (1998) Intercellular communication is involved in the bystander regulation of gene expression in human cells exposed to very low fluences of alpha particles. Radiat. Res., 150, 497504.[Medline]
-
Lorimore,S.A., Kadhim,M.A., Pocock,D.A., Papworth,D., Stevens,D.L., Goodhead,D.T. and Wright,E.G. (1998) Chromosomal instability in the descendants of unirradiated surviving cells after a-particle irradiation. Proc. Natl Acad. Sci. USA, 95, 57305733.[Abstract/Full Text]
-
Prise,K.M., Belyakov,O.V., Folkard,M. and Michael,B.D. (1998) Studies of bystander effects in human fibroblasts using a charged particle microbeam. Int. J. Radiat. Biol., 74, 793798.[Medline]
-
Belyakov,O.V. Prise,K.M. Mothersill,C., Folkard,M. and Michael,B.D. (2000) Studies of bystander effects in primary urothelial cells using a charged particle microbeam. Radiat. Res., vol. 153.
-
Wu,L.-J., Randers-Pehrson,G., Waldren,C.A., Geard,C.R., Yu,Z.L. and Hei,T.K. (1999) Targeted cytoplasmic irradiation with alpha particles induces mutations in mammalian cells. Proc. Natl Acad. Sci. USA, 96, 49594964.[Abstract/Full Text]
-
Mothersill,C. and Seymour,C.B. (1998) Cell-cell contact during gamma irradiation is not required to induce a bystander effect in normal human keratinocytes: evidence for release during irradiation of a signal controlling survival into the medium. Radiat. Res., 149, 256262.[Medline]
-
Seymour,C.B. and Mothersill,C. (1997) Delayed expression of lethal mutations and genomic instability in the progeny of human epithelial cells that survived in a bystander-killing environment. Radiat. Oncol. Invest., 5, 106110.
-
Mothersill,C., Stamato,T.D., Perez,M.L., Mooney,R., Cummins,R. and Seymour,C.B. (2000) A role for mitochondria in the induction of radiation-induced bystander effects? Br. J. Cancer, 82, 17401746.[Medline]
-
Iyer,R. and Lehnert,B.E. (2000) Factors underlying the cell growth-related bystander responses to alpha particles. Cancer Res., 60, 12901298.[Abstract/Full Text]
-
Nagasawa,H. and Little,J.B. (1999) Unexpected sensitivity to the induction of mutations by very low doses of alpha-particle radiation: evidence for a bystander effect. Radiat. Res., 52, 552557.
-
Seymour,C.B. and Mothersill,C. (2000) Relative contribution of bystander and targeted cell killing to the low dose region of the radiation dose-response curve. Radiat. Res., 153, 508511.[Medline]
-
Watson,G.E., Lorimore,S.A., Clutton,S.M., Kadhim,M.A. and Wright,E.G. (1997) Genetic factors influencing alpha-particle induced genomic instability. Int. J. Radiat. Biol., 71, 497503.[Medline]
-
Mothersill,C., O'Malley,K.J., Murphy,D.M., Seymour,C.B., Lorimore,S.A. and Wright,E.G. (1999) Identification and characterisation of three subtypes of radiation response in normal human urothelial cultures exposed to ionising radiation. Carcinogenesis, 20, 22732278.[Abstract/Full Text]
-
Medical Research Council (1993) Responsibility in the Use of Animals for Medical Research. UK Home Office Project Licence number PPL 30/1272.
-
Mothersill,C., Harney,J., Lyng,F., Cottell,D., Parsons,K., Murphy,D. and Seymour,C. (1995) Primary explants of human uroepithelium show an unusual response to low-dose irradiation with cobalt-60 gamma rays. Radiat. Res., 142, 181187.[Medline]
-
Pirisi,L., Yasumoto,S., Feller,S., Doniger,J. and DiPaolo,J. (1988) Transformation of human fibroblasts and keratinocytes with human papillomavirus type 16 DNA. J. Virol., 61, 10611066.
-
Harney,J., Seymour,C.B., Murphy,D. and Mothersill,C. (1995) Variation in the expression of p53, cmyc and bcl-2 oncoproteins in individual patient cultures of normal urothelium exposed to cobalt 60 gamma-rays and n-nitrosodiethanolamine. Cancer Epidemiol. Biomarkers Prev., 4, 617625.[Abstract]
-
Mothersill,C., O'Malley,K., Murhpy,D., Lynch,T., Payne,S., Seymour,C. and Harney,J. (1999) P53 protein expression and increased SSCP mobility shifts in the p53 gene in normal urothelium cultured from smokers. Carcinogenesis, 18, 12411245.[Abstract]
-
Colucci,S., Mothersill,C., Harney,J., Gamble,C., Seymour,C.B. and Arrand,J.E. (1997) Induction of multiple PCR-SSCPE mobility shifts in p53 exons in cultures of normal human urothelium exposed to low-dose gamma radiation. Int. J. Radiat. Biol., 72, 2131.[Medline]
-
Wolff,S. (1998) The adaptive response in radiobiology: evolving insights and implications. Environ. Health Pers., 106, 277283.[Medline]
-
Marples,B. and Joiner,M.C. (1993) The response of v79 cells to low radiation doses; evidence of enhanced sensitivity of the whole cell population. Radiat. Res., 133, 4151.[Medline]
Received December 1, 2000;
revised May 9, 2001;
accepted May 10, 2001.