Identification and characterization of three subtypes of radiation response in normal human urothelial cultures exposed to ionizing radiation

Carmel E. Mothersill3, Kiaran J. O'Malley1, Dennis M. Murphy1, Colin B. Seymour, Sally A. Lorimore2 and Eric G. Wright2,4

Radiation Science Centre, Dublin Institute of Technology,Kevin Street, Dublin and
1 Department of Urology, Beaumont Hospital, Dublin 9, Republic of Ireland and
2 Radiation and Genome Stability Unit, Medical Research Council, Chilton, Didcot, UK


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In an attempt to assess genetic variation underlying the variation in human responses to radiation exposure, measurements of apoptosis, necrosis and induction of key proteins were made in primary explant cultures of human normal urothelium and correlated with growth post- exposure to a range of doses of 60Co. These data were validated by similar experiments using CBA/H and C57/BL6 mouse strains, known to exhibit genetically determined differences in response to radiation. The data for human tissues show a wide variation in response with three broad categories being identifiable. The commonest had a hypersensitive response involving considerable apoptosis in the low dose region, followed by `induction' of a survival response at higher doses involving the persistence of abnormal cells. The pattern of gene expression was consistent with suppression of apoptosis. The second category showed no induction of survival and considerable necrosis was seen in the progeny. The rarest category showed an extremely hypersensitive low dose response and despite induction of a survival response, the sensitivity to higher doses was very severe. Considerable apoptosis and necrosis were seen in these cultures. In the mouse experiments, strain CBA/H (mice known to exhibit genetic instability post-irradiation) had lower levels of delayed cell death and apoptosis than C57/BL6 mice (which exhibit significantly less instability). It is concluded that there is a variation in response to radiation between human patient cultures which is detectable in this system and which is consistent with a pattern of radiation- induced delayed death/apoptosis correlating with long-term genomic stability. The mouse experiments demonstrate the importance of genetic factors in determining these responses.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
It is well known that individual variation in response to accidental radiation exposure of healthy people exists and that patients vary in response to a uniform dose of radiotherapy (1,2). Variation in response among mouse strains and among tumour and normal cell lines is also well documented (3). There is limited information, however, on the mechanisms underlying determination of the ultimate radiation outcome apart from studies on specific gene defects (4,5). Many lines of evidence now suggest that the final determination of outcome following exposure to radiation or other genotoxic stress may depend on the initial handling of the damage (6,7). An important factor seems to be whether the cell dies, repairs all the damage, undergoes defective repair or responds in a way which leads to transformation. The decision whether the damage is dealt with by apoptosis or whether rescue or repair is attempted is critical. Death of the individual cell removes the problem from the tissue but if the cell does not die, it may acquire genomic instability and lead to a population of cells with abnormally high susceptibility to chromosomal instability mutation and other delayed effects (812). This event is considered by many to be a very early predisposing factor in radiation carcinogenesis (1315). A very interesting hypothesis is that patients or mice which have a very acute death response to radiation may in fact be less susceptible to the delayed effects, including genomic instability and carcinogenic initiation. Previous work in the Dublin laboratory, using normal human uroepithelium demonstrated a wide variation in response to radiation. This was detected using a growth end-point and was also detected as differences in expression of p53, bcl-2 and c-myc proteins. The effects were observed in primary cultures derived from fragments of tissue exposed as explants and left to grow post-irradiation for 14 days (1618). The link to genetic predisposition or intrinsic radiosensitivity could only be inferred since patients without cancer are not normally exposed to irradiation. Work in the Radiation and Genome Stability Unit has established that different mouse strains known to vary in their radiation sensitivity have different susceptibilities to chromosomal instability and that the pattern of inheritance is Mendelian recessive (19). This strongly suggests that generation of genomic instability involves genetic factors and further suggests that the strains may differ in their handling of radiation damage. The aim of the work described here was to see if the predictive use of the end-points identified using human uroepithelium could be validated using cultures of uroepithelium from mouse strains known to vary in their genetic predisposition. This might permit identification of subpopulations of people at greater risk from radiation than the mean population.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Tissue sources
Human urothelial specimens.
Pieces of ureter (3–4 cm) and bladder biopsies (2x0.5 cm cold cut) were obtained from patients with no history of cancer. A control group of patients with neoplasms of tissues other than the transitional epithelium were also obtained. All patients had given informed consent and the hospital's ethics committee approved the study. Smoking history is known to confound in these studies (20) and smokers were analysed as a separate group. The breakdown of patients was: normal smokers 79, of which 50 were male; cancer-bearing smokers 12, of which 10 were male; normal non-smokers 61, of which 21 were male; cancer-bearing non-smokers 16, of which 14 were male; normal paediatric patients 15, of which 9 were male. All specimens were collected in cold sterile phosphate-buffered saline 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 MRC colony. Purebred and F1 crosses of both sexes were used. The bladders were opened, rinsed in Earle's balanced salt solution and treated exactly as the human specimens. The animal studies were carried out in compliance with the guidance issued by the Medical Research Council in Responsibility in the Use of Animals for Medical Research (July 1993) and UK Home Office Project Licence no. PPL 30/1272. The samples were coded at the MRC and the cultures were analysed by operators in Dublin, blind with respect to the sex and genetic background of the animals.

Explant culture
The methods have been published in detail previously (17). 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 Chemical Co., Poole, 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, Roskilde, Denmark) containing 2 ml of start-up medium. This medium was RPMI-1640 containing 20% 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, UK). Cultures were maintained in an incubator at 37°C in an atmosphere of 5% CO2 in air. After the explants had attached (~3 days), the medium was carefully removed and replaced with 2 ml Clonetics serum-free formulation for keratinocytes (Clonetics Corp., San Diego, CA). Cultures were then irradiated and left undisturbed (except where indicated in the text) without further medium changes until they were stained 14 days later.

Irradiation
Cultures were irradiated at room temperature using a 60Co teletherapy unit at a flask to source distance of 80 cm. The dose rate during these experiments was ~2 Gy/min.

Immunochemistry
These methods have been described in detail previously (17). Briefly, cultures were fixed in 10% formalin and stored at 4°C until processed. Processing always took place within 7 days of fixation. The explant 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), BAX (Santa Cruz Laboratories, Santa Cruz, CA) and c-myc (clone 9E10, a kind gift from G. Evan, ICRF, UK). Immunochemistry was performed using a Vectastain ABC kit (Vector Laboratories, Burlingame, CA) for mouse monoclonal or rabbit polyclonal antibodies as appropriate. Diaminobendizine was used to express the positive reaction and cultures were lightly counterstained with Harris haematoxylin. Due to limited availability of human normal tissue for culture, only a few representative members of large protein families such as bcl-2 and BAX could be tested. The antibodies were chosen because they gave good staining on normal material in the Pathology Department at the time the study was set up. Two explants were stained for each antibody and >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 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. Due to 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 a dose group (eight in total) were used to assess the extent of growth inhibition relative to the unirradiated control. Numbers of apoptotic and necrotic cells were scored by pooling data from all eight explants per dose group during the immunohistochemical scoring. Morphological criteria under the light microscope were used because of the necessity to conserve the very limited amounts of material and because there is no universally agreed method of detecting apoptosis except morphology. 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 >160 human patients were used in the study. These were grouped according to whether their cultures did or did not show a continuous reduction in growth with increasing dose (discussed previously in ref. 18) and were analysed for protein expression, apoptosis and necrosis over a 14 day growth period post-irradiation. Subgroups of paediatric patients and tumour-bearing patients were analysed separately. Smokers were also considered as a distinct group. Most patient cultures were only exposed to 0, 0.5 and 5 Gy since this allowed sectoring to a group to be done and biopsies could not be used to set up large experiments, but 50 patients where large amounts of ureter were available were analysed over a seven point dose range from 0.1 to 5 Gy to try to define the low dose response. The time course experiments were done on this same set of patients. No attempt was made to analyse these data with respect to patient history since the numbers per group were too low. The experiments with the CBA/H and C57/BL 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 means ± SE and significance was determined using paired or unpaired t-tests as appropriate. 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 standard errors between similarly treated explants from the same patient of <5%.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Human urothelium
Definition of response types.
Using the patient cultures where a full range of doses was tested (50 patients), the three post-irradiation growth response types seen previously were again found. A description of these subtypes is give below: type 1, low dose hypersensitivity, induction of proliferation above threshold, high dose resistance; type 2, monotonic, sensitive response; type 3, hypersensitive response at low doses, induction of proliferation, high dose sensitivity.

In Figure 1aGo–c the corresponding scores for maximum levels of apoptosis and necrosis are graphed along with the data for per cent of control growth achieved on day 14 post-irradiation for a typical representative from each group. The full data set for growth, apoptosis and necrosis are presented in Table IaGo–cGo. Because apoptosis peaks on days 2–3 in these cultures (see data in Table IGo), the numbers of apoptotic cells were scored on days 2 and 3 post-irradiation using phase contrast microscopy and without sacrificing the culture. These data were validated using patient cultures where enough material was available to do light and electron microscopy on fixed and appropriately stained material. Necrosis was measured in stained cultures on day 14. Oncoprotein expression on day 14 post-irradiation is shown in Figure 2aGo–c for the cultures presented in Table IGo. Together these show that there is a distinct pattern of expression of protein and of cell death for each growth response type. Necrosis appears to be the main cell death mechanism post-irradiation for all groups and is predominant at high doses in type 2. A key finding is that in type 1 the necrosis level does not increase with dose. There is an elevation in bcl-2 expression and reduced expression of c-myc and BAX in this group. Apoptosis is detected at low doses but does not increase with dose for any subtype. Since these data are from cultures which developed from explants irradiated up to 14 days previously, they are late effects in the progeny of irradiated cells. Using the data from the range of radiation doses it was decided to define type 1 as cultures having a per cent control growth value after 5 Gy which was greater than the mean for the entire group and a non-monotonic dose–response curve with a low dose breakpoint. Type 2 is defined as having a 5 Gy value less than the mean for the entire group and a monotonic dose–response curve. Type 3 is defined as having both the 0.5 and 5 Gy values <50% of the mean for the entire group together with a non-monotonic dose–response curve.





View larger version (0K):
[in this window]
[in a new window]
 
Fig. 1. (a) Percent of cells showing apoptotic ({blacktriangleup}) or necrotic (x) morphology in type 1 primary cultures of human urothelium which were irradiated as explants. Growth detected at 14 days post-exposure is also shown ({blacklozenge}). (b) Percent of cells showing apoptotic ({blacktriangleup}) or necrotic (x) morphology in type 2 primary cultures of human urothelium which were irradiated as explants. Growth detected at 14 days post-exposure is also shown ({blacklozenge}). (c) Percent of cells showing apoptotic ({blacktriangleup}) or necrotic (x) morphology in type 3 primary cultures of human urothelium which were irradiated as explants. Growth detected at 14 days post-exposure is also shown as a percent of the growth of the unirradiated control ({blacklozenge}).

 

View this table:
[in this window]
[in a new window]
 
Table Ia. Changes in growth and per cent apoptotic-like cells and necrotic cells with time in culture post-exposure to 0, 0.5 or 5 Gy
 

View this table:
[in this window]
[in a new window]
 
Table Ic. Changes in growth and per cent apoptotic-like cells and necrotic cells with time in culture post-exposure to 0, 0.5 or 5 Gy
 




View larger version (0K):
[in this window]
[in a new window]
 
Fig. 2. (a) Percent of cells counted in situ in primary cultures of type 1 human urothelium irradiated as explants which were positive by immunohistochemistry for apoptosis-related proteins. {blacklozenge}, p53-240; {blacksquare}, bcl-2; {blacktriangleup}, BAX; x, c-myc. (b) Percent of cells counted in situ in primary cultures of type 2 human urothelium irradiated as explants which were positive by immunohistochemistry for apoptosis-related proteins. {blacklozenge}, p53-240; {blacksquare}, bcl-2; {blacktriangleup}, BAX; x, c-myc. (c) Percent of cells counted in situ in primary cultures of type 3 human urothelium irradiated as explants which were positive by immunohistochemistry for apoptosis-related proteins. {blacklozenge}, p53-240; {blacksquare}, bcl-2; {blacktriangleup}, BAX; x, c-myc.

 
Effect of smoking and pre-existing tumours
Figure 3Go shows the results for growth of human cultures exposed to 0, 0.5 and 5 Gy {gamma}-radiation and grouped according to whether the tissue came from a smoker, a patient with a distant tumour or was a paediatric sample. The clear result is that all smokers and all tumour-bearing patients had type 1 responses. Non-smokers and children divided about equally between type 1 and type 2/3 responses. The analyses were also performed separately for males and females. Because of low numbers per group there was no statistically significant effect but there was a clear trend in the normal non-smoking group for females to have a type 2 response (67% of the type 2 responders in this group were female).



View larger version (0K):
[in this window]
[in a new window]
 
Fig. 3. Percent of patients with normal urothelium showing type 1, type 2 or type 3 responses to radiation, grouped according to age (adult versus paediatric), smoking (Y/N) and remote site tumour history (+/–).

 
Time course of the response
In the patient cultures where enough material was available we looked at expression of the proteins and cell death over the time period of the culture. These data are presented in Table IaGo–cGo for patients from each subtype. Expression of apoptosis is seen to reach a maximum around day 4 post-irradiation in all cases, while necrosis occurs relatively late. The pattern of protein expression is fixed within 24 h and does not change over the time course of the experiments (data not shown; see ref. 18). Given that the cells are dividing, this means that the signals to maintain high levels of bcl-2 must be continuously produced or transmitted to progeny in some way.

Validation of the data using mouse urothelium
The problems with all human data of this sort are, first, it is usually impossible to carry out replicate studies on the same genotype again, second, human variation is very great so very large numbers of patients are required to see patterns and, third, experimental irradiation of humans cannot be undertaken and therefore the usefulness of the data in terms of classifying human response cannot be validated. In an attempt to overcome this, it was decided to repeat the analysis using mice known to vary in their early and delayed responses to radiation. The data for mouse urothelium are shown in Table IIGo. Clearly, the CBA/H strain, which shows chromosomal instability, at least in bone marrow cells, has reduced apoptosis and high bcl-2 expression. The bcl-2/c-myc ratio, which has been shown to correlate with subtype in humans, is high in cultures from this strain. This would make the CBA/H response type 1. The C57/BL6 mice, which did not show delayed chromosomal instability in marrow following irradiation, had high levels of apoptosis and low bcl-2/c-myc ratios in the urothelial cultures. This makes their response similar to the human type 2/3. The responses of bladder cultures of progeny of crosses of these mouse strains are shown in Table IIIGo. The results are not consistent with a straight dominant or recessive transmission of the characteristic but, when all the data from parents and progeny are ranked in order of increasing apoptosis post-irradiation (Figure 4Go), there is some evidence of male transmission/expression of the apoptosis response.


View this table:
[in this window]
[in a new window]
 
Table II. Characteristics of irradiated cultures of bladder from CBA/H and C57/BL6 mice
 

View this table:
[in this window]
[in a new window]
 
Table III. Characteristics of irradiated cultures of bladder from crosses of CBA/H and C57/BL6 mice
 


View larger version (0K):
[in this window]
[in a new window]
 
Fig. 4. Percentage of cells which showed apoptosis-like morphology in pure bred and F1 cross male and female CBA/H or C57/BL6 mouse urothelium irradiated as explants with 0.5 or 5 Gy.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Retrospective analysis of radiation response data obtained from patients in Dublin attending the Urological Clinic at Beaumont Hospital showed clear differences in response to radiation, measured as the ability of tissue irradiated as explants to give rise to cultures of uroepithelium (1618). These data and the patients involved could not be re-assayed so the current study involves a further series of 160 patients where an attempt was made to study parameters in the cultures and patient histories which might indicate mechanisms involved in expression of the radiation response. Since it is impossible to validate the relevance of end-points directly in humans, two mouse strains were also studied using identical tissue and techniques.

The results confirm the earlier findings that there are three broad types of human response and suggest that a protein profile and pattern of apoptosis and necrosis which is distinctive can be identified for each subtype. In the case of type 1 patients the pattern is of resistance to cell death induced by doses of radiation >0.5 Gy. Cells which proliferate post 5 Gy irradiation from the explant have high bcl-2 levels and many are abnormal. bcl-2 is an anti-apoptotic protein (21) and the high levels may be preventing apoptosis of cells which carry abnormal DNA. In the region 0.1–0.5 Gy apoptosis predominates as a death mechanism and there is very high expression of p53, BAX and c-myc. This could suggest that a threshold exists above which the burden of damage precludes dealing with it through cell death mechanisms. Excessive cell death would lead to tissue collapse and the observation is similar to the phenomenon of accelerated repopulation of clonogens, which can occur in irradiated tumours (22). The relatively low standard errors on the data indicate that the subtypes are quite distinct. This was found previously in a smaller series of patients (18). It is interesting to note that all the smoker samples and all the patients with tumours at distant sites (e.g. kidney, stomach, breast) had the type 1 response, as had the mice whose cultures were chromosomally unstable. This suggests that genetic factors and smoking may predispose to tumour induction in a systemic way in addition to direct initiation

The type 2 response is shown by the chromosomally stable mice (C57/BL6) and roughly half of the non-smoking normal human patients. The association of this `radiosensitive' response with `chromosome stability' would support a role for cell death, both apoptotic and necrotic, in removing damaged cells from the tissue because unlike the type 1 responding cultures, these cells do not appear to induce bcl-2 protein and do not avoid apoptosis.

The data for the CBA/H and C57/BL6 pure strains and F1 crosses are not consistent with a dominant or recessive pattern of inheritance of apoptotic response post-irradiation. Using bone marrow cultures from these strains, the pattern of inheritance of the chromosomally unstable phenotype was clearly recessive (19). The urothelial data, on the other hand, could be interpreted to mean that the phenotypes of resistance or susceptibility to radiation-induced apoptosis are associated with the male gender. The pure CBA/H mice and the progeny with a male CBA/H parent all exhibit supression of apoptosis. The male from the CBA/H femalexC57/BL6 male also shows supressed apoptosis. This latter observation precludes consideration of an X-linked effect and it is probable that genomic imprinting is involved (23), although studies to look for a genomically imprinted site, which are outside the scope of this paper, would be needed to investigate this in mice. In humans and in mice there is some literature to suggest association of leukaemia in progeny with paternal irradiation (24,25). The problem with following this suggestion experimentally or epidemiologically in humans is again one of the need for very large numbers of patients with detailed histories and follow-up for at least two generations.

In conclusion, these data suggest that there are two major subtypes of response to irradiation in human urothelium, a type associated with survival of damaged cells and a type associated with elimination of these cells. The data from mouse urothelium shows that the survival response is found in the strain which exhibits genomic instability.


View this table:
[in this window]
[in a new window]
 
Table Ib. Changes in growth and per cent apoptotic-like cells and necrotic cells with time in culture post-exposure to 0, 0.5 or 5 Gy
 

    Acknowledgments
 
We acknowledge the financial support of the Irish Cancer Research Advancement Board and St Lukes Hospital for access to their radiation facilities.


    Notes
 
3 To whom correspondence should be addressed Email: cmothersill{at}dit.ie Back

4 Present address: University of Dundee, Department of Molecular and Cellular Pathology, Ninewells Hospital and Medical School, Dundee DD1 9SV, Scotland Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

  1. Committee on the Biological Effects of Ionising Radiation (1990) Effects of Exposure to Low Levels of Ionising Radiations. National Academy of Sciences/National Research Council, Washington, DC.
  2. Boice,J.D.Jr, Land,C.E., Shore,R.E., Norman,J.E. and Tokunaga,M. (1979) Risk of breast cancer following low-dose exposure. Radiology, 131, 589–597.[Abstract]
  3. Hall,E. (1994) Radiobiology for the Radiologist, 4th Edn. Lippencott, Philadelphia, PA.
  4. Knudson,A.G. (1971) Mutation and cancer: statistical study of retinoblastoma. Proc. Natl Acad. Sci. USA, 68, 820–823.[Abstract]
  5. Cole,J., Arlett,C.F. and Green,M.H. (1988) Comparative human radiosensitivity. II. The survival following gamma-irradiation of unstimulated (G0) T-lymphocyte lines and fibroblasts from normal donors, from ataxia-telangiectasia patients and from ataxia-telangiectasia heterozygotes. Int. J. Radiat. Biol., 54, 929–943.[ISI][Medline]
  6. Zhan,Q., Bae,I., Fornace,A.J.Jr and Craig,R.W. (1997) Induction of bcl-2 family member, Mcl1 as an early response to DNA damage. Oncogene, 14, 1031–1039.[ISI][Medline]
  7. Sheikh,M.S., Burns,T.F., Huang,Y., Wu,G.S., Amundson,S., Brooks,K.S., Fornace,A.J.Jr and El-Deiry,W.S. (1998) p53 dependent and independent regulation of the death receptor KILLER/DR5 gene expression in response to genotoxic stress and TNF{alpha}. Cancer Res., 58, 1593–1598.[Abstract]
  8. Kadhim,M., Macdonald,D.A., Goodhead,S.A., Lorimore,S.A., Marsden,S.J. and Wright,E.G. (1992) Tramsmission of chromosomal instability after plutonium alpha-particle irradiation. Nature, 355, 738–740.[ISI][Medline]
  9. Kadhim,M.A., Lorimore,S.A., Townsend,K.M.S., Goodhead,D.T., Buckle,V.J. and Wright,E.G. (1995) Radiation-induced genomic instability: delayed cytogenetic aberrations and apoptosis in primary human bone marrow cells. Int. J. Radiat. Biol., 57, 287–293.
  10. Morgan,W.F., Day,J.P., Kaplan,M.I., McGee,E.M. and Limoli,C.L. (1996) Genomic instability induced by ionising radiation. Radiat. Res., 146, 247–258.[ISI][Medline]
  11. Lyng,F., O'Reilly,S., Cottell,D., Seymour,C.B. and Mothersill,C. (1996) Persistent expression of morphological abnormalities in the distant progeny of irradiated cells. Radiat. Environ. Biophys., 35, 273–283.[ISI][Medline]
  12. Mendonca,M.S., Temples,T.M., Farrington,D.L. and Bloch,C. (1998) Evidence for a role of delayed death and genomic instability in radiation-induced neoplastic transformation of human hybrid cells. Int. J. Radiat. Biol., 74, 755–764.[ISI][Medline]
  13. Little,J.B. (1998) Radiation-induced genomic instability. Int. J. Radiat. Biol., 74, 663–671.[ISI][Medline]
  14. Ullrich,R.L. and Ponnaiya,B. (1998) Radiation-induced instability and its relation to radiation carcinogenesis. Int. J. Radiat. Biol., 74, 747–754.[ISI][Medline]
  15. Mothersill,C. and Seymour,C.B. (1998) Delayed death and genomic instability after low dose irradiation: implications for protection [Invited Commentary]. Mutagenesis, 13, 421–426.[Abstract]
  16. Harney,J., Mothersill,C., Seymour,C. and Murphy,D. (1995) Interrelationship between p53, CMYC and BCL-2 oncoprotein expression in cultures of normal human urothelium exposed to cobalt 60 gamma rays and n-nitrosodiethanolamine. Cancer Epidemiol. Biomarkers Prev., 4, 617–625.[Abstract]
  17. Mothersill,C., Harney,J., Lyng,F., Cottell,D. and Seymour,C.B. (1995) Primary explants of normal human uroepithelium show an unusual response to low doses of cobalt 60 gamma rays. Radiat. Res., 142, 181–187.[ISI][Medline]
  18. Mothersill,C., O'Malley,K., Harney,J., Lyng,F., Murphy,D. and Seymour,C. (1997) Further investigations of the response of human uroepithelium to low doses of cobalt 60 gamma radiation. Radiat. Res., 147, 156–165.[ISI][Medline]
  19. Watson,G.E., Lorimore,S.A., Clutton,S.M., Kadhim,M.A. and Wright,E.G. (1997) Genetic factors influencing {alpha}-particle-induced chromosomal instability. Int. J. Radiat. Biol., 71, 497–503.[ISI][Medline]
  20. Mothersill,C., O'Malley,K., Murhpy,D., Lynch,T., Payne,S., Seymour,C. and Harney,J. (1997) P53 protein expression and increased SSCP mobility shifts in the p53 gene in normal urothelium cultured from smokers. Carcinogenesis, 18, 1241–1245.[Abstract]
  21. Kroemer,G. (1997) The proto-oncogene bcl-2 and its role in regulating apoptosis. Nature Med., 3, 614–620.[ISI][Medline]
  22. Withers,H.R., Taylor,J.M.G. and Maciejewski,B. (1988) The hazard of accelerated tumour clonogen repopulation during radiotherapy. Acta Oncol., 27, 131–141.[ISI][Medline]
  23. Schofield,P.N. (1998) Impact of genomic imprinting on genomic instability and radiation-induced mutation. Int. J. Radiat. Biol., 74, 705–710.[ISI][Medline]
  24. Lord,B.I., Woolford,L.B., Wang,L., Stones,V.A., McDonald,D., Lorimore,S.A., Papworth,D., Wright,E.G. and Scott,D. (1998) Tumour induction by methyl-nitroso-urea following preconceptional paternal contamination with plutonium-239. Br. J. Cancer, 78, 301–311.[ISI][Medline]
  25. Lord,B.I., Woolford,L.B., Wang,L., Stones,V.A., McDonald,D., Lorimore,S.A., Wright,E.G. and Scott,D. (1998) Induction of lympho-haemopoietic malignancy: impact of preconception paternal irradiation. Int. J. Radiat. Biol., 74, 721–728.[ISI][Medline]
  26. Gardner,M.J., Snee,M.P., Hall,A.J., Powell,C.A., Downes,S. and Terrell,J.D. (1990) Results of case–control study of leukaemia and lymphoma among young people near Sellafield nuclear plant in West Cumbria. Br. Med. J., 300, 423–429.[ISI][Medline]
Received February 26, 1999; revised August 4, 1999; accepted August 16, 1999.