British Journal of Radiology (2007) 80, S2-S6
© 2007 British Institute of Radiology
doi: 10.1259/bjr/60507340
The genetic basis of tissue responses to ionizing radiation
K J Lindsay, BSc
P J Coates, PhD
S A Lorimore, BSc
and
E G Wright, PhD, FRCPath, FRSE
Division of Pathology and Neuroscience, University of Dundee, Ninewells Hospital and Medical School, Dundee, UK
 |
Abstract
|
|---|
The response of mammalian cells to ionizing radiation can be directly influenced by genetics, and mouse strains can be identified that differ in their cellular radiosensitivity. The C57BL/6 radiation resistant and DBA/2 radiation susceptible mouse strains were utilized to aid the elucidation of the mechanisms involved in the early response to ionizing radiation. Investigation of the p53 pathway revealed differences in the expression and activity of p53 and its downstream targets between these mouse strains. The radiation resistant C57BL/6 strain showed an early p53 response and preferentially upregulated pro-apoptotic Bax, whereas the radiation sensitive DBA/2 strain exhibited a later, more prolonged p53 response and a greater expression of the cyclin dependent kinase inhibitor p21. These two mouse strains also showed significantly different levels of splenic radiation-induced apoptosis, the radiation resistant C57BL/6 scoring twofold more apoptotic cells than its radiation sensitive counterpart. These data provided a quantitative endpoint for an apoptosis genetic linkage analysis. The preliminary results of the linkage analysis indicated that three distinct loci may be involved in driving the different apoptosis phenotypes exhibited by the mouse strains. Moreover, we ascertained whether the mechanisms involved in the response to ionizing radiation may work in a tissue-specific fashion. In the linkage analysis, comparison of apoptosis scores in the colon and small intestine with data from the spleen showed little correlation suggesting that levels of apoptosis are tissue-specific. Tissue-specificity in the colon and small intestine was further illustrated by work with a 2D gel electrophoresis system. This revealed different patterns of p53 phosphorylation between the intestinal tissues both before and after exposure to ionizing radiation. The data discussed here will aid our understanding of the genes and mechanisms involved in radiation responses.
|
Introduction
|
|---|
It has been widely accepted that an individual's genetic background influences the response to genotoxic insults including ionizing radiation. Genetic differences have also been described in inbred mouse strains and provide a valuable tool in understanding the pathology and mechanisms involved in radiation-associated diseases.
In 1963, Roderick [1] published a seminal work in the field of radiation genetics. He described differences in the survival rates of 27 inbred mouse strains following daily low dose X-ray irradiation. Some of these strains exhibited resistance to radiation-induced death whereas others were highly susceptible. These differences can be attributed to inherited genes of low penetrance, mirroring the general human population.
Previously, our group has identified such differences between the C57BL/6 and DBA/2 mouse strains [2]. The C57BL/6 strain also exhibits resistance to the phenomenon of radiation-induced genomic instability which encompasses: gene mutations, chromosome aberrations, malignant transformation and cell death. Conversely, the DBA/2 strain is sensitive to radiation-induced instability [3, 4].
We have continued to use these two inbred mouse strains to help to understand the mechanisms involved in the response to ionizing radiation in the minutes to hours following the insult. Moreover, we wish to identify the extent to which these mechanisms are influenced by the genetic background. In the first instance, we will concentrate particularly on the p53 pathway and investigate its key downstream targets and how they can manipulate the fate of the cell.
The major pathway upregulated in response to ionizing radiation is the p53 pathway. p53 is a tumour suppressor the importance of which can be illustrated by the fact that it is mutated in more than 50% of human cancer, and most probably plays a role in almost all cancers due to other interfering mutations. Following an ionizing radiation insult, protein kinases including ATM and DNA-PK phosphorylate p53, preventing the interaction with its major regulator MDM2. This activated form of p53 is free to upregulate downstream targets such as the cyclin dependent kinase inhibitor p21 and the pro-apoptotic molecules PUMA and Noxa. These two proteins in turn upregulate the Bcl family member Bax. Preferential upregulation of p21 leads the cell to undergo growth arrest, whereby damaged DNA could potentially become integrated into the genome. Conversely, increased Bax expression promotes programmed cell death or apoptosis and restoration of tissue homeostasis.
In addition, as previous in vivo observations in various mouse tissues have shown differences in the response to radiation between tissues [5], we will look at whether these pathways or mechanisms represent global or tissue-specific responses following a radiation insult. This will be achieved by analysis of p53 activity and apoptosis in the splenic and intestinal tissues.
The primary focus of these studies was to understand which genes and pathways drive the response to ionizing radiation and how contrasting genetic backgrounds can modulate this.
The p53 pathway
C57BL/6 and DBA/2 mice were irradiated with 1 Gy 
radiation. Tissues were removed at various timepoints post irradiation for immunohistochemistry and western blotting analysis [2, 6]. These analyses revealed that the response kinetics and expression of p53 are very different between the C57BL/6 and DBA/2 strains [2, 6]. The C57BL/6 strain upregulates p53 just 30 min after 1 Gy irradiation, earlier than DBA/2 which shows minimal p53 expression until 1 h post-irradiation. The C57BL/6 p53 response begins to subside by 4 h, yet is more prolonged in DBA/2 where significant levels of p53 protein are still present at 6 h. Furthermore, immunohistochemistry staining revealed that the pattern of p53 expression showed little similarity between the strains [6]. In C57BL/6, p53 is preferentially upregulated in the white pulp of the spleen, however, in DBA/2 the protein shows most prominence in the red pulp.
The downstream activities of p53 were also subject to genetic modification [2, 6]. The DBA/2 radiation-sensitive strain shows the stronger and more prolonged p21 response (by both immunohistochemistry and western blotting [2, 6]) leading to cell cycle arrest. In contrast, the C57BL/6 radiation-resistant strain preferentially targets Bax for upregulation, promoting apoptosis [6].
Figure 1
shows examples of immunohistochemistry and western blotting for tissues obtained from C57BL/6 and DBA/2 mice. The immunohistochemistry pictures show p53, p21 and Bax expression in C57BL/6 mice at 3 h following 1 Gy radiation. The western blots show the p53 response in C57BL/6 and DBA/2 mice and illustrate the different patterns of upregulation that these strains exhibit.
Our work on the p53 pathway has revealed separate patterns of p53 expression and kinetics of activity in the 2 mouse strains [6]. The radiation-resistant C57BL/6 strain exhibited an early, rapid response in contrast to the later, more prolonged response of the radiation-sensitive DBA/2 strain. Two of the major downstream targets of p53 were also subject to this genetic-dependent bias. The radiation-susceptible DBA/2 strain expressed higher levels of p21, which can lead to cell cycle arrest. Therefore, its radiation sensitivity could in part be explained by the hypothesis that damaged cells enter cell cycle arrest, as opposed to apoptosis more frequently in DBA/2 mice, leading to an increased risk of damage becoming irreversibly integrated into the genome. By contrast, C57BL/6 spleens preferentially upregulate pro-apoptotic Bax post-irradiation. The apoptosis of damaged cells eliminates the possibility of aberrations becoming incorporated into the genome and restores tissue homeostasis. This is consistent with other in vivo mouse experiments which have shown that increased levels of p21 protect against apoptosis [7].
|
The genetic modification of apoptosis
|
|---|
To further the discoveries made regarding the differences in upregulation of p53 targets, the apoptosis rates of these strains were measured in spleen to discern any strain-specific observations [2]. C57BL/6, DBA/2 and DBA/2-related CBA/Ca mice were subjected to a sub-lethal dose of 0.5 Gy irradiation and apoptosis was scored by morphology in 10 fields of splenic white pulp (Figure 2). These three strains exhibited vast differences in their rates of apoptosis. At both 4 h and 6 h post-irradiation, the C57BL/6 radiation-resistant strain showed a twofold higher incidence of apoptosis than the DBA/2 radiation sensitive strain. The apoptosis exhibited by the CBA/Ca strain was certainly lower than that observed in the C57BL/6 mice, but the difference between these strains was not as great as between the C57BL/6 and DBA/2 strains. This strain is also radiation-sensitive and these mice are susceptible to radiation-induced genomic instability and acute myleloid leukaemia induction, in contrast to the C57BL/6 strain [3]. Interestingly, the F1 progeny of the C57BL/6 and DBA/2 strains (denoted as the BDF1 strain) exhibited an apoptotic response intermediate to that of the parental strains. This suggests that the apoptosis phenotype in these strains is not simply dominant or recessive, but most probably involves a number of genes co-operating in a complex network.
View larger version (19K):
[in this window]
[in a new window]
|
Figure 2. Mean number of apoptotic cells counted in 10 fields of splenic white pulp. This graph shows the splenic apoptosis counts of the C57BL/6, DBA/2 and CBA/Ca mouse strains at the indicated timepoints following 0.5 Gy radiation.
|
|
These preliminary results provided a quantifiable endpoint, of scores of apoptotic cells in C57BL/6 and DBA/2 mice, for a genetic linkage analysis. Such an analysis should allow identification of the genetic markers responsible for the different apoptotic phenotypes observed between the strains.
|
Apoptosis genetic linkage analysis
|
|---|
A genetic linkage analysis based on scores of apoptotic cells is currently underway. Using an intercross approach, a population of F2 animals (derived from C57BL/6 and DBA/2 parental strains) underwent splenic apoptosis scoring. According to Mendelian genetics, 1 in 4 of these F2 mice should have a C57BL/6 genotype and a C57BL/6-like phenotype for one locus, and 1 in 4 should exhibit DBA/2-like genetics and apoptosis rates for a single locus. Test cohorts with both the highest (C57BL/6-like) and lowest (DBA/2-like) scores were selected. DNA from this selection underwent PCR analysis with microsatellite markers covering the entire mouse genome. The preliminary results revealed three loci with significant differences between the high apoptosis (C57BL/6-like) and low apoptosis (DBA/2-like) groups of the test cohort. Further investigation of these regions should allow elucidation of particular genes responsible for the different apoptosis phenotypes exhibited by the C57BL/6 and DBA/2 strains.
|
Tissue-specific radiation responses
|
|---|
Previous observations have revealed differences in the rates and patterns of radiation-induced apoptosis in different tissues in our mouse models [6]. Thus, the secondary aim of this work was to investigate possible tissue-specificity with regard to radiation-induced apoptosis and p53 responses.
We have demonstrated tissue-specific apoptosis in the C57BL/6 and DBA/2 strains by comparison of the spleen and intestinal tissues [6]. Apoptosis was measured by TUNEL staining in 20 crypts of colon and small intestine tissue in C57BL/6 and DBA/2 mice. As stated previously, at 4 h and 6 h post 1 Gy irradiation, C57BL/6 splenic cells show a twofold higher rate of apoptosis compared with DBA/2. However, in the small intestine and colon, this pattern is very different. In both tissues, C57BL/6 still shows the stronger apoptosis response, yet in small intestine the C57BL/6 cells showed only 20% more apoptosis than DBA/2 after 4 h. In colon this figure was 200% after 4 h but dropped to just 40% at 6 h post-irradiation [6]. Therefore it is clear that radiation-induced apoptosis is a tissue specific process, yet C57BL/6 shows a higher response throughout the whole body.
The investigation of tissue-specific apoptosis was extended by scoring intestinal apoptosis in the test cohort of mice used in the spleen genetic linkage analysis (Figure 3). Both the patterns of the graphs and the correlation coefficients indicate that the apoptosis response in these mice is different in the spleen and intestine, yet show a degree of similarity between the colon and the small intestine. Differences in the rates and patterns of radiation-induced apoptosis have been reported for some time [8]. Our apoptosis scores in the spleen, small intestine and colon [6], revealed clearly different cell death patterns between these tissues indicating that our mouse models exhibit tissue-specific radiation-induced apoptosis. However, this suggests that, as the data collected for the genetic linkage analysis were based on apoptosis scores in the spleen, the genes and potential mechanisms that the linkage may reveal could be tissue-specific for the spleen and not relate to radiation-induced apoptosis in other tissues or in the individual as a whole.
View larger version (26K):
[in this window]
[in a new window]
|
Figure 3. Mean number of apoptotic cells counted in 10 fields of splenic white pulp, and 20 crypts of the small intestine and the colon on a logarithmic scale.(a) C57BL/6-like high apoptosis group of the test cohort, (b) DBA/2-like low apoptosis group of the test cohort. Table (c) illustrates the correlation coefficients (r) between each of the tissues.
|
|
Tissue-specific radiation responses can be further illustrated by analysis of the phosphorylation patterns of radiation-inducible genes such as p53. Our preliminary work on the closely related intestinal tissues using a 2D gel electrophoresis system has revealed very clear differences in the post-irradiation phosphorylation status of p53 (Figure 4). Small intestine and colon tissues were removed from C57BL/6 mice before and 1 h post 4 Gy radiation. These tissues were lysed and separated electrophoretically in two dimensions. As 2D gel electrophoresis separates proteins by charge as well as size, each of the positively charged phosphorylation sites on the p53 protein can be visualized. The most obvious difference between the tissues is the fact that two bands of p53 are present in the small intestine, whereas only a single band can be seen in the colon. Further work with antibodies specific for the different phosphorylated p53 sites in various tissues in our mouse strains, should allow elucidation of the key differences in the phosphorylation pattern of this protein which result from tissue-specificity and genetic modification. The tissue-specific p53 responses illustrated so vividly by the 2D gel electrophoresis system in colon and small intestine, indicate another level of complexity with regard to the possible elucidation of global radiation responses. These preliminary results reveal stark differences in p53 activity that can occur even between tissues closely related in location and function. Moreover, this indicates that many of the early radiation responses we clarify may show tissue-specific differences rather than represent global events. The most striking observation from the 2D phosphorylation data, is the separate bands of p53 exhibited in the small intestine but not the colon. Recent experimental work [9] has identified a number of human p53 isoforms that can arise from different promoters, contain alternative translation start sites and be differentially spliced. Thus, if mouse p53 is similarly encoded and can be expressed in a number of different isoforms, this could explain the discrepancies between the tissues.
In conclusion, we have provided evidence to confirm that an individual's genetic background influences the response to radiation. Our model mouse strains C57BL/6 and DBA/2 showed strain-specific differences in p53 expression and activity and in the upregulation of downstream targets p21 and Bax. Moreover, these strains exhibited vast differences in levels of apoptosis which prompted an apoptosis-based genetic linkage analysis. The analysis revealed a role for three distinct loci in the mouse genome potentially responsible for the genetic modification of apoptosis. In addition, we also observed a second level of apoptosis modification, namely, in the tissue-specificity of the process.
Work funded by the Department of Health, Leukaemia Research Fund and the Medical Research Council.
|
Acknowledgments
|
|---|
CM5 polyclonal sera was a kind gift from D P Lane. This work was funded by the Department of Health, Leukaemia Research Fund and Medical Research Council.
Received for publication May 12, 2006.
Accepted for publication August 21, 2006.
|
References
|
|---|
- Roderick TH. The response of twenty-seven inbred strains of mice to daily doses of whole body x-irradiation. Radiat Res 1963;20:631–39.[Medline]
- Wallace M, Coates PJ, Wright EG, Ball KL. Differential post-translational modification of the tumour suppressor proteins Rb and p53 modulate the rates of radiation-induced apoptosis in vivo. Oncogene 2001;20:3597–608.[CrossRef][Medline]
- Watson GE, Lorimore SA, Clutton SM, Kadhim MA, Wright EG. Genetic factors influencing alpha-particle-induced chromosomal instability. Int J Radiat Biol 1997;71:497–503.[CrossRef][Medline]
- Wright EG. Radiation-induced genomic instability in haemopoietic cells. Int J Radiat Biol 1998;74:681–7.[CrossRef][Medline]
- MacCallum DE, Hupp TR, Midgley CA, Stuart D, Campbell SJ, Harper A, et al. The p53 response to ionising radiation in adult and developing murine tissues. Oncogene 1996;13:2575–87.[Medline]
- Coates PJ, Lorimore SA, Lindsay KJ, Wright EG. Tissue-specific p53 responses to ionizing radiation and their genetic modification: the key to tissue-specific tumour susceptibility? J Pathol 2003;201:377–88.[CrossRef][Medline]
- Komarova EA, Christov K, Faerman AI, Gudkov AV. Different impact of p53 and p21 on the radiation response of mouse tissues. Oncogene 2000;19:3791–8.[CrossRef][Medline]
- Nomura T, Kinuta M, Hongyo T, Nakajima H, Hatanaka T. Programmed cell death in whole body and organ systems by low dose radiation. J Radiat Res (Tokyo) 1992;33 Suppl:109–23.[Medline]
- Bourdon J-C, Fernandes K, Murray-Zmijewski F, Liu G, Diot A, Xirodimas DP, et al. p53 isoforms can regulate p53 transcriptional activity. Genes Dev 2005;19:2122–37.[Abstract/Free Full Text]
Top
Abstract
Introduction
