Mechanisms Underlying Cellular Responses to Low Dose/Low LET Ionizing Radiation in Primary Haematopoietic Cells.
Stefania Militi1; Deborah Bowler1 Denise Macdonald1; Alex Almasan2; Kevin Prise3 Munira Kadhim1 ( PI)
Radiation and Genome Stability Unit, Medical Research Council, Harwell, UK1;
Depts. Cancer Biology & Radiation Oncology, Cleveland Clinic Foundation, Cleveland, USA2; Centre for Cancer Research and Cell Biology, Queen’s University Belfast, Belfast, UK3
Genomic instability (GI) is characterised by a persistent elevated rate of genetic alterations and is observed in a large fraction of the clones surviving ionizing radiation exposure.
Evidence exists for a genetic basis of susceptibility to radiation-induced genomic instability in both human and mouse cells. Differences in radiosensitivity between several mouse strains have been uncovered by their responses to low doses of high-LET and high doses of low-LET radiation. These differences may be due to several molecular mechanisms including radiation-induced DNA damage recognition and the initiation of signal transduction cascade(s), which can lead to cell-cycle arrest, DNA damage repair, or apoptosis. The earliest signalling molecules, which are known to initiate the signal transduction cascade at damage sites, are the protein kinases ataxia telangiectasia mutated (ATM) and the ATM- and Rad-3-related (ATR). In order to understand the role of genetic predisposition to genomic instability following low-dose, low-LET radiation exposure, two mouse strains that differ in their sensitivity to ionizing radiation, CBA/CaH and C57BL/6J, were selected for further investigation.
METHODS
Lysed bone marrow haematopoietic cells from these strains were exposed to X-ray doses from 0.01 to 3 Gy and several endpoints associated with response to irradiation were measured including changes in the expression levels of ATR, activation of ATM and its downstream target gene Chk2, and expression of apoptotic regulators Bcl-2, Bcl-xL and Bax, immediately, 2 and 24 hours post-irradiation. Chromosomal instability was assessed in a clonal assay of stem cells, at15 population doublings post-irradiation.
RESULTS AND DISCUSSION
Through cytogenetic analysis the current study demonstrated that chromosomal instability was induced in CBA/CaH and C57BL/6J strains of mice following very low doses of low LET irradiation (<0.1 Gy). Although instability was significantly induced in cultures obtained from CBA/CaH and C57BL/6J, we observed qualitative differences relating to more heavily damaged cells after exposure to 0.05Gy and 0.1Gy than 0.01Gy in both strains.
Western blots demonstrated that ATR levels and ATM phosphorylation (Ser-1981) were constitutively high at slightly different levels in unirradiated CBA/CaH and C57BL/6J cells. Following exposure to several doses of X-rays (0.01, 0.05, 0.1, 1 and 3 Gy) results showed differences predominantly in ATM phosphorylation in both strains of mice.
Activation of ATM through phosphorylation was higher in CBA/CaH than in C57BL/6J (2 hours post-irradiation) suggesting that ionizing radiation causes decreased ATM activation in C57BL/6J. Furthermore, as the phosphorylation of Chk2 is largely dependent on ATM, we also examined this event as a function of the DNA damage in order to support the different ATM activity observed in both strains of mice. Chk2 (Thr-68) phosphorylation was maximal after 3Gy at 2 hour post-irradiation in CBA/CaH. To the contrary, the level of Chk2 activity was diminished in C57BL/6J cells at the same time point post irradiation up to 1Gy compared to unirradiated control. Chk2 activity data are concordant with the ATM phosphorylation trend observed in both strains of mice and demonstrated that ATM is required for the phosphorylation of Chk2 kinase. Moreover, these findings are consistent with the cytogenetic analysis which showed a greater percentage of heavily damaged cells in C57BL/6J compared to CBA/CaH. Collectively, the results suggest C57BL/6J mice are less effective in repairing the damage caused by ionizing radiation.
Having previously observed an inverse relationship between apoptosis and chromosome damage induction following high dose radiation exposure in these strain of mice, we investigated the expression of apoptosis at molecular and cellular level following low dose radiation .Preliminary results showed that the expression of Bax was only reduced in C57BL/6J at 24 hours post irradiation. Moreover, the percentage of apoptotic cells at the same time point was higher in CBA/CaH than in C57BL/6J. These results indicate that Bax might be involved in modulating the resistance/susceptibility phenotype of mice to ionizing radiation.
Collectively, these finding suggest a model wherein normal recognition and repair of DNA damage leads to cell death and a consequent reduction in aberrant cells especially with more than one aberration (eg. CBA/H). Conversely, normal recognition of damage, but abnormal repair might lead to increased survival, decreased apoptosis, and increased number of aberrant metaphases with more than one aberration (eg. C57BL/6J).
The current study shows a genetic difference to the susceptibility to irradiation and offers a continuing approach to identifying the responsible gene(s).
This research was supported by the Office of science (BER), U.S. Department of Energy, Grant No. DE-FG02-05ER64079
» Return to top
