Sally A. Amundson, Christine Koch-Paiz, and Albert J. Fornace Jr.,
NCI, Bethesda, MD. Supported by DOE grant ER62683.
During the first several years of our project to study cellular responses to low-dose radiation exposure, we have established that changes in gene expression can be triggered by doses of low LET radiation of 10 cGy and less in human and murine cells. As shown in Fig. 1 where we used a quantitative single-probe hybridization method (6) to accurately measure increases in mRNA levels relative to untreated cells, induction showed a linear non-threshold response for multiple stress genes in the human p53-wt myeloid ML-1 line (2). Similar linear non-threshold responses have also been observed in human peripheral blood lymphocytes (PBL) irradiated ex vivo (3) as well as in mice irradiated in vivo. Significant responses were observed to 2 cGy (2), and more recently we have observed significant induction after 1cGy of either high or low dose-rate radiation in ML-1 cells. While no appreciable apoptosis or cytotoxicity after doses of 10 cGy or less was observed (2), a cellular response could be clearly discerned as illustrated by transient cell-cycle delay (Fig. 2).
We have used cDNA microarray hybridization analysis to identify radiation-regulated genes that could potentially serve as informative biomarkers of radiation exposure (1, 3-5). Our initial studies have identified several genes significantly up-regulated in human PBL between 24 and 72 hours after ex vivo irradiation. Three of these genes, DDB2, CIP1/WAF1 and XPC, were induced in a linear fashion between 0.2 and 2 Gy at 24 and 48 hours after treatment, with less linearity at earlier or later times (3). Interestingly, these and other strongly radiation responsive genes are p53-regulated, which indicates a major role for p53 in mediating radiation gene responses in PBL (and probably other primary cells in vitro and in vivo). We are currently using murine microarrays to examine the in vivo response to whole body irradiation of mice. Doses from 0.2 to 2 Gy result in the induction of a large number of genes in liver, spleen and thymus 24 hours after exposure, with the results in the two hematopoietic tissues, spleen and thymus, being the most similar to each other, and also yielding the largest magnitude responses. These results support the use of peripheral blood cells as an accessible and sensitive indicator of radiation exposure, and begin laying the foundation for expression profiles that may someday provide signatures for past radiation exposure.
We have also found a pattern of changes in gene expression for unirradiated bystander cells that is distinct from that induced in the directly irradiated cells. Concurrent modification of cell survival and mutation are also being monitored in the same cell lines. We are investigating the gene expression aspect of the bystander response using transfer of culture medium to target “bystander” cells following exposure of “inducer” cultures to low LET gamma-rays. Comparison of the genes regulated by direct irradiation with those responding to the bystander effect may identify candidates for genes transmitting and regulating the bystander signal, while an understanding of bystander response at the molecular level has important implications for risk assessment of low radiation doses. Since changes in gene expression are important in many of the cellular responses to ionizing radiation, such as G1 checkpoint activation and apoptosis, these types of studies should provide valuable mechanistic insight into genes involved in cellular processes including the maintenance of genomic stability, bystander effects, adaptive responses, genetic factors that may affect intra-individual susceptibility, and damage processing. They should also contribute to our understanding of signal transduction mechanisms involved in the cellular responses to low doses of ionizing radiation.
References
1. Amundson, S. A., M. Bittner, Y. D. Chen, J. Trent, P. Meltzer, and A. J. Fornace, Jr. 1999. cDNA microarray hybridization reveals complexity and heterogeneity of cellular genotoxic stress responses. Oncogene 18:3666-3672.
2. Amundson, S. A., K. T. Do, and A. J. Fornace, Jr. 1999. Induction of Stress Genes by Low Doses of Gamma Rays. Radiat Res 152:225-231.
3. Amundson, S. A., S. Shahab, M. Bittner, P. Meltzer, J. Trent, and A. J. Fornace, Jr. 2000. Identification of potential mRNA markers in peripheral blood lymphocytes for human exposure to ionizing radiation. Radiation Res. 154:342-346.
4. Bittner, M., Y. Chen, S. A. Amundson, J. Khan, A. J. Fornace, Jr, E. R. Dougherty, P. S. Meltzer, and J. M. Trent. 2000. Obtaining and evaluating gene expression profiles with cDNA microarrays. p. 5-25. In Genomics and Proteomics, S. Suhai (ed.), Kluwer Academic / Plenum Publishers, New York.
5. Fornace, Jr, A. J., S. A. Amundson, M. Bittner, T. G. Myers, P. Meltzer, J. N. Weinstein, and J. Trent. 1999. The complexity of radiation stress responses: analysis by informatics and functional genomics approaches. Gene Expression 7:387-400.
6. Koch-Paiz, C. A., R. Momenan, S. A. Amundson, E. Lamoreaux, and A. J. Fornace, Jr. 2000. Estimation of relative mRNA content by filter hybridization to a polyuridylic probe. Biotechniques 29:708-714.