Abstract
Radiation Leukaemogenesis at Low Doses
DE-FG02-05 ER 63947
Natalie Brown, Rosemary Finnon, John Moody,
Christophe Badie, Simon Bouffler (PI)
Health Protection Agency, Radiation Protection Division,
Chilton, Didcot, Oxfordshire OX11 0RQ, United Kingdom
Introduction
A much better understanding of the mechanisms by which radiation causes leukaemia is needed to allow a complete quantitative estimation of radiation leukaemia risk at low doses and dose rates. Such mechanistic knowledge is most readily obtained through the study of animal model systems as suitable human materials are not generally available. The CBA mouse model of radiation-induced acute myeloid leukaemogenesis has been used extensively for both quantitative and mechanistic studies. One of the central aims of this project is to identify the events that convert a normal cell into myeloid leukaemic cells in the CBA model and explore the dose-response relationships for these events.
Mechanisms of mouse myeloid leukaemogenesis
Following high dose/dose-rate x- or γ-radiation exposure acute myeloid leukaemia (AML) develops after a mean latent period of 18 months in up to ~25% mice (maximal incidence at 3 Gy). Chromosome (chr2) deletions associate with ~95% of AML cases in CBA mice. These large deletions lead to the hemizygous loss of the haemopoietic transcription factor Sfpi1/PU.1 and defines the predominant leukaemogenic pathway. In most AML cases Sfpi1/PU.1 point mutations in exon 5 are present.
Dose-response and time course of Sfpi1/PU.1 loss
Previous conventional cytogenetic analyses suggested that chr2 deletions were early events in leukaemogenesis. A fluorescence in situ hybridization method for quantitation of Sfpi1/PU.1 losses has been developed. This assay confirms that Sfpi1/PU.1 losses in bone marrow occur within 24 hours of in vivo radiation exposure and remain present. Dose-response data down to 0.1 Gy are now available and a time course analysis out to 18 months is being undertaken.
A new system for quantification of Sfpi1/PU.1 events following radiation is being developed. A mouse strain carrying a green fluorescent protein (gfp) gene driven by the Sfpi1/PU.1 promotor has been developed (Nutt et al. 2005). This reporter of Sfpi1/PU.1 expression is being bred on the CBA genetic background. Preliminary results suggest that Sfpi1/PU.1 events can be detected soon after irradiation of such strains.
Identification of other genomic events associated with radiation AML
While Sfpi1/PU.1 loss and mutation defines the predominant leukaemogenic pathway, minor pathways lacking chr2 involvement exist and it is likely that additional genetic events are associated with disease progression in del2 AMLs. A collaborative study has been undertaken with Dr Wei-Wen Cai of Baylor College of Medicine using BAC array based comparative genomic hybridization (CGH) to identify additional regions of DNA sequence gain or loss in chr2 deleted AMLs and the few non-del2 AMLs available from x-irradiation studies. This analysis has identified a common region of amplification on chr6 in del2 AMLs confirming previous cytogenetic studies. Several candidate regions of sequence gain/loss in non del2 AMLs have been identified including losses on chrs12, 16, and 18 and amplification of a chr15 region. The chr18 region is of particular interest as it includes the interval that has been implicated as harbouring an AML susceptibility factor (Darakhshan et al. 2006).
Characterization of AML cell lines
A panel of 6 radiation-induced AML cell lines has been assembled. These are being characterized in terms of Sfpi1/PU.1 status, cell surface markers, and pattern of expression of Sfpi1/PU.1 and downstream target genes. These analyses are being undertaken to help elucidate the nature of the target cell for radiation leukaemogenesis and to characterize the functional consequences of a range of Sfpi1/PU.1 copy number/mutation states. Surface marker and transcriptional analyses will be presented.
Summary
Increasing information on the pathways that lead to radiation-induced AML in the mouse is becoming available. This is allowing the exploration of dose-response relationships for pre-leukaemic events. Ultimately this mechanistic knowledge will allow the development of biologically realistic risk projection models, which may demonstrate the value of such modelling methods.
References
Darakhshan. F, Badie. C, Moody. J, Coster. M, Finnon. R, Finnon. P, Edwards. AA, Szluinska. M, Skidmore. CJ, Yoshida. K, Ullrich. R, Cox. R, and Bouffler. SD (2006). Evidence for complex multigenic inheritance of radiation AML susceptibility in mice revealed using a surrogate phenotypic assay. Carcinogenesis 27: 311-318.
Nutt. SL, Metcalf. D, D’Amico. A, Polli. M, and Wu. L (2005). Dynamic regulation of PU.1 expression in multipotent haemopoietic progrenitors. Journal of Experimental Medicine 201: 221-231
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