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Download Contract No: FIGH-CT-1999-00006
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Contract No: FIGH-CT-1999-00006 Title: Mechanisms and Genetics of Radiation-Induced Tumorigenesis Mouse Models of Leukaemia, Lymphoma and Skin Neoplasia (MAGELLANS) 1. Introduction The development of judgements on post-irradiation cancer risk for the purposes of radiological protection is a complex process which involves evaluation of data on cancer rates in irradiated humans (epidemiology), together with information from animal studies and from investigation of the mechanisms/genetics of cancer induction. The central estimates of radiation cancer risk are derived from epidemiological studies and these allow for the estimation of cancer risk down to doses of around 100-200 mSv (ie a little above the life-time dose from natural background radiation). Since epidemiology does not have the power to directly address cancer risk at the low doses of principal importance in radiological protection, scientifically informed judgements have to be made on the shape of the dose-response for cancer induction. The majority scientific view is that there is a simple proportionate relationship between dose and risk but there are proposals that at low doses there is a dose interval (a dose threshold) where there is no excess risk of cancer. Much of the information that supports a non-threshold type dose-response comes from fundamental understanding of the manner in which the primary cellular target, DNA, is damaged by radiation and the repair processes that act on that damage. Such repair is judged to be subject to some errors and even at low doses there will be induction of rare cancerassociated mutations in critical genes. There is, however, legitimate concern that there is insufficient knowledge on the relationships between post-irradiation cellular damage and cancer development in body (somatic) organs. Therefore, a major objective in radiological protection research is to obtain detailed information on the somatic mechanisms of radiation tumorigenesis to bridge this gap. An additional uncertainty in radiological protection is the extent to which genetic make-up can influence inter-individual differences in radiation cancer risk. There is some knowledge of rare human-genetic disorders that can substantially increase that risk but a larger and more difficult problem is how to judge the extent to which more common variation in human genes might apply. Since radiation-associated human tumours are of very limited availability, research in this whole area is beginning to utilise the increasing power of experimental models of human cancer in mice. The MAGELLANS consortium has used mouse models of acute myeloid leukaemia (AML), lymphoma and skin cancer (with other tumours) to seek further evidence on the mechanisms and genetics of radiation tumorigenesis. This work was conducted under three workpackages (WPs). 1 2. Objectives Workpackage 1: AML and lymphoma The principal objectives of WP1 were: a) To generally compare and contrast the mechanisms of induction/development of these related tumours. b) To gain detailed information on chromosome (chr)2 fragility known to be associated with AML induction and the nature of the critical chr2 gene loss/mutation in these tumours. c) To study germ line factors, possibly involving chr2 fragility, that may determine genetic susceptibility to induced AML. Workpackage 2: Skin (and other) tumours The principal objectives of WP2 were: a) To develop and validate a new mouse model for basal cell skin cancer (BCC) in mice genetically deficient (+/-) in the Ptch cancer susceptibility gene. b) To investigate radiation-associated loss of the wild type Ptch+ allele in BCC and other induced tumours c) To undertake a pilot study to determine whether the expected susceptibility of Ptch+/- mice to radiation tumorigenesis is influenced by other genes d) To seek evidence on the identity and function of variant genes that influence susceptibility to induced skin papilloma/carcinoma and, in some cases, lung tumours. e) To investigate possible genetic associations between susceptibility to induced cancer and inflammatory response. Workpackage 3: DNA damage response/repair genes There is growing appreciation of the close genetic associations between deficiency in DNA damage response/repair, radiosensitivity and cancer susceptibility. Accordingly, the objectives of WP3 were a) To develop a mouse genomic database to relate the genetic map position of DNA damage response/repair genes with regions of the map (loci) that influence tumour susceptibility. b) To seek experimental evidence of variant mouse DNA damage response/repair genes that influence radiation cancer risk. 3. Results Workpackage 1: Much work in WP1 centred on the characterisation of somatic gene/chromosomal losses in AML and lymphoma. These studies provided clear evidence that these tumours, although related, developed via different multistage pathways involving different genes. For AML, studies on post-irradiation chr2 fragility provided evidence of a novel mechanisms of preferential chr2 breakage associated with the remodelling of chromosome structure in a region rich in genes important to the function of bone marrow cells. The critical region of chr2 lost through this fragility mechanism was tracked to the Sfpi1 gene, a key regulator of myeloid cell development; the residual (non-deleted ) Sfpi1 gene copy was shown to be mutated in a high proportion of AMLs. These and other data showed that Sfpi1 loss was most likely to be the critical initiating event for AML induction; evidence for other gene losses associated with AML and lymphoma development was also obtained. Genetic studies provided good evidence that AML susceptibility was linked with the postirradiation expression of chr2 fragility in bone marrow cells. Therefore, this novel form of susceptibility probably involves changes in the control of chromosome remodelling. A candidate locus for this chromosomal trait was mapped but the patterns of genetic expression observed suggest the involvement of more than one gene. 2 Workpackage 2: A major success in WP2 was the development and validation of the new Ptch mouse model. Irradiated Ptch+/- mice showed increased susceptibility to the postirradiation development of BCC and medulloblastoma (a central nervous system tumour); evidence was also obtained of age at irradiation and gender effects on tumour susceptibility. The predicted early loss of the residual Ptch+ allele in BCC and medulloblastoma development was confirmed by molecular analysis and, overall, radiation tumorigenesis was judged to proceed via a conventional multistage mechanism. Studies on the inheritance of skin tumour susceptibility in sensitive (CarS) and resistant (Car R) mouse lines confirmed that multiple interacting genetic loci are involved. Some of these loci were fine mapped and candidate variant genes have been identified; eg Pthlh having a cell migration/adhesion function and Scaa1 specifying a serpin-like protein. The majority of these loci associated with skin tumorigenesis appear to be tissue-specific but one locus was shown to be a genetic determinant of both skin and lung tumorigenesis. Skin tumorigenesis in Ptch+/- mice was found to be strongly influenced by genetic background, again indicating the involvement of multiple genes. Finally, collaborative studies provided evidence of concordance between susceptibility to lung tumorigenesis and resistance to inflammatory response. Workpackage 3: Much of the experimental work of the consortium made use of the rapidly developing databases on the structure of the mouse and human genomes (eg www.ncbi.nlm.nih.gov). Included in these databases is a wealth of information on the genomic map positions of genes associated with DNA damage response/repair. The construction of the MAGELLANS database that relates these genes to regions of the mouse genome encoding tumour susceptibility loci involved utilisation of public databases, review of the scientific literature and some additional experimental work. Following production and circulation of an interim database in January 2002 an updated version of 1.7 MB has been constructed; this will be posted on the NRPB website (www.nrpb.org). In collaborative experimental studies using inbred and cross-bred mice, evidence was obtained that a variant gene (PrkdcBALB encoding DNA PKcs) was not only associated with increased susceptibility to radiogenic breast cancer but also to development of tumours of the small intestine. Finally, work in WP2 also revealed structural variation in Mre11 gene amongst 13 inbred mouse strains; this was a late finding and requires further follow-up. 4. Implications The data obtained by the consortium on somatic mechanisms and genetic factors in radiation tumorigenesis have a number of implications for the further development of biological models of radiation cancer risk. Somatic mechanisms: The work of the partners has more firmly established the value of mouse models in mechanistic studies. A consistent finding has been that acute doses of ionising radiation appear to act primarily at an early phase of development of AML, BCC and medulloblastoma. For lymphoma, the gene loss events recorded could not be related to radiation but it was clear that lymphoma development followed a multistep mutational process that differed from AML. 3 The radiation-associated deletion of tumour suppressor-type genes (Sfpi1 for AML and Ptch+ for BCC and medulloblastoma) in precursor cells appeared to be followed by an unremarkable process of multistage neoplastic development. The overall conclusion was that the data were wholly consistent with conventional models of radiation tumorigenesis (see UNSCEAR 2000) and there were no indications that radiation tumorigenesis proceeded via unusual mechanisms. A major objective was to seek linkage of mouse data with in vitro findings on radiation action in cultured cells. In this respect the consistent finding of radiation-associated loss of tumourrelated genes is important since it follows predictions from in vitro knowledge of DNA damage response mutagenesis (see UNSCEAR 2000). In brief, the error-prone postirradiation repair of sometimes complex DNA double strand (ds) lesions is believed to underlie the predominant DNA deletion mechanism of mutation after irradiation. Such errorprone repair and mutagenic processes now appear to apply to in vivo radiation tumorigenesis and, on this basis, the data argue against the proposition of a dose threshold for cancer risk. The data of WP3 on enhanced radiation tumorigenesis in mice partially deficient (PrkdcBALB) in the repair of DNA ds lesions adds further weight to this conclusion. Overall the mechanistic data obtained tend to weight in favour of a simple low dose relationship between dose to target cells and cancer risk. Genetic factors: The mouse genetic models used in the MAGELLANS project considered the impact on radiogenic cancer of a rare strongly expressing mutant gene (Ptch+/-) and of potentially more common variant genes having a lesser degree of expression. Work with the Ptch+/- mouse strain adds further weight to the view that such rare mutations can substantially increase radiation cancer risk albeit with the potential for modification by other genes. The maximum enhancement of cancer risk at young ages of irradiation is an important issue and requires further follow-up. Studies on the variant gene(s) for susceptibility to AML and skin cancer succeeded in identifying candidate loci and in some cases candidate genes and their mechanisms of action. The most important overall conclusions are: 1) That such variant genes tend to interact in a complex manner. 2) That these genes are usually but not always tissue-specific in their activity. 3) That cross-sensitivity carcinogenic agents is frequent. This proof-of-principle evidence is important but not yet sufficient to act as clear guidance for the modelling of genetic components of radiation cancer risk. Nevertheless, some initial comment is possible. For example, variant germ line DNA damage response genes (eg Prkdc) predominantly affecting radiation risk in a number of tissues might be expected to have a greater impact on the population than genes affecting tumour development from all causes in a single tissue. The potential for such differential effects will need to be considered in the further development of modelling procedures. Co-ordinator: Dr R Cox (National Radiological Protection Board) Partners: Dr R Huiskamp (Nuclear Research and Consultancy Group, NL) Dr A Saran (Ente per Nuove Tecnologie, L’Energia a L’Ambiente, I) Dr T Dragani (Instituto Nazionale per lo Studio et al Cura dei Tumori, I) 4