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Transcript
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