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Cytogenetic and molecular mechanisms of radiation myeloid leukaemogenesis inthe mouse

Objective


The original hypothesis that recombinogenic TLR sequences in murine haemopoietic cells act to direct AML-associated chromosome (ch)2 breakage/rearrangement after radiation has been supported by DNA cloning and subsequent fluorescence in situ hybridization (FISH) cytogenetic studies. A molecular model of specific TLR sequence recombination has been developed in order to explain the genesis of these ch2 events. Ch2 allele loss and the associated deletion breakpoints have been defined by a study of loss of heterozygosity (LOH) for ch2 microsatellite loci in the AMLs of F1 hybrid mice; ch2 allele loss in these F1 AMLs showed no parental specificity. Also, in CBA/H AMLs, somatic telomere rearrangement and limited erosion has been shown to accompany leukaemogenic development. Good progress was made in the identification of murine DNA clones encoding the microsatellites that show close linkage to AML-associated ch2 breakpoints.

Dose-rate effects on CBA/H AML induction by x-rays and 1 MeV fission neutrons have been determined at single doses; these data were broadly consistent with prevailing views in radiological protection on dose-rate and radiation quality effects on radiation leukaemogenesis. Subsequent cytogenetic studies revealed that ch2-mediated leukaemogenesis processes do not differ significantly after high and low LET radiations; also, that there is no obvious contribution to myeloid leukaemogenesis from high LET-induced genomic instability.

Studied on the status of early ch2 events in the haemopoietic systems of x-irradiated mice provided further support for the involvement of telomeric rearrangement and ch2 radiosensitivity in radiation leukaemogenesis but implied that, in the absence of further genomic changes, ch2 deletion/rearrangement does not have major effects on haemopoietic cell proliferation in vivo.

Molecular analysis of germ line TLR sequences provided strong evidence for their germ line hypervariability in the mouse. The initial observation of a possible association between locus-specific TLR polymorphism and AML-susceptibility in one CBA/H colony was however shown by subsequent colony re-screening and by investigation of a satellite colony to be a chance finding determined by the high germ line mutation rate of these sequences.

Finally, it was shown that the tumorigenic radiosensitivity of lymphoma-prone mice deficient in the p53 tumour suppressor gene was likely to be associated with a defect in checkpoint control in a late rather than an early phase of the cell cycle. This defect resulted in the high frequency in vivo induction of whole chromosome gain and loss in the haemopoietic system but appeared not to influence the overall induction of structural chromosome changes.
For the purpose of improving the estimation of cancer risk in low-dose and low dose-rate irradiated human populations it is of major importance to extend knowledge of the cellular, molecular and genetic processes that underlie cancer induction by radiation. Studies with animal models of radiation oncogenesis represent an already productive approach to this problem; in particular, investigations on radiation induced acute myeloid leukemia (AML) in the mouse have yielded evidence on the specific nature of the AML-inducing event. These studies suggest that cytogenetically visible deletion of chromosome (ch)2 is an initiating event for AML, that telomere-like repeat (TRL) DNA sequence at "fragile" chromosomal sites may catalyse ch2 deletion and gene loss following radiation and that leukaemogenic radiosensitivity may be determined by germ line mutations present within certain mouse strains.
Here, it is proposed to extend knowledge on the mechanisms of murine radiation leukaemogenesis. These studies will focus on (a) the isolation and characterisation of TRL sequences at chromosomal fragile sites;(b) the relationships between specific TLR sequence arrays and induced ch2 breakage in AMLs; (c) the characterisation and genetic mapping of the putative AML-predisposing mutation and its distribution amongst different mouse strains; (d) the induction of AMLs in F1 and F2 hybrids of genetically divergent mice in order to facilitate molecular studies of ch2 gene loss events; (e) the characterisation of AMLs induced by low-dose rate exposure to low and high LET radiations; (f) the isolation of mouse embryonic stem cell lines for in vitro and in vivo investigation of myeloid differentiation and leukaemogenic change; (g) the establishment of the homologous recombinant technology to define the in vivo leukaemogenic activity of the ch2 deletions/rearrangements. and (h) the in vitro conversion of pre-leukaemic to leukaemic with growth factors.

Funding Scheme

CSC - Cost-sharing contracts

Coordinator

Vlaamse Instelling voor Technologisch Onderzoek
Address
200,Boeretang
2400 Mol
Belgium

Participants (2)

ENERGY RESEARCH CENTRE OF THE NETHERLANDS
Netherlands
Address
Westerduinweg 3
Petten
NATIONAL RADIOLOGICAL PROTECTION BOARD
United Kingdom
Address

OX11 0RQ Didcot,harwell,chilton