Roger Cox

Molecular mapping of chromosome 2 deletions in murine radiation-induced AML localizes a putative tumor suppressor gene to a 1.0 cM region homologous to human chromosome segment 11p11-12.

Radiation‐induced acute myeloid leukemias (AMLs) in the mouse are characterized by chromosome 2 deletions. Previous studies showed that a minimal deleted region (mdr) of ∼6.5 cM is lost from one homologue in chromosome 2–deleted AMLs. An AML tumor suppressor gene is proposed to map within this mdr. In this study, we refine the mdr to a 1 cM interval between markers D2Mit126 and D2Mit185 by microsatellite analysis of 21 primary radiation‐induced F1 AMLs. The construction of a partial yeast artificial chromosome (YAC) contig spanning the mdr and the location of six known genes indicated that the 1 cM mdr is homologous to human 11p11–12, a region implicated in some human AMLs. Screening of five cell lines derived from primary radiation‐induced AMLs for homozygous loss of microsatellites and genes mapping within the mdr revealed loss of both copies of the hemopoietic tissue‐specific transcription factor Sfpi1 (PU.1/Spi1) in one cell line. Studies of primary and F1 AMLs failed to implicate Sfpi1 as the AML tumor suppressor gene. YAC contig construction, together with data suggesting that the critical gene flanks Sfpi1, represents significant progress toward identifying an AML tumor suppressor gene. Genes Chromosomes Cancer 24:95–104, 1999.

Telomeric sequences, radiation sensitivity and genomic instability

Purpose : To review the basic features of telomeres with particular emphasis on their potential importance in radiation biology. Recent findings suggest that telomere length can influence radiation sensitivity in mouse and that several human radiosensitive disorders also show abnormalities in telomere dynamics. Numerous studies indicate that telomeric sequences may play a role in determining the stability of certain genomic regions both spontaneously and following irradiation. Furthermore, a number of transmissible genomic instability systems have been described in which it appears that telomere metabolism may be contributing to the delayed effects observed. Features of telomeres and telomere biology relevant to these topics are reviewed. Conclusions : The evidence that telomeres and the molecular pathways of telomere maintenance can play a role in determining the outcome of radiation exposure is now substantial. Thus, the field of telomere biology deserves continued attention from radiobiologists.

Molecular mechanisms of radiation oncogenesis

Resolving the molecular mechanisms of radiation oncogenesis represents an important but daunting challenge in radiation research. This brief review outlines the principal oncogenic mechanisms that need to be considered in the context of radiation effects on the genome, how these might relate to specific gene and chromosomal changes relevant to neoplasia and the possible implications of such knowledge for the modelling of cancer risk. The long-term application of this mechanistic knowledge to the determination of tumour causality and for the assessment of individual cancer risk is also briefly discussed.

Loss of heterozygosity in spontaneous and x-ray–induced intestinal tumors arising in F1 hybrid Min mice: Evidence for sequential loss of Apc+ and Dpc4 in tumor development†

Min (multiple intestinal neoplasia) mice carry a mutant allele of the murine Apc (adenomatous polyposis coli) locus and are predisposed to intestinal adenoma formation in the intestinal tract. Early studies have shown complete loss of function of Apc by whole chromosome loss on the tumor‐sensitive C57BL/6J genetic background and in AKR × B6 F1 hybrids. Gamma‐radiation–induced chromosomal losses focus the critical region on wt Apc, but because of the limited number of polymorphic markers used, no other critical regions of loss on chromosome 18 were identified. Using intestinal tumors arising spontaneously and induced by X‐rays in CBA/H × C57BL/6J F1 hybrid mice and high‐resolution microsatellite loss of heterozygosity (LOH) techniques, we provide mapping data for wt Apc loss, which confirms and extends earlier observations. In addition, high‐frequency loss events at the Dpc4 locus were found in both spontaneous and radiation‐induced tumors. These data identified LOH of Dpc4 as a critical secondary event following complete functional loss of Apc. LOH across the Trp53 genomic region of chromosome 11 was not observed. No LOH was recorded for the Mom1 candidate gene Pla2g2a or for 9 out of 10 polymorphic markers from the Mom1 genomic region on murine chromosome 4. One marker mapping distal to Pla2g2a showed LOH in a small minority of spontaneous tumors. These data support the contention that Mom1 does not act as a classical tumor suppressor. Overall, our data indicates a significant role for Dpc4 mutation in intestinal tumor progression in the mouse and provides further evidence for the importance of interstitial chromosome losses in radiation tumorigenesis. Genes, Chromosomes and Cancer 28:387–394, 2000.

Microsatellite analysis of recurrent chromosome 2 deletions in acute myeloid leukaemia induced by radiation in FI hybrid mice

Deletions and/or rearrangements involving one copy of chromosome 2 are consistent and early events in the development of murine acute myeloid leukaemia (AML) by radiation. More than 90% of AMLs induced in the CBA strain of mice express such cytogenetic alterations, with chromosome 2 breakpoints clustering in the C and F regions of the chromosome. In inbred mouse strains, the molecular resolution of these breakpoints is problematic. However, by using x‐ray‐induced AMLs in FI progeny of genetically divergent CBA/H × C57BI, it has been possible to show region‐specific loss of heterozygosity (LOH) in genetically linked sets of chromosome 2 microsatellite alleles from one of the two parental chromosomes. In the majority of cases, an acceptable concordance was shown for AML chromosome 2 deletion, as defined by microsatellites and as revealed by G‐band cytogenetics. A degree of breakpoint clustering was found, but the identification of a number of deletion types, based on the position of proximal and distal breakpoints as defined by microsatellite analysis, strongly supports a leukaemogenic mechanism involving gene deletion. No bias towards loss of CBA or C57BI alleles was observed, and the gender of AML‐presenting animals did not appear to influence the parental origin of the deletions. A molecular map of chromosome 2 breakpoints has now been established in FI AMLs as a first step towards the molecular cloning of breakpoint sequences. Genes Chromosom Cancer 16:238–246 (1996).

The multi-step nature of carcinogenesis and the implications for risk analysis

PURPOSEnFor the purposes of constructing biologically realistic models of radiation cancer risk it is necessary to have detailed knowledge of the post-irradiation mechanisms involved.nnnMETHODSnThis brief review considers selected aspects of DNA damage response, tumour initiation/development, together with attendant factors and provides comment on the implications for cancer risk estimation and some of the uncertainties that remain.nnnCONCLUSIONSnIt is judged from studies with animal models that, in the main, radiation tumorigenesis proceeds in a conventional multi-step fashion following radiation-induced specific gene losses from single-target stem cells.

A major breakpoint cluster domain in murine radiation-induced acute myeloid leukemia*

Cytogenetic and molecular studies have provided evidence of the clustering of chromosome 2 deletion breakpoints in radiation‐induced murine acute myeloid leukemia (AML). Moreover, clustering occurs in at least two fragile domains rich in telomere‐like arrays. Here we describe a physical map of the distal breakpoint cluster and confirm the presence of inverted head‐to‐head telomeric sequence arrays. These potentially recombinogenic sequences were not, however, the direct focus for post‐irradiation chromosome breakage in AML. Instead, the two arrays bordered a 2.5‐kb sequence with properties expected of a nuclear matrix attachment region (MAR). The putative MAR co‐localized in the fragile domain with genes important to the hemopoietic system (leukocyte tyrosine kinase, zinc finger protein 106, erythrocyte protein band 4.2, and β2‐microglobulin (β2m)); the β2m subdomain was a particular focus of breakage. On the basis of these and other data, we suggest that AML‐associated chromosome 2 fragility in the mouse is a consequence of domain‐specific fragility in genomic domains containing numerous genes critical to the hemopoietic system. Copyright

Somatic cell hybrids for high-density mapping of chromosome 2 breakpoints in radiation-induced myeloid leukemia cell lines from inbred mice†

Chromosome 2 (chr 2) deletions are recurrent abnormalities in acute myeloid leukemia (AML) induced by ionizing radiation in the mouse. The localization of deletion sites has proven extremely useful in providing information on the molecular mechanisms of leukemogenesis. The models available for the study of AML are mostly represented by inbred mouse strains, in which the molecular resolution of breakpoints is problematic. In this study, we have examined five leukemic cell lines exhibiting hemizygous chr 2 loss, derived from CBA, C3H, or (C57BL×CBA/H) F1 mice in which AML had been induced by a whole‐body dose of radiation. By application of a somatic cell hybridization technique, we have generated interspecific cell hybrids retaining the deleted murine chr 2 homologue. This strategy permitted a very detailed genetic analysis allowing the utilization of any genetic marker on chr 2 without a requirement for polymorphism. Somatic cell hybrid clones were subjected to a high‐density polymerase chain reaction–based microsatellite screening using 62–106 informative markers for each cell line. Detailed maps accurately defining chr 2 breakpoints were obtained. The identification of critical breakpoint markers allowed the construction of partial yeast artificial chromosome contigs across chr 2 breakpoints. These maps represent an essential resource for cloning of the breakpoint regions. Mol. Carcinog. 27:219–228, 2000. Published 2000 Wiley‐Liss, Inc.

In utero haemopoietic sensitivity to alpha, beta or X-irradiation in CBA/H mice

Purpose : To assess in utero sensitivity to x-rays, α -emissions from plutonium-239 and β -emissions from tritium in terms of induction of chromosomal aberrations in bone marrow cells. Materials and methods : CBA/H mice were exposed to a single dose of X-rays (0.5Gy) on either day 7 or day 14 of pregnancy or given 239 Pu (100 kBq kg -1) by intraperitoneal injection on either day 6 or day 13. Tritium was administered to mice throughout pregnancy as either tritiated water, ad libitum in drinking water (total intake averaged 130 MBq), or as homogenized tritiated cress, administered by gastric intubation (total 60 MBq). Irradiated and unexposed control mice and their offspring were sacrificed at 2-8 weeks after birth. Direct metaphase preparations from femoral bone marrow cells from mothers and offspring were used for G-band analysis. Results : The incidence of stable aberrations was significantly and similarly increased in neonatal and maternal marrow samples after exposure to X-rays, 239 Pu or 3 H. The estimated average bone absorbed doses from 239 Pu in pregnant females were similar to the X-ray dose of 0.5 Gy, suggesting a low RBE for α -irradiation in adults. The similar levels of damage observed in neonates after X-irradiation and 239Pu exposure are indicative of greater in utero sensitivity to α -irradiation since the overall estimated in utero α -particle doses to haemopoietic tissue were much lower. In utero doses from 3 H and corresponding maternal doses were around 0.5Gy, showing no evidence of greater in utero sensitivity, no significant difference between the effects of the two forms of tritium, and were consistent with an RBE value of 1-2. Conclusions : Comparison of stable aberration yields in haemopoietic cells suggests a greater sensitivity to α -particles from 239 Pu than X-rays or β -particles from 3 H for irradiation in utero but a low RBE value in adults.PURPOSEnTo assess in utero sensitivity to x-rays, alpha-emissions from plutonium-239 and beta-emissions from tritium in terms of induction of chromosomal aberrations in bone marrow cells.nnnMATERIALS AND METHODSnCBA/H mice were exposed to a single dose of X-rays (0.5Gy) on either day 7 or day 14 of pregnancy or given (239)Pu (100 kBq kg(-1)) by intraperitoneal injection on either day 6 or day 13. Tritium was administered to mice throughout pregnancy as either tritiated water, ad libitum in drinking water (total intake averaged 130 MBq), or as homogenized tritiated cress, administered by gastric intubation (total 60 MBq). Irradiated and unexposed control mice and their offspring were sacrificed at 2-8 weeks after birth. Direct metaphase preparations from femoral bone marrow cells from mothers and offspring were used for G-band analysis.nnnRESULTSnThe incidence of stable aberrations was significantly and similarly increased in neonatal and maternal marrow samples after exposure to X-rays, (239)Pu or (3)H. The estimated average bone absorbed doses from (239)Pu in pregnant females were similar to the X-ray dose of 0.5 Gy, suggesting a low RBE for alpha-irradiation in adults. The similar levels of damage observed in neonates after X-irradiation and 239Pu exposure are indicative of greater in utero sensitivity to alpha-irradiation since the overall estimated in utero alpha-particle doses to haemopoietic tissue were much lower. In utero doses from (3)H and corresponding maternal doses were around 0.5Gy, showing no evidence of greater in utero sensitivity, no significant difference between the effects of the two forms of tritium, and were consistent with an RBE value of 1-2.nnnCONCLUSIONSnComparison of stable aberration yields in haemopoietic cells suggests a greater sensitivity to alpha-particles from (239)Pu than X-rays or beta-particles from (3)H for irradiation in utero but a low RBE value in adults.

Analysis of loss of heterozygosity in lymphoma and leukaemia arising in F1 hybrid mice locates a common region of chromosome 4 loss.

Previous studies have identified five lymphoma‐related tumour suppressor gene regions on murine chromosome 4. Using detailed allelotype analysis on a range of lympho‐haematopoietic tumour types arising in F1 hybrid mice, we now show a consistent pattern of loss of heterozygosity (LOH) which identifies a common region of loss delineated by microsatellites D4Mit21 and D4Mit53 on proximal chromosome 4. This critical segment corresponds to the thymic lymphoma tumour suppressor region 5 (TLSR5) identified in an earlier study. Tumours of this type have also been reported as showing allelic loss from the Trp53 and Ikaros regions on chromosome 11. In the present study, only a small fraction of tumours showed LOH in the Ikaros region, while a minority of lymphomas, but not acute myeloid leukaemias, showed allelic loss of the chromosome 11 segment encoding Trp53. These and other data indicate strongly that the genomic regions identified as showing recurrent LOH depend on the genetic background of the mice. Overall, the results indicate a key role for a tumour suppressor gene(s) encoded in an ∼3 cM segment on proximal chromosome 4 and provide an experimental basis for the further investigation of the functional role of candidate genes which include Pax5 and Tgfbr1.