Simon Bouffler

Mammalian Ku86 mediates chromosomal fusions and apoptosis caused by critically short telomeres

Here we analyze the functional interaction between Ku86 and telomerase at the mammalian telomere by studying mice deficient for both proteins. We show that absence of Ku86 prevents the end‐to‐end chromosomal fusions that result from critical telomere shortening in telomerase‐deficient mice. In addition, Ku86 deficiency rescues the male early germ cell apoptosis triggered by short telomeres in these mice. Together, these findings define a role for Ku86 in mediating chromosomal instability and apoptosis triggered by short telomeres. In addition, we show here that Ku86 deficiency results in telomerase‐dependent telomere elongation and in the fusion of random pairs of chromosomes in telomerase‐proficient cells, suggesting a model in which Ku86 keeps normal‐length telomeres less accessible to telomerase‐ mediated telomere lengthening and to DNA repair activities.

The involvement of telomeric sequences in chromosomal aberrations.

The genomes of higher eukaryotes are not homogeneous in terms of structure or function. Many examples of chromosomal regions particularly prone to involvement in aberrations have been reported. The molecular structures of some of these regions have now been determined, most notably the folate-sensitive fragile sites and FRA16B-a distamycin A-sensitive fragile site. In addition, a number of cytological studies suggest that telomeric sequences can in some circumstances be involved in chromosomal aberrations more frequently than expected. Here, the roles of telomeric DNA sequences, both terminal and interstitial, and telomerase in chromosomal aberration formation are reviewed.

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.

Chromosome 2 hypersensitivity and clonal development in murine radiation acute myeloid leukaemia

Acute myeloid leukaemias induced by ionizing radiation in mouse are characterized by chromosome (chr) 2 aberrations. While it is known that chr 2 aberrations form early and in abundance post-irradiation, unequivocal evidence for hypersensitivity of chr 2 in the first post-irradiation mitoses is lacking. Here it is established that chromosomal aberrations detected in bone marrow cells by chromosome painting are induced in all mice at an approximately 2-fold greater frequency in chr 2 by comparison with chrs 1 and 3 at 24 and 48 h following in vivo whole-body X-irradiation. Long-term follow up studies (to 15 months post-irradiation) indicated that chromosomal hypersensitivity is accounted for largely by the existence of hot-spots for aberration formation on sensitive chromosomes. Analysis of clonal developments suggested that chr 2 aberrant clones are selected for entry into the proliferating bone marrow cell compartment in preference to cells with other aberrations and that these clones in general have a higher proliferative potential. However, neither the induction of chr 2 aberrations nor the presence of a chr 2 aberrant clone specifically predict the development of AML in an individual irradiated mouse. Nonetheless these events or sub-groups of these events are necessary for AML development.

Chromosomal Abnormalities in Neutron-Induced Acute Myeloid Leukemias in CBA/H Mice

Acute myeloid leukemias (AMLs) induced in CBA/H mice by 1 MeV fission neutrons have been examined for chromosomal abnormalities by G-band analysis. In common with X-ray- and alpha-particle-induced AMLs in CBA/H mice, more than 90% (16/17) of the myeloid leukemias had chromosome 2 abnormalities, in this case, all interstitial deletions. Chromosome 2 breakpoints were not wholly consistent, but clustering in three specific G-band regions was observed. Very distal (H-region) breakpoints were more common in the neutron AMLs than in X-ray- or alpha-particle-induced leukemias. These data indicate that neutron-induced AMLs in CBA/H mice are not characterized by a specific chromosome deletion but that a variety of chromosome 2 deletion types are associated with the disease. Trisomy of chromosome 1(12.5% AMLs) and aneusomy of chromosomes 6 (31% AMLs) and Y (37.5% AMLs) were noted. While chromatid breakage was observed occasionally in neutron-induced AML, no clear indications of persistent chromosomal instability or high levels of stable chromosomal change were apparent.

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.

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).

Long but dysfunctional telomeres correlate with chromosomal radiosensitivity in a mouse AML cell line.

Purpose : To compare the chromosomal radiosensitivity of C3H mouse acute myeloid leukaemia (AML) cell lines 7926 and 8709 and to investigate the mechanistic basis of the radiosensitivity observed in 7926. Materials and methods : Yields of chromosome aberrations following X-irradiation were determined in Giemsa-stained metaphases. Cell cycle phase distributions were determined by BrdU incorporation and microscopy, apoptosis was assessed by caspase assays. Telomerase activity (TRAP assay), telomere length (Q-FISH and Southern blotting) and telomere function (Robertsonian-like fusion formation) were also examined. The expression levels of telomerase components, telomerase regulators and DNA PKcs were determined on Northern blots. Results : A total of 4.5-7.6-fold elevated chromosome aberration yields were found in 7926 by comparison with 8709 3-24h after 0.5 and 1 Gy X-ray exposure. This difference could not be accounted for by differences in chromatid break-rejoining rates, cell cycle phase distribution or the induction of apoptosis. Telomeres and telomerase were dysfunctional in 7926. However, average telomere length was approximately two-fold greater than in 8709. Conclusion : Defective telomere function in 7926 correlates with chromosomal radiosensitivity. This implicates telomere function in addition to telomere length as a determinant of chromosomal radiosensitivity.

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.

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.