Redox imbalance and mutagenesis in spleens of mice harboring a hypomorphic allele of Gpdx(a) encoding glucose 6-phosphate dehydrogenase

Mice harboring the activity-attenuated Gpdx(a-m2Neu) allele and also harboring a chromosomally integrated lacZ reporter gene to study mutagenesis (pUR288) were used to demonstrate that moderate glucose 6-phosphate dehydrogenase (G6PD) deficiency causes elevated mutagenesis and endogenous oxidative stress in the spleen. G6PD-deficient spleens with a residual enzyme activity of 22% exhibited a dramatic shift in the mutational pattern of lacZ (4.6-fold increase in the prevalence of recombination mutations of lacZ) together with a 1.8-fold increase in mutant frequencies in lacZ. A concomitant 3-fold reduction in catalase activity (dependent upon NADPH) indicated that the in vivo supply of G6PD-generated NADPH was insufficient. An additional 3-fold increase in oxidized glutathione suggested that redox control was disturbed in G6PD-deficient spleens. These findings indicate that G6PD is required for limiting oxidative mutagenesis in the mouse spleen. Gpdx(a-m2Neu) is the first hypomorphic allele of a mouse housekeeping gene associated with elevated somatic mutagenesis in vivo.     

Moderate G6PD deficiency increases mutation rates in the brain of mice

Mice that harbored the x-ray-induced low efficiency allele of the major X-linked isozyme of glucose-6-phospate dehydrogenase (G6PD), Gpdx(a-m2Neu), and, in addition, harbored the transgenic shuttle vector for the determination of mutagenesis in vivo, pUR288, were employed to further our understanding of the interdependence of general metabolism, oxidative stress control, and somatic mutagenesis. The Gpdx(a-m2Neu) mutation conferred moderate G6PD deficiency in hemizygous males (Gpdx(a-m2Neu/y)) displaying residual enzyme activities of 27% in red blood cells and 13% in brain (compared to wild-type controls, Gpdx(a/y) males). In spite of this mild phenotype, the brains of G6PD-deficient males exhibited a significant distortion of redox control ( approximately 3-fold decrease in the ratio of reduced glutathione to oxidized glutathione), a considerable accumulation of promutagenic etheno DNA adducts ( approximately 13-fold increase in ethenodeoxyadenosine and approximately 5-fold increase in ethenodeoxycytidine), and a substantial elevation of somatic mutation rates ( approximately 3-fold increase in mutant frequencies in lacZ, the target and reporter gene of mutagenesis in the shuttle vector, pUR288). The mutation pattern in the brain was dominated by illegitimate genetic recombinations, a presumed hallmark of oxidative mutagenesis. These findings suggested a critical function for G6PD in limiting oxidative mutagenesis in the mouse brain.     

Paradoxical decrease in mutant frequencies and chromosomal rearrangements in a transgenic lacZ reporter gene in Ku80 null mice deficient in DNA double strand break repair

Repair of DNA double strand breaks (DSB), either by homologous recombination (HR) or nonhomologous end-joining (NHEJ), is essential to maintain genomic stability. To examine the impact of NHEJ deficiency on genomic integrity in Ku80 null (Ku-) mice, the chromosomally integrated shuttle vector pUR288, which includes a lacZ reporter gene, was used to measure mutations in vivo. Unexpectedly, a significant decrease was found in mutant frequencies of Ku- liver (5.04x10(-5)) and brain (4.55x10(-5)) compared to tissues obtained from normal (Ku+) littermates (7.92x10(-5)and 7.30x10(-5), respectively). No significant difference was found in mutant frequencies in spleen from Ku- (7.21x10(-5)) and Ku+ mice (8.16x10(-5)). The determination of the mutant spectrum in lacZ revealed the almost complete absence of chromosomal rearrangements (R) in Ku- tissues (0.5%, 3/616), a notable distinction from Ku+ controls (16.7%, 104/621). These findings suggest that accurate repair of DSB by HR and elimination of cells with unrepaired DNA damage by apoptosis are capable of maintaining genomic stability of the lacZ reporter in Ku- mice.     

Detecting genotoxic effects of potential clastogens: an in vivo study using the transgenic lacZ plasmid and the MutaMouse model

In the present paper the capacity of the pUR288 plasmid mouse model and the MutaMouse model to detect the clastogens bleomycin, m-AMSA, o-AMSA and camptothecin, was investigated. Ethylnitrosourea (ENU) served as a positive control, methylcellulose as a negative control. Only bleomycin induced a slight but significant increase in lacZ mutant frequency (MF) in bone marrow of pUR288 plasmid mice. Exposure to the other compounds did not result in an increase in the MF in bone marrow and liver in both mouse models. For the MutaMouse this result was expected, for the plasmid mouse an increase in MF after clastogen exposure was expected. The positive control ENU induced statistically significant increases in MF compared with the negative control in both models and in both tissues analyzed. Hybridisation of DNA of mutant colonies derived from plasmid mice with labelled total mouse DNA (Hybridisation Assay) demonstrated an increase in the percentage of colonies hybridised with total mouse DNA as compared with the negative control, which suggests that there was indeed a biological response associated with treatment. The latter results indicate that the plasmid mouse assay may still be a promising model for the detection of clastogens.     

Bcl-2 reduces mutant rates in a transgenic lacZ reporter gene in mouse pre-B lymphocytes

To assess mutagenesis during early B-lymphocyte development in vitro, progenitor B cells (pre-B cells) were obtained from fetal livers of BALB/c mice and DBA/2N mice that harbored the transgenic shuttle vector, pUR288, with a lacZ reporter gene for the determination of mutant frequencies (MFs). Differentiation-arrested pre-B cells demonstrated a marked dose-dependent increase in lacZ mutant levels after exposure to gamma-irradiation with a peak MF of 250 x 10(-5) at 2.5 Gy. Without genotoxic treatment, pre-B cells undergoing spontaneous differentiation into surface IgM expressing immature B cells exhibited lacZ mutant levels of up to 95 x 10(-5). The mutational pattern was dominated in both experiments by illegitimate recombination mutations of lacZ, not point mutations. Likewise, in both experiments, the enforced expression of Bcl-2 resulted in a striking reduction of lacZ mutations. These findings indicated that mouse pre-B cells are prone to accumulate induced and self-inflicted mutations, particularly recombinations. Additionally, our studies revealed a heretofore unknown role of Bcl-2 in inhibiting mutagenesis during early B-cell development in mice.     

Frequent occurrence of large duplications at reciprocal genomic rearrangement breakpoints in multiple myeloma and other tumors

Using a combination of array comparative genomic hybridization, mate pair and cloned sequences, and FISH analyses, we have identified in multiple myeloma cell lines and tumors a novel and recurrent type of genomic rearrangement, i.e. interchromosomal rearrangements (translocations or insertions) and intrachromosomal inversions that contain long (1–4000 kb; median ∼100 kb) identical sequences adjacent to both reciprocal breakpoint junctions. These duplicated sequences were generated from sequences immediately adjacent to the breakpoint from at least one—but sometimes both—chromosomal donor site(s). Tandem duplications had a similar size distribution suggesting the possibility of a shared mechanism for generating duplicated sequences at breakpoints. Although about 25% of apparent secondary rearrangements contained these duplications, primary IGH translocations rarely, if ever, had large duplications at breakpoint junctions. Significantly, these duplications often contain super-enhancers and/or oncogenes (e.g. MYC) that are dysregulated by rearrangements during tumor progression. We also found that long identical sequences often were identified at both reciprocal breakpoint junctions in six of eight other tumor types. Finally, we have been unable to find reports of similar kinds of rearrangements in wild-type or mutant prokaryotes or lower eukaryotes such as yeast.     

Heterogeneous duplications in patients with Pelizaeus-Merzbacher disease suggest a mechanism of coupled homologous and nonhomologous recombination

We describe genomic structures of 59 X-chromosome segmental duplications that include the proteolipid protein 1 gene (PLP1) in patients with Pelizaeus-Merzbacher disease. We provide the first report of 13 junction sequences, which gives insight into underlying mechanisms. Although proximal breakpoints were highly variable, distal breakpoints tended to cluster around low-copy repeats (LCRs) (50% of distal breakpoints), and each duplication event appeared to be unique (100 kb to 4.6 Mb in size). Sequence analysis of the junctions revealed no large homologous regions between proximal and distal breakpoints. Most junctions had microhomology of 1-6 bases, and one had a 2-base insertion. Boundaries between single-copy and duplicated DNA were identical to the reference genomic sequence in all patients investigated. Taken together, these data suggest that the tandem duplications are formed by a coupled homologous and nonhomologous recombination mechanism. We suggest repair of a double-stranded break (DSB) by one-sided homologous strand invasion of a sister chromatid, followed by DNA synthesis and nonhomologous end joining with the other end of the break. This is in contrast to other genomic disorders that have recurrent rearrangements formed by nonallelic homologous recombination between LCRs. Interspersed repetitive elements (Alu elements, long interspersed nuclear elements, and long terminal repeats) were found at 18 of the 26 breakpoint sequences studied. No specific motif that may predispose to DSBs was revealed, but single or alternating tracts of purines and pyrimidines that may cause secondary structures were common. Analysis of the 2-Mb region susceptible to duplications identified proximal-specific repeats and distal LCRs in addition to the previously reported ones, suggesting that the unique genomic architecture may have a role in nonrecurrent rearrangements by promoting instability.     

Different molecular mechanisms causing 9p21 deletions in acute lymphoblastic leukemia of childhood

Deletion of chromosome 9p21 is a crucial event for the development of several cancers including acute lymphoblastic leukemia (ALL). Double strand breaks (DSBs) triggering 9p21 deletions in ALL have been reported to occur at a few defined sites by illegitimate action of the V(D)J recombination activating protein complex. We have cloned 23 breakpoint junctions for a total of 46 breakpoints in 17 childhood ALL (9 B- and 8 T-lineages) showing different size deletions at one or both homologous chromosomes 9 to investigate which particular sequences make the region susceptible to interstitial deletion. We found that half of 9p21 deletion breakpoints were mediated by ectopic V(D)J recombination mechanisms whereas the remaining half were associated to repeated sequences, including some with potential for non-B DNA structure formation. Other mechanisms, such as microhomology-mediated repair, that are common in other cancers, play only a very minor role in ALL. Nucleotide insertions at breakpoint junctions and microinversions flanking the breakpoints have been detected at 20/23 and 2/23 breakpoint junctions, respectively, both in the presence of recombination signal sequence (RSS)-like sequences and of other unspecific sequences. The majority of breakpoints were unique except for two cases, both T-ALL, showing identical deletions. Four of the 46 breakpoints coincide with those reported in other cases, thus confirming the presence of recurrent deletion hotspots. Among the six cases with heterozygous 9p deletions, we found that the remaining CDKN2A and CDKN2B alleles were hypermethylated at CpG islands.     

The prevalence of factor V Leiden,prothrombin G20210A and methylenetetrahydrofolate reductase polymorphism C677T among G6PD deficient individuals from Western Iran

It has been suggested that the allele frequency of thrombophilic mutations is affected by glucose-6-phosphate dehydrogenase (G6PD) deficiency. The prevalence of thrombophilic mutations were studied in sixty G6PD deficient individuals including 57 males and three females with the mean age of 15 ± 3.08 and 110 age and sex matched healthy individuals consisted of 95 males and 15 females with the mean age of 16.19 ± 2.17 from the Kermanshah Province of Iran. Using a combination of PCR-RFLP technique, single strand conformation polymorphism (SSCP) analysis and DNA sequencing polymorphic G6PD mutations were identified. The factor V Leiden, prothrombin G20210A and methylenetetrahydrofolate reductase (MTHFR) C677T were detected by PCR-RFLP method using MnlI, HindIII and HinfI restriction enzymes, respectively. Three mutations, G6PD Mediterranean, G6PD Chatham and G6PD Cosenza were identified in 60 G6PD deficient individuals with highest prevalence of G6PD Mediterranean (91.6%). In G6PD deficient individuals the prevalence of factor V Leiden tended to be higher (5%) compared to healthy individuals (2.7%). The prevalence of prothrombin G20210A mutation in G6PD deficient individuals was 1.7%. However, in normal subjects the prevalence of this mutation was 2.7%. The frequency of T allele in G6PD deficient individuals were insignificantly higher (29.16%) than those in healthy individuals (26.8%). Our finding indicates that the prevalence of factor V Leiden, prothrombin G20210A and MTHFR C677T in G6PD deficient individuals is not statistically different compared to normal subjects and G6PD deficiency is not associated with these thrombophilic mutations in Western Iran.     

Emergence of complex rearrangements at translocation breakpoints in a transgenic mouse; implications for mechanisms involved in the formation of chromosome rearrangements

Cryptic complex rearrangements as a result of a reciprocal chromosome translocation have been characterised in a transgenic mouse strain. Analysis of the breakpoint junctions in our previous studies showed that the ada transgene was integrated at the breakpoint forming a fusion gene with Golga3 (Mea2). In this study, further detailed analysis around the translocation junctions revealed that the surrounding regions were composed of 13 fragments of defined transgenic chromosome origins over approximately 1.9-Mb areas. Exactly the same cluster structure of these 13 breakpoint fragments already existed in the second generation of the transgenic mice. Our results show that this highly complex rearrangement has been conserved as the incipient form without any additional changes for 18 years up to the present generation, suggesting simultaneous occurrence of multiple events in the founder mouse.     

Further delineation of nonhomologous-based recombination and evidence for subtelomeric segmental duplications in 1p36 rearrangements

The mechanisms involved in the formation of subtelomeric rearrangements are now beginning to be elucidated. Breakpoint sequencing analysis of 1p36 rearrangements has made important contributions to this line of inquiry. Despite the unique architecture of segmental duplications inherent to human subtelomeres, no common mechanism has been identified thus far and different nonexclusive recombination–repair mechanisms seem to predominate. In order to gain further insights into the mechanisms of chromosome breakage, repair, and stabilization mediating subtelomeric rearrangements in humans, we investigated the constitutional rearrangements of 1p36. Cloning of the breakpoint junctions in a complex rearrangement and three non-reciprocal translocations revealed similarities at the junctions, such as microhomology of up to three nucleotides, along with no significant sequence identity in close proximity to the breakpoint regions. All the breakpoints appeared to be unique and their occurrence was limited to non-repetitive, unique DNA sequences. Several recombination- or cleavage-associated motifs that may promote non-homologous recombination were observed in close proximity to the junctions. We conclude that NHEJ is likely the mechanism of DNA repair that generates these rearrangements. Additionally, two apparently pure terminal deletions were also investigated, and the refinement of the breakpoint regions identified two distinct genomic intervals ~25-kb apart, each containing a series of 1p36 specific segmental duplications with 90–98% identity. Segmental duplications can serve as substrates for ectopic homologous recombination or stimulate genomic rearrangements.     

Identification of sequence motifs at the breakpoint junctions in three t(1;9)(p36.3;q34) and delineation of mechanisms involved in generating balanced translocations

Although approximately 1 in 500 individuals carries a reciprocal translocation, little is known about the mechanisms that result in their formation. We analyzed the sequences surrounding the breakpoints in three unbalanced translocations of 1p and 9q, all of which were designated t(1;9)(p36.3;q34), to investigate the presence of sequence motifs that might mediate nonhomologous end joining (NHEJ). The breakpoint regions were unique in all individuals. Two of three translocations demonstrated insertions and duplications at the junctions, suggesting NHEJ in the formation of the rearrangements. No homology was identified in the breakpoint regions, further supporting NHEJ. We found translin motifs at the breakpoint junctions, suggesting the involvement of translin in the joining of the broken chromosome ends. We propose a model for balanced translocation formation in humans similar to transposition in bacteria, in which staggered nicks are repaired resulting in duplications and insertions at the translocation breakpoints.     

Chromosome catastrophes involve replication mechanisms generating complex genomic rearrangements

Complex genomic rearrangements (CGRs) consisting of two or more breakpoint junctions have been observed in genomic disorders. Recently, a chromosome catastrophe phenomenon termed chromothripsis, in which numerous genomic rearrangements are apparently acquired in one single catastrophic event, was described in multiple cancers. Here, we show that constitutionally acquired CGRs share similarities with cancer chromothripsis. In the 17 CGR cases investigated, we observed localization and multiple copy number changes including deletions, duplications, and/or triplications, as well as extensive translocations and inversions. Genomic rearrangements involved varied in size and complexities; in one case, array comparative genomic hybridization revealed 18 copy number changes. Breakpoint sequencing identified characteristic features, including small templated insertions at breakpoints and microhomology at breakpoint junctions, which have been attributed to replicative processes. The resemblance between CGR and chromothripsis suggests similar mechanistic underpinnings. Such chromosome catastrophic events appear to reflect basic DNA metabolism operative throughout an organism's life cycle.     

Molecular characterization and delineation of subtle deletions in de novo “balanced” chromosomal rearrangements

To test the hypothesis that the phenotypic abnormalities seen in cases with apparently balanced chromosomal rearrangements are the result of the presence of cryptic deletions or duplications of chromosomal material near the breakpoints, we analyzed three cases with apparently balanced chromosomal rearrangements and phenotypic abnormalities. We characterized the breakpoints in these cases by using microsatellite analysis by polymerase chain reaction and fluorescence in situ hybridization analysis of yeast artificial chromosome clones selected from the breakpoint regions. Molecular characterization of the translocation breakpoint in patient 1 [46,XY,t(2;6)(p22.2;q23.1)] showed the presence of a 4- to 6-Mb cryptic deletion between markers D6S412 and D6S1705 near the 6q23.1 breakpoint. Molecular characterization of the proximal inversion 7q22.1 breakpoint in patient 2 [46,XY,inv(7)(q22.1q32.1)] revealed the presence of a 4-Mb cryptic deletion between D7S651 and D7S515 markers. No deletion or duplication of chromosomal material was found near the breakpoints in patient 3 [46,XX,t(2;6)(q33.1;p12.2)]. Our study suggests that a systematic molecular study of breakpoints should be carried out in cases with apparently balanced chromosomal rearrangements and phenotypic abnormalities, because cryptic deletions near the breakpoints may explain the phenotypic abnormalities in these cases. Received: 9 March 1998 / Accepted: 24 April 1998     

Nuclear positioning,higher-order folding,and gene expression of Mmu15 sequences are refractory to chromosomal translocation

Nuclear localization influences the expression of certain genes. Chromosomal rearrangements can reposition genes in the nucleus and thus could impact the expression of genes far from chromosomal breakpoints. However, the extent to which chromosomal rearrangements influence nuclear organization and gene expression is poorly understood. We examined mouse progenitor B cell lymphomas with a common translocation, der(12)t(12;15), which fuses a gene-rich region of mouse chromosome12 (Mmu12) with a gene-poor region of mouse chromosome15 (Mmu15). We found that sequences 2.3 Mb proximal and 2.7 Mb distal to the der(12)t(12;15) breakpoint had different nuclear positions measured relative to the nuclear radius. However, their positions were similar on unrearranged chromosomes in the same tumor cells and normal progenitor B cells. In addition, higher-order chromatin folding marked by three-dimensional gene clustering was not significantly altered for the 7 Mb of Mmu15 sequence distal to this translocation breakpoint. Translocation also did not correspond to significant changes in gene expression in this region. Thus, any changes to Mmu15 structure and function imposed by the der(12)t(12;15) translocation are constrained to sequences near (<2.5 Mb) the translocation junction. These data contrast with those of certain other chromosomal rearrangements and suggest that significant changes to Mmu15 sequence are structurally and functionally tolerated in the tumor cells examined.     

Induction of lymphomas on implantation of human oral squamous cell carcinomas in nude mice

Cancer cells from five oral cancer patients and pleomorphic adenoma cells from one individual were inoculated as single cell suspension into subcutis of 30 Swiss nude mice and tail vein of additional 30 mice. Further, tumor tissue pieces from three oral cancer patients were xenografted s.c. in 18 nude mice, and 10 mice were kept as controls. In animals implanted with tumor pieces, 7/18 (39%) mice, developed squamous cell carcinoma at the site of inoculation within 8-15 days, while tumors were not observed in mice inoculated with single cell suspension, up to 60/90 days. In 8/68 (12%) mice, white foci were observed in several tissues, with hepatomegaly and splenomegaly noted in 27/68 (39%) mice. Histopathological examination of various tissues revealed presence of large cell lymphoma in several organs in 14/68 (21%) mice. No regional or distant metastasis of the implanted oral tumor cells was detected. Mice injected with cells from pleomorphic adenoma, also demonstrated large cell lymphoma in 2/10 (20%) mice, whereas none of the 10 control animals showed any gross abnormalities or microscopic abnormalities in several organs. 2/16 (12%) lymphomas exhibited positive reaction with mouse B cell antibodies illustrating the murine origin of the lymphomas, and these were immunophenotyed as B cell lymphomas. The lymphomas were also examined with mouse T cell antibodies and none reacted positively with the mouse T cell antibodies. The lymphomas also failed to react with human T cell, B cell and human Leucocyte common antigen (LCA) antibodies, indicating that the induced lymphomas were not of human origin. The tumor specimens from seven of eight oral cancer patients and the pleomorphic adenoma patient induced lymphomas in nude mice. Thus it appears that xenografting oral tumor cells into nude mice may cause induction of the murine lymphomas, and this needs further investigation.     

Glucose-6-phosphate dehydrogenase modulates vascular endothelial growth factor-mediated angiogenesis

Glucose-6-phosphate dehydrogenase (G6PD), the first enzyme of the pentose phosphate pathway, is the principal intracellular source of NADPH. NADPH is utilized as a cofactor by vascular endothelial cell nitric-oxide synthase (eNOS) to generate nitric oxide (NO*). To determine whether G6PD modulates NO*-mediated angiogenesis, we decreased G6PD expression in bovine aortic endothelial cells using an antisense oligodeoxynucleotide to G6PD or increased G6PD expression by adenoviral gene transfer, and we examined vascular endothelial growth factor (VEGF)-stimulated endothelial cell proliferation, migration, and capillary-like tube formation. Deficient G6PD activity was associated with a significant decrease in endothelial cell proliferation, migration, and tube formation, whereas increased G6PD activity promoted these processes. VEGF-stimulated eNOS activity and NO* production were decreased significantly in endothelial cells with deficient G6PD activity and enhanced in G6PD-overexpressing cells. In addition, G6PD-deficient cells demonstrated decreased tyrosine phosphorylation of the VEGF receptor Flk-1/KDR, Akt, and eNOS compared with cells with normal G6PD activity, whereas overexpression of G6PD enhanced phosphorylation of Flk-1/KDR, Akt, and eNOS. In the Pretsch mouse, a murine model of G6PD deficiency, vessel outgrowth from thoracic aorta segments was impaired compared with C3H wild-type mice. In an in vivo Matrigel angiogenesis assay, cell migration into the plugs was inhibited significantly in G6PD-deficient mice compared with wild-type mice, and gene transfer of G6PD restored the wild-type phenotype in G6PD-deficient mice. These findings demonstrate that G6PD modulates angiogenesis and may represent a novel angiogenic regulator.     

Comparative molecular genetic analysis of lymphomas from six inbred mouse strains.

Previous studies of 21 highly lymphomatous AKXD recombinant inbred mouse strains demonstrated correlations between lymphoma type, the somatic proviral DNA content of the lymphoma, and the frequency of virally induced rearrangements in eight common sites of viral integration (Myc, Pim-i, Pvt-1, Mlvi-1, Mlvi-2, Fis-1, Myb, and Evi-1). In this study we analyzed lymphomas from six inbred mouse strains, AKR/J, C58/J, HRS/J (hr/hr and hr/+), SJL/J, SEA/GnJ, and CWD/LeAgl, to determine whether these correlations are also evident in these strains. Mice of the AKR/J, C58/J, and HRS/J strains died exclusively of T-cell lymphomas. In contrast to earlier studies which showed a great disparity in the rate and incidence of lymphomas in HRS/J hr/hr and HRS/J hr/+ mice, we found a high incidence of T-cell lymphomas and the same mean age of onset of disease in both strains. SJL/J mice died primarily of pre-B-cell lymphomas, whereas CWD/LeAgl and SEA/GnJ mice died primarily of B-cell lymphomas. Somatically acquired mink cell focus-forming proviruses were detected only in T-cell lymphomas, whereas ecotropic proviruses were found in lymphomas from all hematopoietic cell lineages. No rearrangements were detected in the Fis-1, Mlvi-2, and Myb loci, whereas rearrangements were detected in the Mlvi-1, Myc, Pim-1, Pvt-1, and Evi-1 loci. Most rearrangements were found in T-cell lymphomas, and many were virally induced. These results are similar to those we obtained previously for lymphomas of 21 highly lymphomatous AKXD recombinant inbred mouse strains.     

Detecting genotoxic effects of potential clastogens: An in vivo study using the transgenic lacZ plasmid and the Muta™Mouse model

In the present paper the capacity of the pUR288 plasmid mouse model and the Muta™Mouse model to detect the clastogens bleomycin, m-AMSA, o-AMSA and camptothecin, was investigated. Ethylnitrosourea (ENU) served as a positive control, methylcellulose as a negative control. Only bleomycin induced a slight but significant increase in lacZ mutant frequency (MF) in bone marrow of pUR288 plasmid mice. Exposure to the other compounds did not result in an increase in the MF in bone marrow and liver in both mouse models. For the Muta™Mouse this result was expected, for the plasmid mouse an increase in MF after clastogen exposure was expected. The positive control ENU induced statistically significant increases in MF compared with the negative control in both models and in both tissues analyzed. Hybridisation of DNA of mutant colonies derived from plasmid mice with labelled total mouse DNA (Hybridisation Assay) demonstrated an increase in the percentage of colonies hybridised with total mouse DNA as compared with the negative control, which suggests that there was indeed a biological response associated with treatment. The latter results indicate that the plasmid mouse assay may still be a promising model for the detection of clastogens.     

Evolutionarily plastic regions at human 3p21.3 coincide with tumor breakpoints identified by the "elimination test"

We have previously found with the microcell hybrid-based "elimination test" that human chromosome 3 transferred into murine or human tumor cells regularly lost certain 3p regions during tumor growth in SCID mice. The most common eliminated region, CER1, is approximately 2.4 Mb at 3p21.3. CER1 breakpoints were clustered in approximately 200-kb regions at both telomeric and centromeric borders. We have also shown, earlier, that tumor-related deletions often coincide with human/mouse synteny breakpoints on 3p12-p22. Here we describe the results of a comparative genomic analysis on the CER1 region in Caenorhabditis elegans, Drosophila melanogaster, Fugu rubripes, Gallus gallus, Mus musculus, Rattus norvegicus, and Canis familiaris. First, four independent synteny breaks were found within the CER1 telomeric breakpoint cluster region, comparing human, dog, and chicken genomes, and two independent synteny breaks within the CER1 centromeric breakpoint cluster region, comparing human, mouse, and chicken genomes, suggesting a nonrandom involvement of tumor breakpoint regions in chromosome evolution. Second, both CER1 breakpoint cluster regions show recent tandem duplications (seven Zn finger protein family genes at the telomeric and eight chemokine receptor genes at the centromeric side). Finally, all genes from these regions underwent horizontal evolution in mammals, with formation of new genes and expansion of gene families, which were displayed in the human genome as tandem gene duplications and pseudogene insertions. In contrast the CER1 middle region contained evolutionarily well-conserved solitary genes and a minimal amount of retroposed genes. The coincidence of evolutionary plasticity with CER1 breakpoints may suggest that regional structural instability is expressed in both evolutionary and cancer-associated chromosome rearrangements.