Spontaneous Chromosomal Instability Research Articles

Spontaneous slow replication fork progression elicits mitosis alterations in homologous recombination-deficient mammalian cells

Homologous recombination deficient (HR(-)) mammalian cells spontaneously display reduced replication fork (RF) movement and mitotic extra centrosomes. We show here that these cells present a complex mitotic phenotype, including prolonged metaphase arrest, anaphase bridges, and multipolar segregations. We then asked whether the replication and the mitotic phenotypes are interdependent. First, we determined low doses of hydroxyurea that did not affect the cell cycle distribution or activate CHK1 phosphorylation but did slow the replication fork movement of wild-type cells to the same level than in HR(-) cells. Remarkably, these low hydroxyurea doses generated the same mitotic defects (and to the same extent) in wild-type cells as observed in unchallenged HR(-) cells. Reciprocally, supplying nucleotide precursors to HR(-) cells suppressed both their replication deceleration and mitotic extra centrosome phenotypes. Therefore, subtle replication stress that escapes to surveillance pathways and, thus, fails to prevent cells from entering mitosis alters metaphase progression and centrosome number, resulting in multipolar mitosis. Importantly, multipolar mitosis results in global unbalanced chromosome segregation involving the whole genome, even fully replicated chromosomes. These data highlight the cross-talk between chromosome replication and segregation, and the importance of HR at the interface of these two processes for protection against general genome instability.

Helqacts in parallel toFanccto suppress replication-associated genome instability

HELQ is a superfamily 2 DNA helicase found in archaea and metazoans. It has been implicated in processing stalled replication forks and in repairing DNA double-strand breaks and inter-strand crosslinks. Though previous studies have suggested the possibility that HELQ is involved in the Fanconi anemia (FA) pathway, a dominant mechanism for inter-strand crosslink repair in vertebrates, this connection remains elusive. Here, we investigated this question in mice using the Helqgt and Fancc− strains. Compared with Fancc−/− mice lacking FANCC, a component of the FA core complex, Helqgt/gt mice exhibited a mild of form of FA-like phenotypes including hypogonadism and cellular sensitivity to the crosslinker mitomycin C. However, unlike Fancc−/− primary fibroblasts, Helqgt/gt cells had intact FANCD2 mono-ubiquitination and focus formation. Notably, for all traits examined, Helq was non-epistatic with Fancc, as Helqgt/gt;Fancc−/− double mutants displayed significantly worsened phenotypes than either single mutant. Importantly, this was most noticeable for the suppression of spontaneous chromosome instability such as micronuclei and 53BP1 nuclear bodies, known consequences of persistently stalled replication forks. These findings suggest that mammalian HELQ contributes to genome stability in unchallenged conditions through a mechanism distinct from the function of FANCC.

Separation of intra-S checkpoint protein contributions to DNA replication fork protection and genomic stability in normal human fibroblasts

The ATR-dependent intra-S checkpoint protects DNA replication forks undergoing replication stress. The checkpoint is enforced by ATR-dependent phosphorylation of CHK1, which is mediated by the TIMELESS-TIPIN complex and CLASPIN. Although loss of checkpoint proteins is associated with spontaneous chromosomal instability, few studies have examined the contribution of these proteins to unchallenged DNA metabolism in human cells that have not undergone carcinogenesis or crisis. Furthermore, the TIMELESS-TIPIN complex and CLASPIN may promote replication fork protection independently of CHK1 activation. Normal human fibroblasts (NHF) were depleted of ATR, CHK1, TIMELESS, TIPIN or CLASPIN and chromosomal aberrations, DNA synthesis, activation of the DNA damage response (DDR) and clonogenic survival were evaluated. This work demonstrates in NHF lines from two individuals that ATR and CHK1 promote chromosomal stability by different mechanisms that depletion of CHK1 produces phenotypes that resemble more closely the depletion of TIPIN or CLASPIN than the depletion of ATR, and that TIMELESS has a distinct contribution to suppression of chromosomal instability that is independent of its heterodimeric partner, TIPIN. Therefore, ATR, CHK1, TIMELESS-TIPIN and CLASPIN have functions for preservation of intrinsic chromosomal stability that are separate from their cooperation for activation of the intra-S checkpoint response to experimentally induced replication stress. These data reveal a complex and coordinated program of genome maintenance enforced by proteins known for their intra-S checkpoint function.

Physical and Functional Interaction Between Fanconi Anemia Group J Helicase and MRE11 Nuclease

FANCJ mutations are linked to the chromosomal instability disorder Fanconi anemia (FA) and increase breast cancer risk. FANCJ encodes a DNA helicase implicated in homologous recombination (HR) repair of double‐strand breaks (DSBs) and interstrand cross‐links (ICLs), but its mechanism of action is not well understood. Live cell imaging demonstrates that FANCJ recruitment to laser‐induced DSBs, but not psoralen‐ICLs, is dependent on nuclease‐active MRE11 and independent of BRCA1. Inhibition of MRE11 nuclease by Mirin adversely affected FANCJ localization to laser‐induced DSBs, but not psoralen‐ICLs. FANCJ is required for timely recruitment of Bloom's syndrome helicase and CtIP to DSBs in vivo. FANCJ directly interacts with MRE11 and inhibits its exonuclease activity in a specific manner, suggesting that FANCJ harnesses MRE11 nuclease to facilitate DSB processing and appropriate end resection. Spontaneous or IR‐induced chromosomal instability in FANCJ‐depleted cells is suppressed when MRE11 is depleted. Interplay between FANCJ and MRE11 insures a normal response to IR‐induced DSBs, whereas FANCJ involvement in ICL repair is regulated by MLH1 and FANCA, but not FANCD2. Thus, MRE11 helps to recruit FANCJ to DSBs and FANCJ can modulate MRE11 nuclease activity at DNA ends, consistent with a collaborative role of FANCJ and BLM helicases with MRE11 in strand resection.

Protective effect of acetyl-l-carnitine and α-lipoic acid against the acute toxicity of diepoxybutane to human lymphocytes

The biotransformation and oxidative stress may contribute to 1,2:3,4-diepoxybutane (DEB)-induced toxicity to human lymphocytes of Fanconi Anemia (FA) patients. Thus, the identification of putative inhibitors of bioactivation, as well as the determination of the protective role of oxidant defenses, on DEB-induced toxicity, can help to understand what is failing in FA cells. In the present work we studied the contribution of several biochemical pathways for DEB-induced acute toxicity in human lymphocyte suspensions, by using inhibitors of epoxide hydrolases, inhibitors of protective enzymes as glutathione S-transferase and catalase, the depletion of glutathione (GSH), and the inhibition of protein synthesis; and a variety of putative protective compounds, including antioxidants, and mitochondrial protective agents. The present study reports two novel findings: (i) it was clearly evidenced, for the first time, that the acute exposure of freshly isolated human lymphocytes to DEB results in severe GSH depletion and loss of ATP, followed by cell death; (ii) acetyl-l-carnitine elicits a significant protective effect on DEB induced toxicity, which was potentiated by α-lipoic acid. Collectively, these findings contribute to increase our knowledge of DEB-induce toxicity and will be very useful when applied in studies with lymphocytes from FA patients, in order to find out a protective agent against spontaneous and DEB-induced chromosome instability.

EZH2 Promotes Expansion of Breast Tumor Initiating Cells through Activation of RAF1-β-Catenin Signaling

It has been proposed that an aggressive secondary cancer stem cell population arises from a primary cancer stem cell population through acquisition of additional genetic mutations and drives cancer progression. Overexpression of Polycomb protein EZH2, essential in stem cell self-renewal, has been linked to breast cancer progression. However, critical mechanism linking increased EZH2 expression to BTIC (breast tumor initiating cell) regulation and cancer progression remains unclear. Here, we identify a mechanism in which EZH2 expression-mediated downregulation of DNA damage repair leads to accumulation of recurrent RAF1 gene amplification in BTICs, which activates p-ERK-β-catenin signaling to promote BTIC expansion. We further reveal that AZD6244, a clinical trial drug that inhibits RAF1-ERK signaling, could prevent breast cancer progression by eliminating BTICs.

Spontaneous and radiation-induced chromosomal instability and persistence of chromosome aberrations after radiotherapy in lymphocytes from prostate cancer patients

The aim of the study was to compare the spontaneous and ex vivo radiation-induced chromosomal damage in lymphocytes of untreated prostate cancer patients and age-matched healthy donors, and to evaluate the chromosomal damage, induced by radiotherapy, and its persistence. Blood samples from 102 prostate cancer patients were obtained before radiotherapy to investigate the excess acentric fragments and dicentric chromosomes. In addition, in a subgroup of ten patients, simple exchanges in chromosomes 2 and 4 were evaluated by fluorescent in situ hybridization (FISH), before the onset of therapy, in the middle and at the end of therapy, and 1 year later. Data were compared to blood samples from ten age-matched healthy donors. We found that spontaneous yields of acentric chromosome fragments and simple exchanges were significantly increased in lymphocytes of patients before onset of therapy, indicating chromosomal instability in these patients. Ex vivo radiation-induced aberrations were not significantly increased, indicating proficient repair of radiation-induced DNA double-strand breaks in lymphocytes of these patients. As expected, the yields of dicentric and acentric chromosomes, and the partial yields of simple exchanges, were increased after the onset of therapy. Surprisingly, yields after 1 year were comparable to those directly after radiotherapy, indicating persistence of chromosomal instability over this time. Our results indicate that prostate cancer patients are characterized by increased spontaneous chromosomal instability. This instability seems to result from defects other than a deficient repair of radiation-induced DNA double-strand breaks. Radiotherapy-induced chromosomal damage persists 1 year after treatment.

Frequency of sister chromatid exchanges and micronuclei monitored over time in patients with early-stage breast cancer: results of an observational study

Spontaneous chromosomal instability correlates with a high risk of cancer. The frequency of spontaneous sister chromatid exchanges (SCE) and micronuclei (MN) in peripheral blood lymphocytes was used for evaluation of spontaneous chromosomal instability in early-stage breast cancer patients to determine whether SCE and MN frequencies are biomarkers of damage from chemotherapy and radiotherapy. In 20 stage I–II breast cancer patients, SCE and MN were measured before surgery and at 4 weeks after. In patients who received adjuvant chemotherapy (CTx), they were also determined before starting radiotherapy (RTx). Other assessments were done 2, 6, and 12 months after RTx in almost all patients and at 18 months in 4 patients. Generalized estimating equations (GEE) were used to estimate population averaged effects at the different treatment and follow-up time points. Moreover, SCE and MN baseline values in patients were compared with those of a control group of 12 healthy women. A significant difference emerged between patients and healthy controls ( P < 0.0001 for SCE and P < 0.0003 for MN; Mann–Whitney test); SCE increased significantly after CTx and MN increased significantly after RTx. In the GEE model, the smoking habit was associated with increased SCE in patients treated with CTx; age significantly affected MN frequencies. Both MN and SCE frequencies are increased in breast cancer patients and are indicators of CTx and RTx damage, respectively. The increased SCE levels in patients treated with CTx may be due to a synergic effect of smoking and chemotherapy.

FANCJ helicase operates in the Fanconi Anemia DNA repair pathway and the response to replicational stress.

Fanconi anemia (FA) is an autosomal recessive disorder characterized by multiple congenital anomalies, progressive bone marrow failure, and high cancer risk. Cells from FA patients exhibit spontaneous chromosomal instability and hypersensitivity to DNA interstrand cross-linking (ICL) agents. Although the precise mechanistic details of the FA/BRCA pathway of ICL-repair are not well understood, progress has been made in the identification of the FA proteins that are required for the pathway. Among the 13 FA complementation groups from which all the FA genes have been cloned, only a few of the FA proteins are predicted to have direct roles in DNA metabolism. One of the more recently identified FA proteins, shown to be responsible for complementation of the FA complementation group J, is the BRCA1 Associated C-terminal Helicase (BACH1, designated FANCJ), originally identified as a protein associated with breast cancer. FANCJ has been proposed to function downstream of FANCD2 monoubiquitination, a critical event in the FA pathway. Evidence supports a role for FANCJ in a homologous recombination (HR) pathway of double strand break (DSB) repair. In this review, we will summarize the current knowledge in terms of FANCJ functions through its enzymatic activities and protein interactions. The molecular roles of FANCJ in DNA repair and the response to replicational stress will be discussed.

FANCD2 Purification and Identification of a Functionally Important Phosphorylation Event at Serine 331.

Fanconi anemia (FA) is an inherited hematological disorder characterized by birth defects, bone marrow failure, and cancer susceptibility. FA cells are characterized by spontaneous chromosomal instability and hypersensitivity to cross linking reagents such as mitomycin C (MMC). Cloned FA genes cooperate in a signaling pathway activated by DNA damage during replication, and monoubiquitination of FANCD2 is the critical downstream event. The check point kinase ATR is required for the efficient monoubiquitination of FANCD2. Inducible ATR-dependent phosphorylation of threonine 691 and serine 717 are reported to enhance the monoubiquitination of FANCD2 and resistance to MMC. In the present study, we have devised means to purify the FANCD2 protein into multiple subcomplexes. In doing so, we have identified at least 3 distinct subcomplexes: a pure multimeric precursor form comprised of FANCD2 polypeptides, a 1 MD form that is chromatin bound, and a 2 MD form that is in the nucleosol. Electron microscopic analysis of the precursor form reveals homogenous appearing complexes. Mass spectroscopic analysis of the 1MD FANCD2 complex has identified the FANCI protein, among others, which confirms recent reports of FANCD2-FANCI interaction. Further mass spectroscopic analysis of FANCD2 has identified a novel phosphorylation site on FANCD2, serine 331, which is further confirmed by immunoblot using phosphospecific antibody against phospho-serine 331. This phosphorylation site is conserved in other species, including mouse, rat, xenopus, and tetraodon nigroviridis. Single amino acid mutation by alanine at this site results in a complete loss of monoubquitination of FANCD2 and resistance to MMC, suggesting that the phosphorylation of S331 is a functionally significant event. Moreover, this site fits the consensus sequence for a CHK1 substrate, which in turn is a substrate of target for CHK1, a substrate of ATR, which is also required for monoubiquitination of FANCD2. These findings further support the correlation between phosphorylation and monoubiquitination of FANCD2 in the DNA damage response with the intervention of CHK1 as a mediator of ATR and suggest dynamic regulation of FANCD2 subcomplexes within the FA pathway.

Relationship between the Hematopoietic Phenotype and the Subtype of Fanconi Anemia Mice.

Fanconi anemia (FA) is a rare inherited disease associated with aplastic anemia and cancer predisposition. Great progress has occurred at a molecular level that allowed the identification of 13 genes involved in the disease. Additionally, several FA mouse models have been developed with the purpose of investigating the biological basis of the disease, and of facilitating the development of new therapies for FA. Because of the genetic heterogeneity of FA, here we have investigated the relevance of different FA genes to preserve the function of the hematopoietic stem cells (HSCs) of the mouse. To this aim, we have comparatively investigated the hematopoietic phenotype of different FA mice: Fanca−/−, Fancc−/−, Fancd1/Brca2Δ27/Δ27and Fancd2−/−. Normal counts of peripheral blood (PB) cells were observed in the different FA mice. Flow cytometry analyses were also incapable of demonstrating significant changes in the hematopoietic subpopulations present in PB, bone marrow (BM) or spleen of the different FA mice. In contrast to these observations, a reduction in the content of the hematopoietic progenitors was observed both in the BM and the spleen of Fancd1/Brca2Δ27/Δ27 and Fancd2−/− mice, either in young or adult. Significantly, a similar reduction in hematopoietic progenitors was only observed in old Fanca−/− and Fancc−/− mice. Moreover, hematopoietic colonies from mice defective in FA proteins downstream the FA core complex were smaller, compared to colonies from wild type, Fanca−/− or Fancc−/− mice. As expected, hematopoietic progenitors from all studied FA mouse models were highly sensitive to the DNA cross-linking agent, mitomycin C (MMC). Concerning the function of the HSCs of these FA mice, reduced competitive repopulation ability was always observed, compared to WT HSCs. However, in animals defective in FA proteins downstream the FA core, CRA values progressively dropped along the transplantation period. Additionally, a spontaneous chromosomal instability was observed in bone marrow cells from Fancd1/Brca2Δ27/Δ27. These animals also showed a marked repopulation defect in their HSCs assessed in their natural physiological environment, when wild type cells were transplanted in unconditioned Brca2Δ27/Δ27 recipients. In summary, our results show a more severe defect in the functionality of the HSC compartment in mice with mutant FA proteins downstream the FA core complex.

Niacin deficiency delays DNA excision repair and increases spontaneous and nitrosourea-induced chromosomal instability in rat bone marrow

We have shown that niacin deficiency impairs poly(ADP-ribose) formation and enhances sister chromatid exchanges and micronuclei formation in rat bone marrow. We designed the current study to investigate the effects of niacin deficiency on the kinetics of DNA repair following ethylation, and the accumulation of double strand breaks, micronuclei (MN) and chromosomal aberrations (CA). Weanling male Long-Evans rats were fed niacin deficient (ND), or pair fed (PF) control diets for 3 weeks. We examined repair kinetics by comet assay in the 36 h following a single dose of ethylnitrosourea (ENU) (30 mg/kg bw). There was no effect of ND on mean tail moment (MTM) before ENU treatment, or on the development of strand breaks between 0 and 8 h after ENU. Repair kinetics between 12 and 30 h were significantly delayed by ND, with a doubling of area under the MTM curve during this period. O 6-ethylation of guanine peaked by 1.5 h, was largely repaired by 15 h, and was also delayed in bone marrow cells from ND rats. ND significantly enhanced double strand break accumulation at 24 h after ENU. ND alone increased chromosome and chromatid breaks (four- and two-fold). ND alone caused a large increase in MN, and this was amplified by ENU treatment. While repair kinetics suggest that ND may be acting by creating catalytically inactive PARP molecules with a dominant-negative effect on repair processes, the effect of ND alone on O 6-ethylation, MN and CA, in the absence of altered comet results, suggests additional mechanisms are also leading to chromosomal instability. These data support the idea that the bone marrow cells of niacin deficient cancer patients may be more sensitive to the side effects of genotoxic chemotherapy, resulting in acute bone marrow suppression and chronic development of secondary leukemias.

Asexual Recombination in a uvsH Mutant of Aspergillus nidulans

Mutations in the gene uvsH of Aspergillus nidulans result in increased spontaneous chromosome instability and increased intragenic and intergenic mitotic recombination in homozygous diploids. The aim of the present work was to obtain a uvs mutant of A. nidulans and to use it for the isolation of asexual recombinants (parameiotic segregants). The mutant uvsH, named B511, showed normal frequency of meiotic recombination in sexual crosses and high frequency of parameiotic segregants in the parasexual crossings with master strains (B511//A757 and B511//A288). Asexual haploid recombinants (parameiotic segregants), diploid and aneuploid segregants were recovered directly from the uvs//uvs+ heterokaryons (B511//A757 and B511// A288). Parameiotic segregants originated through mitotic crossing-over and independent assortment of chromosomes.

Hematopoietic Dysfunction in a Mouse Model for Fanconi Anemia Group D1

We have investigated the hematopoietic phenotype of mice with a hypomorphic mutation in the Brca2/Fancd1 gene (Brca2Δ27/Δ27 mutation). In contrast to observations made in other Fanconi anemia (FA) mouse models, low numbers of hematopoietic colony-forming cells (CFCs) were noted in Brca2Δ27/Δ27 mice, either young or adult. Additionally, a high incidence of spontaneous chromosomal instability was observed in Brca2Δ27/Δ27 bone marrow (BM) cells, but not in Brca2+/Δ27 or Fanca−/− BM cells. Although Brca2Δ27/Δ27 CFCs were not hypersensitive to ionizing radiation, a very severe hematopoietic syndrome was observed in irradiated Brca2Δ27/Δ27 mice. Conventional BM competition experiments showed a marked repopulation defect in Brca2Δ27/Δ27 hematopoietic stem cells (HSCs), compared to wild-type HSCs. Moreover, we have observed for the first time in a DNA repair disease model a very significant proliferation defect in Brca2Δ27/Δ27 HSCs maintained in their natural physiological environment. The progressive repopulation of wild-type HSCs transplanted into unconditioned Brca2Δ27/Δ27 recipients is reminiscent of the somatic mosaicism phenomenon observed in a number of genetic diseases, including FA. The hematopoietic phenotype associated with the Brca2Δ27/Δ27 mutation suggests that this FA-D1 mouse model will constitute an important tool for the development of new therapies for FA, including gene therapy.

Meiotic Behavior of Carica papaya L.: Spontaneous Chromosome Instability and Elimination in Important cvs. in North Indian Conditions

Cytogenetical analysis was done in tropical species of papaya that is widely cultivated for its fruits and papain production. The proper and orderly chromosome behavior of the species is a perquisite to ensure high fertility and seed set in the crop. A study on pre-meiotic and post-meiotic stages was done on commercially important cultivars of India and few exotic cultivars. The meiosis in the flower buds was highly disturbed and many meiotic irregularities were recorded from the material fixed in summar. These ranged from chromosome stickiness (44%), chromatin bridges (21.1%), laggards (4.8), chromosome diminution/degeneration (5.2), and micronucleus (5.9%) formation, in the commercially important varieties. Pollen stickiness accounting for persistent pollen adherence in groups of 3–10 was observed in all the varieties ranging from 85.95% in cv. CO2 to 11.64% in CO5. Callose deposition on tetrads accounting for adhesion of pollen grains (permanent tetrads) was also observed. These were found to have a profound effect on the pollen fertility of the cultivars, wherein most of the commercial papaya cultivars had less than 50% of fertile pollen. Thus the role of meiotic irregularities in poor seed setting and seed production capacities under northern Indian conditions, specifically during the summer season has been established. Genetic or abiotic stress factors could be responsible for inducing such kind of anomalies, which are manifested in the form of pollen sterility and poor seed setting. Crop regulation strategies need to be evolved, so that the maximum seed set and fruit production occurs after this period to off set the restrictions imposed due to irregular meiosis.

Chromosomal Instability in Peripheral Blood Lymphocytes and Risk of Prostate Cancer

Prostate cancer is an extremely complex disease, and it is likely that chromosomal instability is involved in the genetic mechanism of tumorigenesis. Several chromosomes have been labeled as "players" in the development of prostate cancer, among them chromosome 1 and X chromosome have been reported to harbor prostate cancer susceptibility loci. However, there is little information regarding the background levels of chromosome instability in these patients. In this pilot study, we examined spontaneous chromosome instability in short-term lymphocyte cultures from 126 study subjects, 61 prostate cancer patients, and 65 healthy controls. We evaluated chromosomal instability using a fluorescence in situ hybridization assay using two probes targeting specific regions on X chromosome and chromosome 1. Our results showed a significantly higher mean level of spontaneous breaks involving the X chromosome in patients compared with controls (mean +/- SE, 2.41 +/- 0.26 and 0.62 +/- 0.08, respectively; P < 0.001). Similarly, chromosome 1 spontaneous breaks were significantly higher among cases compared with controls (mean +/- SE, 1.95 +/- 0.24 and 1.09 +/- 0.16, respectively; P < 0.001). Using the median number of breaks in the controls as the cutoff value, we observed an odds ratio (95% confidence interval) of 15.53 (5.74 - 42.03; P < 0.001) for spontaneous X chromosome breaks and 3.71 (1.60 - 8.63; P < 0.001) for chromosome 1 breaks and risk of development of prostate cancer. In conclusion, our preliminary results show that spontaneous chromosome instability could be a risk factor for prostate cancer.

Chromosomal instability in two siblings with gonad deficiency: Case report

Non-random de novo autosomal chromosomal rearrangements have not been shown to cause exocrine or gonadal dysfunction. We report on two siblings, a brother and a sister, both with de novo chromosomal rearrangements and gonadal deficiency including premature ovarian failure. They had normal phenotypes without additional manifestations of known chromosomal breakage syndromes (except for the gonadal dysfunction) and normal alpha-fetoprotein dosage level. The association of sperm abnormalities in the brother and ovarian dysfunction in the sister suggested an increased spontaneous chromosomal instability. Since the co-occurrence of chromosomal anomalies and reproductive failures may not be coincidental, we performed repeated chromosomal analysis of peripheral blood lymphocytes prior to proposing ICSI for IVF (for the brother). In both sibs, infertility was associated with random and non-random de novo autosomal chromosomal abnormalities. We discuss the possible relationship between these unusual clinical and cytogenetic features and their potential links to ataxia-telangiectasia.

Association of biallelicBRCA2/FANCD1mutations with spontaneous chromosomal instability and solid tumors of childhood

AbstractThe clinical, cytogenetic, and molecular findings of 2 Fanconi anemia (FA) subtype D1 kindreds, initially identified through a young child with a solid tumor (medullobastoma, Wilms tumor), are described. Each kindred subsequently had a second affected child; one developed Wilms tumor followed by a medulloblastoma, and the other developed T-lineage acute lymphoblastic leukemia. Cytogenetic studies revealed an unusually high spontaneous chromosome aberration rate, contrasting with other FA subtypes. Molecular analysis revealed biallelicBRCA2/FANCD1mutations. The patients did not exhibit bone marrow failure. Our studies suggest that the D1 subtype represents a severe end of the cytogenetic spectrum within FA, consistent with a critical downstream role ofBRCA2in the FA pathway. Furthermore, this FA subgroup may be preferentially associated with an increased predisposition to solid tumors in early childhood. Recognition of this constellation of findings has significant implications for medical management and genetic counseling of FA families.

The function of RecQ helicase gene family (especially BLM) in DNA recombination and joining

Bloom syndrome is a rare autosomal recessive genetic disorder characterized by lupus-like erythematous telangiectasias of the face, sun sensitivity, stunted growth, and immunodeficiency. Chromosome instability syndromes have a common feature, being associated at high frequency with neoplasia. BS is considered as one of the chromosome instability syndromes since the fibroblasts or lymphocytes of BS patients show excessive spontaneous chromosome instability. The causative gene of BS (BLM) was identified as a RecQ helicase homologue. In this review, we showed the characteristic phenotypes of BS, especially two Japanese siblings. In the latter of the review, the functional domains of BLM, those are nuclear localization signal and the interacting proteins such as ATM, are shown. Several lines of reports indicates that BLM helicase is involved in the re-initiation of DNA replication at sites where replication forks have arrested or collapsed. To elucidate the precise function of RecQ helicase in DNA repair and replication aims not only to improve our understanding of the molecular basis for tumorigenesis, but also to extend the range of potential therapeutic targets.

Chromosome instability and nibrin protein variants in NBS heterozygotes.

The frequency of spontaneous chromosome abnormalities in peripheral blood lymphocytes and the X-ray G2 sensitivity in lymphoblastoid cell lines (LCL) have been evaluated in heterozygous subjects from three unrelated Nijmegen Breakage Syndrome (NBS) families, characterised by different mutations in the NBS1 gene. In all the 13 NBS heterozygotes analysed, we found spontaneous chromosome instability consisting in chromosome and chromatid breakages and rearrangements, while radiosensitivity was similar to that of control LCLs in seven out of eight tested NBS heterozygotes. The densitometric analysis of nibrin by immunoblotting indicated only a slight reduction in some of the LCLs from NBS carriers, whereas the immunoprecipitation assay appears a more reliable tool to detect NBS carriers. By means of immunoprecipitation, we investigated two homozygous and four heterozygous subjects. In the cells of the NBS patient 668, with the mutation 900del25, an alternative form of nibrin with a molecular weight of approximately 55 kDa has been detected. This variant protein, together with the normal p95, was also found in the LCL 34 established from a carrier of the same family. Signals of nibrin with a molecular weight lower than 95 kDa, but higher than that observed in LCLs 668 and 34, were detected also in three carriers from the family with mutation 835del4.