Common fragile sites in colon cancer cell lines: role of mismatch repair, RAD51 and poly(ADP-ribose) polymerase-1

Common fragile sites (CFS) are specific chromosomal areas prone to form gaps and breaks when cells are exposed to stresses that affect DNA synthesis, such as exposure to aphidicolin (APC), an inhibitor of DNA polymerases. The APC-induced DNA damage is repaired primarily by homologous recombination (HR), and RAD51, one of the key players in HR, participates to CFS stability. Since another DNA repair pathway, the mismatch repair (MMR), is known to control HR, we examined the influence of both the MMR and HR DNA repair pathways on the extent of chromosomal damage and distribution of CFS provoked by APC and/or by RAD51 silencing in MMR-deficient and -proficient colon cancer cell lines (i.e., HCT-15 and HCT-15 transfected with hMSH6, or HCT-116 and HCT-116/3+6, in which a part of a chromosome 3 containing the wild-type hMLH1 allele was inserted). Here, we show that MMR-deficient cells are more sensitive to APC-induced chromosomal damage particularly at the CFS as compared to MMR-proficient cells, indicating an involvement of MMR in the control of CFS stability. The most expressed CFS is FRA16D in 16q23, an area containing the tumour suppressor gene WWOX often mutated in colon cancer. We also show that silencing of RAD51 provokes a higher number of breaks in MMR-proficient cells with respect to their MMR-deficient counterparts, likely as a consequence of the combined inhibitory effects of RAD51 silencing on HR and MMR-mediated suppression of HR. The RAD51 silencing causes a broader distribution of breaks at CFS than that observed with APC. Treatment with APC of RAD51-silenced cells further increases DNA breaks in MMR-proficient cells. The RNAi-mediated silencing of PARP-1 does not cause chromosomal breaks or affect the expression/distribution of CFS induced by APC. Our results indicate that MMR modulates colon cancer sensitivity to chromosomal breaks and CFS induced by APC and RAD51 silencing.     

How common are common fragile sites: variation of aphidicolin-induced chromosomal fragile sites in a population of the deer mouse (Peromyscus maniculatus)

Aphidicolin (APC)-induced chromosomal gaps and breaks were analyzed for ten deer mice (Peromyscus maniculatus) from a natural population. The FSM statistical methodology was used to identify fragile sites as chromosomal loci exhibiting significantly non-random numbers of gaps/breaks in each individual and enabled an assessment of variation in fragile sites among the individuals. The individual deer mice exhibited as few as 7 to as many as 19 of the populational total of 34 sites. Two sites were fragile in all individuals and 13 sites were fragile in single individuals only. Defined by populational frequencies of greater than 50%, high-frequency fragile sites constituted 26% of the populational total. Approximately 35% of the total fragile sites were fragile in 20–40% of the population (low-frequency fragile sites) and about 38% were fragile in single individuals only. Analysis of the data pooled over all individuals identified significantly non-random breakage at 80 sites, 47 of which were not identified as fragile in any single individual. It appears, therefore, that fragile site identifications from pooled data have fostered an inflated estimate of the numbers and frequencies of common fragile sites. Comparison of the fragile site and spontaneous breakage (control) data suggest that APC-induced fragile sites represent regions of chromosomes that experience elevated levels of somatic mutation. Additionally, the occurrence of APC-induced fragile sites at or near the interstitial breakpoints of two pericentric-inversion polymorphisms in this population supports the hypothesis that fragile sites experience an increased rate of meiotic chromosomal mutation and are predisposed to undergo phylogenetic rearrangement. Received: 22 January 1997 / Accepted: 24 February 1997     

Aphidicolin and bleomycin induced chromosome damage as biomarker of mutagen sensitivity: a twin study

The induction of chromosome damage in cultured human lymphocytes by in vitro treatments with aphidicolin (APC) and bleomycin (BLM) has been proposed as test of sensitivity to mutagens. To assess their validity, we have investigated whether the individual expression of induced chromosome damage has a genetic rather than an environmental basis. Metaphase analysis for chromosomal aberrations (CA) and micronucleus (MN) assay in cytokinesis-blocked cells have been performed in peripheral blood lymphocytes from 19 healthy male twins (9 monozygotic and 10 dizygotic pairs), aged 70-78 years, after APC, BLM and APC+BLM treatments.Concordance between twins revealed a high genetic component in the sensitivity towards clastogenic action of APC both as percentages of CA and MN. The micronucleus assay demonstrated a genetic basis also in the expression of chromosome damage induced by BLM and APC+BLM treatments. Since twins were elderly people, to investigate the possible role of age, CA and MN frequencies were compared with those found in lymphocytes from 11 young male donors. Basal and APC-induced chromosome damage were clearly increased in the former. Following BLM and APC+BLM treatments, age significantly increased mitotic delay, as shown by the mitotic indexes (MI) and by the ratios between binucleated and mononucleated (B/M) cells.     

How common are common fragile sites in humans: interindividual variation in the distribution of aphidicolin-induced fragile sites

To obtain an estimate of the variation in common fragile sites (CFSs) among individuals, aphidicolin (APC)-induced chromosomal breakage data were analyzed for 20 karyotypically normal adult humans. As it is specifically designed to meet the analytical requirements for considering fragile sites as presence/absence characters in single individuals, the FSM methodology (B?hm et al., 1995) was used to statistically distinguish fragile from nonfragile sites. These analyses indicated that the APC-induced fragile sites are not ubiquitous but vary extensively among individuals; the per-individual number of fragile sites ranged from as few as seven to as many as 20. Of the 45 different sites identified as fragile, 19 (42%) occurred in more than half of the individuals, but only two sites (3p14 and 16q23) were fragile in all of the individuals; 12 (27% of the total) were fragile in single individuals only. Although these analyses provide statistical confirmation (and initial estimates of population variation) for 43 of the 88 APC-inducible fragile sites currently recognized as occurring among humans, they are consistent with the hypothesis that many of the currently recognized human CFSs have been erroneously identified. These results indicate the need for per-individual statistical identification of CFSs for larger samples of individuals and that studies of particular fragile sites should be conducted on individuals documented to be fragile at the loci under consideration.     

Expression of aphidicolin, FUdR and caffeine‐induced fragile sites in lymphocytes of healthy Turkish individuals

The expression of common fragile sites (c‐fra) and frequency of chromosomal aberrations were studied in peripheral lymphocytes of 50 healthy Turkish individuals (26 males and 24 females from 1 to 87 years of age) after induction with aphidicolin (APC), 5′‐fluorodeoxyuridine (FUdR), and caffeine. A correlation was seen between age and the frequency of chromosomal aberrations in APC and caffeine treated cultures, but there were no significant differences in the frequencies of chromosomal aberrations between males and females in any of the treatments. The mean frequency of aberrations induced by FUdR was significantly higher than that induced by APC and caffeine. A chromosome aberration is defined as a fragile site when present in 1% of the cells analyzed from each culture and in at least 50% of the individuals studied. Using these criteria, 12 c‐fra were observed in the three treatments: 1p21, 1q21, 2p11‐q11, 3p14, 4q31, 6q26, 7q22, 7q32, 8q24, 11q23, 16q23, and Xp22. Sites 3p14, 16q23, and Xp22 were the most frequently observed c‐fra, with only the frequency of Xp22 being significantly increased in females in APC treated cultures. The results of these studies are important as a base against which the effects of other clastogenic and environmental agents, as well as genetic background, can be compared. This revised version was published online in July 2006 with corrections to the Cover Date.     

Induction of sister chromatid exchanges at common fragile sites.

Experiments were performed to gain further insight into chromosome structure and behavior at common fragile sites by testing the hypothesis that gaps at these sites predispose to intrachromosomal recombination as measured by sister chromatid exchanges (SCEs). Human lymphocytes were concurrently treated with aphidicolin, for determination of fragile site expression, and with 5-bromodeoxy-uridine, for SCE analysis. Aphidicolin induced chromosome gaps nonrandomly, with the great majority of gaps occurring at common fragile sites. On average, 66% of gaps were accompanied by an SCE at the site of the lesion. Analysis of two specific common fragile sites at 3p14 and 16q23 showed the same pattern; that is, on average 70% of gaps at these sites were accompanied by an SCE. These results show that common fragile sites are hot spots not only for chromosomal lesions such as gaps but also for SCE formation.     

Correlated fragile site expression allows the identification of candidate fragile genes involved in immunity and associated with carcinogenesis

Background  

Common fragile sites (cfs) are specific regions in the human genome that are particularly prone to genomic instability under conditions of replicative stress. Several investigations support the view that common fragile sites play a role in carcinogenesis. We discuss a genome-wide approach based on graph theory and Gene Ontology vocabulary for the functional characterization of common fragile sites and for the identification of genes that contribute to tumour cell biology.     

Genetic determination of fragile-site expression.

To explore genetic effects on the expression of chromosomal fragile sites in vitro, we studied the expression of common fragile sites (c-fra) in cultured lymphocytes of a human chimera (Chi46,XX/46,XY). Since the two cell lines in the chimera share the same environment in vitro and in vivo on cell culture preceding chromosome analysis, differences in the expression of c-fra must be due to genetic factors. The peripheral lymphocytes were cultured in medium 199 and in medium RPMI 1640 with and without aphidicolin. All lesions were localized after GTG-banding and mapped to the human idiogram. In the cultures with aphidicolin the XX cells showed, at high (0.4 microM) APC levels, a significantly higher expression of c-fra than did the XY cells. This difference between cell lineages was not confined to certain individual c-fra; rather it was seen for practically all of them. Therefore, we conclude that there are genetic factors which influence the propensity of c-fra to be expressed. Whether sex is one of these factors, or perhaps even the most important one, still has to be elucidated.     

Common fragile sites in man and three closely related primate species

The expression of common fragile sites was studied in peripheral lymphocytes of man, gorilla, chimpanzee, and orangutan after induction with aphidicolin, methotrexate, or fluorodeoxyuridine. As far as the chromosomal localization is concerned, it appears that many of these sites have been highly conserved during primate evolution. However, differences were found in the relative expression of certain sites. In all four species, mapping of approximately 500 lesions disclosed the most breakage-prone common fragile sites, at which about 90% of all induced aberrations were localized. Comparison of chromosome regions involved in evolutionary changes to fragile sites in the four primate species revealed 30 sites that were located at or close to the same chromosomal band. However, no correlation was found between the relative expression of a certain common fragile site in vitro and a potential involvement of this chromosomal site in evolutionary changes.     

Increased common fragile site expression, cell proliferation defects, and apoptosis following conditional inactivation of mouse Hus1 in primary cultured cells

Targeted disruption of the mouse Hus1 cell cycle checkpoint gene results in embryonic lethality and proliferative arrest in cultured cells. To investigate the essential functions of Hus1, we developed a system for the regulated inactivation of mouse Hus1 in primary fibroblasts. Inactivation of a loxP site-flanked conditional Hus1 allele by using a cre-expressing adenovirus resulted in reduced cell doubling, cell cycle alterations, and increased apoptosis. These phenotypes were associated with a significantly increased frequency of gross chromosomal abnormalities and an S-phase-specific accumulation of phosphorylated histone H2AX, an indicator of double-stranded DNA breaks. To determine whether these chromosomal abnormalities occurred randomly or at specific genomic regions, we assessed the stability of common fragile sites, chromosomal loci that are prone to breakage in cells undergoing replication stress. Hus1 was found to be essential for fragile site stability, because spontaneous chromosomal abnormalities occurred preferentially at common fragile sites upon conditional Hus1 inactivation. Although p53 levels increased after Hus1 loss, deletion of p53 failed to rescue the cell-doubling defect or increased apoptosis in conditional Hus1 knockout cells. In summary, we propose that Hus1 loss leads to chromosomal instability during DNA replication, triggering increased apoptosis and impaired proliferation through p53-independent mechanisms.     

Multiple common fragile sites are expressed in the genome of the laboratory rat

Splenic lymphocytes from Sprague Dawley and Fischer 344 rats were exposed to two chemicals known to induce common fragile site expression in man: fluorodeoxyuridine (in conjunction with the enhancing effects of caffeine) and aphidicolin. Of 39 sites that were significantly damaged in excess, 12 meet the criteria for fragility proposed in this investigation. Rat fragile sites appear to differ from those in man in that no common hierarchical frequency of expression is evident from the two methods of induction. In addition, a comparison of published cancer-specific chromosome breakpoints from a variety of rat tumors reveals little or no apparent concordance with the identified fragile sites. The rat is an animal model in which multiple common fragile sites can be induced and, as such, will be valuable for testing hypotheses concerning the biological basis of chromosomal fragility.     

Mutations in the APC tumour suppressor gene cause chromosomal instability

Two forms of genetic instability have been described in colorectal cancer: microsatellite instability and chromosomal instability. Microsatellite instability results from mutations in mismatch repair genes; chromosomal instability is the hallmark of many colorectal cancers, although it is not completely understood at the molecular level. As truncations of the Adenomatous Polyposis Coli (APC) gene are found in most colorectal tumours, we thought that mutations in APC might be responsible for chromosomal instability. To test this hypothesis, we examined mouse embryonic stem (ES) cells homozygous for Min (multiple intestinal neoplasia) or Apc1638T alleles. Here we show that Apc mutant ES cells display extensive chromosome and spindle aberrations, providing genetic evidence for a role of APC in chromosome segregation. Consistent with this, APC accumulates at the kinetochore during mitosis. Apc mutant cells form mitotic spindles with an abundance of microtubules that inefficiently connect with kinetochores. This phenotype is recapitulated by the induced expression of a 253-amino-acid carboxy-terminal fragment of APC in microsatellite unstable colorectal cancer cells. We conclude that loss of APC sequences that lie C-terminal to the beta-catenin regulatory domain contributes to chromosomal instability in colorectal cancer.     

Evidence for a relatively random array of human chromosomes on the mitotic ring

We used fluorescence in situ hybridization (FISH) to study the positions of human chromosomes on the mitotic rings of cultured human lymphocytes, MRC-5 fibroblasts, and CCD-34Lu fibroblasts. The homologous chromosomes of all three cell types had relatively random positions with respect to each other on the mitotic rings of prometaphase rosettes and anaphase cells. Also, the positions of the X and Y chromosomes, colocalized with the somatic homologues in male cells, were highly variable from one mitotic ring to another. Although random chromosomal positions were found in different pairs of CCD-34Lu and MRC-5 late-anaphases, the separations between the same homologous chromosomes in paired late-anaphase and telophase chromosomal masses were highly correlated. Thus, although some loose spatial associations of chromosomes secondary to interphase positioning may exist on the mitotic rings of some cells, a fixed order of human chromosomes and/or a rigorous separation of homologous chromosomes on the mitotic ring are not necessary for normal mitosis. Furthermore, the relative chromosomal positions on each individual metaphase plate are most likely carried through anaphase into telophase.     

Membrane-bound CD95 ligand expressed on human antigen-presenting cells prevents alloantigen-specific T cell response without impairment of viral and third-party T cell immunity

Genetically modified antigen-presenting cells (APC) represent an attractive strategy for in vitro immunomodulation. In the human system, APC expressing HLA-A1 and a membrane-bound form of CD95L (m-CD95L) were used for selective depletion of HLA-A1-specific T cells. In short-term assays, m-CD95L-expressing APC-induced apoptosis in activated T cells and the constitutive presence of m-CD95L and HLA-A1 expressing APC in long-term T cell cultures prevented the expansion of CD4(+) and CD8(+) HLA-A1-specific T cells and the development of HLA-A1-specific cytotoxicity. However, immunity towards third party, viral and bacterial antigens was maintained and T cells spared from depletion could be induced to develop cytotoxicity towards unrelated antigens. Interestingly, inhibition of HLA-A1-specific T cell response absolutely requires the coexpression of m-CD95L and HLA-A1 antigen on the same APC. Thus, m-CD95L expressing APC might be used in clinical settings to obtain tolerance induction in allogeneic transplantation systems or autoimmune diseases.     

Increased genetic instability of the common fragile site at 3p14 after integration of exogenous DNA.

We determined previously that the selectable marker pSV2neo is preferentially inserted into chromosomal fragile sites in human x hamster hybrid cells in the presence of an agent (aphidicolin) that induces fragile-site expression. In contrast, cells transfected without fragile-site induction showed an essentially random integration pattern. To determine whether the integration of marker DNA at fragile sites affects the frequency of fragile-site expression, the parental hybrid and three transfectants (two with pSV2neo integrated at the fragile site at 3p14.2 [FRA3B] and specific hamster fragile sites [chromosome 1, bands q26-31, or mar2, bands q11-13] and one with pSV2neo integrated at sites that are not fragile sites) were treated with aphidicolin. After 24 h the two cell lines with plasmid integration at FRA3B showed structural rearrangements at that site; these rearrangements accounted for 43%-67% of the total deletions and translocations observed. Structural rearrangements were not observed in the parental cell line. After 5 d aphidicolin treatment, the observed excess in frequency of structural rearrangements at FRA3B in the cell lines with pSV2neo integration at 3p14 over that in the two lines without FRA3B integration was less dramatic, but nonetheless significant. Fluorescent in situ hybridization (FISH) analysis of these cells, using a biotin-labeled pSV2neo probe, showed results consistent with the gross rearrangements detected cytogenetically in the lines with FRA3B integration; however, the pSV2neo sequences were frequently deleted concomitantly with translocations.(ABSTRACT TRUNCATED AT 250 WORDS)     

Arm-specific telomere dynamics of each individual chromosome in induced pluripotent stem cells revealed by quantitative fluorescence in situ hybridization

We have reported that telomere fluorescence units (TFUs) of established induced pluripotent stem cells (iPSCs) derived from human amnion (hAM933) and fetal lung fibroblasts (MRC-5) were significantly longer than those of the parental cells, and that the telomere extension rates varied quite significantly among clones without chromosomal instability, although the telomeres of other iPSCs derived from MRC-5 became shorter as the number of passages increased along with chromosomal abnormalities from an early stage. In the present study we attempted to clarify telomere dynamics in each individual chromosomal arm of parental cells and their derived clonal human iPSCs at different numbers of passages using quantitative fluorescence in situ hybridization (Q-FISH). Although no speci?c arm of any particular chromosome appeared to be consistently shorter or longer than most of the other chromosomes in any of the cell strains, telomere elongation in each chromosome of an iPSC appeared to be random and stochastic. However, in terms of the whole genome of any specific cell, the telomeres showed overall elongation associated with iPSC generation. We have thus demonstrated the specific telomere dynamics of each individual chromosomal arm in iPSCs derived from parental cells, and in the parental cells themselves, using Q-FISH.     

Cytoskeletal Polarization of T Cells Is Regulated by an Immunoreceptor Tyrosine-based Activation Motif–dependent Mechanism

Abstract. Binding of a T cell to an appropriate antigen-presenting cell (APC) induces the rapid reorientation of the T cell cytoskeleton and secretory apparatus towards the cell–cell contact site in a T cell antigen receptor (TCR) and peptide/major histocompatibility complex–dependent process. Such T cell polarization directs the delivery of cytokines and cytotoxic mediators towards the APC and contributes to the highly selective and specific action of effector T cells. To study the signaling pathways that regulate cytoskeletal rearrangements in T lymphocytes, we set up a conjugate formation assay using Jurkat T cells as effectors and cell-sized latex beads coated with various antibodies as artificial APCs. Here, we report that beads coated with antibodies specific for the TCR-CD3 complex were sufficient to induce T cell polarization towards the bead attachment site, as judged by reorientation of the microtubule-organizing center (MTOC) and localized actin polymerization. Thus, these cytoskeletal changes did not depend on activation of additional coreceptors. Moreover, single subunits of the TCR complex, namely TCR-ζ and CD3ε, were equally effective in inducing cytoskeletal polarization. However, mutagenesis of the immunoreceptor tyrosine-based activation motifs (ITAMs), present three times in TCR-ζ and once in CD3ε, revealed that the induction of cytoskeletal rearrangements required the presence of at least one intact ITAM. In agreement with this result, lack of functional Lck, the protein tyrosine kinase responsible for ITAM phosphorylation, abolished both MTOC reorientation and polarized actin polymerization. Both inhibitor and transient overexpression studies demonstrated that MTOC reorientation could occur in the absence of Ras activation. Our results suggest that APC-induced T cell polarization is a TCR-mediated event that is coupled to the TCR by the same signaling motif as TCR-induced gene activation, but diverges in its distal signaling requirements.     

Segments of Djungarian hamster chromosomes, specific for translocation of integration of amplified mdr1 genes, do not possess increased instability]

Earlier we have revealed in Djungarian hamster DM-15 cells three chromosomal segments (2p22, 5p1, 7q23-25) that are specific for transposition of amplified mdr1 genes from the site of location of resident gene copy (it was mapped to 4q21-23). In situ hybridization revealed in wild type cells no mdr1 homologous sequences in all these three segments. In this work we studied distribution of chromosomal breakages induced in DM-15 cells by aphidicolin, 5-fluorodeoxyuridine or N-phosphonacetyl-L-aspartate. The data obtained indicate that two of above mentioned segments. 2p22 and 7q23-25, contain no fragile sites. So, specificity of these segments for the transposition of amplified mdr1 genes is not due to their particular instability or to the presence in them of sequences homologous to amplifiable selectable gene.     

Epigallocatechin gallate induces telomere fragmentation in HeLa and 293 but not in MRC-5 cells

Telomeres are the tandem repetitive sequence at the end of chromosomes and its integrity is crucial for cell vitality. We studied the effect of (-)-epigallocatechin-3-gallate (EGCG), one of the major tea polyphenols, on telomeres in HeLa, 293 cells and MRC-5 fibroblasts. At concentrations of above 50 microM, EGCG was found to causes telomere fragmentation in HeLa cells as a result of single-strand breaks in a dose-dependent manner. Treatment of EGCG also caused telomere fragmentation in 293 cells but had little or only marginal effect on MRC-5 fibroblasts. The telomere fragments detected by electrophoresis showed a unique size distribution that seems to suggest that the strand breaks were not produced randomly, but with preference at some specific sites. We speculate that the differential effect of EGCG in inducing telomere fragmentation in HeLa and 293 verse MRC-5 cells might be relevant to the apoptosis-inducing effect of EGCG on cancerous cells but not on normal cells.     

Assessment of the genotoxicity of atenolol in human peripheral blood lymphocytes: Correlation between chromosomal fragility and content of micronuclei

The antihypertensive drug atenolol was found to induce chromosome loss, detected as micronuclei in the peripheral lymphocytes of treated patients. The fundamental question which chromosomes the micronuclei were derived from remains to be answered. Analysis of structural chromosomal aberrations (CAs) and expression of fragile sites (FS) were pursued in this study. They revealed a significantly higher incidence of chromosomal aberrations (chromatid and chromosome breaks) in patients compared with controls, where 10 FS emerged as specific. Also, the band 17q12–21, where known fragile sites have not been reported, was only expressed in atenolol-treated patients. Fluorescence in situ hybridization using chromosome-specific probes revealed the preferential involvement of chromosomes 7, 11, 17 and X in the micronuclei (MN) of patients. The results also suggest a correlation between chromosomal fragility and content of MN, and support the findings for a linkage between hypertension and a locus on chromosome 17.