BRCA1 knock-down causes telomere dysfunction in mammary epithelial cells.

A breast cancer predisposing gene, BRCA1, is a major suppressor of chromosomal instability and its dysfunction affects multiple pathways involved in DNA damage response. There is increasing evidence that the mechanisms involved in maintenance of telomeres, specialized structures at chromosome ends, are linked with DNA damage response. We therefore investigated whether BRCA1 dysfunction affects telomere maintenance. To achieve this we knocked-down BRCA1 in two mammary epithelial cell lines using RNA interference. Subsequent analysis by immunofluorescence, RT-PCR and Western blotting revealed that a short interfering RNA oligonucleotide used was able to knock-down BRCA1 efficiently. This knock-down did not have any effect on telomerase enzyme activity and telomere length. However, BRCA1 knock-down correlated with the increase in chromatin bridges in anaphase cells which usually reflect telomere dysfunction. Therefore, this study suggests that BRCA1 knockdown in mammary epithelial cells causes telomere dysfunction.

Effects of hTERT on metal ion-induced genomic instability.

There is currently a great interest in delayed chromosomal and other damaging effects of low-dose exposure to a variety of pollutants which appear collectively to act through induction of stress-response pathways related to oxidative stress and ageing. These have been studied mostly in the radiation field but evidence is accumulating that the mechanisms can also be triggered by chemicals, especially heavy metals. Humans are exposed to metals, including chromium (Cr) (VI) and vanadium (V) (V), from the environment, industry and surgical implants. Thus, the impact of low-dose stress responses may be larger than expected from individual toxicity projections. In this study, a short (24 h) exposure of human fibroblasts to low doses of Cr (VI) and V (V) caused both acute chromosome damage and genomic instability in the progeny of exposed cells for at least 30 days after exposure. Acutely, Cr (VI) caused chromatid breaks without aneuploidy while V (V) caused aneuploidy without chromatid breaks. The longer-term genomic instability was similar but depended on hTERT positivity. In telomerase-negative hTERT- cells, Cr (VI) and V (V) caused a long lasting and transmissible induction of dicentric chromosomes, nucleoplasmic bridges, micronuclei and aneuploidy. There was also a long term and transmissible reduction of clonogenic survival, with an increased beta-galactosidase staining and apoptosis. This instability was not present in telomerase-positive hTERT+ cells. In contrast, in hTERT+ cells the metals caused a persistent induction of tetraploidy, which was not noted in hTERT- cells. The growth and survival of both metal-exposed hTERT+ and hTERT- cells differed if they were cultured at subconfluent levels or plated out as colonies. Genomic instability is considered to be a driving force towards cancer. This study suggests that the type of genomic instability in human cells may depend critically on whether they are telomerase-positive or -negative and that their sensitivities to metals could depend on whether they are clustered or diffuse.

Shortened telomeres in murine scid cells expressing mutant hRAD54 coincide with reduction in recombination at telomeres.

Murine severe combined immunodeficiency (scid) cells are characterized by defective Prkdc (DNA-PKcs), one of the key genes involved in the repair of DNA double-strand breaks. Interestingly, scid mice are not null mutants and their cells are likely to show low DNA-PKcs activity. Prkdc is also involved in telomere maintenance and in contrast to mice genetically engineered to lack Prkdc (i.e. null mutants), which show complete absence of DNA-PKcs activity, loss of telomere capping function and normal telomere length, cells from scid mice show not only loss of telomere capping function but also abnormally elongated telomeres. Here we demonstrate that telomere elongation observed in murine scid cells can be reversed by expressing mutant hRAD54, a protein involved in homologous recombination. In addition, we measured recombination rates at telomeres using chromosome orientation fluorescence in situ hybridization (CO-FISH) and found that these are elevated in scid cells in comparison with control cells, or significantly reduced in scid cells expressing mutant hRAD54. Similarly, recombination rates at telomeres are reduced in scid cells following introduction of functional Prkdc. Since expression of mutant hRAD54 and restoration of functional Prkdc in scid cells cause the same effects, i.e. telomere shortening and reduced recombination rates at telomeres, these results argue that telomere elongation in scid cells is a complex trait resulting from interactions between homologous recombination mechanisms and DNA-PKcs.

Accelerated telomere shortening and telomere abnormalities in radiosensitive cell lines.

We examined telomere maintenance in cells of 11 primary fibroblast cell lines with differing genetic defects that confer sensitivity to ionizing radiation. These included cell lines derived from patients with ataxia telangiectasia, Nijmegen breakage syndrome, Fanconi anemia, defective Artemis, DNA ligase I and DNA ligase IV, an immunodeficient patient with a defect in DNA double-strand break repair, and a patient diagnosed with xeroderma pigmentosum who, in addition, showed severe clinical sensitivity to ionizing radiation. Our results, based on Southern blot, flow-FISH and Q-FISH (quantitative FISH) measurements, revealed an accelerated rate of telomere shortening in most cell lines derived from the above patients compared to cell lines from normal individuals or a cell line isolated from a heterozygotic parent of one radiosensitive patient. This accelerated telomere shortening was accompanied by the formation of chromatin bridges in anaphase cells, indicative of the early loss of telomere capping function and in some cases low levels of chromosome abnormalities in metaphase cells. We also analyzed telomere maintenance in mouse embryonic stem cells deficient in Brca1, another defect that confers radiosensitivity. Similarly, these cells showed accelerated telomere shortening and mild telomere dysfunction in comparison to control cells. Our results suggest that mechanisms that confer sensitivity to ionizing radiation may be linked with mechanisms that cause telomere dysfunction.

Chromosomal aberrations involving telomeres in BRCA1 deficient human and mouse cell lines.

Cells defective in BRCA1 show genomic instability as evidenced by increased radiosensitivity, the presence of chromosomal abnormalities and the loss of heterozygosity at many loci. Reported chromosomal abnormalities in BRCA1 deficient cells include dicentric chromosomes. Dicentric chromosomes, in some cases, may arise as a result of end-to-end chromosome fusions, which represent signatures of telomere dysfunction. In this study we examined BRCA1 deficient human and mouse cells for the presence of chromosomal aberrations indicative of telomere dysfunction. We identified a lymphoblastoid cell line, GM14090, established from a BRCA1 carrier that showed elevated levels of dicentric chromosomes. Molecular cytogenetic analysis revealed that these dicentric chromosomes result from end-to-end chromosome fusions. The frequency of end-to-end chromosome fusions did not change after exposure of GM14090 cells to bleomycin but we observed elevated levels of chromosomal abnormalities involving interactions between DNA double strand breaks and uncapped telomeres in this cell line. We observed similar chromosomal abnormalities involving telomeres in the breast cancer cell line, HCC1937, homozygous for BRCA1 mutation. Finally, we analyzed mouse embryonic stem cells lacking functional Brca1 and observed the presence of telomere dysfunction following exposure of these cells to bleomycin. Our results reveal cytogenetic evidence of telomere dysfunction in BRCA1 deficient cells.

Telomere length measurement in mouse chromosomes by a modified Q-FISH method.

Telomeres are physical ends of mammalian chromosomes that dynamically change during the lifetime of a cell or organism. In order to understand mechanisms responsible for telomere dynamics, it is necessary to develop methods for accurate telomere length measurement. The most sensitive method for measuring telomere length in mouse chromosomes is quantitative fluorescence in situ hybridization (Q-FISH). The usual protocol for Q-FISH requires plasmids with variable numbers of telomeric repeats and fluorescence beads as calibration standards. Here, we describe a Q-FISH protocol in which two mouse lymphoma cell lines with well-defined telomere lengths are used as calibration standards. Using this protocol we demonstrate that reproducible results can be obtained in a set of four different mouse cell lines. This method can be adapted so that any pair of mammalian cell lines can serve as an internal calibration standard.

Lack of spontaneous and radiation-induced chromosome breakage at interstitial telomeric sites in murine scid cells.

Interstitial telomeric sites (ITSs) in chromosomes from DNA repair-proficient mammalian cells are sensitive to both spontaneous and radiation-induced chromosome breakage. Exact mechanisms of this chromosome breakage sensitivity are not known. To investigate factors that predispose ITSs to chromosome breakage we used murine scid cells. These cells lack functional DNA-PKcs, an enzyme involved in the repair of DNA double-strand breaks. Interestingly, our results revealed lack of both spontaneous and radiation-induced chromosome breakage at ITSs found in scid chromosomes. Therefore, it is possible that increased sensitivity of ITSs to chromosome breakage is associated with the functional DNA double-strand break repair machinery. To investigate if this is the case we used scid cells in which DNA-PKcs deficiency was corrected. Our results revealed complete disappearance of ITSs in scid cells with functional DNA-PKcs, presumably through chromosome breakage at ITSs, but their unchanged frequency in positive and negative control cells. Therefore, our results indicate that the functional DNA double-strand break machinery is required for elevated sensitivity of ITSs to chromosome breakage. Interestingly, we observed significant differences in mitotic chromosome condensation between scid cells and their counterparts with restored DNA-PKcs activity suggesting that lack of functional DNA-PKcs may cause a defect in chromatin organization. Increased condensation of mitotic chromosomes in the scid background was also confirmed in vivo. Therefore, our results indicate a previously unanticipated role of DNA-PKcs in chromatin organisation, which could contribute to the lack of ITS sensitivity to chromosome breakage in murine scid cells.

Telomere shortening in mouse strains with constitutional chromosomal aberrations.

PURPOSE: To compare telomere length in mouse strains with constitutional chromosomal aberrations generated either by exposure of parents to ionizing radiation, a chemical mutagen or arising spontaneously with that of the karyotypically normal mouse from the same genetic background. MATERIALS AND METHODS: Telomere length was assessed in five independently derived strains of mouse with constitutional chromosomal aberrations and in the karyotypically normal control mouse using quantitative fluorescence in situ hybridization (Q-FISH). Bone marrow cells obtained directly from the animals were used for the analysis. RESULTS: Chromosomal aberrations, one in each mouse strain, included three reciprocal translocations induced by ionizing radiation, one insertion induced by a chemical mutagen and one spontaneous Robertsonian translocation. There was no cytogenetically detectable loss of material in any of the strains and most mice were phenotypically normal. Telomeres were significantly shorter in all mouse strains with constitutional chromosomal aberrations in comparison with those originating from the karyatypically normal mouse from the same genetic background. Telomeres were significantly shorter at p-arms than at q-arms in all animals. The telomere length in individual chromosomes was variable and there was no single chromosome with consistently short telomeres in all animals. CONCLUSIONS: The presence of stable chromosomal aberrations, such as translocations or insertions, in the mouse genome may generate telomere shortening. This might have implications for understanding biological consequences or radiation-induced stable chromosomal aberrations.

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.

Telomere length measurement by Q-FISH.

Telomeres are essential functional elements of eukaryotic chromosomes involved in genome stability maintenance. The most important indicator of correct telomere function is telomere length maintenance within the range typical for each species. Telomere length can be estimated by the classical methodology based on Southern blot. However, this methodology is relatively crude and can provide estimate of average telomere length only. To overcome disadvantages of classical telomere length estimate, a new technique termed Q-FISH has been invented. Q-FISH provides estimate of telomere length in each individual chromosome with the resolution of 200 base pairs. In addition, Q-FISH may be used to estimate telomere length in species containing interstitial telomeric sites in their genomes. The classical methodology is non-informative in these cases. Finally, Q-FISH has been essential in estimating telomere length in the mouse, a species with ultra-long telomeres difficult to measure using classical methods. Principles of Q-FISH and its applications are briefly described in this article.

Telomere length abnormalities in mammalian radiosensitive cells.

Telomere lengths in radiosensitive murine lymphoma cells L5178Y-S and parental radioresistant L5178Y cells were measured by quantitative fluorescence in situ hybridization. Results revealed a 7-fold reduction in telomere length in radiosensitive cells (7 kb) in comparison with radioresistant cells (48 kb). Therefore, it was reasoned that telomere length might be used as a marker for chromosomal radiosensitivity. In agreement with this hypothesis, a significant inverse correlation between telomere length and chromosomal radiosensitivity was observed in lymphocytes from 24 breast cancer patients and 5 normal individuals. In contrast, no chromosomal radiosensitivity was observed in mouse cell lines that showed shortened telomeres, possibly reflecting differences in radiation responses between primary cells and established cell lines. Telomere length abnormalities observed in radiosensitive cells suggest that these two phenotypes may be linked.

Chromosome-specific telomeric associations in Chinese hamster embryonic cells.

Telomeric associations (TAs) represent an important cytogenetic marker of human tumor cells. It has been thought that the primary cause of TAs is telomere shortening. However, we report here a surprising aspect of telomere maintenance in primary Chinese hamster embryonic (CHE) cells: relatively high frequencies of TAs in spite of normal telomere length. These TAs are present in both interphase and metaphase cells, suggesting that metaphase TAs may be relics of interphase chromosome organization. In addition, some TAs observed here are chromosome-specific and recurrent in at least three consecutive cell cycles in two different CHE cell strains. In spite of relatively high frequencies of TAs, none of the CHE strains show chromosome instability resulting from breakage-fusion-bridge cycles, as would be expected from tumor cell studies. It appears that TAs in CHE cells may be reversible events. These results are discussed in light of current understanding of telomere biology.

Mammalian Ku86 protein prevents telomeric fusions independently of the length of TTAGGG repeats and the G-strand overhang.

Ku86 together with Ku70, DNA-PKcs, XRCC4 and DNA ligase IV forms a complex involved in repairing DNA double-strand breaks (DSB) in mammals. Yeast Ku has an essential role at the telomere; in particular, Ku deficiency leads to telomere shortening, loss of telomere clustering, loss of telomeric silencing and deregulation of the telomeric G-overhang. In mammals, Ku proteins associate to telomeric repeats; however, the possible role of Ku in regulating telomere length has not yet been addressed. We have measured telomere length in different cell types from wild-type and Ku86-deficient mice. In contrast to yeast, Ku86 deficiency does not result in telomere shortening or deregulation of the G-strand overhang. Interestingly, Ku86-/- cells show telomeric fusions with long telomeres (>81 kb) at the fusion point. These results indicate that mammalian Ku86 plays a fundamental role at the telomere by preventing telomeric fusions independently of the length of TTAGGG repeats and the integrity of the G-strand overhang.

Chinese hamster telomeres are comparable in size to mouse telomeres.

We report here the results of a telomere length analysis in four male Chinese hamsters by quantitative fluorescence in situ hybridization (Q-FISH). We were able to measure telomere length of 64 (73%) of 88 Chinese hamster telomeres. We could not measure telomere length in chromosome 10 or in the short arms of chromosomes 5, 6, 7 and 8 because of the overlaps between the interstitial and terminal telomeric signals. Our analysis in the 73% of Chinese hamster telomeres indicate that their average length is approximately 38 kb. Therefore, Chinese hamster telomeres are comparable in length to mouse telomeres, but are much longer than human telomeres. Similar to previous Q-FISH studies on human and mouse chromosomes, our results indicate that individual Chinese hamster chromosomes may have specific telomere lengths, suggesting that chromosome-specific factors may be involved in telomere length regulation.

Elongated telomeres in scid mice.

Severe combined immunodeficiency (scid) mice are deficient in the enzyme DNA-PK (DNA-dependent protein kinase) as a result of the mutation in the gene encoding the catalytic subunit (DNA-PKcs) of this enzyme. DNA-PKcs is a member of the phosphatidylinositol 3-kinase superfamily, which includes the human protein ATM (ataxia telangiectasia mutated) and the yeast protein Tel1. Using Q-FISH (quantitative fluorescence in situ hybridization), we show here that scid mice from four different genetic backgrounds have, on average, 1.5-2 times longer telomeres than those of corresponding wild-type mice. Our results point to the possibility that DNA-PKcs may, directly or indirectly, be involved in telomere length regulation in mammalian cells.

Telomere length regulation--a view from the individual chromosome perspective.

Telomeres are specialized structures at chromosome termini implicated in oncogenesis and cellular aging. Since both phenomena are related to variations in telomere length it is of interest to understand mechanisms responsible for telomere length regulation. Recent studies in mammalian cells indicate that specific chromosomes may have specific telomere lengths, suggesting the existence of chromosome-specific factors involved in telomere length regulation. Although these chromosome-specific factors are largely unknown at present, in the mouse evidence suggests a possible role of centromere position in telomere length regulation-telomeres closer to centromeres (i.e., p-arm telomeres) are significantly shorter than their counterparts more distant from centromeres (i.e., q-arm telomeres). The mouse may be a special case because its karyotype consists almost exclusively of acrocentric chromosomes in which p-arm telomeres and centromeres are located immediately adjacent to each other. However, a weak correlation between telomere length and centromere position is observed in the case of nonacrocentric human and Chinese hamster chromosomes, suggesting that the putative centromere position effect might be evolutionarily conserved. Alternatively, telomere length in individual nonacrocentric chromosomes may be affected by the sequence organization of subtelomeric chromosome regions or by some other, currently unknown, factors.

Telomere length and telomere-centromere relationships?

The quantitative fluorescence in situ hybridization (Q-FISH) technique enables an accurate estimate of individual telomere lengths, a possibility beyond the resolution of conventional techniques. So far, Q-FISH has been used for the estimate of individual telomere lengths in human, mouse and Chinese hamster chromosomes. This analysis revealed large variations in the size of individual telomeres and a specific intra-chromosomal distribution of telomere lengths; telomeres closer to centromeres appear to be shorter than their counterparts more distant from centromeres. This observation suggests that individual telomere length may be affected by centromere position, a possibility consistent with the theory of chromosome field postulated more than 40 years ago by Lima-de-Faria. The link between the theory of chromosome field and the role of telomere-centromere relationships in the regulation of telomere length is discussed in this article.

Telomeres and mechanisms of Robertsonian fusion.

The Robertsonian (Rb) fusion, a chromosome rearrangement involving centric fusion of two acro-(telo)centric chromosomes to form a single metacentric, is one of the most frequent events in mammalian karyotype evolution. Since one of the functions of telomeres is to preserve chromosome integrity, a prerequisite for the formation of Rb fusions should be either telomere loss or telomere inactivation. Possible mechanisms underlying the formation of various types of Rb fusion are discussed here. For example, Rb fusion in wild mice involves complete loss of p-arm telomeres by chromosome breakage within minor satellite sequences. By contrast, interstitial telomeric sites are found in the pericentromeric regions of chromosomes originating from a number of vertebrate species, suggesting the occurrence of Rb-like fusion without loss of telomeres, a possibility consistent with some form of telomere inactivation. Finally, a recent study suggests that telomere shortening induced by the deletion of the telomerase RNA gene in the mouse germ-line leads to telomere loss and high frequencies of Rb fusion in mouse somatic cells. Thus, at least three mechanisms in mammalian cells lead to the formation of Rb fusions.

Telomeres and radiation-induced chromosome breakage.

Molecular characterization of some cytologically apparent terminal deletions in human tumours revealed that these were subtelomeric cryptic translocations undetectable by classical cytogenetic methods. To determine whether subtelomeric cryptic translocations occur following exposure of mammalian cells to ionizing radiation we used four cell lines exhibiting variable telomere lengths. Our G1 and G2 radiation experiments revealed that a small percentage of broken chromosomes exhibited telomeric signals. This occurred exclusively in cell lines exhibiting FISH-detectable telomeres, suggesting that telomeric signals at radiation-induced chromosome breaks were the result of subtelomeric cryptic translocations. In addition, telomeric signals at G2 chromatid breaks were usually paired with telomeres of intact sister chromatids, further supporting the notion that subtelomeric cryptic translocations are responsible for the presence of telomeric sequences at radiation-induced chromosome breaks. In one of the cell lines we identified what looked like de novo telomeric signals at derived chromatid breaks observed 20 h following irradiation. Our previous study suggested that these signals may be the result of amplification of interstitial telomeric sites in the first cell cycle and spontaneous breakage of interstitial telomeric sites in subsequent cell cycles. Taken together our results suggest that a small percentage of radiation-induced chromosome/chromatid breaks may be modified by subtelomeric cryptic translocations and that interstitial telomeric sites may be involved in radiation-induced chromosome instability.

Chromosome healing, telomere capture and mechanisms of radiation-induced chromosome breakage.

BACKGROUND: It is generally assumed that radiation-induced chromosome breaks are the result of a cell's inability to rejoin DNA double-strand breaks (dsb), but the exact mechanisms underlying the failure to rejoin some dsb and the conversion of these lesions into chromosome breaks are poorly understood at present. It has been speculated that the conversion of dsb into chromosome breaks, following exposure of mammalian cells to ionizing radiation, may be mediated by the enzyme telomerase. Telomerase is a reverse transcriptase that has two distinct functions, to replicate pre-existing chromosome ends (telomeres) and to heal broken chromosomes by de novo addition of telomeric sequences directly on to non-telomeric DNA. Alternatively, dsb may be converted into chromosome breaks by a telomerase-independent mechanism termed telomere capture. PURPOSE: To review telomere biology and to examine the significance of chromosome healing and telomere capture mechanisms tor radiation cytogenetics. CONCLUSION: The currently available literature suggests that telomere capture may be a more frequent mechanism for stabilization of broken chromosomes in mammalian cells than telomerase-mediated chromosome healing. However, a definitive conclusion must await improvements in the resolution of molecular cytogenetic techniques to a degree which allows telomerase products to be clearly distinguishable from subtelomeric cryptic translocations indicative of telomere capture.