Telomeric Associations Research Articles

The telomere profile distinguishes two classes of genetically distinct cutaneous squamous cell carcinomas

The incidence of skin cancer is increasing worldwide and cutaneous squamous cell carcinomas (SCCs) are associated with considerable morbidity and mortality, particularly in immunosuppressed individuals ('carcinomatous catastrophy'). Yet, molecular mechanisms are still insufficiently understood. Besides ultraviolet (UV)-indicative mutations, chromosomal aberrations are prominent. As telomeres are essential in preserving chromosome integrity, and telomere erosion as well as aberrant spatial telomere distribution contribute to genomic instability, we first established telomere length profiles across the whole tissue and identified normal skin (10/30) harboring discrete epidermal sites (stem cell territories) of evenly short telomeres. Precancerous actinic keratoses (AKs) (17) and SCCs (27) expressed two telomere phenotypes: (i) tissue-wide evenly short to intermediate and (ii) longer and tissue-wide heterogeneous telomere lengths, suggesting two modes of initiation, with one likely to originate in the epidermal stem cells. Although tumor histotype, location, patient gender or age failed to distinguish the two SCC telomere phenotypes, as did telomerase activity, we found a trend for a higher degree of aberrant p53 and cyclin D1 expression with long/heterogeneous telomeres. In addition, we established an association for the short/homogeneous telomeres with a simpler and the heterogeneous telomeres with a more complex karyotype correlating also with distinct chromosomal changes. SCCs (13) from renal transplant recipients displayed the same telomere dichotomy, suggesting that both telomere subtypes contribute to 'carcinomatous catastrophy' under immunosuppression by selecting for a common set (3, 9p and 17q) and subtype-specific aberrations (e.g., 6p gain, 13q loss). As a second mechanism of telomere-dependent genomic instability, we investigated changes in telomere distribution with its most severe form of telomeric aggregates (TAs). We identified a telomere length-independent but progression-dependent increase in cells with small telomere associations in AKs (17/17) and additional TAs in SCCs (24/32), basal cell carcinomas (30/31) and malignant melanomas (15/15), and provide evidence for a reactive oxygen species-dependent mechanism in this UV-induced telomere organization-dependent genomic instability.

Cytogenetic study of secondary malignancy in giant cell tumor

Giant cell tumor (GCT) is classified as a benign bone tumor, but it is locally aggressive, and sometimes metastasizes in a benign state. In addition, malignant transformation occurs once in a while. Most of the secondary malignancies in GCT occur after treatment of benign GCT that has included radiation therapy [1, 2]. As a cytogenetic characteristic of GCT, telomeric associations (tas) were reported [3, 4]. Tas may generate dicentric chromosomes (dic) and chromatoid breakagefusion-bridges, which lead to chromosomal instability and tumorigenesis [5, 6]. Recently, the relationship between cytogenetic abnormalities and clinical behavior in GCT has begun to be elucidated. For example, the DNA ploidy pattern may predict the recurrence potential of GCT, chromosomal abnormalities superimposed on tas are responsible for an aggressive clinical course [7], and centrosome amplification may be useful in predicting the clinical behavior of GCT [8]. Here we report a case of secondary malignancy in GCT. Malignant transformation occurred in a relatively early period, and any radiation therapy was not administered to the primary lesion. Malignant transformation was demonstrated not only by histopathological study but also by cytogenetic analysis. The recurrent tumor, which was a secondary malignancy in GCT, had a near-triploid karyotype with multiple structural abnormalities as observed in pleomorphic sarcoma, while the primary benign GCT had a near-diploid karyotype with tas and dic.

3D organization of telomeres in porcine neutrophils and analysis of LPS-activation effect

BackgroundWhile the essential role of 3D nuclear architecture on nuclear functions has been demonstrated for various cell types, information available for neutrophils, essential components of the immune system, remains limited. In this study, we analysed the spatial arrangements of telomeres which play a central role in cell fate. Our studies were carried out in swine, which is an excellent model organism for both biomedical research and agronomic applications. We isolated bacterial artificial chromosome (BAC)-containing subtelomeric p and q sequences specific to each porcine chromosome. This allowed us to study the behaviour of p and q telomeres of homologous chromosomes for seven pairs chosen for their difference in length and morphology. This was performed using 3D-FISH on structurally preserved neutrophils, and confocal microscopy. Resting and lipopolysaccharide (LPS)-activated states were investigated to ascertain whether a response to a pathogen aggression modifies this organization.ResultsThe positions of the p and q telomeres relative to the nuclear outer border were determined in the two states. All p telomeres changed their position significantly during the activation process, although the effect was less pronounced for the q telomeres. The patterns of telomeric associations between homologs and their frequencies were analysed for 7 pairs of chromosomes. This analysis revealed that the distribution of pp, qq and pq associations differs significantly among the 7 chromosomes. This distribution does not fit with the theoretical distribution for each chromosome, suggesting that preferential associations occur between subtelomeres.ConclusionsThe percentage of nuclei harbouring at least one telomeric association between homologs varies significantly among the chromosomes, the smallest metacentric chromosome SSC12, which is also the richest in gene-density, harbouring the highest value. The distribution of types of telomeric associations is highly dependent on the chromosomes and is not affected by the activation process. The frequencies of telomeric associations are also highly dependent on the type of association and the type of chromosome. Overall, the LPS-activation process induces only minor changes in these patterns of associations. When telomeric associations occur, the associations of p and q arms from the same chromosome are the most frequent, suggesting that “chromosome bending” occurs in neutrophils as previously observed in gametes.

The chromatin remodelling complex NoRC safeguards genome stability by heterochromatin formation at telomeres and centromeres

Constitutive heterochromatin is crucial for the integrity of chromosomes and genomic stability. Here, we show that the chromatin remodelling complex NoRC, known to silence a fraction of rRNA genes, also establishes a repressive heterochromatic structure at centromeres and telomeres, preserving the structural integrity of these repetitive loci. Knockdown of NoRC leads to relaxation of centromeric and telomeric heterochromatin, abnormalities in mitotic spindle assembly, impaired chromosome segregation and enhanced chromosomal instability. The results demonstrate that NoRC safeguards genomic stability by coordinating enzymatic activities that establish features of repressive chromatin at centromeric and telomeric regions, and this heterochromatic structure is required for sustaining genomic integrity.

Mixed gonadal dysgenesis in a patient with de novo tas(Y;19)(p11.3;q13.4) and 45,X mosaicism

We report a patient with a de novo telomeric association between chromosomes 19 and Y in conjunction with mixed gonadal dysgenesis. The patient was first admitted to the clinic because of abnormal external genitalia. Laparoscopic evaluation revealed (1) a rudimentary uterus, one fallopian tube, and a small gonad resembling an ovary on the right side, and (2) an immature fallopian tube, a vas deferens, and a gonad resembling a testis on the left side. Conventional cytogenetic analysis performed on cultivated peripheral blood cells, and tissue obtained from the phallus and a gonadal structure which resembled a testis revealed two different cell lines with the 46,X,tas (Y;19)(p11.3;q13.4) and 45,X karyotype. Y chromosome microdeletion analysis showed that the patient did not have any genomic deletions in the AZFa, b, c, or SRY regions on the long arm of the Y chromosome. This is the first report of a patient with mixed gonadal dysgenesis that is accompanied by a telomeric association between chromosomes 19 and Y with 45,X mosaicism.

Constitutional telomeric association (Y;7) in a patient with a female phenotype

Telomeric associations (TAs) are fusions between two telomeres of two different chromosomes without visible loss of chromosomal material. Constitutional telomeric associations are rare chromosomal anomalies. We report on the cytogenetic and molecular analyses of a TA involving chromosomes Y and 7 in a child with a female phenotype. Prenatal cytogenetic analysis showed a 45,X chromosome complement in all cells. No fetal abnormality was identified at ultrasound examinations and the pregnancy went to term. During childhood, the proband had gonadal dysgenesis but no other phenotypic manifestations of Turner syndrome. Molecular genetic analyses showed the presence of genomic DNA of the SRY gene without any mutation. Karyotyping and fluorescent in situ hybridization (FISH) analyses on blood showed two cell lines: one cell line with a TA involving chromosomes Y and 7 [46,X,tas(Y;7)(p11.32;q36.3)] and a second cell line with a 45,X pattern. A human pantelomeric repeat TTAGGG probe hybridized to the junction of the TA within the derivative chromosome. FISH and array comparative genomic hybridization (aCGH) analyses demonstrated that tas(Y;7) occurred without detectable loss of any sequence at the derivative chromosome. SNP array analysis excluded an uniparental isodisomy of chromosome 7. Knowing more about TAs will help geneticists to deliver accurate genetic counseling.

Mammalian DNA2 helicase/nuclease cleaves G-quadruplex DNA and is required for telomere integrity

Efficient and faithful replication of telomeric DNA is critical for maintaining genome integrity. The G-quadruplex (G4) structure arising in the repetitive TTAGGG sequence is thought to stall replication forks, impairing efficient telomere replication and leading to telomere instabilities. However, pathways modulating telomeric G4 are poorly understood, and it is unclear whether defects in these pathways contribute to genome instabilities in vivo. Here, we report that mammalian DNA2 helicase/nuclease recognizes and cleaves telomeric G4 in vitro. Consistent with DNA2's role in removing G4, DNA2 deficiency in mouse cells leads to telomere replication defects, elevating the levels of fragile telomeres (FTs) and sister telomere associations (STAs). Such telomere defects are enhanced by stabilizers of G4. Moreover, DNA2 deficiency induces telomere DNA damage and chromosome segregation errors, resulting in tetraploidy and aneuploidy. Consequently, DNA2-deficient mice develop aneuploidy-associated cancers containing dysfunctional telomeres. Collectively, our genetic, cytological, and biochemical results suggest that mammalian DNA2 reduces replication stress at telomeres, thereby preserving genome stability and suppressing cancer development, and that this may involve, at least in part, nucleolytic processing of telomeric G4.

A Role for the Nucleoporin Nup170p in Chromatin Structure and Gene Silencing

Embedded in the nuclear envelope, nuclear pore complexes (NPCs) not only regulate nuclear transport but also interface with transcriptionally active euchromatin, largely silenced heterochromatin, as well as the boundaries between these regions. It is unclear what functional role NPCs play in establishing or maintaining these distinct chromatin domains. We report that the yeast NPC protein Nup170p interacts with regions of the genome that contain ribosomal protein and subtelomeric genes, where it functions in nucleosome positioning and as a repressor of transcription. We show that the role of Nup170p in subtelomeric gene silencing is linked to its association with the RSC chromatin-remodeling complex and the silencing factor Sir4p, and that the binding of Nup170p and Sir4p to subtelomeric chromatin is cooperative and necessary for the association of telomeres with the nuclear envelope. Our results establish the NPC as an active participant in silencing and the formation of peripheral heterochromatin.

A Newborn with Genital Ambiguity, 45,X/46,XY Mosaicism, a Jumping Chromosome Y, and Congenital Adrenal Hyperplasia

Disorders of sex development (DSD), formerly termed “intersex” conditions, arise from numerous causes. CAH secondary to 21-hydroxylase deficiency is the most common cause of DSD. Sex chromosome disorders, including sex chromosome mosaicism, are the second most common cause of DSD. We discuss a medically complex neonate with DSD presenting with ambiguous genitalia. Hormone levels suggested 21-hydroxylase deficiency. Molecular analysis revealed compound heterozygous mutations in the 21-hydroxylase gene (CYP21A2), confirming the diagnosis of CAH. Chromosome analysis revealed sex chromosome mosaicism with three cell lines: 45,X[8]/45,X,tas(Y;16)(p11.32;p13.3)[8]/45,X,t(Y;8)(p11.32;p23.3)[4] with the Y chromosome in telomere association with chromosomes 8p and 16p in different cell lines, a “jumping translocation.” Histologically, the right gonad had irregular, distended seminiferous tubules with hyperplastic germ cells contiguous with ovarian stroma and primordial follicles. The left gonad had scant ovarian stroma and embryonic remnants. Chromosome analyses showed mosaicism in both gonads: 45,X[17]/45,X,tas(Y;8)(p11.32;p23.3)[3]. This is the first case of coexisting CAH and 45,X/46,XY mosaicism reported in the English literature and the third case of a constitutional chromosome Y “jumping translocation.” Our report documents the medical and genetic complexity of children such as this one with ambiguous genitalia and discusses the need for a multidisciplinary team approach.

Expression profile of telomere‐associated genes in multiple myeloma

To further contribute to the understanding of multiple myeloma, we have focused our research interests on the mechanisms by which tumour plasma cells have a higher survival rate than normal plasma cells. In this article, we study the expression profile of genes involved in the regulation and protection of telomere length, telomerase activity and apoptosis in samples from patients with monoclonal gammopathy of undetermined significance, smouldering multiple myeloma, multiple myeloma (MM) and plasma cell leukaemia (PCL), as well as several human myeloma cell lines (HMCLs). Using conventional cytogenetic and fluorescence in situ hybridization studies, we identified a high number of telomeric associations (TAs). Moreover, telomere length measurements by terminal restriction fragment (TRF) assay showed a shorter mean TRF peak value, with a consistent correlation with the number of TAs. Using gene expression arrays and quantitative PCR we identified the hTERT gene together with 16 other genes directly involved in telomere length maintenance: HSPA9, KRAS, RB1, members of the Small nucleolar ribonucleoproteins family, A/B subfamily of ubiquitously expressed heterogeneous nuclear ribonucleoproteins, and 14-3-3 family. The expression levels of these genes were even higher than those in human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs), which have unlimited proliferation capacity. In conclusion, the gene signature suggests that MM tumour cells are able to maintain stable short telomere lengths without exceeding the short critical length, allowing cell divisions to continue. We propose that this could be a mechanism contributing to MM tumour cells expansion in the bone marrow (BM).

Telomere length is associated with ACE I/D polymorphism in hypertensive patients with left ventricular hypertrophy

Short telomeres are often associated with cardiovascular risk factors and age-related diseases, while the angiotensin converting enzyme (ACE) gene insertion/deletion polymorphism (DD, ID, II) has shown such associations less consistently. We hypothesized that telomere length and association of telomere length with cardiovascular risk is affected by ACE (I/D) genotype. We measured leucocyte telomere length (LTL) by Southern blot and analysed ACE I/D genotypes in 1249 subjects with hypertension and left ventricular hypertrophy (LVH). We examined interactions of ACE I/D genotype with LTL and cardiovascular risk. Mean LTL in DD or ID genotype was shorter (8.15 and 8.14 kb, respectively), than in II genotype (8.27 kb, p=0.0005). This difference was significant in the younger subjects (55-64 years, p=0.02) but not in the older group (65-80 years, p=0.56 ). In DD but not I/D or II genotype, proportion of short telomeres (<5 kb) was related to Framingham risk score. Shorter LTL in genotypes DD or ID suggests a negative effect of the D allele on telomere length. Homozygocity for the D allele appears to strengthen the association of telomere length with increased cardiovascular risk in elderly hypertensive subjects with LVH.

Characterization of telomeric repeats in metaphase chromosomes and interphase nuclei of Syrian Hamster Fibroblasts

BackgroundRodents have been reported to contain large arrays of interstitial telomeric sequences (TTAGGG)n (ITS) located in pericentromeric heterochromatin. The relative sizes of telomeric sequences at the ends of chromosomes (TS) and ITS in Syrian hamster (Mesocricetus auratus) cells have not been evaluated yet, as well as their structural organization in interphase nuclei.ResultsFISH signal distribution analysis was performed on DAPI-banded metaphase chromosomes of Syrian hamster fibroblasts, and relative lengths of telomere signals were estimated. Besides well-distinguished FISH signals from ITS located on chromosomes ##2, 4, 14, 20 and X that we reported earlier, low-intensity FISH signals were visualized with different frequency of detection on all other metacentric chromosomes excluding chromosome #21. The analysis of 3D-distribution of TS in interphase nuclei demonstrated that some TS foci formed clearly distinguished associations (2–3 foci in a cluster) in the nuclei of cells subjected to FISH or transfected with the plasmid expressing telomeric protein TRF1 fused with GFP. In G0 and G1/early S-phase, the average total number of GFP-TRF1 foci per nucleus was less than that of PNA FISH foci in the corresponding cell cycle phases suggesting that TRF1 overexpression might contribute to the fusion of neighboring telomeres. The mean total number of GFP-TRF1 and FISH foci per nucleus was increased during the transition from G0 to G1/early S-phase that might be the consequence of duplication of some TS.ConclusionsThe relative lengths of TS in Syrian hamster cells were found to be moderately variable. All but one metacentric chromosomes contain ITS in pericentromeric heterochromatin indicating that significant rearrangements of ancestral genome occurred in evolution. Visualization of GFP-TRF1 fibrils that formed bridges between distinct telomeric foci allowed suggesting that telomere associations observed in interphase cells are reversible. The data obtained in the study provide the further insight in the structure and dynamics of telomeric sequences in somatic mammalian cells.

GENETIC OF THYROID CANCER FAMILIAL NON MEDULLARY THYROID CANCER

Differentiated non-medullary thyroid cancer (NMTC) is mostly sporadic, but the recurrence of familial form of the disease has been reported. Short or dysfunctional telomeres have been associated with familial benign diseases and familial breast cancer. We aimed to study the telomere-telomerase complex in familial NMTC (FNMTC). The genetic analysis included the measurement in the peripheral blood of relative telomere length (RTL), telomerase reverse transcriptase (hTERT) gene amplification, hTERT mRNA expression, telomerase protein activity and search of hTERT or TERC (telomerase RNA component) gene mutations. We, also, studied telomeric fusions and associations as well as other chromosomal fragility features by conventional and molecular cytogenetic analyses, in phytohemagglutinin stimulated T-lymphocytes from familial patients, unaffected family members, sporadic PTC patients and healthy subjects. We found that, telomere lenght was significantly shorter in the blood of familial patients compared to sporadic PTCs, healthy subjects, nodular goiter and unaffected siblings. hTERT gene amplification was significantly higher in FNMTC patients compared to the other groups and, in particular, it was significantly greater in offspring with respect to parents. hTERT mRNA expression as well as telomerase activity were significantly higher in FNMTC patients compared to sporadic In addition, we demonstrated that familial patients have a significant increase in spontaneous telomeric associations and telomeric fusions compared to healthy subjects and sporadic cases. Q-FISH analysis demonstrated that familial cases display a significant decrease in the telomeric PNA-FISH signal intensity in metaphase chromsome. Our study demonstrates that patients with FNMTC display an imbalance of the telomeretelomerase complex in the peripheral blood.

GENETIC OF THYROID CANCER FAMILIAL NON MEDULLARY THYROID CANCER

Differentiated non-medullary thyroid cancer (NMTC) is mostly sporadic, but the recurrence of familial form of the disease has been reported. Short or dysfunctional telomeres have been associated with familial benign diseases and familial breast cancer. We aimed to study the telomere-telomerase complex in familial NMTC (FNMTC). The genetic analysis included the measurement in the peripheral blood of relative telomere length (RTL), telomerase reverse transcriptase (hTERT) gene amplification, hTERT mRNA expression, telomerase protein activity and search of hTERT or TERC (telomerase RNA component) gene mutations. We, also, studied telomeric fusions and associations as well as other chromosomal fragility features by conventional and molecular cytogenetic analyses, in phytohemagglutinin stimulated T-lymphocytes from familial patients, unaffected family members, sporadic PTC patients and healthy subjects. We found that, telomere lenght was significantly shorter in the blood of familial patients compared to sporadic PTCs, healthy subjects, nodular goiter and unaffected siblings. hTERT gene amplification was significantly higher in FNMTC patients compared to the other groups and, in particular, it was significantly greater in offspring with respect to parents. hTERT mRNA expression as well as telomerase activity were significantly higher in FNMTC patients compared to sporadic In addition, we demonstrated that familial patients have a significant increase in spontaneous telomeric associations and telomeric fusions compared to healthy subjects and sporadic cases. Q-FISH analysis demonstrated that familial cases display a significant decrease in the telomeric PNA-FISH signal intensity in metaphase chromsome. Our study demonstrates that patients with FNMTC display an imbalance of the telomeretelomerase complex in the peripheral blood.

Analysis of giant cell tumour of bone cells for Noonan syndrome/Cherubism‐related mutations

Giant cell tumour of bone (GCTB) is an osteolytic tumour which contains numerous osteoclast-like giant cells and a proliferation of mononuclear stromal cells (MSC). Giant cell-rich osteolytic lesions can also develop in the jaw bones in Noonan syndrome, a cherubism-like developmental abnormality that is transmitted in an autosomal dominant fashion, often because of mutation in the PTPN11 or BRAF genes. We screened GCTBs for mutations in PTPN11 and BRAF to determine whether GCTBs develop through alterations of genes involved in Noonan syndrome. MSC were isolated from 10 GCTBs. Chromosome banding analysis of these cells revealed telomeric associations (tas) in 7 of the 10 cases. Thus, the cultured cells expressed a cytogenetic abnormality typically found in short-term cultures from GCTBs. Sequencing of DNA extracted from the seven GCTB-derived MSC cultures displaying tas did not identify any mutation in PTPN11 or in exons 9-15 of BRAF. Our findings indicate that the molecular pathways involved in GCTB development are different from those causing Noonan syndrome. The method for isolating and culturing GCTB stromal cells described in this study generated a population of MSC that contained tas, indicating that it is useful for obtaining stromal cells from GCTB and other giant cell-rich lesions, such as giant cell reparative granuloma, for genetic and other studies.

Beyond average: potential for measurement of short telomeres

The length of telomeres, and in particular the abundance of short telomeres, has been proposed as a biomarker of aging and of general health status. A wide variety of studies show the association of short telomeres with age related pathologies and cancer, as well as with lifespan and mortality. These facts highlight the importance of measuring telomere length in human populations and by using reliable methods to uncover the association between telomere length and human disease. This review discusses the advantages and drawbacks of current telomere length measurement methods. Most of these methods provide mean telomere length values per cell or per sample and very few of them are able to measure the abundance of short telomeres, which are the ones indicative of telomere dysfunction. The information provided by each method and their suitability for different studies is discussed here.

12q amplification defines a subtype of extraskeletal osteosarcoma with good prognosis that is the soft tissue homologue of parosteal osteosarcoma

Extraskeletal osteosarcomas are rare tumors with neoplastic cells synthesizing bone, usually associated with poor prognosis. We present the case of a 40-year-old man with an extraskeletal osteosarcoma that was treated by surgery and adjuvant radiotherapy. Thirteen years after the diagnosis, he remains disease-free, without any recurrences or metastases. Histopathological analysis favored the diagnosis of chondroblastic extraskeletal osteosarcoma grade II. G-banding, comparative genomic hybridization (CGH), and real-time PCR for the MDM2 and CDK4 genes were performed to describe the genetic profile of this tumor and revealed aberrations that are common findings of parosteal osteosarcomas. Ring chromosomes, giant marker chromosomes, and a telomeric association were found with G-banding. CGH revealed that 12q was amplified in the ring and giant markers identified by G-banding. Real-time PCR for MDM2 and CDK4 confirmed the amplification of these genes located in 12q. Our findings suggest that a variant of extraskeletal osteosarcoma, which is genotypically similar to parosteal osteosarcoma, exists and is associated with good prognosis.

Telomere Abnormalities and Chromosome Fragility in Patients Affected by Familial Papillary Thyroid Cancer

Genomic instability has been proposed to play a role in cancer development and can occur through different mechanisms including telomere association and telomere loss. Studies carried out in our unit have demonstrated that familial papillary thyroid cancer (fPTC) patients display an imbalance, at the germinal level, in telomere-telomerase complex. We aimed to verify whether familial fPTC patients show an increased spontaneous chromosome fragility. To this purpose, we compared telomeric fusions and associations as well as other chromosomal fragility features by conventional and molecular cytogenetic analyses, in phytohemagglutinin stimulated T-lymphocytes from fPTC patients, unaffected family members, sporadic papillary thyroid cancer patients, and healthy subjects. We demonstrate that fPTC patients have a significant increase in spontaneous telomeric associations and telomeric fusions compared with healthy subjects and sporadic cases in the frame of an otherwise common spontaneous chromosome fragility pattern. A quantitative fluorescence in situ hybridization analysis demonstrates that familial cases display a significant decrease in the telomeric peptide nucleic acid-fluorescence in situ hybridization signal intensity in the metaphase chromosome. Moreover, three copies of the hTERT gene were found only in familial cases, although the result was not statistically significant. These results contribute in defining familial thyroid cancer as a clinical entity characterized by an altered telomere stability, which may be associated with the predisposition to develop the familial form of thyroid cancer.

Abstract SY23-02: A telomere-dependent DNA damage checkpoint induced by prolonged mitotic arrest

Abstract Telomere shortening and disruption of telomeric components are pathways that induce telomere deprotection. Here we describe another pathway, where prolonged mitotic arrest induces damage signals at telomeres. Exposure to microtubule drugs, kinesin inhibitors, proteasome inhibitors or the disruption of proper chromosome alignment resulted in the formation of damage-foci at telomeres. Induction of mitotic telomere deprotection coincided with dissociation of TRF2 from telomeres, telomeric 3′-overhang degradation and ATM activation, and could be suppressed by TRF2 overexpression or inhibition of Aurora B kinase. Normal cells that escape from prolonged mitotic arrest halted in the following G1 phase, whereas cells lacking p53 continued to cycle and became aneuploid. We propose a telomere dependent mitotic duration checkpoint that ensures elimination of cells, which fail to progress through mitosis. To avoid unwanted checkpoint activation by natural chromosome ends, cells have evolved telomeres. Human telomeres are composed of double stranded TTAGGG repeats and a single stranded G rich 3′ overhang, which are covered and protected by shelterin. Among the six shelterin components TRF2 and POT1 have predominantly been implicated in chromosome end protection by preventing ATM- and ATR-dependent checkpoint activation. Upon disruption of TRF2 or POT1 telomeres are recognized as sites of DNA damage, resulting in phosphorylation of histone H2AX (γ-H2AX) within the telomeric and sub-telomeric chromatin and association of 53BP1 with the chromosome ends. The co-localization of DNA-damage response factors and chromosome ends can be visualized as telomere dysfunction-induced foci (TIF). TIF have also been intimately linked to replicative senescence and shown to occur spontaneously in cancer cell lines. Cells arrested in mitosis are known to either die during mitotic arrest, or skip cytokinesis and “slip” into the subsequent G1 phase of the cell cycle. Mitotic slippage occurs through the degradation of Cyclin B1 in the presence of the active spindle assembly checkpoint (SAC). Cells that exit from prolonged mitotic arrest or progress through mitotic slippage exhibit various fates, including apoptosis or p53-dependent cell cycle arrest. In both normal and cancer cells, cell death during mitotic arrest, or apoptosis or senescence after escape from prolonged mitotic arrest are crucial for preventing chromosome instability. A failure to remove cells from the cycling population following prolonged mitotic arrest may allow cells to continue propagating with an abnormal number of chromosomes. However, despite intense research, the molecular mechanisms that trigger growth arrest or death in mitotically arrested cultures have not yet been identified. In the course of experimentation to explore putative telomeric functions for cohesin, we found that mitotic arrest per se induces telomere deprotection in primary and transformed human cells. Telomere deprotection during mitotic arrest associated with loss of the telomeric 3′-overhangs, led to ATM activation and was ATM dependent. TRF2 was dissociated from telomeres during prolonged mitotic arrest, providing the molecular basis for overhang loss and ATM activation, which was emphasized by the finding that TRF2 overexpression protected telomeres from the damage machinery during mitotic arrest. Inhibition of Aurora B kinase suppressed the telomere deprotection phenotype, suggesting the involvment of the SAC. Cells suffering from mitotic telomere deprotection underwent p53 dependent cell cycle arrest in the following G1 phase after mitotic release, while cells lacking p53 function continued to cycle and became aneuploid. Our findings provide a molecular mechanism explaining the induction of DNA damage signaling, cell cycle arrest or apoptosis following prolonged mitotic arrest, and explain the mechanism of action of therapeutic drugs, such as Taxol, Vinblastine and Velcade, which all inhibit mitotic progression. We propose that telomeric destabilization during mitotic arrest induces DNA damage signaling and serves as a mitotic duration checkpoint, responsible for eliminating cells that fail to progress through mitosis properly. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr SY23-02. doi:1538-7445.AM2012-SY23-02

Abstract 3884: A novel, in vitro tankyrase 1 trans-ribosylation assay

Abstract The poly (ADP-ribose) polymerase, tankyrase 1, is an encouraging pharmacological target for cancer therapy. First discovered by its association with the TRF1, tankyrase 1 plays an important role in the maintenance of telomere length, sister telomere association, and mitotic spindle organization. More recently, interactions between tankyrase 1 and axin were shown to stabilize β-catenin and modulate Wnt signaling, enabling selective targeting of the Wnt pathway with tankyrase inhibitors. In addition, tankyrase 1 has also demonstrated potential as a target for synthetic lethality for BRCA-deficient cancers. Given the effect on these cancer-associated pathways, there is a growing interest for identifying tankyrase-specific inhibitors. Historical methods to evaluate tankyrase activity are not amenable for high throughput screening. Limited availability of the full length enzyme required radioactivity to attain the needed sensitivity. Truncation of tankyrase 1 improved solubility and increased availability, but the physiological response may not be equivalent to the full length enzyme. Many of these assays have also used modified substrates where molecules such as fluorescein or biotin are linked to NAD without characterizing the effects on enzyme activity. Most historical assays have also relied on measuring autoribosylation of tankyrase 1; whereas, transribosylation may be more relevant for assessing the activity of tankyrase 1 on other molecules. To address these issues, we have developed a novel, in vitro, high throughput assay for evaluating the transribosylation activity of tankyrase 1. This is accomplished using an ELISA format which semi-quantitatively detects poly (ADP-ribose) (PAR) deposited onto immobilized histone proteins by tankyrase 1. An anti-PAR monoclonal antibody, goat anti-mouse IgG-HRP conjugate, and HRP substrate generate a colorimetric or chemiluminescent signal, and the conversion of substrate correlates with tankyrase 1 activity. This assay ensures physiological significance by utilizing the full length enzyme and unmodified substrate in a transribosylation format, providing sensitivity down to pmols of enzyme. This assay is utilized to demonstrate the specificity of the tankyrase 1 inhibitor, XAV939, for tankyrase 1 compared to PARP1 and to reveal differential effects of activated DNA for full-length tankyrase 1 compared to the truncated enzyme. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3884. doi:1538-7445.AM2012-3884