Telomere dysfunction as a cause of genomic instability in Werner syndrome

Werner syndrome (WS) is a rare human premature aging disease caused by mutations in the gene encoding the RecQ helicase WRN. In addition to the aging features, this disorder is marked by genomic instability, associated with an elevated incidence of cancer. Several lines of evidence suggest that telomere dysfunction is associated with the aging phenotype of the syndrome; however, the origin of the genomic instability observed in WS cells and the reason for the high incidence of cancer in WS have not been established. We previously proposed that WRN helicase activity was necessary to prevent dramatic telomere loss during DNA replication. Here we demonstrate that replication-associated telomere loss is responsible for the chromosome fusions found in WS fibroblasts. Moreover, using metaphase analysis we show that telomere elongation by telomerase can significantly reduce the appearance of new chromosomal aberrations in cells lacking WRN, similar to complementation of WS cells with WRN. Our results suggest that the genome instability in WS cells depends directly on telomere dysfunction, linking chromosome end maintenance to chromosomal aberrations in this disease.     

Telomere dysfunction and its role in haematological cancer

Observations in human tumours, as well as mouse models, have indicated that telomere dysfunction may be a key event driving genomic instability and disease progression in many solid tumour types. In this scenario, telomere shortening ultimately results in telomere dysfunction, fusion and genomic instability, creating the large-scale rearrangements that are characteristic of these tumours. It is now becoming apparent that this paradigm may also apply to haematological malignancies; indeed these conditions have provided some of the most convincing evidence of telomere dysfunction in any malignancy. Telomere length has been shown in several malignancies to provide clinically useful prognostic information, implicating telomere dysfunction in disease progression. In these malignancies extreme telomere shortening, telomere dysfunction and fusion have all been documented and correlate with the emergence of increased genomic complexity. Telomeres may therefore represent both a clinically useful prognostic tool and a potential target for therapeutic intervention.     

Telomere dysfunction in hypertension

Aging is a major risk factor for hypertension and associated cardiovascular disease. In most proliferative tissues, aging is characterized by shortening of the DNA component of telomeres, the specialized genetic segments that cap the end of eukaryotic chromosomes and protect them from end-to-end fusions. By inducing genomic instability, replicative senescence and apoptosis, telomere shortening is thought to contribute to organismal aging and to the development of age-related diseases. Here, we review animal and human studies that have investigated the possible links between telomere ablation and the pathogenesis of hypertension and related target organ damage. Although evidence is mounting that alterations in telomerase activity and telomere shortening may play a role in the pathogenesis of hypertension, additional studies are required to understand the molecular mechanisms by which telomere dysfunction and hypertension are functionally connected. As our knowledge on this emerging field grows, the challenge will be to ascertain whether all this information might translate into clinical applications.     

Activation of antitumor cytotoxic T lymphocytes by fusions of human dendritic cells and breast carcinoma cells

We have reported that fusions of murine dendritic cells (DCs) and murine carcinoma cells reverse unresponsiveness to tumor-associated antigens and induce the rejection of established metastases. In the present study, fusions were generated with primary human breast carcinoma cells and autologous DCs. Fusion cells coexpressed tumor-associated antigens and DC-derived costimulatory molecules. The fusion cells also retained the functional potency of DCs and stimulated autologous T cell proliferation. Significantly, the results show that autologous T cells are primed by the fusion cells to induce MHC class I-dependent lysis of autologous breast tumor cells. These findings demonstrate that fusions of human breast cancer cells and DCs activate T cell responses against autologous tumors.     

Telomere dysfunction alters the chemotherapeutic profile of transformed cells

Telomerase inhibition has been touted as a novel cancer-selective therapeutic goal based on the observation of high telomerase levels in most cancers and the importance of telomere maintenance in long-term cellular growth and survival. Here, the impact of telomere dysfunction on chemotherapeutic responses was assessed in normal and neoplastic cells derived from telomerase RNA null (mTERC(-/-)) mice. Telomere dysfunction, rather than telomerase per se, was found to be the principal determinant governing chemosensitivity specifically to agents that induced double-stranded DNA breaks (DSB). Enhanced chemosensitivity in telomere dysfunctional cells was linked to therapy-induced fragmentation and multichromosomal fusions, whereas telomerase reconstitution restored genomic integrity and chemoresistance. Loss of p53 function muted the cytotoxic effects of DSB-inducing agents in cells with telomere dysfunction. Together, these results point to the combined use of DSB-inducing agents and telomere maintenance inhibition as an effective anticancer therapeutic approach particularly in cells with intact p53-dependent checkpoint responses.     

Ku is required for telomeric C-rich strand maintenance but not for end-to-end chromosome fusions in Arabidopsis

Telomere dysfunction arising from mutations in telomerase or in telomere capping proteins leads to end-to-end chromosome fusions. Paradoxically, the Ku7080 heterodimer, essential for nonhomologous end-joining double-strand break repair, is also found at telomeres, and in mammals it is required to prevent telomere fusion. Previously, we showed that inactivation of Ku70 in Arabidopsis results in telomere lengthening. Here, we have demonstrated that this telomere elongation is telomerase dependent. Further, we found that the terminal 3' G overhang was significantly extended in ku70 mutants and in plants deficient in both Ku70 and the catalytic subunit of telomerase (TERT), implying that Ku is needed for proper maintenance of the telomeric C-rich strand. Consistent with inefficient C-strand maintenance, telomere shortening was accelerated in ku70 tert double mutants, and the onset of a terminal sterile phenotype was reached two to three times faster than in tert single mutants. Unexpectedly, abundant anaphase bridges were found in terminal plants harboring critically shortened telomeres, indicating that Ku is not required for the formation of end-to-end chromosome fusions in telomerase-deficient Arabidopsis. Together, these findings define Ku70 as a gene in higher eukaryotes required for maintenance of the telomeric C-rich strand and underscore the complexity and diversity of molecular interactions at telomeres.     

Telomere dynamics in Fancg-deficient mouse and human cells

A number of DNA repair proteins also play roles in telomere metabolism. To investigate whether the accelerated telomere shortening reported in Fanconi anemia (FA) hematopoietic cells relates to a direct role of the FA pathway in telomere maintenance, we have analyzed telomere dynamics in Fancg-deficient mouse and human cells. We show here that both hematopoietic (stem and differentiated bone marrow cells, B and T lymphocytes) and nonhematopoietic (germ cells, mouse embryonic fibroblasts [MEFs]) Fancg(-/-) mouse cells display normal telomere length, normal telomerase activity, and normal chromosome end-capping, even in the presence of extensive clastogen-induced cytogenetic instability (mitomycin C [MMC], gamma-radiation). In addition, telomerase-deficient MEFs with humanlike telomere length and decreased Fancg expression (G5 Terc(-/-)/Fancg shRNA3 MEFs) display normal telomere maintenance. Finally, early-passage primary fibroblasts from patients with FA of complementation group G as well as primary human cells with reduced FANCG expression (FANCG shRNA IMR90 cells) show no signs of telomere dysfunction. Our observations indicate that accelerated telomere shortening in patients with FA is not due to a role of FANCG at telomeres but instead may be secondary to the disease. These findings suggest that telomerase-based therapies could be useful prophylactic agents in FA aplastic anemia by preserving their telomere reserve in the context of the disease.     

Three‐dimensional telomere architecture of esophageal squamous cell carcinoma: comparison of tumor and normal epithelial cells

Telomeres are repetitive nucleotide sequences (TTAGGG)n located at the ends of chromosomes that function to preserve chromosomal integrity and prevent terminal end‐to‐end fusions. Telomere loss or dysfunction results in breakage–bridge–fusion cycles, aneuploidy, gene amplification and chromosomal rearrangements, which can lead to genomic instability and promote carcinogenesis. Evaluating the hypothesis that changes in telomeres contribute to the development of esophageal squamous cell carcinoma (ESCC) and to determine whether there are differences between young and old patients, we compared the three‐dimensional (3D) nuclear telomere architecture in ESCC tumor cells with that of normal epithelial cells obtained from the same patient. Patients were equally divided by age into two groups, one comprising those less than 45 years of age and the other consisting of those over 80 years of age. Tumor and normal epithelial cells located at least 10 cm from the border of the tumor were biopsied in ESCC patients. Hematoxylin and eosin staining was performed for each sample to confirm and identify the cancer and normal epithelial cells. This study was based on quantitative 3D fluorescence in situ hybridization (Q‐FISH), 3D imaging and 3D analysis of paraffin‐embedded slides. The 3D telomere architecture data were computer analyzed using 100 nuclei per slide. The following were the main parameters compared: the number of signals (number of telomeres), signal intensity (telomere length), number of telomere aggregates, and nuclear volume. Tumor and normal epithelial samples from 16 patients were compared. The normal epithelial cells had more telomere signals and higher intensities than the tumor cells, with P‐values of P < 0.0001 and P = 0.0078, respectively. There were no statistically significant differences in the numbers of telomere aggregates or the nuclear volumes between the tumor and normal epithelial cells. Secondary analyses examined the effects of age on 3D telomere architecture and found no statistically significant differences in any parameter tested between the young and old patients in either the tumor or epithelial cells. The 3D nuclear telomeric signature was able to detect differences in telomere architecture between the ESCC and normal epithelial tissues. However, there were no differences observed between the young and old patients.     

Model of human aging: Recent findings on Werner’s and Hutchinson-Gilford progeria syndromes

The molecular mechanisms involved in human aging are complicated. Two progeria syndromes, Werner’s syndrome (WS) and Hutchinson-Gilford progeria syndrome (HGPS), characterized by clinical features mimicking physiological aging at an early age, provide insights into the mechanisms of natural aging. Based on recent findings on WS and HGPS, we suggest a model of human aging. Human aging can be triggered by two main mechanisms, telomere shortening and DNA damage. In telomere-dependent aging, telomere shortening and dysfunction may lead to DNA damage responses which induce cellular senescence. In DNA damage-initiated aging, DNA damage accumulates, along with DNA repair deficiencies, resulting in genomic instability and accelerated cellular senescence. In addition, aging due to both mechanisms (DNA damage and telomere shortening) is strongly dependent on p53 status. These two mechanisms can also act cooperatively to increase the overall level of genomic instability, triggering the onset of human aging phenotypes.     

DNA-PKcs is critical for telomere capping

The DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is critical for DNA repair via the nonhomologous end joining pathway. Previously, it was reported that bone marrow cells and spontaneously transformed fibroblasts from SCID (severe combined immunodeficiency) mice have defects in telomere maintenance. The genetically defective SCID mouse arose spontaneously from its parental strain CB17. One known genomic alteration in SCID mice is a truncation of the extreme carboxyl terminus of DNA-PKcs, but other as yet unidentified alterations may also exist. We have used a defined system, the DNA-PKcs knockout mouse, to investigate specifically the role DNA-PKcs specifically plays in telomere maintenance. We report that primary mouse embryonic fibroblasts (MEFs) and primary cultured kidney cells from 6-8 month-old DNA-PKcs-deficient mice accumulate a large number of telomere fusions, yet still retain wild-type telomere length. Thus, the phenotype of this defect separates the two-telomere related phenotypes, capping, and length maintenance. DNA-PKcs-deficient MEFs also exhibit elevated levels of chromosome fragments and breaks, which correlate with increased telomere fusions. Based on the high levels of telomere fusions observed in DNA-PKcs deficient cells, we conclude that DNA-PKcs plays an important capping role at the mammalian telomere.     

DNA double-strand break repair proteins are required to cap the ends of mammalian chromosomes

Recent findings intriguingly place DNA double-strand break repair proteins at chromosome ends in yeast, where they help maintain normal telomere length and structure. In the present study, an essential telomere function, the ability to cap and thereby protect chromosomes from end-to-end fusions, was assessed in repair-deficient mouse cell lines. By using fluorescence in situ hybridization with a probe to telomeric DNA, spontaneously occurring chromosome aberrations were examined for telomere signal at the points of fusion, a clear indication of impaired end-capping. Telomeric fusions were not observed in any of the repair-proficient controls and occurred only rarely in a p53 null mutant. In striking contrast, chromosomal end fusions that retained telomeric sequence were observed in nontransformed DNA-PK(cs)-deficient cells, where they were a major source of chromosomal instability. Metacentric chromosomes created by telomeric fusion became even more abundant in these cells after spontaneous immortalization. Restoration of repair proficiency through transfection with a functional cDNA copy of the human DNA-PK(cs) gene reduced the number of fusions compared with a negative transfection control. Virally transformed cells derived from Ku70 and Ku80 knockout mice also displayed end-to-end fusions. These studies demonstrate that DNA double-strand break repair genes play a dual role in maintaining chromosomal stability in mammalian cells, the known role in repairing incidental DNA damage, as well as a new protective role in telomeric end-capping.     

Telomere shortening of epithelial cells characterises the adenoma-carcinoma transition of human colorectal cancer

BACKGROUND: and aims: Chromosomal instability is one of the most consistent markers of sporadic colorectal cancer in humans. There is growing evidence that telomere shortening is one of the mechanisms leading to chromosomal instability and cancer initiation. METHODS: To test this hypothesis, the telomere length of colorectal epithelial cells and cells from connective tissue was determined at the adenoma-carcinoma transition at the cellular level by quantitative fluorescence in situ hybridisation. RESULTS: Our study showed that the telomere fluorescence intensity of epithelial cells was significantly weaker at the earliest morphologically definable stage of carcinoma-high grade dysplasia with minimal invasive growth-compared with the surrounding adenoma. In contrast, cells from connective tissue had a similar telomere signal intensity at the carcinoma stage compared with the adenoma, and in turn cells from connective tissue had overall significantly stronger telomere fluorescence signals compared with epithelial cells. CONCLUSIONS: These results demonstrate that short telomeres of epithelial cells characterise the adenoma-carcinoma transition during human colorectal carcinogenesis, suggesting that carcinomas arise from cells with critical short telomeres within the adenoma. Since the adenoma-carcinoma transition in colorectal cancer is characterised by an increase in chromosomal instability and anaphase bridges, our data support the hypothesis that short telomeres initiate colorectal cancer by induction of chromosomal instability.     

Hepatocellular telomere shortening correlates with chromosomal instability and the development of human hepatoma

The telomere hypothesis of cancer initiation indicates that telomere shortening initiates cancer by induction of chromosomal instability. To test whether this hypothesis applies to human hepatocellular carcinoma (HCC), we analyzed the telomere length of hepatocytes in cytological smears of fine-needle biopsies of liver tumors from patients with cirrhosis (n = 39). The tumors consisted of 24 HCC and 15 regenerative nodules as diagnosed by combined histological and cytological diagnostics. In addition, we analyzed the telomere length of hepatocytes in HCC and surrounding noncancerous liver tissue within individual patients in another cohort of 10 patients with cirrhosis. Telomere length analysis of hepatocytes was correlated with tumor pathology and ploidy grade of the tumors, which was analyzed by cytophotometry. Telomeres were significantly shortened in hepatocytes of HCC compared to hepatocytes in regenerative nodules or surrounding noncancerous liver tissue. Hepatocyte telomere shortening in HCC was independent of the patient's age. There was no overlap in mean telomere lengths of individual samples when comparing HCC with regenerative nodules or noncancerous surrounding liver. Within the HCC group, telomeres were significantly shorter in hepatocytes of aneuploid tumors compared to diploid tumors. In conclusion, our data suggest that the telomere hypothesis of cancer initiation applies to human HCC and that cell type-specific telomere length analysis might indicate the risk of HCC development.     

Determining the influence of telomere dysfunction and DNA damage on stem and progenitor cell aging – what markers can we use?

The decline in organ maintenance and function is one of the major problems limiting quality of life during aging. The accumulation of telomere dysfunction and DNA damage appears to be one of the underlying causes. Uncapping of chromosome ends in response to critical telomere shortening limits the proliferative capacity of human cells by activation of DNA damage checkpoints inducing senescence or apoptosis. Telomere shortening occurs in the vast majority of human tissues during aging and in chronic diseases that increase the rate of cell turnover. There is emerging evidence that telomere shortening can limit the maintenance and function of adult stem cells -- a cell type of utmost importance for organ maintenance and regeneration. In mouse models, telomere dysfunction leads to a depletion of adult stem cell compartments suggesting that stem cells are very sensitive to DNA damage. Both the rarity of stem and progenitor cells in adult organs and their removal in response to damage make it difficult to assess the impact of telomere dysfunction and DNA damage on stem and progenitor cell aging. Such approaches require the development of sensitive biomarkers recognizing low levels of telomere dysfunction and DNA damage in stem and progenitor cells. Here, we review experimental data on the prevalence of telomere dysfunction and DNA damage during aging and its possible impact on stem and progenitor cell aging.     

Insights into Cdc13 dependent telomere length regulation

Cdc13 is a single stranded telomere binding protein that specifically localizes to the telomere ends of budding yeasts and is essential for cell viability. It caps the ends of chromosomes thus preventing chromosome end-to-end fusions and exonucleolytic degradation, events that could lead to genomic instability and senescence, the hallmark of aging. Cdc13 is also involved in telomere length regulation by recruiting or preventing access of telomerase to the telomeric overhang. Recruitment of telomerase to the telomeres for G-strand extension is required for continuous cell division, while preventing its access to the telomeres through capping the chromosome ends prevents mitotic events that could lead to cell immortality, the hall mark of carcinogenesis. Cdc13 and its putative homologues human CTC1 and POT1 are therefore key to many biological processes directly associated with life extension and cancer prevention and can be viewed as an ideal target for cancer and age related therapies.     

Estrogen mediates Aurora-A overexpression, centrosome amplification, chromosomal instability, and breast cancer in female ACI rats

Estrogens play a crucial role in the causation and development of sporadic human breast cancer (BC). Chromosomal instability (CIN) is a defining trait of early human ductal carcinoma in situ (DCIS) and is believed to precipitate breast oncogenesis. We reported earlier that 100% of female ACI (August/Copenhagen/Irish) rats treated with essentially physiological serum levels of 17beta-estradiol lead to mammary gland tumors with histopathologic, cellular, molecular, and ploidy changes remarkably similar to those seen in human DCIS and invasive sporadic ductal BC. Aurora-A (Aur-A), a centrosome kinase, and centrosome amplification have been implicated in the origin of aneuploidy via CIN. After 4 mo of estradiol treatment, levels of Aur-A and centrosomal proteins, gamma-tubulin and centrin, rose significantly in female ACI rat mammary glands and remained elevated in mammary tumors at 5-6 mo of estrogen treatment. Centrosome amplification was initially detected at 3 mo of treatment in focal dysplasias, before DCIS. At 5-6 mo, 90% of the mammary tumor centrosomes were amplified. Comparative genomic hybridization revealed nonrandom amplified chromosome regions in seven chromosomes with a frequency of 55-82% in 11 primary tumors each from individual rats. Thus, we report that estrogen is causally linked via estrogen receptor alpha to Aur-A overexpression, centrosome amplification, CIN, and aneuploidy leading to BC in susceptible mammary gland cells.     

Centrosome amplification drives chromosomal instability in breast tumor development

Earlier studies of invasive breast tumors have shown that 60-80% are aneuploid and approximately 80% exhibit amplified centrosomes. In this study, we investigated the relationship of centrosome amplification with aneuploidy, chromosomal instability, p53 mutation, and loss of differentiation in human breast tumors. Twenty invasive breast tumors and seven normal breast tissues were analyzed by fluorescence in situ hybridization with centromeric probes to chromosomes 3, 7, and 17. We analyzed these tumors for both aneuploidy and unstable karyotypes as determined by chromosomal instability. The results were then tested for correlation with three measures of centrosome amplification: centrosome size, centrosome number, and centrosome microtubule nucleation capacity. Centrosome size and centrosome number both showed a positive, significant, linear correlation with aneuploidy and chromosomal instability. Microtubule nucleation capacity showed no such correlation, but did correlate significantly with loss of tissue differentiation. Centrosome amplification was detected in in situ ductal carcinomas, suggesting that centrosome amplification is an early event in these lesions. Centrosome amplification and chromosomal instability occurred independently of p53 mutation, whereas p53 mutation was associated with a significant increase in centrosome microtubule nucleation capacity. Together, these results demonstrate that independent aspects of centrosome amplification correlate with chromosomal instability and loss of tissue differentiation and may be involved in tumor development and progression. These results further suggest that aspects of centrosome amplification may have clinical diagnostic and/or prognostic value and that the centrosome may be a potential target for cancer therapy.     

Ink4a/Arf tumor suppressor does not modulate the degenerative conditions or tumor spectrum of the telomerase-deficient mouse

The Rb/p16(Ink4a) and p53/p19Arf tumor suppressor pathways have been linked to diverse cancer-relevant processes, including those governing the cellular responses to telomere dysfunction. In this study, we sought to provide direct genetic evidence of a role for the Ink4a/Arf tumor suppressor gene, encoding both p16(Ink4a) and p19(Arf), in modulating the cellular and tissue phenotypes associated with telomere dysfunction by using the mTerc Ink4a/Arf mouse model. In contrast to the rescue associated with p53 deficiency, Ink4a/Arf deficiency did not attenuate the degenerative phenotypes elicited by telomere dysfunction in the late-generation mTerc-/- mice. Furthermore, in contrast to accelerated cancer onset and increased epithelial cancers of late-generation mTerc-/- p53 mutant mice, late-generation mTerc-/- Ink4a/Arf mutant mice experienced a delayed tumor onset and maintained the lymphoma and sarcoma spectrum. Consistent with the negligible role of Ink4a/Arf in the telomere checkpoint response in vivo, late-generation mTerc-/- Ink4a/Arf-/- tissues show activated p53, and derivative tumor cell lines sustain frequent loss of p53 function, whereas all early generation mTerc Ink4a/Arf-/- tumor cell lines remain intact for p53. In addition, the late-generation mTerc-/- Ink4a/Arf-/- tumors showed activation of the alternative lengthening of telomere mechanism, underscoring the need for adaptation to the presence of telomere dysfunction in the absence of p16(Ink4a) and p19(Arf). These observations highlight the importance of genetic context in dictating whether telomere dysfunction promotes or suppresses age-related degenerative conditions as well as the rate of initiation and type of spontaneous cancers.     

Quantitative determination of telomerase activity in breast cancer and benign breast diseases.

Telomerase plays an important role in maintaining the stability of chromosomes. This ribonucleoprotein prevents chromosome ends (telomeres) from gradual loss with each cell division. It enables tumor cells to maintain telomere length, allowing indefinite replicative capacity. Telomerase activity has been detected in the majority of tumor and germ cells and in immortalized cell lines. Quantitative telomerase PCR-ELISA (TeloTAGGG Telomerase PCR ELISA(PLUS)) was evaluated for distinguishing benign and malignant breast tissue. Activity of telomerase was determined in 27 samples of fibrocystic and dysplastic tissues, 28 fibroadenomas and phylloid tumors, and 154 breast cancer tissues; 59 specimens were analyzed retrospectively. Analytical precision and linearity of the assay was tested using breast carcinoma cell line ZR-75-1 and breast tumor tissue extracts. About 4% of tumor samples were excluded from analysis due to interferences in the PCR reaction. Relative telomerase activity differed significantly in the groups of dysplastic tissues, fibroadenomas and carcinomas. The highest activity was found in breast cancer tissue. This method can identify breast cancer tissue with 73% clinical sensitivity and 93% specificity as compared to benign breast tumors. We did not find a correlation between telomerase activity and the tissue levels of estrogen and progesterone receptors, HER-2/neu oncoprotein concentration, tumor size, and lymph node positivity. Probability of disease-free survival was significantly lower for patients with telomerase activity higher than median value. As the assay for telomerase activity has very high analytical sensitivity and high specificity for cancer cells, this routinely used method may prove useful for distinguishing malignant phenotype of breast tissues.