Phenotypic Diversity Caused by Differential Expression of SFTPC-Cre-Transgenic Alleles.

Type II alveolar epithelial cells (AEC2s) play an essential role in the function and maintenance of the pulmonary epithelium. Several transgenic mice have been developed to study the function of these cells in vivo by using the human SFTPC promoter to drive expression of Cre recombinase. The precise activity of each of these transgenic alleles has not been studied, and previous reports suggest that their activity can depend on breeding strategies. We bred mice with a conditional allele of the essential telomere capping protein TRF2 with two different SFTPC-Cre-transgenic strains and observed opposite phenotypes (100% lethality vs. 100% viability). We characterized the Cre recombinase activity in these two transgenic lines and found that the contrasting phenotypes were driven by difference in embryonic expression of the two transgenes, likely due to position effects or differences in the transgenic constructs. We also tested if SFTPC-Cre activity was dependent on maternal or paternal inheritance. When paternally inherited, both SFTPC-Cre alleles produced offspring with constitutive reporter activity independent of the inheritance of the Cre allele, suggesting that Cre recombinase was expressed in the male germline before meiosis. Immunohistochemical analysis of the testis showed reporter activity during spermatogenesis. Analysis of single-cell RNA sequencing data from murine and human testis demonstrated SFTPC expression uniquely during human spermatogenesis, suggesting that use of the human promoter in these constructs is responsible for male germline activity. Our data highlight the importance of careful analysis of transgenic allele activity and identify an SFTPC-Cre allele that is useful for panepithelial targeting in the mouse.

Role of TRF2 and TPP1 regulation in idiopathic recurrent pregnancy loss.

Telomeres are the tandem repeats (TTAGGG) present at the ends of the chromosomes that ensure chromosome stability and protect chromosomes from degradation. Telomeres in somatic human cells shorten after every cellular division and are linked to the cellular senescence. In this study we have investigated telomere length and expression of shelterin genes in aborted fetus material from idiopathic recurrent pregnancy losses. Telomere length was measured using Telomere Restriction Fragment Length (TRF) analysis. The gene expression levels for important shelterin complex proteins (TRF1, TRF2, POT1, and TPP1) were determined by Real-time Quantitative Reverse Transcriptase PCR (qRT-PCR). Our results demonstrated down regulation of TRF2 and TPP1 and a strong decline in average telomere length in abort material from women suffering from idiopathic recurrent pregnancy loss. We suggest that shorter telomere length and downregulation of the major shelterin components TRF2 and TPP1 leading to "telomere uncapping", might play a critical role in recurrent pregnancy loss.

Mechanism of Action of G-Quadruplex-Forming Oligonucleotide Homologous to the Telomere Overhang in Melanoma.

T-oligo, a guanine-rich oligonucleotide homologous to the 3'-telomeric overhang of telomeres, elicits potent DNA-damage responses in melanoma cells; however, its mechanism of action is largely unknown. Guanine-rich oligonucleotides can form G-quadruplexes (G4), which are stabilized by the hydrogen bonding of guanine residues. In this study, we confirmed the G4-forming capabilities of T-oligo using nondenaturing PAGE, nuclear magnetic resonance, and immunofluorescence. Using an anti-G-quadruplex antibody, we showed that T-oligo can form G4 in the nuclei of melanoma cells. Furthermore, using DNase I in a nuclease degradation assay, G4-T-oligo was found to be more stable than single-stranded T-oligo. G4-T-oligo had decreased antiproliferative effects compared with single-stranded T-oligo. However, G4-T-oligo has similar cellular uptake as single-stranded T-oligo, as shown by FACS analysis. Inhibition of JNK, which causes DNA damage-induced apoptosis, partially reversed the antiproliferative activity of T-oligo. T-oligo also inhibited mRNA expression of human telomerase reverse transcriptase, a catalytic subunit of telomerase that was reversed by JNK inhibition. Furthermore, two shelterin complex proteins TRF2/POT1 were found to be up-regulated and bound by T-oligo, suggesting that T-oligo may mediate dissociation of these proteins from the telomere overhang. These studies show that T-oligo can form a G-quadruplex and that the antitumor effects of T-oligo may be mediated through POT1/TRF2 and via human telomerase reverse transcriptase inhibition through JNK activation.

The differential spatiotemporal expression pattern of shelterin genes throughout lifespan.

Shelterin forms the core complex of telomere proteins and plays critical roles in protecting telomeres against unwanted activation of the DNA damage response and in Emaintaining telomere length homeostasis. Although shelterin expression is believed to be ubiquitous for stabilization of chromosomal ends. Evidences suggest that some shelterin subunits have tissue-specific functions. However, very little is known regarding how shelterin subunit gene expression is regulated during development and aging. Using two different animal models, the mouse and zebrafish, we reveal herein that shelterin subunits exhibit distinct spatial and temporal expression patterns that do not correlate with the proliferative status of the organ systems examined. Together, this work shows that the shelterin subunits exhibit distinct spatiotemporal expression patterns, suggesting important tissue-specific functions during development and aging.

The telomere-binding protein TRF2 is required for metronomic therapeutic effects of gemcitabine and capecitabine.

Gemcitabine and capecitabine are two effective anticancer agents against solid tumors. The pharmacological mechanisms have been known as incorporation into DNA and thereby inhibition of DNA synthesis. When used as metronomic chemotherapy, they may inhibit angiogenesis and induce immunity. In our previous study, we showed that low-dose gemcitabine caused telomere shortening by stabilizing TRF2 that was required for XPF-dependent telomere loss. In this report, we established a SKOV3.ip1 ascites cell model. Tumor-bearing mice were treated with low-dose gemcitabine (GEM) or capecitabine (CAP). Both GEM and CAP caused telomere shortening and increased expression of TRF2 with improved ascites in nude mice and decreased in vitro clonogenic activity. TRF2 knockdown altered telomeres to a shortened but new status that may evade XPF-dependent telomere loss and conferred resistance of SKOV3.ip1 ascites cells to low-dose GEM and CAP. Our study provides a new mechanism of metronomic chemotherapy i.e. TRF2 is required for metronomic therapeutic effects of gemcitabine and capecitabine.

HULC cooperates with MALAT1 to aggravate liver cancer stem cells growth through telomere repeat-binding factor 2.

The dysregulation of lncRNAs has increasingly been linked to many human diseases, especially in cancers. Our results demonstrate HULC, MALAT1 and TRF2 are highly expressed in human hepatocellular carcinoma tissues, and HULC plus MALAT1 overexpression drastically promotes the growth of liver cancer stem cells. Mechanistically, both HULC and MALAT1 overexpression enhanced RNA polII, P300, CREPT to load on the promoter region of telomere repeat-binding factor 2(TRF2), triggering the overexpression, phosphorylation and SUMOylation of TRF2. Strikingly, the excessive TRF2 interacts with HULC or MALAT1 to form the complex that loads on the telomeric region, replacing the CST/AAF and recruiting POT1, pPOT1, ExoI, SNM1B, HP1 α. Accordingly, the telomere is greatly protected and enlonged. Furthermore, the excessive HULC plus MALAT1 reduced the methylation of the TERC promoter dependent on TRF2, increasing the TERC expression that causes the increase of interplay between TRET and TERC. Ultimately, the interaction between RFC and PCNA or between CDK2 and CyclinE, the telomerase activity and the microsatellite instability (MSI) are significantly increased in the liver cancer stem cells. Our demonstrations suggest that haploinsufficiency of HULC/MALAT1 plays an important role in malignant growth of liver cancer stem cell.

Telomeric repeat-binding factor 2: a marker for survival and anti-EGFR efficacy in oral carcinoma.

Oral Squamous Cell Carcinoma (OSCC) is the most common oral cancer worldwide. Treatments including surgery, radio- and chemo-therapies mostly result in debilitating side effects. Thus, a more accurate evaluation of patients at risk of recurrence after radio/chemo treatment is important for preserving their quality of life. We assessed whether the Telomeric Repeat-binding Factor 2 (TERF2) influences tumor aggressiveness and treatment response. TERF2 is over-expressed in many cancers but its correlation to patient outcome remains controversial in OSCC. Our retrospective study on sixty-two patients showed that TERF2 overexpression has a negative impact on survival time. TERF2-dependent survival time was independent of tumor size in a multivariate analysis. In vitro, TERF2 knockdown by RNA interference had no effect on cell proliferation, migration, senescence and apoptosis. Instead, TERF2 knockdown increased the expression of cytokines implicated in inflammation and angiogenesis, except for vascular endothelial growth factor. TERF2 knockdown resulted in a decrease vascularization and growth of xenograft tumors. Finally, response to erlotinib/Tarceva and cetuximab/Erbitux treatment was increased in TRF2 knocked-down cells. Hence, TERF2 may represent an independent marker of survival for OSCC and a predictive marker for cetuximab/Erbitux and erlotinib/Tarceva efficacy.

Knockdown of telomeric repeat binding factor 2 enhances tumor radiosensitivity regardless of telomerase status.

PURPOSE: To investigate the effects of TRF2 depletion on radiosensitivity in both the telomerase-positive cell lines (A549) and alternative lengthening of telomere (ALT) cell lines (U2OS). METHODS: X-ray irradiation was used to establish two radioresistant cancer models (A549R and U2OSR) from A549 and U2OS. Colony formation assay was applied to examine the radiosensitivity of radioresistant A549R and U2OSR cells and TRF2 low-expression cells. Real-time PCR and TeloTAGGG Telomerase PCR ELISA Kit were performed to examine telomere length and telomerase activity separately. γ-H2AX was detected by immunofluorescence to assess the radiation-induced DSBs. RESULTS: Radioresistant cancer models were established, in which TRF2 was significantly over-expressed. Low expression of TRF2 protein could enhance the radiosensitivity and induce telomere length of A549 and U2OS cell shortening. In A549 cells with TRF2 down-regulated, the telomerase activity was inhibited, too. TRF2 deficiency increases γ-H2AX foci and fails to protect telomere from radiation. CONCLUSION: The data suggest that TRF2 is a radioresistant protein in A549 and U2OS cells, and could potentially be a target for radiosensitization of both telomerase-positive and ALT cells in radiotherapy.

Suppression of Ku80 correlates with radiosensitivity and telomere shortening in the U2OS telomerase-negative osteosarcoma cell line.

Ku70/80 heterodimer is a central element in the nonhomologous end joining (NHEJ) DNA repair pathway, Ku80 playing a key role in regulating the multiple functions of Ku proteins. It has been found that the Ku80 protein located at telomeres is a major contributor to radiosensitivity in some telomerase positive human cancer cells. However, in ALT human osteosarcoma cells, the precise function in radiosensitivity and telomere maintenance is still unknown. The aim of this study was to investigate the effects of Ku80 depletion in the U2OS ALT cell line cell line. Suppression of Ku80 expression was performed using a vector-based shRNA and stable Ku80 knockdown in cells was verified by Western blotting. U2OS cells treated with shRNA-Ku80 showed lower radiobiological parameters (D0, Dq and SF2) in clonogenic assays. Furthermore, shRNA-Ku80 vector transfected cells displayed shortening of the telomere length and showed less expression of TRF2 protein. These results demonstrated that down-regulation of Ku80 can sensitize ALT cells U2OS to radiation, and this radiosensitization is related to telomere length shortening.

Anti-aging effects of vitamin C on human pluripotent stem cell-derived cardiomyocytes.

Human pluripotent stem cells (hPSCs) have arisen as a source of cells for biomedical research due to their developmental potential. Stem cells possess the promise of providing clinicians with novel treatments for disease as well as allowing researchers to generate human-specific cellular metabolism models. Aging is a natural process of living organisms, yet aging in human heart cells is difficult to study due to the ethical considerations regarding human experimentation as well as a current lack of alternative experimental models. hPSC-derived cardiomyocytes (CMs) bear a resemblance to human cardiac cells and thus hPSC-derived CMs are considered to be a viable alternative model to study human heart cell aging. In this study, we used hPSC-derived CMs as an in vitro aging model. We generated cardiomyocytes from hPSCs and demonstrated the process of aging in both human embryonic stem cell (hESC)- and induced pluripotent stem cell (hiPSC)-derived CMs. Aging in hESC-derived CMs correlated with reduced membrane potential in mitochondria, the accumulation of lipofuscin, a slower beating pattern, and the downregulation of human telomerase RNA (hTR) and cell cycle regulating genes. Interestingly, the expression of hTR in hiPSC-derived CMs was not significantly downregulated, unlike in hESC-derived CMs. In order to delay aging, vitamin C was added to the cultured CMs. When cells were treated with 100 µM of vitamin C for 48 h, anti-aging effects, specifically on the expression of telomere-related genes and their functionality in aging cells, were observed. Taken together, these results suggest that hPSC-derived CMs can be used as a unique human cardiomyocyte aging model in vitro and that vitamin C shows anti-aging effects in this model.

Stem cell expansion during carcinogenesis in stem cell-depleted conditional telomeric repeat factor 2 null mutant mice.

To examine the role of telomeric repeat-binding factor 2 (TRF2) in epithelial tumorigenesis, we characterized conditional loss of TRF2 expression in the basal layer of mouse epidermis. These mice exhibit some characteristics of dyskeratosis congenita, a human stem cell depletion syndrome caused by telomere dysfunction. The epidermis in conditional TRF2 null mice exhibited DNA damage response and apoptosis, which correlated with stem cell depletion. The stem cell population in conditional TRF2 null epidermis exhibited shorter telomeres than those in control mice. Squamous cell carcinomas induced in conditional TRF2 null mice developed with increased latency and slower growth due to reduced numbers of proliferating cells as the result of increased apoptosis. TRF2 null epidermal stem cells were found in both primary and metastatic tumors. Despite the low-grade phenotype of the conditional TRF2 null primary tumors, the number of metastatic lesions was similar to control cancers. Basal cells from TRF2 null tumors demonstrated extreme telomere shortening and dramatically increased numbers of telomeric signals by fluorescence in situ hybridization due to increased genomic instability and aneuploidy in these cancers. DNA damage response signals were detected at telomeres in TRF2 null tumor cells from these mice. The increased genomic instability in these tumors correlated with eightfold expansion of the transformed stem cell population compared with that in control cancers. We concluded that genomic instability resulting from loss of TRF2 expression provides biological advantages to the cancer stem cell population.

Expression of telomere binding proteins in gastric cancer and correlation with clinicopathological parameters.

AIM: The aim of this study was to investigate the expression of telomere repeat binding factor 1 (TRF1), TRF2 and protection of telomeres 1 (POT1) in gastric cancer and their relationships with clinicopathological features and telomerase activity. METHODS: In total 36 gastric cancer tissue and paired adjacent normal tissue were analyzed. The mRNA expression of telomere binding proteins TRF1, TRF2 and POT1 were measured using quantitative reverse transcription polymerase chain reaction, and telomerase activity was assessed by the telomeric repeat amplification protocol/enzyme linked immunosorbent assay method. RESULTS: The expression of POT1 was significantly higher in tumor tissue than in adjacent normal tissue (P < 0.001). Levels of TRF2 mRNA were significantly higher in bigger tumors (diameter ≥ 5 cm) than in small tumors (diameter < 5 cm) (P = 0.043). POT1 mRNA transcription levels were higher in tumors with lymph nodes metastases than in those without lymph nodes metastases (P = 0.048). POT1 expression was significantly correlated with tumor stage (P = 0.008). A higher level of expression of POT1 was observed in late-stage tumors (III, IV) than in early stage tumors (I, II). Telomerase activity was significantly higher in gastric cancers than in corresponding normal tissue (P < 0.001). Moreover, POT1 expression was significantly positive correlated with telomerase activity (r = 0.572, P < 0.01). CONCLUSION: POT1 was overexpressed in gastric cancer and may be associated with stomach carcinogenesis and gastric cancer progression.

Elevated TRF2 in advanced breast cancers with short telomeres.

Telomere repeat binding factor 2 (TRF2) binds directly to telomeres and preserves the structural integrity of chromosome ends. In vitro models suggest that expression of TRF2 protein increases during mammary cancer progression. However, a recent study has reported that TRF2 mRNA levels tend to be lower in clinical specimens of malignant breast tissue. Here, we conduct the first large-scale investigation to assess the levels and cellular localization of the TRF2 protein in normal, pre-malignant and malignant breast tissues. Breast tissue arrays, containing normal, ductal carcinoma in situ (DCIS) and invasive carcinoma specimens, were used to assess the expression and localization of TRF2 protein. Telomere lengths were semi-quantitatively measured using a pantelomeric peptide nucleic acid probe. A mixed effects modeling approach was used to assess the relationship between TRF2 expression and telomeric signal scores across disease states or clinical staging. We demonstrate that TRF2 is exclusively nuclear with a trend toward lower expression with increased malignancy. More case-to-case variability of TRF2 immunostaining intensity was noted amongst the invasive carcinomas than the other disease groups. Invasive carcinomas also displayed variable telomere lengths while telomeres in normal mammary epithelium were generally longer. Statistical analyses revealed that increased TRF2 immunostaining intensity in invasive carcinomas is associated with shorter telomeres and shorter telomeres correlate with a higher TNM stage. All immortalized and cancer cell lines within the array displayed strong, nuclear TRF2 expression. Our data indicate that elevated expression of TRF2 is not a frequent occurrence during the transformation of breast cancer cells in vivo, but higher levels of this telomere-binding protein may be important for protecting advanced cancer cells with critically short telomeres. Our findings also reinforce the concept that serially propagated cancer cells, although tumor-derived, may not model all types of authentic tumors especially those demonstrating genetic heterogeneity.

Expression of TRF1, TRF2, TIN2, TERT, KU70, and BRCA1 proteins is associated with telomere shortening and may contribute to multistage carcinogenesis of gastric cancer.

PURPOSE: Telomere dysfunction is believed to be a significant factor in carcinogenesis. To elucidate the carcinogenesis mechanism in gastric cancer, the expression of telomeric proteins and changes in telomere length were investigated during multistage carcinogenesis of gastric cancer. METHODS: Tissue samples were obtained during surgical operations from the normal gastric mucosa of 10 patients, the precancerous lesions of 15 patients, the gastric cancer tissues (GC) of 20 patients, and of tumors due to gastric cancer with lymph node metastasis (GCLM) from 5 patients. The expression of TRF1, TRF2, and TIN2 proteins was measured by Western blotting, while the expression of TERT, KU70, and BRCA1 proteins was detected using the immunohistochemical method. The mean telomere length was determined by Southern blotting. RESULTS: Compared with normal gastric mucosa tissues, the expression of TRF1, TRF2, and TIN2 proteins was significantly higher in precancerous lesions, GC, and GCLM (P < 0.01). The expression of TRF1, TRF2, and TIN2 proteins was significantly higher in GC and GCLM than in precancerous lesions (P < 0.01). The expression of TERT and Ku70 proteins in precancerous lesions and GC tissues was significantly higher than that in normal gastric mucosa tissues (P < 0.01). The expression of TERT and Ku70 proteins in GC tissues was significantly higher than in precancerous lesions (P < 0.01). In normal gastric mucosa, the BRCA1 protein was primarily located in the cell nucleus. In precancerous lesions and GC, the expression of the BRCA1 protein was apparent in the cell cytoplasm. The mean telomere length in precancerous lesions, GC, and GCLM was significantly shorter than that in normal gastric mucosa tissues (P < 0.05). The mean telomere length in GC and GCLM was significantly shorter than that in precancerous lesions (P < 0.05). The mean telomere length in all tissue samples was inversely correlated with the level of TRF1, TRF2, TIN2, TERT, and Ku70 proteins. CONCLUSIONS: Our results suggest that the over-expression of telomeric proteins, TRF1, TRF2, TIN2, TERT, and Ku70, and the transposition of the BRCA1 protein may work together to reduce the telomere length in precancerous lesions and gastric cancer, and could contribute to the multistage carcinogenesis of gastric cancer. These findings offer new insight into the mechanism of carcinogenesis in gastric cancer.

Altered mRNA expression of telomere binding proteins (TPP1, POT1, RAP1, TRF1 and TRF2) in ulcerative colitis and Crohn's disease.

AIMS: To determine mRNA expression of telomeric binding proteins in inflammatory bowel disease (IBD), and to note any effects of pharmacotherapy on telomere binding protein expression. METHODS: Peripheral blood mononuclear cells (PBMC) obtained from 31 IBD patients and 13 controls were activated with phytohaemagglutinin and purified to yield activated (CD25+) T lymphocytes. TPP1, POT1, RAP1, TRF1 and TRF2 mRNA expression in PBMC and activated T lymphocytes was measured with RT-PCR. RESULTS: In activated (CD25+) T lymphocytes, mean TRF2 mRNA levels were lower in both UC (6.6 vs 10, p=0.004) and CD subjects (6.9 vs 10; p=0.004). Similarly. in activated (CD25+) T lymphocytes mean RAP1 mRNA expression was significantly lower in UC subjects (4.5 vs 9.8, p=0.029) but not in CD subjects. In resting PBMC, mean TRF1 mRNA levels were lower in both UC (2.6 vs 3.5; p=0.008) and CD subjects (1.0 vs 3.5; p=0.04). No difference in PBMC and activated (CD25+) T lymphocytes mRNA levels of TPP1 and POT1 were noted in either UC or CD subjects. An association with 5-aminosalicylate therapy (R(2)=0.4) was only detected with RAP1 mRNA expression. TRF2 mRNA expression was inversely associated with disease duration only in UC subjects (p=0.05; R(2)=-0.6). CONCLUSIONS: The downregulation of TRF2 and RAP1 mRNA expression in CD25+ T-lymphocytes in IBD suggests that these telomere binding proteins play a role in telomere regulation and may contribute to the telomeric fusions and chromosomal abnormalities observed in UC. These findings may also indicate a systemic process of telomere uncapping which could represent a biomarker for IBD associated cancer risk.

Sp1 upregulates expression of TRF2 and TRF2 inhibition reduces tumorigenesis in human colorectal carcinoma cells.

EXPERIMENTAL DESIGN: Telomere repeat binding factor 2 (TRF2) plays a key role in the protective activity of telomere and is overexpression in several kinds of solid cancer cells. However, the role of overexpressed TRF2 in colorectal carcinoma remains unclear. The aim of this study was to determine the expression of TRF2, address the mechanism of TRF2 overexpression in human colorectal carcinoma. In present study, we examined the expression of TRF2 in colorectal cancer tissues from 39 patients, peritumoral normal tissues from 21 patients, and colon carcinoma SW480 cell line by quantitative PCR, immunohistochemistry and western blot. After siRNA silencing TRF2 expression in SW480, tumorigenesis of TRF2 was tested by cell proliferation, soft agar assay, cytofluorimetric analysis and cytogenetic analysis. To discover transcription factor that mediated TRF2 expression, Chromatin Immunoprecipitation (Chip) Assay and Electrophoretic mobility shift assays (EMSA) were employed. RESULTS: Overexpression of TRF2 protein was detected in SW480 cells and 19 of 39 colorectal carcinoma tissues (49%), no overexpression was observed in 21 of 21 adjacent peritumoral normal colorectal tissues. After siRNA silencing TRF2 expression, the proliferation and colony formation of SW480 cells were significantly inhibited. Defective TRF2 induced apoptosis and increased chromosomal instability in SW480 cells, in which there were more end-to-end fusions and ring chromosomes. Chip assay and EMSA showed that transcription factor Sp1 is involved in upregulation of TRF2. These results indicate that TRF2 is overexpressed in colorectal carcinoma, Sp1 upregulates TRF2 expression, TRF2 inhibition reduces tumorigenesis of colorectal cancer, which suggests that TRF2 and SP1 may become new targets for the development of anti-cancer therapy in colorectal carcinoma.

Telomere shortening is correlated with the DNA damage response and telomeric protein down-regulation in colorectal preneoplastic lesions.

BACKGROUND: A relation between telomere attrition in early carcinogenesis and activation of DNA damage response (DDR) has been proposed. We explored telomere length and its link with DDR in colorectal multistep carcinogenesis. PATIENTS AND METHODS: We studied normal mucosa, low-grade dysplasia (LGD) and high-grade dysplasia (HGD) and invasive carcinoma (IC) in matched human colon specimens by evaluating p-ataxia telangiectasia mutated (ATM), p-checkpoint kinase 2 (Chk2), c-H2AX, TRF1 and TRF2 expressions by immunohistochemistry. FISH was used to assess telomere length. RESULTS: Telomeres shortened significantly from normal (N) to LGD and HGD (P < 0.0001; P = 0.012), then increased in length in IC (P = 0.006). TRF1 and TRF2 expressions were diminished from N to LGD and HGD (P = 0.004, P < 0.0001, ns) and were reexpressed at the invasive stage (P = 0.053 and P = 0.046). Phosphorylated ATM, Chk2 and H2AX appeared already in LGD (respectively, P = 0.001, P = 0.002 and P = 0.02). Their expression decreased from HGD to IC (respectively, P = 0.03, P = 0.02 and P = 0.37). These activating phosphorylations were inversely correlated with telomere length and TRF1/2 expression. CONCLUSION: In a model of colon multistep carcinogenesis, our data indicate that telomeric length and protein expression levels are inversely correlated with the activation of the DDR pathway.

hTERT, the catalytic component of telomerase, is downregulated in the haematopoietic stem cells of patients with chronic myeloid leukaemia.

Telomere shortening is associated with disease progression in chronic myeloid leukaemia (CML). To investigate the biology and regulation of telomerase in CML, we evaluated expression of the telomerase components, its regulators and several telomeric-associated proteins. Quantitative real-time-polymerase chain reaction (PCR) was used to compare gene expression in the CD34+/leukaemic blast cells of 22 CML patient samples to the CD34+ cell population of healthy individuals. hTERT, the catalytic component of telomerase, was downregulated in eight of 12 chronic phase (CP) patients (P = 0.0387). Furthermore, hTERT was significantly downregulated in two of three patients in accelerated phase (AP) and seven of seven patients in blast crisis (BC), P = 0.0017. Expression of hTR and telomeric-associated proteins TEP1, TRF1, TRF2, tankyrase and PinX1 was high in the majority of CP and AP patients. With the exceptions of TEP1 and hTR, expression of these factors was highest in CP and decreased during disease progression. Expression of c-Myc, a positive regulator of hTERT transcription, correlated with hTERT expression and decreased with disease progression, falling below control levels in BC. hTERT levels were increased in CP patients following successful treatment with imatinib, relative to untreated CP patients. We suggest that reduced hTERT expression directly causes the shortened telomeres observed in CML.

Accumulation and altered localization of telomere-associated protein TRF2 in immortally transformed and tumor-derived human breast cells.

We have used cultured human mammary epithelial cells (HMEC) and breast tumor-derived lines to gain information on defects that occur during breast cancer progression. HMEC immortalized by a variety of agents (the chemical carcinogen benzo(a)pyrene, oncogenes c-myc and ZNF217, and/or dominant negative p53 genetic suppressor element GSE22) displayed marked upregulation (10-15 fold) of the telomere-binding protein, TRF2. Upregulation of TRF2 protein was apparently due to differences in post-transcriptional regulation, as mRNA levels remained comparable in finite lifespan and immortal HMEC. TRF2 protein was not upregulated by the oncogenic agents alone in the absence of immortalization, nor by expression of exogenously introduced hTERT genes. We found TRF2 levels to be at least twofold higher than in control cells in 11/15 breast tumor cell lines, suggesting that elevated TRF2 levels are a frequent occurrence during the transformation of breast tumor cells in vivo. The dispersed distribution of TRF2 throughout the nuclei in some immortalized and tumor-derived cells indicated that not all the TRF2 was associated with telomeres in these cells. The process responsible for accumulation of TRF2 in immortalized HMEC and breast tumor-derived cell lines may promote tumorigenesis by contributing to the cells' ability to maintain an indefinite lifespan.

Statins enhance migratory capacity by upregulation of the telomere repeat-binding factor TRF2 in endothelial progenitor cells.

BACKGROUND: Cultivation of endothelial progenitor cells (EPCs) leads to premature replicative senescence, limiting ex vivo expansion for potential clinical cell therapy. Recent studies have linked senescence to the dysfunction of telomeres, the "ends" of chromosomes, via the so-called mitotic clock or culture-induced stress. The purpose of this study was to elucidate a possible role of telomere biology in the functional augmentation of EPCs by statins. METHODS AND RESULTS: Human EPCs were isolated from peripheral blood. Using flow cytometry after fluorescence in situ hybridization with a telomere-specific (C3TA2)3 peptide nucleic acid probe (Flow-FISH), we found mean telomere length in untreated EPCs from healthy subjects to range between 8.5+/-0.2 and 11.1+/-0.5 kb with no change over 6 days of culture, excluding telomere erosion as one cause for premature senescence. Although mean telomere length did not differ between statin-treated and untreated EPCs, atorvastatin (0.1 micromol/L) and mevastatin (1.0 micromol/L) both led to a more than 3-fold increase in the expression of the telomere capping protein TRF2 (telomere repeat-binding factor), as shown by immunoblotting, whereas quantitative reverse transcription-polymerase chain reaction demonstrated no increase in TRF2 mRNA. Telomere dysfunction of EPCs was also paralleled by a 4-fold increase in the DNA damage checkpoint-kinase 2 (Chk2). Conversely, statin cotreatment or overexpression of TRF2 completely suppressed Chk2 induction. Finally, overexpression of a dominant negative mutant of the TRF2 protein abrogated statin-induced enhancement of migratory activity down to baseline values. CONCLUSIONS: Ex vivo culturing of EPCs leads to "uncapping" of telomeres, indicated by the loss of TRF2. Statin cotreatment of EPCs prevents impairment of their functional capacity by a TRF2-dependent, posttranscriptional mechanism. This is the first time a beneficial effect of statins on telomere biology has been described.