Apoptosis ; physiology ; Cell Cycle ; physiology ; Humans ; Neoplasms ; etiology ; metabolism ; Tankyrases ; genetics ; physiology ; Telomerase ; metabolism ; physiology ; Telomere ; genetics ; metabolism ; Telomere-Binding Proteins ; genetics ; physiology ; Telomeric Repeat Binding Protein 1 ; genetics ; physiology ; Telomeric Repeat Binding Protein 2 ; genetics ; physiology

OBJECTIVE: To investigate the effect of RNA interference silencing telomere repeat factor 2 by observing Hep-2 cells' proliferation and apoptosis in larynx carcinoma cell line. METHOD: A recombinant plasmid containing a single shRNA (shTRF2) was constructed. The expression of TRF2 gene was detected by PCR and cell proliferation was examined using CCK-8. Hep-2 cells' apoptosis was detected by flow cytometer (FCM). RESULT: After treatment with shTRF2, the expression of TRF2 was distinctively depressed, and Hep-2 cells proliferation was obviously inhibited. Compared with control and negative group, cells with treatment of RNAi exhibited significantly more apoptosis. CONCLUSION Using RNA interference technique to silence TRF2 gene is effective on inhibiting cancer cells' proliferation and help to induce cancer cells' apoptosis.
Apoptosis ; genetics ; Cell Line, Tumor ; Cell Proliferation ; Humans ; RNA Interference ; RNA, Small Interfering ; genetics ; Telomeric Repeat Binding Protein 2 ; genetics

With the smooth move towards the coming expected clinical reports of anticancer pharmaceutical molecules targeting telomeres and telomerase, and also with the exciting success in the extension of lifespan by regulating telomerase activity without increased onset of oncogenesis in laboratory mouse models (Garcia-Cao et al., 2006; Jaskelioff et al., 2011), we are convinced that targeting telomeres based on telomerase will be a potential approach to conquer both aging and cancer and the idea of longevity seems to be no more mysterious. More interestingly, emerging evidences from clinical research reveal that other telomeric factors, like specific telomeric binding proteins and nonspecific telomere associated proteins also show crucial importance in aging and oncogenesis. This stems from their roles in the stability of telomere structure and in the inhibition of DNA damage response at telomeres. Uncapping these proteins from chromosome ends leads to dramatic telomere loss and telomere dysfunction which is more abrupt than those induced by telomerase inactivation. Abnormal expression of these factors results in developmental failure, aging and even oncogenesis evidenced by several experimental models and clinical cases, indicating telomere specific proteins and its associated proteins have complimentary roles to telomerase in telomere protection and controlling cellular fate. Thus, these telomeric factors might be potential clinical biomarkers for early detection or even therapeutic targets of aging and cancer. Future studies to elucidate how these proteins function in telomere protection might benefit patients suffering aging or cancer who are not sensitive to telomerase mediation.
Aging ; Biomarkers ; metabolism ; Humans ; Longevity ; Neoplasms ; metabolism ; pathology ; Telomerase ; metabolism ; Telomere ; metabolism ; ultrastructure ; Telomeric Repeat Binding Protein 2 ; metabolism

This study was aimed to investigate the expression levels of TRF1, TRF2 and RAP1 mRNA in peripheral blood mononuclear cells of patients with acquired aplastic anemia, and to explore their relation with onset of acquired aplastic anemia. Peripheral blood mononuclear cells of 40 patients with acquired aplastic anemia and 20 normal subjects as control were collected to detect mRNA expression of TRF1, TRF2 and RAP1 by using real-time quantitative polymerase chain reaction. The results showed that the expression levels of TRF1 and RAP1 in peripheral blood mononuclear cells of patients with acquired aplastic anemia were significantly higher than that in normal controls (P < 0.05), while the expression level of TRF2 was lower than that in normal controls (P < 0.01). There was significant correlation between TRF2 and RAP1 expressions level (r = 0.522, P = 0.001). It is concluded that the changes in expression levels of TRF1, TRF2 and RAP1 may play a role in the pathogenesis of acquired aplastic anemia.
Adolescent ; Adult ; Aged ; Aged, 80 and over ; Anemia, Aplastic ; blood ; Case-Control Studies ; Child ; Female ; Humans ; Leukocytes, Mononuclear ; metabolism ; Male ; Middle Aged ; RNA, Messenger ; genetics ; Telomeric Repeat Binding Protein 1 ; metabolism ; Telomeric Repeat Binding Protein 2 ; metabolism ; Young Adult ; rap1 GTP-Binding Proteins ; metabolism

OBJECTIVETo detect the expression levels of telomere binding factor 2 (TRF2) on leukemia cell lines and primary leukemia cells.

METHODSThe expression of TRF2 mRNA was detected with quantitative real-time RT-PCR in leukemia cell lines and primary leukemia cells. The Western blot analysis was used for the detection of TRF2 protein expression.

RESULTTRF2 was overexpressed in T-cell leukemia cell lines but not in myelogenous leukemia cell lines. Significant higher expression levels of TRF2 were observed in primary leukemia cells from patients with M0 and M1 subtypes of acute myelogenous leukemia (AML) compared with normal control and other subtypes of AML.

CONCLUSIONIncreased TRF2 expression levels are found in T-cell leukemia cell lines and AML patients with poor prognosis, which suggests that TRF2 expression might be related to the prognosis of leukemia.

Adolescent ; Adult ; Female ; HL-60 Cells ; Humans ; Jurkat Cells ; K562 Cells ; Leukemia, Myeloid, Acute ; metabolism ; Leukemia, T-Cell ; metabolism ; pathology ; Male ; Middle Aged ; RNA, Messenger ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Telomeric Repeat Binding Protein 2 ; metabolism ; Young Adult

OBJECTIVETo clarify the regulation of p53 through telomere pathway by investigating the molecular interaction between p53 and the main telomere-associated protein Telomeric Repeat Factor 2 (TRF2) in vitro.

METHODSFour different p53-GST (glutathione S-transferase) fusion proteins and GST were expressed in E. coli and purified through glutathione sepharose 4B beads. The human recombinant p53s included wild type p53 (1-393), N terminus-truncated form p53 2C (95-393), C terminus-truncated form p53 N5 (2-293) and single amino acid mutant p53 R175H (175 arginine to histidine). Purified p53-GST fusion proteins and GST were mixed with cellular protein extracts of human breast cancer cells MCF-7 in vitro by pull down. The molecular interaction between p53 and TRF2 were detected by Western blot.

RESULTSSDS-PAGE and Coomassie brilliant blue staining showed that the molecular weights of all purified proteins were as expected, with purities over 90%. Western blot of TRF2 indicated that both wild type p53 and p53 R175H could bind with TRF2 of MCF-7 cells in similar capacity, while GST alone failed to do so. The molecular interaction between p53 2C and TRF2 was enhanced. In contrast, the interaction between p53 N5 and TRF2 was significantly reduced.

CONCLUSIONSp53 can interact with TRF2 directly and specifically in vitro, with C terminus of p53 (293-393) being the binding region for their interaction. This C terminus-dependent interaction between p53 and TRF2 may be related to the cellular activities induced by telomere alterations.

Breast Neoplasms ; metabolism ; pathology ; Cell Line, Tumor ; DNA-Binding Proteins ; metabolism ; Escherichia coli ; genetics ; metabolism ; Female ; Glutathione Transferase ; genetics ; metabolism ; Humans ; Protein Binding ; Recombinant Fusion Proteins ; isolation & purification ; metabolism ; Telomeric Repeat Binding Protein 2 ; metabolism ; Transformation, Genetic ; Tumor Suppressor Protein p53 ; genetics ; metabolism

To investigate the mechanisms of the telomerase regulations during the apoptosis of the human MDS-RAEB cell line MUTZ-1 cells induced by arsenic trioxide (As(2)O(3)), telomerase activity was detected by TRAP-ELISA and the expressions of mRNAs of hTERT, TRF1 (TTAGGG repeat binding factor 1), TRF2 (TTAGGG repeat binding factor 2), bcl-2, and bax genes were detected by RT-PCR. Apoptosis was detected by translocation of phosphatidylserine (PS) by flow cytometry. The results showed that 1 - 8 micromol/L of As(2)O(3) induced typical apoptosis of MUIZ-1 cells in the dose-and time-dependent manners, the telomerase activity could be down-regulated at this concentration and negatively correlated with increased apoptosis (r = -0.938, P = 0.018). The expression of telomerase activity was positively related to the expression of hTERT (r = 0.783, P = 0.022), but As(2)O(3) had no effect on the mRNA expression of TRF1 and TRF2 genes. The inhibition of telomerase activity by As(2)O(3) on MUTZ-1 cells was accompanied with the low expression of bcl-2 gene and the decrease of bcl-2/bax ratio. It is concluded that the apoptosis of MUTZ-1cells induced by As(2)O(3) may occur via the inhibition of telomerase activity and down-regulation of the expression of hTERT mRNA, and this may be one of the mechanisms inducing apoptosis in MUTZ-1 cells treated by As(2)O(3).
Antineoplastic Agents ; pharmacology ; Apoptosis ; drug effects ; Arsenicals ; pharmacology ; Cell Line ; Dose-Response Relationship, Drug ; Flow Cytometry ; Gene Expression ; drug effects ; Humans ; Myelodysplastic Syndromes ; genetics ; metabolism ; pathology ; Oxides ; pharmacology ; Proto-Oncogene Proteins c-bcl-2 ; genetics ; RNA, Messenger ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Telomerase ; genetics ; metabolism ; Telomeric Repeat Binding Protein 2 ; genetics ; bcl-2-Associated X Protein ; genetics

OBJECTIVETo identify genes associated with metastasis suppression and to investigate the molecular mechanism of osteosarcoma metastasis.

METHODSThe subtracted cDNA library of low metastatic human osteosarcoma cell line SOSP-9607 was constructed using suppression subtractive hybridization. Partial clones were sequenced. The acquired sequence data were aligned against the GenBank nucleotide database using Blastn to search for sequence matches. The interested clone was used to perform Northern blot and reverse transcriptase-PCR (RT-PCR) analysis on mRNA isolated from low metastatic cell line SOSP-9607 and OS-9901, high metastatic cell line SOSP-M and three pulmonic metastatic nodules of nude mice.

RESULTSA cDNA clone from low metastatic cell line SOSP-9607 subtracted cDNA library was identified as telomeric repeat binding factor 2 (TERF2) by sequence analysis and Blastn search. Northern blot and RT-PCR analysis demonstrated that TERF2 expressed highly in low metastatic cell line SOSP-9607 and OS-9901, but not in high metastatic cell line SOSP-M and three pulmonic metastatic nodules.

CONCLUSIONTERF2 may be important for suppressing metastasis of osteosarcoma.

Animals ; Base Sequence ; Blotting, Northern ; DNA-Binding Proteins ; genetics ; Gene Expression Regulation, Neoplastic ; Humans ; Mice ; Mice, Nude ; Molecular Sequence Data ; Neoplasm Metastasis ; genetics ; Neoplasm Transplantation ; Nucleic Acid Hybridization ; methods ; Osteosarcoma ; genetics ; pathology ; RNA, Messenger ; genetics ; metabolism ; Telomeric Repeat Binding Protein 2 ; Transplantation, Heterologous ; Tumor Cells, Cultured

OBJECTIVETo detect the expression levels of telomere binding factor 2 (TRF2) mRNA in tumor tissue of non-Hodgkin lymphoma (NHL) patients using quantitative real-time RT-PCR.

METHODSThe target gene mRNA was amplified with RT-PCR, then was sequentially electrophoresed and purified as standards, and the standard curves of gene expression were established. The expression levels of TRF2 mRNA of lymphoid tissue from NHL and reactive lymphoadenopathy were detected with real-time RT-PCR.

RESULTSThe correlation coefficient was 0.996 between the amount of template cDNA and the intensity of fluorescence signal when gene expression standard curves were established. The correlation coefficient of template cDNA amount and grey density of bands derived from gel electrophoresis of real-time RT-PCR final products was 0.779 (P<0.05). Of all NHL patients, expression levels of TRF2 mRNA of follicular lymphoma, mantle cell lymphoma and diffuse large B cell lymphoma were(22.943 +/-9.424) amol, (23.181 +/-5.983) amol and (18.339 +/-7.910) amol, respectively, which had no significant difference compared with reactive lymphoadenopathy [(21.796 +/-4.800) amol, P>0.05]. The expression level of TRF2 mRNA of Burkitt lymphoma was (33.170 +/-12.841) amol, which was significantly higher than that of reactive lymphoadenopathy and other types of NHL (P<0.05).

CONCLUSIONAlcohol drinking isn't one of the risk factors of colorectal cancer among Jiashan County population.

Adult ; Aged ; Burkitt Lymphoma ; genetics ; metabolism ; Female ; Humans ; Lymphoma, B-Cell ; genetics ; metabolism ; Lymphoma, Non-Hodgkin ; genetics ; metabolism ; pathology ; Male ; Middle Aged ; RNA, Neoplasm ; analysis ; biosynthesis ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; methods ; Telomeric Repeat Binding Protein 2 ; analysis ; biosynthesis ; genetics

OBJECTIVETo explore the effect of combined gene therapy with interference hTERT and TRF2 gene on the treatment of breast cancer.

METHODSRecombinant adenovirus rAd-hTERT and rAd-TRF2 expressing siRNA-hTERT and siRNA-TRF2 was constructed, and the vectors were transfected into MCF-7 cells. Than the expressions of hTERT and TRF2 proteins were detected by Western blot, the inhibition of MCF-7 cell proliferation by MTT colorimetry, and the changes of MCF-7 cell cycle by flow cytometry and the colony forming ability of MCF-7 cells by clone form test.

RESULTSAt 48 h after transfection, the relative expression amounts of hTERT protein of the PBS control group, rAd-blank group, rAd-HK control group, rAd-hTERT group, rAd-TRF2 group and rAd-hTERT and rAd-TRF2 group were 1.00, 0.94 +/- 0.02, 0.95 +/- 0.04, 0.18 +/- 0.04, 0.95 +/- 0.01 and 0.18 +/- 0.04, respectively. The relative expression amounts of TRF2 protein were 1.00, 1.01 +/- 0.08, 0.96 +/- 0.02, 0.95 +/- 0.08, 0.22 +/- 0.01 and 0.26 +/- 0.02, respectively. After transfection of rAd-hTERT or rAd-TRF2 into MCF-7 cells separately, the inhibition rate of cell proliferation was only 54.6% and 48.4%, there was 8.9% +/- 1.2% or 9.2% +/- 2.3% of MCF-7 cells into M phase, 66.4% +/- 1.5% or 64.6% +/- 1.9% of MCF-7 cells was arrested at G(0)/G(1) phase, and the cell colony forming ability was decreased significantly (cell colony number from 100 in PBS control group down to 41.3 +/- 5.1 and 43.7 +/- 6.4). But after transfection by rAd-hTERT and rAd-TRF2 simultaneously, the inhibition rate of cell proliferation was about 82.1%, and M phase cells was significantly reduced to 4.4% +/- 1.2%. Large numbers of cells were arrested at G(0)/G(1) phase (81.4% +/- 1.3%), and the cell colony forming ability was more significantly decreased (cell colony number there were only 29.2 +/- 3.9).

CONCLUSIONMore effective effect of tumor gene therapy can be achieved by combination of interference hTERT and TRF2 genes as compared with interference by either of the single gene alone.

Adenoviridae ; genetics ; Breast Neoplasms ; genetics ; metabolism ; pathology ; Cell Cycle ; Cell Line, Tumor ; Cell Proliferation ; Female ; Gene Expression Regulation, Neoplastic ; Genetic Therapy ; Genetic Vectors ; Humans ; RNA Interference ; RNA, Small Interfering ; genetics ; Telomerase ; genetics ; metabolism ; Telomeric Repeat Binding Protein 2 ; genetics ; metabolism ; Transfection ; Tumor Stem Cell Assay