Development and characterization of polyclonal antibodies against the linker region of the telomere-binding protein TRF2

Background: TRF2 (telomeric repeat binding factor 2) is an essential component of the telomere-binding protein complex shelterin. TRF2 induces the formation of a special structure of telomeric DNA and counteracts activation of DNA damage-response pathways telomeres. TRF2 has a poorly characterized linker region (udTRF2) between its homodimerization and DNA-binding domains. Some lines of evidence have shown that this region could be involved in TRF2 interaction with nuclear lamina. Results: In this study, the fragment of the TERF2 gene encoding udTRF2 domain of telomere-binding protein TRF2 was produced by PCR and cloned into the pET32a vector. The resulting plasmid pET32a-udTRF2 was used for the expression of the recombinant udTRF2 in E. coli RosettaBlue (DE3). The protein was isolated and purified using ammonium sulfate precipitation followed by ion-exchange chromatography. The purified recombinant protein udTRF2 was injected into guinea pigs to generate polyclonal antibodies. The ability of anti-udTRF2 antibodies to bind endogenous TRF2 in human skin fibroblasts was tested by western blotting and immunofluorescent staining. Conclusions: In this study, the recombinant protein udTRF2 and antibodies to it were generated. Both protein and antibodies will provide a useful tool for investigation of the functions of the udTRF2 domain and its role in the interaction between TRF2 and nuclear lamina.

Expression of TRF2 and its prognostic relevance in advanced stage cervical cancer patients

BACKGROUND: Telomeres are protective caps consisted of specific tandem repeats (5'-TTAGGG-3'). Shortening of telomeres at each cell division is known as "mitotic clock" of the cells, which renders telomeres as important regulators of lifespan. TRF2 is one of the critical members of shelterin complex, which is a protein complex responsible from the preservation of cap structure, and loss or mutation of TRF2 results in DNA damage, senescence or apoptosis. Since cancer is frequently associated with aberrant cell cycle progression, defective DNA repair or apoptosis pathways, TRF2 could be one likely candidate for cancer therapy. Here we investigated the prognostic role of TRF2 levels in cervical cancer patients. Fold-induction rates were evaluated with respect to median values after real-time PCR analysis. Overall survival, distant disease-free and local recurrence-free survival rates were calculated using Kaplan-Meier long rank test. RESULTS: Both five year overall- and disease-free survival rates were longer in patients with higher TRF2 expression compared to lower expression, but results were not statistically significant (69.2% vs 28.9%, respectively). Mean local recurrence-free survivals (LRF) were very close ( 58.6, CI: 44.3-72.9 vs 54.5, CI: 32.1-76.9 months) for high and low expressions, respectively. Cumulative proportion of LRF at the end of five year period was 76.9% for high and 57.1% for low TRF2 expression (P = 0.75). Statistically significant difference was found between survival ratios and Bcl-xL and p53 gene expressions, but not with TRF2. A respectable correlation between TRF2 expression and apoptosis along with distant metastasis was noted (P = 0.045 and 0.036, respectively). Additionally, high TRF2 expression levels had a positive impact in five year survival rate of stage IIIB-IVA patients (P = 0.04). CONCLUSIONS: Our results support the role of TRF2 in apoptosis and imply a positive relation with distant metastases and survival in advanced stage patients. The remarkable difference in survival periods of patients with different TRF2 expressions suggest that TRF2 may be a candidate factor to estimate survival for cervical cancer, a preliminary observation which should further be verified with a larger cohort.

How will telomeric complex be further contributed to our longevity? - the potential novel biomarkers of telomere complex counteracting both aging and cancer

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.

Expression of TRF1, TRF2 and RAP1 mRNA in peripheral blood mononuclear cells of patients with acquired aplastic anemia / 中国实验血液学杂志

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.

Inhibitory effect of interference hTERT and TRF2 gene on the growth of breast cancer MCF-7 cells / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To explore the effect of combined gene therapy with interference hTERT and TRF2 gene on the treatment of breast cancer.</p><p><b>METHODS</b>Recombinant 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.</p><p><b>RESULTS</b>At 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).</p><p><b>CONCLUSION</b>More 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.</p>

Experiment studies on growth and proliferation of Hep-2 cells by silence of TRF2 gene / 临床耳鼻咽喉头颈外科杂志

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.

Expression of telomere binding factor 2 (TRF2) on leukemia cell lines and primary leukemia cells / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To detect the expression levels of telomere binding factor 2 (TRF2) on leukemia cell lines and primary leukemia cells.</p><p><b>METHODS</b>The 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.</p><p><b>RESULT</b>TRF2 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.</p><p><b>CONCLUSION</b>Increased 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.</p>

In vitro binding of p53 and telomeric repeat factor 2 / 中华病理学杂志

<p><b>OBJECTIVE</b>To 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.</p><p><b>METHODS</b>Four 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.</p><p><b>RESULTS</b>SDS-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.</p><p><b>CONCLUSIONS</b>p53 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.</p>

Study on the mechanisms of telomerase regulations during apoptosis of the human MDS-RAEB cell line MUTZ-1 cells induced by arsenic trioxide / 中国实验血液学杂志

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).

Quantitative detection of telomere binding factor 2 gene expression in non-Hodgkin lymphoma with a real-time reverse transcription-polymerase chain reaction assay / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To 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.</p><p><b>METHODS</b>The 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.</p><p><b>RESULTS</b>The 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).</p><p><b>CONCLUSION</b>Alcohol drinking isn't one of the risk factors of colorectal cancer among Jiashan County population.</p>

Identification of genes associated with human osteosarcoma metastasis suppression using suppression subtractive hybridization / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To identify genes associated with metastasis suppression and to investigate the molecular mechanism of osteosarcoma metastasis.</p><p><b>METHODS</b>The 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.</p><p><b>RESULTS</b>A 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.</p><p><b>CONCLUSION</b>TERF2 may be important for suppressing metastasis of osteosarcoma.</p>