9 research outputs found
Additional file 1: Figure S1. of Human umbilical cord-derived mesenchymal stromal cells protect against premature renal senescence resulting from oxidative stress in rats with acute kidney injury
showing telomerase activity determination. A Assay that combines PCR and ELISA techniques, by mixing total kidney protein with Taq DNA polymerase, nucleotides and two primers (one biotinylated TTAGGG primer, working as a telomerase substrate, and one anti-sense primer to amplify the reaction). B The amplified biotinylated sequences were then hybridised to a digoxigenin-labelled telomeric-specific detection probe and immobilised in a streptavidin-coated microplate. C The immobilised PCR product was detected with an antibody against digoxigenin, which was conjugated to peroxidase. D When the reaction occurs, it forms a coloured reaction product. (TIF 9425 kb
Telomerase Variant A279T Induces Telomere Dysfunction and Inhibits Non-Canonical Telomerase Activity in Esophageal Carcinomas
<div><p>Background</p><p>Although implicated in the pathogenesis of several chronic inflammatory disorders and hematologic malignancies, telomerase mutations have not been thoroughly characterized in human cancers. The present study was performed to examine the frequency and potential clinical relevance of telomerase mutations in esophageal carcinomas.</p><p>Methods</p><p>Sequencing techniques were used to evaluate mutational status of <i>telomerase reverse transcriptase (TERT)</i> and <i>telomerase RNA component (TERC)</i> in neoplastic and adjacent normal mucosa from 143 esophageal cancer (EsC) patients. MTS, flow cytometry, time lapse microscopy, and murine xenograft techniques were used to assess proliferation, apoptosis, chemotaxis, and tumorigenicity of EsC cells expressing either wtTERT or TERT variants. Immunoprecipitation, immunoblot, immunofluorescence, promoter-reporter and qRT-PCR techniques were used to evaluate interactions of TERT and several TERT variants with BRG-1 and β-catenin, and to assess expression of cytoskeletal proteins, and cell signaling. Fluorescence in-situ hybridization and spectral karyotyping techniques were used to examine telomere length and chromosomal stability.</p><p>Results</p><p>Sequencing analysis revealed one deletion involving <i>TERC (TERC del 341-360)</i>, and two non-synonymous <i>TERT</i> variants [A279T (2 homozygous, 9 heterozygous); A1062T (4 heterozygous)]. The minor allele frequency of the A279T variant was five-fold higher in EsC patients compared to healthy blood donors (p<0.01). Relative to wtTERT, A279T decreased telomere length, destabilized TERT-BRG-1-β-catenin complex, markedly depleted β-catenin, and down-regulated canonical Wnt signaling in cancer cells; these phenomena coincided with decreased proliferation, depletion of additional cytoskeletal proteins, impaired chemotaxis, increased chemosensitivity, and significantly decreased tumorigenicity of EsC cells. A279T expression significantly increased chromosomal aberrations in mouse embryonic fibroblasts (MEFs) following Zeocin™ exposure, as well as Li Fraumeni fibroblasts in the absence of pharmacologically-induced DNA damage.</p><p>Conclusions</p><p>A279T induces telomere dysfunction and inhibits non-canonical telomerase activity in esophageal cancer cells. These findings warrant further analysis of A279T expression in esophageal cancers and premalignant esophageal lesions.</p></div
Gene expression levels of A279T-transduced cells normalized to wtTERT-transduced cells.
<p>*ND: Not detected.</p
Effects of A279T on genomic stability in normal cells.
<p>A. SKY assay demonstrating that A279T-induces genomic instability in Zeocin™-treated MEF-1 cells. Upper panel: translocations, dicentric, and rearranged chromosomes are present in cells expressing A279T compared to wtTERT. Lower left panel: a multi-centric chromosome observed in cells harboring A279T. Lower right panel: a ring chromosome is formed and every chromosome is rearranged in cells transfected with A279T. See text for additional details. B. Upper panel: representative results of SKY analysis Li Fraumeni fibroblasts constitutively expressing wtTERT or A279T-TERT. Lower panel: close-up of chromosomes 1 and 16. C. Summary of results of two independent experiments demonstrating that A279T expression increases genomic instability in Li Fraumeni cells.</p
Effects of A279T on tumorigenicity and telomere length of esophageal cancer cells in vivo.
<p>A. A279T significantly inhibits growth of subcutaneous EsC2 xenografts in athymic nude mice. B. Representative results of telomere-specific FISH analysis of ExC2 xenografts depicting shortened telomere length in xenografts from EsC2-A279T cells compared to those derived from EsC2-TERT cells; Red: telomere; green: centromere; blue: DAPI.</p
A279T inhibits proliferation of esophageal cancer cells.
<p>(*p<0.05; **p<0.01). A. MTS assay demonstrating inhibition of EsC1 (left) and EsC2 (right) proliferation by A279T relative to wtTERT. B. Immunofluorescence analysis (left panel) with corresponding summary (right panel) of Ki67 expression in esophageal cancer cells expressing wtTERT or A279T (Red: Ki67; blue: DAPI). EsC1 and EsC2 cells expressing A279T exhibit decreased Ki67 levels relative to respective cells expressing wtTERT. C. Annexin V-FITC assay demonstrating A279T-induces apoptosis in EsC2 but not EsC1 cells. Results are expressing as mean ± SD of triplicate experiments. D. Graphic summarization of immunofluorescence analysis of β-galactosidase expression in EsC1 and EsC2 following constitutive expression of wtTERT or A279T. Red: β-galactosidase; blue: DAPI.</p
A279T down-regulates β-catenin independent of telomerase activity.
<p>A. Telomerase enzymatic activity of TERT and TERC mutations in VA13 cells, measured by TRAPeze assay. Telomerase activity is defined as 100% of wtTERT. B. Quantitative PCR analysis of telomere lengths in EsC1, EsC2 and OE21 esophageal cancer cells transfected with empty vector, wtTERT, or A279T-TERT. C. Immunoblot analysis of TERT and related shelterin protein levels in EsC1 and EsC2 cells transduced with wtTERT, A279T-TERT or empty vector. Expression of A279T depletes several shelterin proteins in esophageal cancer cells. D. Upper panel: Immunoprecipitation experiments demonstrating that A279T disrupts TERT-BRG-1-β-catenin complex. This phenomenon was not seen in cells expressing G260D. Lower panel: immunoblot experiments demonstrating decreased β-catenin levels in EsC1 and EsC2 expressing A279T. E. Representative immunofluorescence analysis of β-catenin expression in EsC1 and EsC2 cells cultured in normal media in the presence or absence of proteasome inhibitors (red: β-catenin; blue: DAPI).</p
Effects of A279T on Wnt signaling and chemosensitivity in cancer cells.
<p>*p<0.05, **p<0.01. A. TOP-flash promoter-reporter assay demonstrating that relative to wtTERT, A279T inhibits Wnt signaling in HeLa cells. This phenomenon was not seen in cells expressing G260D or A1062T. B. Immunoblot of β-catenin levels in cytoplasmic and nuclear extracts of EsC1 and EsC2 cells following constitutive expression of wtTERT or A279T-TERT. C. MTS assay demonstrating that relative to EsC1 and EsC2 cells expressing wtTERT, EsC1 and EsC2 cells expressing A279T are more sensitive to cisplatin (2 day treatment) and paclitaxel (3 hour treatment) measured on day 3.</p
Mutations of TERT in Esophageal Cancers among 143 Cases.
†<p>codon 279 GCC/ACC (Ala/Thr).</p>‡<p>codon 1062 GCC/ACC (Ala/Thr)].</p><p>*mAF (minor allele frequency): frequency of the less frequent allele in a given population.</p><p>**p value: compared to both esophageal cancers combined. Fisher's exact test.</p><p>***NS: not significant.</p