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eloped progressive hepatic insufficiency, and died approximately four months later. Subsequent analysis revealed a germ-line deletion in telomerase RNA component ; this loss-of-function mutation was also identified in the proband’s son, who at 30 years of age exhibited premature aging, mild anemia, and early cirrhosis. The present study was undertaken to examine the frequency and potential clinical relevance of telomerase complex mutations in sporadic esophageal cancers. and mouse embryonic fibroblast cell lines were obtained from American Type Culture Collection. All cells were maintained in RPMI 1640 media at 37uC in 5% CO2. Li Fraumeni fibroblasts were generously provided by Michael Tainsky, and were cultured as described. The proteasome inhibitors, MG132 and ALLN were obtained from Sigma, reconstituted in DMSO, and stored at 220uC. Cisplatin and paclitaxel were purchased from the Clinical Center Pharmacy at the NCI. Cell Proliferation Assays EsC1 cells and EsC2 cells were plated in 96-well plates in 100 mL media. Cell viability was quantitated by MTS colorimetric techniques using the Cell Titer 96 Aqueous One Solution Cell Proliferation Assay. For chemosensitivity experiments, responses to cisplatin or paclitaxel were plotted as fractions of viable cells relative to untreated controls. Each experiment was performed in triplicate at least twice. Annexin V-FITC assay Apoptosis was assessed using the Annexin V-FITC kit according to vendor protocols. Telomerase activity by telomerase repeats amplification protocol Two micrograms of pcDNA3-Flag-TERC or -Terc 341360 del were co-transfected with either 2 mg of pcDNA3-Flag vector, pcDNA3-Flag- wtTERT, or -A279T into VA-13 cells at 60 percent confluency in 6-well polystyrene dishes using Superfect Transfection Reagent, according to manufacturer’s instructions. Telomerase activity in transfected cells was determined using the fluorescent telomerase repeat amplification kit as previously described. Materials and Methods Ethics Statement All human tissues were procured on IRB-approved protocols. All mouse experiments were approved by the 16177223 National Cancer Institute Animal Care and Use Committee, and were in accordance with the NIH Guide for Care and Use of Laboratory Animals. Telomere length assay Mean telomere length in esophageal cancer cells constitutively expressing wtTERT, A279T, or vector control sequences were analyzed by quantitative polymerase chain reaction techniques. PCR was SCD-inhibitor biological activity conducted in triplicate in a Rotor-Gene Q real-time instrument with the Rotor-Gene SYBR Green Kit. The telomere length for each sample was determined using the telomere to single copy gene ratio with the calculation of the DCt. The T/S ratio for each sample was normalized to the mean T/S ratio of reference sample, which was used for the standard curve, both as a reference sample and as a validation sample. Patient samples Genomic DNA was isolated as described from snap-frozen esophageal cancers and adjacent normal mucosa from 80 patients undergoing potentially curative resections at the National Cancer Institute, University of Michigan, and Dalhousie University. In addition, genomic DNA was extracted from formalin-fixed paraffin embedded tissues from 63 esophageal cancer patients from Cornell University Medical Center, using PicoPure DNA Extraction Kit, and later purified with DNeasy Blood 17628524 & Tissue Kit. PCR products from snap-frozen tissues were purified with a QIAquick PCR purification kit, followed by direct seq

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Author: HMTase- hmtase