Telomerase Inhibitor Imetelstat (GRN163L) Limits the Lifespan of Human Pancreatic Cancer Cells
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{"title"=>"Telomerase inhibitor imetelstat (GRN163L) limits the lifespan of human pancreatic cancer cells", "type"=>"journal", "authors"=>[{"first_name"=>"Katrina M.", "last_name"=>"Burchett", "scopus_author_id"=>"56085457400"}, {"first_name"=>"Ying", "last_name"=>"Yan", "scopus_author_id"=>"36889053000"}, {"first_name"=>"Michel M.", "last_name"=>"Ouellette", "scopus_author_id"=>"7102723767"}], "year"=>2014, "source"=>"PLoS ONE", "identifiers"=>{"pui"=>"372701525", "sgr"=>"84896952141", "issn"=>"19326203", "pmid"=>"24409321", "scopus"=>"2-s2.0-84896952141", "doi"=>"10.1371/journal.pone.0085155", "isbn"=>"1932-6203 (Electronic) 1932-6203 (Linking)"}, "id"=>"cf447249-d3e4-322c-aa90-e5a2d128dca8", "abstract"=>"Telomerase is required for the unlimited lifespan of cancer cells. The vast majority of pancreatic adenocarcinomas overexpress telomerase activity and blocking telomerase could limit their lifespan. GRN163L (Imetelstat) is a lipid-conjugated N3'→P5' thio-phosphoramidate oligonucleotide that blocks the template region of telomerase. The aim of this study was to define the effects of long-term GRN163L exposure on the maintenance of telomeres and lifespan of pancreatic cancer cells. Telomere size, telomerase activity, and telomerase inhibition response to GRN163L were measured in a panel of 10 pancreatic cancer cell lines. The cell lines exhibited large differences in levels of telomerase activity (46-fold variation), but most lines had very short telomeres (2-3 kb in size). GRN163L inhibited telomerase in all 10 pancreatic cancer cell lines, with IC50 ranging from 50 nM to 200 nM. Continuous GRN163L exposure of CAPAN1 (IC50 = 75 nM) and CD18 cells (IC50 = 204 nM) resulted in an initial rapid shortening of the telomeres followed by the maintenance of extremely short but stable telomeres. Continuous exposure to the drug eventually led to crisis and to a complete loss of viability after 47 (CAPAN1) and 69 (CD18) doublings. Crisis In these cells was accompanied by activation of a DNA damage response (γ-H2AX) and evidence of both senescence (SA-β-galactosidase activity) and apoptosis (sub-G1 DNA content, PARP cleavage). Removal of the drug after long-term GRN163L exposure led to a reactivation of telomerase and re-elongation of telomeres in the third week of cultivation without GRN163L. These findings show that the lifespan of pancreatic cancer cells can be limited by continuous telomerase inhibition. These results should facilitate the design of future clinical trials of GRN163L in patients with pancreatic cancer.", "link"=>"http://www.mendeley.com/research/telomerase-inhibitor-imetelstat-grn163l-limits-lifespan-human-pancreatic-cancer-cells", "reader_count"=>60, "reader_count_by_academic_status"=>{"Researcher"=>13, "Student > Doctoral Student"=>4, "Student > Ph. D. Student"=>8, "Student > Postgraduate"=>1, "Student > Master"=>15, "Student > Bachelor"=>16, "Lecturer > Senior Lecturer"=>1, "Professor"=>1, "Other"=>1}, "reader_count_by_user_role"=>{"Researcher"=>13, "Student > Doctoral Student"=>4, "Student > Ph. D. Student"=>8, "Student > Postgraduate"=>1, "Student > Master"=>15, "Student > Bachelor"=>16, "Lecturer > Senior Lecturer"=>1, "Professor"=>1, "Other"=>1}, "reader_count_by_subject_area"=>{"Unspecified"=>1, "Engineering"=>1, "Biochemistry, Genetics and Molecular Biology"=>22, "Materials Science"=>1, "Agricultural and Biological Sciences"=>16, "Medicine and Dentistry"=>9, "Pharmacology, Toxicology and Pharmaceutical Science"=>2, "Chemistry"=>5, "Immunology and Microbiology"=>3}, "reader_count_by_subdiscipline"=>{"Engineering"=>{"Engineering"=>1}, "Materials Science"=>{"Materials Science"=>1}, "Medicine and Dentistry"=>{"Medicine and Dentistry"=>9}, "Chemistry"=>{"Chemistry"=>5}, "Immunology and Microbiology"=>{"Immunology and Microbiology"=>3}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>16}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>22}, "Unspecified"=>{"Unspecified"=>1}, "Pharmacology, Toxicology and Pharmaceutical Science"=>{"Pharmacology, Toxicology and Pharmaceutical Science"=>2}}, "reader_count_by_country"=>{"United States"=>2, "United Kingdom"=>2, "France"=>1, "India"=>2}, "group_count"=>1}

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Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/1340060"], "description"=>"<p>Cells were treated every 2–3 days with no drug (CTR), mismatch oligo (MIS) or GRN163L (GRN). A–B) Southern blot analysis of the telomeres. At the indicated population doubling (PD), genomic DNA samples were collected and subsequently processed for telomere size measurements, as described in <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085155#pone-0085155-g001\" target=\"_blank\">Figure 1A</a>. C–D) Telomere size measurements. Median telomere sizes are shown for the drug- and control-treated CAPAN1 and CD18 cells. E) Detection of ALT by quantitative PCR. Samples tested included CAPAN1 and CD18 cells treated with no drug (CTR), mismatch oligo (MIS) and GRN163L (GRN), all of which harvested at the end of the growth curves presented in <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085155#pone-0085155-g003\" target=\"_blank\">Figure 3</a>. Also included were stocks of CAPAN1, CD18 and VA13 cells. Samples were tested in triplicate with (+) and without (−) the Φ29 DNA polymerase. A representative dot blot is shown.</p>", "links"=>[], "tags"=>["Molecular cell biology", "Chromosome biology", "telomeres", "Nucleic acids", "dna", "Cell death", "cell division", "Cell growth", "Cellular stress responses", "Drugs and devices", "Drug research and development", "drug discovery", "oncology", "Cancer treatment", "Chemotherapy and drug treatment", "Cancers and neoplasms", "Gastrointestinal tumors", "Pancreatic cancer", "grn163l", "telomere"], "article_id"=>895265, "categories"=>["Biological Sciences", "Medicine"], "users"=>["Katrina M. Burchett", "Ying Yan", "Michel M. Ouellette"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0085155.g005", "stats"=>{"downloads"=>0, "page_views"=>5, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Effects_of_chronic_GRN163L_on_telomere_maintenance_/895265", "title"=>"Effects of chronic GRN163L on telomere maintenance.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-01-07 02:59:43"}
  • {"files"=>["https://ndownloader.figshare.com/files/1340068"], "description"=>"<p>CD18 treated with GRN163L (GRN) or with no drug (CTR) were harvested at the end of the growth curve presented in <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085155#pone-0085155-g003\" target=\"_blank\">Figure 3</a>. Cells were fixed and stained with antibodies against TRF2 (green) and γ-H2AX (red) and were counter-stained with DAPI (blue). Images were visualized on a confocal microscope (Zeiss 510 Meta Confocal Laser Scanning Microscope). Three representative images of each sample are shown.</p>", "links"=>[], "tags"=>["Molecular cell biology", "Chromosome biology", "telomeres", "Nucleic acids", "dna", "Cell death", "cell division", "Cell growth", "Cellular stress responses", "Drugs and devices", "Drug research and development", "drug discovery", "oncology", "Cancer treatment", "Chemotherapy and drug treatment", "Cancers and neoplasms", "Gastrointestinal tumors", "Pancreatic cancer", "grn163l-treated", "cd18"], "article_id"=>895273, "categories"=>["Biological Sciences", "Medicine"], "users"=>["Katrina M. Burchett", "Ying Yan", "Michel M. Ouellette"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0085155.g006", "stats"=>{"downloads"=>0, "page_views"=>24, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Immunofluorescence_analysis_of_telomeres_in_the_GRN163L_treated_CD18_cells_/895273", "title"=>"Immunofluorescence analysis of telomeres in the GRN163L-treated CD18 cells.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-01-07 02:59:43"}
  • {"files"=>["https://ndownloader.figshare.com/files/1340070"], "description"=>"<p>After 63 days of continuous GRN163L exposure, CAPAN1 cells were sub-cultivated for 3 weeks in the presence or absence of GRN163L. A, B) Quantification of telomerase activity. Telomerase activity was measured using the TRAP assay at time 0 and after 1, 2, and 3 weeks. Relative telomerase activity was calculated as the ratio of the intensity of the telomerase ladder over the intensity of the ITAS. Each measurement is the Mean ± S.D. of triplicate samples (n = 3). C, D) Telomere size measurements. Telomeres were analyzed at time 0 and after 1, 2, and 3 weeks. Median telomere size was estimated as the size corresponding to half the cumulative sum of the intensities. E) Levels of the γ-H2AX. The indicated samples were Western blotted with antibodies against phosphorylated H2AX (γ-H2AX) and actin. F) Growth rate measurements. Once a week, cells were counted and the number of population doublings done was plotted as a function of time.</p>", "links"=>[], "tags"=>["Molecular cell biology", "Chromosome biology", "telomeres", "Nucleic acids", "dna", "Cell death", "cell division", "Cell growth", "Cellular stress responses", "Drugs and devices", "Drug research and development", "drug discovery", "oncology", "Cancer treatment", "Chemotherapy and drug treatment", "Cancers and neoplasms", "Gastrointestinal tumors", "Pancreatic cancer", "grn163l"], "article_id"=>895275, "categories"=>["Biological Sciences", "Medicine"], "users"=>["Katrina M. Burchett", "Ying Yan", "Michel M. Ouellette"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0085155.g007", "stats"=>{"downloads"=>0, "page_views"=>13, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Recovery_from_chronic_GRN163L_exposure_after_removal_of_the_drug_/895275", "title"=>"Recovery from chronic GRN163L exposure after removal of the drug.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-01-07 02:59:43"}
  • {"files"=>["https://ndownloader.figshare.com/files/1340071", "https://ndownloader.figshare.com/files/1340072", "https://ndownloader.figshare.com/files/1340073", "https://ndownloader.figshare.com/files/1340074", "https://ndownloader.figshare.com/files/1340075"], "description"=>"<div><p>Telomerase is required for the unlimited lifespan of cancer cells. The vast majority of pancreatic adenocarcinomas overexpress telomerase activity and blocking telomerase could limit their lifespan. GRN163L (Imetelstat) is a lipid-conjugated N3′→P5′ thio-phosphoramidate oligonucleotide that blocks the template region of telomerase. The aim of this study was to define the effects of long-term GRN163L exposure on the maintenance of telomeres and lifespan of pancreatic cancer cells. Telomere size, telomerase activity, and telomerase inhibition response to GRN163L were measured in a panel of 10 pancreatic cancer cell lines. The cell lines exhibited large differences in levels of telomerase activity (46-fold variation), but most lines had very short telomeres (2–3 kb in size). GRN163L inhibited telomerase in all 10 pancreatic cancer cell lines, with IC<sub>50</sub> ranging from 50 nM to 200 nM. Continuous GRN163L exposure of CAPAN1 (IC<sub>50</sub> = 75 nM) and CD18 cells (IC<sub>50</sub> = 204 nM) resulted in an initial rapid shortening of the telomeres followed by the maintenance of extremely short but stable telomeres. Continuous exposure to the drug eventually led to crisis and to a complete loss of viability after 47 (CAPAN1) and 69 (CD18) doublings. Crisis In these cells was accompanied by activation of a DNA damage response (γ-H2AX) and evidence of both senescence (SA-β-galactosidase activity) and apoptosis (sub-G1 DNA content, PARP cleavage). Removal of the drug after long-term GRN163L exposure led to a reactivation of telomerase and re-elongation of telomeres in the third week of cultivation without GRN163L. These findings show that the lifespan of pancreatic cancer cells can be limited by continuous telomerase inhibition. These results should facilitate the design of future clinical trials of GRN163L in patients with pancreatic cancer.</p></div>", "links"=>[], "tags"=>["Molecular cell biology", "Chromosome biology", "telomeres", "Nucleic acids", "dna", "Cell death", "cell division", "Cell growth", "Cellular stress responses", "Drugs and devices", "Drug research and development", "drug discovery", "oncology", "Cancer treatment", "Chemotherapy and drug treatment", "Cancers and neoplasms", "Gastrointestinal tumors", "Pancreatic cancer", "inhibitor", "imetelstat", "limits", "lifespan", "pancreatic", "cancer"], "article_id"=>895276, "categories"=>["Biological Sciences", "Medicine"], "users"=>["Katrina M. Burchett", "Ying Yan", "Michel M. Ouellette"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0085155.s001", "https://dx.doi.org/10.1371/journal.pone.0085155.s002", "https://dx.doi.org/10.1371/journal.pone.0085155.s003", "https://dx.doi.org/10.1371/journal.pone.0085155.s004", "https://dx.doi.org/10.1371/journal.pone.0085155.s005"], "stats"=>{"downloads"=>4, "page_views"=>37, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Telomerase_Inhibitor_Imetelstat_GRN163L_Limits_the_Lifespan_of_Human_Pancreatic_Cancer_Cells_/895276", "title"=>"Telomerase Inhibitor Imetelstat (GRN163L) Limits the Lifespan of Human Pancreatic Cancer Cells", "pos_in_sequence"=>0, "defined_type"=>4, "published_date"=>"2014-01-07 02:59:43"}
  • {"files"=>["https://ndownloader.figshare.com/files/1340052"], "description"=>"<p>A) Telomere size measurements. Genomic DNA was digested with restriction enzymes, resolved by electrophoresis in agarose gels and detected by in situ hybridization to [<sup>32</sup>P]-(CCCTAA)<sub>4</sub>. B) Quantification of telomere size. Gel in A was scanned and the intensity of each lane was plotted as a function of telomere size, as described in the Material and Methods section. The median telomere size was estimated as the size corresponding to half the cumulative sum of the intensities. C) Detection of telomerase activity by the TRAP assay. Cell extracts containing 300 cells each were assayed using a Cy5-labeled TS oligo substrate. After PCR with the same oligo and a telomeric comeback primer, products were resolved by electrophoresis and detected with a Typhon PhosphoImager. Buffer was lysis buffer only. ITAS, Internal Telomerase Assay Standard. D) Quantification of relative telomerase activity. Relative telomerase activity was calculated as the ratio of the intensity of the telomerase ladder over the intensity of the ITAS. Each measurement is the Mean ± S.D. of triplicate samples (n = 3). Telomerase activity in each line is expressed as a percent of HeLa cells’ activity.</p>", "links"=>[], "tags"=>["Molecular cell biology", "Chromosome biology", "telomeres", "Nucleic acids", "dna", "Cell death", "cell division", "Cell growth", "Cellular stress responses", "Drugs and devices", "Drug research and development", "drug discovery", "oncology", "Cancer treatment", "Chemotherapy and drug treatment", "Cancers and neoplasms", "Gastrointestinal tumors", "Pancreatic cancer", "telomerase", "pancreatic", "cancer"], "article_id"=>895257, "categories"=>["Biological Sciences", "Medicine"], "users"=>["Katrina M. Burchett", "Ying Yan", "Michel M. Ouellette"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0085155.g001", "stats"=>{"downloads"=>1, "page_views"=>18, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Telomere_size_and_telomerase_activity_among_pancreatic_cancer_cell_lines_/895257", "title"=>"Telomere size and telomerase activity among pancreatic cancer cell lines.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-01-07 02:59:43"}
  • {"files"=>["https://ndownloader.figshare.com/files/1340054"], "description"=>"<p>A) Dose-Response curve of telomerase inhibition by GRN163L. HPAF cells were treated in triplicate with increasing concentrations of GRN163L (50 nM to 4 µM). Twenty-four hours later, telomerase activity was measured using the TRAP assay. Samples treated with no drug were set to 100%. B) IC<sub>50</sub> of each line for the inhibition of telomerase by GRN163L. Dose-response curves were performed as in panel A. Non-linear curve fitting allowed calculation of an IC<sub>50</sub> for each line. Results are expressed as the IC<sub>50</sub> (middle bar) and its 95% interval of confidence (flanking bars).</p>", "links"=>[], "tags"=>["Molecular cell biology", "Chromosome biology", "telomeres", "Nucleic acids", "dna", "Cell death", "cell division", "Cell growth", "Cellular stress responses", "Drugs and devices", "Drug research and development", "drug discovery", "oncology", "Cancer treatment", "Chemotherapy and drug treatment", "Cancers and neoplasms", "Gastrointestinal tumors", "Pancreatic cancer", "responses", "pancreatic", "cancer", "lines"], "article_id"=>895259, "categories"=>["Biological Sciences", "Medicine"], "users"=>["Katrina M. Burchett", "Ying Yan", "Michel M. Ouellette"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0085155.g002", "stats"=>{"downloads"=>2, "page_views"=>3, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Short_term_responses_of_pancreatic_cancer_cell_lines_to_GRN163L_/895259", "title"=>"Short-term responses of pancreatic cancer cell lines to GRN163L.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-01-07 02:59:43"}
  • {"files"=>["https://ndownloader.figshare.com/files/1340055"], "description"=>"<p>A) Pancreatic cancer cell lines CAPAN1 and CD18 were selected for these studies. Every 2–3 days, each lines was given either no drug (vehicle), GRN163L (1 µM) or the Mismatched oligo (1 µM). Once a week, cells were counted and the results are plotted as the number of population doublings achieved as a function of time. Every other week, samples were put aside for TRF analysis (DNA) or frozen down as backup. B,C) Growth curves of the drug-treated CAPAN1 (B) and CD18 (C) cells are shown.</p>", "links"=>[], "tags"=>["Molecular cell biology", "Chromosome biology", "telomeres", "Nucleic acids", "dna", "Cell death", "cell division", "Cell growth", "Cellular stress responses", "Drugs and devices", "Drug research and development", "drug discovery", "oncology", "Cancer treatment", "Chemotherapy and drug treatment", "Cancers and neoplasms", "Gastrointestinal tumors", "Pancreatic cancer", "grn163l", "cellular"], "article_id"=>895260, "categories"=>["Biological Sciences", "Medicine"], "users"=>["Katrina M. Burchett", "Ying Yan", "Michel M. Ouellette"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0085155.g003", "stats"=>{"downloads"=>2, "page_views"=>12, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Effects_of_chronic_GRN163L_on_cellular_lifespan_/895260", "title"=>"Effects of chronic GRN163L on cellular lifespan.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-01-07 02:59:43"}
  • {"files"=>["https://ndownloader.figshare.com/files/1340057"], "description"=>"<p>At the end of the growth curves presented in <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085155#pone-0085155-g003\" target=\"_blank\">Figure 3</a>, cells treated with no drug (CTR), mismatch oligo (MIS) and GRN163L (GRN) were analyzed for SA-β-galactosidase activity. A) Histological analysis of SA-β-galactosidase activity. Histochemical staining reveals SA-β-galactosidase activity in the GRN163L-treated CAPAN1 and CD18 cells, as evidenced by the deposition of insoluble blue pigments (Right panels). B) Percent of cells in each sample that stained positive for SA-β-galactosidase activity. Measurements were done in triplicates (Mean ± S.D., n = 3). C) Western blot analysis. Samples were probed with antibodies against histone H2AX, phosphorylated H2AX (γ-H2AX), PARP and actin. D) Flow cytometric analysis of DNA content. Cells were stained with propidium iodide and analyzed for DNA content. Measurements were made twice at one week interval for the CAPAN1 (Mean ± S.D., n = 2). In the case of the CD18, one measurement only could be made before the GRN163L-treated cells were lost to crisis (n = 1).</p>", "links"=>[], "tags"=>["Molecular cell biology", "Chromosome biology", "telomeres", "Nucleic acids", "dna", "Cell death", "cell division", "Cell growth", "Cellular stress responses", "Drugs and devices", "Drug research and development", "drug discovery", "oncology", "Cancer treatment", "Chemotherapy and drug treatment", "Cancers and neoplasms", "Gastrointestinal tumors", "Pancreatic cancer", "senescence", "grn163l-treated", "capan1", "cd18"], "article_id"=>895262, "categories"=>["Biological Sciences", "Medicine"], "users"=>["Katrina M. Burchett", "Ying Yan", "Michel M. Ouellette"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0085155.g004", "stats"=>{"downloads"=>0, "page_views"=>28, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Markers_of_apoptosis_senescence_and_DNA_damage_response_in_the_GRN163L_treated_CAPAN1_and_CD18_cells_/895262", "title"=>"Markers of apoptosis, senescence and DNA damage response in the GRN163L-treated CAPAN1 and CD18 cells.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-01-07 02:59:43"}

PMC Usage Stats | Further Information

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Relative Metric

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