title: Telomerase downregulation induces proapoptotic genes expression and initializes breast cancer cells apoptosis followed by DNA fragmentation in a cell type dependent manner.
abstract: The aim of the study was to analyze the consequence of silencing genes coding for the key subunits of the telomerase complex, i.e. TERT, TERC and TP1 in human breast cancer MCF7 and MDA-MB-231cells. The transfection was performed using Lipofectamine2000 and pooled siRNAs. The cytotoxic and/or antiproliferative effect of siRNA was measured by the SRB assay, the cell cycle was analysed by flow cytometry and DNA fragmentation by TUNEL analysis. Telomerase activity was assessed by TRAP, followed by PAGE and ELISA assays. Telomerase downregulation was also assessed using qPCR in order to estimate the changes in the expression profile of genes engaged in apoptosis. It was revealed that treatment of breast cancer cells with different siRNAs (100 nM) resulted in a cell type and time-dependent effects. The downregulation of telomerase subunits was followed by reduction of telomerase activity down to almost 60% compared to control cells. However, a significant effect was only observed when the TERT subunit was downregulated. Its silencing resulted in a significant (p<0.05) increase of apoptosis (over 10% in MCF7 and about 5% in MDA-MB-231 cells, corresponding to the Annexin V assay) and DNA fragmentation (almost 30% in MCF7 and over 25% in MDA-MB-231 cells). Interestingly, also several proapoptotic genes were induced after the downregulation of the key telomerase subunit, including Bax, Bik or caspase-1 and caspase-14, as well as NGFR and TNFSF10 which were upregulated twice and more.
authors.affiliation: Department of Clinical Chemistry and Molecular Diagnostics, Poznan University of Medical Sciences, ul. Przybyszewskiego 49, 60-355, Poznan, Poland. blazejr@ump.edu.pl
authors.forename: Blazej
authors.initials: B
authors.lastname: Rubis
authors.forename: Hanna
authors.initials: H
authors.lastname: Holysz
authors.forename: Marta
authors.initials: M
authors.lastname: Gladych
authors.forename: Ewa
authors.initials: E
authors.lastname: Toton
authors.forename: Anna
authors.initials: A
authors.lastname: Paszel
authors.forename: Natalia
authors.initials: N
authors.lastname: Lisiak
authors.forename: Mariusz
authors.initials: M
authors.lastname: Kaczmarek
authors.forename: Johann
authors.initials: J
authors.lastname: Hofmann
authors.forename: Maria
authors.initials: M
authors.lastname: Rybczynska
mesh_terms: D017209:Apoptosis / Q000502:physiology*; D051017:Apoptosis Regulatory Proteins / Q000378:metabolism*; D001943:Breast Neoplasms / Q000201:enzymology*; D053938:DNA Fragmentation; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D005434:Flow Cytometry; D020869:Gene Expression Profiling; D015971:Gene Expression Regulation, Enzymologic / Q000235:genetics / Q000502:physiology*; D006801:Humans; D020287:In Situ Nick-End Labeling; D061986:MCF-7 Cells; D021122:Protein Subunits / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics; D019098:Telomerase / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
chemical_list: D051017:Apoptosis Regulatory Proteins; D021122:Protein Subunits; D034741:RNA, Small Interfering; C509186:TERT protein, human; D019098:Telomerase