Telomere Transcripts Target Telomerase in Human Cancer Cells
<p>Telomeric repeat-containing RNA (TERRA) transcript expression levels in tumor cell models. (<b>A</b>) Bars depict mean and error bars represent standard deviation (SD) of TERRA relative quantity (RQ) values as determined by quantitative PCR (qPCR). RQs are normalized to <span class="html-italic">36B4</span> as reference gene and Saos-2; (<b>B</b>) Scatter blot of cell lines positive for telomerase activity (TA) (<span class="html-italic">n</span> = 6 from colon tumors: SW620, LT97, HT29, HCT116, Vaco, Caco; <span class="html-italic">n</span> = 11 from brain tumors: CRL-1718, CRL-2020, KG-MH, KM-YH, LN-140, MGC, MR1, U-373MG, YU-PM, HTB138, HTB-186) and alternative lengthening of telomeres (ALT) (GM-639, GM-847, G-292). RQ value of Saos-2 is indicated by dashed line. Lines depict median and interquartile range.</p> "> Figure 2
<p>Recombinant TERRA expression constructs in human cancer cells. (<b>A</b>) Expression cassettes of adeno- and lentivirus constructs (AV and LV) with specific exo-5′ and exo-3′ primer pairs indicated for detection of recombinant TERRA by qPCR. TERRA expression constructs contain ≈130 telomere hexanucleotide repeats of TTAGGG and TTGGGG variant sequences under control of human RNase P RNA H1 RNA polymerase III promoter (hH1) or human cytomegalovirus RNA polymerase II promoter (CMV). Transcription start point (TSP) and orientation is indicated at hH1 and CMV promoters marked by green and red boxes, respectively. Polymerase III termination site (term), SV40 polyadenylation signal (pA) and multi-cloning-site (mcs) are indicated; (<b>B</b>) Recombinant 5′- and 3′-TERRA levels in tumor cell lines 48 h after AV infection with multiplicity of infection (MOI) 10. Recombinant TERRA expression levels from hH1 (AV hH1-TERRA, upper panel) and CMV (CMV-TERRA, lower panel) promoter construct were determined by qPCR in relation to 36B4. Bars depict mean and error bars depict SD.</p> "> Figure 3
<p>Increase of TERRA levels by recombinant expression. CMV and hH1 TERRA promoter constructs for expression of polyadenylated and non-polyadenylated transcripts, respectively. (<b>A</b>,<b>B</b>) RQ values for TERRA expression were analyzed by qPCR and compared to <span class="html-italic">36B4</span> as reference gene and to enhanced green fluorescent protein (eGFP) infected cells as control; (<b>A</b>) Cells were infected with AV at MOI 10 and analyzed after 48 h; (<b>B</b>) Cells were infected with LV and single cell clones were isolated. Representative results in one of three clones per cell line at passage four are shown; (<b>C</b>) TERRA expression detected by telomeric RNA- fluorescence in situ hybridization (FISH) with fluorescein isothiocyanate (FITC) label in interphase nucleus (4′,6-diamidino-2-phenylindole, DAPI) of LV clones from SW480 cells at passage four. Cells were treated with 200 U/mL DNase A. Scale bars of fluorescence micrographs represent 10 μm. Inlays depict a representative nucleus.</p> "> Figure 4
<p>Telomeres and in vitro cell growth properties of tumor cells with recombinant TERRA expression. Bars depict mean and error bars depict SD of two independent experiments performed in triplicates. (<b>A</b>) Senescence-associated (SA) β-galactosidase (βgal) positive cells (upper panel) and relative TL analyzed for telomeric content by qPCR as telomere-specific/single-copy gene (T/S) quantity in cells infected with AV at MOI 10 after 48 h (middle panel). Relative TL was analyzed in single LV cell clones at passage four (lower panel). Results for TL were normalized on Saos-2 infected with eGFP (middle panel) and on LV cell clones expressing eGFP (lower panel); (<b>B</b>) LV cell clones at passage four were simultaneously grown for six passages in culture and cumulative population doubling levels (PDLs) are shown (upper panel). Cell viability and colony formation values were normalized on LV cell clones expressing eGFP marked by dashed lines (middle and lower panel).</p> "> Figure 5
<p>TA in tumor cell lines with recombinant TERRA expression and with TERT-blocking AVs. Bars depict mean and error bars depict SD of two independent experiments performed in triplicates. (<b>A</b>) LV cell clones at passage four (upper panel) and cell lines infected with AV at MOI 10 after 72 h (middle and lower panels) analyzed for TA in total product generated (TPG) units by telomeric repeat amplification protocol (TRAP) assay. Dashed lines represent the detection limit; (<b>B</b>) Human (HeLa) and canine (MBSa, CoFSa, PSTS) tumor cells infected with AV at MOI 10 were analyzed after 72 h for eGFP expression (green) by fluorescence microscopy and for SA β-galactosidase activity (blue) by phase-contrast microscopy. Scale bars depict 500 μm; (<b>C</b>) Human and canine tumor cells analyzed for TA by TRAP assay and normalized to AV eGFP as control.</p> ">
Abstract
:1. Introduction
2. Materials and Methods
2.1. Cell Culture
2.2. Adeno- and Lentivirus Constructs
2.3. Relative Telomere Length and Expression
2.4. Telomerase Activity
2.5. Cell Growth and Viability
2.6. Clonogenicity Assay
2.7. Senescence-Associated β-Galactosidase Activity
2.8. Statistical Analysis
3. Results
3.1. TERRA Expression in Human Cell Models
3.2. Transient and Stable Expression of Recombinant TERRA in Human Cell Lines
3.3. Telomere Length and Cell Growth Behavior
3.4. Telomerase Activity in Cell Models with Recombinant TERRA
4. Discussion
Acknowledgments
Author Contributions
Conflicts of Interest
References
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Kreilmeier, T.; Mejri, D.; Hauck, M.; Kleiter, M.; Holzmann, K. Telomere Transcripts Target Telomerase in Human Cancer Cells. Genes 2016, 7, 46. https://doi.org/10.3390/genes7080046
Kreilmeier T, Mejri D, Hauck M, Kleiter M, Holzmann K. Telomere Transcripts Target Telomerase in Human Cancer Cells. Genes. 2016; 7(8):46. https://doi.org/10.3390/genes7080046
Chicago/Turabian StyleKreilmeier, Theresa, Doris Mejri, Marlene Hauck, Miriam Kleiter, and Klaus Holzmann. 2016. "Telomere Transcripts Target Telomerase in Human Cancer Cells" Genes 7, no. 8: 46. https://doi.org/10.3390/genes7080046