The enormous sequence heterogeneity of telomerase RNA (TR) subunits has thus far complicated their characterization in a wider phylogenetic range. Our recent finding that land plant TRs are, similarly to known ciliate TRs, transcribed by RNA polymerase III and under the control of the type-3 promoter, allowed us to design a novel strategy to characterize TRs in early diverging Viridiplantae taxa, as well as in ciliates and other Diaphoretickes lineages. Starting with the characterization of the upstream sequence element of the type 3 promoter that is conserved in a number of small nuclear RNAs, and the expected minimum TR template region as search features, we identified candidate TRs in selected Diaphoretickes genomes. Homologous TRs were then used to build covariance models to identify TRs in more distant species. Transcripts of the identified TRs were confirmed by transcriptomic data, RT-PCR and Northern hybridization. A templating role for one of our candidates was validated in Physcomitrium patens. Analysis of secondary structure demonstrated a deep conservation of motifs (pseudoknot and template boundary element) observed in all published TRs. These results elucidate the evolution of the earliest eukaryotic TRs, linking the common origin of TRs across Diaphoretickes, and underlying evolutionary transitions in telomere repeats.
- MeSH
- genetická transkripce MeSH
- konformace nukleové kyseliny MeSH
- molekulární evoluce * MeSH
- mutace MeSH
- RNA rostlin biosyntéza chemie genetika MeSH
- RNA-polymerasa II metabolismus MeSH
- RNA-polymerasa III metabolismus MeSH
- RNA biosyntéza chemie genetika MeSH
- sekvenční seřazení MeSH
- telomerasa biosyntéza chemie genetika MeSH
- telomery chemie MeSH
- transkriptom MeSH
- Viridiplantae genetika MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, U.S. Gov't, Non-P.H.S. MeSH
We have previously shown that PMEG diphosphate (PMEGpp) and PMEDAP diphosphate (PMEDAPpp) inhibit the enzymatic activity of human telomerase in a cell-free assay. Here, we investigated the ability of PMEG and PMEDAP to induce telomere shortening and telomerase inhibition at both transcriptional and activity level in T-lymphoblastic leukemia cells CCRF-CEM and MOLT-4. At defined time points (3days and 9weeks), the telomerase activity and relative levels of hTERT and c-myc mRNA were determined using real-time RT-PCR. Telomere length was measured by the flow-FISH method. Both PMEDAP and PMEG induced telomere shortening in CCRF-CEM cells after 9weeks of exposure by 50% and 20%, respectively, without major impairment of telomerase activity. The effect of the tested compounds on telomere length in MOLT-4 cells was the opposite, with telomere elongation by 50% and 40% after 9-week treatment with PMEDAP and PMEG, respectively. At this time point, telomerase activity in MOLT-4 cells appeared to be slightly higher than that of CCRF-CEM cells, nevertheless no correlation between telomerase activity and telomere length was found. Both compounds down-regulated the expression of hTERT and c-myc mRNA in CCRF-CEM and MOLT-4 cells at 72h in a concentration-dependent manner while prolonged exposure to PMEG or PMEDAP for 9weeks had weaker effects. In conclusion, PMEDAP and PMEG are able to modulate telomere length in leukemic cells and this effect is cell-type specific. It is neither due to direct telomerase inhibition nor impairment of hTERT expression and it is likely to be telomerase-independent.
- MeSH
- adenin analogy a deriváty farmakologie MeSH
- buněčné kultury MeSH
- časové faktory MeSH
- elektroforéza v polyakrylamidovém gelu MeSH
- guanin analogy a deriváty farmakologie MeSH
- hybridizace in situ fluorescenční MeSH
- lidé MeSH
- nádorové buněčné linie MeSH
- organofosforové sloučeniny farmakologie MeSH
- polymerázová řetězová reakce s reverzní transkripcí MeSH
- protinádorové látky farmakologie MeSH
- protoonkogenní proteiny c-myc biosyntéza MeSH
- RNA metabolismus MeSH
- telomerasa antagonisté a inhibitory biosyntéza MeSH
- telomery účinky léků MeSH
- vztah mezi dávkou a účinkem léčiva MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
DNA methylation inhibitors are being extensively studied as potential anticancer agents. In the present study, we compared the capability of alpha anomer of 5-aza-2'-deoxycytidine (alpha-5-azadCyd) to induce down-regulation of hTERT expression in HL-60 cells with other nucleoside analogs that act as DNA methylation inhibitors: beta-5-azadCyd (decitabine), (S)-9-(2,3-dihydroxypropyl)adenine [(S)-DHPA], isobutyl ester of (R,S)-3-(adenin-9-yl)-2-hydroxypropanoic acid [(R,S)-AHPA-ibu] and prospective DNA methylation inhibitors (S)-1-[3-hydroxy-2-(phosphonomethoxy)propyl]-5-azacytosine [(S)-HPMPazaC] and 5-fluoro-zebularine (F-PymRf). Exposure to alpha-5-azadCyd induced the down-regulation of hTERT expression in low micromolar concentrations (0.05-50 microM). A more cytotoxic beta anomer caused a transient up-regulation of hTERT and a subsequent reduction in hTERT mRNA levels at concentrations more than 10 times below its GIC50 value. In this respect, (S)-DHPA and (R,S)-AHPA-ibu were less efficient, since a similar effect was achieved at concentrations above their GIC(50). In contrast, F-PymRf treatment resulted in up to a three-fold induction of hTERT expression within a broad range of concentrations. In all cases, the down-regulation of hTERT expression was concentration-dependent. The correlation was found between c-myc overexpression and transiently elevated hTERT expression after treatment with all tested compounds except for alpha-5-azadCyd and (S)-HPMPazaC. Although the primary task of hypomethylating agents in anticancer therapy lies in reactivation of silenced tumour-suppressor genes, the inhibition of hTERT expression might also be a fruitful clinical effect of this approach.
- MeSH
- azacytidin analogy a deriváty farmakologie chemie MeSH
- DNA metabolismus MeSH
- down regulace MeSH
- financování organizované MeSH
- HL-60 buňky MeSH
- lidé MeSH
- messenger RNA biosyntéza MeSH
- metylace DNA účinky léků MeSH
- polymerázová řetězová reakce s reverzní transkripcí MeSH
- protinádorové antimetabolity farmakologie MeSH
- S-adenosylhomocystein metabolismus MeSH
- S-adenosylmethionin metabolismus MeSH
- stereoizomerie MeSH
- telomerasa biosyntéza MeSH
- vztah mezi dávkou a účinkem léčiva MeSH
- Check Tag
- lidé MeSH
- MeSH
- alternativní sestřih MeSH
- finanční podpora výzkumu jako téma MeSH
- imunohistochemie metody MeSH
- kolorektální nádory diagnóza enzymologie farmakoterapie MeSH
- lidé MeSH
- lymfatické metastázy MeSH
- messenger RNA metabolismus MeSH
- polymerázová řetězová reakce s reverzní transkripcí MeSH
- telomerasa biosyntéza metabolismus MeSH
- Check Tag
- lidé MeSH
