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Down regulation of human telomerase reverse transcriptase (hTERT) expression by BIBR1532 in human glioblastoma LN18 cells.

ABSTRACT: Increased telomerase activity can be blocked by targeting the hTERT activity at both RNA and catalytic subunits. Various inhibitors had been used to regulate hTERT activity in glioblastoma cell lines and showed promising results. The present study hypothesized that the telomerase specific inhibitor BIBR1532 can effectively down-regulate the telomerase activity in LN18 glioblastoma cell line. LN18 glioblastoma cell line was treated with various concentrations of BIBR1532 at different time intervals. MTT assay was performed to determine cell viability after BIBR1532 treatment. hTERT mRNA and protein expression were determined by qRT-PCR and western blotting, respectively. Flow cytometry and TRAP assay was performed to detect the rate of apoptosis and telomerase activity in treated and control samples. One-way ANOVA was performed to compare the mean values of variables in control and BIBR1532 treated groups. LN18 cells showed a significant dose dependent cytotoxic effect after treatment with BIBR1532. hTERT mRNA expression in cells treated with 25, 100 and 200 μM BIBR1532 treated groups was decreased ~ 21, ~ 61.2, and ~ 77%, respectively (p < 0.05). We also observed that, BIBR1532 treatment reduced the expression of hTERT protein in LN18 cells in a dose dependent manner. The Flow cytometry data showed that, the drug induced significant increase in the total percentage of apoptotic cells with 200 μM concentration of BIBR1532 at all time points. BIBR1532 exhibited potent inhibition of telomerase activity in a dose-dependent manner in LN18 cells. BIBR1532 could induce apoptosis in LN18 cells through the downregulation of telomerase activity at transcriptional and translational level. We conclude that BIBR1532 may be a therapeutic agent to suppress telomerase activity, however, further efforts are necessary in order to explore this therapeutic strategy.

SUBMITTER: Lavanya C

PROVIDER: S-EPMC6081925 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundTelomerase is a reverse transcriptase that maintains the telomeres of linear chromosomes and preserves genomic integrity. The core components are a catalytic protein subunit, the telomerase reverse transcriptase (TERT), and an RNA subunit, the telomerase RNA (TR). Telomerase is unique in its ability to catalyze processive DNA synthesis, which is facilitated by telomere-specific DNA-binding domains in TERT called anchor sites. A conserved glutamine residue in the TERT N-terminus is important for anchor site interactions in lower eukaryotes. The significance of this residue in higher eukaryotes, however, has not been investigated.Methodology/principal findingsTo understand the significance of this residue in higher eukaryotes, we performed site-directed mutagenesis on human TERT (hTERT) Q169 to create neutral (Q169A), conservative (Q169N), and non-conservative (Q169D) mutant proteins. We show that these mutations severely compromise telomerase activity in vitro and in vivo. The functional defects are not due to abrogated interactions with hTR or telomeric ssDNA. However, substitution of hTERT Q169 dramatically impaired the ability of telomerase to incorporate nucleotides at the second position of the template. Furthermore, Q169 mutagenesis altered the relative strength of hTERT-telomeric ssDNA interactions, which identifies Q169 as a novel residue in hTERT required for optimal primer binding. Proteolysis experiments indicate that Q169 substitution alters the protease-sensitivity of the hTERT N-terminus, indicating that a conformational change in this region of hTERT is likely critical for catalytic function.Conclusions/significanceWe provide the first detailed evidence regarding the biochemical and cellular roles of an evolutionarily-conserved Gln residue in higher eukaryotes. Collectively, our results indicate that Q169 is needed to maintain the hTERT N-terminus in a conformation that is necessary for optimal enzyme-primer interactions and nucleotide incorporation. We show that Q169 is critical for the structure and function of human telomerase, thereby identifying a novel residue in hTERT that may be amenable to therapeutic intervention.

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Down regulation of human telomerase reverse transcriptase (hTERT) expression by BIBR1532 in human glioblastoma LN18 cells.

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