Browsing by Subject "Telomerase"

Now showing 1 - 5 of 5

Open Access
The effect of telomerase template antagonist GRN163L on Bone-Marrow-Derived rat mMesenchymal stem cells is reversible and associated with altered expression of cyclin d1, cdk4 and cdk6
(Springer Science+Business Media, 2010) Tokcaer-Keskin, Z.; Dikmen, Z. G.; Ayaloglu-Butun, F.; Gultekin, S.; Gryaznov, S. M.; Akcali, K. C.
Telomerase activity is essential for the continued growth and survival of malignant cells, therefore inhibition of this activity presents an attractive target for anti-cancer therapy. The telomerase inhibitor GRN163L, was shown to inhibit the growth of cancer cells both in vitro and in vivo. Mesenchymal stem cells (MSCs) also show telomerase activity in maintaining their self-renewal; therefore the effects of telomerase inhibitors on MSCs may be an issue of concern. MSCs are multipotent cells and are important for the homeostasis of the organism. In this study, we sought to demonstrate in vitro effects of GRN163L on rat MSCs. When MSCs were treated with 1 μM GRN163L, their phenotype changed from spindle-shaped cells to rounded ones and detached from the plate surface, similar to cancer cells. Quantitative-RT-PCR and immunoblotting results revealed that GRN163L holds MSCs at the G1 state of the cell cycle, with a drastic decrease in mRNA and protein levels of cyclin D1 and its cdk counterparts, cdk4 and cdk6. This effect was not observed when MSCs were treated with a mismatch control oligonucleotide. One week after GRN163L was removed, mRNA and protein expressions of the genes, as well as the phenotype of MSCs returned to those of untreated cells. Therefore, we concluded that GRN163L does not interfere with the self-renewal and differentiation of MSCs under short term in vitro culture conditions. Our study provides additional support for treating cancers by administrating GRN163L without depleting the body's stem cell pools. © 2010 Springer Science+Business Media, LLC.
Open Access
Imetelstat (a telomerase antagonist) exerts off target effects on the cytoskeleton
(2013) Mender I.; Senturk, S.; Ozgunes, N.; Can Akcali, K.; Kletsas, D.; Gryaznov, S.; Can, A.; Shay J.W.; Dikmen, Z.G.
Telomerase is a cellular ribonucleoprotein reverse transcriptase that plays a crucial role in telomere maintenance. This enzyme is expressed in approximately 90% of human tumors, but not in the majority of normal somatic cells. Imetelstat sodium (GRN163L), is a 13-mer oligonucleotide N3'→P5' thio-phosphoramidate lipid conjugate, which represents the latest generation of telomerase inhibitors targeting the template region of the human functional telomerase RNA (hTR) subunit. In preclinical trials, this compound has been found to inhibit telomerase activity in multiple cancer cell lines, as well as in vivo xenograft mouse models. Currently, GRN163L is being investigated in several clinical trials, including a phase II human non small cell lung cancer clinical trial, in a maintenance setting following standard doublet chemotherapy. In addition to the inhibition of telomerase activity in cancer cell lines, GRN163L causes morphological cell rounding changes, independent of hTR expression or telomere length. This leads to the loss of cell adhesion properties; however, the mechanism underlying this effect is not yet fully understood. In the present study, we observed that GRN163L treatment leads to the loss of adhesion in A549 lung cancer cells, due to decreased E-cadherin expression, leading to the disruption of the cytoskeleton through the alteration of actin, tubulin and intermediate filament organization. Consequently, the less adherent cancer cells initially cease to proliferate and are arrested in the G1 phase of the cell cycle, accompanied by decreased matrix metalloproteinase-2 (MMP-2) expression. These effects of GRN163L are independent of its telomerase catalytic activity and may increase the therapeutic efficacy of GRN163L by decreasing the adhesion, proliferation and metastatic potential of cancer cells in vivo.
Open Access
Investigating the mechanisms of telomere maintenance in zebrafish tissues and human brain cancer cell lines
(2019-09) Şerifoğlu, Naz
Telomeres are nucleoprotein complexes formed at each end of the chromosomes to protect these ends from deterioration. In each round of cellular division, telomeric sequences shorten due to the end replication problem of DNA polymerase. Progressive telomere shortening results in replicative senescence in healthy somatic cells. To evade replicative senescence, cells need to maintain their telomere length either by activating the telomerase enzyme or through the alternative lengthening of telomeres (ALT). Telomerase is a holoenzyme, which is composed of dyskerin, telomerase RNA subunit (TR or TERC), and telomerase catalytic subunit (TERT). Dyskerin and TR are constitutively expressed in all cells but TERT expression is silenced in adult somatic cells. Thus, telomerase activity is dependent on the expression of TERT. Current studies show that TERT re-activation is a common feature of cancer cells and 85-90% of cancers utilize telomerase enzyme in maintaining telomeres to become immortal. Remaining of cancer cells maintain their telomeres by the alternative lengthening of telomeres (ALT), which is a DNA repair pathway dependent mechanism. Current models suggest that ALT is achieved by homology-directed DNA repair, through the interaction of multiple proteins. DNA methylation is regarded as a key player in epigenetic silencing of transcription. DNA methyltransferase inhibitors are currently being used in cancer treatments. Recent studies show that DNA methyltransferases and their expression levels impact both telomerase- and ALT mediated lengthening of telomeres, and have different outcomes in different tissue types. In this study, we worked on the zebrafish brain and human brain cancer cell lines. In zebrafish brain, we observed differences in methylated regions at Sp1 binding site between young and old that can be associated with telomere shortening. By silencing DNMT1 and DNMT3B in brain cancer cell lines, we investigated the changes in gene expression levels of telomerase and ALT related genes, telomerase activity, population doubling time and replicative senescence status. To further investigate TERT regulation, we introduced mutations to the Sp1 binding sites in the promoter region and measured the promoter activity with luciferase assay. Our results show that Sp1 methylation sites in the telomerase promoter region are critical in brain aging, dependent on their position. We propose a therapeutical option for brain aging and tumorigenesis.
Open Access
Reprogramming of replicative senescence in hepatocellular carcinoma-derived cells
(National Academy of Sciences, 2006) Ozturk, N.; Erdal, E.; Mumcuoglu, M.; Akcali, K. C.; Yalcin, O.; Senturk, S.; Arslan-Ergul, A.; Gur, B.; Yulug, I.; Cetin Atalay, R.; Yakicier, C.; Yagci, T.; Tez, M.; Ozturk, M.
Tumor cells have the capacity to proliferate indefinitely that is qualified as replicative immortality. This ability contrasts with the intrinsic control of the number of cell divisions in human somatic tissues by a mechanism called replicative senescence. Replicative immortality is acquired by inactivation of p53 and p16INK4a genes and reactivation of hTERT gene expression. It is unknown whether the cancer cell replicative immortality is reversible. Here, we show the spontaneous induction of replicative senescence in p53-and p16 INK4a-deficient hepatocellular carcinoma cells. This phenomenon is characterized with hTERT repression, telomere shortening, senescence arrest, and tumor suppression. SIP1 gene (ZFHX1B) is partly responsible for replicative senescence, because short hairpin RNA-mediated SIP1 inactivation released hTERT repression and rescued clonal hepatocellular carcinoma cells from senescence arrest. © 2006 by The National Academy of Sciences of the USA.
Open Access
TERT distal promoter GC islands are critical for telomerase and together with DNMT3B silencing may serve as a senescence-inducing agent in gliomas
(Taylor & Francis Inc., 2022-08-23) Şerifoğlu, Naz; Adams, Michelle; Erbaba, Begün; Arslan-Ergül, Ayça
Telomerase is reactivated in the majority of cancers. For instance, in gliomas, it is common that the TERT promoter is mutated. Research on telomere promoter GC islands have been focused primarily on proximal TERT promoter but little is known about the distal promoter. Therefore, in this study, we investigated the proximal and distal TERT promoter, in terms of DNA methylation. We did bisulfite sequencing in zebrafish tissue samples for the distal tert promoter. In the zebrafish brain tissues, we identified a hypomethylation site in the tert promoter, and found that this hypomethylation was associated with aging and shortened telomeres. Through site directed mutagenesis in glioma cell lines, we changed 10 GC spots individually, cloned into a reporter vector, and measured promoter activity. Finally, we silenced DNMT3B and measured telomerase activity along with vidaza and adriamycin treatments. Site directed mutagenesis of glioma cell lines revealed that each of the 10 GC spots are critical for telomerase activity. Changing GC to AT abolished promoter activity in all spots when transfected into glioma cell lines. Then, through silencing of DNMT3B, we observed a reduction in hTERT expression levels, while hTR remained the same, and a major increase in senescence-associated beta-galactosidase activity. Finally, we propose a model regarding the efficacy of two chemotherapeutic drugs, adriamycin and azacytidine, on gliomas. Here, we show that distal TERT promoter is critical; changing even one GC to AT abolishes TERT promoter activity. DNMT3B, a de novo methyltransferase, together with GC islands in distal TERT promoter plays an important role in regulation of telomerase expression and senescence.