scholarly journals Characterization of Interactions between PinX1 and Human Telomerase Subunits hTERT and hTR

2004 ◽  
Vol 279 (50) ◽  
pp. 51745-51748 ◽  
Author(s):  
Soma S. R. Banik ◽  
Christopher M. Counter
Keyword(s):  
Telomerase Activity ◽  
Protein Subunit ◽  
Human Telomerase Reverse Transcriptase ◽  
Telomere Elongation ◽  
Cellular Context ◽  
Core Components ◽  
Human Telomerase

The addition of telomeric repeats to chromosome ends by the enzyme telomerase is a highly orchestrated process. Although much is known regarding telomerase catalytic activityin vitro, less is known about how this activity is regulatedin vivoto ensure proper telomere elongation. One protein that appears to be involved in negatively regulating telomerase functionin vivois PinX1 because overexpression of PinX1 inhibits telomerase activity and causes telomere shortening. To understand the nature of this repression, we characterized the interactions among PinX1 and the core components of telomerase, the human telomerase reverse transcriptase (hTERT) and associated human telomerase RNA (hTR). We now show thatin vitroPinX1 binds directly to the hTERT protein subunit, primarily to the hTR-binding domain, as well as to the hTR subunit. However, in a cellular context, the association of PinX1 with hTR is dependent on the presence of hTERT. Taken together, we suggest that PinX1 represses telomerase activityin vivoby binding to the assembled hTERT·hTR complex.

scholarly journals Functional Multimerization of the Human Telomerase Reverse Transcriptase

2001 ◽  
Vol 21 (18) ◽  
pp. 6151-6160 ◽  
Author(s):  
Tara L. Beattie ◽  
Wen Zhou ◽  
Murray O. Robinson ◽  
Lea Harrington
Keyword(s):  
Reverse Transcriptase ◽  
Telomerase Activity ◽  
Telomerase Reverse Transcriptase ◽  
Telomerase Rna ◽  
Human Telomerase Reverse Transcriptase ◽  
Catalytic Core ◽  
Cell Extracts ◽  
Human Telomerase

ABSTRACT The telomerase enzyme exists as a large complex (∼1,000 kDa) in mammals and at minimum is composed of the telomerase RNA and the catalytic subunit telomerase reverse transcriptase (TERT). In Saccharomyces cerevisiae, telomerase appears to function as an interdependent dimer or multimer in vivo (J. Prescott and E. H. Blackburn, Genes Dev. 11:2790–2800, 1997). However, the requirements for multimerization are not known, and it remained unclear whether telomerase exists as a multimer in other organisms. We show here that human TERT (hTERT) forms a functional multimer in a rabbit reticulocyte lysate reconstitution assay and in human cell extracts. Two separate, catalytically inactive TERT proteins can complement each other in trans to reconstitute catalytic activity. This complementation requires the amino terminus of one hTERT and the reverse transcriptase and C-terminal domains of the second hTERT. The telomerase RNA must associate with only the latter hTERT for reconstitution of telomerase activity to occur. Multimerization of telomerase also facilitates the recognition and elongation of substrates in vitro and in vivo. These data suggest that the catalytic core of human telomerase may exist as a functionally cooperative dimer or multimer in vivo.

Bioflavonoids protect cells against halogenated boroxine-induced genotoxic damage by upregulation of hTERT expression

2019 ◽  
Vol 74 (5-6) ◽  
pp. 125-129 ◽  
Author(s):  
Maida Hadzic ◽  
Sanin Haveric ◽  
Anja Haveric ◽  
Naida Lojo-Kadric ◽  
Borivoj Galic ◽  
...  
Keyword(s):  
Cell Cycle Progression ◽  
Housekeeping Genes ◽  
Inhibitory Effects ◽  
Human Telomerase Reverse Transcriptase ◽  
Cycle Progression ◽  
Genotoxic Damage ◽  
Malignant Skin ◽  
Human Telomerase

Abstract Plant bioflavonoids are widely present in the human diet and have various protective properties. In this study, we have demonstrated the capacity of delphinidin and luteolin to increase human telomerase reverse transcriptase (hTERT) expression level and act as protective agents against halogenated boroxine-induced genotoxic damage. Halogenated boroxine K2(B3O3F4OH) (HB), is a novel compound with potential for the treatment of both benign and malignant skin changes. In vivo and in vitro studies have confirmed the inhibitory effects of HB on carcinoma cell proliferation and cell cycle progression as well as enzyme inhibition. However, minor genotoxic effects of HB are registered in higher applied concentrations, but those can be suppressed by in vitro addition of delphinidin and luteolin in appropriate concentrations. Fresh peripheral blood samples were cultivated for 72 h followed by independent and concomitant treatments of HB with luteolin or delphinidin. We analyzed the differences in relative hTERT expression between series of treatments compared with controls, which were based on normalized ratios with housekeeping genes. The obtained results have shown that selected bioflavonoids induce upregulation of hTERT that may contribute to the repair of genotoxic damage in vitro.

Effect of hTR Antisense Oligodeoxynucleotides on Telomerase Acitvity and Ciplatin -Sensitivity of Primary Acute Leukemia Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4359-4359
Author(s):  
Yang Xiao
Keyword(s):  
Gel Electrophoresis ◽  
Telomerase Activity ◽  
Leukemic Cells ◽  
Morphological Observation ◽  
Antisense Oligodeoxynucleotide ◽  
Human Telomerase Reverse Transcriptase ◽  
Significant Difference ◽  
Initiator Codon ◽  
Human Telomerase

Abstract BACKGROUND: Between the three key components part of human telomerase, human telomerase RNA (hTR) and human telomerase reverse transcriptase (hTERT) have significant correlation with telomerase activity. The previous study has identified that the telomerase activity of K562 and HL-60 cells was special suppresssed significantly by phosphorothoate antisense oligodeoxynucleotide (ASODN) complementary to the initiator codon of hTR. This study was designed to evalutae the effection of hTR ASODN on telomerase activity and apoptosis of primary acute leukemic cells. To research whether hTR ASODN could enhance apoptosis rates of primary leukemic cells to cisplatin. METHODS: Primary leukemic cells were treated with phosphorothoate ASODN complementary to the initiator codon of hTR in vivo. The changing of telomerase activity was assayed by telomeric repeat amplification protocol(TRAP) and polymerase chain reaction enzyme-linked immunoassay(PCR-ELISA). The survival rates of cells was measured by trypan blue exclusion. Apoptosis was assayed by morphological observation (Giemsa and PI), DNA gel electrophoresis and flow cytometry analysis technology. RESULTS: Primary acute leukemic cells expressed high level of telomerase activity which decreased as the cells treated by hTR ASODN. The telomerase activity was suppresssed significantly by 10umol/L ASODN and the effecttion was most significantly at 72h; Apoptotic bodies of primary leukemic cells were observed easily by fluorescence micrscope when cisplatin was added 48h after ASODN treatment for 24h; Agarose gel electrophoresis of genomic DNA from primary leukemic cells treated with ASODN and cisplatin combination for 72h showed typical DNA ladder; neither did DNA from primary leukemic cells treated with sense oligodeoxynucleotide (SODN) plus cisplatin nor cisplatin alone. In addition, apoptosis rates of primary leukemic cells treated with ASODN for 24h and then with cisplatin for 72h were 41.36±9.28%. There were statistically significant difference in the percentage of apoptotic cells between hTR ASODN plus cisplatin and SODN plus cisplatin (14.51±4.78%) or cisplatin alone group (12.61±2.56)% (P<0.01). CONCLUSIONS: ASODN complementary to the region of hTR could significantly inhibit the telomerase activity of primary acute leukemic cells, and increased the cisplatin-induced apoptosis and enhanced cisplatin-sensitivity in vivo, indicating telomerase may be a new target of treatment to leukemia.

scholarly journals Polymerization Defects within Human Telomerase Are Distinct from Telomerase RNA and TEP1 Binding

2000 ◽  
Vol 11 (10) ◽  
pp. 3329-3340 ◽  
Author(s):  
Tara L. Beattie ◽  
Wen Zhou ◽  
Murray O. Robinson ◽  
Lea Harrington
Keyword(s):  
Telomerase Activity ◽  
Amino Terminus ◽  
Telomerase Rna ◽  
Carboxy Terminus ◽  
Active Core ◽  
Human Telomerase ◽  
Precise Role

The minimal, active core of human telomerase is postulated to contain two components, the telomerase RNA hTER and the telomerase reverse transcriptase hTERT. The reconstitution of human telomerase activity in vitro has facilitated the identification of sequences within the telomerase RNA and the RT motifs of hTERT that are essential for telomerase activity. However, the precise role of residues outside the RT domain of hTERT is unknown. Here we have delineated several regions within hTERT that are important for telomerase catalysis, primer use, and interaction with the telomerase RNA and the telomerase-associated protein TEP1. In particular, certain deletions of the amino and carboxy terminus of hTERT that retained an interaction with telomerase RNA and TEP1 were nonetheless completely inactive in vitro and in vivo. Furthermore, hTERT truncations lacking the amino terminus that were competent to bind the telomerase RNA were severely compromised for the ability to elongate telomeric and nontelomeric primers. These results suggest that the interaction of telomerase RNA with hTERT can be functionally uncoupled from polymerization, and that there are regions outside the RT domain of hTERT that are critical for telomerase activity and primer use. These results establish that the human telomerase RT possesses unique polymerization determinants that distinguish it from other RTs.

scholarly journals Regulation of poly(a)-specific ribonuclease activity by reversible lysine acetylation

2020 ◽  
Vol 295 (30) ◽  
pp. 10255-10270
Author(s):  
Eden A. Dejene ◽  
Yixuan Li ◽  
Zahra Showkatian ◽  
Hongbo Ling ◽  
Edward Seto
Keyword(s):  
Enzymatic Activity ◽  
Noncoding Rna ◽  
Critical Role ◽  
Telomere Maintenance ◽  
Post Translational Modifications ◽  
Telomere Elongation ◽  
The Stability ◽  
Human Telomerase

Poly(A)-specific ribonuclease (PARN) is a 3′-exoribonuclease that plays an important role in regulating the stability and maturation of RNAs. Recently, PARN has been found to regulate the maturation of the human telomerase RNA component (hTR), a noncoding RNA required for telomere elongation. Specifically, PARN cleaves the 3′-end of immature, polyadenylated hTR to form the mature, nonpolyadenylated template. Despite PARN's critical role in mediating telomere maintenance, little is known about how PARN's function is regulated by post-translational modifications. In this study, using shRNA- and CRISPR/Cas9-mediated gene silencing and knockout approaches, along with 3′-exoribonuclease activity assays and additional biochemical methods, we examined whether PARN is post-translationally modified by acetylation and what effect acetylation has on PARN's activity. We found PARN is primarily acetylated by the acetyltransferase p300 at Lys-566 and deacetylated by sirtuin1 (SIRT1). We also revealed how acetylation of PARN can decrease its enzymatic activity both in vitro, using a synthetic RNA probe, and in vivo, by quantifying endogenous levels of adenylated hTR. Furthermore, we also found that SIRT1 can regulate levels of adenylated hTR through PARN. The findings of our study uncover a mechanism by which PARN acetylation and deacetylation regulate its enzymatic activity as well as levels of mature hTR. Thus, PARN's acetylation status may play a role in regulating telomere length.

scholarly journals N-Terminal Domains of the Human Telomerase Catalytic Subunit Required for Enzyme Activity in Vivo

2001 ◽  
Vol 21 (22) ◽  
pp. 7775-7786 ◽  
Author(s):  
Blaine N. Armbruster ◽  
Soma S. R. Banik ◽  
Chuanhai Guo ◽  
Allyson C. Smith ◽  
Christopher M. Counter
Keyword(s):  
Enzyme Activity ◽  
Catalytic Activity ◽  
Cellular Proliferation ◽  
Telomere Maintenance ◽  
Biological Functions ◽  
Sequence Alignments ◽  
Telomere Elongation ◽  
Human Telomerase

ABSTRACT Most tumor cells depend upon activation of the ribonucleoprotein enzyme telomerase for telomere maintenance and continual proliferation. The catalytic activity of this enzyme can be reconstituted in vitro with the RNA (hTR) and catalytic (hTERT) subunits. However, catalytic activity alone is insufficient for the full in vivo function of the enzyme. In addition, the enzyme must localize to the nucleus, recognize chromosome ends, and orchestrate telomere elongation in a highly regulated fashion. To identify domains of hTERT involved in these biological functions, we introduced a panel of 90 N-terminal hTERT substitution mutants into telomerase-negative cells and assayed the resulting cells for catalytic activity and, as a marker of in vivo function, for cellular proliferation. We found four domains to be essential for in vitro and in vivo enzyme activity, two of which were required for hTR binding. These domains map to regions defined by sequence alignments and mutational analysis in yeast, indicating that the N terminus has also been functionally conserved throughout evolution. Additionally, we discovered a novel domain, DAT, that “dissociates activities of telomerase,” where mutations left the enzyme catalytically active, but was unable to function in vivo. Since mutations in this domain had no measurable effect on hTERT homomultimerization, hTR binding, or nuclear targeting, we propose that this domain is involved in other aspects of in vivo telomere elongation. The discovery of these domains provides the first step in dissecting the biological functions of human telomerase, with the ultimate goal of targeting this enzyme for the treatment of human cancers.

scholarly journals Establishment and Characterization of a Reliable Xenograft Model of Hodgkin Lymphoma Suitable for the Study of Tumor Origin and the Design of New Therapies

Cancers ◽  
2018 ◽  
Vol 10 (11) ◽  
pp. 414 ◽  
Author(s):  
Radhia M’kacher ◽  
Monika Frenzel ◽  
Mustafa Al Jawhari ◽  
Steffen Junker ◽  
Corina Cuceu ◽  
...  
Keyword(s):  
Animal Model ◽  
Histone Deacetylase ◽  
High Frequency ◽  
Histone Deacetylase Inhibitor ◽  
Telomerase Activity ◽  
Nsg Mice ◽  
Deacetylase Inhibitor

To identify the cells responsible for the initiation and maintenance of Hodgkin lymphoma (HL) cells, we have characterized a subpopulation of HL cells grown in vitro and in vivo with the aim of establishing a reliable and robust animal model for HL. To validate our model, we challenged the tumor cells in vivo by injecting the alkylating histone-deacetylase inhibitor, EDO-S101, a salvage regimen for HL patients, into xenografted mice. Methodology: Blood lymphocytes from 50 HL patients and seven HL cell lines were used. Immunohistochemistry, flow cytometry, and cytogenetics analyses were performed. The in vitro and in vivo effects of EDO-S101 were assessed. Results: We have successfully determined conditions for in vitro amplification and characterization of the HL L428-c subline, containing a higher proportion of CD30−/CD15− cells than the parental L428 cell line. This subline displayed excellent clonogenic potential and reliable reproducibility upon xenografting into immunodeficient NOD-SCID-gamma (−/−)(NSG) mice. Using cell sorting, we demonstrate that CD30−/CD15− subpopulations can gain the phenotype of the L428-c cell line in vitro. Moreover, the human cells recovered from the seventh week after injection of L428-c cells into NSG mice were small cells characterized by a high frequency of CD30−/CD15− cells. Cytogenetic analysis demonstrated that they were diploid and showed high telomere instability and telomerase activity. Accordingly, chromosomal instability emerged, as shown by the formation of dicentric chromosomes, ring chromosomes, and breakage/fusion/bridge cycles. Similarly, high telomerase activity and telomere instability were detected in circulating lymphocytes from HL patients. The beneficial effect of the histone-deacetylase inhibitor EDO-S101 as an anti-tumor drug validated our animal model. Conclusion: Our HL animal model requires only 103 cells and is characterized by a high survival/toxicity ratio and high reproducibility. Moreover, the cells that engraft in mice are characterized by a high frequency of small CD30−/CD15− cells exhibiting high telomerase activity and telomere dysfunction.

scholarly journals Dual Regulation of c-Myc by p300 via Acetylation-Dependent Control of Myc Protein Turnover and Coactivation of Myc-Induced Transcription

2005 ◽  
Vol 25 (23) ◽  
pp. 10220-10234 ◽  
Author(s):  
Francesco Faiola ◽  
Xiaohui Liu ◽  
Szuying Lo ◽  
Songqin Pan ◽  
Kangling Zhang ◽  
...  
Keyword(s):  
Dna Binding ◽  
Cell Fate ◽  
Mammalian Cells ◽  
Dependent Manner ◽  
Dual Roles ◽  
Human Telomerase Reverse Transcriptase ◽  
Lysine Residues ◽  
Human Telomerase

ABSTRACT The c-Myc oncoprotein (Myc) controls cell fate by regulating gene transcription in association with a DNA-binding partner, Max. While Max lacks a transcription regulatory domain, the N terminus of Myc contains a transcription activation domain (TAD) that recruits cofactor complexes containing the histone acetyltransferases (HATs) GCN5 and Tip60. Here, we report a novel functional interaction between Myc TAD and the p300 coactivator-acetyltransferase. We show that p300 associates with Myc in mammalian cells and in vitro through direct interactions with Myc TAD residues 1 to 110 and acetylates Myc in a TAD-dependent manner in vivo at several lysine residues located between the TAD and DNA-binding domain. Moreover, the Myc:Max complex is differentially acetylated by p300 and GCN5 and is not acetylated by Tip60 in vitro, suggesting distinct functions for these acetyltransferases. Whereas p300 and CBP can stabilize Myc independently of acetylation, p300-mediated acetylation results in increased Myc turnover. In addition, p300 functions as a coactivator that is recruited by Myc to the promoter of the human telomerase reverse transcriptase gene, and p300/CBP stimulates Myc TAD-dependent transcription in a HAT domain-dependent manner. Our results suggest dual roles for p300/CBP in Myc regulation: as a Myc coactivator that stabilizes Myc and as an inducer of Myc instability via direct Myc acetylation.

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