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

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.

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

Journal of Biological Chemistry ◽

10.1074/jbc.m408131200 ◽

2004 ◽

Vol 279 (50) ◽

pp. 51745-51748 ◽

Cited By ~ 61

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.

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Mechanism of Human Telomerase Reverse Transcriptase (hTERT) Regulation and Clinical Impacts in Leukemia

Genes ◽

10.3390/genes12081188 ◽

2021 ◽

Vol 12 (8) ◽

pp. 1188

Author(s):

Mot Yee Yik ◽

Adam Azlan ◽

Yaashini Rajasegaran ◽

Aliaa Rosli ◽

Narazah Mohd Yusoff ◽

...

Keyword(s):

Reverse Transcriptase ◽

Diagnostic Value ◽

Telomerase Activity ◽

Biological Molecules ◽

Leukemic Cells ◽

Telomerase Reverse Transcriptase ◽

Human Telomerase Reverse Transcriptase ◽

Elevated Expression ◽

Human Telomerase ◽

Next Generation Sequencing Ngs

The proliferative capacity and continuous survival of cells are highly dependent on telomerase expression and the maintenance of telomere length. For this reason, elevated expression of telomerase has been identified in virtually all cancers, including leukemias; however, it should be noted that expression of telomerase is sometimes observed later in malignant development. This time point of activation is highly dependent on the type of leukemia and its causative factors. Many recent studies in this field have contributed to the elucidation of the mechanisms by which the various forms of leukemias increase telomerase activity. These include the dysregulation of telomerase reverse transcriptase (TERT) at various levels which include transcriptional, post-transcriptional, and post-translational stages. The pathways and biological molecules involved in these processes are also being deciphered with the advent of enabling technologies such as next-generation sequencing (NGS), ribonucleic acid sequencing (RNA-Seq), liquid chromatography-mass spectrometry (LCMS/MS), and many others. It has also been established that TERT possess diagnostic value as most adult cells do not express high levels of telomerase. Indeed, studies have shown that prognosis is not favorable in patients who have leukemias expressing high levels of telomerase. Recent research has indicated that targeting of this gene is able to control the survival of malignant cells and therefore offers a potential treatment for TERT-dependent leukemias. Here we review the mechanisms of hTERT regulation and deliberate their association in malignant states of leukemic cells. Further, we also cover the clinical implications of this gene including its use in diagnostic, prognostic, and therapeutic discoveries.

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Identification of Two RNA-binding Proteins Associated with Human Telomerase RNA

Molecular Biology of the Cell ◽

10.1091/mbc.11.3.999 ◽

2000 ◽

Vol 11 (3) ◽

pp. 999-1010 ◽

Cited By ~ 88

Author(s):

Siyuan Le ◽

Rolf Sternglanz ◽

Carol W. Greider

Keyword(s):

Rna Binding ◽

Rna Binding Proteins ◽

Telomerase Activity ◽

Telomere Maintenance ◽

Telomerase Rna ◽

Human Telomerase Reverse Transcriptase ◽

Ribonucleoprotein Complexes ◽

Cell Extracts ◽

Human Telomerase

Telomerase plays a crucial role in telomere maintenance in vivo. To understand telomerase regulation, we have been characterizing components of the enzyme. To date several components of the mammalian telomerase holoenzyme have been identified: the essential RNA component (human telomerase RNA [hTR]), the catalytic subunit human telomerase reverse transcriptase (hTERT), and telomerase-associated protein 1. Here we describe the identification of two new proteins that interact with hTR: hStau and L22. Antisera against both proteins immunoprecipitated hTR, hTERT, and telomerase activity from cell extracts, suggesting that the proteins are associated with telomerase. Both proteins localized to the nucleolus and cytoplasm. Although these proteins are associated with telomerase, we found no evidence of their association with each other or with telomerase-associated protein 1. Both hStau and L22 are more abundant than TERT. This, together with their localization, suggests that they may be associated with other ribonucleoprotein complexes in cells. We propose that these two hTR-associated proteins may play a role in hTR processing, telomerase assembly, or localization in vivo.

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Telomerase activity and cell apoptosis in colon cancer cell by human telomerase reverse transcriptase gene antisense oligodeoxynucleotide

World Journal of Gastroenterology ◽

10.3748/wjg.v9.i9.1981 ◽

2003 ◽

Vol 9 (9) ◽

pp. 1981 ◽

Cited By ~ 10

Author(s):

Ying-An Jiang

Keyword(s):

Colon Cancer ◽

Reverse Transcriptase ◽

Cell Apoptosis ◽

Telomerase Activity ◽

Colon Cancer Cell ◽

Antisense Oligodeoxynucleotide ◽

Telomerase Reverse Transcriptase ◽

Reverse Transcriptase Gene ◽

Human Telomerase Reverse Transcriptase ◽

Human Telomerase

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Effect of hTERT Antisense on Cisplatin Sensitivity of Acute Lymphoblastic Leukemic Cells.

Blood ◽

10.1182/blood.v104.11.4358.4358 ◽

2004 ◽

Vol 104 (11) ◽

pp. 4358-4358

Author(s):

Wenyu Li ◽

Yuan Zhang

Keyword(s):

Leukemic Cells ◽

Morphological Observation ◽

Blue Dye ◽

Double Staining ◽

Hoechst 33258 ◽

Human Telomerase Reverse Transcriptase ◽

Cisplatin Sensitivity ◽

Staining Techniques ◽

Induced Apoptosis ◽

Human Telomerase

Abstract AIM: To investigate the effect of human telomerase reverse transcriptase(hTERT) antisense phosphorothioate oligodeoxynucleotide(AS PS-ODN) on enhancing cisplatin sensitivity in acute lymphoblastic leukemic cells. METHODS: The expression levels of hTERT protein were detected by immunofluorescence using fluoresce isothiocyanate(FITC) lable. Cell surviving fraction was determined using the trypan blue dye exclusion assay. Apoptosis was detected by morphological observation and flow cytomertric cell cycle analysis. RESULTS: The expression of hTERT protein was inhibited after treated by hTERT AS PS-ODN. Treatment with cisplatin after 24h of exposure to AS PS-ODN had significantly reduced the number of viable ALL cells. However, there was no difference on ALL cells survival between sense oligodeoxynucleotide(S PS-ODN)/CDDP combination and CDDP treated cells alone. In morphological observation of apoptotic cells using Hoechst 33258 and PI double staining techniques, cells displayed classic apoptotic changes treated with CDDP or CDDP combined with hTERT AS PS-ODN or S PS-ODN at 48h. Apoptotic rates of cells added CDDP and AS PS-ODN were higher than that of cells added CDDP only(P<0.05). Apoptotic rates of cells added CDDP and S PS-ODN were similar with that of cells added CDDP only(P>0.05). CONCLUSION: hTERT AS-ODN inhibited the expression of hTERT protein and increased the CDDP induced apoptosis in primer acute lymphoblastic leukemic cells.

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Functional Multimerization of the Human Telomerase Reverse Transcriptase

Molecular and Cellular Biology ◽

10.1128/mcb.21.18.6151-6160.2001 ◽

2001 ◽

Vol 21 (18) ◽

pp. 6151-6160 ◽

Cited By ~ 92

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.

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Telomerase Activity Reconstitutedin Vitrowith Purified Human Telomerase Reverse Transcriptase and Human Telomerase RNA Component

Journal of Biological Chemistry ◽

10.1074/jbc.m000622200 ◽

2000 ◽

Vol 275 (29) ◽

pp. 22568-22573 ◽

Cited By ~ 47

Author(s):

Kenkichi Masutomi ◽

Shuichi Kaneko ◽

Naoyuki Hayashi ◽

Tatsuya Yamashita ◽

Yukihiro Shirota ◽

...

Keyword(s):

Reverse Transcriptase ◽

Telomerase Activity ◽

Telomerase Reverse Transcriptase ◽

Telomerase Rna ◽

Human Telomerase Reverse Transcriptase ◽

Human Telomerase

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Cisplatin-induced apoptotic endonuclease EndoG inhibits telomerase activity and causes malignant transformation of human CD4+ T lymphocytes

Biomeditsinskaya Khimiya ◽

10.18097/pbmc20176301013 ◽

2017 ◽

Vol 63 (1) ◽

pp. 13-26 ◽

Cited By ~ 5

Author(s):

D.D. Zhdanov ◽

D.A. Vasina ◽

E.V. Orlova ◽

V.S. Orlova ◽

V.S. Pokrovsky ◽

...

Keyword(s):

T Cells ◽

Alternative Splicing ◽

Malignant Transformation ◽

Cd4 T Cells ◽

Replicative Senescence ◽

Telomerase Activity ◽

Full Length ◽

Leukemic Cells ◽

Proliferative Potential ◽

Human Telomerase Reverse Transcriptase

Alternative splicing of telomerase catalytic subunit hTERT pre-mRNA (human Telomerase Reverse Transcriptase) regulates telomerase activity. Increased expression of non-active splice variant hTERT results in inhibition of telomerase. Apoptotic endonuclease EndoG is known to participate in hTERT alternative splicing. Expression of EndoG can be induced in response to DNA damages. The aim of this study was to determine the ability of a DNA-damaging compound, cisplatin, to induce EndoG and its influence on alternative splicing of hTERT and telomerase activity in human CD4+ Т lymphocytes. Overexpression of EndoG in CD4+ T cells downregulated the expression of active full-length hTERT variant and upregulated its non-active spliced variant. Reduction of full-length hTERT caused downregulation of telomerase activity, shortening of telomeres length during cell divisions, converting cells to the replicative senescence state, activation of apoptosis and finally cell death. Few cells survived and underwent malignant transformation. Transformed cells have increased telomerase activity and proliferative potential compare to initial CD4+ T cells. These cells have phenotype of T lymphoblastic leukemic cells and are able to form tumors and cause death in experimental mice.

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