Nonradioactive telomerase activity assay by microchip electrophoresis: Privileges to the classical gel electrophoresis assay

2005 ◽  
Vol 26 (15) ◽  
pp. 3021-3024 ◽  
Author(s):  
Zhivko Zhelev ◽  
Rumiana Bakalova ◽  
Ashraf Ewis ◽  
Hideki Ohba ◽  
Mitsuru Ishikawa ◽  
...  
Keyword(s):  
Gel Electrophoresis ◽  
Telomerase Activity ◽  
Microchip Electrophoresis ◽  
Activity Assay

scholarly journals Zinc Protoporphyrin Binding to Telomerase Complexes and Inhibition of Telomerase Activity

2021 ◽  
Author(s):  
Zhaowen Zhu ◽  
Huy Tran ◽  
Meleah M. Mathahs ◽  
Brian D. Fink ◽  
John A. Albert ◽  
...  
Keyword(s):  
Dna Synthesis ◽  
Gel Electrophoresis ◽  
Cellular Proliferation ◽  
Telomerase Activity ◽  
Zinc Protoporphyrin ◽  
Agarose Gel ◽  
Agarose Gel Electrophoresis ◽  
Metaphase Chromosomes ◽  
Intact Cells ◽  
Confocal Fluorescence

Abstract Background: Zinc protoporphyrin (ZnPP) is a naturally occurring metalloprotoporphyrin (MPP) that is currently under development as a chemotherapeutic agent although its mechanism is unclear. Similar to natural and synthetic porphyrins, MPPs are thought to bind DNA and stabilize secondary structures such as guanine quadruplexes (G-4) and thus potentially impact telomerase activity and DNA synthesis which are important targets for chemotherapy. Interactions of MPPs with telomerase have not been previously reported. Methods: We wished to evaluate the effects of common MPPs, i.e., ZnPP, tin protoporphyrin (SnPP), and iron protoporphyrin (FePP), on cellular proliferation, apoptosis, and telomerase activity in hepatoma cells. The cytotoxicities of porphyrins were determined by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay. Native agarose gel electrophoresis was used to identify ZnPP binding of telomerase complexes. Inhibition of telomerase activity by ZnPP was assessed by conventional telomeric repeat amplification protocol (TRAP) and direct telomerase activity assays. Colocalization of ZnPP with telomerase was analyzed with immunofluorescence staining and confocal microscopic analysis. Results: ZnPP was the most effective MPP for decreasing DNA synthesis and cellular proliferation, while promoting apoptosis in cultured hepatocytes. Concurrently, ZnPP down-regulated telomerase expression and was the best overall inhibitor of telomerase activity in intact cells and in vitro assays, with IC50 and EC50 values of ca 2.5 and 6 µM respectively. The natural fluorescence properties of ZnPP enabled direct imaging in cellular fractions using non-denaturing agarose gel electrophoresis, western blots, and confocal fluorescence microscopy. ZnPP localized to large cellular complexes (> 600kD) that contained telomerase and dyskerin as confirmed with immunocomplex mobility shift, immunoprecipitation, and immunoblot analyses. Confocal fluorescence studies showed that ZnPP co-localized with telomerase reverse transcriptase (TERT) and telomeres in the nucleus of synchronized S-phase cells. ZnPP also co-localized with TERT in the perinuclear regions of log phase cells but did not co-localize with telomeres on the ends of metaphase chromosomes, a site known to be devoid of telomerase complexes. Taken together, these results suggest that ZnPP does not bind to telomeric sequences per se, but alternatively, interacts with other structural components of the telomerase complex to inhibit telomerase enzymatic activity. Conclusions. ZnPP can actively interfere with telomerase activity in neoplastic cells, thus eliciting pro-apoptotic and anti-proliferative properties. These data support further development of natural or synthetic protoporphyrins for use as chemotherapeutic agents to augment current treatment protocols for a number of neoplasms.

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 A Highly Sensitive Telomerase Activity Assay that Eliminates False-Negative Results Caused by PCR Inhibitors

Molecules ◽  
2013 ◽  
Vol 18 (10) ◽  
pp. 11751-11767 ◽  
Author(s):  
Hidenobu Yaku ◽  
Takashi Murashima ◽  
Daisuke Miyoshi ◽  
Naoki Sugimoto
Keyword(s):  
False Negative ◽  
Telomerase Activity ◽  
Activity Assay ◽  
Pcr Inhibitors ◽  
Negative Results ◽  
False Negative Results ◽  
Highly Sensitive

Multi-code magnetic beads based on DNAzyme-mediated double-cycling amplification for a point-of-care assay of telomerase activity

The Analyst ◽  
2019 ◽  
Vol 144 (14) ◽  
pp. 4241-4249 ◽  
Author(s):  
Chunxue Liu ◽  
Susu Zhang ◽  
Xia Li ◽  
Qingwang Xue ◽  
Wei Jiang
Keyword(s):  
Medical Care ◽  
Magnetic Beads ◽  
Point Of Care ◽  
High Specificity ◽  
Telomerase Activity ◽  
Activity Assay ◽  
Double Cycling ◽  
Specificity And Sensitivity ◽  
Point Of Care Assay

Development of a reliable and facile telomerase activity assay with high specificity and sensitivity is a central challenge to make telomerase testing a routine part of medical care with respect to cancer.

Telomerase Activity Assay via 3,3′,5,5′-Tetramethylbenzidine Dilation Etching of Gold Nanorods

2022 ◽  
Author(s):  
Xiao Feng Yang ◽  
Wei He ◽  
Yuan Fang Li ◽  
Peng Fei Gao ◽  
Cheng Zhi Huang
Keyword(s):  
Gold Nanorods ◽  
Telomerase Activity ◽  
Activity Assay
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