TRAP-silver staining, a highly sensitive assay for measuring telomerase activity in tumor tissue and cell lines

Torre, Cristiane Arruda Dalla; Maciel, Rui M. B.; Pinheiro, Nídia Alice; Andrade, Joyce Anderson Duffles; Toledo, Sílvia Regina Caminada de; Villa, Luisa L.; Cerutti, Janete M.

doi:10.1590/s0100-879x2002000100009

Cited by 24 publications

(14 citation statements)

References 8 publications

(9 reference statements)

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“…The PCR products were separated by 12.5% nondenaturing PAGE in 0.5 Â TBE. Silver staining was carried out as previously described (Dalla Torre et al, 2002). After electrophoresis, the gels were fixed with a solution containing 0.5% acetic acid and 10% ethanol for 15 min and stained with 0.2% AgNO 2 for 10 min.…”

Section: Immunoprecipitation and Western Blot Analysesmentioning

confidence: 99%

Hdm2 negatively regulates telomerase activity by functioning as an E3 ligase of hTERT

Lee

et al. 2010

Oncogene

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In this study, we identified posttranslational regulation of human telomerase reverse-transcriptase (hTERT) by the E3 ligase Hdm2. The telomerase activity generated by exogenous hTERT in U2OS cells was reduced on adriamycin treatment. The overexpressed levels of hTERT were also decreased under the same conditions. These processes were reversed by treatment with a proteasome inhibitor or depletion of Hdm2. Furthermore, intrinsic telomerase activity was increased in HCT116 cells with ablation of Hdm2. Immunoprecipitation analyses showed that hTERT and Hdm2 bound to each other in multiple domains. Ubiquitination analyses showed that Hdm2 could polyubiquitinate hTERT principally at the N-terminus, which was further degraded in a proteasomedependent manner. An hTERT mutant with all five lysine residues at the N-terminus of hTERT that mutated to arginine became resistant to Hdm2-mediated ubiquitination and degradation. In U2OS cells, depletion of Hdm2 or addition of the Hdm2-resistant hTERT mutant strengthened the cellular protective effects against apoptosis. Similar results were obtained with the Hdm2-stable H1299 cell line. These observations indicate that Hdm2 is an E3 ligase of hTERT.

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Section: Immunoprecipitation and Western Blot Analysesmentioning

confidence: 99%

Hdm2 negatively regulates telomerase activity by functioning as an E3 ligase of hTERT

Lee

et al. 2010

Oncogene

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“…The PCR products were resolved by 12.5% non-denaturing polyacrylamide gel electrophoresis in 0.5ϫ TBE. Silver staining was carried out as previously described (Dalla Torre et al, 2002). Positive control was purchased from Chemicon International (Temicula, CA).…”

Section: Biochemical Analysesmentioning

confidence: 99%

PML-IV functions as a negative regulator of telomerase by interacting with TERT

Ghim

Lee

et al. 2009

Journal of Cell Science

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Maintaining proper telomere length requires the presence of the telomerase enzyme. Here we show that telomerase reverse transcriptase (TERT), a catalytic component of telomerase, is recruited to promyelocytic leukemia (PML) nuclear bodies through its interaction with PML-IV. Treatment of interferon-α (IFNα) in H1299 cells resulted in the increase of PML proteins with a concurrent decrease of telomerase activity, as previously reported. PML depletion, however, stimulated telomerase activity that had been inhibited by IFNα with no changes in TERT mRNA levels. Upon treatment with IFNα, exogenous TERT localized to PML nuclear bodies and binding between TERT and PML increased. Immunoprecipitation and immunofluorescence analyses showed that TERT specifically bound to PML-IV. Residues 553-633 of the C-terminal region of PML-IV were required for its interaction with the TERT region spanning residues 1-350 and 595-946. The expression of PML-IV and its deletion mutant, 553-633, suppressed intrinsic telomerase activity in H1299. TERT-mediated immunoprecipitation of PML or the 553-633 fragment demonstrated that these interactions inhibited telomerase activity. H1299 cell lines stably expressing PML-IV displayed decreased telomerase activity with no change of TERT mRNA levels. Accordingly, telomere length of PML-IV stable cell lines was shortened. These results indicate that PML-IV is a negative regulator of telomerase in the post-translational state.

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“…Several developed quantitative assays with the TRAP assay as a foundation [19,20] required use of harmful radioactive or expensive fluorescent substances. There are a number of reports overcoming those drawbacks by incorporating silver staining techniques with TRAP [21][22][23] , however they have the disadvantage of being quite time consuming and laborious. Hereby, it is represented that the modified assay overcomes those drawbacks with use of less equipment and chemicals, and shortens assay time.…”

mentioning

confidence: 99%

Effect of Doxorubicin on Telomerase Activity and Apoptotic Gene Expression in Doxorubicin-Resistant and -Sensitive MCF-7 Cells

Eskiocak

İşeri²,

Kars³

et al. 2008

Chemotherapy

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Background: Dose- and time-dependent effects of doxorubicin on telomerase activity (TA) and expression levels of hTERT, Bcl-2, Bcl-x_L and Bax were investigated in doxorubicin-resistant and -sensitive MCF-7 cells. Methods: Doxorubicin-resistant MCF-7/R was developed from sensitive MCF-7 breast carcinoma cell line and acquired resistance was demonstrated by XTT and mRNA analysis of MDR1 and MRP1 genes. Expression levels were determined by RT-PCR. Newly developed rapid and simple TRAP-silver staining assay was used to assess TA levels. Results: Doxorubicin-selected MCF-7 cells were 107-fold resistant to the drug and overexpress MDR1 and MRP1 genes. 72 h doxorubicin incubation caused a decrease in TA in parallel with a small decrease in hTERT level in both sensitive and resistant cells. Bcl-2 expression level decreased upon doxorubicin application in sensitive cells. However, the Bcl-x_Llevel increased in sensitive cells after 72 h of doxorubicin incubation. Conclusion: This report demonstrates the inhibitory effects of doxorubicin on TA in both resistant and sensitive MCF-7 cells possibly through modulation of the apoptotic pathway genes.

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TRAP-silver staining, a highly sensitive assay for measuring telomerase activity in tumor tissue and cell lines

Cited by 24 publications

References 8 publications

Hdm2 negatively regulates telomerase activity by functioning as an E3 ligase of hTERT

Hdm2 negatively regulates telomerase activity by functioning as an E3 ligase of hTERT

PML-IV functions as a negative regulator of telomerase by interacting with TERT

Effect of Doxorubicin on Telomerase Activity and Apoptotic Gene Expression in Doxorubicin-Resistant and -Sensitive MCF-7 Cells

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