Methylation Status and Expression of Human Telomerase Reverse Transcriptase mRNA in Relation to Hypermethylation of the p16 gene in Colorectal Cancers as Analyzed by Bisulfite PCR-SSCP

Nomoto, Kiyoaki

doi:10.1093/jjco/hyf001

Cited by 40 publications

(33 citation statements)

References 17 publications

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“…This leads to progression into the S phase (31). Studies have indicated that telomerase activity is correlated with high expression of cyclin D and low expression of p16INK4 (32)(33)(34). In the present study, inhibition of hTERT expression significantly altered the cell cycle profile of H1299 cells; the proportion of cells in G1 was increased (to 73.9%), while the percentage of cells in S and G2 phases was decreased.…”

Section: Discussionsupporting

confidence: 41%

Effect of plasmid-mediated RNA interference targeting telomerase reverse transcriptase on lung cancer cells

Deng

2011

Oncol Rep

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Abstract. In the present study, a plasmid-mediated siRNA interference vector targeting the hTERT gene was constructed and stably transfected into H1299 lung cancer cells. Using realtime quantitative fluorescent PCR technology, Western blotting and flow cytometry-based cell cycle profiling, the silencing effect of this vector and its inhibitory effect on proliferation in lung cancer cells were explored. Based upon the results of our previous study, a pair of siRNA sequences was selected, and a DNA template primer was designed and synthesized. After cloning of the template primer into the promoter of the pGenesil-1.1 expression vector, the constructed interference vector was validated using enzyme digestion and gene sequencing. The recombinant interference vector and empty vector were separately transfected into H1299 lung cancer cells with cationic liposomes, and stable monoclonally transfected cells were obtained after selection with G418. After stable transfection, hTERT mRNA and protein expression levels were detected using real-time RT-PCR technology and Western blotting. Using the MTT method and a colony formation assay, the growth and proliferation of the stably transfected lung cancer cells were determined. Changes in the cell cycle profile of the stably transfected lung cancer cells were detected using flow cytometry. An interference vector targeting the hTERT gene (pGenesil.1-hTERT) was successfully constructed. Enzyme digestion and gene sequencing confirmed that the sequence insertion met the criteria of the design. After transfection of H1299 cells with pGenesil

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Section: Discussionsupporting

confidence: 41%

Effect of plasmid-mediated RNA interference targeting telomerase reverse transcriptase on lung cancer cells

Deng

2011

Oncol Rep

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“…In body fluids, including CSF, proliferating lymphocytes can be the cause of hTERT expression (16)(17)(18), which limits its use as a diagnostic tool in cancer. In contrast, hTERT promoter methylation is correlated with hTERT expression in most telomerase-positive tumors (19)(20)(21)(22), whereas the promoter is unmethylated in telomerase-positive-activated lymphocytes (23). Therefore, the use of hTERT methylation as a cancer biomarker might circumvent the confounding effect of activated lymphocytes.…”

Section: Introductionmentioning

confidence: 99%

Methylation of the hTERT Promoter: A Novel Cancer Biomarker for Leptomeningeal Metastasis Detection in Cerebrospinal Fluids

Bougel

Lhermitte

Gallagher

et al. 2013

Clinical Cancer Research

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Purpose: The diagnosis of leptomeningeal metastases is usually confirmed by the finding of malignant cells by cytologic examination in the cerebrospinal fluid (CSF). More sensitive and specific cancer biomarkers may improve the detection of tumor cells in the CSF. Promoter methylation of the human telomerase reverse transcriptase (hTERT) gene characterizes most cancer cells. The aim of this study was to develop a sensitive method to detect hTERT methylation and to explore its use as a cancer biomarker in CSF.Experimental Design: In 77 CSF specimens from 67 patients, hTERT promoter methylation was evaluated using real-time methylation-sensitive high-resolution melting (MS-HRM) and real-time TaqMan PCR and MS-HRM in a single-tube assay.Results: Real-time MS-HRM assay was able to detect down to 1% hTERT-methylated DNA in a background of unmethylated DNA. PCR products were obtained from 90% (69/77) of CSF samples. No false positive hTERT was detected in the 21 non-neoplastic control cases, given to the method a specificity of 100%. The sensitivity of the real-time MS-HRM compared with the cytologic gold standard analysis was of 92% (11/12). Twenty-six CSFs from 22 patients with an hTERT-methylated primary tumor showed cytologic results suspicious for malignancy; in 17 (65%) of them, a diagnosis of leptomeningeal metastases could be confirmed by the hTERT methylation test.Conclusion: The hTERT real-time MS-HRM approach is fast, specific, sensitive, and could therefore become a valuable tool for diagnosis of leptomeningeal metastases as an adjunct to the traditional examination of CSF.

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“…Hypermethylation of hTERT was found to be necessary for its expression in cancer cells (3,8). The apparent opposition to the classic model of regulation was recently clarified by the observation that methylation plays a dual role (9,10).…”

Section: Introductionmentioning

confidence: 99%

hTERT methylation is necessary but not sufficient for telomerase activity in colorectal cells

Valls¹,

Bougel²,

Piñol-Felis³

et al. 2011

Oncology Letters

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Abstract. Colorectal cancers exhibit a high telomerase activity, usually correlated with the hypermethylation of the promoter of its hTERT catalytic subunit. Although telomerase is not expressed in normal tissue, certain proliferative somatic cells such as intestinal crypt cells have demonstrated telomerase activity. The aim of this study was to determine whether a correlation exists between telomerase activity, levels of hTERT methylation and telomere length in tumoral and normal colorectal tissues. Tumor, transitional and normal tissues were obtained from 11 patients with a colorectal cancer. After bisulfite modification of genomic DNA, hTERT promoter methylation was analyzed by methylation-sensitive single-strand conformation analysis (MS-SSCA). Telomerase activity and telomere length were measured by a fluorescenttelomeric repeat amplification protocol assay and by Southern blotting, respectively. A significant increase of hTERT methylation and telomerase activity, and a reduction of the mean telomere length were observed in the tumor tissues compared to the transitional and normal mucosa. In the transitional and normal mucosa, telomerase activity was significantly lower than that in tumor tissues, even with high levels of hTERT methylation. Nevertheless, hTERT promoter methylation was not linearly correlated to telomerase activity. These data indicate that hTERT promoter methylation is a necessary event for hTERT expression, as is telomerase activity. However, methylation is not sufficient for hTERT activation, particularly in normal colorectal cells.

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Methylation Status and Expression of Human Telomerase Reverse Transcriptase mRNA in Relation to Hypermethylation of the p16 gene in Colorectal Cancers as Analyzed by Bisulfite PCR-SSCP

Abstract: Increased expression of hTERT is related to hypermethylation of hTERT in colorectal cancerous tissues as well as some cancer cell lines and disconcordant with hypermethylation of p16.

Cited by 40 publications

References 17 publications

Effect of plasmid-mediated RNA interference targeting telomerase reverse transcriptase on lung cancer cells

Effect of plasmid-mediated RNA interference targeting telomerase reverse transcriptase on lung cancer cells

Methylation of the hTERT Promoter: A Novel Cancer Biomarker for Leptomeningeal Metastasis Detection in Cerebrospinal Fluids

hTERT methylation is necessary but not sufficient for telomerase activity in colorectal cells

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