Ferrocenylnaphthalene diimide‐based electrochemical hybridization assay via a multi‐electrode chip was applied to detect the methylation frequency in the promoter region of human telomerase reverse transcriptase (hTERT) gene for clinical samples from tissues, local exfoliated oral cells from a lesion, or from entire oral cavity after their methylation specific PCRs. These methylation frequencies were increased with cancer progress as the following order: healthy volunteers, oral leukoplakia as precancerous lesion, and oral squamous cell carcinoma (OSCC). Operating characteristic analysis of the obtained current data doesn't only give excellent discrimination ability of OSCC, but also of oral leukoplakia from healthy volunteers for all samples. Sensitivity and specificity was 95 % and 90 %, respectively, which is a comparable with methods in practical use.
Telomerase activity is present in most cancers and is tightly regulated by the expression of human telomerase reverse transcriptase (hTERT). Hypermethylation in the promoter region of hTERT contributes to the regulation of hTERT expression. In this study, we investigated the methylation and expression of hTERT in oral squamous cell carcinoma (OSCC), oral leukoplakia, and normal oral mucosa. Furthermore, we investigated the significance of hTERT to the clinicopathological findings of OSCC. 35 OSCC, 50 oral leukoplakia (epithelial dysplasia n = 25, squamous cell hyperplasia n = 25), and 10 normal oral mucosa samples were investigated through methylation-specific PCR. Immunohistochemistry was analyzed in 35 OSCC, 50 oral leukoplakia, and 4 normal oral mucosa samples. The methylation and expression of hTERT increased from normal oral mucosa to oral leukoplakia to OSCC. In OSCC, all samples were methylated. However, partial methylation (20%) or unmethylation (80%), but never complete methylation, was observed in normal oral mucosa. Additionally, hTERT expression correlated with cervical lymph node metastasis. These results suggested that the methylation and expression of hTERT is high in oral carcinogenesis and may play an important role in oral cancer. hTERT expression may also be predictive of cervical lymph node metastasis.
Since aberrant methylation at CpG sites is linked to the silencing of tumor suppressor genes, DNA methylation analysis is important for cancer diagnosis. We developed ferrocenylnaphthalene diimide (FND), which has two ferrocenyl moieties at the substituent termini, as an electrochemical indicator for hybridized DNA duplexes. In this study, we attempted to detect aberrant methylation of human telomerase reverse transcriptase gene (hTERT), an efficient cancer marker, using FND-based hybridization coupled with electrochemical detection via a multi-electrode chip.
1IntroductionTe lomeric DNAc onsists of repetitive DNAs equences (TTAGGG)a tt he ends of chromosomes and contributes to genomic stability.T elomerase is ar ibonucleoprotein composed of the catalytic subunit of humant elomerase reverse transcriptase (hTERT)a nd telomerase RNA component and elongates telomeric DNA [ 1].I th as been known that telomerase is involvedi ne stablishing cellular immortalization [2] and is describedt oc ontribute to oncogenic event because of expression of telomerase in cancerc ell. Its activityi so bserved in more than 80 %o f clinicals amples from head and neck squamous cell carcinomas using telomeric repeat amplificationp rotocol (TRAP) assay [3][4][5][6][7]. TRAPw as tested in tissueo r mouthwash from patients carrying leukoplakia as ap recancerous lesion [4,5].M oreover, hTERT mRNAe xpression is elevated in cancerc ells having telomerase activity [6].T herefore,t elomerase activity is expected to be auseful markeri nc ancer detection.Although TRAP assay is conventional method to detect at elomerase activity,t his method requires several tediouss teps and it is difficult to provide objective evaluations of its activity [8].T oo vercome this problem, novel telomerase detecting methods have been developing and these are containing PCR-and/org el electrophoresis-free technique [9].F or example,t elomerase activity detection was monitored by the wavelength change of Surface Plasmon Resonance on the telomerase substrate (TS) primerimmobilizedo ns ilicon microsensing chip before and after telomerase treatment [10].S ince electrochemicalt echnique is expected to provide as imple and rapid method [11],e lectrochemicalt elomerase assay has been studied by severalr esearchers [9,12,13].I th as been reported that electrochemical signal was decreaseda fter telomerase elongation of TS primer-immobilized electrode hybridizedw ith its complementary ferrocenyloligonucleotide [12].T he former or latter techniquep rovides high sensitivity of 10 HeLa cells/mLo r0 .1 HeLa cells/mL( 100 HeLac ells/mL),r espectively.W eh ave been developing an electrochemical telomerase assay( ECTA), which consists TS primer-immobilized electrode coupled with ferrocenylnaphthalene diimide as telomere DNAl igand (Scheme 1) [13].T his method providess imple and rapid screening with the detection limit of 0.4 HeLa cells/mL (10 cells in 25 mLo fs ample solution) [13].E xfoliated cell and tissue fromh ealthy and cancer individuals were tested by ECTAa nd high positive rate of 85 %a nd 90 %, respectively. In clinical site,i ti sr equired the discrimination of cancer patient from patient suffered from oral disease rathert han healthyo ne evaluating by patients with ad isease who test positive and patients without ad isease who test negative.F or example, detectiono fg astric and cervical cancers is generally carried out using gastric photofluorography with barium [14] and cytology [ 15],r espectively.T hese detectionm ethods have ar elativeh igh sensitivity and specificity from 80 %t o9 5%.One benefito fd iagnosing oral lesions f...
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