Background: Telomerase is a promising biomarker in cancer diagnosis and therapy. The elongation of telomeric repeats catalyzed by telomerase is accompanied by release of six PPi for each TTAGGG repeat (1 pmol PPi/310 pg telomeric repeats). We developed a novel method to measure telomerase activity by use of an enzymatic luminometric PPi assay (ELIPA). Methods: Extracts of cell lines and tissues were incubated with primer at 30℃ for 30min. Released PPi was converted to ATP by sulfurylase, and ATP was detected by a luciferase bioluminescence system. The ELIPA results were compared with results obtained with the conventional telomeric repeat amplification (TRAP)-ELISA in 42 lung carcinoma tissues and 27 control tissues without malignancy. Results: The lower detection limits of ELIPA and TRAP-ELISA were 5 and 10 cells, respectively. The within-run imprecision (CV) of ELIPA was <12%. When compared with TRAP-ELISA, the correlation coefficient (r) was 0.79. When we used the cutoff value from ROC analysis to distinguish malignant and nonmalignant tissues, the sensitivity and specificity of ELIPA were 83% and 96%, respectively, whereas the sensitivity and specificity of TRAP-ELISA were 71% and 96%, respectively. Conclusion: ELIPA is a simple and sensitive homogeneous method to quantify telomerase activity.

The aromatic hydrocarbon receptor (AhR) is a ligand-activated transcription factor that mediates many of the biological and toxicological effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin) and related chemicals. Here we developed a novel method to detect the presence of AhR ligands using Exonuclease Protection Mediated PCR bioassay (EPM-PCR). This assay measures the ability of a chemical to activate AhR DNA binding in vitro. In the presence of AhR ligand, an expected length PCR product was observed on electrophoresis, but no signal was detected in the absence of ligand. Real-time quantitative PCR was performed to quantifyDNA bound to ligand:AhR complex. We obtained a standard curve with TCDD concentration to bound DNA copies in the range of 0.01pM-10nM of TCDD. Minimal detection limit of the assay was below 0.01pM TCDD, and the whole detection time was less than 5h. In comparison to the chemical-activated luciferase gene expression (CALUX) bioassay, EPM-PCR bioassay is more sensitive and easier to perform. These results suggest that this assay is useful for detection and quantification of TCDD and related AhR ligands in a cell-free system without the use of radioactivity.

Background: Telomerase maintains telomere length and is considered to be necessary for the indefinite proliferation of human cells. Activation of telomerase plays a key role in the malignant transformation process. The aim of this study was to study the regulation of telomerase, and to explore the possibility of telomerase as a biomarker in squamous carcinogenesis of the esophagus. Methods: Twenty-nine esophageal squamous cell carcinomas (ESCC) and its corresponding adjacent normal tissues, and 47 epithelial squamous dysplasia tissues were analyzed by the reverse transcriptase-polymerase chain reaction (RT-PCR) technique for the mRNA expression of three major telomerase subunits: human telomerase RNA (hTR), telomerase protein component 1 (TP1), and human telomerase reverse transcriptase (hTERT) and by telomeric repeat amplification protocol assay (TRAP) for telomerase activity. Results: For the expression of hTR and TP1 mRNA, there were no significant differences among ESCC, dysplasia and normal tissues (P>0.05). In contrast, hTERT mRNA expression was detected in 28 of 29 ESCC (96.6%), in 23 of 47 dysplasia (48.9%), and only in two of 29 normal tissues (7.5%). Telomerase activity was positive in 25 of 29 ESCC (86.2%), in 21 of 47 (44.7%) epithelial dysplasia tissues, and in none of normal tissue. All together, 95 of 105 cases (90.48%) were concordant for both results, i.e., telomerase activity positive and hTERT positive or telomerase activity negative and hTERT negative tissues, and telomerase activity correlated with hTERT mRNA expression (P<0.001). Conclusions: Higher telomerase activity and hTERT mRNA expression were shown during an early stage in the esophageal carcinogenesis. Activation of telomerase activity was strongly correlated with hTERT mRNA expression, suggesting hTERT is a major regulator of telomerase activity, and telomerase activation may play a critical role in esophageal carcinogenesis. Therefore, telomerase, especially hTERT can be used as a potential molecular biomarker of ESCC.

Telomerase is expected to be a new biomarker for cancer diagnosis. The telomeric repeat amplification protocol (TRAP) is a sensitive method to detect telomerase activity. However, TRAP and its modified protocols are not always suitable for measuring telomerase activity of a large number of clinical samples to diagnosis cancer because these methods generally require a time-consuming detection step such as gel electrophoresis. To improve the procedure for mass diagnosis, we applied bioluminescence to replace the detection step. Telomerase activity is measured by evaluating the amount of inorganic pyrophosphate generated in PCR amplification of telomerase elongation product, with use of the sensitive enzymatic luminometric inorganic pyrophosphate detection assay (ELIDA). TRAP connected with ELIDA (TRAP-ELIDA) can quantitatively detect telomerase activity within linearity from 2 to 1000 cell equivalents. The ELIDA signals accorded with results of TRAP-SYBR green staining, and the results of ELIDA were significantly correlated to those of TRAP connected with an enzymelinked immunosorbent assay (TRAP-ELISA) (250.992, P<0.001). TRAP-ELIDA is a simple and sensitive method to quantify telomerase activity without time-consuming gel electrophoresis. Because TRAP-ELIDA measures telomerase activity with a luminometer, it could be applied to a large number of clinical samples at the same time.

Polymorphisms of the nucleotide excision repair gene XPD are candidates for influencing cancer susceptibility. To determine the effect of XPD genetic polymorphisms on the risk of esophageal squamous cell carcinoma (ESCC) and its interaction with carcinogen exposure, XPD polymorphisms at codon 312 (Asp→Asn) and codon 751 (Lys→Gln) were determined in 135 ESCC patients and 152 normal controls. Polymorphism at codon 312 made no contribution to genetic risk for ESCC. Our results showed that there was a significant difference between frequencies for XPD 751 Gln/Gln genotype in ESCC patients (8.9%) and normal cases (1.3%), and that Gln/Gln genotype was associated with an increased risk of ESCC (odds ratio [OR]=6.71; 95% confidence interval [CI]: 1.90-23.73). The results of the logistic regression model showed that XPD 751 Gln/Gln genotype and drinking were candidates for influencing the risk of ESCC. Among smokers, the risk of ESCC in XPD 751 Gln/Gln genotype increased 8-fold than that XPD 751 Lys/Lys genotype (OR=8.42, 95% CI: 1.02-69.58). The results indicated that XPD 751 Gln/Gln genotype may be contributing factors in the risk of ESCC and may modify risk attributable to environmental exposures.