Rapid, quantitative nonisotopic assay for telomerase activity in human tumors.
Telomerase is a ribonucleoprotein enzyme that adds TTAGGG repeats onto human telomeres, preventing their shortening. The activation of this enzyme is an important step in cell immortalization and carcinogenesis and seems to represent a new and promising marker in cancer diagnosis and management. Telomerase activity is usually detected in cellular protein extract by the telomeric repeat amplification protocol (TRAP) assay, which can provide only a qualitative (presence/absence) evaluation. Here we present a modification of this method that can provide quantitative information without requiring time-consuming post-PCR procedures such as gel electrophoresis with radioactive materials and autoradiography. The detection and measurement of telomerase activity is performed by evaluating the amount of double-stranded DNA generated in the telomerase reaction and PCR amplification, with the use of the sensitive DNA fluorescent dye PicoGreen. In a subset of tumors, the presence of telomerase activity was confirmed by the conventional TRAP assay. By this method we evaluated telomerase activity in unselected groups of breast (n = 15), ovarian (n = 12), endometrial (n = 12), gastric (n = 20), and renal (n = 12) carcinomas, in meningiomas (n = 8), and in pheochromocitomas (n = 10). The results indicate substantial differences of telomerase activity among cancer groups; however, a large variability among patients of the same group is observed. Kidney, ovarian, and breast carcinomas showed the highest mean values (31.8 +/- 28.9, 29.2 +/- 26.7, and 35.3 +/- 15.9 ng DNA/microg protein, respectively, mean +/- SD), whereas gastric and endometrial cancers had a lower activity (17.2 +/- 8.8 and 13.5 +/- 7.9 ng DNA/microg protein, respectively). Very low or no detectable telomerase activity was found in meningiomas (with the exception of one malignant atypical variant) and pheochromocitomas (9.7 +/- 12.9 and 2.8 +/- 2.1 ng DNA/microg protein, respectively). In conclusion, our method seems to be an accurate and reasonable procedure for measuring telomerase activity in human cancers.
Department of Clinical Physiopathology, University of Florence, Italy.
S
S
Gelmini
A
A
Caldini
L
L
Becherini
S
S
Capaccioli
M
M
Pazzagli
C
C
Orlando
D014408:Biomarkers, Tumor / Q000032:analysis*; D004273:DNA, Neoplasm / Q000032:analysis*; D005456:Fluorescent Dyes; D006801:Humans; D007700:Kinetics; D009369:Neoplasms / Q000737:chemistry; D009930:Organic Chemicals; D016133:Polymerase Chain Reaction / Q000379:methods; D013050:Spectrometry, Fluorescence; D019098:Telomerase / Q000032:analysis*; D014407:Tumor Cells, Cultured
D014408:Biomarkers, Tumor; D004273:DNA, Neoplasm; D005456:Fluorescent Dyes; D009930:Organic Chemicals; C099476:PicoGreen; D019098:Telomerase