Improved TRAP-silver staining versus conventional radioactive TRAP assays: quantification of telomerase activity during immortalization and in pathological human endometrium.
OBJECTIVE
To develop a sensitive telomeric repeat amplification protocol (TRAP)-silver staining assay for telomerase activity quantification.


METHODS
TRAP assays were performed by using a TRAPeze telomerase kit with or without [alpha-32P]-dCTP. Amplification products were electrophoresed in polyacrylamide gels and detected by autoradiography or a modified silver staining protocol. Telomerase activity was quantified from radioactive counts or optical density of telomerase products from test extracts and controls.


RESULTS
TRAP-silver staining assay was at least as sensitive as radioactive TRAP assay and quantified telomerase activity within linearity from 10 to 3,000 cell equivalents. Both methods quantified a weak telomerase activity in normal endometrial glandular epithelial cells (GEC) and a strong increase in immortalized GEC. In human pathologic endometria (n=24), telomerase activity was correlated with lesion seriousness and distinguished simple hyperplasias from nonhyperplasic or cancerous lesions.


CONCLUSIONS
TRAP-silver staining assay is suitable for cell and tissue telomerase activity routine quantification.
Nemos
C
C
Laboratoire de Biochimie Biologie Moléculaire, Université de Franche-Comté, UFR Sciences et Techniques, Besançon, France.
Rémy-Martin
J P
JP
Adami
P
P
Arbez-Gindre
F
F
Schaal
J P
JP
Jouvenot
M
M
Delage-Mourroux
R
R
D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000818:Animals; D004717:Endometrium / Q000737:chemistry* / Q000201:enzymology* / Q000473:pathology; D005260:Female; D006168:Guinea Pigs; D006367:HeLa Cells; D006801:Humans; D008875:Middle Aged; D016622:Silver Staining / Q000379:methods*; D019098:Telomerase / Q000032:analysis* / Q000378:metabolism*
D019098:Telomerase