Immunodetection of human telomerase reverse-transcriptase (hTERT) re-appraised: nucleolin and telomerase cross paths.
The involvement of telomerase in cellular immortalization and senescence has often been assessed by means of telomerase expression at the RNA level and quantification of telomerase activity by the telomeric repeat amplification protocol assay. However, these methods either neglected the existence of various telomerase splice variants, or ignored the nonconventional functions of telomerase independent of its ability to elongate and maintain telomere length. Immunodetection of telomerase is now being recognized as a necessary approach to precisely elucidate its roles in oncogenesis and senescence. A few antibodies directed against the catalytic subunit of the human telomerase (hTERT) are currently used but their specificity is not always demonstrated. A survey of the literature showed inconsistencies and led us to comparatively re-evaluate the most frequently used antibodies. Surprisingly, mass spectrometry, two-dimensional gel analysis and immunofluorescent experiments revealed that the most frequently used hTERT immunoprobe, a mouse monoclonal antibody that was claimed to be directed against an hTERT protein epitope, in fact recognizes nucleolin rather than telomerase. Our findings have interesting implications regarding the biology of nucleolin and telomerase in the context of pathophysiological investigations recently carried out.
Wu
Ying-Li
YL
INSERM U685, Hôpital Saint-Louis, Institut d'Hématologie, 1 avenue Claude Vellefaux, 75010 Paris, France.
Dudognon
Charles
C
Nguyen
Eric
E
Hillion
Josette
J
Pendino
Frédéric
F
Tarkanyi
Ilona
I
Aradi
Janos
J
Lanotte
Michel
M
Tong
Jian-Hua
JH
Chen
Guo-Qiang
GQ
Ségal-Bendirdjian
Evelyne
E
D000906:Antibodies / Q000378:metabolism; D000918:Antibody Specificity; D002454:Cell Differentiation; D002478:Cells, Cultured; D003429:Cross Reactions; D004268:DNA-Binding Proteins / Q000032:analysis* / Q000172:deficiency; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D005455:Fluorescent Antibody Technique; D020869:Gene Expression Profiling; D006367:HeLa Cells; D006801:Humans; D047468:Immunoprecipitation; D013058:Mass Spectrometry; D010449:Peptide Mapping; D010750:Phosphoproteins / Q000032:analysis*; D012333:RNA, Messenger / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000032:analysis*; D019098:Telomerase / Q000032:analysis* / Q000172:deficiency; D000097796:Nucleolin
D000906:Antibodies; D004268:DNA-Binding Proteins; D010750:Phosphoproteins; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D019098:Telomerase; D000097796:Nucleolin