Immunodetection of human telomerase reverse-transcriptase (hTERT) re-appraised: nucleolin and telomerase cross paths

Abstract

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.

Quantitation of HER2 and telomerase biomarkers in solid tumors with IgY antibodies and nanocrystal detection

“…22 Some prior IHC studies have used a telomerase antibody that detects nucleolin, not telomerase. 7 Although not conclusive, the trends in the present study warrant further investigation of a quantitative relationship between IHCdetected telomerase and clinical status of prostate cancer patients and their tumors.…”

“…2,3 Analytical and clinical validation of cancer biomarkers has suffered from bias in the design, conduct and interpretation of such research, 4 incompletely validated imaging, 5 and lack of affinity standards 6 and antibodies that did not, in fact, detect correct targets. 7 Here, we describe a novel approach to cell-based bioimaging with relative quantitation for biomarker validation. We report characterization of two new IgY antibodies for quantitation of model cancer biomarker systems, HER2 and telomerase, 8 and explore analytical improvements, including low cross-reactivity IgY-isotype chicken polyclonal antibodies raised against recombinant polypeptides; digital quantification of antibody signals with streptavidin-conjugated semiconductor nanocrystals to obviate photobleaching of organic fluorescent dyes; complete z-plane fluorescence image capture using 3D-deconvolution microscopy; high-throughput, automated, robotic slide processing; and quantitative, massively parallel, high-throughput analysis of peptide antigen-antibody interactions by layered peptide array (LPA) technology.…”

Pediatric Ependymoma: Biological Perspectives

“…However, re-evaluation of the antibody (NCL-hTERT) used to detect hTERT in this study has verified the actual target to be Nucleolin, a phosphoprotein that acts as a chaperone for hTERT during its transport from cytoplasm to nucleolus (136).…”

Telomerase stimulates ribosomal DNA transcription under hyperproliferative conditions

“…These results demonstrated that TERT binds the active fraction of the rDNA. Moreover, precipitating chromatin from nontransfected HEK cells with an established commercially available anti-TERT antibody 11 revealed that endogenous TERT binds the rDNA promoter and internal gene sequences (Fig. 1c).…”