Flow cytometric detection of human telomerase reverse transcriptase (hTERT) expression in a subpopulation of bone marrow cells

Abstract

Telomerase activity has been found in most common cancers, thus indicating that telomerase detection may be a useful marker in cancer diagnosis. The telomeric amplification protocol (TRAP) assay and RT-PCR are customarily used to detect telomerase activity and the expression of the associated genes in cells. However, these methods do not provide any information about telomerase activation at an individual cell level. To analyze cells separately, those cells have to be isolated by sometimes complicated method. The immunohistochemical detection of human telomerase reverse transcriptase (hTERT) is useful to detect telomerase positive cells in a background of non-cancerous cells. A method has been developed for the detection of intranuclear hTERT protein, in a subpopulation of hematopoietic cells, using concurrent staining of a cell surface antigen and multicolor flow cytometry. Only mouse monoclonal anti-hTERT antibody demonstrated the specific positivity in immunocytochemistry and immunofluorescent flow cytometry. Human leukemia and myeloma cell lines showed 100% positivity, whereas normal neutrophils showed 0% positivity. hTERT expression was analyzed in hematopoietic precursor cells of bone marrow samples using concurrent staining of surface CD34 antigen and intracellular hTERT protein and multi-parameter flow cytometry. CD34 positive cells demonstrated higher expression of hTERT than CD34 negative cells. A quick, easy and sensitive assay for determining the hTERT protein expression has been developed. Using this method and the multi-parameter nature of flow cytometry and its ability to identify cellular subpopulations will provide a better understanding of the mechanisms regarding the activation of telomerase.