Abstract
Telomerase is a telomere-specific DNA polymerase consisting of protein and RNA components, which is activated in germline cells and the majority of cancers and serves to counter the consequences of telomere shortening. Recently, it has been reported that telomerase activity level was correlated with the progression and prognosis of hematopoietic malignancies, thus, it is important to analyze telomerase activity. The protein component, hTERT, is believed to be the catalytic subunit of human telomerase and its expression at the mRNA level correlates well with telomerase activity in vitro. Current techniques for assaying telomerase activity detect only the mean activity in a sample and unable to isolate specific cell sub-populations. We developed immuno-fluorescence flow cytometry based assay to detect hTERT expression using a monoclonal antibody. This method allows sub-population of cells to be separated according to hTERT expression more easily, combining antibody against cell surface antigen. Expression level of hTERT in T cell leukemia cell line Jurkat and myeloma cell line RPMI8226, KMS12PE, KMS28PE, were very high and well correlated in between this method and real time quantitative PCR. Human mature granulocytes supposed to have no telomerase activity did not show any hTERT expression in this flow-cytometric assay. Immuno-histochemistry demonstrated the specific nuclear expression of hTERT. Bone marrow samples were obtained from 8 MDS patients (5 RA, 1 RAEB, 2 RAEB-t) and analyzed hTERT isolating MDS blast cells. In the blast population, cells expressing hTERT were 33.1% (21.4–40.3) in RA patient sample, while those in RAEB and RAEB-t patients sample were significantly higher at 75.1% (68.2–81.9). When CD34 positive hematopoietic stem cells were analyzed, the cells expressing hTERT were higher in RAEB and RAEB-t than in RA. Mature granulocyte population in bone marrow cells did not show any positivity. Our result suggests that hTERT expression and telomerase activity in the MDS blasts or CD34 positive stem cells were up-regulated during the disease progression and the high expression level of hTERT and telomerase activity were not due to the blast expansion. This technique allows us to easily analyze hTERT expression in hematopoietic malignancy even though the malignant cells were very small in bone marrow sample, e.g. MDS, MM or MGUS of which telomerase activity were supposed to be correlated with the disease progression and prognosis.
Apoptotic endonuclease EndoG regulates alternative splicing of human telomerase catalytic subunit hTERT
