Quantitative relationship between functionally active telomerase and major telomerase components (hTERT and hTR) in acute leukaemia cells

Abstract

Functionally active telomerase is affected at various steps including transcriptional and post-transcriptional levels of major telomerase components (hTR and human telomerase reverse transcriptase (hTERT)). We therefore developed a rapid and sensitive method to quantify hTERT and its splicing variants as well as the hTR by a Taqman real-time reverse transcriptase -polymerase chain reaction to determine whether their altered expression may contribute to telomere attrition in vivo or not. Fresh leukaemia cells obtained from 38 consecutive patients were used in this study. The enzymatic level of telomerase activity measured by TRAP assay was generally associated with the copy numbers of full-length hTERT þ a þ b mRNA (P ¼ 0.0024), but did not correlate with hTR expression (P ¼ 0.6753). In spite of high copy numbers of full-length hTERT mRNA, telomerase activity was low in some cases correlating with low copy numbers of hTR, raising the possibility that alteration of the hTR : hTERT ratio may affect functionally active telomerase activity in vivo. The spliced nonactive hTERT mRNA tends to be lower in patients with high telomerase activity, suggesting that this epiphenomenon may play some role in telomerase regulation. An understanding of the complexities of telomerase gene regulation in biologically heterogeneous leukaemia cells may offer new therapeutic approaches to the treatment of acute leukaemia.

Telomere Targeting with a New G4 Ligand Enhances Radiation-Induced Killing of Human Glioblastoma Cells

“…Quantitative real-time reverse transcriptase PCR (qRT-PCR) was carried out with the LightCycler system (Roche Diagnostics). The primers used for amplification of hTERT, TRF1, and TRF2 (telomeric repeat binding factors 1 and 2) have been described elsewhere (16,17). In addition, the amount of ABL transcripts was quantified in all samples as an internal control to normalize the results (18).…”

“…1D) indicate that this compound has a preference for quadruplex structures. These experiments compare the amount of dye bound to a variety of structures (typically [12][13][14][15][16][17][18][19][20], each trapped in a small dialysis chamber immerged in a large bath containing 1 mmol/L TAC. Binding to the 2 quadruplex samples [22AG and 24G20, corresponding to the human telomeric motif [A(GGGTTA) 3 GGG] and T 2 G 20 T 2 sequence, respectively] was stronger than that for duplexes and DNA and RNA single strands [poly(rA), poly(rU), poly(dT), and poly(dU)] or i-motifs (formed with C-rich sequences).…”

Transcriptional profiling of Epstein–Barr virus (EBV) genes and host cellular genes in nasal NK/T-cell lymphoma and chronic active EBV infection

“…To confirm the results of microarray, we performed RT-PCR by ABI PRISM 7700 Sequence Detection System (Applied Biosystems, Foster City, CA, USA) as reported elsewhere (Ohyashiki et al, 2005b). For relative quantification of EBV gene expression, the level of gene expression was expressed as the percent of those in SNK-6* cells.…”

Real-Time RT-PCR Quantification of Human Telomerase Reverse Transcriptase Splice Variants in Tumor Cell Lines and Non–Small Cell Lung Cancer

“…Alternative splicing of TERT may be a novel mechanism of telomerase regulation (22 ), and TERT mRNA splicing patterns have been studied in immortal human cells (23 ) and benign and malignant breast tumors (24 ) in an attempt to elucidate their biological role. The quantitative relationship between functionally active telomerase and the major telomerase components TERT and hTR in acute leukemia cells was recently studied (25 ). The expression of the ␣-negative splice variant can inhibit telomerase activity (26 ) in telomerasepositive cells and causes telomere shortening and eventually cell death.…”