Received on May 3, 2006
Accepted on October 24, 2006

Cancer Diagnostics

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

¹ Laboratory of Analytical Chemistry, University of Athens, Athens, Greece
² "Sotiria" General Hospital for Chest Diseases, Athens, Greece
³ Department of Pathology, Onassis Cardiac Surgery Center, Athens, Greece

^* To whom correspondence should be addressed. E-mail: lianidou{at}chem.uoa.gr.

Background: We developed and validated a real-time reverse transcription (RT)-PCR for the quantification of 4 individual human telomerase reverse transcriptase (TERT) splice variants (++, -+, +-, --) in tumor cell lines and non-small cell lung cancer (NSCLC).

Methods: We used in silico designed primers and a common TaqMan probe for highly specific amplification of each TERT splice variant, PCR transcript-specific DNA external standards as calibrators, and the MCF-7 cell line for the development and validation of the method. We then quantified TERT splice variants in 6 tumor cell lines and telomerase activity and TERT splice variant expression in cancerous and paired noncancerous tissue samples from 28 NSCLC patients.

Results: In most tumor cell lines, we observed little variation in the proportion of TERT splice variants. The +- splice variant showed the highest expression and -+ and -- the lowest. Quantification of the 4 TERT splice variants in NSCLC and surrounding nonneoplastic tissues showed the highest expression percentage for the +- variant in both NSCLC and adjacent nonneoplastic tissue samples, followed by ++, with the -+ and -- splice variants having the lowest expression. In the NSCLC tumors, the ++ variant had higher expression than other splice variants, and its expression correlated with telomerase activity, overall survival, and disease-free survival.

Conclusions: Real-time RT-PCR quantification is a specific, sensitive, and rapid method that can elucidate the biological role of TERT splice variants in tumor development and progression. Our results suggest that the expression of the TERT ++ splice variant may be an independent negative prognostic factor for NSCLC patients.