Rapid quantification of telomerase activity in liver tissue samples to detect human hepatocellular carcinoma

Telomerase, a ribonucleoprotein with reverse transcriptase activity, enables human cells to maintain chromosome stability and to proliferate without limits. Various studies demonstrated telomerase reactivation in human hepatocellular carcinoma (HCC). Therefore, quantification of telomerase activity has been proposed to detect and classify HCC. However, up until now conventional PCR-based assays have demonstrated only limited applicability due to complex and time-consuming post-amplification procedures. In this study, a novel rapid closed-tube system, based on the telomeric repeat amplification protocol (TRAP), has been optimized and evaluated to quantitate telomerase activity in human liver tissue samples. Altogether, 15 liver tissue samples were obtained from the human tissue depository at our institution (4 normal liver, 1 liver adenoma, 8 HCC, and 2 liver metastases) and evaluated following confirmation of RNA integrity by gel analysis. The RNA subcomponent of the telomerase enzyme is essential for telomerase activity and thus crucial to allow valid quantification. For the real-time quantitative TRAP, telomerase-positive 293T cells were employed in a SYBR Green assay as standards to calculate the relative telomerase activity (RTA, mean±SEM) per µg of protein. RNAse- and heat-inactivated samples served as negative controls. Telomerase positive samples were added to heat-inactivated controls to rule out interference by PCR inhibitors. Low RTA levels were detected in normal liver (8,3±3,1) and liver adenoma, whereas HCC samples showed significantly higher levels (88,3±24,9, p=0,015). A tendency for higher RTA levels was observed in G3 (n=3) compared to G2 (n=4) tumors (111,8±39,9 vs. 50,75±31,3). Presence of telomerase activity was confirmed by a conventional polyacrylamid gel. In summary, the real-time quantitative TRAP allows valid quantification of telomerase activity in liver tissue samples and might be employed to assess liver biopsies in patients with suspected HCC.

Key words

TRAP - hepatocellular carcinoma - telomerase