No abstract

The sensitive telomeric repeat amplification protocol (TRAP) permits telomerase detection in mammalian cell and tissue extracts with very low telomerase activity levels. Unfortunately, conventional TRAP assays require complex post-amplification procedures, such as polyacrylamide gel electrophoresis and densitometry, to measure telomerase products. Therefore, a real-time quantitative TRAP assay (RQ-TRAP) was optimized in the present study and evaluated in comparison with a commercially available quantitative TRAP kit and by monitoring telomerase activity in human hepatocyte cultures, human hepatoma cell lines and telomerase reconstitution experiments. The novel real-time telomerase detection method has many advantages. Other than sample extraction and real-time cycling, no additional time-consuming steps have to be performed for telomerase quantification; reliable and linear telomerase quantification is possible down to single-cell dilutions without the interference of primer-dimer artifacts, and the costs are less. Moreover, the precision is similar to other amplification-based telomerase quantification assays and the results are comparable to data obtained with two commercially available assays. The closed-tube system reduces the risk of carryover contamination and supports high throughput. In conclusion, RQ-TRAP provides a new tool for the rapid and reliable quantification of telomerase activity.

Currently, different techniques to expand human hepatocytes in vitro are being investigated to generate enough cells for liver-directed cell therapies. However, based on observations in fibroblasts and other cell types, telomere attrition limits the proliferative capacity of normal somatic cells. Therefore, we explored whether telomere-dependent replicative aging restricts the in vitro proliferation of human hepatocytes. Subpopulations of cells isolated from a neonatal liver and characterized as hepatocyte derived by RT-PCR and flow cytometry started to proliferate 5-7 days after plating and were termed proliferating human hepatocytes (PHH). Following retroviral-mediated transduction of the catalytic telomerase subunit, telomerase reverse transcriptase (hTERT), telomerase activity increased from almost undetectable levels to levels as high as in HepG2 and other telomerase-positive cell lines. As expected, untransduced PHH progressively lost telomeric repeats and arrested after 30-35 cell divisions with telomeres of less than 5 kilo bases. In comparison, telomerasereconstituted PHH maintained elongated telomeres and continued to proliferate as shown by colorimetric assays and cell counts. In this study, telomere stabilization extended the proliferative capacity of in vitro proliferating human neonatal hepatocytes. Therefore, telomere attrition needs to be addressed when developing techniques to expand human hepatocytes.