RESEARCH AND METHODOLOGY ...
The identification of C0057684 an inducer of hTERT Expression and Telomerase Activity
Summary of Laboratory Practice
The long term goal of Sierra Sciences is to find a compound that induces hTERT expression and telomerase activity for basic research purposes as well as anti-aging therapeutic applications. As a result of our cell-based high throughput screening effort utilizing a transient hTERT minimal promoter and luciferase reporter expression system, 1,040 compounds from a diversified small molecule library of over 130,000 compounds were found to induce luciferase expression.
Cells treated with the active compounds from our primary screen are further being examined for hTERT mRNA expression through real-time RT-PCR. While approximately 80% of these active compounds are still under evaluation in cell-based assays, we have identified C0057684 as an inducer of hTERT mRNA expression in several cell lines.
Furthermore, C0057684 treated cells were evaluated for telomerase enzyme activity through the Telomere Repeat Amplification Protocol (TRAP). In a series of such studies, C0057684 was able to consistently induce hTERT expression and telomerase activity. Further investigation of C0057684 and its analogs is currently in progress at Sierra Sciences.
Materials and Methods
A plasmid containing the hTERT minimal promoter driving expression of the luc2 reporter gene was transfected, on a large scale, into MRC5 cells using Lipofectamine 2000 reagent (Invitrogen) and then plated in 96-well plate format. Small molecule compounds (ChemDiv) were then added to transfected cells. Luciferase expression was detected using CellTiter-Glo (Promega). Real-Time RT-PCR Total RNA was isolated using PureLink Micro-to-Midi Total RNA Purification kit (Invitrogen).
Researchers use PCR Analysis to analyze the efficacy
of Telomere Lengthening Methods.
The Polymerase Chain Reaction
The Polymerase Chain Reaction is an elaborate technique for identifying a specific sequence of DNA. It works by generating millions of copies of a "suspect" sequence of DNA using thermal techniques. From here, scientists can observe, through UV spectroscopy, an amplified sequence of DNA
Summary
C0057684, identified through a transient expression system using the hTERT minimal promoter expressing luciferase, induces endogenous hTERT mRNA transcription leading to functional telomerase activity. Since expression of hTERT and telomere length are the rate-limiting factors in replicative life span (1), the results of these initial experiments illustrate why compound C0057684 is of significant interest. While the mechanism of action of C0057684-induced telomerase expression remains unknown, research is currently in progress to understand its influence on cellular activity.
Acknowledgements
Special thanks to Richard Offerdahl and Pierluigi Zappacosta for their support of our research. We also want to thank our Scientific Advisory Board and consultants for their invaluable advice and discussions. And finally, cheers to the entire Sierra Sciences staff for all your hard work.