Establishing cell-based reporter systems for the analysis of hTERT expression.
Telomeres are the protective structures at the end of eukaryotic chromosomes. Telomerase is a ribonucleoprotein that contains both an RNA and a protein component for the maintenance of telomere length. Telomerase activity is detected in the majority of malignant tumors, but not in normal somatic cells, suggesting that telomerase reactivation is a crucial step in cell immortality and carcinogenesis. The mechanism of how telomerase is activated during tumorigenesis remains unclear. However, the expression of the human telomerase reverse transcriptase (hTERT) gene, which encodes the catalytic protein subunit of human telomerase, has been shown to be the major determining factor. To gain insight into the mechanisms regulating hTERT expression and to facilitate the screening of agents that affect hTERT expression, we have established cell-based systems for monitoring hTERT expression. We linked the hTERT promoter to two different reporter genes encoding green fluorescence protein (GFP) and secreted alkaline phosphatase (SEAP), respectively. These constructs were then transfected into H1299 and hTERT-BJ1 cells. Stable clones harboring these DNA constructs were isolated. In these cells, hTERT expression can be monitored through the quantification of GFP or SEAP activity on an automatic plate reader. Using these systems, we have identified several small molecule compounds that affect the expression of telomerase.
Huang
Yi-Yuan
YY
Institute of Biopharmaceutical Science, National Yang-Ming University, Taipei, Taiwan.
Shih
Jing-Wen
JW
Lin
Jing-Jer
JJ
D000469:Alkaline Phosphatase / Q000378:metabolism; D045744:Cell Line, Tumor; D002477:Cells / Q000378:metabolism*; D020869:Gene Expression Profiling / Q000379:methods*; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D008856:Microscopy, Fluorescence; D008967:Molecular Biology / Q000379:methods*; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D019098:Telomerase / Q000235:genetics*
D049452:Green Fluorescent Proteins; C509186:TERT protein, human; D019098:Telomerase; D000469:Alkaline Phosphatase