Human telomerase is the subject of intense research because of its critical function in cellular immortality via telomere length maintenance. Presence of telomerase activity is strongly associated with immortality of embryonic stem and cancer cells. The human telomerase complex is composed of the human Telomerase Reverse Transcriptase protein (hTERT) and the human Telomerase RNA (hTR). Expression of telomerase activity is controlled, mainly at the level of hTERT transcription. However, the specific mechanisms of repression or silencing of the hTERT promoter region remain unclear. Therefore, the purpose of our screen is to find small molecules which will relieve repression of the hTERT promoter. This should allow for transcription of hTERT mRNA and consequently the assembly of active telomerase.
Two cell lines were engineered by infecting human fetal lung fibroblasts (MRC5) with lentiviral vectors containing either a mutant hTR (CTAGCG) or a wildtype hTR (TTAGGG) template sequence. Both viral constructs also express an siRNA sequence that inactivates the endogenous wildtype hTR. The hTR sequences contained within the lentiviruses are not affected by the siRNA sequence because they contain two additional base changes around the template region. When hTERT is expressed in mutant hTR cells dysfunctional CTAGCG telomeric repeats are added at the ends of telomeres. The addition of mutated telomeric sequences cause the cells to become unhealthy and inhibit their growth. At the same time, the wildtype hTR cells with induced telomerase activity proliferate as normal. Currently we have screened 49,524 compounds and have detected 618 (1.25%) active compounds (primary hits).