Saquinavir AZD1480 mw treatment was able to increase the binding of nuclear proteins to the E-Box sequence (Figure 3A). Pooled data from 3 different experiments confirmed the positive modulation Nutlin-3a of saquinavir on the binding to the E-Box portion of hTERT promoter (Figure 3B).

To explore the role of c-Myc in saquinavir-mediated up-regulation of hTERT transcription, we analyzed the effect of the protease inhibitor on the expression and cellular distribution of the oncogene product, principal responsible for hTERT gene transcription. We found that saquinavir increased c-Myc expression in the nuclei of saquinavir-treated Jurkat cells (Figure 3C). This observation strongly supports a role for this transcription factor in saquinavir mediated up-regulation of hTERT levels and telomerase activity in Jurkat cells. Results relative to 3 separate experiments are shown in Figure 3D. Figure 3 Role of c-Myc in saquinavir activity. A. Representative gel showing the binding of nuclear extracts of Selleck PCI 32765 Jurkat cells to the oligonucleotide 5’- TCCTGCTGCGCACGTGGGAAGCCCT-3’, containing the downstream “CACGTG” E-Box sequence localized at position −34 of hTERT promoter, 24 h following exposure to saquinavir determined using EMSA. Saquinavir up-regulates the binding of nuclear proteins to the E-Box sequence.

B. Graph shows the mean ± SD of the OD obtained from 3 EMSA independent experiments. C. Representative experiment showing the effect of saquinavir on c-Myc transcription factor expression tested on nuclear and cytoplasmic extracts of 2×106 viable Jurkat cells after 24 h of treatment (Western Blot). Quality of nuclear

extracts was tested using anti Histone H1 Ab. D. Graphs show the mean ± SD of c-Myc OD values obtained from 3 experiments of and all p values were calculated using Student’s t-test. E. Representative experiment showing the role of c-Myc on saquinavir-mediated hTERT up-regulation. Jurkat cells were transfected AMP deaminase with siRNA targeting c-Myc mRNA as described in Material and Methods. c-Myc silencing induces marked down-regulation of c-Myc protein and hTERT which is a target of the transcriptional factor. Saquinavir restores c-Myc and hTERT expression to control levels. F. Pooled results relative to 2 separate experiments of c-Myc silencing. Asterisk indicates p < 0.05. Role of c-Myc in saquinavir-induced hTERT up-regulation In order to better understand the role of c-Myc in the observed saquinavir-induced hTERT up-regulation, we transfected transiently Jurkat cells with siRNA targeting c-Myc mRNA. The results shown in Figure 3E point out that a marked down-regulation of c-Myc protein occurred in c-Myc silenced cells.