The antiretroviral agent saquinavir enhances hTERT expression and telomerase activity in human T leukaemia cells in vitro

Abstract

Background: Saquinavir, a protease inhibitor utilized in HIV infection, shows antitumor activity in various experimental models. In previous studies performed in our laboratory the drug was found to induce a substantial increase of telomerase activity in normal peripheral blood mononuclear cells. Aim of the present investigation was to test whether saquinavir was able to increase telomerase activity and the expression of the catalytic subunit of telomerase, hTERT, in human malignant hematopoietic cells.

Methods: Human Jurkat CD4+ T cell leukaemia cell line was used throughout the present study. The antiproliferative effect of saquinavir was tested by the MTT assay. Telomerase activity was determined according to the telomeric repeat amplification protocol. The expression of hTERT mRNA was semi-quantitative evaluated by RT-PCR amplification and quantitative Real Time PCR. The binding of the transcription factor c-Myc to its specific E-Box DNA binding-site of hTERT promoter was analyzed by Electophoretic Mobility Shift Assay (EMSA). The amount of c-Myc in cytoplasm and nucleus of leukemia cells was determined by Western Blot analysis, and c-Myc down-regulation was obtained by siRNA transfection.

Results: Saquinavir produced a substantial increase of telomerase activity in Jurkat cells in vitro without increasing but rather reducing target cell proliferation rate. Telomerase up-regulation appeared to be the result of enhanced expression of hTERT. Saquinavir-mediated up-regulation of hTERT gene was the result of the increased binding of proteins to the E-Box sequence of the promoter. Moreover, saquinavir amplified the expression of c-Myc especially in the nuclear cell fraction. The direct influence of saquinavir on this transcription factor was also demonstrated by the antagonistic effect of the drug on siRNA induced c-Myc suppression. Since c-Myc is the main responsible for hTERT transcription, these findings suggest that the main mechanism underlying saquinavir-induced telomerase activation is mediated by c-Myc up-regulation.

Conclusions: Saquinavir augments hTERT expression while inhibiting leukemic cell growth. Experimental evidences show that this effect is mediated by saquinavir-influenced increase of c-Myc levels. This could have relevance in terms of enhanced hTERT-dependent tumor cell immunogenicity and suggests new paharmacological approaches interfering with c-Myc dependent pathways.

Figure 1

Effect of saquinavir on cell growth and telomerase activity. A. After 96 h, of culture MTT assay was performed as described in “Materials and Methods”, on Jurkat cells treated with saquinavir 3.75, 7.5 and 15 μM or DMSO as control. Saquinavir concentration which inhibited significantly cell viability (15 μM, p < 0,005), was close to the IC50 (i.e. 17. 36 μM, see “Results” section). The data are represented as percentage cell viability of the untreated cells. Each bar represents the mean ± SD of determinations from 3 independent experiments. Asterisk indicates p < 0.05. B. Representative blot of telomerase activity (TRAP Assay) of whole cell extracts from 500 viable Jurkat cells determined 24, 48 and 72 h following treatment with saquinavir. Graph shows the mean ± SD of OD obtained from pooled results of the effect of saquinavir (15 μM) on telomerase activity of Jurkat cell line from 3 separate experiments. All p values were calculated using Student’s t-test. Asterisk indicates p < 0.05.

Figure 2

Effect of saquinavir on hTERT expression. A. Representative experiment showing the effect of saquinavir (15 μM) on hTERT expression tested on whole cell extracts from 2×10⁶ viable CD4⁺ Jurkat cells 48 h following treatment (Western Blot). Gel loading control was based on GAPDH expression. Saquinavir increases hTERT levels in Jurkat cells. B. Graph shows the mean ± SD of the ratio hTERT/GAPDH band intensity obtained by pooling the results from 3 independent experiments. C. Representative gel showing the effect of saquinavir on hTERT mRNA in Jurkat cell line, determined after 24 and 48 h of treatment, using RT-PCR. GAPDH was used as internal control. Saquinavir up-regulates hTERT mRNA transcription. D. Graphs show the mean ± SD of OD for 3 independent RT-PCR experiments. E. Effect of saquinavir on hTERT mRNA expression of Jurkat cells 24 hours following treatment analysed by quantitative real-time RT-PCR. Levels of hTERT are normalized against GAPDH housekeeping expression. The graph shows the difference in terms of gene expression working out the Delta Delta CT algorithm between TERT and the housekeeping GAPDH. Data shown are representative of 2 independent experiments. All p values were calculated using one-way paired Student’s t-test. Asterisk indicates p < 0.05.

Figure 3

Role of c-Myc in saquinavir activity. A. Representative gel showing the binding of nuclear extracts of 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 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.