05. Results Effect of saquinavir #Selleck GDC-0449 randurls[1|1|,|CHEM1|]# on “in vitro” Jurkat cell growth Saquinavir has shown dose- and time-related anti-proliferative and pro-apoptotic effects on different tumors [3, 4]. Graded concentrations of saquinavir (from 3.75 to 15 μM) were added to Jurkat cell suspension as described in Material and Methods. The effect of saquinavir on Jurkat cell growth has been evaluated using the MTT assay, performed after 96 h of incubation with the antiretroviral agent. The results obtained from 3 pooled independent experiments and shown in Figure 1A, indicate that the IC 50 was 17.36 μM, with a confidence interval corresponding to 8.93 and 25.79 μM. 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 PFT�� 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. Influence of saquinavir on telomerase activity of Jurkat cell line Telomerase is a specialized RNA template-containing reverse transcriptase able to compensate for telomeric loss occurring at each cell replication, which is reactivated in tumor cells . In previous studies we found that saquinavir was able to increase telomerase in T cells [8, 9]. Here we analyzed the effect of saquinavir DOK2 on telomerase activity of Jurkat cells after 24, 48 and 72 h of treatment.
Based on the results obtained in terms of cell growth inhibition, we decided to use the concentration of 15 μM of the agent throughout the next steps of our study. We found that the protease inhibitor was able to induce up-regulation of telomerase activity, from 24 h to 72 h of cell exposure (Figure 1). Similar results were obtained by pooling data obtained from 3 independent experiments in correspondence of all analyzed time intervals (Figure 1B). Influence of saquinavir on telomerase catalytic subunit hTERT expression A major mechanism regulating telomerase activity in human cells is transcriptional control of the telomerase catalytic subunit gene, hTERT . Several transcription factors, including oncogene products (e.g. c-Myc) and tumor suppressor gene products (e.g.