| Description: |
At both concentrations, everolimus caused a G1 cell cycle arrest in all tested cells and effects were more pronounced in the HTLV‐I‐negative cells CEM and Jurkat .On 20 nM everolimus treatment for 72 hr, the percentage of cycling cells (S + G2 + M) decreased by 55% in CEM and only by 34% in HuT‐102 cells. Everolimus did not cause any increases in the pre‐G1 region, presumably representing apoptotic cells, in all tested cells even at a 2000 nM treatment up to 72 hr. HuT‐102 cells treated with 20 nM everolimus for 4, 8 or 12 days were positively stained with SA‐β‐Gal, a hallmark of senescent cells, as early as 4 days post‐treatment. Indeed, HuT‐102 cells treated with 20 nM everolimus showed 56% and 82% SA‐β‐Gal positivity at 8 and 12 days, respectively. The senescent cells also assumed a characteristic enlarged, granular and flattened appearance in culture (data not shown). HTLV‐I transformed C91 cells were also tested for β‐galactosidase positivity and by day 8 post‐treatment with 20 nM everolimus, 72% of cells were SA‐β‐Gal positive, compared to 14% in control cells . However, MT2 cells were less sensitive to everolimus‐induced senescence as treatment for 12 days with 20 nM everolimus caused 27% SA‐β‐Gal positivity only, which was increased to 90% at day 16. Everolimus‐treated HuT‐102 and MT2 cells exhibited upregulation of p21 starting day 1 while p21 protein levels remained undetectable in HTLV‐I‐negative CEM and Jurkat cells. |