(right) Graph represents the percentage of cells that stained positive for \galactosidase activity (stained blue\green in left panel). of senescent cells over time can become deleterious leading to diseases and physiological decline. Our data reveal a novel interplay between senescence and the stress response that affects both the progression of senescence and the behavior of senescent cells. We show that constitutive exposure to stress induces the formation of stress granules (SGs) in proliferative and presenescent cells, but not in fully senescent cells. Stress granule assembly alone is sufficient to decrease the number of senescent cells without affecting the expression of bona fide senescence markers. SG\mediated inhibition of senescence is associated with the recruitment of the plasminogen activator inhibitor\1 (PAI\1), a known promoter of senescence, to these entities. PAI\1 localization to SGs increases the translocation of cyclin D1 to the nucleus, promotes RB phosphorylation, and maintains a proliferative, non\senescent state. Together, our data indicate that SGs may be targets of intervention to modulate senescence in order to impair or prevent its deleterious effects. senescence modulators. In addition, this AS\mediated effect was not due to the activation of apoptosis in these cells, since no caspase\3 cleavage products, a well\established marker of apoptosis\induced cell death 45, was detected at any stages of the senescence process in either the presence or the absence of AS (Fig?1C). While these observations clearly show that AS, a well\known promoter of oxidative stress, interferes with the commitment of cells to the senescence process, the mechanisms behind this effect remains unknown. Open in a separate window Figure 1 Repeated exposure to arsenite decreases the number of cells which commit to the senescence process (left) IDH4 cells were treated daily post\induction of senescence for 30?min with (AS) or without (UNT) 0.5?mM sodium arsenite. Proliferating (PRO, Days 0C3), presenescent (PRE, days 4C6) and senescent (SEN, days 7C10) cells were subsequently subjected to staining for \galactosidase activity. Phase contrast images demonstrate the \galactosidase staining of the IDH4 cells at the various stages CP-690550 (Tofacitinib citrate) of the senescence process. Scale bars, 400?m. (right) Graph represents the percentage of cells that stained positive for \galactosidase activity (stained blue\green) in the phase contrast images shown in (left panel). The percentage of senescent cells in each experiment was calculated using three random fields. Data are represented as a mean of three independent experiments??SE (error bars). *inhibitor of SG formation 20, 32, 50, 51. Given that the doses of CHX previously used to prevent SG formation (~100?g/ml) also affect general translation to levels that are similar to AS treatment (Fig?2B) 32, 49, 51, 52, 53, we first determined the minimum dose of CHX that could prevent SG assembly without affecting the levels of newly synthesized proteins. We observed that 0.5?g/ml of CHX for 30?min was sufficient to prevent both the formation CP-690550 (Tofacitinib citrate) of SGs (Fig?4A) and the AS\mediated impairment of senescence in human fibroblasts (Fig?4B and Appendix?Fig S6). However, treatment of fibroblasts with puromycin (Puro), an inhibitor of translation elongation that has no impact on the assembly of AS\induced SGs 54, 55, was not able to prevent SG formation nor Rabbit Polyclonal to DOK5 did it rescue the effect of AS on senescence (Appendix?Fig S7). To further confirm the role of SGs in the CP-690550 (Tofacitinib citrate) inhibition of senescence, we exposed human fibroblasts at the PRO stage of senescence to a single dose of pateamine A (PatA), a natural compound that was previously shown to trigger SG assembly independently of eIF2 phosphorylation 51, 56. Interestingly, this single dose of PatA not only led to sustained formation of SGs throughout the 3?days of the PRO phase (Fig?5A), but it also caused the same impairment in senescence that was seen with repeated exposure to AS. The PatA\mediated inhibition of senescence was also reversed by 0.5?g/ml CHX (Fig?5B). Open in a separate window Figure 3 Knockdown of G3BP1 prevents the assembly of stress granules and the arsenite\mediated effect of SGs on senescence (left) Proliferating IDH4 cells were transfected with a control (Ctl) or a G3BP1\specific siRNA, and senescence was induced 24?h post\transfection. Western blots were performed using whole\cell extracts from PRO, PRE, and SEN cells, and antibodies specific for G3BP1 and tubulin (loading control) proteins. (right) ImageJ was used to quantify the levels of G3BP1 protein, which were normalized to those of tubulin protein. The graph represents the normalized G3BP1 protein levels in the siG3BP1 condition relative to siCtl in PRO, PRE, and SEN cells. (left) IDH4 cells, transfected.

(right) Graph represents the percentage of cells that stained positive for \galactosidase activity (stained blue\green in left panel)