Institute of Cytology, St. Petersburg, Russia.
Cell Cycle. 2012 Jun 15;11(12):2402-7. doi: 10.4161/cc.20882.
The TOR (target of rapamycin) pathway is involved in aging in diverse organisms from yeast to mammals. We have previously demonstrated in human and rodent cells that mTOR converts stress-induced cell cycle arrest to irreversible senescence (geroconversion), whereas rapamycin decelerates or suppresses geroconversion during cell cycle arrest. Here, we investigated whether rapamycin can suppress replicative senescence of rodent cells. Mouse embryonic fibroblasts (MEFs) gradually acquired senescent morphology and ceased proliferation. Rapamycin decreased cellular hypertrophy, and SA-β-Gal staining otherwise developed by 4-6 passages, but it blocked cell proliferation, masking its effects on replicative lifespan. We determined that rapamycin inhibited pS6 at 100-300 pM and inhibited proliferation with IC(50) around 30 pM. At 30 pM, rapamycin partially suppressed senescence. However, the gerosuppressive effect was balanced by the cytostatic effect, making it difficult to suppress senescence without causing quiescence. We also investigated rat embryonic fibroblasts (REFs), which exhibited markers of senescence at passage 7, yet were able to slowly proliferate until 12-14 passages. REFs grew in size, acquired a large, flat cell morphology, SA-β-Gal staining and components of DNA damage response (DDR), in particular, γH2AX/53BP1 foci. Incubation of REFs with rapamycin (from passage 7 to passage 10) allowed REFs to overcome the replicative senescence crisis. Following rapamycin treatment and removal, a fraction of proliferating REFs gradually increased and senescent phenotype disappeared completely by passage 24.
TOR（雷帕霉素靶蛋白）途径参与从酵母到哺乳动物等多种生物的衰老过程。我们之前已经在人类和啮齿动物细胞中证明，mTOR 将应激诱导的细胞周期停滞转化为不可逆的衰老（衰老转化），而雷帕霉素在细胞周期停滞时会减缓或抑制衰老转化。在这里，我们研究了雷帕霉素是否可以抑制啮齿动物细胞的复制性衰老。小鼠胚胎成纤维细胞（MEFs）逐渐获得衰老的形态并停止增殖。雷帕霉素减少了细胞肥大，否则通过 4-6 个传代会发展出 SA-β-Gal 染色，但它阻止了细胞增殖，掩盖了其对复制寿命的影响。我们确定雷帕霉素在 100-300 pM 时抑制 pS6，并在 IC50 约为 30 pM 时抑制增殖。在 30 pM 时，雷帕霉素部分抑制衰老。然而，gerosuppressive 效应被细胞静止效应所平衡，使得在不引起静止的情况下难以抑制衰老。我们还研究了大鼠胚胎成纤维细胞（REFs），它们在传代 7 时表现出衰老的标志物，但能够缓慢增殖至 12-14 代。REFs 增大，获得大而扁平的细胞形态，SA-β-Gal 染色和 DNA 损伤反应（DDR）的成分，特别是 γH2AX/53BP1 焦点。从传代 7 到传代 10 孵育 REF 时，REF 能够克服复制性衰老危机。雷帕霉素处理和去除后，一部分增殖的 REF 逐渐增加，衰老表型在传代 24 时完全消失。
