Macrophage and microglia polarization: focus on autophagy-dependent reprogramming.

The approach to the study of autophagy has been undergoing considerable change lately: from investigations of the protein components of autophagic machinery to its regulation at different molecular levels. Autophagy is being examinated not only as a separated degradative process per se in cells but as an executor mechanism of certain signaling pathways that converge on it, being activated under specific conditions. Additionally, autophagy is beginning to be observed as a key integral part of cellular reprogramming, the transition from one phenotypic state to another associated with rapid degradation of the previous proteostasis. Macrophages and microglia demonstrate a diversity of phenotypes reflecting their effective capability to phenotypic plasticity. Therefore, understanding the role of autophagy in macrophage and microglia functions needs to be addressed. In this review, we focus on autophagy as a fundamental intracellular process underlying macrophages and microglia polarization.

mTOR kinase inhibitor pp242 causes mitophagy terminated by apoptotic cell death in E1A-Ras transformed cells.

mTOR is a critical target for controlling cell cycle progression, senescence and cell death in mammalian cancer cells. Here we studied the role of mTOR-dependent autophagy in implementating the antiprolifrative effect of mTORC1-specific inhibitor rapamycin and ATP-competitive mTOR kinase inhibitor pp242. We carried out a comprehensive analysis of pp242- and rapamycin-induced autophagy in ERas tumor cells. Rapamycin exerts cytostatic effect on ERas tumor cells, thus causing a temporary and reversible cell cycle arrest, activation of non-selective autophagy not accompanied by cell death. The rapamycin-treated cells are able to continue proliferation after drug removal. The ATP-competitive mTORC1/mTORC2 kinase inhibitor pp242 is highly cytotoxic by suppressing the function of mTORC1-4EBP1 axis and mTORC1-dependent phosphorylation of mTORC1 target--ULK1-Ser757 (Atg1). In contrast to rapamycin, pp242 activates the selective autophagy targeting mitochondria (mitophagy). The pp242-induced mitophagy is accompanied by accumulation of LC3 and conversion of LC3-I form to LC3-II. However reduced degradation of p62/SQSTM indicates abnormal flux of autophagic process. According to transmission electron microscopy data, short-term pp242-treated ERas cells exhibit numerous heavily damaged mitochondria, which are included in single membrane-bound autophagic/autolysophagic vacuoles (mitophagy). Despite the lack of typical for apoptosis features, ERas-treated cells with induced mitophagy revealed the activation of caspase 3, 9 and nucleosomal DNA fragmentation. Thus, pp242 activates autophagy with suppressed later stages, leading to impaired recycling and accumulation of dysfunctional mitochondria and cell death. Better understanding of how autophagy determines the fate of a cell--survival or cell death, can help to development of new strategy for cancer therapy.

Sustained activation of DNA damage response in irradiated apoptosis-resistant cells induces reversible senescence associated with mTOR downregulation and expression of stem cell markers.

Cells respond to genotoxic stress by activating the DNA damage response (DDR). When injury is severe or irreparable, cells induce apoptosis or cellular senescence to prevent transmission of the lesions to the daughter cells upon cell division. Resistance to apoptosis is a hallmark of cancer that challenges the efficacy of cancer therapy. In this work, the effects of ionizing radiation on apoptosis-resistant E1A + E1B transformed cells were investigated to ascertain whether the activation of cellular senescence could provide an alternative tumor suppressor mechanism. We show that irradiated cells arrest cell cycle at G 2/M phase and resume DNA replication in the absence of cell division followed by formation of giant polyploid cells. Permanent activation of DDR signaling due to impaired DNA repair results in the induction of cellular senescence in E1A + E1B cells. However, irradiated cells bypass senescence and restore the population by dividing cells, which have near normal size and ploidy and do not express senescence markers. Reversion of senescence and appearance of proliferating cells were associated with downregulation of mTOR, activation of autophagy, mitigation of DDR signaling, and expression of stem cell markers.

Rapamycin induces pluripotent genes associated with avoidance of replicative senescence.

Primary rodent cells undergo replicative senescence, independent from telomere shortening. We have recently shown that treatment with rapamycin during passages 3-7 suppressed replicative senescence in rat embryonic fibroblasts (REFs), which otherwise occurred by 10-14 passages. Here, we further investigated rapamycin-primed cells for an extended number of passages. Rapamycin-primed cells continued to proliferate without accumulation of senescent markers. Importantly, these cells retained the ability to undergo serum starvation- and etoposide-induced cell cycle arrest. The p53/p21 pathway was functional. This indicates that rapamycin did not cause either transformation or loss of cell cycle checkpoints. We found that rapamycin activated transcription of pluripotent genes, oct-4, sox-2, nanog, as well as further upregulated telomerase (tert) gene. The rapamycin-derived cells have mostly non-rearranged, near-normal karyotype. Still, when cultivated for a higher number of passages, these cells acquired a chromosomal marker within the chromosome 3. We conclude that suppression mTORC1 activity may prevent replicative senescence without transformation of rodent cells.

Suppression of replicative senescence by rapamycin in rodent embryonic cells.

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-ß-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-ß-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.

p21Waf1 is required for complete oncogenic transformation of mouse embryo fibroblasts by E1Aad5 and c-Ha-ras oncogenes.

Cyclin-dependent kinase inhibitor p21(Waf1) is known to have alternative functions associated with positive regulation of proliferation, actin cytoskeleton remodeling and suppression of apoptosis. The goal of the present study was to assess the role of p21(Waf1) in the establishment of the transformed phenotype of mouse embryo fibroblasts with stable expression of E1Aad5 and c-Ha-ras complementary oncogenes. Herein, we demonstrate that E1A/c-Ha-Ras-transformed p21(Waf1)-null fibroblasts possess some characteristic features of transformed cells, such as loss of contact inhibition, high saturation density, shortened cell cycle, inability to undergo cell-cycle arrest after DNA damage and serum deprivation, but, at the same time, they are not completely transformed in that they are unable to proliferate at clonal density, are anchorage-dependent, retain a fibroblast-like morphology with pronounced actin cytoskeleton and show reduced migration and invasion. Our data support the concept of p21(Waf1) "tumor suppressor" having alternative oncogenic functions in the cytoplasm and for the first time indicate that p21(Waf1) can be indispensable for complete oncogenic transformation.

Rapamycin decelerates cellular senescence.

When the cell cycle is arrested but cellular growth is not, then cells senesce, permanently losing proliferative potential. Here we demonstrated that the duration of cell cycle arrest determines a progressive loss of proliferative capacity. In human and rodent cell lines, rapamycin (an inhibitor of mTOR) dramatically decelerated loss of proliferative potential caused by ectopic p21, p16 and sodium butyrate-induced p21. Thus, when the cell cycle was arrested by these factors in the presence of rapamycin, cells retained the capacity to resume proliferation, once p21, p16 or sodium butyrate were removed. While rapamycin prevented the permanent loss of proliferative potential in arrested cells, it did not force the arrested cells into proliferation. During cell cycle arrest, rapamycin transformed the irreversible arrest into a reversible condition. Our data demonstrate that senescence can be pharmacologically suppressed.