The upregulation of Ulk1-dependent autophagy does not require the p53 activity in mouse embryonic stem cells.

Autophagy is known to play a critical role in the early stages of embryogenesis including the formation of blastocyst. The existence of p53 protein-deficient mice may identify that p53 is not indispensable for the activation of autophagy in pluripotent cells derived from the inner cell mass of the blastocyst. We utilized a p53-knockout (KO) mouse embryonic stem cell (mESC) line to investigate the contribution of p53 in autophagy. We showed that lack of p53 has no effect on cell pluripotency but significantly hinders the differentiation process induced by retinoic acid. Using MRT68921, we revealed that Ulk1-dependent autophagy is activated in response to serum deprivation despite the deletion of p53 in mESCs. However, under retinoic acid-induced differentiation, the accumulation of autophagosomes and lysosomes is impaired in p53 KO mESCs, indicating a critical role of p53 in the regulation of autophagy upon differentiation.

Inducible Ulk1 expression activates the p53 protein in mouse embryonic stem cells.

Defective pluripotent cells are removed from embryos prior to differentiation, presumably due to upregulation of the p53 pathway. However, the mechanism underlying p53 protein activation is still unknown. Embryonic stem cells (ESCs), corresponding to cells of the preimplantation blastocyst, likely have similar mechanisms for abnormal cell elimination. Using a mouse ESC cell line with inducible ulk1 gene expression, we showed that Ulk1 upregulation is accompanied by p53 phosphorylation on Ser15. ESCs tolerated the activated p53 and did not undergo apoptosis or cell cycle blockade upon Ulk1 overexpression. However, massive cell death was observed after retinoic acid treatment, suggesting a role of Ulk1-induced p53 activation in the elimination of defective pluripotent cells prior to differentiation.

Resveratrol-induced p53 activation is associated with autophagy in mouse embryonic stem cells.

Resveratrol is a natural polyphenol with several therapeutic effects, in particular, inducing p53-dependent cell cycle arrest and/or apoptosis in tumor cells. Resveratrol-induced p53 activation may trigger differentiation and apoptosis in embryonic stem cells (ESCs). We show that resveratrol activates p53 that is negatively regulated by SIRT1 deacetylation on Lys379 and positively by AMPK phosphorylation on Ser15 in mouse ESCs (mESCs). Surprisingly, the resveratrol-activated p53 is not associated with either G1/S cell cycle checkpoint or apoptosis in mESCs. Instead, it stimulates autophagy in a transcriptional-dependent manner involving up-regulation of dram1 gene expression. This study demonstrates a novel mechanism of resveratrol-dependent p53 activation in mESCs.

mTOR pathway occupies a central role in the emergence of latent cancer cells.

The current focus in oncology research is the translational control of cancer cells as a major mechanism of cellular plasticity. Recent evidence has prompted a reevaluation of the role of the mTOR pathway in cancer development leading to new conclusions. The mechanistic mTOR inhibition is well known to be a tool for generating quiescent stem cells and cancer cells. In response to mTOR suppression, quiescent cancer cells dynamically change their proteome, triggering alternative non-canonical translation mechanisms. The shift to selective translation may have clinical relevance, since quiescent tumor cells can acquire new phenotypical features. This review provides new insights into the patterns of mTOR functioning in quiescent cancer cells, enhancing our current understanding of the biology of latent metastasis.

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.

The mTOR Pathway in Pluripotent Stem Cells: Lessons for Understanding Cancer Cell Dormancy.

Currently, the success of targeted anticancer therapies largely depends on the correct understanding of the dormant state of cancer cells, since it is increasingly regarded to fuel tumor recurrence. The concept of cancer cell dormancy is often considered as an adaptive response of cancer cells to stress, and, therefore, is limited. It is possible that the cancer dormant state is not a privilege of cancer cells but the same reproductive survival strategy as diapause used by embryonic stem cells (ESCs). Recent advances reveal that high autophagy and mTOR pathway reduction are key mechanisms contributing to dormancy and diapause. ESCs, sharing their main features with cancer stem cells, have a delicate balance between the mTOR pathway and autophagy activity permissive for diapause induction. In this review, we discuss the functioning of the mTOR signaling and autophagy in ESCs in detail that allows us to deepen our understanding of the biology of cancer cell dormancy.

Resveratrol enhances pluripotency of mouse embryonic stem cells by activating AMPK/Ulk1 pathway.

Resveratrol, a natural polyphenolic compound, shows many beneficial effects in various animal models. It increases efficiency of somatic cell reprograming into iPSCs and contributes to cell differentiation. Here, we studied the effect of resveratrol on proliferation and pluripotency of mouse embryonic stem cells (mESCs). Our results demonstrate that resveratrol induces autophagy in mESCs that is provided by the activation of the AMPK/Ulk1 pathway and the concomitant suppression of the activity of the mTORC1 signaling cascade. These events correlate with the enhanced expression of pluripotency markers Oct3/4, Sox2, Nanog, Klf4, SSEA-1 and alkaline phosphatase. Pluripotency is retained under resveratrol-caused retardation of cell proliferation. Given that the Ulk1 overexpression enhances pluripotency of mESCs, the available data evidence that mTOR/Ulk1/AMPK-autophagy network provides the resveratrol-mediated regulation of mESC pluripotency. The capability of resveratrol to support the mESC pluripotency provides a new approach for developing a defined medium for ESC culturing as well as for better understanding signaling events that govern self-renewal and pluripotency.

G1 checkpoint is compromised in mouse ESCs due to functional uncoupling of p53-p21Waf1 signaling.

Mouse embryonic stem cells (mESCs) lack of G1 checkpoint despite that irradiation (IR) activates ATM/ATR-mediated DDR signaling pathway. The IR-induced p53 localizes in the nuclei and up-regulates p21/Waf1 transcription but that does not lead to accumulation of p21/Waf1 protein. The negative control of the p21Waf1 expression appears to occur at 2 levels of regulation. First, both p21/Waf1 gene transcription and the p21/Waf1 protein content increase in mESCs treated with histone-deacetylase inhibitors, implying its epigenetic regulation. Second, proteasome inhibitors cause the p21/Waf1 accumulation, indicating that the protein is a subject of proteasome-dependent degradation in ESСs. Then, the dynamics of IR-induced p21Waf1 protein show its accumulation at long-term time points (3 and 5 days) that coincides with an increase in the proportion of G1-phase cells, down-regulation of Oct4 and Nanog pluripotent gene transcription and activation of endoderm-specific genes sox17 and afp. In addition, nutlin-dependent stabilization of p53 in mESC was also accompanied by the accumulation of p21/Waf1 as well as restoration of G1 checkpoint and an onset of differentiation. Thus, the lack of functional p21/Waf1 is indispensable for maintaining self-renewal and pluripotency of mESCs.

New insights into cell cycle regulation and DNA damage response in embryonic stem cells.

Embryonic stem cells (ESCs) have unlimited proliferative potential, while retaining the ability to differentiate into descendants of all three embryonic layers. High proliferation rate of ESCs is accompanied by a shortening of the G(1) phase and the lack of G(1) checkpoint following DNA damage. The absence of G(1) arrest in ESCs after DNA damage is likely caused by a dysfunction of the p53-dependent p21Waf1 pathway that is a key event for the maintenance of pluripotency. There are controversial data on the functional status of p53, but it is well established that one of the key p53 target-p21Waf1-is expressed in ESCs at a very low level. Despite the lack of G(1) checkpoint, ESCs are capable to repair DNA defects; moreover the DNA damage response (DDR) signaling operates very effectively throughout the cell cycle. This review covers also the results obtained with the reprogramming of somatic cells into the induced pluripotent stem cells, for which have been shown that a partial dysfunction of the p53Waf1 pathway increases the frequency of generation of pluripotent cells. In summary, these results indicate that the G(1) checkpoint control and DDR are distinct from somatic cells and their status is tightly connected with maintaining of pluripotency and self-renewal.