RESUMEN
Embryonic stem cells (ESCs) have the capacity for self-renewal and pluripotency. Due to high proliferative activity, ESCs use a specific pathway of the formation of ATP molecules, which can lead to the development of the adaptive metabolic response under the conditions of energy deficiency (which is different from the response of differentiated cells). It is known that metabolic signals are integrated with the cell cycle progression; however, the signaling pathways that connect the availability of nutrients with the regulation of cell cycle in ESCs are insufficiently studied. We have studied the effect of the AICAR agent, which imitates an increase in AMP level and induces the activation of the metabolic sensor AMPK, on proliferation, cell cycle distribution, and pluripotency of mouse ESCs (mESCs). It has been demonstrated that cells treated with AICAR do not stop at the control G1/S point of the cell cycle, since they do not accumulate P21/WAF1 (G1/S checkpoint regulator), despite P53 activation. On the contrary, AICAR increases the rate of mESC proliferation, which correlates with increased expression of pluripotency marker genes (OCT3/4, NANOG, SOX2, KLF4, ESRRB, PRDM14). In addition, an increase in the transcription of the HIFlα gene (a key regulator of the cell proliferation and viability, as well as glucose metabolism under stress) was detected. An increase in the expression of glycolytic enzyme genes (LDHA, ALDOA, PCK2, GLUT4) under the effect of AICAR indicates a change in mESC metabolism towards increased glycolysis. Thus, AICAR-dependent AMPK activation as one of possible mechanisms of the mESC adaptive response to the emergence of energetic imbalance is not accompanied by a cell cycle arrest at the G1/S checkpoint, but involves the processes of increasing glycolytic activity.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Aminoimidazol Carboxamida/análogos & derivados , Antígenos de Diferenciación/biosíntesis , Puntos de Control de la Fase G1 del Ciclo Celular/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Ribonucleótidos/farmacología , Puntos de Control de la Fase S del Ciclo Celular/efectos de los fármacos , Aminoimidazol Carboxamida/farmacología , Animales , Activación Enzimática/efectos de los fármacos , Factor 4 Similar a Kruppel , Ratones , Células Madre Embrionarias de RatonesRESUMEN
Somatic cells in response to DNA damage activate two important protective mechanisms: G1 checkpoint control and a program for recognizing and repairing DNA defects (DDR signaling). Both mechanisms are triggered by the activation of common sensor kinases ATM and ATR, which in turn phosphorylate downstream targets. Mouse embryonic stem cells (mESCs) lack of G1 checkpoint and undergo only temporary G2 delay after DNA damage. We have analyzed the ability of mESCs to detect DNA damage and to form repair foci after irradiation. We showed irradiation-induced activation of ATM and ATR is followed by formation of γH2AX foci co-localized with DNA repair proteins Rad51, DNA-PK and adapter protein 53BP1. Furthermore, we checked contribution of ATM/Chk2 and ATR/Chk1 cascades to cell cycle control and viability of mESCs after DNA damage. Inhibition of ATR/Chk1 cascade leads to accumulation of G1 phase cells, whereas perturbation of ATM/Chk2 activity causes no such effect. Moreover, inhibition of ATR/Chk1 activity, but not ATM/Chk2, substantially augments the killing effect of ionizing radiation on mESCs. In summary, our results indicate that mESCs are capable of recognizing DNA damage and forming repair foci, but their DDR signaling it seems to be distinct from somatic cells and tightly connected with maintaining of pluripotency and self-renewal.
Asunto(s)
Reparación del ADN/efectos de la radiación , Células Madre Embrionarias/efectos de la radiación , Puntos de Control de la Fase G2 del Ciclo Celular/efectos de la radiación , Animales , Proteínas de la Ataxia Telangiectasia Mutada/genética , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Línea Celular , Supervivencia Celular/efectos de la radiación , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Daño del ADN , Reparación del ADN/genética , Proteína Quinasa Activada por ADN/genética , Proteína Quinasa Activada por ADN/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Rayos gamma , Regulación de la Expresión Génica , Histonas/genética , Histonas/metabolismo , Ratones , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosforilación , Recombinasa Rad51/genética , Recombinasa Rad51/metabolismo , Transducción de Señal , Proteína 1 de Unión al Supresor Tumoral P53RESUMEN
Embryonic stem cells (ESCs) are the progenitors of all adult cells so any disruption in their genome can have disastrous consequences for the developing organism. ESCs are characterized by a high proliferation activity and do not undergo checkpoints upon DNA-damage executing only G2/M delay after DNA damage. ATM and ATR kinase are key sensors of DNA double strands breaks and activate downstream signaling pathways involving checkpoints, DNA repair and apoptosis. We estimated ATM/ATR signaling pathway activation in human ESCs and have revealed that irradiation induced ATM, ATR Chk2 phosphorylation, γH2AX foci formation and their co-localization with 53BP1 and Rad51 proteins. Interestingly, human ESCs display non-induced yH2AX foci co-localized with Rad51 and marking DNA single-strand breaks. Next we have revealed the substantial contribution of ATM, Chk1 and Chk2 kinases to G2/M block after irradiation of human ESCs and ATM-dependent activation (phosphorylation) of p53. However p53 activation and subsequent induction of p21 gene expression after DNA damage do not result in p21 protein accumulation due to proteasomal degradation.
Asunto(s)
Proteínas de la Ataxia Telangiectasia Mutada/genética , Daño del ADN/genética , Células Madre Embrionarias/metabolismo , Transducción de Señal/genética , Apoptosis/genética , Reparación del ADN/genética , Células Madre Embrionarias/patología , Histonas/genética , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Recombinasa Rad51/genética , Proteína p53 Supresora de Tumor/genética , Proteína 1 de Unión al Supresor Tumoral P53RESUMEN
Numerous studies have already established that autophagy plays a central role in the survival of all cells, including malignant ones. Autophagy is a central cog in the general mechanism that provides the intracellular proteostasis determining cellular physiological and phenotypic characteristics. The accumulated data show that autophagy largely contributes to cancer cell stemness. Thus, autophagy modulation is considered one of the promising pharmacological targets in therapy aimed at cancer stem cell elimination. However, autophagy is a multi-stage intracellular process that involves numerous protein participants. In addition, the process can be activated simultaneously by various signaling modules. Therefore, it is no small feat to select an effective pharmacological drug against autophagy. What's more, the search for potential chemotherapeutic agents that could eliminate cancer stem cells through pharmacological inhibition of autophagy is still under way. In the present work, we selected a panel of autophagy inhibitors (Autophinib, SBI-0206965, Siramesine, MRT68921, and IITZ-01), some of whom have been recently identified as effective autophagy inhibitors in cancer cells. Using A549 cancer cells, which express the core stem factors Oct4 and Sox2, we evaluated the effect of these drugs on the survival and preservation of the original properties of cancer stem cells. Among the agents selected, only Autophinib demonstrated a significant toxic effect on cancer stem cells. The obtained results demonstrate that autophagy inhibition by Autophinib downregulates the expression of the Sox2 protein in A549 cells, and that this downregulation correlates with a pronounced induction of apoptosis. Moreover, Autophinib-treated A549 cells are unable to form spheroids, which indicates a reduction in stemness. Thus, among the drugs studied, only Autophinib can be considered a potential agent against cancer stem cells.