Cell Cycle Dysregulation and Renal Fibrosis

Precise regulation of cell cycle is essential for tissue homeostasis and development, while cell cycle dysregulation is associated with many human diseases including renal fibrosis, a common process of various chronic kidney diseases progressing to end-stage renal disease. Under normal physiological conditions, most of the renal cells are post-mitotic quiescent cells arrested in the G0 phase of cell cycle and renal cells turnover is very low. Injuries induced by toxins, hypoxia, and metabolic disorders can stimulate renal cells to enter the cell cycle, which is essential for kidney regeneration and renal function restoration. However, more severe or repeated injuries will lead to maladaptive repair, manifesting as cell cycle arrest or overproliferation of renal cells, both of which are closely related to renal fibrosis. Thus, cell cycle dysregulation of renal cells is a potential therapeutic target for the treatment of renal fibrosis. In this review, we focus on cell cycle regulation of renal cells in healthy and diseased kidney, discussing the role of cell cycle dysregulation of renal cells in renal fibrosis. Better understanding of the function of cell cycle dysregulation in renal fibrosis is essential for the development of therapeutics to halt renal fibrosis progression or promote regression.

Novel Roles of Small Extracellular Vesicles in Regulating the Quiescence and Proliferation of Neural Stem Cells

Neural stem cell (NSC) quiescence plays pivotal roles in avoiding exhaustion of NSCs and securing sustainable neurogenesis in the adult brain. The maintenance of quiescence and transition between proliferation and quiescence are complex processes associated with multiple niche signals and environmental stimuli. Exosomes are small extracellular vesicles (sEVs) containing functional cargos such as proteins, microRNAs, and mRNAs. The role of sEVs in NSC quiescence has not been fully investigated. Here, we applied proteomics to analyze the protein cargos of sEVs derived from proliferating, quiescent, and reactivating NSCs. Our findings revealed fluctuation of expression levels and functional clusters of gene ontology annotations of differentially expressed proteins especially in protein translation and vesicular transport among three sources of exosomes. Moreover, the use of exosome inhibitors revealed exosome contribution to entrance into as well as maintenance of quiescence. Exosome inhibition delayed entrance into quiescence, induced quiescent NSCs to exit from the G0 phase of the cell cycle, and significantly upregulated protein translation in quiescent NSCs. Our results suggest that NSC exosomes are involved in attenuating protein synthesis and thereby regulating the quiescence of NSCs.

CBIO-12. KAT5 ACTIVITY CONTROLS GLIOBLASTOMA CELL CYCLE DYNAMICS AND TUMOR GROWTH

Current standard of care therapy for glioblastoma (GB) includes cytoreduction followed by ablative therapies that target rapidly dividing cell types. However, non-cycling, quiescent-like states (G0 phase cells) are present in both normal tissue and tumors and play important roles in maintaining heterogeneity and cellular hierarchies. The presence of quiescent-like/G0 states therefore represents a natural reservoir of tumor cells that are resistant to current treatments. Quiescence or G0 phase is a reversible state of “stasis” cells enter in response to developmental or environmental cues. However, it remains largely unclear to what degree or by what mechanisms tumor cells enter into or exit from quiescent-like states. To gain insight into how GB cells might regulate G0-like states, we performed a genome-wide CRISPR-Cas9 screen in patient-derived GB stem-like cells (GSCs) harboring a G0 reporter construct, which is stabilized when cells enter a G0-like state. Among the top screen hits were members of the Tip60/KAT5 histone acetyltransferase complex, including KAT5 itself. Remarkably, we show that knockout of KAT5 in vitro and in vivo dramatically increases G0 subpopulations in GSC cultures and GSC-induced tumors. Using genetically engineered GSC harboring KAT5 under the control of a Doxycyclin-titratable promoter, we establish that incrementally down regulating KAT5 activity is sufficient to slow cell cycle dynamics causing a build-up G0-like cells; and that partial inhibition of KAT5 leads to extended (mouse) patient survival. Further, in primary tumors, cell-based KAT5 activity assays revealed that high grade tumors harbor larger cell subpopulations with higher KAT5 activity than lower grade tumors. In summary, our results suggest that Tip60/KAT5 activity plays key roles in G0 ingress/egress for GBM tumors, may contribute to tumor progression, and may provide novel therapeutic opportunities.

Parthenolide Induces Apoptosis and Cell Cycle Arrest by the Suppression of miR-375 Through Nucleolin in Prostate Cancer

Aims: To investigate the effect of parthenolide on nucleolin in controlling the expression of miR-375 that induces apoptosis and cell cycle arrest in prostate cancer. Study Design: This study is an experimental study. Methodology: The cytotoxicity effect of parthenolide was tested by MTT assay for 48 h. Microscopic techniques were used to identify the morphological changes of the cell line. The expression of apoptotic and cell cycle regulatory genes was analyzed by the Real-Time PCR. The phase of cell cycle arrest was identified by Flow cytometry. Results: The obtained results indicated that parthenolide induced cytotoxicity and suppressed the proliferation by reducing the growth of LNCaP cells in 48 h. The microscopic analysis showed the alteration of cell morphology and increase of cytoplasmic reactive oxygen species. Parthenolide promotes apoptosis by the downregulation of nucleolin, Bcl-2, and up-regulation of Bax gene. Moreover, the flow cytometry assay showed the G1/G0 phase of cell cycle arrest. Conclusion: Parthenolide induces apoptosis and cell cycle arrest through nucleolin by the suppression of miR-375 in prostate cancer cells.

The fate of notch-1 transcript is linked to cell cycle dynamics by activity of a natural antisense transcript

A core imprint of metazoan life is that perturbations of cell cycle are offset by compensatory changes in successive cellular generations. This trait enhances robustness of multicellular growth and requires transmission of signaling cues within a cell lineage. Notably, the identity and mode of activity of transgenerational signals remain largely unknown. Here we report the discovery of a natural antisense transcript encoded in exon 25 of notch-1 locus (nAS25) by which mother cells control the fate of notch-1 transcript in daughter cells to buffer against perturbations of cell cycle. The antisense transcript is transcribed at G1 phase of cell cycle from a bi-directional E2F1-dependent promoter in the mother cell where the titer of nAS25 is calibrated to the length of G1. Transmission of the antisense transcript from mother to daughter cells stabilizes notch-1 sense transcript in G0 phase of daughter cells by masking it from RNA editing and resultant nonsense-mediated degradation. In consequence, nAS25-mediated amplification of notch-1 signaling reprograms G1 phase in daughter cells to compensate for the altered dynamics of the mother cell. The function of nAS25/notch-1 in integrating G1 phase history of the mother cell into that of daughter cells is compatible with the predicted activity of a molecular oscillator, slower than cyclins, that coordinates cell cycle within cell lineage.

Novel roles of small extracellular vesicles in regulating the quiescence and proliferation of neural stem cells

Neural stem cells (NSCs) quiescence plays pivotal roles in securing sustainable neurogenesis and avoiding stemness exhaustion in the adult brain. The maintenance of quiescence and transition between proliferation and quiescence are complex processes associated with multiple niche signals, and environmental stimuli. Though the mechanisms of the transitions between NSC states have been extensively investigated, they remain to be fully elucidated. Exosomes are small extracellular vesicles (sEVs) containing functional units such as proteins, microRNAs, and mRNAs. It has already been demonstrated that sEVs actively participate in cancer cell proliferation and metastasis. However, the role of sEVs in NSC quiescence has not been investigated. Here, we applied proteomics to analyze the protein cargos of sEVs derived from proliferating, quiescent, and reactivating NSCs. Our findings revealed expression level fluctuations of NSCs sEV protein cargo at different proliferative conditions. We also identified functional clusters of gene ontology annotations from differentially expressed proteins in three sources of exosomes. Moreover, the use of exosome inhibitors revealed the contribution of exosomes to NSC quiescence at the entrance into quiescence, as well as in quiescence maintenance. Exosome inhibition delayed the entrance into quiescence by proliferating NSCs and allowed quiescent NSCs to exit from the G0 phase of the cell cycle. Protein translation was significantly upregulated in both quiescent NSCs and quiescent-induced NSCs via the exosome inhibition. Our results demonstrated that NSC exosomes are involved in regulating the quiescence of NSCs and provide a functional prediction of NSCs exosome protein cargos in terms of cell-cycle regulation and protein synthesis.

Large-Scale Quantitatively Detecting and Analyzing of ceRNAs reveal NOTCH3 mRNA, miR-369-3p and rno-Rmdn2_0006 Together Regulate the Hepatocytes in G0 Phase and in G1 Phase During the Initiation Stage of Rat Liver Regeneration

Aims:Generally, key events of the liver regeneration initiation (LRI) are how the hepatocytes in G0 phase change to G1 phase. We use the data from large-scale quantitatively detecting and analyzing (LQDA) to reveal ceRNAs together regulate the hepatocytes in G0 phase or in G1 phase during LRI.Main methods:The hepatocytes of the rat liver right lobe were isolated at 0 hour, 6 hour and 24 hour after partial hepatectomy (PH), their ceRNA expression abundance was measured by LQDA, and the correlation of their expression, interaction and role were revealed by ceRNA comprehensive analysis. Key findings:It was found that expression of NOTCH3 mRNA is up-regulated at 0 h, but miR-369-3p and rno-Rmdn2_0006 of hepatocytes not change significantly at that, meanwhile, the G0 phase-related gene CDKN1c promoted by NOTCH3 up-regulation, and the G1 phase-related gene PSEN2 inhibited by NOTCH3 down-regulation at that. On the contrary, NOTCH3 mRNA and rno-Rmdn2_0006 were up-regulated at 6 h, but miR-136-3p was down-regulated at that, the G1 phase-related genes CHUK, DDX24, HES1, NET1 and STAT3 promoted by NOTCH3 up-regulated, and the G0 phase-related gene CDKN1a inhibited by NOTCH3 down-regulated at that. These results suggested that the ceRNAs and the NOTCH3-regulated G0 phase- and G1 phase-related genes show a correlation in expression, interaction and role, they together regulate the hepatocytes in G0 phase at 0 h and in G1 phase at 6 h. Significance:These discovers are helpful to understand the mechanism which ceRNA together regulate the hepatocytes in G0 phase or G1 phase.

Advanced Oxidation Protein Products Induce G1/G0-Phase Arrest in Ovarian Granulosa Cells via the ROS-JNK/p38 MAPK-p21 Pathway in Premature Ovarian Insufficiency

The mechanism underlying the role of oxidative stress and advanced oxidation protein products (AOPPs) in the aetiology of premature ovarian insufficiency (POI) is poorly understood. Here, we investigated the plasma AOPP level in POI patients and the effects of AOPPs on granulosa cells both in vitro and in vivo. KGN cells were treated with different AOPP doses, and cell cycle distribution, intracellular reactive oxygen species (ROS), and protein expression levels were measured. Sprague–Dawley (SD) rats were treated daily with PBS, rat serum albumin, AOPP, or AOPP+ N-acetylcysteine (NAC) for 12 weeks to explore the effect of AOPPs on ovarian function. Plasma AOPP concentrations were significantly higher in both POI and biochemical POI patients than in controls and negatively correlated with anti-Müllerian hormone and the antral follicle count. KGN cells treated with AOPP exhibited G1/G0-phase arrest. AOPP induced G1/G0-phase arrest in KGN cells by activating the ROS-c-Jun N-terminal kinase (JNK)/p38 mitogen-activated protein kinase (MAPK)-p21 pathway. Pretreatment with NAC, SP600125, SB203580, and si-p21 blocked AOPP-induced G1/G0-phase arrest. In SD rats, AOPP treatment increased the proportion of atretic follicles, and NAC attenuated the adverse effects of AOPPs in the ovary. In conclusion, we provide mechanistic evidence that AOPPs may induce cell cycle arrest in granulosa cells via the ROS-JNK/p38 MAPK-p21 pathway and thus may be a novel biomarker of POI.

α-Tocopherol Attenuates Oxidative Phosphorylation of CD34+ Cells, Enhances Their G0 Phase Fraction and Promotes Hematopoietic Stem and Primitive Progenitor Cell Maintenance

Alpha tocopherol acetate (αTOA) is an analogue of alpha tocopherol (αTOC) that exists in the form of an injectable drug. In the context of the metabolic hypothesis of stem cells, we studied the impact of αTOA on the metabolic energetic profile and functional properties of hematopoietic stem and progenitor cells. In ex vivo experiments performed on cord blood CD34+ cells, we found that αTOA effectively attenuates oxidative phosphorylation without affecting the glycolysis rate. This effect concerns complex I and complex II of the mitochondrial respiratory chain and is related to the relatively late increase (3 days) in ROS (Reactive Oxygen Species). The most interesting effect was the inhibition of Hypoxia-Inducible Factor (HIF)-2α (Hexpression, which is a determinant of the most pronounced biological effect—the accumulation of CD34+ cells in the G0 phase of the cell cycle. In parallel, better maintenance of the primitive stem cell activity was revealed by the expansion seen in secondary cultures (higher production of colony forming cells (CFC) and Severe Combined Immunodeficiency-mice (scid)-repopulating cells (SRC)). While the presence of αTOA enhanced the maintenance of Hematopoietic Stem Cells (HSC) and contained their proliferation ex vivo, whether it could play the same role in vivo remained unknown. Creating αTOC deficiency via a vitamin E-free diet in mice, we found an accelerated proliferation of CFC and an expanded compartment of LSK (lineagenegative Sca-1+cKit+) and SLAM (cells expressing Signaling Lymphocytic Activation Molecule family receptors) bone marrow cell populations whose in vivo repopulating capacity was decreased. These in vivo data are in favor of our hypothesis that αTOC may have a physiological role in the maintenance of stem cells. Taking into account that αTOC also exhibits an effect on proliferative capacity, it may also be relevant for the ex vivo manipulation of hematopoietic stem cells. For this purpose, low non-toxic doses of αTOA should be used.

Towards a Framework for Better Understanding of Quiescent Cancer Cells

Quiescent cancer cells (QCCs) are cancer cells that are reversibly suspended in G0 phase with the ability to re-enter the cell cycle and initiate tumor growth, and, ultimately, cancer recurrence and metastasis. QCCs are also therapeutically challenging due to their resistance to most conventional cancer treatments that selectively act on proliferating cells. Considering the significant impact of QCCs on cancer progression and treatment, better understanding of appropriate experimental models, and the evaluation of QCCs are key questions in the field that have direct influence on potential pharmacological interventions. Here, this review focuses on existing and emerging preclinical models and detection methods for QCCs and discusses their respective features and scope for application. By providing a framework for selecting appropriate experimental models and investigative methods, the identification of the key players that regulate the survival and activation of QCCs and the development of more effective QCC-targeting therapeutic agents may mitigate the consequences of QCCs.