A truncated form of the p27 CDK inhibitor translated from pre-mRNA causes cell cycle arrest at G2 phase

Pre-mRNA splicing is indispensable for eukaryotic gene expression. Splicing inhibition causes cell cycle arrest and cell death, which are the reasons of potent anti-tumor activity of splicing inhibitors. Here, we found that truncated proteins are involved in cell cycle arrest and cell death upon splicing inhibition. We analyzed pre-mRNAs accumulated in the cytoplasm where translation occurs, and found that a truncated form of the p27 CDK inhibitor, named p27*, is translated from pre-mRNA and accumulated in G2 arrested cells. Overexpression of p27* caused G2 phase arrest through inhibiting CDK-cyclin complexes. Conversely, knockout of p27* accelerated resumption of cell proliferation after washout of splicing inhibitor. Interestingly, p27* was resistant to proteasomal degradation. We propose that cells produce truncated proteins with different nature to the original proteins via pre-mRNA translation only under splicing deficient conditions to response to the splicing deficient conditions.

Shift in G1-Checkpoint from ATM-Alone to a Cooperative ATM Plus ATR Regulation with Increasing Dose of Radiation

The current view of the involvement of PI3-kinases in checkpoint responses after DNA damage is that ATM is the key regulator of G1-, S- or G2-phase checkpoints, that ATR is only partly involved in the regulation of S- and G2-phase checkpoints and that DNA-PKcs is not involved in checkpoint regulation. However, further analysis of the contributions of these kinases to checkpoint responses in cells exposed to ionizing radiation (IR) recently uncovered striking integrations and interplays among ATM, ATR and DNA-PKcs that adapt not only to the phase of the cell cycle in which cells are irradiated, but also to the load of DNA double-strand breaks (DSBs), presumably to optimize their processing. Specifically, we found that low IR doses in G2-phase cells activate a G2-checkpoint that is regulated by epistatically coupled ATM and ATR. Thus, inhibition of either kinase suppresses almost fully its activation. At high IR doses, the epistatic ATM/ATR coupling relaxes, yielding to a cooperative regulation. Thus, single-kinase inhibition suppresses partly, and only combined inhibition suppresses fully G2-checkpoint activation. Interestingly, DNA-PKcs integrates with ATM/ATR in G2-checkpoint control, but functions in its recovery in a dose-independent manner. Strikingly, irradiation during S-phase activates, independently of dose, an exclusively ATR-dependent G2 checkpoint. Here, ATM couples with DNA-PKcs to regulate checkpoint recovery. In the present work, we extend these studies and investigate organization and functions of these PI3-kinases in the activation of the G1 checkpoint in cells irradiated either in the G0 or G1 phase. We report that ATM is the sole regulator of the G1 checkpoint after exposure to low IR doses. At high IR doses, ATM remains dominant, but contributions from ATR also become detectable and are associated with limited ATM/ATR-dependent end resection at DSBs. Under these conditions, only combined ATM + ATR inhibition fully abrogates checkpoint and resection. Contributions of DNA-PKcs and CHK2 to the regulation of the G1 checkpoint are not obvious in these experiments and may be masked by the endpoint employed for checkpoint analysis and perturbations in normal progression through the cell cycle of cells exposed to DNA-PKcs inhibitors. The results broaden our understanding of organization throughout the cell cycle and adaptation with increasing IR dose of the ATM/ATR/DNA-PKcs module to regulate checkpoint responses. They emphasize notable similarities and distinct differences between G1-, G2- and S-phase checkpoint regulation that may guide DSB processing decisions.

Control of G2 phase duration by CDC25B modulates the switch from direct to indirect neurogenesis in the neocortex

During development, cortical neurons are produced in a temporally regulated sequence from apical progenitors, directly, or indirectly through the production of intermediate basal progenitors. The balance between these major progenitors types is determinant for the production of the proper number and types of neurons and it is thus important to decipher the cellular and molecular cues controlling this equilibrium. Here we address the role of a cell cycle regulator, the CDC25B phosphatase, in this process. We show that deleting CDC25B in apical progenitors leads to a transient increase of the production of TBR1+ neurons at the expense of TBR2+ basal progenitors in mouse neocortex. This phenotype is associated with lengthening of the G2 phase of the cell cycle, the total cell cycle length being unaffected. Using in utero electroporation and cortical slice cultures, we demonstrate that the defect in TBR2+ basal progenitor production requires interaction with CDK1 and is due to the G2 phase lengthening in CDC25B mutants. Altogether, this study identifies a new role for CDC25B and the length of the G2 phase in direct versus indirect neurogenesis at early stages of the cortical development.

Telomerase subunit Est2 marks internal sites that are prone to accumulate DNA damage

Background The main function of telomerase is at the telomeres but under adverse conditions telomerase can bind to internal regions causing deleterious effects as observed in cancer cells. Results By mapping the global occupancy of the catalytic subunit of telomerase (Est2) in the budding yeast Saccharomyces cerevisiae, we reveal that it binds to multiple guanine-rich genomic loci, which we termed “non-telomeric binding sites” (NTBS). We characterize Est2 binding to NTBS. Contrary to telomeres, Est2 binds to NTBS in G1 and G2 phase independently of Est1 and Est3. The absence of Est1 and Est3 renders telomerase inactive at NTBS. However, upon global DNA damage, Est1 and Est3 join Est2 at NTBS and telomere addition can be observed indicating that Est2 occupancy marks NTBS regions as particular risks for genome stability. Conclusions Our results provide a novel model of telomerase regulation in the cell cycle using internal regions as “parking spots” of Est2 but marking them as hotspots for telomere addition.

FBXO47 is essential for preventing the synaptonemal complex from premature disassembly in mouse male meiosis

Meiotic prophase is a prolonged G2 phase that ensures the completion of numerous meiosis-specific chromosome events. During meiotic prophase, homologous chromosomes undergo synapsis to facilitate meiotic recombination yielding crossovers. It remains largely elusive how homolog synapsis is temporally maintained and destabilized during meiotic prophase. Here we show that FBXO47 is the stabilizer of synaptonemal complex during male meiotic prophase. Disruption of FBXO47 shows severe impact on homologous chromosome synapsis and DSB repair processes, leading to male infertility. Notably, in the absence of FBXO47, although once homologous chromosomes are synapsed, the synaptonemal complex is precociously disassembled before progressing beyond pachytene. Remarkably, Fbxo47 KO spermatocytes remain in earlier stage of meiotic prophase and lack crossovers, despite apparently exhibiting diplotene-like chromosome morphology. We propose that FBXO47 functions independently of SCF E3 ligase, and plays a crucial role in preventing synaptonemal complex from premature disassembly during cell cycle progression of meiotic prophase.

Ethanol Extract of Croton Kongensis Promotes Cell Cycle Arrest and Consequently Inhibits Anchorage-Independent Growth of Cervical Cancer Hela Cells

Extracts from Croton kongenis present anticancer activities on various cancers. However, there is no research conducted to investigate the effects of Croton kongenis extracts on cervical cancer as well as on zebrafish. In this study, we demonstrated that Croton kongenis ethanol extract expressed high toxicity to cervical cancer Hela cells with an IC50 dose of 20.4 µg/mL and to zebrafish embryos with malformations, lethality and hatching inhibition at 72-hpf at effective dose of 125 µg/mL. Interestingly, treatment with Croton kongenis ethanol extract caused cell-cycle-arrest at the G2 phase. Particularly, percentages of Croton kongenis ethanol extract-treated cells in G1, S, G2/M were 70%, 6% and 23%, while percentages of control cells in G1, S, G2/M were 65%, 15% and 18%, respectively. Consistent with cell-cycle-arrest, the expressions of CDKN1A, CDNK2A and p53 in Croton kongenis ethanol extract-treated cells were up-regulated 2.0-, 1.65- and 1.8-fold, respectively. Significantly, treatment with Croton kongenis ethanol extract inhibited anchorage-independent growth of Hela cells; the number of colonies formed in soft-agar of Croton kongenis ethanol extract-treated cells was only one-fourth of that of control cells. In conclusion, we suggest that Croton kongenis ethanol extract could be able to use as a traditional medicine for treatment of cervical cancer.

Combined Scutellarin and C18H17NO6 Imperils the Survival of Glioma: Partly Associated With the Repression of PSEN1/PI3K-AKT Signaling Axis

Glioma, the most common intracranial tumor, harbors great harm. Since the treatment for it has reached the bottleneck stage, the development of new drugs becomes a trend. Therefore, we focus on the effect of scutellarin (SCU) and its combination with C18H17NO6 (abbreviated as combination) on glioma and its possible mechanism in this study. Firstly, SCU and C18H17NO6 both suppressed the proliferation of U251 and LN229 cells in a dose-dependent manner, and C18H17NO6 augmented the inhibition effect of SCU on U251 and LN229 cells in vitro. Moreover, there was an interactive effect between them. Secondly, SCU and C18H17NO6 decreased U251 cells in G2 phase and LN229 cells in G2 and S phases but increased U251 cells in S phase, respectively. Meanwhile, the combination could further reduce U251 cells in G2 phase and LN229 cells in G2 and S phases. Thirdly, SCU and C18H17NO6 both induced the apoptosis of U251 and LN229. The combination further increased the apoptosis rate of both cells compared with the two drugs alone. Furthermore, SCU and C18H17NO6 both inhibited the lateral and vertical migration of both cells, which was further repressed by the combination. More importantly, the effect of SCU and the combination was better than positive control-temozolomide, and the toxicity was low. Additionally, SCU and C18H17NO6 could suppress the growth of glioma in vivo, and the effect of the combination was better. Finally, SCU and the combination upregulated the presenilin 1 (PSEN1) level but inactivated the phosphatidylinositol 3−kinase (PI3K)-protein kinase B (AKT) signaling in vitro and in vivo. Accordingly, we concluded that scutellarin and its combination with C18H17NO6 suppressed the proliferation/growth and migration and induced the apoptosis of glioma, in which the mechanism might be associated with the repression of PSEN1/PI3K-AKT signaling axis.

Primary Cilia–Related Pathways Moderate the Development and Therapy Resistance of Glioblastoma

As microtubule-based structures, primary cilia are typically present on the cells during the G0 or G1-S/G2 phase of the cell cycle and are closely related to the development of the central nervous system. The presence or absence of this special organelle may regulate the central nervous system tumorigenesis (e.g., glioblastoma) and several degenerative diseases. Additionally, the development of primary cilia can be regulated by several pathways. Conversely, primary cilia are able to regulate a few signaling transduction pathways. Therefore, development of the central nervous system tumors in conjunction with abnormal cilia can be regulated by up- or downregulation of the pathways related to cilia and ciliogenesis. Here, we review some pathways related to ciliogenesis and tumorigenesis, aiming to provide a potential target for developing new therapies at genetic and molecular levels.

MicroRNA Profiling Reveals an Abundant ssc-miRNA-148a-3p that Promotes Proliferation and Differentiation during Intramuscular Adipogenesis in Chinese Guizhou Congjiang Pigs Breeds by targeting PPARGC1A

BackgroundIntramuscular fat development is regulated by a series of complicated processes, and non-coding RNA (ncRNA) such as microRNA (miRNA) plays a critical role during intramuscular preadipocyte proliferation and differentiation development in pigs. In present research, we detected the expression profiles of miRNA during different differentiation stages, namely, day 0 (D0), day 4 (D4), and day 8 (D8), of intramuscular preadipocytes from the longissimus dorsi muscle of Chinese Guizhou Congjiang pigs to provide first insights into their potential involvement in intramuscular preadipocyte development. And we investigated the function of miR-148a-3p in adipocyte proliferation, apoptosis, and differentiation. ResultsA total of 67, 95, and 16 differentially expressed (DE) miRNAs were detected between D4 and D0, between D8 and D0, and between D8 and D4, respectively. We further characterized the role of miR-148a-3p which was differentially expressed and highest expressed abundance in D0, D4, and D8. To explore the role of miR-148a-3p in porcine intramuscular preadipocyte, miR-148a-3p mimics and inhibitors were used to perform miR-148a-3p overexpression and knockdown, respectively. Overexpression of miRNA-148a-3p increased the number of intramuscular preadipocytes in the S/G2 phase of the cell cycle and decreased the proportion of cells in the G0/G1 phase. Moreover, it promoted proliferation by regulation of cyclin B, cyclin G1, cyclin D1, CDK2, CDK3, and CDK4 and inhibited apoptosis of intramuscular preadipocyte by regulating the expression of Caspase-3, Bax, and Bcl-2. Meanwhile, the mimics of miR-148a-3p dramatically promoted intramuscular preadipocyte differentiation and upregulated the expression levels of adipogenic marker genes PPARγ, FASN, FABP4, HSL, APOE, LPL, and CEBPα. Furthermore, miR-148a-3p promoted intramuscular preadipocyte differentiation via restraining the AMPK/ACC/CPT1C signaling pathway. PPARGC1A was identified as a target gene of miR-148a-3p by luciferase activity and western blotting assays. ConclusionOur study provides novel insights into the regulatory mechanisms underlying intramuscular preadipocyte development and identified amount of miRNAs whose regulatory potential will need to be explored in the future. Our results establish that miR-148a-3p promoted adipocyte differentiation by targeting PPARGC1A.