Microcephaly Gene Mcph1 Deficiency Induces p19ARF-Dependent Cell Cycle Arrest and Senescence.

MCPH1 has been identified as the causal gene for primary microcephaly type 1, a neurodevelopmental disorder characterized by reduced brain size and delayed growth. As a multifunction protein, MCPH1 has been reported to repress the expression of TERT and interact with transcriptional regulator E2F1. However, it remains unclear whether MCPH1 regulates brain development through its transcriptional regulation function. This study showed that the knockout of Mcph1 in mice leads to delayed growth as early as the embryo stage E11.5. Transcriptome analysis (RNA-seq) revealed that the deletion of Mcph1 resulted in changes in the expression levels of a limited number of genes. Although the expression of some of E2F1 targets, such as Satb2 and Cdkn1c, was affected, the differentially expressed genes (DEGs) were not significantly enriched as E2F1 target genes. Further investigations showed that primary and immortalized Mcph1 knockout mouse embryonic fibroblasts (MEFs) exhibited cell cycle arrest and cellular senescence phenotype. Interestingly, the upregulation of p19ARF was detected in Mcph1 knockout MEFs, and silencing p19Arf restored the cell cycle and growth arrest to wild-type levels. Our findings suggested it is unlikely that MCPH1 regulates neurodevelopment through E2F1-mediated transcriptional regulation, and p19ARF-dependent cell cycle arrest and cellular senescence may contribute to the developmental abnormalities observed in primary microcephaly.

CDC45 promotes the stemness and metastasis in lung adenocarcinoma by affecting the cell cycle.

OBJECTIVE: This study aimed to assess the functions of cell division cycle protein 45 (CDC45) in Non-small cell lung cancer (NSCLC) cancer and its effects on stemness and metastasis. METHODS: Firstly, differentially expressed genes related to lung cancer metastasis and stemness were screened by differential analysis and lasso regression. Then, in vitro, experiments such as colony formation assay, scratch assay, and transwell assay were conducted to evaluate the impact of CDC45 knockdown on the proliferation and migration abilities of lung cancer cells. Western blotting was used to measure the expression levels of related proteins and investigate the regulation of CDC45 on the cell cycle. Finally, in vivo model with subcutaneous injection of lung cancer cells was performed to verify the effect of CDC45 on tumor growth. RESULTS: This study identified CDC45 as a key gene potentially influencing tumor stemness and lymph node metastasis. Knockdown of CDC45 not only suppressed the proliferation and migration abilities of lung cancer cells but also caused cell cycle arrest at the G2/M phase. Further analysis revealed a negative correlation between CDC45 and cell cycle-related proteins, stemness-related markers, and tumor mutations. Mouse experiments confirmed that CDC45 knockdown inhibited tumor growth. CONCLUSION: As a novel regulator of stemness, CDC45 plays a role in regulating lung cancer cell proliferation, migration, and cell cycle. Therefore, CDC45 may serve as a potential target for lung cancer treatment and provide a reference for further mechanistic research and therapeutic development.

Gulp1 regulates chondrocyte growth arrest and differentiation via the TGF-ß/SMAD2/3 pathway.

Chondrocyte differentiation is crucial for cartilage formation. However, the complex processes and mechanisms coordinating chondrocyte proliferation and differentiation remain incompletely understood. Here, we report a novel function of the adaptor protein Gulp1 in chondrocyte differentiation. Gulp1 expression is upregulated during chondrogenic differentiation. Gulp1 knockdown in chondrogenic ATDC5 cells reduces the expression of chondrogenic and hypertrophic marker genes during differentiation. Furthermore, Gulp1 knockdown impairs cell growth arrest during chondrocyte differentiation and reduces the expression of the cyclin-dependent kinase inhibitor p21. The activation of the TGF-ß/SMAD2/3 pathway, which is associated with p21 expression in chondrocytes, is impaired in Gulp1 knockdown cells. Collectively, these results demonstrate that Gulp1 contributes to cell growth arrest and chondrocyte differentiation by modulating the TGF-ß/SMAD2/3 pathway.

DNA hypomethylation activates Cdk4/6 and Atr to induce DNA replication and cell cycle arrest to constrain liver outgrowth in zebrafish.

Coordinating epigenomic inheritance and cell cycle progression is essential for organogenesis. UHRF1 connects these functions during development by facilitating maintenance of DNA methylation and cell cycle progression. Here, we provide evidence resolving the paradoxical phenotype of uhrf1 mutant zebrafish embryos which have activation of pro-proliferative genes and increased number of hepatocytes in S-phase, but the liver fails to grow. We uncover decreased Cdkn2a/b and persistent Cdk4/6 activation as the mechanism driving uhrf1 mutant hepatocytes into S-phase. This induces replication stress, DNA damage and Atr activation. Palbociclib treatment of uhrf1 mutants prevented aberrant S-phase entry, reduced DNA damage, and rescued most cellular and developmental phenotypes, but it did not rescue DNA hypomethylation, transposon expression or the interferon response. Inhibiting Atr reduced DNA replication and increased liver size in uhrf1 mutants, suggesting that Atr activation leads to dormant origin firing and prevents hepatocyte proliferation. Cdkn2a/b was downregulated pro-proliferative genes were also induced in a Cdk4/6 dependent fashion in the liver of dnmt1 mutants, suggesting DNA hypomethylation as a mechanism of Cdk4/6 activation during development. This shows that the developmental defects caused by DNA hypomethylation are attributed to persistent Cdk4/6 activation, DNA replication stress, dormant origin firing and cell cycle inhibition.

Protein phosphatase 1 regulatory subunit 15 A promotes translation initiation and induces G2M phase arrest during cuproptosis in cancers.

Copper ions play a crucial role as cofactors for essential enzymes in cellular processes. However, when the intracellular concentration of copper ions exceeds the homeostatic threshold, they become toxic to cells. In our study, we demonstrated that elesclomol, as a carrier of copper ions, caused an upregulation of protein phosphatase 1 regulatory subunit 15 A (PPP1R15A), which plays a role in regulating substrate selectivity of protein phosphatase 1 during cuproptosis. Mechanistically, we investigated that PPP1R15A activated translation initiation by dephosphorylating eukaryotic translation initiation factor 2 subunit alpha at the S51 residue through protein phosphatase 1 and phosphorylating eukaryotic translation initiation factor 4E binding protein 1 at the T70 residue. In addition, PPP1R15A reduced H3K4 methylation by altering the phosphorylation of histone methyltransferases, which led to the silencing of MYC and G2M phase arrest.

Loss-of-function of kinesin-5 KIF11 causes microcephaly, chorioretinopathy, and developmental disorders through chromosome instability and cell cycle arrest.

Kinesin motors play a fundamental role in development by controlling intracellular transport, spindle assembly, and microtubule organization. In humans, patients carrying mutations in KIF11 suffer from an autosomal dominant inheritable disease called microcephaly with or without chorioretinopathy, lymphoedema, or mental retardation (MCLMR). While mitotic functions of KIF11 proteins have been well documented in centrosome separation and spindle assembly, cellular mechanisms underlying KIF11 dysfunction and MCLMR remain unclear. In this study, we generate KIF11-inhibition chick and zebrafish models and find that KIF11 inhibition results in microcephaly, chorioretinopathy, and severe developmental defects in vivo. Notably, loss-of-function of KIF11 causes the formation of monopolar spindle and chromosome misalignment, which finally contribute to cell cycle arrest, chromosome instability, and cell death. Our results demonstrate that KIF11 is crucial for spindle assembly, chromosome alignment, and cell cycle progression of progenitor stem cells, indicating a potential link between polyploidy and MCLMR. Our data have revealed that KIF11 inhibition cause microcephaly, chorioretinopathy, and development disorders through the formation of monopolar spindle, polyploid, and cell cycle arrest.

Skp2-Cyclin A Interaction Is Necessary for Mitotic Entry and Maintenance of Diploidy.

Skp2, the substrate recognition component of the SCFSkp2 ubiquitin ligase, has been implicated in the targeted destruction of a number of key cell cycle regulators and the promotion of S-phase. One of its critical targets is the Cyclin dependent kinase (Cdk) inhibitor p27, and indeed the overexpression of Skp2 in a number of cancers is directly correlated with the premature degradation of p27. Skp2 was first identified as a protein that interacts with Cyclin A in transformed cells, but its role in this complex has remained unclear. In this paper, we demonstrate that Skp2 interacts with Cyclin A in Drosophila and is required to maintain Cyclin A levels and permit mitotic entry. Failure of mitotic entry in Skp2 mutant cells results in polyploidy. If these cells enter mitosis again they are unable to properly segregate their chromosomes, leading to checkpoint dependent cell cycle arrest or apoptosis. Thus, Skp2 is required for mitosis and for maintaining diploidy and genome stability.

COX6C expression driven by copy amplification of 8q22.2 regulates cell proliferation via mediation of mitosis by ROS-AMPK signaling in lung adenocarcinoma.

Copy number variations (CNVs) play a vital role in regulating genes expression and tumorigenesis. We explored the copy number alterations in early-stage lung adenocarcinoma using high-throughput sequencing and nucleic acid flight mass spectrometry technology, and found that 8q22.1-22.2 is frequently amplified in lung adenocarcinoma tissues. COX6C localizes on the region and its expression is notably enhanced that driven by amplification in lung adenocarcinoma. Knockdown of COX6C significantly inhibits the cell proliferation, and induces S-G2/M cell cycle arrest, mitosis deficiency and apoptosis. Moreover, COX6C depletion causes a deficiency in mitochondrial fusion, and impairment of oxidative phosphorylation. Mechanistically, COX6C-induced mitochondrial deficiency stimulates ROS accumulation and activates AMPK pathway, then leading to abnormality in spindle formation and chromosome segregation, activating spindle assemble checkpoint, causing mitotic arrest, and ultimately inducing cell apoptosis. Collectively, we suggested that copy amplification-mediated COX6C upregulation might serves as a prospective biomarker for prognosis and targeting therapy in patients with lung adenocarcinoma.

Targeting Cyclin-Dependent Kinase 1 Induces Apoptosis and Cell Cycle Arrest of Activated Hepatic Stellate Cells.

Liver fibrosis is the integral process of chronic liver diseases caused by multiple etiologies and characterized by excessive deposition of extracellular matrix (ECM). During liver fibrosis, hepatic stellate cells (HSCs) transform into a highly proliferative, activated state, producing various cytokines, chemokines, and ECM. However, the precise mechanisms that license HSCs into the highly proliferative state remain unclear. Cyclin-dependent kinase 1 (CDK1) is a requisite event for the transition of the G1/S and G2/M phases in eukaryotic cells. In this study, it is demonstrated that CDK1 and its activating partners, Cyclin A2 and Cyclin B1, are upregulated in both liver fibrosis/cirrhosis patient specimens and the murine hepatic fibrosis models, especially in activated HSCs. In vitro, CDK1 is upregulated in spontaneously activated HSCs, and inhibiting CDK1 with specific small-molecule inhibitors (CGP74514A, RO-3306, or Purvalanol A) orshort hairpin RNAs (shRNAs) resulted in HSC apoptosis and cell cycle arrest by regulating Survivin expression. Above all, it is illustrated that increased CDK1 expression licenses the HSCs into a highly proliferative state and can serve as a potential therapeutic target in liver fibrosis.

Depletion of DNTTIP2 Induces Cell Cycle Arrest in Pancreatic Cancer Cells.

BACKGROUND/AIM: Pancreatic cancer is one of the most lethal malignant cancers worldwide and the seventh most common cause of cancer-related death in both sexes. Herein, we analyzed open access data and discovered that expression of a gene called deoxynucleotidyltransferase terminal-interacting protein 2 (DNTTIP2) is linked to prognosis of pancreatic ductal adenocarcinoma (PDAC). We then elucidated the role of DNTTIP2 in the proliferation of pancreatic cancer cells in vitro. MATERIALS AND METHODS: A WST-8 assay, cell cycle analysis, Annexin-V staining, quantitative reverse transcription-PCR, and western blot analysis were conducted to assess cell proliferation, cell cycle, apoptosis, and expression of DNTTIP2 mRNA and protein, respectively, in DNTTIP2-depleteted MIA-PaCa-2 and PK-1 cells. RESULTS: Depletion of DNTTIP2 induced G1 arrest in MIA-PaCa-2 cells by decreasing expression of special AT-rich sequence binding protein 1 (SATB1) and cyclin-dependent kinase 6 (CDK6). In addition, depletion of DNTTIP2 induced G2 arrest in PK-1 cells by decreasing expression of CDK1. Depletion of DNTTIP2 did not induce apoptosis in MIA-PaCa-2 or PK-1 cells. CONCLUSION: DNTTIP2 is involved in proliferation of pancreatic cancer cells. Thus, DNTTIP2 is a potential target for inhibiting progression of pancreatic cancers.

CPEB2 inhibit cell proliferation through upregulating p21 mRNA stability in glioma.

Glioma is the most common primary malignant brain tumor in adults and remains an incurable disease at present. Thus, there is an urgent need for progress in finding novel molecular mechanisms that control the progression of glioma which could be used as therapeutic targets for glioma patients. The RNA binding protein cytoplasmic polyadenylate element-binding protein 2 (CPEB2) is involved in the pathogenesis of several tumors. However, the role of CPEB2 in glioma progression is unknown. In this study, the functional characterization of the role and molecular mechanism of CPEB2 in glioma were examined using a series of biological and cellular approaches in vitro and in vivo. Our work shows CPEB2 is significantly downregulated in various glioma patient cohorts. Functional characterization of CPEB2 by overexpression and knockdown revealed that it inhibits glioma cell proliferation and promotes apoptosis. CPEB2 exerts an anti-tumor effect by increasing p21 mRNA stability and inducing G1 cell cycle arrest in glioma. Overall, this work stands as the first report of CPEB2 downregulation and involvement in glioma pathogenesis, and identifies CPEB2 as an important tumor suppressor gene through targeting p21 in glioma, which revealed that CPEB2 may become a promising predictive biomarker for prognosis in glioma patients.

KLHL29-mediated DDX3X degradation promotes chemosensitivity by abrogating cell cycle checkpoint in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is a heterogeneous breast cancer subtype and accounts for approximately 15-20% of breast cancer cases. In this study, we identified KLHL29, which is an understudied member of the Kelch-like gene family, as a crucial tumor suppressor that regulates chemosensitivity in TNBC. KLHL29 expression was significantly downregulated in breast cancer tissues compared with adjacent normal tissues, and low levels of KLHL29 were associated with unfavorable prognoses. Ectopic KLHL29 suppressed, while depleting KLHL29 promoted, the growth, proliferation, migration, and invasion of TNBC. Mechanistically, KLHL29 recruited the CUL3 E3-ligase to the RNA-binding protein DDX3X, leading to the proteasomal degradation of the latter. This downregulation of DDX3X resulted in the destabilization of CCND1 mRNA and the consequent cell cycle arrest at G0/G1 phase. Remarkably, the DDX3X inhibitor RK33 combined with platinum-based chemotherapy can synergistically suppress TNBC that usually expresses low levels of KLHL29 and high levels of DDX3X using cancer cell-derived xenograft and patient-derived organoids models. Altogether, we uncovered the potential role for the KLHL29-DDX3X signaling cascade in the regulation of TNBC progression, thus providing a promising combination strategy for overcoming TNBC chemoresistance.

Uncovering NOTCH1 as a Promising Target in the Treatment of MLL-Rearranged Leukemia.

MLL rearrangement (MLLr) is responsible for the development of acute leukemias with poor outcomes. Therefore, new therapeutic approaches are urgently needed. The NOTCH1 pathway plays a critical role in the pathogenesis of many cancers including acute leukemia. Using a CRISPR/Cas9 MLL-AF4/-AF9 translocation model, the newly developed NOTCH1 inhibitor CAD204520 with less toxic side effects allowed us to unravel the impact of NOTCH1 as a pathogenic driver and potential therapeutic target in MLLr leukemia. RNA sequencing (RNA-seq) and RT-qPCR of our MLLr model and MLLr cell lines showed the NOTCH1 pathway was overexpressed and activated. Strikingly, we confirmed this elevated expression level in leukemia patients. We also demonstrated that CAD204520 treatment of MLLr cells significantly reduces NOTCH1 and its target genes as well as NOTCH1 receptor expression. This was not observed with a comparable cytarabine treatment, indicating the specificity of the small molecule. Accordingly, treatment with CAD204520 resulted in dose-dependent reduced proliferation and viability, increased apoptosis, and the induction of cell cycle arrest via the downregulation of MLL and NOTCH1 target genes. In conclusion, our findings uncover the oncogenic relevance of the NOTCH1 pathway in MLLr leukemia. Its inhibition leads to specific anti-leukemic effects and paves the way for further evaluation in clinical settings.

Licochalcone A induces cell cycle arrest and apoptosis via suppressing MAPK signaling pathway and the expression of FBXO5 in lung squamous cell cancer.

Lung squamous cell carcinoma (LSCC) is a highly heterogeneous malignancy with high mortality and few therapeutic options. Licochalcone A (LCA, PubChem ID: 5318998) is a chalcone extracted from licorice and possesses anticancer and anti­inflammatory activities. The present study aimed to elucidate the anticancer effect of LCA on LSCC and explore the conceivable molecular mechanism. MTT assay revealed that LCA significantly inhibited the proliferation of LSCC cells with less cytotoxicity towards human bronchial epithelial cells. 5­ethynyl­2'­deoxyuridine (EdU) assay demonstrated that LCA could reduce the proliferation rate of LSCC cells. The flow cytometric assays indicated that LCA increased the cell number of the G1 phase and induced the apoptosis of LSCC cells. LCA downregulated the protein expression of cyclin D1, cyclin E, CDK2 and CDK4. Meanwhile, LCA increased the expression level of Bax, cleaved poly(ADP­ribose)polymerase­1 (PARP1) and caspase 3, as well as downregulated the level of Bcl­2. Proteomics assay demonstrated that LCA exerted its antitumor effects via inhibiting mitogen­activated protein kinase (MAPK) signaling pathways and the expression of F­box protein 5 (FBXO5). Western blot analysis showed that LCA decreased the expression of p­ERK1/2, p­p38MAPK and FBXO5. In the xenograft tumors of LSCC, LCA significantly inhibited the volumes and weight of tumors in nude mice with little toxicity in vital organs. Therefore, the present study demonstrated that LCA effectively inhibited cell proliferation and induced apoptosis in vitro, and suppressed xenograft tumor growth in vivo. LCA may serve as a future therapeutic candidate of LSCC.

SETD4 inhibits prostate cancer development by promoting H3K27me3-mediated NUPR1 transcriptional repression and cell cycle arrest.

The suppressor of variegation enhancer of zeste-trithorax (SET) domain methyltransferases have been reported to function as key regulators in multiple tumor types by catalyzing histone lysine methylation. Nevertheless, our understanding on the role of these lysine methyltransferases, including SETD4, in prostate cancer (PCa) remains limited. Hence, the specific role of SETD4 in PCa was investigated in this study. The expression of SETD4 in PCa cells and tissue samples was downregulated in PCa cells and tissue specimens, and decreased SETD4 expression led to inferior clinicopathological characteristics in patients with PCa. knockdown of SETD4 facilitated the proliferation of PCa cells and accelerated cell cycle progression. Mechanistically, SETD4 repressed NUPR1 transcription by methylating H3K27 to generate H3K27me3, subsequently inactivated Akt pathway and impeded the tumorigenesis of PCa. Our results highlight that SETD4 prevents the development of PCa by catalyzing the methylation of H3K27 and suppressing NUPR1 transcription, subsequently inactivating the Akt signaling pathway. The findings suggest the potential application of SETD4 in PCa prognosis and therapeutics.

Cell-cycle arrest at the G1/S boundary enhances transient voltage-gated ion channel expression in human and insect cells.

Heterologous expression of recombinant ion channel subunits in cell lines is often limited by the presence of a low number of channels at the cell surface level. Here, we introduce a combination of two techniques: viral expression using the baculovirus system plus cell-cycle arrest at the G1/S boundary using either thymidine or hydroxyurea. This method achieved a manifold increase in the peak current density of expressed ion channels compared with the classical liposome-mediated transfection methods. The enhanced ionic current was accompanied by an increase in the density of gating charges, confirming that the increased yield of protein and ionic current reflects the functional localization of channels in the plasma membrane. This modified method of viral expression coordinated with the cell cycle arrest will pave the way to better decipher the structure and function of ion channels and their association with ion channelopathies.

BACH1 Loss Exerts Antitumor Effects on Mantle Cell Lymphoma Cells via Inducing a Tumor-Intrinsic Innate Immune Response and Cell-Cycle Arrest.

BTB and CNC homology 1 (BACH1) is a transcription repressor that regulates multiple physiological processes, including intracellular heme homeostasis and immune responses. Increasing lines of evidence indicate that BACH1 reshapes metastasis and metabolism of human solid tumors. However, its potential roles in mantle cell lymphoma (MCL) remain largely unknown. Here, we found that silencing BACH1 in MCL cells induced markedly cell-cycle arrest and cell apoptosis, whereas overexpression of BACH1 exhibited the opposite patterns. Increased BACH1 levels not only promoted tumor growth and dispersal in xenografts, but also conferred a long-term poor prognosis in patients with MCL. Interestingly, RNA sequencing analysis revealed noncanonical function of BACH1 in regulation of type I interferon (IFNI) response, DNA replication and repair, and cell cycle. Mechanistically, zinc finger and BTB domain containing 20 (ZBTB20) and HMG-box transcription factor 1 (HBP1) were for the first time identified as two novel downstream targets repressed by BACH1 in MCL cells. Further double-knockdown functional assays confirmed that loss of BACH1 induced ZBTB20-mediated IFNα production and HBP1-mediated cell-cycle arrest, indicating that BACH1-centered regulatory network may be a novel targetable vulnerability in MCL cells. IMPLICATIONS: BACH1 serves as a pleotropic regulator of tumor-intrinsic innate immune response and cell-cycle progression, disruption of which may offer a promising therapeutic strategy for MCL treatment.

Cell Cycle Regulation by NF-YC in Drosophila Eye Imaginal Disc: Implications for Synchronization in the Non-Proliferative Region.

Cell cycle progression during development is meticulously coordinated with differentiation. This is particularly evident in the Drosophila 3rd instar eye imaginal disc, where the cell cycle is synchronized and arrests at the G1 phase in the non-proliferative region (NPR), setting the stage for photoreceptor cell differentiation. Here, we identify the transcription factor Nuclear Factor-YC (NF-YC) as a crucial player in this finely tuned progression, elucidating its specific role in the synchronized movement of the morphogenetic furrow. Depletion of NF-YC leads to extended expression of Cyclin A (CycA) and Cyclin B (CycB) from the FMW to the NPR. Notably, NF-YC knockdown resulted in decreased expression of Eyes absent (Eya) but did not affect Decapentaplegic (Dpp) and Hedgehog (Hh). Our findings highlight the role of NF-YC in restricting the expression of CycA and CycB in the NPR, thereby facilitating cell-cycle synchronization. Moreover, we identify the transcriptional cofactor Eya as a downstream target of NF-YC, revealing a new regulatory pathway in Drosophila eye development. This study expands our understanding of NF-YC's role from cell cycle control to encompass developmental processes.

[Overexpression of connexin 40 (Cx40) inhibits the proliferation of H9c2 cardiomyocytes in rats by cell cycle arrest].

Objective To establish a stable strain of H9c2 cardiomyocytes overexpressing Cx40 and preliminarily investigate the effect of lentiviral vector-mediated Cx40 protein overexpression on the proliferation of H9c2 cells and its related mechanisms. Methods The Cx40 gene fragment was cloned from H9c2 cells by PCR and linked with lentivirus vector pLVX-IRES-Puro to obtain the recombinant plasmid pLVX-Flag-Cx40. Recombinant lentiviral particles carrying Flag-Cx40 were obtained by cotransfection with packaging plasmids into HEK293T cells. A stable expression strain (H9c2-Flag-Cx40 cell) was screened from infected H9c2 cells by purinomycin. The expression of Cx40 protein was detected by Western blot analysis, and the effect of Cx40 on H9c2 cells proliferation was determined by CCK-8 assay; cell cycle changes were measured by flow cytometry; the expression of the cell cycle protein cyclin D1 was detected by qRT-PCR and Western blot analysis. Co-immunoprecipitation (Co-IP) immunoprecipitation and Western blot analysis were used to identify the binding of Cx40 and Yes associated protein (YAP) in H9c2 cells; cytoplasmic and cytosolic proteins were isolated to detect the effect of Cx40 on the localization of YAP using Western blot analysis. Results Sequencing results showed that the recombinant pLVX-Flag-Cx40 expression vector was successfully established. A stable transfected cell line containing recombinant Flag-Cx40 lentivirus (H9c2-Flag-Cx40 cell) was successfully constructed from H9c2 cells. Compared with the control group, overexpression of Cx40 significantly reduced the proliferation of H9c2 cells, arrested the cell cycle at G0/G1 and reduced cyclin D1 expression. A significant increase in YAP expression was observed in the cytoplasm of the H9c2-Flag-Cx40 stable cell line, while the expression in the nucleus was significantly reduced. Cx40 bound to YAP in the cytoplasm and prevented it from entering the nucleus to play the role of transcriptional coactivation. Conclusion Overexpression of Cx40 induces cell-cycle arrest at G0/G1 phase and inhibits the proliferation in H9c2 cells.

Cell cycle arrest and p53 prevent ON-target megabase-scale rearrangements induced by CRISPR-Cas9.

The CRISPR-Cas9 system has revolutionized our ability to precisely modify the genome and has led to gene editing in clinical applications. Comprehensive analysis of gene editing products at the targeted cut-site has revealed a complex spectrum of outcomes. ON-target genotoxicity is underestimated with standard PCR-based methods and necessitates appropriate and more sensitive detection methods. Here, we present two complementary Fluorescence-Assisted Megabase-scale Rearrangements Detection (FAMReD) systems that enable the detection, quantification, and cell sorting of edited cells with megabase-scale loss of heterozygosity (LOH). These tools reveal rare complex chromosomal rearrangements caused by Cas9-nuclease and show that LOH frequency depends on cell division rate during editing and p53 status. Cell cycle arrest during editing suppresses the occurrence of LOH without compromising editing. These data are confirmed in human stem/progenitor cells, suggesting that clinical trials should consider p53 status and cell proliferation rate during editing to limit this risk by designing safer protocols.