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OBJECTIVE: PDZ-binding kinase (PBK) has been reported as a poor prognostic factor and is a promising molecular target for anticancer therapeutics. Here, we aimed to investigate the effect of specific PBK inhibitor OTS514 on the survival of OSCC cells. METHODS: Four OSCC cell lines (HSC-2, HSC-3, SAS, and OSC-19) were used to examine the effect of OTS514 on cell survival and apoptosis. DNA microarray analysis was conducted to investigate the effect of OTS514 on gene expression in OSCC cells. Gene set enrichment analysis was performed to identify molecular signatures related to the antiproliferative effect of OTS514. RESULTS: OTS514 decreased the cell survival of OSCC cells dose-dependently, and administration of OTS514 readily suppressed the HSC-2-derived tumor growth in immunodeficient mice. Treatment with OTS514 significantly increased the number of apoptotic cells and caspase-3/7 activity. Importantly, OTS514 suppressed the expression of E2F target genes with a marked decrease in protein levels of E2F1, a transcriptional factor. Moreover, TP53 knockdown attenuated OTS514-induced apoptosis. CONCLUSION: OTS514 suppressed the proliferation of OSCC cells by downregulating the expression of E2F target genes and induced apoptosis by mediating the p53 signaling pathway. These results highlight the clinical application of PBK inhibitors in the development of molecular-targeted therapeutics against OSCC.
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Carcinoma de Células Escamosas , Quinases de Proteína Quinase Ativadas por Mitógeno , Neoplasias Bucais , Quinolonas , Tiofenos , Animais , Camundongos , Linhagem Celular Tumoral , MAP Quinases Reguladas por Sinal Extracelular , Carcinoma de Células Escamosas/tratamento farmacológico , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Neoplasias Bucais/tratamento farmacológico , Neoplasias Bucais/genética , Neoplasias Bucais/metabolismo , Apoptose , Proliferação de Células/genéticaRESUMO
BACKGROUND: Targeted knock-in assisted by the CRISPR/Cas9 system is an advanced technology with promising applications in various research fields including medical and agricultural sciences. However, improvements in the efficiency, precision, and specificity of targeted knock-in are prerequisites to facilitate the practical application of this technology. To improve the efficiency of targeted knock-in, it is necessary to have a molecular system that allows sensitive monitoring of targeted knock-in events with simple procedures. METHODS AND RESULTS: We developed an assay, named CD55 correction assay, with which to monitor CD55 gene correction accomplished by targeted knock-in. To create the reporter clones used in this assay, we initially introduced a 7.7-kb heterozygous deletion covering CD55 exons 2-5, and then incorporated a truncating mutation within exon 4 of the remaining CD55 allele in human cell lines. The resultant reporter clones that lost the CD55 protein on the cell membrane were next transfected with Cas9 constructs along with a donor plasmid carrying wild-type CD55 exon 4. The cells were subsequently stained with fluorescence-labeled CD55 antibody and analyzed by flow cytometry to detect CD55-positive cells. These procedures allow high-throughput, quantitative detection of targeted gene correction events occurring in an endogenous human gene. CONCLUSIONS: The current study demonstrated the utility of the CD55 correction assay to sensitively quantify the efficiency of targeted knock-in. When used with the PIGA correction assay, the CD55 correction assay will help accurately determine the efficiency of targeted knock-in, precluding possible experimental biases caused by cell line-specific and locus-specific factors.
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There has been a great deal of research on cell division and its mechanisms; however, its processes still have many unknowns. To find novel proteins that regulate cell division, we performed the screening using siRNAs and/or the expression plasmid of the target genes and identified leucine zipper protein 1 (LUZP1). Recent studies have shown that LUZP1 interacts with various proteins and stabilizes the actin cytoskeleton; however, the function of LUZP1 in mitosis is not known. In this study, we found that LUZP1 colocalized with the chromosomal passenger complex (CPC) at the centromere in metaphase and at the central spindle in anaphase and that these LUZP1 localizations were regulated by CPC activity and kinesin family member 20A (KIF20A). Mass spectrometry analysis identified that LUZP1 interacted with death-associated protein kinase 3 (DAPK3), one regulator of the cleavage furrow ingression in cytokinesis. In addition, we found that LUZP1 also interacted with myosin light chain 9 (MYL9), a substrate of DAPK3, and comprehensively inhibited MYL9 phosphorylation by DAPK3. In line with a known role for MYL9 in the actin-myosin contraction, LUZP1 suppression accelerated the constriction velocity at the division plane in our time-lapse analysis. Our study indicates that LUZP1 is a novel regulator for cytokinesis that regulates the constriction velocity of the contractile ring.
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Citocinese , Zíper de Leucina , Citocinese/genética , Constrição , Citoesqueleto de Actina , MitoseRESUMO
5' adenosine monophosphate-activated protein kinase-related kinase 5 (ARK5) is involved in mitochondrial ATP production and associated with poor prognosis of multiple myeloma (MM). However, the molecular mechanisms of ARK5 in MM remain largely unknown. This study examined the pathogenic role of ARK5 in mitochondria by using genetically modified isogenic cell clones with or without ARK5 in human myeloma cell lines, KMS-11 and Sachi, which overexpress ARK5. The biallelic knockout of ARK5 (ARK5-KO) inhibited cell proliferation, colony formation, and migration with increased apoptosis. Mitochondrial fusion was enhanced in ARK5-KO cells, unlike in ARK5 wild-type (ARK5-WT) cells, which exhibited increased mitochondrial fission. Furthermore, ARK5-KO cells demonstrated a lower phosphorylated dynamin-related protein 1 at serine 616, higher protein expression of mitofusin-1 (MFN1) and MFN2, optic atrophy 1 with a lower level of ATP, and higher levels of lactate and reactive oxygen species than ARK5-WT cells. Our findings suggest that ARK5-enhanced myeloma cells can survive associated mitochondrial fission and activity. This study first revealed the relationship between ARK5 and mitochondrial morphological dynamics. Thus, our outcomes show novel aspects of mitochondrial biology of ARK5, which can afford a more advanced treatment approach for unfavorable MM expressing ARK5.
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CRISPR/Cas9 is a powerful genome editing system that has remarkably facilitated gene knockout and targeted knock-in. To accelerate the practical use of CRISPR/Cas9, however, it remains crucial to improve the efficiency, precision, and specificity of genome editing, particularly targeted knock-in, achieved with this system. To improve genome editing efficiency, researchers should first have a molecular assay that allows sensitive monitoring of genome editing events with simple procedures. In the current study, we demonstrate that genome editing events occurring in L1CAM, an X-chromosome gene encoding a cell surface protein, can be readily monitored using flow cytometry (FCM) in multiple human cell lines including neuroblastoma cell lines. The abrogation of L1CAM was efficiently achieved using Cas9 nucleases which disrupt exons encoding the L1CAM extracellular domain, and was easily detected by FCM using anti-L1CAM antibodies. Notably, L1CAM-abrogated cells could be quantified by FCM in four days after transfection with a Cas9 nuclease, which is much faster than an established assay based on the PIGA gene. In addition, the L1CAM-based assay allowed us to measure the efficiency of targeted knock-in (correction of L1CAM mutations) accomplished through different strategies, including a Cas9 nuclease-mediated method, tandem paired nicking, and prime editing. Our L1CAM-based assay using FCM enables rapid and sensitive quantification of genome editing efficiencies and will thereby help researchers improve genome editing technologies.
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Edição de Genes , Molécula L1 de Adesão de Célula Nervosa , Humanos , Edição de Genes/métodos , Citometria de Fluxo , Sistemas CRISPR-Cas/genética , Molécula L1 de Adesão de Célula Nervosa/genética , Linhagem CelularRESUMO
Malignant mesothelioma (MMe) is a rare but aggressive malignancy. Although the molecular genetics of MMe is known, including BRCA1-associated protein-1 (BAP1) gene alterations, the prognosis of MMe patients remains poor. Here, we generated BAP1 knockout (BAP1-KO) human mesothelial cell clones to develop molecular-targeted therapeutics based on genetic alterations in MMe. cDNA microarray and quantitative RT-PCR (qRT-PCR) analyses revealed high expression of a calcium/calmodulin-dependent protein kinase type II subunit delta (CAMK2D) gene in the BAP1-KO cells. CAMK2D was highly expressed in 70% of the human MMe tissues (56/80) and correlated with the loss of BAP1 expression, making it a potential diagnostic and therapeutic target for BAP1-deficient MMe. We screened an anticancer drugs library using BAP1-KO cells and successfully identified a CaMKII inhibitor, KN-93, which displayed a more potent and selective antiproliferative effect against BAP1-deficient cells than cisplatin or pemetrexed. KN-93 significantly suppressed the tumor growth in mice xenografted with BAP1-deficient MMe cells. This study is the first to provide a potential molecular-targeted therapeutic approach for BAP1-deficient MMe.
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The hilly and rural areas' people of Bangladesh have a great history of putting into use numerous traditional medicinal plants to cure diseases. Therefore, with ethanol extract of Molineria capitulata (EEMC), methanol extract of Trichosanthes tricuspidata (METT), and methanol extract of Amorphophallus campanulatus (MEAC), we mandate evaluation of in vitro α-amylase inhibition, antioxidants, and molecular docking, and ADMET/T analysis. According to iodine starch methods, α-amylase inhibition was performed, and quantitative total phenolic and flavonoid content was determined by established methods, whereas DPPH free radical scavenging and reducing power assays were performed in previously established protocols, respectively. A comparative study among three plants (EEMC, METT, and MEAC) possessed a significant (p < 0.01) effect but EEMC showed the highest impact on enzyme inhibition. Plants in the measuring phenolic content METT and flavonoid measurement MEAC displayed most potent in the same way in the DPPH test was METT, and in reducing power capability MEAC has showed the highest effect between three extracts. Docking's study also reveals the compounds of METT (Cyclotricuspidoside A and Cyclotricuspidoside C) exhibit the superior score among all the compounds. This finding indicates that EEMC, METT, and MEAC substantially impact α-amylase inhibition along with antioxidants. In silico study also reveals the potency of these plants, but further in-depth, precise molecular studies are needed.
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Background and aim: This study evaluated the anxiolytic, antidepressant, and antioxidant activity of the methanol extract of Canarium resiniferum (MECR) leaves, and determined the total phenolic and flavonoid contents in this extract. Experimental procedure: The anxiolytic effect of MECR (100, 200, 400 mg/kg, p. o.) was tested in mice using the elevated plus-maze (EPM) test, the hole-board test (HBT), and the light-dark box (LDB) test. Its antidepressant effect was evaluated in the tail suspension (TST) and the forced swim (FST) tests. The total phenolic (TPC) and flavonoid (TFC) content was measured using standard colorimetric assays. Antioxidant activity was determined using the DPPH radical scavenging and ferric reducing antioxidant power (FRAP) assays. Results and conclusion: MECR, at all doses, showed dose-dependent anxiolytic activity. At 400 mg/kg, it significantly increased the time spent and number of entries in the open arms (EPM test), the number of head-dips (HBT), and the time spent into the light compartment (LDB) test compared to the control. In the TST and FST, MECR dose-dependently reduced the duration of immobility compared to untreated animals. This was significant for all doses except for 100 mg/kg in the FST model. MECR showed high TPC and TFC (90.94 ± 0.75 mg GAE/g and 51.54 ± 0.78 mg QE/g of dried extract, respectively) and displayed potent activity in the DPPH radical scavenging (IC50 = 177.82 µg/mL) and FRAP assays. These findings indicate that C. resiniferum has the potential to alleviate anxiety and depression disorders, which merits further exploration.
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Tandem paired nicking (TPN) is a method of genome editing that enables precise and relatively efficient targeted knock-in without appreciable restraint by p53-mediated DNA damage response. TPN is initiated by introducing two site-specific nicks on the same DNA strand using Cas9 nickases in such a way that the nicks encompass the knock-in site and are located within a homologous region between a donor DNA and the genome. This nicking design results in the creation of two nicks on the donor DNA and two in the genome, leading to relatively efficient homology-directed recombination between these DNA fragments. In this study, we sought to identify the optimal design of TPN experiments that would improve the efficiency of targeted knock-in, using multiple reporter systems based on exogenous and endogenous genes. We found that efficient targeted knock-in via TPN is supported by the use of 1700-2000-bp donor DNAs, exactly 20-nt-long spacers predicted to be efficient in on-target cleavage, and tandem-paired Cas9 nickases nicking at positions close to each other. These findings will help establish a methodology for efficient and precise targeted knock-in based on TPN, which could broaden the applicability of targeted knock-in to various fields of life science.
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Sistemas CRISPR-Cas , DNA/análise , RNA Guia de Cinetoplastídeos/genética , Linhagem Celular Tumoral , Quebras de DNA de Cadeia Dupla , Edição de Genes , Técnicas de Introdução de Genes , Marcação de Genes/métodos , Genes Reporter , Engenharia Genética , Células HCT116 , Recombinação Homóloga , Humanos , Plasmídeos/metabolismo , Recombinação GenéticaRESUMO
Targeted knock-in supported by the CRISPR/Cas systems enables the insertion, deletion, and substitution of genome sequences exactly as designed. Although this technology is considered to have wide range of applications in life sciences, one of its prerequisites for practical use is to improve the efficiency, precision, and specificity achieved. To improve the efficiency of targeted knock-in, there first needs to be a reporter system that permits simple and accurate monitoring of targeted knock-in events. In the present study, we created such a system using the PIGP gene, an autosomal gene essential for GPI-anchor biosynthesis, as a reporter gene. We first deleted a PIGP allele using Cas9 nucleases and then incorporated a truncating mutation into the other PIGP allele in two near-diploid human cell lines. The resulting cell clones were used to monitor the correction of the PIGP mutations by detecting GPI anchors distributed over the cell membrane via flow cytometry. We confirmed the utility of these reporter clones by performing targeted knock-in in these clones via a Cas9 nickase-based strategy known as tandem paired nicking, as well as a common process using Cas9 nucleases, and evaluating the efficiencies of the achieved targeted knock-in. We also leveraged these reporter clones to test a modified procedure for tandem paired nicking and demonstrated a slight increase in the efficiency of targeted knock-in by the new procedure. These data provide evidence for the utility of our PIGP-based assay system to quantify the efficiency of targeted knock-in and thereby help improve the technology of targeted knock-in.
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Proteína 9 Associada à CRISPR/genética , Sistemas CRISPR-Cas , Membrana Celular/genética , Citometria de Fluxo , Proteínas Ligadas por GPI/genética , Técnicas de Introdução de Genes , Genes Reporter , Hexosiltransferases/genética , Proteínas de Membrana/genética , Proteína 9 Associada à CRISPR/metabolismo , Membrana Celular/metabolismo , Proteínas Ligadas por GPI/biossíntese , Regulação da Expressão Gênica , Células HCT116 , Hexosiltransferases/metabolismo , Humanos , Proteínas de Membrana/metabolismo , MutaçãoRESUMO
Internal tandem duplication (ITD) of FMS-like tyrosine kinase 3 (FLT3) confers poor prognosis and is found in approximately 25% of cases of acute myeloid leukemia (AML). Although FLT3 inhibitors have shown clinical benefit in patients with AML harboring FLT3-ITD, the therapeutic effect is limited. Here, to explore alternative therapeutics, we established a cellular model of monoallelic FLT3ITD/WT cells using the CRISPR-Cas9 system in a human myeloid leukemia cell line, K562. cDNA microarray analysis revealed elevated CD52 expression in K562-FLT3ITD/WT cells compared to K562-FLT3WT/WT cells, an observation that was further confirmed by quantitative real-time-PCR and flow cytometric analyses. The elevated expression of CD52 in K562-FLT3ITD/WT cells was decreased in wild-type FLT3 (FLT3-WT) knock-in K562-FLT3ITD/WT cells. In K562-FLT3ITD/WT cells, a STAT5 inhibitor, pimozide, downregulated CD52 protein expression while an AKT inhibitor, afuresertib, did not affect CD52 expression. Notably, an anti-CD52 antibody, alemtuzumab, induced significant antibody-dependent cell-mediated cytotoxicity (ADCC) in K562-FLT3ITD/WT cells compared to K562-FLT3WT/WT cells. Furthermore, alemtuzumab significantly suppressed the xenograft tumor growth of K562-FLT3ITD/WT cells in severe combined immunodeficiency (SCID) mice. Taken together, our data suggested that genetically modified FLT3-ITD knock-in human myeloid leukemia K562 cells upregulated CD52 expression via activation of STAT5, and alemtuzumab showed an antitumor effect via induction of ADCC in K562-FLT3ITD/WT cells. Our findings may allow establishment of a new therapeutic option, alemtuzumab, to treat leukemia with the FLT3-ITD mutation.
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Malignant pleural mesothelioma (MPM) is an aggressive malignancy of the pleura that is currently incurable due to the lack of an effective early diagnostic method and specific medication. The CDKN2A (p16) and NF2 genes are both frequently mutated in MPM. To understand how these mutations contribute to MPM tumor growth, we generated NF2/p16 double-knockout (DKO) cell clones using human MeT-5A and HOMC-B1 mesothelial cell lines. Cell growth and migration activities were significantly increased in DKO compared with parental cells. cDNA microarray analysis revealed differences in global gene expression profiles between DKO and parental cells. Quantitative PCR and western blot analyses showed upregulation of CD24 concomitant with increased phosphorylation of AKT, p70S6K, and c-Jun in DKO clones. This upregulation was abrogated by exogenous expression of NF2 and p16. CD24 knockdown in DKO cells significantly decreased TGF-ß1 expression and increased expression of E-cadherin, an epithelial-mesenchymal transition marker. CD24 was highly expressed in human mesothelioma tissues (28/45 cases, 62%) and associated with the loss of NF2 and p16. Public data analysis revealed a significantly shorter survival time in MPM patients with high CD24 gene expression levels. These results strongly indicate the potential use of CD24 as a prognostic marker as well as a novel diagnostic and therapeutic target for MPM.