scholarly journals EPEN-34. THE CRISPR-Cas9 SYSTEM-MEDIATED ENDOGENOUS GENE REARRANGEMENT INDUCED C11orf95-RELA FUSION IN VITRO AND IN VIVO THAT LED TO THE DEVELOPMENT OF EPENDYMOMA-LIKE TUMOR

2020 ◽  
Vol 22 (Supplement_3) ◽  
pp. iii314-iii315
Author(s):  
Mutsumi Takadera ◽  
Tatsuya Ozawa ◽  
Kaishi Satomi ◽  
Tetsuya Yamamoto ◽  
Koichi Ichimura
Keyword(s):  
Large Scale ◽  
Gene Rearrangement ◽  
Cultured Cells ◽  
Fusion Gene ◽  
Tumor Formation ◽  
Fusion Genes ◽  
Endogenous Gene ◽  
Human And Mouse

Abstract Recent large-scale genomic studies of ependymal tumors have identified recurrent RELA and YAP1 fusion genes in supratentorial ependymomas. The formation of the C11orf95-RELA fusion gene has been attributed to massive genomic rearrangement involving chromosome 11q termed Chromothripsis in many cases. However, the causal relationship has not been clarified experimentally. In this study, we developed a system to reproduce the oncogenic gene rearrangement using the CRISPR-Cas9 system and examined whether consequent endogenous ependymoma fusion genes are competent to form brain tumors in mice. Initially, to investigate whether C11orf95-RELA fusion can be formed by inducing the relevant gene rearrangement in vitro, we designed multiple guide RNAs on the human and mouse genomic loci and introduced them into cultured cells. RT-PCR and immunoblot analyses detected endogenous C11orf95-RELA fusion transcript and protein in both human and mouse cultured cells. Subsequently, we lentivirally introduced the gRNAs into a mouse brain. Brain tumor formation was observed from around 2 months after the lentivirus injection, thus indicating successful gene rearrangement followed by C11orf95-RELA fusion expression in vivo. Analysis of the tumor tissue confirmed the expression of the endogenous C11orf95-RELA fusion gene. These results suggested that a gene rearrangement is a primary mechanism to form the C11orf95-RELA fusion which is the direct driver of tumorigenesis. Our system to simulate a genomic event will provide significant insights into the understanding of the tumorigenic mechanism in ependymomas.

scholarly journals PDTM-27. THE CRISPR-Cas9 SYSTEM-MEDIATED ENDOGENOUS GENE-REARRANGEMENT INDUCED C11orf95-RELA FUSION IN VITRO AND IN VIVO THAT LED TO DEVELOPMENT OF BRAIN TUMORS

2019 ◽  
Vol 21 (Supplement_6) ◽  
pp. vi193-vi193
Author(s):  
Mutsumi Takadera ◽  
Tatsuya Ozawa ◽  
Tetsuya Yamamoto ◽  
Koichi Ichimura
Keyword(s):  
Brain Tumor ◽  
Gene Transfer ◽  
Gene Rearrangement ◽  
Fusion Gene ◽  
Tumor Formation ◽  
Transfer System ◽  
Fusion Genes ◽  
Guide Rnas ◽  
Gene Transfer System

Abstract Recent large-scale genomic studies of ependymal tumors have identified recurrent RELA and YAP1 fusion genes in supratentorial ependymomas. We have so far shown that C11orf95-RELA fusion was a potent oncogene capable of inducing human ependymoma-like tumors in the RCAS/tv-a retroviral gene transfer system. However, molecular mechanism underlying the tumor formation has not been fully investigated. Given that C11orf95-RELA fusion genes are caused by gene rearrangement between C11orf95 and RELA gene due to chromothripsis involving 11q, it has been suggested that expression of these fusion genes is regulated under the C11orf95 promoter. In this study, we developed a system to reproduce the oncogenic gene rearrangement using the CRISPR-Cas9 system and examined if consequent endogenous ependymoma fusion genes are competent to form brain tumor in mice. Initially, we designed guide RNAs on the human and mouse genomic loci corresponding to breakpoints observed in human RELA fusion positive ependymomas and introduced them into HEK293T or NIH3T3 cells. RT-PCR and immunoblot analyses revealed that a combination of multiple guide RNAs efficiently induced gene rearrangement between C11orf95 and RELA gene, resulting in C11orf95-RELA fusion protein. Subsequently, we introduced the same guide RNAs using a lentiviral gene transfer system into mouse brain. Brain tumor formation was observed around 2 months after the guide RNA delivery, indicating successful gene rearrangement followed by C11orf95-RELA fusion expression in vivo. These results thus firmly establish that a gene rearrangement is the mechanism to form the C11orf95-RELA fusion which is the direct driver of tumorigenesis. Furthermore, we found that endogenous YAP1-FAM118B fusion gene can be also induced with a similar experimental strategy to that of C11orf95-RELA fusion. Our system to simulate genomic event will provide insights into the tumorigenic mechanism of ependymomas that harbor various fusion genes.

scholarly journals Synaptopodin: An Actin-associated Protein in Telencephalic Dendrites and Renal Podocytes

1997 ◽  
Vol 139 (1) ◽  
pp. 193-204 ◽  
Author(s):  
Peter Mundel ◽  
Hans W. Heid ◽  
Thomas M. Mundel ◽  
Meike Krüger ◽  
Jochen Reiser ◽  
...  
Keyword(s):  
Dendritic Spines ◽  
Cultured Cells ◽  
Rat Kidney ◽  
Amino Acid Sequences ◽  
Cdna Clones ◽  
Globular Domain ◽  
Postnatal Maturation ◽  
Human And Mouse

Synaptopodin is an actin-associated protein of differentiated podocytes that also occurs as part of the actin cytoskeleton of postsynaptic densities (PSD) and associated dendritic spines in a subpopulation of exclusively telencephalic synapses. Amino acid sequences determined in purified rat kidney and forebrain synaptopodin and derived from human and mouse brain cDNA clones show no significant homology to any known protein. In particular, synaptopodin does not contain functional domains found in receptor-clustering PSD proteins. The open reading frame of synaptopodin encodes a polypeptide with a calculated Mr of 73.7 kD (human)/74.0 kD (mouse) and an isoelectric point of 9.38 (human)/9.27 (mouse). Synaptopodin contains a high amount of proline (∼20%) equally distributed along the protein, thus virtually excluding the formation of any globular domain. Sequence comparison between human and mouse synaptopodin revealed 84% identity at the protein level. In both brain and kidney, in vivo and in vitro, synaptopodin gene expression is differentiation dependent. During postnatal maturation of rat brain, synaptopodin is first detected by Western blot analysis at day 15 and reaches maximum expression in the adult animal. The exclusive synaptopodin synthesis in the telencephalon has been confirmed by in situ hybridization, where synaptopodin mRNA is only found in perikarya of the olfactory bulb, cerebral cortex, striatum, and hippocampus, i.e., the expression is restricted to areas of high synaptic plasticity. From these results and experiments with cultured cells we conclude that synaptopodin represents a novel kind of proline-rich, actin-associated protein that may play a role in modulating actin-based shape and motility of dendritic spines and podocyte foot processes.

scholarly journals Paired CRISPR/Cas9 guide-RNAs enable high-throughput deletion scanning (ScanDel) of a Mendelian disease locus for functionally critical non-coding elements

2016 ◽  
Author(s):  
Molly Gasperini ◽  
Gregory M. Findlay ◽  
Aaron McKenna ◽  
Jennifer H. Milbank ◽  
Choli Lee ◽  
...  
Keyword(s):  
Large Scale ◽  
Transcriptional Start Site ◽  
Regulatory Elements ◽  
Mendelian Disease ◽  
Regulatory Sequences ◽  
Coding Region ◽  
Endogenous Gene ◽  
Distal Regulatory Elements

AbstractThe extent to which distal non-coding mutations contribute to Mendelian disease remains a major unknown in human genetics. Given that a gene’s in vivo function can be appropriately modeled in vitro, CRISPR/Cas9 genome editing enables the large-scale perturbation of distal non-coding regions to identify functional elements in their native context. However, early attempts at such screens have relied on one individual guide RNA (gRNA) per cell, resulting in sparse mutagenesis with minimal redundancy across regions of interest. To address this, we developed a system that uses pairs of gRNAs to program thousands of kilobase-scale deletions that scan across a targeted region in a tiling fashion (“ScanDel”). As a proof-of-concept, we applied ScanDel to program 4,342 overlapping 1- and 2- kilobase (Kb) deletions that tile a 206 Kb region centered on HPRT1, the gene underlying Lesch-Nyhan syndrome, with median 27-fold redundancy per base. Programmed deletions were functionally assayed by selecting for loss of HPRT1 function with 6-thioguanine. HPRT1 exons served as positive controls, and all were successfully identified as functionally critical by the screen. Remarkably, HPRT1 function appeared robust to deletion of any intergenic or deeply intronic non-coding region across the 206 Kb locus, indicating that proximal regulatory sequences are sufficient for its expression. A sparser mutagenesis screen of the same 206 Kb with individual gRNAs also failed to identify critical distal regulatory elements. Although our screen did find programmed deletions and individual gRNAs with putative functional consequences that targeted exon-proximal non-coding sequences (e.g. the promoter), long-read sequencing revealed that this signal was driven almost entirely by rare, unexpected deletions that extended into exonic sequence. These targeted validation experiments defined a small region surrounding the transcriptional start site as the only non-coding sequence essential to HPRT1 function. Overall, our results suggest that distal regulatory elements are not critical for HPRT1 expression, and underscore the necessity of comprehensive edited-locus genotyping for validating the results of CRISPR screens. The application of ScanDel to additional loci will enable more insight into the extent to which the disruption of distal non-coding elements contributes to Mendelian diseases. In addition, dense, redundant, large-scale deletion scanning with gRNA pairs will facilitate a deeper understanding of endogenous gene regulation in the human genome.

Morin Inhibits Ovarian Cancer Growth through the Inhibition of NF-κB Signaling Pathway

2020 ◽  
Vol 19 (18) ◽  
pp. 2243-2250 ◽  
Author(s):  
Meimei Xu ◽  
Yan Zhang
Keyword(s):  
Ovarian Cancer ◽  
Colony Formation ◽  
Cultured Cells ◽  
Tumor Formation ◽  
Cancer Growth ◽  
Ovarian Cancer Cells ◽  
Therapeutic Drugs ◽  
Underlying Mechanisms

Background &Objective: Ovarian cancer has the highest mortality in gynecological tumors without effective therapeutic drugs as a result of drug-resistance for long-term utilization. Morin has been reported to possess powerful anti-tumor effects in several cancers. The present study aims to investigate whether Morin could influence ovarian cancer growth and underlying mechanisms. Methods: Morin was administered to cultured cells in vitro and formed tumors in vivo. MTT and colony formation assays were performed to explore the effects of Morin on the proliferation and colony formation of OVCAR3 and SKOV3 ovarian cancer cells. Western blot, RT-qPCR, immunofluorescence as well as ELISA were used to detect protein and mRNA expression of target factors. Tumor formation was performed to investigate tumorigenesis ability of drug-treated cells. Results: The proliferation and colony size of OVCAR3 and SKOV3 were significantly decreased after Morin administration. The expression of NF-κB and inflammatory cytokine IL6/8 induced by TNF-α can be inhibited by Morin. Furthermore, Morin inhibited the volume of ovarian cancer tumors in nude mice. Conclusion: Morin effectively alleviates ovarian cancer growth, inhibits the inflammatory response, and reduces tumor size via modulation of the NF-κB pathway.

Novel PDGFRA Point Mutations in Hypereosinophilic Syndrome Induce Growth Factor Independence and Leukemia in Vitro and In Vivo.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 767-767
Author(s):  
Christian Elling ◽  
Philipp Erben ◽  
Christoph Walz ◽  
Marie Frickenhaus ◽  
Mirle Schemionek ◽  
...  
Keyword(s):  
Growth Factor ◽  
Conflicts Of Interest ◽  
Fusion Gene ◽  
Hypereosinophilic Syndrome ◽  
Point Mutations ◽  
Fusion Genes ◽  
Double Mutation ◽  
Expression Levels

Abstract Abstract 767 Considerable progress has been achieved in our understanding of the pathogenesis of hypereosinophilic syndrome (HES) and chronic eosinophilic leukemia (CEL) by identification of constitutively activated tyrosine kinase fusion genes, e.g. FIP1L1-PDGFRA or ETV6-PDGFRB. However, the overall incidence of those fusion genes in HES/CEL is below 15%, and the molecular pathogenesis of the remaining cases remains elusive. We therefore established generic quantitative RT-PCR assays (RQ-PCR) to detect overexpression of 3'-regions of PDGFRA or PDGFRB as a possible indicator of an underlying fusion gene or point mutation. Patients with known fusion genes involving PDGFRA (n=5, 51 patients) or PDGFRB (n=5; 7 patients) showed significantly increased normalized expression levels compared to 191 patients with fusion gene-negative eosinophilia or healthy individuals (PDGFRA/ABL: 0.73 vs. 0.0066 vs. 0.0064, p<0.0001; PDGFRB/ABL: 196 vs. 3.8 vs. 5.85, p<0.0001). In all patients with significantly increased expression levels who were negative for fusion genes, functionally relevant regions of PDGFRA were sequenced. Several novel mutations (R481G, I562M, H570R, M628T, L705P, G729D) as well as a double mutation (H650Q and R748G) were identified. When cloned into 32D cells, M628T, H650Q, and R748G mutants separately induced growth factor-independent proliferation and clonogenic growth, and this was associated with constitutive phosphorylation of downstream targets STAT5, ERK, and AKT. Low doses of imatinib antagonized all of these effects in vitro. M628T and R748G but not H650Q 32D cell mutants induced acute leukemia after injection into congenic C3H/HeJ mice, similar to FIP1L1-PDGFRA. Interestingly, these two mutants showed a significantly higher propensity to invade the lymph nodes than the FIP1L1-PDGFRA fusion. Oral administration of imatinib to injected mice significantly decreased leukemic growth in vivo and significantly prolonged survival of the recipients. In conclusion, we demonstrate that novel point mutations of the PDGFRA gene found in patients with HES/CEL induce growth factor independence and leukemia in vitro and in vivo and suggest that these patients may benefit from treatment with imatinib. *CE, PE, AR, and SK contributed equally to this work. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Tumor Energy Metabolism and Potential of 3-Bromopyruvate as an Inhibitor of Aerobic Glycolysis: Implications in Tumor Treatment

Cancers ◽  
2019 ◽  
Vol 11 (3) ◽  
pp. 317 ◽  
Author(s):  
Tengjiao Fan ◽  
Guohui Sun ◽  
Xiaodong Sun ◽  
Lijiao Zhao ◽  
Rugang Zhong ◽  
...  
Keyword(s):  
Energy Metabolism ◽  
Tumor Cells ◽  
Cultured Cells ◽  
Aerobic Glycolysis ◽  
Underlying Mechanism ◽  
Human Case ◽  
Tumor Formation ◽  
Antitumor Effects

Tumor formation and growth depend on various biological metabolism processes that are distinctly different with normal tissues. Abnormal energy metabolism is one of the typical characteristics of tumors. It has been proven that most tumor cells highly rely on aerobic glycolysis to obtain energy rather than mitochondrial oxidative phosphorylation (OXPHOS) even in the presence of oxygen, a phenomenon called “Warburg effect”. Thus, inhibition of aerobic glycolysis becomes an attractive strategy to specifically kill tumor cells, while normal cells remain unaffected. In recent years, a small molecule alkylating agent, 3-bromopyruvate (3-BrPA), being an effective glycolytic inhibitor, has shown great potential as a promising antitumor drug. Not only it targets glycolysis process, but also inhibits mitochondrial OXPHOS in tumor cells. Excellent antitumor effects of 3-BrPA were observed in cultured cells and tumor-bearing animal models. In this review, we described the energy metabolic pathways of tumor cells, mechanism of action and cellular targets of 3-BrPA, antitumor effects, and the underlying mechanism of 3-BrPA alone or in combination with other antitumor drugs (e.g., cisplatin, doxorubicin, daunorubicin, 5-fluorouracil, etc.) in vitro and in vivo. In addition, few human case studies of 3-BrPA were also involved. Finally, the novel chemotherapeutic strategies of 3-BrPA, including wafer, liposomal nanoparticle, aerosol, and conjugate formulations, were also discussed for future clinical application.

scholarly journals Real-Time Monitoring of Nuclear Factor κB Activity in Cultured Cells and in Animal Models

2009 ◽  
Vol 8 (5) ◽  
pp. 7290.2009.00026 ◽  
Author(s):  
Christian E. Badr ◽  
Johanna M. Niers ◽  
Lee-Ann Tjon-Kon-Fat ◽  
David P. Noske ◽  
Thomas Wurdinger ◽  
...  
Keyword(s):  
Real Time ◽  
Cultured Cells ◽  
Tumor Development ◽  
Nuclear Factor Κb ◽  
Tumor Formation ◽  
Nuclear Factor ◽  
Reporter System ◽  
Human Disorders

Nuclear factor κB (NF-κB) is a transcription factor that plays a major role in many human disorders, including immune diseases and cancer. We designed a reporter system based on NF-κB responsive promoter elements driving expression of the secreted Gaussia princeps luciferase (Gluc). We show that this bioluminescent reporter is a highly sensitive tool for noninvasive monitoring of the kinetics of NF-κB activation and inhibition over time, both in conditioned medium of cultured cells and in the blood and urine of animals. NF-κB activation was successfully monitored in real time in endothelial cells in response to tumor angiogenic signaling, as well as in monocytes in response to inflammation. Further, we demonstrated dual blood monitoring of both NF-κB activation during tumor development as correlated to tumor formation using the NF-κB Gluc reporter, as well as the secreted alkaline phosphatase reporter. This NF-κB reporter system provides a powerful tool for monitoring NF-κB activity in real time in vitro and in vivo.

scholarly journals Antiparkinson Drug Benztropine Suppresses Tumor Growth, Circulating Tumor Cells, and Metastasis by Acting on SLC6A3 and Reducing STAT3

2020 ◽  
Author(s):  
Chiharu Sogawa ◽  
Manh Tien Tran ◽  
Masayuki Ishige ◽  
Kilian Trin ◽  
Yuka Okusha ◽  
...  
Keyword(s):  
Tumor Growth ◽  
Cultured Cells ◽  
Genetic Alterations ◽  
Tumor Formation ◽  
Beta Catenin ◽  
Screening System ◽  
Antiparkinson Drug ◽  
Pharmacologically Active

Tumor growth, progression, and therapy resistance are crucial factors in the prognosis of cancer. Properties of three-dimensional tumor-like organoids (tumoroids) more closely resemble in vivo tumors compared to two-dimensionally cultured cells and are therefore effectively used for assays and drug screening. We here established a repurposed drug for novel anticancer research and therapeutics using a tumoroid-based screening system. We screened 6 pharmacologically active compounds by using an original tumoroid-based multiplex phenotypic screening system with matrix metalloproteinase 9 (MMP9) promoter-driven fluorescence reporter for the evaluation of both tumoroid formation and progression. The effects of one of the hit compounds were examined on tumor formation and progression in vitro and in vivo. Antiparkinson drug benztropine was the most effective compound uncovered by the screen. Benztropine significantly inhibited in vitro tumoroid formation, cancer cell survival, and MMP9 promoter activity. Benztropine also reduced the activity of oncogenic signaling transducers and trans-activators for MMP9, including STAT3, NF-&kappa;B, and &beta;-catenin, and properties of cancer stem cells / cancer-initiating cells. Benztropine and GBR-12935 directly targeted the dopamine transporter DAT/SLC6A3, whose genetic alterations such as amplification were correlated with poor prognosis for cancer patients. Benztropine also inhibited tumor growth, circulating tumor cell (CTC) number, and rate of metastasis in a tumor allograft model in mice. In conclusion, we propose the repurposing of benztropine for anticancer research and therapeutics that can suppress tumor progression, CTC, and metastasis of aggressive cancers by reducing key pro-tumorigenic factors.

scholarly journals Aurora B Overexpression Causes Aneuploidy and p21Cip1Repression during Tumor Development

2015 ◽  
Vol 35 (20) ◽  
pp. 3566-3578 ◽  
Author(s):  
Alejandra González-Loyola ◽  
Gonzalo Fernández-Miranda ◽  
Marianna Trakala ◽  
David Partida ◽  
Kumiko Samejima ◽  
...  
Keyword(s):  
Chromosome Segregation ◽  
Cultured Cells ◽  
Tumor Development ◽  
Inverse Correlation ◽  
Aurora Kinase ◽  
Tumor Formation ◽  
Aurora B ◽  
Aurora Kinase B

Aurora kinase B, one of the three members of the mammalian Aurora kinase family, is the catalytic component of the chromosomal passenger complex, an essential regulator of chromosome segregation in mitosis. Aurora B is overexpressed in human tumors although whether this kinase may function as an oncogenein vivois not established. Here, we report a new mouse model in which expression of the endogenousAurkblocus can be inducedin vitroandin vivo. Overexpression of Aurora B in cultured cells induces defective chromosome segregation and aneuploidy. Long-term overexpression of Aurora Bin vivoresults in aneuploidy and the development of multiple spontaneous tumors in adult mice, including a high incidence of lymphomas. Overexpression of Aurora B also results in a reduced DNA damage response and decreased levels of the p53 target p21Cip1in vitroandin vivo, in line with an inverse correlation between Aurora B and p21Cip1expression in human leukemias. Thus, overexpression of Aurora B may contribute to tumor formation not only by inducing chromosomal instability but also by suppressing the function of the cell cycle inhibitor p21Cip1.

scholarly journals Inhibition of protein translation by the DISC1-Boymaw fusion gene from a Scottish family with major psychiatric disorders

2014 ◽  
Author(s):  
Baohu Ji ◽  
Kerin K. Higa ◽  
Minjung Kim ◽  
Lynn Zhou ◽  
Jared W. Young ◽  
...  
Keyword(s):  
Psychiatric Disorders ◽  
Fusion Gene ◽  
Protein Translation ◽  
Chromosome Translocation ◽  
Humanized Mice ◽  
Fusion Genes ◽  
Chromosome 11 ◽  
Brain Functions

AbstractThe t(1; 11) translocation appears to be the causal genetic lesion with 70% penetrance for schizophrenia, major depression, and other psychiatric disorders in a Scottish family. Molecular studies identified the disruption of the DISC1 (disrupted-in-schizophrenia 1) gene by chromosome translocation at chromosome 1q42. Our previous studies, however, revealed that the translocation also disrupted another gene, Boymaw (also termed DISC1FP1), on chromosome 11. After translocation, two fusion genes (the DISC1-Boymaw (DB7) and the Boymaw-DISC1 (BD13)) are generated between the DISC1 and Boymaw genes. In the present study, we report that expression of the DB7 fusion gene inhibits both intracellular NADH oxidoreductase activities and protein translation. We generated humanized DISC1-Boymaw mice with gene targeting to examine the in vivo functions of the fusion genes. Consistent with the in vitro studies on the DB7 fusion gene, protein translation activity is decreased in the hippocampus and in cultured primary neurons from the brains of the humanized mice. Expression of Gad67, Nmdar1, and Psd95 proteins are also reduced. The humanized mice display prolonged and increased responses to the NMDA receptor antagonist, ketamine, on various mouse genetic backgrounds. Abnormal information processing of acoustic startle and depressive-like behaviors are also observed. In addition, the humanized mice display abnormal erythropoiesis, which was reported to associate with depression in humans. Expression of the DB7 fusion gene may reduce protein translation to impair brain functions and thereby contribute to the pathogenesis of major psychiatric disorders.

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