RESUMEN
Japanese encephalitis (JE), caused by the Japanese encephalitis virus (JEV) infection, continues to pose significant public health challenges worldwide despite efficient vaccines. The virus is classified into five genotypes, among which genotype V (GV) was not detected for a long period after its initial isolation in 1952, until reports emerged from China and the Republic of Korea (ROK) since 2009. The characteristics of the virus are crucial in estimating its potential epidemiological impact. However, characterization of GV JEVs has so far been limited to two strains: Muar, the original isolate, and XZ0934, isolated in China. Two additional ROK GV JEV isolates, NCCP 43279 and NCCP 43413, are currently available, but their characteristics have not been explored. Our phylogenetic analysis revealed that GV virus sequences from the ROK segregate into two clades. NCCP 43279 and NCCP 43413 belong to different clades and exhibit distinct in vitro phenotypes. NCCP 43279 forms larger plaques but demonstrates inefficient propagation in cell culture compared to NCCP 43413. In vivo, NCCP 43279 induces higher morbidity and mortality in mice than NCCP 43413. Notably, NCCP 43279 shows more severe blood-brain barrier damage, suggesting superior brain invasion capabilities. Consistent with its higher virulence, NCCP 43279 displays more pronounced histopathological and immunopathological outcomes. In conclusion, our study confirms that the two ROK isolates are not only classified into different clades but also exhibit distinct in vitro and in vivo characteristics.
Asunto(s)
Virus de la Encefalitis Japonesa (Especie) , Encefalitis Japonesa , Genotipo , Filogenia , Virus de la Encefalitis Japonesa (Especie)/genética , Virus de la Encefalitis Japonesa (Especie)/aislamiento & purificación , Virus de la Encefalitis Japonesa (Especie)/clasificación , Animales , República de Corea/epidemiología , Encefalitis Japonesa/virología , Encefalitis Japonesa/veterinaria , Encefalitis Japonesa/epidemiología , Ratones , Humanos , Virulencia , Línea Celular , FemeninoRESUMEN
Effectively targeting cancer stemness is essential for successful cancer therapy. Recent studies have revealed that SOX2, a pluripotent stem cell factor, significantly contributes to cancer stem cell (CSC)-like characteristics closely associated with cancer malignancy. However, its contradictory impact on patient survival in specific cancer types, including lung adenocarcinoma (LUAD), underscores the need for more comprehensive research to clarify its functional effect on cancer stemness. In this study, we demonstrate that SOX2 is not universally required for the regulation of CSC-like properties in LUAD. We generated SOX2 knockouts in A549, H358, and HCC827 LUAD cells using the CRISPR/Cas9 system. Our results reveal unchanged CSC characteristics, including sustained proliferation, tumor sphere formation, invasion, migration, and therapy resistance, compared to normal cells. Conversely, SOX2 knockdown using conditional shRNA targeting SOX2, significantly reduced CSC traits. However, these loss-of-function effects were not rescued by SOX2 resistant to shRNA, underscoring the potential for SOX2 protein level-independent results in prior siRNA- or shRNA-based research. Ultimately, our findings demonstrate that SOX2 is not absolutely essential in LUAD cancer cells. This emphasizes the necessity of considering cancer subtype-dependent and context-dependent factors when targeting SOX2 overexpression as a potential therapeutic vulnerability in diverse cancers.
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Adenocarcinoma del Pulmón , Neoplasias Pulmonares , Células Madre Neoplásicas , Factores de Transcripción SOXB1 , Humanos , Adenocarcinoma del Pulmón/patología , Neoplasias Pulmonares/patología , Células Madre Neoplásicas/patología , ARN Interferente Pequeño/metabolismo , Factores de Transcripción SOXB1/genética , Factores de Transcripción SOXB1/metabolismoRESUMEN
The cell-cycle phase is a major determinant of repair pathway choice at DNA double strand breaks, non-homologous end joining (NHEJ), or homologous recombination (HR). Chk1 responds to genotoxic stress in S/G2 phase, but here, we report a role of Chk1 in directly promoting NHEJ repair in G1 phase. ASF1A is a histone chaperone, but it promotes NHEJ through a pathway independent of its histone-chaperone activity. Chk1 activated by ataxia telangiectasia mutated (ATM) kinase on DNA breaks in G1 promotes NHEJ through direct phosphorylation of ASF1A at Ser-166. ASF1A phosphorylated at Ser-166 interacts with the repair protein MDC1 and thus enhances MDC1's interaction with ATM and the stable localization of ATM at DNA breaks. Chk1 deficiency suppresses all steps downstream of MDC1 following a DNA break in G1, namely histone ubiquitination, 53BP1 localization to the DNA break, and NHEJ. Thus, ASF1A phosphorylation by Chk1 is essential for DNA break repair by NHEJ in G1.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/metabolismo , Reparación del ADN por Unión de Extremidades , Chaperonas Moleculares/metabolismo , Fase G1 , Humanos , FosforilaciónRESUMEN
Pancreatic ductal adenocarcinoma (PDAC) remains one of the most challenging cancers to treat. Due to the asymptomatic nature of the disease and lack of curative treatment modalities, the 5-y survival rate of PDAC patients is one of the lowest of any cancer type. The recurrent genetic alterations in PDAC are yet to be targeted. Therefore, identification of effective drug combinations is desperately needed. Here, we performed an in vivo CRISPR screen in an orthotopic patient-derived xenograft (PDX) model to identify gene targets whose inhibition creates synergistic tumor growth inhibition with gemcitabine (Gem), a first- or second-line chemotherapeutic agent for PDAC treatment. The approach revealed protein arginine methyltransferase gene 5 (PRMT5) as an effective druggable candidate whose inhibition creates synergistic vulnerability of PDAC cells to Gem. Genetic depletion and pharmacological inhibition indicate that loss of PRMT5 activity synergistically enhances Gem cytotoxicity due to the accumulation of excessive DNA damage. At the molecular level, we show that inhibition of PRMT5 results in RPA depletion and impaired homology-directed DNA repair (HDR) activity. The combination (Gem + PRMT5 inhibition) creates conditional lethality and synergistic reduction of PDAC tumors in vivo. The findings demonstrate that unbiased genetic screenings combined with a clinically relevant model system is a practical approach in identifying synthetic lethal drug combinations for cancer treatment.
Asunto(s)
Antineoplásicos/farmacología , Desoxicitidina/análogos & derivados , Neoplasias Pancreáticas/metabolismo , Proteína-Arginina N-Metiltransferasas , Animales , Sistemas CRISPR-Cas/genética , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Desoxicitidina/farmacología , Desarrollo de Medicamentos , Técnicas de Inactivación de Genes , Humanos , Ratones Desnudos , Proteína-Arginina N-Metiltransferasas/antagonistas & inhibidores , Proteína-Arginina N-Metiltransferasas/genética , Proteína-Arginina N-Metiltransferasas/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto , GemcitabinaRESUMEN
BACKGROUND: While clinical factors such as age, grade, stage, and histological subtype provide physicians with information about patient prognosis, genomic data can further improve these predictions. Previous studies have shown that germline variants in known cancer driver genes are predictive of patient outcome, but no study has systematically analyzed multiple cancers in an unbiased way to identify genetic loci that can improve patient outcome predictions made using clinical factors. METHODS: We analyzed sequencing data from the over 10,000 cancer patients available through The Cancer Genome Atlas to identify germline variants associated with patient outcome using multivariate Cox regression models. RESULTS: We identified 79 prognostic germline variants in individual cancers and 112 prognostic germline variants in groups of cancers. The germline variants identified in individual cancers provide additional predictive power about patient outcomes beyond clinical information currently in use and may therefore augment clinical decisions based on expected tumor aggressiveness. Molecularly, at least 12 of the germline variants are likely associated with patient outcome through perturbation of protein structure and at least five through association with gene expression differences. Almost half of these germline variants are in previously reported tumor suppressors, oncogenes or cancer driver genes with the other half pointing to genomic loci that should be further investigated for their roles in cancers. CONCLUSIONS: Germline variants are predictive of outcome in cancer patients and specific germline variants can improve patient outcome predictions beyond predictions made using clinical factors alone. The germline variants also implicate new means by which known oncogenes, tumor suppressor genes, and driver genes are perturbed in cancer and suggest roles in cancer for other genes that have not been extensively studied in oncology. Further studies in other cancer cohorts are necessary to confirm that germline variation is associated with outcome in cancer patients as this is a proof-of-principle study.
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Biomarcadores de Tumor/genética , Mutación de Línea Germinal , Neoplasias/genética , Pruebas Genéticas/estadística & datos numéricos , Humanos , Neoplasias/patología , Proteínas Oncogénicas/genética , Polimorfismo de Nucleótido Simple , Sitios de Carácter Cuantitativo , Proteínas Supresoras de Tumor/genéticaRESUMEN
High-risk human papilloma viruses (HPVs) cause cervical, anal, and oropharyngeal cancers, unlike the low-risk HPVs, which cause benign lesions. E6 oncoproteins from the high-risk strains are essential for cell proliferation and transformation in HPV-induced cancers. We report that a cellular deubiquitinase, USP46, is selectively recruited by the E6 of high-risk, but not low-risk, HPV to deubiqutinate and stabilize Cdt2/DTL. Stabilization of Cdt2, a component of the CRL4Cdt2 E3 ubiquitin ligase, limits the level of Set8, an epigenetic writer, and promotes cell proliferation. USP46 is essential for the proliferation of HPV-transformed cells, but not of cells without HPV. Cdt2 is elevated in human cervical cancers and knockdown of USP46 inhibits HPV-transformed tumor growth in xenografts. Recruitment of a cellular deubiquitinase to stabilize key cellular proteins is an important activity of oncogenic E6, and the importance of E6-USP46-Cdt2-Set8 pathway in HPV-induced cancers makes USP46 a target for the therapy of such cancers.
Asunto(s)
Endopeptidasas/genética , Papillomavirus Humano 16/genética , Papillomavirus Humano 18/genética , Proteínas Nucleares/genética , Infecciones por Papillomavirus/genética , Neoplasias del Cuello Uterino/genética , Animales , Ciclo Celular , Línea Celular Tumoral , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Endopeptidasas/metabolismo , Femenino , Regulación de la Expresión Génica , Células HeLa , N-Metiltransferasa de Histona-Lisina/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Interacciones Huésped-Patógeno/genética , Papillomavirus Humano 16/metabolismo , Papillomavirus Humano 16/patogenicidad , Papillomavirus Humano 18/metabolismo , Papillomavirus Humano 18/patogenicidad , Humanos , Inyecciones Intralesiones , Ratones , Proteínas Nucleares/metabolismo , Proteínas Oncogénicas Virales/genética , Proteínas Oncogénicas Virales/metabolismo , Infecciones por Papillomavirus/enzimología , Infecciones por Papillomavirus/patología , Infecciones por Papillomavirus/virología , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Transducción de Señal , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Neoplasias del Cuello Uterino/enzimología , Neoplasias del Cuello Uterino/patología , Neoplasias del Cuello Uterino/virología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Double-strand breaks (DSBs) of DNA in eukaryotic cells are predominantly repaired by non-homologous end joining (NHEJ). The histone chaperone anti-silencing factor 1a (ASF1a) interacts with MDC1 and is recruited to sites of DSBs to facilitate the interaction of phospho-ATM with MDC1 and phosphorylation of MDC1, which are required for the recruitment of RNF8/RNF168 histone ubiquitin ligases. Thus, ASF1a deficiency reduces histone ubiquitination at DSBs, decreasing the recruitment of 53BP1, and decreases NHEJ, rendering cells more sensitive to DSBs. This role of ASF1a in DSB repair cannot be provided by the closely related ASF1b and does not require its histone chaperone activity. Homozygous deletion of ASF1A is seen in 10%-15% of certain cancers, suggesting that loss of NHEJ may be selected in some malignancies and that the deletion can be used as a molecular biomarker for cancers susceptible to radiotherapy or to DSB-inducing chemotherapy.
Asunto(s)
Proteínas de la Ataxia Telangiectasia Mutada/genética , Proteínas de Ciclo Celular/genética , Reparación del ADN por Unión de Extremidades , ADN de Neoplasias/genética , Regulación Neoplásica de la Expresión Génica , Proteínas Nucleares/genética , Transactivadores/genética , Proteínas Adaptadoras Transductoras de Señales , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Proteínas de Ciclo Celular/metabolismo , Línea Celular Transformada , Línea Celular Tumoral , Cromatina/química , Cromatina/metabolismo , ADN/genética , ADN/metabolismo , Roturas del ADN de Doble Cadena , ADN de Neoplasias/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Eliminación de Gen , Células HEK293 , Células HeLa , Histonas/genética , Histonas/metabolismo , Humanos , Chaperonas Moleculares , Proteínas Nucleares/metabolismo , Fosforilación , Transducción de Señal , Transactivadores/metabolismo , Proteína 1 de Unión al Supresor Tumoral P53/genética , Proteína 1 de Unión al Supresor Tumoral P53/metabolismo , Ubiquitina/genética , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , UbiquitinaciónRESUMEN
MCM8-9 complex is required for homologous recombination (HR)-mediated repair of double-strand breaks (DSBs). Here we report that MCM8-9 is required for DNA resection by MRN (MRE11-RAD50-NBS1) at DSBs to generate ssDNA. MCM8-9 interacts with MRN and is required for the nuclease activity and stable association of MRN with DSBs. The ATPase motifs of MCM8-9 are required for recruitment of MRE11 to foci of DNA damage. Homozygous deletion of the MCM9 found in various cancers sensitizes a cancer cell line to interstrand-crosslinking (ICL) agents. A cancer-derived point mutation or an SNP on MCM8 associated with premature ovarian failure (POF) diminishes the functional activity of MCM8. Therefore, the MCM8-9 complex facilitates DNA resection by the MRN complex during HR repair, genetic or epigenetic inactivation of MCM8 or MCM9 are seen in human cancers, and genetic inactivation of MCM8 may be the basis of a POF syndrome.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Roturas del ADN de Doble Cadena , Enzimas Reparadoras del ADN/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas de Mantenimiento de Minicromosoma/metabolismo , Proteínas Nucleares/metabolismo , Transportadoras de Casetes de Unión a ATP/metabolismo , Ácido Anhídrido Hidrolasas , Animales , Proteínas Portadoras/metabolismo , Inmunoprecipitación de Cromatina , Daño del ADN , Reparación del ADN , ADN de Cadena Simple , Células HEK293 , Células HeLa , Humanos , Proteína Homóloga de MRE11 , Ratones , Reparación del ADN por Recombinación , Proteínas Supresoras de Tumor/metabolismo , Xenopus , Proteínas de Xenopus/metabolismoRESUMEN
Phosphorylation of Orc2, one of the six subunits of the origin recognition complex (ORC), by cyclin A/CDK2 during S phase leads to the dissociation of Orc2, Orc3, Orc4, and Orc5 subunits (Orc2-5) from human chromatin and replication origins. Dephosphorylation of the phosphorylated Orc2 by protein phosphatase 1 (PP1) is accompanied by the binding of the dissociated subunits to chromatin. Here we show that PP1 physically interacts with Orc2. The binding of PP1 to Orc2 and the dephosphorylation of Orc2 by PP1 occurred in a cell cycle-dependent manner through an interaction with 119-KSVSF-123, which is the consensus motif for the binding of PP1, of Orc2. The dephosphorylation of Orc2 by PP1 is required for the binding of Orc2 to chromatin. These results support that PP1 dephosphorylates Orc2 to promote the binding of ORC to chromatin and replication origins for the subsequent round of the cell cycle.
Asunto(s)
Cromatina/metabolismo , Complejo de Reconocimiento del Origen/metabolismo , Proteína Fosfatasa 1/metabolismo , Sustitución de Aminoácidos , Ciclo Celular , Línea Celular , Cromatina/genética , Células HEK293 , Células HeLa , Humanos , Mutagénesis Sitio-Dirigida , Complejo de Reconocimiento del Origen/química , Complejo de Reconocimiento del Origen/genética , Fosforilación , Unión Proteica , Dominios y Motivos de Interacción de Proteínas/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Origen de Réplica , Técnicas del Sistema de Dos HíbridosRESUMEN
Long noncoding RNAs (lncRNAs) have emerged as a major regulator of cell physiology, but many of which have no known function. CDKN1A/p21 is an important inhibitor of the cell-cycle, regulator of the DNA damage response and effector of the tumor suppressor p53, playing a crucial role in tumor development and prevention. In order to identify a regulator for tumor progression, we performed an siRNA screen of human lncRNAs required for cell proliferation, and identified a novel lncRNA, APTR, that acts in trans to repress the CDKN1A/p21 promoter independent of p53 to promote cell proliferation. APTR associates with the promoter of CDKN1A/p21 and this association requires a complementary-Alu sequence encoded in APTR. A different module of APTR associates with and recruits the Polycomb repressive complex 2 (PRC2) to epigenetically repress the p21 promoter. A decrease in APTR is necessary for the induction of p21 after heat stress and DNA damage by doxorubicin, and the levels of APTR and p21 are anti-correlated in human glioblastomas. Our data identify a new regulator of the cell-cycle inhibitor CDKN1A/p21 that acts as a proliferative factor in cancer cell lines and in glioblastomas and demonstrate that Alu elements present in lncRNAs can contribute to targeting regulatory lncRNAs to promoters.
Asunto(s)
Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Complejo Represivo Polycomb 2/metabolismo , Regiones Promotoras Genéticas , ARN Largo no Codificante/genética , Secuencia de Bases , Línea Celular , Proliferación Celular , Inmunoprecipitación de Cromatina , Cartilla de ADN , Silenciador del Gen , Humanos , Reacción en Cadena de la Polimerasa , ARN Largo no Codificante/metabolismo , ARN Interferente Pequeño/genéticaRESUMEN
Many agents used for chemotherapy, such as doxorubicin, interfere with DNA replication, but the effect of this interference on transcription is largely unknown. Here we show that doxorubicin induces the firing of dense clusters of neoreplication origins that lead to clusters of stalled replication forks in gene-rich parts of the genome, particularly on expressed genes. Genes that overlap with these clusters of stalled forks are actively dechromatinized, unwound, and repressed by an ATR-dependent checkpoint pathway. The ATR checkpoint pathway causes a histone chaperone normally associated with the replication fork, ASF1a, to degrade through a CRL1(ßTRCP)-dependent ubiquitination/proteasome pathway, leading to the localized dechromatinization and gene repression. Therefore, a globally active checkpoint pathway interacts with local clusters of stalled forks to specifically repress genes in the vicinity of the stalled forks, providing a new mechanism of action of chemotherapy drugs like doxorubicin. Finally, ASF1a-depleted cancer cells are more sensitive to doxorubicin, suggesting that the 7%-10% of prostate adenocarcinomas and adenoid cystic carcinomas reported to have homozygous deletion or significant underexpression of ASF1a should be tested for high sensitivity to doxorubicin.
Asunto(s)
Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Regulación Neoplásica de la Expresión Génica , Origen de Réplica/genética , Ubiquitina-Proteína Ligasas/metabolismo , Antineoplásicos/farmacología , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Puntos de Control del Ciclo Celular/genética , Línea Celular Tumoral , Células/efectos de los fármacos , Replicación del ADN/genética , Doxorrubicina/farmacología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/genética , Técnicas de Silenciamiento del Gen , Células HeLa , Histonas/metabolismo , Humanos , Chaperonas Moleculares , ARN Polimerasa II/metabolismoRESUMEN
Phosphorylation of Thr(116) and Thr(226) on Orc2, one of the six subunits of the origin recognition complex (ORC), by cyclin A/CDK2 during S phase leads to the dissociation of Orc2, Orc3, Orc4, and Orc5 subunits (Orc2-5) from human chromatin and replication origins. The phosphorylated Orc2 becomes dephosphorylated in the late M phase of the cell cycle. Here we show that protein phosphatase 1 (PP1) dephosphorylates Orc2. Dephosphorylation of Orc2 was accompanied by associating the dissociated Orc subunits with chromatin. Inhibitors of PP1 preferentially inhibited the dephosphorylation of Orc2. The overexpression of the α, ß and γ PP1 isoforms decreased the amount of phosphorylated Orc2, and the depletion of these isoforms by RNA interference increased the amount of phosphorylated Orc2. These results suggest that PP1 dephosphorylates Orc2 to promote the binding of ORC to chromatin.
Asunto(s)
Cromatina/metabolismo , Complejo de Reconocimiento del Origen/metabolismo , Proteína Fosfatasa 1/metabolismo , Células HeLa , Humanos , Isoenzimas/metabolismo , Complejo de Reconocimiento del Origen/genética , Fosforilación , Unión Proteica , Proteína Fosfatasa 1/antagonistas & inhibidores , Proteína Fosfatasa 1/genética , Treonina/genética , Treonina/metabolismoRESUMEN
Serine protease inhibitors from bumblebee venom have been shown to block plasmin activity. In this study, we identified the protein BiVSPI from the venom of Bombus ignitus to be a serine protease inhibitor and an antimicrobial factor. BiVSPI is a 55-amino acid mature peptide with ten conserved cysteine residues and a P1 methionine residue. BiVSPI is expressed in the venom gland and also found in the venom as an 8-kDa peptide. Recombinant BiVSPI that was expressed in baculovirus-infected insect cells exhibited inhibitory activity against chymotrypsin but not trypsin. BiVSPI also inhibited microbial serine proteases, such as subtilisin A (Ki=6.57nM) and proteinase K (Ki=7.11nM). In addition, BiVSPI was shown to bind directly to Bacillus subtilis, Bacillus thuringiensis, and Beauveria bassiana but not to Escherichia coli. Consistent with these results, BiVSPI exhibited antimicrobial activity against Gram-positive bacteria and fungi. These findings provide evidence for a novel serine protease inhibitor in bumblebee venom that has antimicrobial functions.
Asunto(s)
Antiinfecciosos/farmacología , Bacterias/enzimología , Venenos de Abeja/enzimología , Abejas , Hongos/enzimología , Serina Proteasas/metabolismo , Inhibidores de Serina Proteinasa/farmacología , Secuencia de Aminoácidos , Animales , Antiinfecciosos/química , Bacterias/efectos de los fármacos , Secuencia de Bases , Clonación Molecular , Hongos/efectos de los fármacos , Bacterias Grampositivas/efectos de los fármacos , Humanos , Concentración 50 Inhibidora , Datos de Secuencia Molecular , Inhibidores de Serina Proteinasa/química , Inhibidores de Serina Proteinasa/genéticaRESUMEN
The minichromosome maintenance protein homologs MCM8 and MCM9 have previously been implicated in DNA replication elongation and prereplication complex (pre-RC) formation, respectively. We found that MCM8 and MCM9 physically associate with each other and that MCM8 is required for the stability of MCM9 protein in mammalian cells. Depletion of MCM8 or MCM9 in human cancer cells or the loss of function MCM9 mutation in mouse embryo fibroblasts sensitizes cells to the DNA interstrand cross-linking (ICL) agent cisplatin. Consistent with a role in the repair of ICLs by homologous recombination (HR), knockdown of MCM8 or MCM9 significantly reduces HR repair efficiency. Chromatin immunoprecipitation analysis using human DR-GFP cells or Xenopus egg extract demonstrated that MCM8 and MCM9 proteins are rapidly recruited to DNA damage sites and promote RAD51 recruitment. Thus, these two metazoan-specific MCM homologs are new components of HR and may represent novel targets for treating cancer in combination with DNA cross-linking agents.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Daño del ADN/genética , Proteínas de Unión al ADN/metabolismo , Recombinación Homóloga , Recombinasa Rad51/metabolismo , Reparación del ADN por Recombinación , Animales , Proteínas de Ciclo Celular/genética , Línea Celular Tumoral , Supervivencia Celular , Inmunoprecipitación de Cromatina , Cisplatino/farmacología , Proteínas de Unión al ADN/genética , Proteínas Fluorescentes Verdes , Células HEK293 , Células HeLa , Humanos , Ratones , Proteínas de Mantenimiento de Minicromosoma , Interferencia de ARN , ARN Interferente Pequeño , XenopusRESUMEN
During the late M to the G(1) phase of the cell cycle, the origin recognition complex (ORC) binds to the replication origin, leading to the assembly of the prereplicative complex for subsequent initiation of eukaryotic chromosome replication. We found that the cell cycle-dependent phosphorylation of human ORC2, one of the six subunits of ORC, dissociates ORC2, -3, -4, and -5 (ORC2-5) subunits from chromatin and replication origins. Phosphorylation at Thr-116 and Thr-226 of ORC2 occurs by cyclin-dependent kinase during the S phase and is maintained until the M phase. Phosphorylation of ORC2 at Thr-116 and Thr-226 dissociated the ORC2-5 from chromatin. Consistent with this, the phosphomimetic ORC2 protein exhibited defective binding to replication origins as well as to chromatin, whereas the phosphodefective protein persisted in binding throughout the cell cycle. These results suggest that the phosphorylation of ORC2 dissociates ORC from chromatin and replication origins and inhibits binding of ORC to newly replicated DNA.
Asunto(s)
Cromatina/metabolismo , Complejo de Reconocimiento del Origen/metabolismo , Procesamiento Proteico-Postraduccional , Origen de Réplica , Secuencia de Aminoácidos , Secuencia Conservada , Células HeLa , Humanos , Datos de Secuencia Molecular , Complejo de Reconocimiento del Origen/química , Fosforilación , Fosfotreonina/metabolismo , Unión Proteica , Fase SRESUMEN
Since human mesenchymal stem cells (MSCs) are therapeutically attractive for tissue regeneration and repair, we examined the physiological responses of human umbilical cord blood-derived MSCs (hUCB-MSCs) to genotoxic stress. We found that that sublethal doses of reactive oxygen species (ROS) and ionizing radiation cause DNA damage and reduce DNA synthesis and cell proliferation in hUCB-MSCs, resulting in cellular senescence. In contrast, these physiological changes were limited in human fibroblast and cancer cells. Our data show that reduced activities of antioxidant enzymes, which may occur due to low gene expression levels, cause hUCB-MSCs to undergo cellular senescence in response to oxidative stress and ionizing radiation. Resistance of hUCB-MSCs to oxidative stresses was restored by increasing the intracellular antioxidant activity in hUCB-MSCs via exogenous addition of antioxidants. Therefore, the proliferation and fate of hUCB-MSCs can be controlled by exposure to oxidative stresses.
Asunto(s)
Senescencia Celular , Sangre Fetal/citología , Rayos gamma , Células Madre Mesenquimatosas/citología , Estrés Oxidativo , Antioxidantes/metabolismo , Antioxidantes/farmacología , Catalasa/farmacología , Muerte Celular , Proliferación Celular/efectos de los fármacos , Ensayo Cometa , Daño del ADN , Relación Dosis-Respuesta a Droga , Activación Enzimática , Células HeLa , Humanos , Peróxido de Hidrógeno/farmacología , Células Madre Mesenquimatosas/efectos de los fármacos , Células Madre Mesenquimatosas/metabolismo , Polietilenglicoles/farmacología , Especies Reactivas de Oxígeno/metabolismoRESUMEN
TopBP1 plays important roles in chromosome replication, DNA damage response, and other cellular regulatory functions in vertebrates. Although the roles of TopBP1 have been studied mostly in cancer cell lines, its physiological function remains unclear in mice and untransformed cells. We generated conditional knock-out mice in which exons 5 and 6 of the TopBP1 gene are flanked by loxP sequences. Although TopBP1-deficient embryos developed to the blastocyst stage, no homozygous mutant embryos were recovered at E8.5 or beyond, and completely resorbed embryos were frequent at E7.5, indicating that mutant embryos tend to die at the peri-implantation stage. This finding indicated that TopBP1 is essential for cell proliferation during early embryogenesis. Ablation of TopBP1 in TopBP1(flox/flox) mouse embryonic fibroblasts and 3T3 cells using Cre recombinase-expressing retrovirus arrests cell cycle progression at the G(1), S, and G(2)/M phases. The TopBP1-ablated mouse cells exhibit phosphorylation of H2AX and Chk2, indicating that the cells contain DNA breaks. The TopBP1-ablated mouse cells enter cellular senescence. Although RNA interference-mediated knockdown of TopBP1 induced cellular senescence in human primary cells, it induced apoptosis in cancer cells. Therefore, TopBP1 deficiency in untransformed mouse and human primary cells induces cellular senescence rather than apoptosis. These results indicate that TopBP1 is essential for cell proliferation and maintenance of chromosomal integrity.
Asunto(s)
Apoptosis/fisiología , Blastocisto/metabolismo , Proteínas Portadoras/metabolismo , Ciclo Celular/fisiología , Proteínas de Unión al ADN/metabolismo , Desarrollo Embrionario/fisiología , Proteínas Nucleares/metabolismo , Células 3T3 , Animales , Blastocisto/citología , Proteínas Portadoras/genética , Línea Celular Tumoral , Senescencia Celular/fisiología , Quinasa de Punto de Control 2 , Inestabilidad Cromosómica/fisiología , Roturas del ADN , Proteínas de Unión al ADN/genética , Pérdida del Embrión/genética , Pérdida del Embrión/metabolismo , Técnicas de Silenciamiento del Gen , Histonas/genética , Histonas/metabolismo , Humanos , Ratones , Ratones Transgénicos , Proteínas Nucleares/genética , Fosforilación/fisiología , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismoRESUMEN
Human TopBP1 with eight BRCA1 C terminus domains has been mainly reported to be involved in DNA damage response pathways. Here we show that TopBP1 is also required for G(1) to S progression in a normal cell cycle. TopBP1 deficiency inhibited cells from entering S phase by up-regulating p21 and p27, resulting in down-regulation of cyclin E/CDK2. Although co-depletion of p21 and p27 with TopBP1 restored the cyclin E/CDK2 kinase activity, however, cells remained arrested at the G(1)/S boundary, showing defective chromatin-loading of replication components. Based on these results, we suggest a dual role of TopBP1 necessary for the G(1)/S transition: one for activating cyclin E/CDK2 kinase and the other for loading replication components onto chromatin to initiate DNA synthesis.