RESUMEN
Cyclin-dependent kinase 7 (Cdk7) is required in cell-cycle and transcriptional regulation owing to its function as both a CDK-activating kinase (CAK) and part of transcription factor TFIIH. Cdk7 forms active complexes by associating with Cyclin H and Mat1, and is regulated by two phosphorylations in the activation segment (T loop): the canonical activating modification at T170 and another at S164. Here we report the crystal structure of the human Cdk7/Cyclin H/Mat1 complex containing both T-loop phosphorylations. Whereas pT170 coordinates basic residues conserved in other CDKs, pS164 nucleates an arginine network unique to the ternary Cdk7 complex, involving all three subunits. We identify differential dependencies of kinase activity and substrate recognition on the individual phosphorylations. CAK function is unaffected by T-loop phosphorylation, whereas activity towards non-CDK substrates is increased several-fold by T170 phosphorylation. Moreover, dual T-loop phosphorylation stimulates multisite phosphorylation of the RNA polymerase II (RNAPII) carboxy-terminal domain (CTD) and SPT5 carboxy-terminal repeat (CTR) region. In human cells, Cdk7 activation is a two-step process wherein S164 phosphorylation precedes, and may prime, T170 phosphorylation. Thus, dual T-loop phosphorylation can regulate Cdk7 through multiple mechanisms, with pS164 supporting tripartite complex formation and possibly influencing processivity, while pT170 enhances activity towards key transcriptional substrates.
Asunto(s)
Quinasa Activadora de Quinasas Ciclina-Dependientes , Quinasas Ciclina-Dependientes , Fosforilación , Humanos , Quinasas Ciclina-Dependientes/metabolismo , Quinasas Ciclina-Dependientes/química , Quinasas Ciclina-Dependientes/genética , Ciclina H/metabolismo , Ciclina H/química , Ciclina H/genética , Cristalografía por Rayos X , ARN Polimerasa II/metabolismo , ARN Polimerasa II/química , Factor de Transcripción TFIIH/metabolismo , Factor de Transcripción TFIIH/química , Factor de Transcripción TFIIH/genética , Modelos Moleculares , Factores de Transcripción/metabolismo , Factores de Transcripción/química , Factores de Transcripción/genética , Dominios Proteicos , Proteínas de Ciclo CelularRESUMEN
The infection of human cytomegalovirus (HCMV) is strongly determined by the host-cell interaction in a way that the efficiency of HCMV lytic replication is dependent on the regulatory interplay between viral and cellular proteins. In particular, the activities of protein kinases, such as cyclin-dependent kinases (CDKs) and the viral CDK ortholog (vCDK/pUL97), play an important role in both viral reproduction and virus-host interaction. Very recently, we reported on the complexes formed between vCDK/pUL97, human cyclin H, and CDK7. Major hallmarks of this interplay are the interaction between cyclin H and vCDK/pUL97, which is consistently detectable across various conditions and host cell types of infection, the decrease or increase in pUL97 kinase activity resulting from cyclin H knock-down or elevated levels, respectively, and significant trans-stimulation of human CDK7 activity by pUL97 in vitro. Due to the fact that even a ternary complex of vCDK/pUL97-cyclin H-CDK7 can be detected by coimmunoprecipitation and visualized by bioinformatic structural modeling, we postulated a putative impact of the respective kinase activities on the patterns of transcription in HCMV-infected cells. Here, we undertook a first vCDK/pUL97-specific transcriptomic analysis, which combined conditions of fully lytic HCMV replication with those under specific vCDK/pUL97 or CDK7 drug-mediated inhibition or transient cyclin H knockout. The novel results were further strengthened using bioinformatic modeling of the involved multi-protein complexes. Our data underline the importance of these kinase activities for the C-terminal domain (CTD) phosphorylation-driven activation of host RNA polymerase in HCMV-infected cells. The impact of the individual experimental conditions on differentially expressed gene profiles is described in detail and discussed.
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Ciclinas , Infecciones por Herpesviridae , Humanos , Ciclinas/metabolismo , Citomegalovirus/genética , Ciclina H/metabolismo , Quinasas Ciclina-Dependientes/genética , Quinasas Ciclina-Dependientes/metabolismo , FosforilaciónRESUMEN
Human cytomegalovirus (HCMV) infection is shaped by a tightly regulated interplay between viral and cellular proteins. Distinct kinase activities, such as the viral cyclin-dependent kinase ortholog (vCDK) pUL97 and cellular CDK7 are both crucial for efficient viral replication. Previously, we reported that both kinases, vCDK/pUL97 and CDK7, interact with cyclin H, thereby achieving an enhanced level of kinase activity and overall functionality in viral replication. Here we provide a variety of novel results, as generated on a methodologically extended basis, and present a concept for the codetermination of viral replication efficiency through these kinase activities: (i) cyclin H expression, in various human cell types, is substantially upregulated by strains of HCMV including the clinically relevant HCMV Merlin; (ii) vCDK/pUL97 interacts with human cyclin H in both HCMV-infected and plasmid-transfected cell systems; (iii) a doxycycline-inducible shRNA-dependent knock-down (KD) of cyclin H significantly reduces pUL97 activity (qSox in vitro kinase assay); (iv) accordingly, pUL97 in vitro kinase activity is seen significantly increased upon addition of recombinant cyclin H; (v) as a point of specific importance, human CDK7 activity shows an increase by vCDK/pUL97-mediated trans-stimulation (whereas pUL97 is not stimulated by CDK7); (vi) phosphosite-specific antibodies indicate an upregulated CDK7 phosphorylation upon HCMV infection, as mediated through a pUL97-specific modulatory effect (i.e. shown by pUL97 inhibitor treatment or pUL97-deficient viral mutant); (vii) finally, an efficient KD of cyclin H in primary fibroblasts generally results in an impaired HCMV replication efficiency as measured on protein and genomic levels. These results show evidence for the codetermination of viral replication by vCDK/pUL97, cyclin H and CDK7, thus supporting the specific importance of cyclin H as a central regulatory factor, and suggesting novel targeting options for antiviral drugs.
Asunto(s)
Quinasas Ciclina-Dependientes , Citomegalovirus , Humanos , Antivirales , Ciclina H , Quinasas Ciclina-Dependientes/genética , Citomegalovirus/genética , FosforilaciónRESUMEN
The complex host interaction network of human cytomegalovirus (HCMV) involves the regulatory protein kinase pUL97, which represents a viral cyclin-dependent kinase (CDK) ortholog. pUL97 interacts with the three human cyclin types T1, H, and B1, whereby the binding region of cyclin T1 and the pUL97 oligomerization region were both assigned to amino acids 231-280. We further addressed the question of whether HCMVs harboring mutations in ORF-UL97, i.e., short deletions or resistance-conferring point mutations, are affected in the interaction with human cyclins and viral replication. To this end, clinically relevant UL97 drug-resistance-conferring mutants were analyzed by whole-genome sequencing and used for genetic marker transfer experiments. The recombinant HCMVs indicated conservation of pUL97-cyclin interaction, since all viral UL97 point mutants continued to interact with the analyzed cyclin types and exerted wild-type-like replication fitness. In comparison, recombinant HCMVs UL97 Δ231-280 and also the smaller deletion Δ236-275, but not Δ241-270, lost interaction with cyclins T1 and H, showed impaired replication efficiency, and also exhibited reduced kinase activity. Moreover, a cellular knock-out of cyclins B1 or T1 did not alter HCMV replication phenotypes or pUL97 kinase activity, possibly indicating alternative, compensatory pUL97-cyclin interactions. In contrast, however, cyclin H knock-out, similar to virus deletion mutants in the pUL97-cyclin H binding region, exhibited strong defective phenotypes of HCMV replication, as supported by reduced pUL97 kinase activity in a cyclin H-dependent coexpression setting. Thus, cyclin H proved to be a very relevant determinant of pUL97 kinase activity and viral replication efficiency. As a conclusion, the results provide evidence for the functional importance of pUL97-cyclin interaction. High selective pressure on the formation of pUL97-cyclin complexes was identified by the use of clinically relevant mutants.
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Ciclina H , Citomegalovirus , Proteínas Virales , Aminoácidos/metabolismo , Ciclina H/genética , Ciclina H/metabolismo , Ciclina T/genética , Ciclina T/metabolismo , Quinasas Ciclina-Dependientes/genética , Quinasas Ciclina-Dependientes/metabolismo , Citomegalovirus/fisiología , Marcadores Genéticos , Humanos , Fosforilación , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Proteínas Virales/genética , Replicación Viral/genéticaRESUMEN
INTRODUCTION: Studies have previously shown that Cyclin H (CCNH) is involved in the tumorigenesis and development of many cancers. The increasing research in CCNH is associated with the poor prognosis of most human cancers. Early diagnosis and clinical treatment are still the main challenges for lung cancer treatment. However, the exact role of CCNH in the tumorigenesis of lung cancer remains unclear. METHODS: The Tumor Genome Atlas (TCGA) database and the Clinical Proteomics Tumor Analysis Association (CPTAC) database were analyzed to detect key genes that might play an important role in lung cancer. The biological functions of CCNH were further revealed through bioinformatics experiments. The Kaplan-Meier method was applied to explore the relationship between CCNH expression and prognosis. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was used to detect the expression levels of CCNH in 6 lung cancer tissues and 3 cancer cell lines. The effect of CCNH expression on lung cancer progression was studied by in vitro functional experiments. RESULTS: Database analysis screened out candidate oncogenes, and CCNH was of great significance to the tumorigenesis of lung cancer. The higher the expression of CCNH was, the lower the survival rate of lung cancer patients were. The qRT-PCR data illustrated that the CCNH expression level was largely increased in lung cancer tissues and cells. The reduction of CCNH inhibited cell proliferation, invasion, and migration. CONCLUSION: CCNH was related to poor prognosis, suggesting that CCNH regulated the tumorigenesis and development of lung cancer. Our study suggested that CCNH was a promising biomarker and target in the treatment of lung cancer.
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Ciclina H/genética , Neoplasias Pulmonares/genética , Adenocarcinoma del Pulmón/genética , Adenocarcinoma del Pulmón/metabolismo , Adenocarcinoma del Pulmón/patología , Biomarcadores de Tumor/genética , Carcinogénesis/genética , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patología , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular/genética , Biología Computacional , Ciclina H/metabolismo , Bases de Datos Genéticas/estadística & datos numéricos , Progresión de la Enfermedad , Regulación Neoplásica de la Expresión Génica , Humanos , Estimación de Kaplan-Meier , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Invasividad Neoplásica/genética , Invasividad Neoplásica/patología , Pronóstico , Mapas de Interacción de Proteínas , ProteómicaRESUMEN
Cyclin-dependent kinase 7 (CDK7), Cyclin H, and the RING-finger protein MAT1 form the heterotrimeric CDK-activating kinase (CAK) complex which is vital for transcription and cell-cycle control. When associated with the general transcription factor II H (TFIIH) it activates RNA polymerase II by hyperphosphorylation of its C-terminal domain (CTD). In the absence of TFIIH the trimeric complex phosphorylates the T-loop of CDKs that control cell-cycle progression. CAK holds a special position among the CDK branch due to this dual activity and the dependence on two proteins for activation. We solved the structure of the CAK complex from the model organism Chaetomium thermophilum at 2.6-Å resolution. Our structure reveals an intricate network of interactions between CDK7 and its two binding partners MAT1 and Cyclin H, providing a structural basis for the mechanism of CDK7 activation and CAK activity regulation. In vitro activity measurements and functional mutagenesis show that CDK7 activation can occur independent of T-loop phosphorylation and is thus exclusively MAT1-dependent by positioning the CDK7 T-loop in its active conformation.
Asunto(s)
Ciclina H , Quinasas Ciclina-Dependientes , Ciclo Celular , Chaetomium/química , Chaetomium/enzimología , Ciclina H/química , Ciclina H/metabolismo , Quinasas Ciclina-Dependientes/química , Quinasas Ciclina-Dependientes/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Fosforilación , Transcripción Genética , Quinasa Activadora de Quinasas Ciclina-DependientesRESUMEN
The incidence of thyroid cancer (TC), particularly well-differentiated forms (DTC), has been rising and remains the highest among endocrine malignancies. Although ionizing radiation (IR) is well established on DTC aetiology, other environmental and genetic factors may also be involved. DNA repair single nucleotide polymorphisms (SNPs) could be among the former, helping in explaining the high incidence. To further clarify the role of DNA repair SNPs in DTC susceptibility, we analyzed 36 SNPs in 27 DNA repair genes in a population of 106 DTCs and corresponding controls with the aim of interpreting joint data from previously studied isolated SNPs in DNA repair genes. Significant associations with DTC susceptibility were observed for XRCC3 rs861539, XPC rs2228001, CCNH rs2230641, MSH6 rs1042821 and ERCC5 rs2227869 and for a haplotype block on chromosome 5q. From 595 SNP-SNP combinations tested and 114 showing relevance, 15 significant SNP combinations (p < 0.01) were detected on paired SNP analysis, most of which involving CCNH rs2230641 and mismatch repair variants. Overall, a gene-dosage effect between the number of risk genotypes and DTC predisposition was observed. In spite of the volume of data presented, new studies are sought to provide an interpretability of the role of SNPs in DNA repair genes and their combinations in DTC susceptibility.
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Reparación del ADN , Predisposición Genética a la Enfermedad , Polimorfismo de Nucleótido Simple , Neoplasias de la Tiroides/genética , Adulto , Anciano , Cromosomas Humanos Par 5/genética , Ciclina H/genética , Proteínas de Unión al ADN/genética , Endonucleasas/genética , Femenino , Haplotipos , Humanos , Masculino , Persona de Mediana Edad , Proteínas Nucleares/genética , Factores de Transcripción/genéticaRESUMEN
RT-qPCR is the gold standard for candidate gene expression analysis. However, the interpretation of RT-qPCR results depends on the proper use of internal controls, i.e., reference genes. Japanese quail is an agronomic species also used as a laboratory model, but little is known about RT-qPCR reference genes for this species. Thus, we investigated 10 putative reference genes (ACTB, GAPDH, PGK1, RPS7, RPS8, RPL19, RPL32, SDHA, TBP and YWHAZ) in three different female and male quail tissues (liver, brain and pectoral muscle). Gene expression stability was evaluated with three different algorithms: geNorm, NormFinder and BestKeeper. For each tissue, a suitable set of reference genes was defined and validated by a differential analysis of gene expression between females and males (CCNH in brain and RPL19 in pectoral muscle). Collectively, our study led to the identification of suitable reference genes in liver, brain and pectoral muscle for Japanese quail, along with recommendations for the identification of reference gene sets for this species.
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Coturnix/genética , Ciclina H/genética , Perfilación de la Expresión Génica/normas , Reacción en Cadena en Tiempo Real de la Polimerasa/normas , Proteínas Ribosómicas/genética , Algoritmos , Animales , Proteínas Aviares/genética , Encéfalo/metabolismo , Femenino , Regulación de la Expresión Génica , Masculino , Músculo Esquelético/química , Especificidad de Órganos , Estándares de ReferenciaRESUMEN
Human cytomegalovirus (HCMV) is a common ß-herpesvirus causing life-long latent infections. HCMV replication interferes with cell cycle regulation in host cells because the HCMV-encoded cyclin-dependent kinase (CDK) ortholog pUL97 extensively phosphorylates the checkpoint regulator retinoblastoma protein. pUL97 also interacts with cyclins B1, T1, and H, and recent findings have strongly suggested that these interactions influence pUL97 substrate recognition. Interestingly, here we detected profound mechanistic differences among these pUL97-cyclin interactions. Our study revealed the following. (i) pUL97 interacts with cyclins B1 and H in a manner dependent on pUL97 activity and HCMV-specific cyclin modulation, respectively. (ii) The phosphorylated state of both proteins is an important determinant of the pUL97-cyclin B1 interaction. (iii) Activated phospho-Thr-315 cyclin H is up-regulated during HCMV replication. (iv) Thr-315 phosphorylation is independent of intracellular pUL97 or CDK7 activity. (v) pUL97-mediated in vitro phosphorylation is detectable for cyclin B1 but not H. (vi) Mutual transphosphorylation between pUL97 and CDK7 is not detectable, and an MS-based phosphosite analysis indicated that pUL97 might unexpectedly not be phosphorylated in its T-loop. (vii) The binary complexes pUL97-cyclin H and CDK7-cyclin H as well as the ternary complex pUL97-cyclin-H-CDK7 are detectable in an assembly-based CoIP approach. (viii) pUL97 self-interaction can be bridged by the transcriptional cyclins T1 or H but not by the classical cell cycle-regulating B1 cyclin. Combined, our findings unravel a number of cyclin type-specific differences in pUL97 interactions and suggest a multifaceted regulatory impact of cyclins on HCMV replication.
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Ciclina B1/metabolismo , Ciclina H/metabolismo , Ciclina T/metabolismo , Citomegalovirus/fisiología , Proteínas Virales/metabolismo , Replicación Viral/fisiología , Ciclina B1/genética , Ciclina H/genética , Ciclina T/genética , Células HEK293 , Humanos , Fosforilación , Dominios Proteicos , Estructura Cuaternaria de Proteína , Proteínas Virales/genéticaRESUMEN
PURPOSE: Digestive tract cancer patients treated with oxaliplatin are often associated with the development of peripheral neuropathy. The aim of the present study is to identify the influence of single-nucleotide polymorphisms (SNPs) in genes involved in oxaliplatin metabolism, cell cycle control, detoxification or excretion pathways with the development of oxaliplatin-induced acute peripheral neuropathy (acute OXAIPN) and its severity among digestive tract cancer patients treated with oxaliplatin-based chemotherapy. PATIENTS AND METHODS: A total of 228 digestive tract cancer patients undergoing with the oxaliplatin-based chemotherapy between November 2014 and December 2016 were included in the current study. Genomic DNA was extracted from peripheral blood by standard phenol-chloroform method. Genotyping of five SNPs in four genes [GSTP1 (rs1965), ABCG2 (rs3114018), CCNH (rs2230641, rs3093816), AGXT (rs4426527)] was carried out by Real-Time TaqMan SNP genotyping assay. RESULTS: We found that the two genetic variants rs2230641 and rs3093816 in cyclin H (CCNH) gene were significantly associated with both the incidence and severity of acute OXAIPN. For CCNH-rs2230641 (AA vs AG+GG; dominant model) Incidence: OR 2.62, 95% CI 1.44-4.75, p = 0.001, severity; OR 4.64, 95% CI 1.58-13.62, p = 0.002. For CCNH-rs3093816 (AA vs AG+GG; dominant model); incidence: OR 3.43, 95% CI 1.57-7.50, p = 0.001; severity: OR 2.36, 95% CI 1.05-5.30, p = 0.033. CONCLUSIONS: The results of the present study found significant association between CCNH polymorphisms and acute OXAIPN development. However, further studies are warranted from independent groups to validate our study results.
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Ciclina H/genética , Neoplasias del Sistema Digestivo/tratamiento farmacológico , Oxaliplatino/efectos adversos , Enfermedades del Sistema Nervioso Periférico/inducido químicamente , Enfermedades del Sistema Nervioso Periférico/genética , Adulto , Anciano , Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/farmacocinética , Ciclo Celular/efectos de los fármacos , Ciclo Celular/genética , Estudios de Cohortes , Neoplasias del Sistema Digestivo/genética , Neoplasias del Sistema Digestivo/metabolismo , Femenino , Predisposición Genética a la Enfermedad , Humanos , Masculino , Persona de Mediana Edad , Oxaliplatino/administración & dosificación , Oxaliplatino/farmacocinética , Polimorfismo de Nucleótido Simple , Estudios Prospectivos , Adulto JovenRESUMEN
The avian reovirus p17 protein is a nucleocytoplasmic shuttling protein. Although we have demonstrated that p17 causes cell growth retardation via activation of p53, the precise mechanisms remain unclear. This is the first report that avian reovirus p17 possesses broad inhibitory effects on cell cycle CDKs, cyclins, CDK-cyclin complexes, and CDK-activating kinase activity in various mammalian, avian, and cancer cell lines. Suppression of CDK activity by p17 occurs by direct binding to CDKs, cyclins, and CDK-cyclin complexes; transcriptional down-regulation of CDKs; cytoplasmic retention of CDKs and cyclins; and inhibition of CDK-activating kinase activity by promoting p53-cyclin H interaction. p17 binds to CDK-cyclin except for CDK1-cyclin B1 and CDK7-cyclin H complexes. We have determined that the negatively charged 151LAVXDVDA(E/D)DGADPN165 motif in cyclin B1 interacts with a positively charged region of CDK1. p17 mimics the cyclin B1 sequence to compete for CDK1 binding. The PSTAIRE motif is not required for interaction of CDK1-cyclin B1, but it is required for other CDK-cyclin complexes. p17 interacts with cyclins by its cyclin-binding motif, 125RXL127 Sequence and mutagenic analyses of p17 indicated that a 140WXFD143 motif and residues Asp-113 and Lys-122 in p17 are critical for CDK2 and CDK6 binding, leading to their sequestration in the cytoplasm. Exogenous expression of p17 significantly enhanced virus replication, whereas p17 mutants with low binding ability to cell cycle CDKs had no effect on virus yield, suggesting that p17 inhibits cell growth and the cell cycle, benefiting virus replication. An in vivo tumorigenesis assay also showed a significant reduction in tumor size.
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Ciclina H/metabolismo , Quinasas Ciclina-Dependientes/metabolismo , Ciclinas/metabolismo , Orthoreovirus Aviar/fisiología , Proteína p53 Supresora de Tumor/metabolismo , Proteínas Virales/metabolismo , Animales , Ciclo Celular , Embrión de Pollo , Chlorocebus aethiops , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Ciclina H/genética , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Ciclinas/antagonistas & inhibidores , Humanos , Infecciones por Reoviridae/virología , Células Tumorales Cultivadas , Proteína p53 Supresora de Tumor/genética , Células Vero , Proteínas Virales/genéticaRESUMEN
BACKGROUND/AIM: The aim of the present study was to investigate the radio-sensitizing efficacy of curcumin, a traditional Chinese medicine (TCM) on colon cancer cells in vitro and in vivo. MATERIALS AND METHODS: Human colon cancer HT-29 cells were treated with curcumin (2.5 µM), irradiation (10 Gy) and the combination of irradiation and curcumin. Cell proliferation was assessed using the MTT assay. Apoptotic cells were detected by Annexin V-PE/7-AAD analysis. PCR was performed to determine differential-expression profiling of 95 DNA-repair genes in irradiated cells and cells treated with both irradiation and curcumin. Differentially-expressed genes were confirmed by Western blotting. In vivo radio-sensitizing efficacy of curcumin was assessed in a xenograft mouse model of HT-29 colon cancer. Curcumin was administrated daily by intraperitoneal injection at 20 mg/kg/dose. Mice received irradiation (10 Gy) twice weekly. Apoptosis of the cancer cells following treatment was determined by TUNEL staining. RESULTS: Irradiation induced proliferation inhibition and apoptosis of HT-29 cells in vitro. Concurrent curcumin treatment sensitized the HT-29 tumor to irradiation (p<0.01). DNA repair-related genes CCNH and XRCC5 were upregulated and LIG4 and PNKP downregulated by the combination of curcumin and irradiation compared with irradiation alone (p<0.05). Combined treatment of curcumin and irradiation resulted in a significantly greater tumor-growth inhibition and apoptosis compared to irradiation treatment alone (p<0.01). CONCLUSION: Curcumin sensitizes human colon cancer in vitro and in vivo to radiation. Downregulation of LIG4 and PNKP and upregulation of XRCC5 and CCNH DNA-repair-related genes were involved in the radio-sensitizing efficacy of curcumin in colon cancer.
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Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/radioterapia , Curcumina/farmacología , Curcumina/uso terapéutico , Fármacos Sensibilizantes a Radiaciones/farmacología , Fármacos Sensibilizantes a Radiaciones/uso terapéutico , Animales , Apoptosis/efectos de los fármacos , Apoptosis/efectos de la radiación , Proliferación Celular/efectos de los fármacos , Proliferación Celular/efectos de la radiación , Neoplasias del Colon/metabolismo , Neoplasias del Colon/patología , Ciclina H/genética , Ciclina H/metabolismo , ADN Ligasa (ATP)/genética , ADN Ligasa (ATP)/metabolismo , Reparación del ADN/genética , Enzimas Reparadoras del ADN/genética , Enzimas Reparadoras del ADN/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de la radiación , Células HT29 , Humanos , Autoantígeno Ku/genética , Autoantígeno Ku/metabolismo , Medicina Tradicional China , Ratones Endogámicos BALB C , Ratones Desnudos , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Carga Tumoral/efectos de los fármacosRESUMEN
BACKGROUND: Genomic analysis of drug response can provide unique insights into therapies that can be used to match the "right drug to the right patient." However, the process of discovering such therapeutic insights using genomic data is not straightforward and represents an area of active investigation. EDDY (Evaluation of Differential DependencY), a statistical test to detect differential statistical dependencies, is one method that leverages genomic data to identify differential genetic dependencies. EDDY has been used in conjunction with the Cancer Therapeutics Response Portal (CTRP), a dataset with drug-response measurements for more than 400 small molecules, and RNAseq data of cell lines in the Cancer Cell Line Encyclopedia (CCLE) to find potential drug-mediator pairs. Mediators were identified as genes that showed significant change in genetic statistical dependencies within annotated pathways between drug sensitive and drug non-sensitive cell lines, and the results are presented as a public web-portal (EDDY-CTRP). However, the interpretability of drug-mediator pairs currently hinders further exploration of these potentially valuable results. METHODS: In this study, we address this challenge by constructing evidence networks built with protein and drug interactions from the STITCH and STRING interaction databases. STITCH and STRING are sister databases that catalog known and predicted drug-protein interactions and protein-protein interactions, respectively. Using these two databases, we have developed a method to construct evidence networks to "explain" the relation between a drug and a mediator. RESULTS: We applied this approach to drug-mediator relations discovered in EDDY-CTRP analysis and identified evidence networks for ~70% of drug-mediator pairs where most mediators were not known direct targets for the drug. Constructed evidence networks enable researchers to contextualize the drug-mediator pair with current research and knowledge. Using evidence networks, we were able to improve the interpretability of the EDDY-CTRP results by linking the drugs and mediators with genes associated with both the drug and the mediator. CONCLUSION: We anticipate that these evidence networks will help inform EDDY-CTRP results and enhance the generation of important insights to drug sensitivity that will lead to improved precision medicine applications.
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Preparaciones Farmacéuticas/metabolismo , Proteínas/metabolismo , Línea Celular , Ciclina H/química , Ciclina H/genética , Ciclina H/metabolismo , Reparación del ADN , Bases de Datos Factuales , Proteínas Quinasas Asociadas a Muerte Celular/química , Proteínas Quinasas Asociadas a Muerte Celular/genética , Proteínas Quinasas Asociadas a Muerte Celular/metabolismo , Redes Reguladoras de Genes , Humanos , Imidazoles/química , Imidazoles/metabolismo , Preparaciones Farmacéuticas/química , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas/química , Proteínas/genética , Triazinas/química , Triazinas/metabolismoRESUMEN
All well-studied eukaryotic cell cycles are driven by cyclins, which activate cyclin-dependent kinases (CDKs), and these protein kinase complexes are viable drug targets. The regulatory control of the Plasmodium falciparum cell division cycle remains poorly understood, and the roles of the various CDKs and cyclins remain unclear. The P. falciparum genome contains multiple CDKs, but surprisingly, it does not contain any sequence-identifiable G1-, S-, or M-phase cyclins. We demonstrate that P. falciparum Cyc1 (PfCyc1) complements a G1 cyclin-depleted Saccharomyces cerevisiae strain and confirm that other identified malaria parasite cyclins do not complement this strain. PfCyc1, which has the highest sequence similarity to the conserved cyclin H, cannot complement a temperature-sensitive yeast cyclin H mutant. Coimmunoprecipitation of PfCyc1 from P. falciparum parasites identifies PfMAT1 and PfMRK as specific interaction partners and does not identify PfPK5 or other CDKs. We then generate an endogenous conditional allele of PfCyc1 in blood-stage P. falciparum using a destabilization domain (DD) approach and find that PfCyc1 is essential for blood-stage proliferation. PfCyc1 knockdown does not impede nuclear division, but it prevents proper cytokinesis. Thus, we demonstrate that PfCyc1 has a functional divergence from bioinformatic predictions, suggesting that the malaria parasite cell division cycle has evolved to use evolutionarily conserved proteins in functionally novel ways.IMPORTANCE Human infection by the eukaryotic parasite Plasmodium falciparum causes malaria. Most well-studied eukaryotic cell cycles are driven by cyclins, which activate cyclin-dependent kinases (CDKs) to promote essential cell division processes. Remarkably, there are no identifiable cyclins that are predicted to control the cell cycle in the malaria parasite genome. Thus, our knowledge regarding the basic mechanisms of the malaria parasite cell cycle remains unsatisfactory. We demonstrate that P. falciparum Cyc1 (PfCyc1), a transcriptional cyclin homolog, complements a cell cycle cyclin-deficient yeast strain but not a transcriptional cyclin-deficient strain. We show that PfCyc1 forms a complex in the parasite with PfMRK and the P. falciparum MAT1 homolog. PfCyc1 is essential and nonredundant in blood-stage P. falciparum PfCyc1 knockdown causes a stage-specific arrest after nuclear division, demonstrating morphologically aberrant cytokinesis. This work demonstrates a conserved PfCyc1/PfMAT1/PfMRK complex in malaria and suggests that it functions as a schizont stage-specific regulator of the P. falciparum life cycle.
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Ciclina H/metabolismo , Citocinesis , Malaria Falciparum/parasitología , Plasmodium falciparum/genética , Proteínas Protozoarias/metabolismo , Ciclo Celular/genética , Ciclina H/química , Ciclina H/genética , Quinasas Ciclina-Dependientes/genética , Quinasas Ciclina-Dependientes/metabolismo , Citocromos c/genética , Citocromos c/metabolismo , Citocinesis/genética , Estadios del Ciclo de Vida/genética , Mutación , Plasmodium falciparum/crecimiento & desarrollo , Proteínas Protozoarias/genética , Reproducción Asexuada/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Cyclin-dependent kinase 7 in conjunction with CyclinH and Mat1 activates cell cycle CDKs and is a part of the general transcription factor TFIIH. Role of Cdk7 is well characterized in model eukaryotes however its relevance in protozoan parasites has not been investigated. This important regulator of key processes warrants closer examination particularly in this parasite given its unique cell cycle progression and flexible mode of replication. We report functional characterization of TgCdk7 and its partners TgCyclinH and TgMat1. Recombinant Cdk7 displays kinase activity upon binding its cyclin partner and this activity is further enhanced in presence of Mat1. The activated kinase phosphorylates C-terminal domain of TgRPB1 suggesting its role in parasite transcription. Therefore, the function of Cdk7 in CTD phosphorylation and RPB1 mediated transcription was investigated using Cdk7 inhibitor. Unphosphorylated CTD binds promoter DNA while phosphorylation by Cdk7 triggers its dissociation from DNA with implications for transcription initiation. Inhibition of Cdk7 in the parasite led to strong reduction in Serine 5 phosphorylation of TgRPB1-CTD at the promoters of constitutively expressed actin1 and sag1 genes with concomitant reduction of both nascent RNA synthesis and 5'-capped transcripts. Therefore, we provide compelling evidence for crucial role of TgCdk7 kinase activity in mRNA synthesis.
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Quinasas Ciclina-Dependientes/genética , ARN Polimerasa II/genética , ARN Mensajero/biosíntesis , Toxoplasma/genética , Ciclo Celular/genética , Ciclina H/genética , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Proteínas de Unión al ADN/genética , Fosforilación , Regiones Promotoras Genéticas , ARN Mensajero/genética , Proteínas Recombinantes/genética , Factor de Transcripción TFIIH/genética , Transcripción GenéticaRESUMEN
PURPOSE: CDK-activating kinase (CAK) is required for the regulation of the cell cycle and is a trimeric complex consisting of cyclin-dependent kinase 7 (CDK7), Cyclin H, and the accessory protein, MAT1. CDK7 also plays a critical role in regulating transcription, primarily by phosphorylating RNA polymerase II, as well as transcription factors such as estrogen receptor-α (ER). Deregulation of cell cycle and transcriptional control are general features of tumor cells, highlighting the potential for the use of CDK7 inhibitors as novel cancer therapeutics. EXPERIMENTAL DESIGN: mRNA and protein expression of CDK7 and its essential cofactors cyclin H and MAT1 were evaluated in breast cancer samples to determine if their levels are altered in cancer. Immunohistochemical staining of >900 breast cancers was used to determine the association with clinicopathologic features and patient outcome. RESULTS: We show that expressions of CDK7, cyclin H, and MAT1 are all closely linked at the mRNA and protein level, and their expression is elevated in breast cancer compared with the normal breast tissue. Intriguingly, CDK7 expression was inversely proportional to tumor grade and size, and outcome analysis showed an association between CAK levels and better outcome. Moreover, CDK7 expression was positively associated with ER expression and in particular with phosphorylation of ER at serine 118, a site important for ER transcriptional activity. CONCLUSIONS: Expressions of components of the CAK complex, CDK7, MAT1, and Cyclin H are elevated in breast cancer and correlate with ER. Like ER, CDK7 expression is inversely proportional to poor prognostic factors and survival. Clin Cancer Res; 22(23); 5929-38. ©2016 AACR.
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Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Proteínas Portadoras/genética , Ciclina H/genética , Quinasas Ciclina-Dependientes/genética , Expresión Génica/genética , Receptores de Estrógenos/genética , Adulto , Proteínas de Ciclo Celular , Femenino , Humanos , Persona de Mediana Edad , Fosforilación/genética , Pronóstico , Transducción de Señal/genética , Factores de Transcripción , Transcripción Genética/genética , Quinasa Activadora de Quinasas Ciclina-DependientesRESUMEN
Cigarette smoke derivatives like NNK (4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone) and NNAL (4-(methylnitrosamino)-1-(3-pyridyl)-1-butan-1-ol) are well-known carcinogens. We analyzed the interaction of enzymes involved in the NER (nucleotide excision repair) pathway with ligands (NNK and NNAL). Binding was characterized for the enzymes sharing equivalent or better interaction as compared to +Ve control. The highest obtained docking energy between NNK and enzymes RAD23A, CCNH, CDK7, and CETN2 were -7.13 kcal/mol, -7.27 kcal/mol, -8.05 kcal/mol and -7.58 kcal/mol respectively. Similarly the highest obtained docking energy between NNAL and enzymes RAD23A, CCNH, CDK7, and CETN2 were -7.46 kcal/mol, -7.94 kcal/mol, -7.83 kcal/mol and -7.67 kcal/mol respectively. In order to find out the effect of NNK and NNAL on enzymes involved in the NER pathway applying protein-protein interaction and protein-complex (i.e. enzymes docked with NNK/NNAL) interaction analysis. It was found that carcinogens are well capable to reduce the normal functioning of genes like RAD23A (HR23A), CCNH, CDK7 and CETN2. In silico analysis indicated loss of functions of these genes and their corresponding enzymes, which possibly might be a cause for alteration of DNA repair pathways leading to damage buildup and finally contributing to cancer formation.
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Algoritmos , Proteínas de Unión al Calcio/metabolismo , Carcinógenos/metabolismo , Proteínas de Ciclo Celular/metabolismo , Ciclina H/metabolismo , Quinasas Ciclina-Dependientes/metabolismo , Enzimas Reparadoras del ADN/metabolismo , Proteínas de Unión al ADN/metabolismo , Nitrosaminas/metabolismo , Piridinas/metabolismo , Proteínas de Unión al Calcio/química , Carcinógenos/química , Proteínas de Ciclo Celular/química , Cromatografía Líquida de Alta Presión , Biología Computacional/métodos , Ciclina H/química , Quinasas Ciclina-Dependientes/química , Reparación del ADN , Enzimas Reparadoras del ADN/química , Proteínas de Unión al ADN/química , Humanos , Modelos Moleculares , Simulación del Acoplamiento Molecular , Nitrosaminas/química , Dominios y Motivos de Interacción de Proteínas , Piridinas/química , Fumar , Quinasa Activadora de Quinasas Ciclina-DependientesRESUMEN
CtBP2, as a transcriptional corepressor of epithelial-specific genes, has been reported to promote tumor due to upregulating epithelial-mesenchymal transition (EMT) in cancer cells. CtBP2 was also demonstrated to contribute to the proliferation of esophageal squamous cell carcinoma (ESCC) cells through a negative transcriptional regulation of p16(INK4A). In this study, for the first time, we reported that CtBP2 expression, along with CCNH/CDK7, was higher in ESCC tissues with lymph node metastases than in those without lymph node metastases. Moreover, both CtBP2 and CCNH/CDK7 were positively correlated with E-cadherin, tumor grade, and tumor metastasis. However, the concrete mechanism of CtBP2's role in enhancing ESCC migration remains incompletely understood. We confirmed that CCNH/CDK7 could directly interact with CtBP2 in ESCC cells in vivo and in vitro. Furthermore, our data demonstrate for the first time that CtBP2 enhanced the migration of ESCC cells in a CCNH/CDK7-dependent manner. Our results indicated that CCNH/CDK7-CtBP2 axis may augment ESCC cell migration, and targeting the interaction of both may provide a novel therapeutic target of ESCC.
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Oxidorreductasas de Alcohol/biosíntesis , Carcinoma de Células Escamosas/genética , Ciclina H/biosíntesis , Quinasas Ciclina-Dependientes/biosíntesis , Neoplasias Esofágicas/genética , Proteínas del Tejido Nervioso/biosíntesis , Anciano , Oxidorreductasas de Alcohol/genética , Carcinoma de Células Escamosas/patología , Movimiento Celular/genética , Proliferación Celular/genética , Proteínas Co-Represoras , Ciclina H/genética , Inhibidor p16 de la Quinasa Dependiente de Ciclina/biosíntesis , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Quinasas Ciclina-Dependientes/genética , Transición Epitelial-Mesenquimal , Neoplasias Esofágicas/patología , Carcinoma de Células Escamosas de Esófago , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Metástasis Linfática , Masculino , Persona de Mediana Edad , Proteínas del Tejido Nervioso/genética , Quinasa Activadora de Quinasas Ciclina-DependientesAsunto(s)
Enfermedades de las Arterias Carótidas/enzimología , Proliferación Celular , Ciclina H/metabolismo , Quinasas Ciclina-Dependientes/metabolismo , Células Endoteliales/enzimología , Células Endoteliales de la Vena Umbilical Humana/enzimología , MicroARNs/metabolismo , Transcripción Genética , Animales , Humanos , MasculinoRESUMEN
Alterations in the regulation of the cell cycle are strongly linked to tumorigenesis, so genetic variants in genes critical to control of the cycle are good candidates to have their association with susceptibility to oral cancer assessed. In this hospital-based, case-control study of 445 patients who had been newly-diagnosed with oral cancer and 449 unaffected controls, we used a multigenic approach to examine the associations among a panel of 10 selected polymorphisms in the pathway of the cell cycle that were possibly susceptible to oral cancer. Six of 9 single nucleotide polymorphisms in the cell cycle showed significant risks for oral cancer, the highest risk being evident for p27 (rs34329; Odds ratio 3.05, 95% CI 2.12 to 4.40). A significant risk of oral cancer was also evident for individual polymorphisms of cyclin E (rs1406), cyclin H (rs3093816), cyclin D1-1 (rs647451), cyclin D2 (rs3217901) and Rb1-2 (rs3092904). The risk of oral cancer increased significantly as the number of unfavourable genotypes in the pathway increased, and so the results point to a stronger combined effect of polymorphisms in important cell cycle regulatory genes on predisposition to oral cancer.