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1.
Mol Oncol ; 17(7): 1228-1245, 2023 07.
Artigo em Inglês | MEDLINE | ID: mdl-37081792

RESUMO

Cyclin-dependent kinases (CDKs), together with their cyclin partners, are the master cell cycle regulators. Remarkably, the cyclin family was extended to include atypical cyclins, characterized by distinctive structural features, but their partner CDKs remain elusive. Here, we conducted a yeast two-hybrid screen to identify new atypical cyclin-CDK complexes. We identified 10 new complexes, including a complex between CDK6 and cyclin I (CCNI), which was found to be active against retinoblastoma protein. CCNI upregulation increased the proliferation of breast cancer cells in vitro and in vivo, with a magnitude similar to that seen upon cyclin D upregulation, an effect that was abrogated by CDK6 silencing or palbociclib treatment. In line with these findings, CCNI downregulation led to a decrease in cell number and a reduction in the percentage of cells reaching S phase. Finally, CCNI upregulation correlated with the high expression of E2F target genes in large panels of cancer cell lines and tissue samples from breast cancer patients. In conclusion, we unveil CCNI as a new player in the pathways that activate CDK6, enriching the wiring of cell cycle control.


Assuntos
Neoplasias da Mama , Ciclina I , Humanos , Feminino , Ciclina I/genética , Ciclinas/genética , Ciclinas/metabolismo , Proliferação de Células/genética , Neoplasias da Mama/genética , Expressão Gênica , Proteínas de Ciclo Celular/genética , Ciclo Celular , Quinase 6 Dependente de Ciclina/genética
2.
Diabetes ; 72(1): 85-96, 2023 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-36201618

RESUMO

A recent discovery effort resulted in identification of novel splice variant and secretory granule antigens within the HLA class I peptidome of human islets and documentation of their recognition by CD8+ T cells from peripheral blood and human islets. In the current study, we applied a systematic discovery process to identify novel CD4+ T cell epitopes derived from these candidate antigens. We predicted 145 potential epitopes spanning unique splice junctions and within conventional secretory granule antigens and measured their in vitro binding to DRB1*04:01. We generated HLA class II tetramers for the 35 peptides with detectable binding and used these to assess immunogenicity and isolate T cell clones. Tetramers corresponding to peptides with verified immunogenicity were then used to label T cells specific for these putative epitopes in peripheral blood. T cells that recognize distinct epitopes derived from a cyclin I splice variant, neuroendocrine convertase 2, and urocortin-3 were detected at frequencies that were similar to those of an immunodominant proinsulin epitope. Cells specific for these novel epitopes predominantly exhibited a Th1-like surface phenotype. Among the three epitopes, responses to the cyclin I peptide exhibited a distinct memory profile. Responses to neuroendocrine convertase 2 were detected among pancreatic infiltrating T cells. These results further establish the contribution of unconventional antigens to the loss of tolerance in autoimmune diabetes.


Assuntos
Linfócitos T CD4-Positivos , Diabetes Mellitus Tipo 1 , Humanos , Ciclina I/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Epitopos de Linfócito T , Peptídeos/metabolismo , Vesículas Secretórias , Processamento Alternativo
3.
Microvasc Res ; 142: 104348, 2022 07.
Artigo em Inglês | MEDLINE | ID: mdl-35245516

RESUMO

BACKGROUND: Retinoblastoma protein (Rb) supports vasoprotective E2F Transcription Factor 1 (E2f1)/Dihydrofolate Reductase (Dhfr) pathway activity in endothelial cells. Cyclin I (Ccni) promotes Cyclin-Dependent Kinase-5 (Cdk5)-mediated Rb phosphorylation. Therefore, we hypothesized that endothelial Ccni may regulate cardiovascular homeostasis, vessel remodeling, and abdominal aortic aneurysm (AAA) formation. METHODS: Aortic CCNI mRNA expression was analyzed in the Gene Expression Omnibus (GEO) GSE57691 cohort consisting of AAA patients (n = 39) and healthy controls (n = 10). We employed wild-type (WT) mice and endothelial Ccni knockout (Ccnifl/flTie2-Cre) mice to conduct in vivo and ex vivo experimentation using an Angiotensin (Ang) II hypertension model and a CaCl2 AAA model. Mice were assessed for Rb/E2f1/Dhfr signaling, biopterin (i.e., biopterin [B], dihydrobiopterin [BH2], and tetrahydrobiopterin [BH4]) production, cardiovascular homeostasis, vessel remodeling, and AAA formation. RESULTS: Aortic CCNI mRNA expression was downregulated in AAA patients. Both Ang II- and CaCl2-induced WT mice showed aortic Ccni upregulation coupled with vasculoprotective upregulation of Rb/E2f1/Dhfr signaling and biopterins. Endothelial Ccni knockout downregulated medial Rb/E2f1/Dhfr signaling and biopterins in Ang II-induced hypertensive mice, which exacerbated eNos uncoupling and H2O2 production. Endothelial Ccni knockout impaired in vivo hemodynamic responses and endothelium-dependent vasodilatation in ex vivo mesenteric arteries in response to Ang II. Endothelial Ccni knockout exacerbated mesenteric artery remodeling and AAA risk in response to Ang II and CaCl2. CONCLUSIONS: Endothelial Ccni acts as a critical negative regulator of eNos uncoupling-mediated ROS generation and thereby reduces vulnerability to hypertension-induced vascular remodeling and AAA development in mice.


Assuntos
Angiotensina II , Aneurisma da Aorta Abdominal , Hipertensão , Remodelação Vascular , Angiotensina II/farmacologia , Animais , Aneurisma da Aorta Abdominal/induzido quimicamente , Aneurisma da Aorta Abdominal/genética , Aneurisma da Aorta Abdominal/prevenção & controle , Cloreto de Cálcio/metabolismo , Ciclina I/metabolismo , Proteína Rica em Cisteína 61/metabolismo , Modelos Animais de Doenças , Células Endoteliais/metabolismo , Endotélio/metabolismo , Humanos , Peróxido de Hidrogênio/metabolismo , Hipertensão/genética , Hipertensão/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , RNA Mensageiro/metabolismo , Tetra-Hidrofolato Desidrogenase/genética , Tetra-Hidrofolato Desidrogenase/metabolismo
4.
ESC Heart Fail ; 9(2): 1370-1379, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-35128826

RESUMO

AIMS: Heart failure (HF) is a chronic heart disease with a high incidence and mortality. Due to the regulatory complexity of gene coexpression networks, the underlying hub genes regulation in HF remain incompletely appreciated. We aimed to explore potential key modules and genes for HF using weighted gene coexpression network analysis (WGCNA). METHODS AND RESULTS: The expression profiles by high throughput sequencing of heart tissues samples from HF and non-HF samples were obtained from the Gene Expression Omnibus database. Differentially expressed genes (DEGs) between HF and non-HF samples were firstly identified. Then, a coexpression network was constructed to identify key modules and potential hub genes. The biological functions of potential hub genes were analysed by Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses. Finally, a protein-protein interaction (PPI) network was constructed using the STRING online tool. A total of 135 DEGs (133 up-regulated and 2 down-regulated DEGs) between HF and non-HF samples were identified in the GSE135055 and GSE123976 datasets. Moreover, a total of 38 modules were screened based on WGCNA in the GSE135055 dataset, and six potential hub genes (UCK2, ASB1, CCNI, CUX1, IRX6, and STX16) were screened from the key module by setting the gene significance over 0.2 and the module membership over 0.8. Furthermore, 78 potential hub genes were obtained by taking the intersection of the 135 DEGs and all genes in the key module, and enrichment analysis revealed that they were mainly involved in the MAPK and PI3K-AKT signalling pathways. Finally, in a PPI network constructed with the 78 potential hub genes, CUX1 and ASB1 were identified as hub genes in HF because they were also identified as potential hub genes in the WGCNA. CONCLUSIONS: To the best of our knowledge, our study is the first to employ WGCNA to identify the key module and hub genes for HF. Our study identified a module and two genes that might play important roles in HF, which may provide potential biomarkers for the diagnosis of HF and improve our knowledge of the molecular mechanisms underlying HF.


Assuntos
Insuficiência Cardíaca , Fosfatidilinositol 3-Quinases , Biomarcadores/metabolismo , Ciclina I , Perfilação da Expressão Gênica/métodos , Redes Reguladoras de Genes , Insuficiência Cardíaca/genética , Proteínas de Homeodomínio , Humanos , Fatores de Transcrição/genética
5.
Cancer Med ; 10(6): 1913-1924, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33620152

RESUMO

Colorectal cancer (CRC) is one of the most common malignancies and most of the patients diagnosed with advanced CRC have unsatisfactory treatment effect and poor prognosis. The purpose of this study was to investigate the effect of CCNI2 on the development of CRC. In this sutdy, immunohistochemical staining was used to detect CCNI2 expression levels in clinical samples, meanwhile, the Kaplan-Meier survival analysis was conducted. Celigo cell counting assay was used for screening shCCNI2s. QPCR and WB were performed to verify knockdown efficiency of CCNI2. Cell proliferation, colony formation, cell cycle, apoptosis, and mechanism investigation of CCNI2 knockdown were investigated by MTT assay, colony formation assay, fluorescence-activated cell sorting, and human apoptosis antibody array, respectively. Otherwise, the mouse model of CCNI2 knockdown was also constructed. The results of immunohistochemical staining and qPCR indicated that CCNI2 had a high expression level in the CRC tissues and cell lines. Kaplan-Meier survival analysis manifested that the high expression of CCNI2 suggested poor prognosis. The expression of CCNI2 was significantly reduced by CCNI2-siRNAs, and the downregulated expression level of CCNI2 inhibited CRC cell proliferation and colony formation, arrested cell cycle in G2 phase, as well as promoted cell apoptosis. The various indexes of solid tumor in mice models indicated that CCNI2 knockdown could suppress the growth of CRC tumor. Based on the comprehensive analysis of the above results, CCNI2 was contributed to the progression of CRC and could serve as a prognostic marker for CRC.


Assuntos
Neoplasias Colorretais/metabolismo , Ciclina I/metabolismo , Idoso , Animais , Apoptose/genética , Ciclo Celular/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Neoplasias Colorretais/genética , Neoplasias Colorretais/mortalidade , Neoplasias Colorretais/patologia , Ciclina I/genética , Modelos Animais de Doenças , Progressão da Doença , Regulação para Baixo , Feminino , Fase G2 , Técnicas de Silenciamento de Genes , Humanos , Estimativa de Kaplan-Meier , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Reação em Cadeia da Polimerase/métodos , Ensaio Tumoral de Célula-Tronco
6.
J Cell Mol Med ; 25(4): 1896-1910, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33465281

RESUMO

Extracellular vesicles (Evs) participate in the development of rheumatoid arthritis (RA), but the mechanisms remain unclear. This study aimed to determine the mechanism by which microRNA-34a (miR-34a) contained in bone marrow mesenchymal stem cell (BM-MSC)-derived Evs functions in RA fibroblast-like synoviocytes (RA-FLSs). BM-MSC-derived Evs and an Evs inhibitor were extracted. A rat model of RA was established. miR-34a gain- and loss-of-function experiments were performed, and the inflammation in rat synovial fluid and tissues was detected. The role of miR-34a in RA-FLSs was also measured in vitro. The target gene of miR-34a was predicted using the online software TargetScan and identified using a dual-luciferase reporter gene assay, and the activation of the ATM/ATR/p53 signalling pathway was assessed. BM-MSC-derived Evs mainly elevated miR-34a expression, which reduced RA inflammation in vivo and inhibited RA-FLS proliferation and resistance to apoptosis in vitro, while inhibited miR-34a expression enhanced RA development. In addition, miR-34a could target cyclin I to activate the ATM/ATR/p53 signalling pathway, thus inhibiting abnormal RA-FLS growth and RA inflammation. Our study showed that miR-34a contained in BM-MSC-derived Evs could reduce RA inflammation by inhibiting the cyclin I/ATM/ATR/p53 signalling pathway.


Assuntos
Artrite Reumatoide/genética , Artrite Reumatoide/metabolismo , Vesículas Extracelulares/metabolismo , Regulação da Expressão Gênica , Células-Tronco Mesenquimais/metabolismo , MicroRNAs/genética , Transdução de Sinais , Animais , Apoptose/genética , Artrite Reumatoide/patologia , Biomarcadores , Biópsia , Células Cultivadas , Ciclina I/metabolismo , Masculino , MicroRNAs/metabolismo , Ratos , Sinoviócitos/metabolismo , Sinoviócitos/patologia , Proteína Supressora de Tumor p53/metabolismo
7.
Medicine (Baltimore) ; 99(8): e19097, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-32080083

RESUMO

Liquid biopsy is an emerging technique for noninvasive detection of various cancers. Majority of liquid biopsy tests still, however, use solitary type of biomarkers with unsatisfactory sensitivity and specificity. To this end, a combined approach of circulating tumor cells (CTCs) and salivary mRNA biomarkers was evaluated for discriminating non-small-cell lung cancer (NSCLC) from healthy controls.Our study included a discovery phase to find multiple biomarkers, and an independent validation phase to confirm the applicability of the selected biomarkers. In the discovery phase, CTC level in blood and 5 mRNA biomarkers in saliva (i.e., CCNI, Epidermal growth factor receptor [EGFR], FGF19, FRS2, and GREB1) were measured for 140 NSCLC patients and 140 healthy controls, followed by developing a predictive model. Next, this panel of biomarkers was applied to another patient cohort consisted of 60 patients with NSCLC and 60 healthy controls in the validation phase.We found that our novel biomarker panel could differentiate patients with NSCLC from healthy controls with high sensitivity (92.1%) and high specificity (92.9%) in the discovery phase. In the validation phase, we achieved sensitivity of 88.3% and specificity of 90.0%.To our best knowledge, it is the first time that a combined use of CTC and salivary mRNA biomarkers were applied for noninvasive detection of NSCLC.


Assuntos
Biomarcadores Tumorais/sangue , Carcinoma Pulmonar de Células não Pequenas/diagnóstico , Células Neoplásicas Circulantes/metabolismo , RNA Mensageiro/metabolismo , Saliva/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Adulto , Idoso , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Ciclina I/metabolismo , Receptores ErbB/metabolismo , Feminino , Fatores de Crescimento de Fibroblastos/metabolismo , Humanos , Biópsia Líquida/métodos , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Masculino , Proteínas de Membrana/metabolismo , Pessoa de Meia-Idade , Proteínas de Neoplasias/metabolismo , Células Neoplásicas Circulantes/patologia , Valor Preditivo dos Testes , Sensibilidade e Especificidade
8.
Nat Commun ; 9(1): 3919, 2018 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-30254248

RESUMO

In addition to genomic mutations, RNA editing is another major mechanism creating sequence variations in proteins by introducing nucleotide changes in mRNA sequences. Deregulated RNA editing contributes to different types of human diseases, including cancers. Here we report that peptides generated as a consequence of RNA editing are indeed naturally presented by human leukocyte antigen (HLA) molecules. We provide evidence that effector CD8+ T cells specific for edited peptides derived from cyclin I are present in human tumours and attack tumour cells that are presenting these epitopes. We show that subpopulations of cancer patients have increased peptide levels and that levels of edited RNA correlate with peptide copy numbers. These findings demonstrate that RNA editing extends the classes of HLA presented self-antigens and that these antigens can be recognised by the immune system.


Assuntos
Antígenos de Neoplasias/imunologia , Epitopos/imunologia , Sistema Imunitário/imunologia , Neoplasias/imunologia , Edição de RNA/imunologia , Apresentação de Antígeno/imunologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Linhagem Celular Tumoral , Células Cultivadas , Ciclina I/genética , Ciclina I/imunologia , Ciclina I/metabolismo , Citotoxicidade Imunológica/imunologia , Antígenos HLA/imunologia , Humanos , Neoplasias/genética , Neoplasias/metabolismo , Peptídeos/genética , Peptídeos/imunologia , Peptídeos/metabolismo , Proteogenômica/métodos
9.
Sci Rep ; 7: 40979, 2017 01 23.
Artigo em Inglês | MEDLINE | ID: mdl-28112194

RESUMO

In contrast to conventional cyclin-dependent kinases that are important for mitotic cell division, cyclin-dependent kinase 5 (CDK5) is predominantly activated in post-mitotic cells and is involved in various cellular events. The kinase activity of CDK5 is tightly regulated by specific activators including p35, p39, and cyclin I (CCNI). Here we show that cyclin I-like (CCNI2), a homolog of CCNI, interacts with CDK5 and activates the kinase activity of CDK5. Different from CCNI, which colocalizes with CDK5 in the nuclei in transfected cells, CCNI2 mainly retains CDK5 in the cytoplasm as well as on the cell membrane. Furthermore, although the expression level of CCNI2 mRNA and CCNI2 protein do not change significantly during cell cycle, depletion of CCNI2 with siRNA affects cell cycle progression as well as cell proliferation. In conclusion, our data strongly suggest that CCNI2 is a novel CDK5 activator and is involved in cell cycle regulation.


Assuntos
Ciclo Celular , Ciclina I/metabolismo , Quinase 5 Dependente de Ciclina/metabolismo , Regulação da Expressão Gênica , Células A549 , Western Blotting , Proliferação de Células , Perfilação da Expressão Gênica , Células HeLa , Humanos , Mapeamento de Interação de Proteínas , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Técnicas do Sistema de Duplo-Híbrido
10.
Eur Rev Med Pharmacol Sci ; 19(23): 4533-41, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26698249

RESUMO

OBJECTIVE: Cisplatin (cis-diamminedichloroplatinum II, CDDP) is one of the most effective chemotherapeutic agents and is widely used in the treatment of cervical cancer (CC), but cancer cell acquired resistance to this drug during the course of its treatment. The aim of this study was to investigate the role of cyclin I to cisplatin resistance in CC cell. PATIENTS AND METHODS: Cervical tumor specimens from 30 patients were recruited in this study. We analyzed the expression of cyclin I by real-time polymerase chain reaction (qRT-PCR), Western blotting examination of downstream effectors. Cell proliferation assay and xenograft experiments were performed for cisplatin cytotoxicity assay. Lentivirus-mediated and siRNA-mediated genes overexpression or knockdown were applied to investigate the role of cyclin I to cisplatin resistance in CC cell. RESULTS: We found that high level of cyclin I was associated with cisplatin resistance in CC. Here, we described that cyclin I protein becomes highly expressed in human CC patients resistant to cisplatin chemotherapy. Stable overexpressed cyclin I promotes Hela cell resistance to higher concentrations of cisplatin. In addition, upregulated level of cyclin I increased tumor cells growth in vitro and enhanced tumor resistance to cisplatin in vivo. The further mechanism investigated showed that cyclin I upregulated the expression of cyclin-dependent kinase 5 (Cdk5) promoting cisplatin resistance by preventing apoptosis in CC cell line. Consistently, the cyclin I overexpressed Hela cell lines produce increased sensitivity to cisplatin treatment through knockdown of Cdk5 protein with siRNA. CONCLUSIONS: These data suggest that a cyclin I-Cdk5 complex forms a critical antiapoptotic factor in the process of generating cisplatin resistance in cervical cancer.


Assuntos
Antineoplásicos/uso terapêutico , Carcinoma de Células Escamosas/genética , Cisplatino/uso terapêutico , Ciclina I/genética , Quinase 5 Dependente de Ciclina/genética , Resistencia a Medicamentos Antineoplásicos/genética , Neoplasias do Colo do Útero/genética , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Carcinoma de Células Escamosas/tratamento farmacológico , Ciclina I/antagonistas & inibidores , Quinase 5 Dependente de Ciclina/metabolismo , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células HeLa , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , RNA Interferente Pequeno/farmacologia , Neoplasias do Colo do Útero/tratamento farmacológico , Ensaios Antitumorais Modelo de Xenoenxerto
11.
Anticancer Res ; 35(2): 1115-9, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25667501

RESUMO

BACKGROUND/AIM: Ovarian cancer is the second most common gynecological malignancy after cancer of the uterine corpus, and the fifth leading cause of cancer-related death among women. It has been discovered that cyclin I (CCNI) protein expression correlates with the proliferation of cancer cells and expression of angiogenesis-related proteins, such as vascular endothelial growth factor (VEGF) and VEGF receptor 2/kinase insert domain receptor (VEGFR2/KDR). We examined whether any association exists between mRNA expression of CCNI and KDR genes in epithelial ovarian cancer (EOC) tissues, clinicopathological parameters and patients' response to chemotherapy. MATERIALS AND METHODS: Expression of CCNI and KDR genes was analyzed by quantitative real-time reverse transcription PCR in 40 human primary EOC tissues and four human ovarian cancer cell lines (TOV-112D, OV-90, OVCAR-3 and Caov-3). RESULTS: CCNI and KDR mRNA expression was detected in all EOC tissues and ovarian cancer cell lines. The mRNA levels of both genes were significantly higher in EOC than in ovarian cancer cell lines (p<0.001). Neither CCNI nor KDR mRNA expression in EOC tissues was significantly associated with variables such as age, menopausal status, International Federation of Gynecology and Obstetrics (FIGO) stage, residual disease, patients' response to chemotherapy, tumor histology, grade or sensitivity to chemotherapy. However, we demonstrated a significant positive correlation between the mRNA expression of KDR and CCNI in EOC tissues (R=0.530, p<0.001). CONCLUSION: Neither CCNI nor KDR mRNA expression predicts response of patients with EOC to platinum-based first-line chemotherapy. Cyclin I may be involved in angiogenesis in EOC, which needs further investigation.


Assuntos
Ciclina I/genética , Neoplasias Epiteliais e Glandulares/genética , Neoplasias Ovarianas/genética , RNA Mensageiro/genética , Sequência de Bases , Carcinoma Epitelial do Ovário , Primers do DNA , Feminino , Humanos , Neoplasias Epiteliais e Glandulares/metabolismo , Neoplasias Ovarianas/metabolismo
12.
Am J Physiol Cell Physiol ; 308(4): C339-47, 2015 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-25500740

RESUMO

The atypical cyclin-dependent kinase 5 (Cdk5) serves an array of different functions in cell biology. Among these are axonal guidance, regulation of intercellular contacts, cell differentiation, and prosurvival signaling. The variance of these functions suggests that Cdk5 activation comes to pass in different cellular compartments. The kinase activity, half-life, and substrate specificity of Cdk5 largely depend on specific activators, such as p25, p35, p39, and cyclin I. We hypothesized that the subcellular distribution of Cdk5 activators also determines the localization of the Cdk5 protein and sets the stage for targeted kinase activity within distinct cellular compartments to suit the varying roles of Cdk5. Cdk5 localization was analyzed in murine kidney and brain slices of wild-type and cyclin I- and/or p35-null mice by immunohistochemistry and in cultured mouse podocytes using immunofluorescence labeling, as well as cell fractionation experiments. The predominance of cyclin I mediates the nuclear localization of Cdk5, whereas the predominance of p35 results in a membranous localization of Cdk5. These findings were further substantiated by overexpression of cyclin I and p35 with altered targeting characteristics in human embryonic kidney 293T cells. These studies reveal that the subcellular localization of Cdk5 is determined by its specific activators. This results in the directed Cdk5 kinase activity in specific cellular compartments dependent on the activator present and allows Cdk5 to serve multiple independent roles.


Assuntos
Ciclina I/metabolismo , Quinase 5 Dependente de Ciclina/metabolismo , Fosfotransferases/metabolismo , Podócitos/enzimologia , Animais , Membrana Celular/enzimologia , Núcleo Celular/enzimologia , Ciclina I/deficiência , Ciclina I/genética , Retículo Endoplasmático/enzimologia , Ativação Enzimática , Células HEK293 , Humanos , Camundongos Knockout , Fosfotransferases/deficiência , Fosfotransferases/genética , Transporte Proteico , Células de Purkinje/enzimologia , Transfecção
13.
Avian Dis ; 58(3): 468-73, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25518444

RESUMO

Tibial dyschondroplasia (TD) is a skeletal disease characterized by the disruption ofendochondral bone formation in avian species. The aim of this study was to determine the expression of Matrilin-3 and Cyclin-I genes in chicken growth plate during impairment and recovery from TD. Gene expressions were analyzed by polymerase chain reaction, and proteins by immunohistochemistry and in situ hybridizations. Expression of genes encoding Matrilin-3 and Cyclin-I were diminished with parallel decrease in proteins during TD, with fewer signs of cartilage cell differentiation. In contrast, there was an increase in mRNA expressions and protein levels of both genes during the recovery phase. These findings suggest that the Matrilin-3 and Cyclin-I genes also play a role in chondrocyte differentiation during the impairment and recovery of growth plate in TD.


Assuntos
Proteínas Aviárias/genética , Galinhas/genética , Ciclina I/genética , Lâmina de Crescimento/metabolismo , Proteínas Matrilinas/genética , Osteocondrodisplasias/veterinária , Doenças das Aves Domésticas/genética , Animais , Proteínas Aviárias/metabolismo , Diferenciação Celular , Galinhas/metabolismo , Condrócitos/citologia , Condrócitos/metabolismo , Ciclina I/metabolismo , Proteínas Matrilinas/metabolismo , Osteocondrodisplasias/genética , Osteocondrodisplasias/metabolismo , Doenças das Aves Domésticas/metabolismo , Recuperação de Função Fisiológica , Tíbia/metabolismo
14.
Proteins ; 82(11): 3117-31, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25136968

RESUMO

A-to-I RNA editing has been recently shown to be a widespread phenomenon with millions of sites spread in the human transcriptome. However, only few are known to be located in coding sequences and modify the amino acid sequence of the protein product. Here, we used high-throughput data, variant prediction tools, and protein structural information in order to find structural and functional preferences for coding RNA editing. We show that RNA editing has a unique pattern of amino acid changes characterized by enriched stop-to-tryptophan changes, positive-to-neutral and neutral-to-positive charge changes. RNA editing tends to have stronger structural effect than equivalent A-to-G SNPs but weaker effect than random A-to-G mutagenesis events. Sites edited at low level tend to be located at conserved positions with stronger predicted deleterious effect on proteins comparing to sites edited at high frequencies. Lowly edited sites tend to destabilize the protein structure and affect amino acids with larger number of intra-molecular contacts. Still, some highly edited sites are predicted also to prominently affect structure and tend to be located at critical positions of the protein matrix and are likely to be functionally important. Using our pipeline, we identify and discuss several novel putative functional coding changing editing sites in the genes COPA (I164V), GIPC1 (T62A), ZN358 (K382R), and CCNI (R75G).


Assuntos
Evolução Biológica , Proteínas/química , Proteínas/genética , Edição de RNA , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Aminoácidos/genética , Ciclina I/química , Ciclina I/genética , Bases de Dados de Proteínas , Humanos , Canal de Potássio Kv1.1/química , Canal de Potássio Kv1.1/genética , Mutagênese , Polimorfismo de Nucleotídeo Único , Domínios e Motivos de Interação entre Proteínas , Receptores de AMPA/química , Receptores de AMPA/genética , Triptofano
15.
Cell Cycle ; 12(16): 2617-24, 2013 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-23907122

RESUMO

Cyclins control cell cycle progression by regulating the activity of cyclin-dependent kinases (Cdks). Cyclin I is a member of the cyclin family because of the presence of a cyclin box motif. It has been suggested that Cyclin I is involved in various biological processes, such as cell survival, angiogenesis, and cell differentiation. However, whether or not Cyclin I has a role in regulating the cell cycle similarly to other cyclins has yet to be clarified. Therefore, we investigated the role for Cyclin I in cell cycle progression. We showed that the protein level of Cyclin I oscillated during the cell cycle, and that Cyclin I was subjected to ubiquitination and degradation in cells. The interaction between Cyclin I and Cdk5 was detected in cells overexpressed with both proteins. Furthermore, depletion of Cyclin I by siRNAs prevented cell proliferation, suggesting the positive role of Cyclin I for the cell cycle progression. In addition, flow cytometric analysis revealed that cells depleted of Cyclin I were accumulated at G2/M phases. By using HeLa.S-Fucci (fluorescent ubiquitination-based cell cycle indicator) cells, we further confirmed that knockdown of Cyclin I induced cell cycle arrest at S/G2/M phases. These results strongly suggest that Cyclin I has the role in the regulation of cell cycle progression.


Assuntos
Ciclo Celular/fisiologia , Ciclina I/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Proliferação de Células , Ciclina I/genética , Quinase 5 Dependente de Ciclina/metabolismo , Citometria de Fluxo , Fluorescência , Técnicas de Silenciamento de Genes , Células HeLa , Humanos , Proteólise , Ubiquitinação
16.
Gynecol Oncol ; 127(1): 217-22, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22772062

RESUMO

OBJECTIVE: Ovarian cancer is the most lethal of all gynecologic malignancies. It is characterized by the spread of intraperitoneal tumors, accumulation of ascites, and formation of tumor blood vessels. Cyclin I has been linked with angiogenesis-related proteins, like vascular endothelial growth factor (VEGF) and VEGF receptor 2 (VEGFR-2), in human breast cancer. We examined whether an association exists between expression of cyclin I, VEGFR-2, clinicopathologic parameters and survival of patients with epithelial ovarian cancer (EOC). METHODS: Cyclin I and VEGFR-2 expressions were analyzed by immunohistochemistry in 55 human primary EOC tissue specimens. RESULTS: Cyclin I immunoreactivity was significantly correlated with VEGFR-2 (R=0.4587, P=0.0004), and immunolabeling of cyclin I and VEGFR-2 significantly correlated with cancer cells' proliferative activity evaluated using cyclin A labeling index as a marker (R=0.3107, P=0.0209 and R=0.4183, P=0.0015, respectively). VEGFR-2 immunostaining was significantly higher in advanced, poorly differentiated, and suboptimally resected EOCs compared to their counterparts (P<0.05). Finally, higher VEGFR-2 expression was significantly associated with shorter disease-free survival (P=0.0437). CONCLUSIONS: Our results indicate that elevated expression of cyclin I and VEGFR-2 is likely to provide a proliferative advantage to the EOC cells, and that cyclin I may be linked with angiogenesis in EOC. Higher expression of VEGFR-2 is associated with more advanced disease. Further investigation of cyclin I in ovarian cancer is needed to evaluate if cyclin I may become a novel target for an anticancer therapy.


Assuntos
Ciclina I/metabolismo , Neoplasias Epiteliais e Glandulares/metabolismo , Neoplasias Ovarianas/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma Epitelial do Ovário , Processos de Crescimento Celular/fisiologia , Ciclina I/biossíntese , Feminino , Humanos , Imuno-Histoquímica , Pessoa de Meia-Idade , Neoplasias Epiteliais e Glandulares/irrigação sanguínea , Neoplasias Epiteliais e Glandulares/patologia , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Neoplasias Ovarianas/irrigação sanguínea , Neoplasias Ovarianas/patologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/biossíntese , Adulto Jovem
17.
Am J Physiol Renal Physiol ; 302(9): F1161-71, 2012 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-22262481

RESUMO

Cyclin-dependent kinase (Cdk)-5 is activated by both cyclin I and the noncyclin activator p35 in terminally differentiated cells such as kidney podocytes and neurons. Cyclin I and p35 are restricted to podocytes in the kidney, and each limit podocyte apoptosis by activating Cdk5. To determine whether both activators are necessary, or whether they serve backup roles, a double cyclin I-p35 null mouse was generated. Experimental glomerular disease characterized by podocyte apoptosis was then induced by administering an anti-podocyte antibody. The results showed that under nonstressed conditions double mutants had normal kidney structure and function and were indistinguishable from wild-type, cyclin I(-/-), or p35(-/-) mice. In contrast, when stressed with disease, podocyte apoptosis increased fourfold compared with diseased cyclin I(-/-) or p35(-/-) mice. This resulted in a more pronounced decrease in podocyte number, proteinuria, and glomerulosclerosis. Under normal states and nephritic states, levels for the prosurvival protein Bcl-2 were lower in double cyclin I(-/-) p35(-/-) mice than the other mice. Similarly, levels of Bcl-xL, another prosurvival member, were lower in normal and nephritic double cyclin I(-/-) p35(-/-) mice but similar to single-cyclin I(-/-) mice. Moreover, levels of ERK1/2 and MEK1/2 activation were lower in nephritic double cyclin I(-/-) p35(-/-) mice but similar to single-cyclin I(-/-) mice. The results demonstrate that the activators of Cdk5, p35, and cyclin I are not required for normal kidney function. However, they play pivotal coordinated roles in maintaining podocyte survival during stress states in disease.


Assuntos
Apoptose/fisiologia , Ciclina I/metabolismo , Quinase 5 Dependente de Ciclina/metabolismo , Fosfotransferases/metabolismo , Podócitos/metabolismo , Podócitos/patologia , Animais , Anticorpos Anti-Idiotípicos/efeitos adversos , Sobrevivência Celular/fisiologia , Ciclina I/deficiência , Ciclina I/genética , Modelos Animais de Doenças , Nefropatias/induzido quimicamente , Nefropatias/metabolismo , Nefropatias/patologia , MAP Quinase Quinase 1/metabolismo , MAP Quinase Quinase 2/metabolismo , Camundongos , Camundongos Knockout , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fosfotransferases/deficiência , Fosfotransferases/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteína bcl-X/metabolismo
18.
Int J Mol Med ; 26(1): 17-25, 2010 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-20514417

RESUMO

The prostate is composed mainly of epithelial and stromal cells, whose dynamic interaction is vital to a broad array of cellular processes, including proliferation, differentiation, growth, and apoptosis. To understand intercellular communication in the development and progression of prostatic diseases, we examined gene expression in tissues from five patients diagnosed with benign prostatic hyperplasia (BPH). Fibroblasts and epithelial cells derived from these tissues were grown in a primary co-culture system that retains many characteristics of the intact human prostate. The mRNA levels of expressed genes as assessed by differential-display reverse transcription-PCR revealed that 110 genes were differentially expressed in co-cultured fibroblasts and epithelial cells, compared with expression in separately cultured cells. Eighty-four of these were confirmed by reverse Northern blotting, and 68 were successfully sequenced. Of the sequenced genes, 43 were differentially expressed in epithelial cells (37 upregulated, 6 downregulated), and 25 were differentially expressed in fibroblasts (6 upregulated, 19 downregulated) in co-cultures versus separate cultures. Semi-quantitative RT-PCR analysis of 12 genes with known functions showed that five of these were differentially expressed in co-cultured cells. Human kallikrein gene 7 (KLK7) was markedly upregulated in co-cultured compared with separately cultured epithelial cells (P<0.001), whereas S100 calcium binding protein A11, tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, cyclin I, and latexin were significantly downregulated in co-cultured fibroblasts (P<0.05). Quantitative real-time RT-PCR and Western blot analysis confirmed KLK7 up-regulation at both the mRNA and protein levels, respectively. Thus, epithelial-stromal cell interaction and communication are likely to be important in BPH. Epithelial cells and fibroblasts may interplay coordinately or collaboratively to influence cellular growth and death through dynamically differential gene expression in response to physiological and pathophysiological changes.


Assuntos
Células Epiteliais/metabolismo , Fibroblastos/metabolismo , Perfilação da Expressão Gênica , Hiperplasia Prostática/genética , Proteínas 14-3-3/genética , Antígenos/genética , Northern Blotting , Western Blotting , Comunicação Celular/genética , Células Cultivadas , Técnicas de Cocultura , Ciclina I/genética , Regulação para Baixo , Células Epiteliais/patologia , Fibroblastos/patologia , Humanos , Calicreínas/genética , Calicreínas/metabolismo , Masculino , Hiperplasia Prostática/metabolismo , Hiperplasia Prostática/patologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas S100/genética , Regulação para Cima
19.
Cell Cycle ; 9(9): 1729-31, 2010 May.
Artigo em Inglês | MEDLINE | ID: mdl-20404535

RESUMO

Cdk5 has long been recognized to play an important role in development, maturation and apoptosis of postmitotic and terminally differentiated cells. Activation of Cdk5 is tightly regulated by specific activators. Cyclin I was recently characterized as the first cyclin protein that binds to and activates Cdk5. Cyclin I-Cdk5 activates the MEK-ERK pathway and results in increased Bcl-2 and Bcl-X(L) mRNA and protein levels. Lack of Cyclin I renders podocytes more susceptible to apoptosis. Interestingly, activation of Cdk5 by p35 is also involved in the podocytes' response to injury. In the absence of p35, podocytes are more prone to undergo apoptosis. Here, we propose a new model where Cdk5 plays a central role in the cellular response machinery against injury-induced apoptosis of post-mitotic cells. While Cyclin I-Cdk5 regulates Bcl-2 family proteins through activation of the MEK-ERK pathway, p35-Cdk5 directly phosphorylates and stabilizes Bcl-2.


Assuntos
Ciclina I/metabolismo , Quinase 5 Dependente de Ciclina/metabolismo , Animais , Apoptose , Sobrevivência Celular , Sistema de Sinalização das MAP Quinases , Camundongos , Mitose , Fosfotransferases/metabolismo , Podócitos/metabolismo , Ligação Proteica , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteína bcl-X/genética , Proteína bcl-X/metabolismo
20.
Liver Int ; 30(1): 119-25, 2010 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19845855

RESUMO

BACKGROUND: The mammalian cyclin kinase subunit (Cks) family has two members, Cks1 and Cks2, which were identified based on the protein sequence homology to yeast Cks. Overexpression of Cks1 and Cks2 has been reported to be associated with high aggressiveness and a poor prognosis in various malignancies, including gastric, breast and prostate carcinomas. Yet, whether Cks1 and Cks2 are overexpressed in hepatocellular carcinoma (HCC) remains uncharacterized. AIMS: To investigate whether overexpression of the Cks family is clinically relevant to HCC, and whether expression patterns of Cks1 and Cks2 in HCC have diagnostic and prognostic value. METHODS: Real-time quantitative reverse transcriptase polymerase chain reaction, immunostaining and Western blot analyses were used to detect the expression of Cks1 and Cks2 at the mRNA and protein levels respectively. The associations between Cks1 and Cks2 expressions and clinical features, as well as the association between Cks1 or Cks2 and p27(kip1) expressions in HCC, were analysed. RESULTS: Expressions of Cks1 and Cks2 at both mRNA and protein levels were significantly higher in HCC than those in the adjacent noncancerous tissues (including chronic hepatitis and cirrhosis) and normal liver tissues. Overexpressions of Cks1 and Cks2 in HCC were closely associated with poor differentiation features. The expressions of both Cks1 and Cks2 were negatively associated with p27(kip1) at the protein level. CONCLUSIONS: Overexpression of Cks1 and Cks2 is associated with the aggressive tumour behaviours of HCC, and thus has diagnostic and prognostic value. Further efforts are needed to develop novel biomarkers for HCC based on CKs1 and Cks2 expressions.


Assuntos
Carcinoma Hepatocelular/enzimologia , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/metabolismo , Ciclina I/metabolismo , Neoplasias Hepáticas/enzimologia , Proteínas Quinases/metabolismo , Biomarcadores Tumorais/metabolismo , Quinases relacionadas a CDC2 e CDC28 , Carcinoma Hepatocelular/diagnóstico , Carcinoma Hepatocelular/genética , Proteínas de Transporte/genética , Contagem de Células , Proteínas de Ciclo Celular/genética , Ciclina I/genética , Inibidor de Quinase Dependente de Ciclina p27/genética , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Hepatite Crônica/diagnóstico , Hepatite Crônica/enzimologia , Hepatite Crônica/genética , Humanos , Cirrose Hepática/diagnóstico , Cirrose Hepática/enzimologia , Cirrose Hepática/genética , Neoplasias Hepáticas/diagnóstico , Neoplasias Hepáticas/genética , Prognóstico , Proteínas Quinases/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa
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