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OBJECTIVE: To explore the mechanism by which lobaplatin, as monotherapy and in combination with paclitaxel, inhibits the proliferation of human gastric cancer SGC-7901 cells. METHODS: After treatment, the MTT assay was used to assess cell viability; cell cycle distribution was evaluated flow-cytometrically. Western blot was used to quantitate cyclin D1, E1, B1, and Cdk2/4 protein levels. RESULTS: Lobaplatin and paclitaxel inhibited SGC-7901 cell growth in a concentration and timedependent manner, with IC25 values at 48h of 1.97±0.17µg/ml and 1.98±0.19 ng/ml, respectively. Interestingly, both drugs synergistically inhibited SGC-7901 cells (combination index [CI]<0.95). Lobaplatin did not affect cyclin D1 and CDK4 protein expression, while cyclin E1 and CDK2 levels were significantly increased, with cyclin B1 amounts markedly decreased (p<0.05). More S phase cells were observed after lobaplatin treatment compared with controls (60.03±1.25 vs. 18.69±0.96%; p<0.05). After treatment with paclitaxel, cyclin D1 and CDK4 protein levels were similar to control values; meanwhile, cylinE1 and CDK2 protein amounts were reduced, with increased cyclin B1 levels, compared with control values (p<0.05). More G2/M cells were obtained after treatment with paclitaxel compared with control values (74.54±0.92 vs. 18.62±0.44% (p<0.05). Lobaplatin and paclitaxel combination did not affect cyclin D1 and CDK4 protein levels (p>0.05); meanwhile, cyclin E1 and CDK2 levels were increased, with reduced cyclin B1 amounts, compared with control values (p<0.05). Notably, more S (43.23±0.81 vs. 22.32±0.86%) and G2/M (31.22±0.96 vs. 25.81±2.08%) phase cells were obtained after combined treatment compared with control values. CONCLUSION: Lobaplatin and paclitaxel synergistically inhibit SGC-7901 cells.
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Antineoplásicos/farmacología , Ciclo Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Ciclobutanos/farmacología , Compuestos Organoplatinos/farmacología , Paclitaxel/farmacología , Neoplasias Gástricas/tratamiento farmacológico , Antineoplásicos/administración & dosificación , Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Línea Celular Tumoral , Ciclina B1/análisis , Ciclina B1/metabolismo , Ciclina E/análisis , Ciclina E/metabolismo , Quinasa 2 Dependiente de la Ciclina/análisis , Quinasa 2 Dependiente de la Ciclina/metabolismo , Ciclobutanos/administración & dosificación , Humanos , Proteínas Oncogénicas/análisis , Proteínas Oncogénicas/metabolismo , Compuestos Organoplatinos/administración & dosificación , Paclitaxel/administración & dosificación , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologíaRESUMEN
Cyclin E and its co-activator, phospho-cyclin-dependent kinase 2 (p-CDK2), regulate G1 to S phase transition and their deregulation induces oncogenesis. Immunohistochemical assessments of these proteins in cancer have been reported but were based only on their nuclear expression. However, the oncogenic forms of cyclin E (low molecular weight cyclin E or LMW-E) in complex with CDK2 are preferentially mislocalized to the cytoplasm. Here, we used separate nuclear and cytoplasmic scoring systems for both cyclin E and p-CDK2 expression to demonstrate altered cellular accumulation of these proteins using immunohistochemical analysis. We examined the specificity of different cyclin E antibodies and evaluated their concordance between immunohistochemical and Western blot analyses in a panel of 14 breast cell lines. Nuclear versus cytoplasmic staining of cyclin E readily differentiated full-length from LMW-E, respectively. We also evaluated the expression of cyclin E and p-CDK2 in 1676 breast carcinoma patients by immunohistochemistry. Cytoplasmic cyclin E correlated strongly with cytoplasmic p-CDK2 (P < 0.0001), high tumor grade, negative estrogen/progesterone receptor status, and human epidermal growth factor receptor 2 positivity (all P < 0.0001). In multivariable analysis, cytoplasmic cyclin E plus phosphorylated CDK2 (as one variable) predicted breast cancer recurrence-free and overall survival. These results suggest that cytoplasmic cyclin E and p-CDK2 can be readily detected with immunohistochemistry and used as clinical biomarkers for aggressive breast cancer.
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Biomarcadores de Tumor/análisis , Neoplasias de la Mama/patología , Ciclina E/análisis , Quinasa 2 Dependiente de la Ciclina/análisis , Adulto , Anciano , Anciano de 80 o más Años , Animales , Western Blotting , Línea Celular Tumoral , Núcleo Celular/química , Núcleo Celular/metabolismo , Citoplasma/química , Citoplasma/metabolismo , Femenino , Humanos , Inmunohistoquímica , Ratones , Ratones Transgénicos , Persona de Mediana Edad , Análisis de Matrices TisularesRESUMEN
OBJECTIVE: To study the proliferation and apoptosis of tetramethylpyrazine (TMP) on leukemic U937 cells and its possible mechanism. METHOD: The inhibitory effect of TMP on the proliferation of U937 cells was detected by CCK-8 assay. The cell apoptosis and cycle distribution were examined by the flow cytometry. The mRNA expressions of bcl-2 and P27 were determined by the Real-time PCR. Western blot was carried out to detect bcl-2, caspase-3, cyclin E1, CDK2 and P27 expressions. RESULT: TMP inhibited the proliferation of U937 cells in a dose-and-time dependent manner, with IC50 value of 160 mg x L(-1) at 48 h. In addition, TMP could induce the apoptosis of U937 cells and block the cell cycle in G0/G1 phase. According to the results of Real-time PCR and Western blot, TMP could down-regulate the expression of apoptosis-related molecule bcl-2, cycle-related protein cyclin E1 and CDK2 and up-regulate caspase-3 and P27. CONCLUSION: TMP shows the effects in inhibiting the proliferation of leukemic U937 cells and inducing the apoptosis. Its mechanism may be related to the impacts on the cell cycle distribution, down-regulation of the bcl-2 expression, which finally activates caspase-3, starts the apoptosis path and causes the cell apoptosis.
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Apoptosis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Leucemia/tratamiento farmacológico , Pirazinas/farmacología , Ciclo Celular/efectos de los fármacos , Quinasa 2 Dependiente de la Ciclina/análisis , Humanos , Proteínas Proto-Oncogénicas c-bcl-2/análisis , Pirazinas/uso terapéutico , Células U937RESUMEN
BACKGROUND: Attenuated endogenous protein levels of cyclin-dependent kinase 2 associated protein 1 (p12(CDK2AP1)) and its active homodimer p25(CDK2AP1) were found in myxofibrosarcoma-derived cell lines. Clinical and biological significances of this putative tumor suppressor in myxofibrosarcoma were studied. METHODS: Plasmids carrying the CDK2AP1 gene and small hairpin RNA interference (shRNAi) targeting CDK2AP1 were transfected into NMFH-1 and/or OH931 cells to evaluate the effects on the CDK2, active caspase 3 (CASP3), cleaved-CASP8 and -CASP9 levels, cell cycle regulation, and/or apoptotic responses. Immunostaining of p12(CDK2AP1) was interpretable in 102 primary myxofibrosarcomas and correlated with clinicopathological variables, CDK2, Ki-67 and active CASP3 protein levels, and disease-specific survival. RESULTS: Exogenous expression of p12(CDK2AP1) in NMFH-1 and OH931 cells significantly induced G0/G1 cell cycle arrest and down-regulated CDK2 protein level. In NMFH-1 cells, these aspects were reversed by shRNAi targeting CDK2AP1 gene. Increased active CASP3 and cleaved-CASP9, but not -CASP8, were detected after CDK2AP1 overexpression, suggesting the cellular apoptosis were induced through the mitochondrial pathway. Immunostains of p12(CDK2AP1) were aberrantly decreased in 56.9 % of cases; positively and negatively correlated with protein levels of CDK2 (p = 0.023), Ki-67 (p = 0.001) and active CASP3 (p < 0.001), respectively. Following by high histological grades, p12(CDK2AP1) down-regulation was predictive of worse disease-specific survival in univariate (p = 0.003) and multivariate (p = 0.004) analyses. CONCLUSIONS: Through down-regulation of CDK2, high p12(CDK2AP1) level induced cell cycle arrest and the mitochondrial-dependent apoptotic pathway. Low p12(CDK2AP1) level represents a poor prognostic factor in patients with myxofibrosarcoma.
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Fibrosarcoma/enzimología , Fibrosarcoma/genética , Mitocondrias/metabolismo , Mixosarcoma/enzimología , Mixosarcoma/genética , Proteínas Supresoras de Tumor/genética , Apoptosis/genética , Caspasa 3/análisis , Caspasa 3/metabolismo , Caspasa 8/metabolismo , Caspasa 9/metabolismo , Puntos de Control del Ciclo Celular/genética , Línea Celular Tumoral , Supervivencia Celular , Homólogo de la Proteína Chromobox 5 , Quinasa 2 Dependiente de la Ciclina/análisis , Quinasa 2 Dependiente de la Ciclina/metabolismo , Femenino , Fibrosarcoma/química , Humanos , Antígeno Ki-67/análisis , Masculino , Persona de Mediana Edad , Índice Mitótico , Mixosarcoma/química , Plásmidos , ARN Mensajero/metabolismo , ARN Interferente Pequeño/genética , Tasa de Supervivencia , TransfecciónRESUMEN
OBJECTIVE: The protein 14-3-3 sigma plays a role in cell cycle arrest by sequestering cyclin-dependent kinase 1 cyclin B1 complexes, as well as cyclin-dependent kinases 2 and 4, hence its definition as a cyclin-dependent kinase inhibitor. However, the nature of the interaction between these biological markers in nasopharyngeal carcinoma is unknown. This study aimed to investigate whether altered expression of these markers contributes to nasopharyngeal carcinogenesis. METHODS: The study population consisted of 30 nasopharyngeal carcinoma patients and 10 patients without nasopharyngeal carcinoma. The nasopharyngeal carcinoma cell lines TW02, TW04 and Hone-1 were also assessed. We analysed levels of messenger RNA and protein for the p16 gene and the 14-3-3 sigma, Epstein-Barr nuclear antigen 1, and cyclin-dependent kinase 2 and 4 proteins, in nasopharyngeal carcinoma tissue specimens and cell lines and in normal nasopharyngeal tissue. RESULTS: Protein and messenger RNA levels for cyclin-dependent kinase 2 and Epstein-Barr nuclear antigen 1 were significantly higher in nasopharyngeal carcinoma compared with normal tissue, while levels of cyclin-dependent kinase 4 generally were not; results for 14-3-3 sigma varied. Nasopharyngeal carcinoma patients had diminished p16 gene expression, compared with normal tissue. CONCLUSION: Levels of cyclin-dependent kinase 2 and Epstein-Barr nuclear antigen 1 were significantly higher in nasopharyngeal carcinoma than in normal tissue, while p16 gene expression was diminished. These three proteins may contribute to nasopharyngeal carcinogenesis.
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Proteínas 14-3-3/análisis , Biomarcadores de Tumor/análisis , Quinasa 2 Dependiente de la Ciclina/análisis , Quinasa 4 Dependiente de la Ciclina/análisis , Antígenos Nucleares del Virus de Epstein-Barr/análisis , Exorribonucleasas/análisis , Neoplasias Nasofaríngeas/química , Proteínas de Neoplasias/análisis , Adulto , Anciano , Carcinoma , Estudios de Casos y Controles , Línea Celular Tumoral/química , Inhibidor p16 de la Quinasa Dependiente de Ciclina , Femenino , Humanos , Masculino , Persona de Mediana Edad , Carcinoma Nasofaríngeo , Nasofaringe/química , Adulto JovenRESUMEN
Multiple myeloma is a hematological cancer of plasma B cells and remains incurable. Two major subtypes of myeloma, hyperdiploid MM (HMM) and non-hyperdiploid MM (NHMM), have distinct chromosomal alterations and different survival outcomes. Transcription factors (TrFs) have been implicated in myeloma oncogenesis, but their dysregulation in myeloma subtypes are less studied. Here, we developed a TrF-pathway coexpression analysis to identify altered coexpression between two sample types. We apply the method to the two myeloma subtypes and the cell cycle arrest pathway, which is significantly differentially expressed between the two subtypes. We find that TrFs MYC, nuclear factor-κB and HOXA9 have significantly lower coexpression with cell cycle arrest in HMM, co-occurring with their overactivation in HMM. In contrast, TrFs ESR1 (estrogen receptor 1), SP1 and E2F1 have significantly lower coexpression with cell cycle arrest in NHMM. SP1 chromatin immunoprecipitation targets are enriched by cell cycle arrest genes. These results motivate a cooperation model of ESR1 and SP1 in regulating cell cycle arrest, and a hypothesis that their overactivation in NHMM disrupts proper regulation of cell cycle arrest. Cotargeting ESR1 and SP1 shows a synergistic effect on inhibiting myeloma proliferation in NHMM cell lines. Therefore, studying TrF-pathway coexpression dysregulation in human cancers facilitates forming novel hypotheses toward clinical utility.
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Puntos de Control del Ciclo Celular , Receptor alfa de Estrógeno/fisiología , Mieloma Múltiple/patología , Factor de Transcripción Sp1/fisiología , Teorema de Bayes , Quinasa 2 Dependiente de la Ciclina/análisis , Factor de Transcripción E2F1/fisiología , Proteínas de Homeodominio/fisiología , Humanos , Interleucina-6/fisiología , Sistema de Señalización de MAP Quinasas , Factor de Transcripción Sp1/análisis , Factores de Transcripción/fisiologíaRESUMEN
This study aimed to analyze the expression, clinical significance of cyclin G2 (CCNG2) in esophageal carcinoma, and the biological effect in its cell line by CCNG2 overexpression. Immunohistochemistry and western blot were used to analyze CCNG2 protein expression in 73 cases of esophageal cancer and normal tissues to study the relationship between CCNG2 expression and clinical factors. CCNG2 lentiviral vector and empty vector were respectively transfected into esophageal cancer Eca-109 cell line. Reverse transcription-polymerase chain reaction and western blot were used to detect the mRNA level and protein of CCNG2. MTT assay and cell cycle were also conducted as to the influence of the upregulated expression of CCNG2 that might be found on Eca-109 cell's biological effect. Immunohistochemistry: The level of CCNG2 protein expression was found to be significantly lower in esophageal cancer tissue than normal tissues (P < 0.05). Western blot: The relative amount of CCNG2 protein in esophageal cancer tissue was respectively found to be significantly lower than in normal tissues (P < 0.05). The level of CCNG2 protein expression was not correlated with gender, age, and tumor size (P > 0.05), but it was correlated with lymph node metastasis, clinic stage, and histological grades (P < 0.05). Loss of CCNG2 expression correlated significantly with poor overall survival time by Kaplan-Meier analysis. The result of the biological function showed that Eca-109 cell-transfected CCNG2 had a lower survival fraction, more percentage of the G0/G1 phases (P < 0.05), and lower cyclin-dependent kinase 2 (CDK2) protein expression. CCNG2 expression decreased in esophageal cancer and correlated significantly with lymph node metastasis, clinic stage, histological grade, and poor overall survival, suggesting that CCNG2 may play important roles as a negative regulator to esophageal cancer cell by promoting degradation of CDK2.
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Ciclina G2/fisiología , Neoplasias Esofágicas/patología , Adulto , Anciano , Ciclina G2/análisis , Ciclina G2/genética , Quinasa 2 Dependiente de la Ciclina/análisis , Neoplasias Esofágicas/química , Neoplasias Esofágicas/mortalidad , Esófago/química , Femenino , Humanos , Metástasis Linfática , Masculino , Persona de Mediana Edad , Estadificación de Neoplasias , PronósticoRESUMEN
3,3'-Diindolylmethane (DIM) is a major in vivo derivative of indole-3-carbinol, which is present in cruciferous vegetables and has been reported to possess anti-carcinogenic properties. In the present study, we examined whether DIM inhibits the development of prostate cancer using the transgenic adenocarcinoma mouse prostate (TRAMP) model. DIM feeding inhibited prostate carcinogenesis in TRAMP mice, reduced the number of cells expressing the SV40 large tumor antigen and proliferating cell nuclear antigen, and increased the number of terminal dUTP nick-end labeling-positive cells in the dorsolateral lobes of the prostate. Additionally, DIM feeding reduced the expression of cyclin A, cyclin-dependent kinase (CDK)2, CDK4, and Bcl-xL, and increased p27 and Bax expression. To assess the mechanisms by which DIM induces apoptosis, LNCaP and DU145 human prostate cancer cells were cultured with various concentrations of DIM. DIM induced a substantial reduction in the numbers of viable cells and induced apoptosis in LNCaP and DU145 cells. DIM increased the cleavage of caspase-9, -7, -3, and poly (ADP-ribose) polymerase (PARP). DIM increased mitochondrial membrane permeability and the translocation of cytochrome c and Smac/Diablo from the mitochondria. Additionally, DIM induced increases in the levels of cleaved caspase-8, truncated Bid, Fas, and Fas ligand, and the caspase-8 inhibitor Z-IETD-FMK was shown to mitigate DIM-induced apoptosis and the cleavage of caspase-3, PARP, and Bid. These results indicate that DIM inhibits prostate carcinogenesis via induction of apoptosis and inhibition of cell cycle progression. DIM induces apoptosis in prostate cancer cells via the mitochondria- and death receptor-mediated pathways.
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Adenocarcinoma/tratamiento farmacológico , Anticarcinógenos/uso terapéutico , Transformación Celular Neoplásica/efectos de los fármacos , Indoles/uso terapéutico , Neoplasias de la Próstata/tratamiento farmacológico , Animales , Antígenos Transformadores de Poliomavirus/análisis , Apoptosis/efectos de los fármacos , Proteínas Reguladoras de la Apoptosis , Proteínas Portadoras/análisis , Caspasas/análisis , Línea Celular Tumoral , Ciclina A/análisis , Quinasa 2 Dependiente de la Ciclina/análisis , Quinasa 4 Dependiente de la Ciclina/análisis , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/análisis , Citocromos c/análisis , Modelos Animales de Enfermedad , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Membranas Mitocondriales/efectos de los fármacos , Proteínas Mitocondriales/análisis , Oligopéptidos/uso terapéutico , Péptidos , Permeabilidad/efectos de los fármacos , Poli(ADP-Ribosa) Polimerasas/análisis , Antígeno Nuclear de Célula en Proliferación/análisis , Transporte de Proteínas/efectos de los fármacos , Proteína bcl-X/análisisRESUMEN
Human cadmium (Cd) exposure is associated with cancers of the lung and kidney. Using cDNA microarray analysis, we have recently reported that the expression of E2F1 is reduced by Cd in human lung fibroblasts, indicating the possibility of G1-phase arrest. To test this hypothesis, we investigated the effects of Cd on the cyclin-dependent kinase (CDK2) and retinoblastoma protein (Rb) regulatory pathways in WI38 human lung fibroblasts. We demonstrate here that G1-phase accumulation was induced by Cd in WI38 (wild-type for p53 and Rb), but not in the SV40 large T antigen-transformed variant WI38-VA13 (p53- and Rb-defective). Cd-induced cell-cycle arrest was associated with a decrease in CDK2 protein and with increase in p21 expression and p53 phosphorylation. Cd treatment caused a distinct increase in the formation of p21-cyclin E-CDK2 complex, as revealed by immunoprecipitation. The level of Rb-E2F1 complexes was increased, and the translocation of E2F1 to the nucleus was decreased by Cd treatment. Consequently, the transcriptional activity of E2F1 and the expression of the E2F1 target genes were also decreased by Cd. These results clearly demonstrate that Cd-mediated G1 arrest in WI38 cells is associated with the suppression of Rb phosphorylation and with the inhibition of E2F1 transcriptional activity.
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Cadmio/toxicidad , Factor de Transcripción E2F1/antagonistas & inhibidores , Fase G1/efectos de los fármacos , Pulmón/efectos de los fármacos , Transcripción Genética/efectos de los fármacos , Línea Celular , Quinasa 2 Dependiente de la Ciclina/análisis , Quinasa 2 Dependiente de la Ciclina/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/análisis , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Factor de Transcripción E2F1/metabolismo , Fibroblastos/efectos de los fármacos , Humanos , Fosforilación , Proteína de Retinoblastoma/análisis , Proteína de Retinoblastoma/metabolismo , Proteína p53 Supresora de Tumor/análisis , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
BACKGROUND: Cyclins drive cell-cycle progression by associating with their kinase partners, cyclin-dependent kinases (CDK). We investigated cyclin D1/CDK6, cyclin E/CDK2 complexes, and the cell-cycle negative regulators p15 and p27 in an incisional skin wound model. METHODS: Wounds were produced on the back of female diabetic mice and their normoglycemic littermates. Animals were treated with polydeoxyribonucleotide (PDRN, 8 mg/kg/i.p.), an agonist of adenosine A2(A) receptors, or its vehicle daily. Granulation tissue proliferation by Ki67 immunostaining, cyclin D/CDK6 and cyclin E/CDK2 complexes, and p21 and p16 proteins (Western blot analysis), and the histologic changes were assessed at different days (3, 6, and 12 days after injury). RESULTS: Numerous Ki67 positive cells were observed at day 3 and day 6 in the granulation tissue of normoglycemic mice. Ki67 positive cells were fewer in diabetic than in normoglycemic mice. PDRN increased Ki67 positive cells in diabetic mice. Normoglycemic mice showed the greatest upregulation of cyclin D1, CDK6, cyclin E, and CDK2 at day 6. Diabetic mice had a markedly lower expression of cyclin D1, CDK6, cyclin E, and CDK2 at day 6. They also showed a greater expression of p15 and p27 at day 6. PDRN administration in diabetic mice increased cyclin D1/CDK6 and cyclin E/CDK2 expression and reduced p15 and p27 inhibitors at day 6 after injury; moreover, it improved the impaired wound healing at day 12. CONCLUSION: Our results suggest that adenosine A2(A) receptor activation by PDRN might represent a therapeutic strategy to overcome the diabetes-impaired cell-cycle machinery.
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Ciclo Celular , Diabetes Mellitus/fisiopatología , Receptor de Adenosina A2A/fisiología , Cicatrización de Heridas/fisiología , Animales , Proliferación Celular , Ciclina D1/análisis , Ciclina E/análisis , Quinasa 2 Dependiente de la Ciclina/análisis , Quinasa 6 Dependiente de la Ciclina/análisis , Inhibidor p27 de las Quinasas Dependientes de la Ciclina , Diabetes Mellitus/patología , Femenino , Tejido de Granulación/patología , Péptidos y Proteínas de Señalización Intracelular/análisis , Ratones , Ratones Endogámicos C57BL , Polidesoxirribonucleótidos/farmacologíaRESUMEN
Cyclin E-associated CDK2 activity is required for the initiation of DNA synthesis in human cells. CDK2 activity is tightly regulated; CDK2 must be in the nucleus, bound to a cyclin, phosphorylated on T160, and dephosphorylated on T14/Y15 for complete kinase activation. Nuclear localization exposes CDK2 to activating enzymes (CAK, Cdc25A) in stimulated cells. Previous studies from our lab indicate CDK2 nuclear localization and cyclin E co-expression are insufficient to cause CDK2 activation or T160 phosphorylation in stimulated IIC9 cells; these activities still require serum stimulation and ERK kinase activity. Recent studies have implicated a role for origin of replication (ORC) licensing proteins in the activation of G1/S Cdks. In this study, we show that CDK2 associates with chromatin and Cdc6 in an ERK-dependent manner following stimulation of IIC9 CHEF cells. We show that nuclear-localized CDK2 (CDK2-NLS) ectopically expressed with cyclin E requires mitogenic stimulation and ERK activation for chromatin association, in addition to previously shown kinase activation and T160 phosphorylation in IIC9 cells. Additionally, we show that expression of Cdc6 in stimulated IIC9 cells treated with ERK inhibitor rescues CDK2-NLS chromatin association, kinase activation, and T160 phosphorylation. From the above data, we deduce ERK-dependent CDK2 activation is due in part to ERK-dependent Cdc6 expression. To examine the role of Cdc6 directly in stimulated primary human fibroblasts, we used RNA interference to attenuate the expression of Cdc6. We show that Cdc6 expression is required for CDK2 chromatin association and kinase activation in stimulated primary human fibroblasts. Additionally, we show that Cdc6 expression is required for the initiation of DNA synthesis and S phase entry in stimulated primary human fibroblasts. Ultimately, this data implicates Cdc6 expression as an important mitogen-induced mechanism in the activation of CDK2/cyclin E, the initiation of DNA synthesis, and the regulation of G1-S phase progression.
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Proteínas de Ciclo Celular/metabolismo , Ciclina E/metabolismo , Quinasa 2 Dependiente de la Ciclina/metabolismo , Proteínas Nucleares/metabolismo , Animales , Butadienos/farmacología , Proteínas de Ciclo Celular/genética , Línea Celular , Cromatina/metabolismo , Cricetinae , Cricetulus , Ciclina E/análisis , Ciclina E/genética , Quinasa 2 Dependiente de la Ciclina/análisis , Quinasa 2 Dependiente de la Ciclina/genética , Quinasas MAP Reguladas por Señal Extracelular/antagonistas & inhibidores , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Fase G1 , Humanos , Nitrilos/farmacología , Proteínas Nucleares/genética , Fosforilación , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Fase SRESUMEN
Tuberous sclerosis complex (TSC) is an autosomally inherited disorder that causes tumors to form in many organs. It is frequently caused by inactivating mutations in the TSC2 tumor-suppressor gene. TSC2 negatively regulates the activity of the GTPase Rheb and thereby inhibits mammalian target of rapamycin complex 1 (mTORC1) signaling. Activation of mTORC1 as a result of lack of TSC2 function is observed in TSC and sporadic lymphangioleiomyomatosis (LAM). TSC2 deficiency has recently been associated with elevated AMP-activated protein kinase (AMPK) activity, which in turn correlated with cytoplasmic localization of p27Kip1 (p27), a negative regulator of cyclin-dependent kinase 2 (Cdk2). How AMPK in the absence of TSC2 is stimulated is not fully understood. In this study, we demonstrate that Rheb activates AMPK and reduces p27 levels in Tsc2-null cells. Importantly, both effects occur largely independent of mTORC1. Furthermore, increased p27 levels following Rheb depletion correlated with reduced Cdk2 activity and cell proliferation in vitro, and with inhibition of tumor formation by Tsc2-null cells in vivo. Taken together, our data suggest that Rheb controls proliferation of TSC2-deficient cells by a mechanism that involves regulation of AMPK and p27, and that Rheb is a potential target for TSC/LAM therapy.
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Proteínas Quinasas Activadas por AMP/metabolismo , Proliferación Celular , Transformación Celular Neoplásica/metabolismo , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Proteínas de Unión al GTP Monoméricas/metabolismo , Neuropéptidos/metabolismo , Proteínas/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Animales , Línea Celular Tumoral , Neoplasias del Colon/metabolismo , Neoplasias del Colon/patología , Quinasa 2 Dependiente de la Ciclina/análisis , Quinasa 2 Dependiente de la Ciclina/metabolismo , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/análisis , Femenino , Humanos , Diana Mecanicista del Complejo 1 de la Rapamicina , Ratones , Ratones Desnudos , Complejos Multiproteicos , Proteína Homóloga de Ras Enriquecida en el Cerebro , Serina-Treonina Quinasas TOR , Proteína 2 del Complejo de la Esclerosis Tuberosa , Proteínas Supresoras de Tumor/análisis , Regulación hacia ArribaRESUMEN
Platinum-based chemotherapies such as cisplatin are used as first-line treatment for many cancers. Although there is often a high initial responsiveness, the majority of patients eventually relapse with platinum-resistant disease. For example, a subset of testicular cancer patients still die even though testicular cancer is considered a paradigm of cisplatin-sensitive solid tumors, but the mechanisms of chemoresistance remain elusive. Here, we have shown that one key determinant of cisplatin-resistance in testicular embryonal carcinoma (EC) is high cytoplasmic expression of the cyclin-dependent kinase (CDK) inhibitor p21. The EC component of the majority of refractory testicular cancer patients exhibited high cytoplasmic p21 expression, which protected EC cell lines against cisplatin-induced apoptosis via CDK2 inhibition. Localization of p21 in the cytoplasm was critical for cisplatin resistance, since relocalization of p21 to the nucleus by Akt inhibition sensitized EC cell lines to cisplatin. We also demonstrated in EC cell lines and human tumor tissue that high cytoplasmic p21 expression and cisplatin resistance of EC were inversely associated with the expression of Oct4 and miR-106b seed family members. Thus, targeting cytoplasmic p21, including by modulation of the Oct4/miR-106b/p21 pathway, may offer new strategies for the treatment of chemoresistant testicular and other types of cancer.
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Antineoplásicos/farmacología , Cisplatino/farmacología , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/análisis , Citoplasma/química , Neoplasias Testiculares/tratamiento farmacológico , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Cromonas/farmacología , Quinasa 2 Dependiente de la Ciclina/análisis , Resistencia a Antineoplásicos , Humanos , Masculino , MicroARNs/fisiología , Morfolinas/farmacología , Factor 3 de Transcripción de Unión a Octámeros/fisiología , Proteínas Proto-Oncogénicas c-akt/fisiología , Neoplasias Testiculares/patologíaRESUMEN
Proteins mediate the bulk of biological activity and are powerfully assayed in the diagnosis of diseases. Protein detection relies largely on antibodies, which have significant technical limitations especially when immobilized on two-dimensional surfaces. Here, we report the integration of peptide aptamers with extended gate metal-oxide-semiconductor field-effect transistors (MOSFETs) to achieve label-free sub-picomolar target protein detection. Specifically, peptide aptamers that recognize highly related protein partners of the cyclin-dependent kinase (CDK) family are immobilized on the transistor gate to enable human CDK2 to be detected at 100 fM or 5 pg/mL, well within the clinically relevant range. The target specificity, ease of fabrication, and scalability of these FET arrays further demonstrate the potential application of the multiplexable field effect format to protein sensing.
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Técnicas Biosensibles/métodos , Proteínas/química , Transistores Electrónicos , Quinasa 2 Dependiente de la Ciclina/análisis , Humanos , Proteínas/análisis , Espectroscopía Infrarroja por Transformada de FourierRESUMEN
Many cell cycle regulatory proteins have been shown to be able to regulate cell death. Activation of Cdk2 has been shown to be necessary for apoptosis of quiescent cells such as thymocytes, neurons, and endothelial cells. This activation is stimulus-specific because it occurs in glucocorticoid and DNA damage but not in CD95-induced apoptosis in thymocytes. Apoptotic Cdk2 activation in lymphoid cells is controlled by a recently identified protein, cyclin O, and its activity is modulated by p53 and members of the Bcl-2 protein family. In this chapter, we describe methods for measuring changes in Cdk2 activity during apoptosis. In addition, we also show the details of the generation of an antibody able to immunoprecipitate the cyclin O complexes from apoptotic cells in native conditions and its use to measure the kinase activity associated with this proapoptotic cyclin.
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Apoptosis , Quinasa 2 Dependiente de la Ciclina/metabolismo , Inmunoprecipitación/métodos , Animales , Ciclo Celular , Células Cultivadas , Quinasa 2 Dependiente de la Ciclina/análisis , Ciclinas/inmunología , Ciclinas/metabolismo , Humanos , Linfocitos T/citologíaRESUMEN
OBJECTIVE: To elucidate the role of microRNA (miRNA) in the pathogenesis of rheumatoid arthritis (RA), we analyzed synoviocytes from RA patients for their miRNA expression. METHODS: Synoviocytes derived from surgical specimens obtained from RA patients were compared with those obtained from osteoarthritis (OA) patients for their expression of a panel of 156 miRNA with quantitative stem-loop reverse transcription-polymerase chain reaction. The miRNA whose expression decreased or increased in RA synoviocytes as compared with OA synoviocytes were identified, and their target genes were predicted by computer analysis. We used an in vitro system of enhancing the expression of specific miRNA by transfection of precursors into synoviocytes, and then we performed proliferation, cell cycle, and apoptosis assays, as well as enzyme-linked immunosorbent assays for cytokine production. The effects of transfection on predicted target protein and messenger RNA (mRNA) were then examined by Western blot analysis and luciferase reporter assay. RESULTS: We found that miR-124a levels significantly decreased in RA synoviocytes as compared with OA synoviocytes. Transfection of precursor miR-124a into RA synoviocytes significantly suppressed their proliferation and arrested the cell cycle at the G1 phase. We identified a putative consensus site for miR-124a binding in the 3'-untranslated region of cyclin-dependent kinase 2 (CDK-2) and monocyte chemoattractant protein 1 (MCP-1) mRNA. Induction of miR-124a in RA synoviocytes significantly suppressed the production of the CDK-2 and MCP-1 proteins. Luciferase reporter assay demonstrated that miR-124a specifically suppressed the reporter activity driven by the 3'-untranslated regions of CDK-2 and MCP-1 mRNA. CONCLUSION: The results of this study suggest that miR-124a is a key miRNA in the posttranscriptional regulatory mechanisms of RA synoviocytes.
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Artritis Reumatoide/fisiopatología , Quimiocina CCL2/metabolismo , MicroARNs/fisiología , Membrana Sinovial/fisiopatología , Apoptosis/fisiología , Artritis Reumatoide/etiología , Ciclo Celular/fisiología , Proliferación Celular , Células Cultivadas , Quimiocina CCL2/análisis , Quinasa 2 Dependiente de la Ciclina/análisis , Quinasas Ciclina-Dependientes/análisis , Ensayo de Inmunoadsorción Enzimática , Fibroblastos/fisiología , Humanos , MicroARNs/análisis , Osteoartritis/fisiopatología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Membrana Sinovial/citología , TransfecciónRESUMEN
Alternative lengthening of telomeres (ALT) is a recombination-mediated process that maintains telomeres in telomerase-negative cancer cells. In asynchronously dividing ALT-positive cell populations, a small fraction of the cells have ALT-associated promyelocytic leukemia nuclear bodies (APBs), which contain (TTAGGG)n DNA and telomere-binding proteins. We found that restoring p53 function in ALT cells caused p21 up-regulation, growth arrest/senescence, and a large increase in cells containing APBs. Knockdown of p21 significantly reduced p53-mediated induction of APBs. Moreover, we found that heterochromatin protein 1 (HP1) is present in APBs, and knockdown of HP1alpha and/or HP1gamma prevented p53-mediated APB induction, which suggests that HP1-mediated chromatin compaction is required for APB formation. Therefore, although the presence of APBs in a cell line or tumor is an excellent qualitative marker for ALT, the association of APBs with growth arrest/senescence and with "closed" telomeric chromatin, which is likely to repress recombination, suggests there is no simple correlation between ALT activity level and the number of APBs or APB-positive cells.
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Proteínas Cromosómicas no Histona/fisiología , Telómero/metabolismo , Proteína p53 Supresora de Tumor/fisiología , Proteínas Adaptadoras Transductoras de Señales , Antígenos Virales de Tumores/metabolismo , Línea Celular , Senescencia Celular/genética , Senescencia Celular/fisiología , Homólogo de la Proteína Chromobox 5 , Proteínas Cromosómicas no Histona/metabolismo , Quinasa 2 Dependiente de la Ciclina/análisis , Quinasa 2 Dependiente de la Ciclina/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/fisiología , Humanos , Proteínas Nucleares/análisis , Proteínas Nucleares/metabolismo , Interferencia de ARN , Proteína 1 de Unión a Repeticiones Teloméricas/análisis , Proteína 2 de Unión a Repeticiones Teloméricas/análisis , Proteína p53 Supresora de Tumor/metabolismo , Regulación hacia ArribaRESUMEN
Human hepatocellular carcinoma (HCC) has an elevated requirement for arginine in vitro, and pegylated recombinant human arginase I (rhArg-PEG), an arginine-depleting enzyme, can inhibit the growth of arginine-dependent tumors. While supplementation of the culture medium with ornithine failed to rescue Hep3B cells from growth inhibition induced by rhArg-PEG, citrulline successfully restored cell growth. The data support the roles previously proposed for ornithine transcarbamylase (OTC) in the arginine auxotrophy and rhArg-PEG sensitivity of HCC cells. Expression profiling of argininosuccinate synthetase (ASS), argininosuccinate lyase (ASL) and OTC in 40 HCC tumor biopsy specimens predicted that 16 of the patients would be rhArg-sensitive, compared with 5 who would be sensitive to arginine deiminase (ADI), another arginine-depleting enzyme with anti-tumor activity. Furthermore, rhArg-PEG-mediated deprivation of arginine from the culture medium of different HCC cell lines produced cell cycle arrests at the G(2)/M or S phase, possibly mediated by transcriptional modulation of cyclins and/or cyclin dependent kinases (CDKs). Based on these results, together with further validation of the in vivo efficacy of rhArg-PEG against HCC, we propose that the application of rhArg-PEG alone or in combination with existing chemotherapeutic drugs may represent a specific and effective therapeutic strategy against HCC.
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Antineoplásicos/farmacología , Arginasa/farmacología , Carcinoma Hepatocelular/tratamiento farmacológico , Ciclo Celular/efectos de los fármacos , Neoplasias Hepáticas/tratamiento farmacológico , Animales , Arginasa/uso terapéutico , Carcinoma Hepatocelular/enzimología , Carcinoma Hepatocelular/patología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Citrulina/metabolismo , Citrulinemia/epidemiología , Quinasa 2 Dependiente de la Ciclina/análisis , Ciclinas/análisis , Humanos , Neoplasias Hepáticas/enzimología , Neoplasias Hepáticas/patología , Ratones , Enfermedad por Deficiencia de Ornitina Carbamoiltransferasa/epidemiología , Proteínas Recombinantes/farmacología , Ensayos Antitumor por Modelo de XenoinjertoAsunto(s)
Anexina A4/análisis , Quinasa 2 Dependiente de la Ciclina/análisis , Fosfatidilinositol 3-Quinasas/análisis , Proteínas Serina-Treonina Quinasas/análisis , Anexina A4/metabolismo , Supervivencia Celular , Clonación Molecular , Quinasa 2 Dependiente de la Ciclina/metabolismo , Células HeLa , Humanos , Fosfatidilinositol 3-Quinasas/metabolismo , Unión Proteica , Proteínas Serina-Treonina Quinasas/metabolismo , Células Tumorales Cultivadas , Quinasa de Factor Nuclear kappa BRESUMEN
All-trans retinoic acid (ATRA) and sodium butyrate (SB) have shown growth-inhibitory and differentiation-inducing properties to tumor cells when used as single agents or in combination, but the exact molecular mechanism still remains to be determined. In order to determine the mechanism of the synergy in treatment with RA and SB, we evaluated the growth inhibition capability of ATRA and SB, alone or in combination, in human oral squamous carcinoma cell lines SCC-1 and SCC-9, and identified the expression of cell cycle-related genes. ATRA and SB inhibited cell growth and induced cell cycle G1 arrest. The inhibition effect was more pronounced with SB than with ATRA (p = 0.000). There were interactions between ATRA and SB (p = 0.000). Consistent with the inhibition effect and G1 arrest, ATRA and SB, alone or in combination, induced the expression of G1 phase markers cyclin-dependent kinase (CDK) 6, p21, and p27; inhibited the expression of S-G2 phase proteins CDK2; and decreased Rb phosphorylation. Cyclin D1 expression was increased in the SB- and ATRA + SB-treated groups, but inhibited in the ATRA-treated group. Cyclin B1 and cyclin E expression was slightly decreased in the SB- and ATRA + SB-treated groups, but did not change in the ATRA-treated group. These results indicate that the growth inhibition and G1 arrest of oral squamous carcinoma cells in response to ATRA and/or SB correlates with the induction of G1 phase cell cycle regulatory proteins CDK6, p21, and p27 and the inhibition of S-G2 phase cell cycle regulatory protein CDK2.