RESUMEN
Cervical cancer is one of the most common malignancies of the female reproductive system. Therefore, it is critical to investigate the molecular mechanisms involved in the development and progression of cervical cancer. In this study, we stimulated cervical cancer cells with 5-aza-2'-deoxycytidine (5-Aza-dC) and found that this treatment inhibited cell proliferation and induced apoptosis; additionally, methylation of p16 and O-6-methylguanine-DNA methyltransferase (MGMT) was reversed, although their expression was suppressed. 5-Aza-dC inhibited E6 and E7 expression and up-regulated p53, p21, and Rb expression. Cells transfected with siRNAs targeting p16 and MGMT as well as cells stimulated with 5-Aza-dC were arrested in S phase, and the expression of p53, p21, and Rb was up-regulated more significantly. However, when cells were stimulated with 5-Aza-dC after transfection with siRNAs targeting p16 and MGMT, proliferation decreased significantly, and the percentage of cells in the sub-G1 peak and in S phase was significantly increased, suggesting a marked increase in apoptosis. But E6 and E7 overexpression could rescue the observed effects in proliferation. Furthermore, X-ray radiation caused cells to arrest in G2/M phase, but cells transfected with p16- and MGMT-targeted siRNAs followed by X-ray radiation exhibited a significant decrease in proliferation and were shifted toward the sub-G1 peak, also indicating enhanced apoptosis. In addition, the effects of 5-Aza-dC and X-ray radiation were most pronounced when MGMT expression was down-regulated. Therefore, down-regulation of p16 and MGMT expression enhances the anti-proliferative effects of 5-Aza-dC and X-ray radiation. This discovery may provide novel ideas for the treatment of cervical cancer.
Asunto(s)
Apoptosis/efectos de los fármacos , Azacitidina/análogos & derivados , Inhibidor p18 de las Quinasas Dependientes de la Ciclina/antagonistas & inhibidores , Metilasas de Modificación del ADN/antagonistas & inhibidores , Enzimas Reparadoras del ADN/antagonistas & inhibidores , Regulación hacia Abajo/efectos de los fármacos , Proteínas Supresoras de Tumor/antagonistas & inhibidores , Neoplasias del Cuello Uterino/tratamiento farmacológico , Neoplasias del Cuello Uterino/patología , Azacitidina/química , Azacitidina/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Inhibidor p16 de la Quinasa Dependiente de Ciclina , Inhibidor p18 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p18 de las Quinasas Dependientes de la Ciclina/metabolismo , Metilasas de Modificación del ADN/genética , Metilasas de Modificación del ADN/metabolismo , Enzimas Reparadoras del ADN/genética , Enzimas Reparadoras del ADN/metabolismo , Decitabina , Regulación hacia Abajo/genética , Femenino , Humanos , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Neoplasias del Cuello Uterino/metabolismo , Rayos XRESUMEN
Nonalcoholic steatohepatitis (NASH) is characterized by excess accumulation of lipids in liver, accompanied with hepatocyte injury, cell death and inflammation. Although p16 is known as tumor suppressor in multiple cancer types, it remains unclear whether p16 plays a critical role in NASH. To determine whether p16 could play a role in the pathogenesis of NASH, wild-type mice and p16-/- mice were fed on a methionine and choline-deficient (MCD) diet for 3 weeks, and liver steatosis, fibrosis, and inflammation were evaluated. Our data show that p16-/- mice fed with MCD diet displayed more significant hepatic steatosis, hepatocyte damage, increased oxidative stress and inflammatory cell infiltration compared to MCD-fed WT mice. It was also clear that the increased ROS and the accumulation of lipid in BEL-7402 cells occurred when p16 expression was depleted with siRNA. These findings indicate that p16 may play a critical role in the development of NASH by reining in ROS production and by inhabiting inflammatory response.
Asunto(s)
Deficiencia de Colina/genética , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Inhibidor p18 de las Quinasas Dependientes de la Ciclina/genética , Hepatocitos/metabolismo , Cirrosis Hepática/genética , Enfermedad del Hígado Graso no Alcohólico/genética , Animales , Línea Celular Tumoral , Deficiencia de Colina/etiología , Deficiencia de Colina/metabolismo , Deficiencia de Colina/patología , Inhibidor p16 de la Quinasa Dependiente de Ciclina/deficiencia , Inhibidor p18 de las Quinasas Dependientes de la Ciclina/antagonistas & inhibidores , Inhibidor p18 de las Quinasas Dependientes de la Ciclina/metabolismo , Modelos Animales de Enfermedad , Alimentos Formulados/efectos adversos , Regulación de la Expresión Génica , Hepatocitos/patología , Humanos , Hígado/metabolismo , Hígado/patología , Cirrosis Hepática/etiología , Cirrosis Hepática/metabolismo , Cirrosis Hepática/patología , Masculino , Metionina/deficiencia , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Enfermedad del Hígado Graso no Alcohólico/etiología , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Enfermedad del Hígado Graso no Alcohólico/patología , Estrés Oxidativo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismoRESUMEN
The expansion of cells for regenerative therapy will require the genetic dissection of complex regulatory mechanisms governing the proliferation of non-transformed human cells. Here, we report the development of a high-throughput RNAi screening strategy specifically for use in primary cells and demonstrate that silencing the cell cycle-dependent kinase inhibitors CDKN2C/p18 or CDKN1A/p21 facilitates cell cycle entry of quiescent adult human pancreatic beta cells. This work identifies p18 and p21 as novel targets for promoting proliferation of human beta cells and demonstrates the promise of functional genetic screens for dissecting therapeutically relevant state changes in primary human cells.
Asunto(s)
Inhibidor p18 de las Quinasas Dependientes de la Ciclina/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Células Secretoras de Insulina/metabolismo , Adolescente , Adulto , Anciano , Alberta , Biomarcadores/metabolismo , Proliferación Celular , Células Cultivadas , Inhibidor p18 de las Quinasas Dependientes de la Ciclina/antagonistas & inhibidores , Inhibidor p18 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/antagonistas & inhibidores , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Estudios de Factibilidad , Femenino , Genómica/métodos , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Ensayos Analíticos de Alto Rendimiento , Humanos , Células Secretoras de Insulina/citología , Masculino , Microscopía Fluorescente , Persona de Mediana Edad , Proyectos Piloto , Interferencia de ARN , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Donantes de Tejidos , Adulto JovenRESUMEN
Hematopoietic stem cells (HSCs) have emerged as promising therapeutic cell sources for high-risk hematological malignancies and immune disorders. However, their clinical use is limited by the inability to expand these cells ex vivo. Therefore, there is an urgent need to identify specific targets and effective probes that can expand HSCs. Here we report a novel class of INK4C (p18(INK4C) or p18) small molecule inhibitors (p18SMIs), which were initially found by in silico 3D screening. We identified a lead p18 inhibitor, XIE18-6, confirmed its p18-targeting specificity and bioactivity of promoting HSCs expansion, and then performed structure-activity relationship (SAR) studies by synthesizing a series of analogs of XIE18-6. Among these, compound 40 showed the most potent bioactivity in HSCs expansion (ED50 = 5.21 nM). We confirmed that compound 40 promoted expansion of both murine and human HSCs, and also confirmed its p18-targeting specificity. Notably, compound 40 did not show significant cytotoxicity toward 32D cells or HSCs, nor did it augment leukemia cell proliferation. Taken together, our newly discovered p18SMIs represent novel chemical agents for murine and human HSCs ex vivo expansion and also can be used as valuable chemical probes for further HSC biology research towards promising utility for therapeutic purposes.
Asunto(s)
Cumarinas/química , Inhibidor p18 de las Quinasas Dependientes de la Ciclina/metabolismo , Bibliotecas de Moléculas Pequeñas/química , Ácidos Sulfanílicos/química , Animales , Sitios de Unión , Células de la Médula Ósea/citología , Células de la Médula Ósea/metabolismo , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Cumarinas/metabolismo , Cumarinas/toxicidad , Quinasa 4 Dependiente de la Ciclina/metabolismo , Quinasa 6 Dependiente de la Ciclina/metabolismo , Inhibidor p18 de las Quinasas Dependientes de la Ciclina/antagonistas & inhibidores , Inhibidor p18 de las Quinasas Dependientes de la Ciclina/genética , Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Humanos , Ratones , Ratones Endogámicos C57BL , Simulación del Acoplamiento Molecular , Estructura Terciaria de Proteína , Bibliotecas de Moléculas Pequeñas/metabolismo , Bibliotecas de Moléculas Pequeñas/toxicidad , Relación Estructura-Actividad , Ácidos Sulfanílicos/metabolismo , Ácidos Sulfanílicos/toxicidadRESUMEN
UNLABELLED: Gammaherpesviruses (GHVs) carry homologs of cellular genes, including those encoding a viral cyclin that promotes reactivation from latent infection. The viral cyclin has reduced sensitivity to host cyclin-dependent kinase inhibitors in vitro; however, the in vivo significance of this is unclear. Here, we tested the genetic requirement for the viral cyclin in mice that lack the host inhibitors p27(Kip1) and p18(INK4c), two cyclin-dependent kinase inhibitors known to be important in regulating B cell proliferation and differentiation. While the viral cyclin was essential for reactivation in wild-type mice, strikingly, it was dispensable for reactivation in mice lacking p27(Kip1) and p18(INK4c). Further analysis revealed that genetic ablation of only p18(INK4c) alleviated the requirement for the viral cyclin for reactivation from latency. p18(INK4c) regulated reactivation in a dose-dependent manner so that the viral cyclin was dispensable in p18(INK4c) heterozygous mice. Finally, treatment of wild-type cells with the cytokine BAFF, a known attenuator of p18(INK4c) function in B lymphocytes, was also able to bypass the requirement for the viral cyclin in reactivation. These data show that the gammaherpesvirus viral cyclin functions specifically to bypass the cyclin-dependent kinase inhibitor p18(INK4c), revealing an unanticipated specificity between a GHV cyclin and a single cyclin-dependent kinase inhibitor. IMPORTANCE: The gammaherpesviruses (GHVs) cause lifelong infection and can cause chronic inflammatory diseases and cancer, especially in immunosuppressed individuals. Many GHVs encode a conserved viral cyclin that is required for infection and disease. While a common property of the viral cyclins is that they resist inhibition by normal cellular mechanisms, it remains unclear how important it is that the GHVs resist this inhibition. We used a mouse GHV that either contained or lacked a viral cyclin to test whether the viral cyclin lost importance when these inhibitory pathways were removed. These studies revealed that the viral cyclin was required for optimal function in normal mice but that it was no longer required following removal or reduced function of a single cellular inhibitor. These data define a very specific role for the viral cyclin in bypassing one cellular inhibitor and point to new methods to intervene with viral cyclins.
Asunto(s)
Inhibidor p18 de las Quinasas Dependientes de la Ciclina/metabolismo , Ciclinas/metabolismo , Gammaherpesvirinae/metabolismo , Activación Viral/fisiología , Latencia del Virus/fisiología , Animales , Factor Activador de Células B/farmacología , Inhibidor p18 de las Quinasas Dependientes de la Ciclina/antagonistas & inhibidores , Inhibidor p18 de las Quinasas Dependientes de la Ciclina/deficiencia , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/deficiencia , Ciclinas/farmacología , Cartilla de ADN/genética , Citometría de Flujo , Gammaherpesvirinae/genética , Immunoblotting , Ratones , Pruebas de Neutralización , Reacción en Cadena de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Activación Viral/efectos de los fármacosRESUMEN
Among cyclin-dependent kinase inhibitors that control the G1 phase in cell cycle, only p18 and p27 can negatively regulate haematopoietic stem cell (HSC) self-renewal. In this manuscript, we demonstrate that p18 protein is a more potent inhibitor of HSC self-renewal than p27 in mouse models and its deficiency promoted HSC expansion in long-term culture. Single-cell analysis indicated that deleting p18 gene favoured self-renewing division of HSC in vitro. Based on the structure of p18 protein and in-silico screening, we further identified novel smallmolecule inhibitors that can specifically block the activity of p18 protein. Our selected lead compounds were able to expand functional HSCs in a short-term culture. Thus, these putative small-molecule inhibitors for p18 protein are valuable for further dissecting the signalling pathways of stem cell self-renewal and may help develop more effective chemical agents for therapeutic expansion of HSC.
Asunto(s)
Técnicas de Cultivo de Célula , Inhibidor p18 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p18 de las Quinasas Dependientes de la Ciclina/metabolismo , Regulación de la Expresión Génica , Células Madre Hematopoyéticas/citología , Animales , Apoptosis , Ciclo Celular , Proliferación Celular , Separación Celular , Técnicas de Cocultivo , Quinasa 6 Dependiente de la Ciclina/metabolismo , Inhibidor p18 de las Quinasas Dependientes de la Ciclina/antagonistas & inhibidores , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Femenino , Eliminación de Gen , Células Madre Hematopoyéticas/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neutrófilos/metabolismo , Fenotipo , Transducción de Señal , Análisis de la Célula Individual , Células Madre/citologíaRESUMEN
Resistance to BRAF(V600E) inhibitors is associated with reactivation of mitogen-activated protein kinase (MAPK) signaling at different levels in melanoma. To identify downstream effectors of MAPK signaling that could be used as potential additional therapeutic targets for BRAF(V600E) inhibitors, we used hTERT/CDK4R24C/p53DD-immortalized primary human melanocytes genetically modified to ectopically express BRAF ( V600E ) or NRAS ( G12D ) and observed induction of the AP-1 transcription factor family member c-Jun. Using a dominant negative approach, in vitro cell proliferation assays, western blots, and flow cytometry showed that MAPK signaling via BRAF(V600E) promotes melanoma cell proliferation at G1 through AP-1-mediated negative regulation of the INK4 family member, cyclin-dependent kinase inhibitor 2C (CDKN2C), and the CIP/KIP family member, cyclin-dependent kinase inhibitor 1A (CDKN1A). These effects were antagonized by pharmacological inhibition of CDKN2C and CDKN1A targets CDK2 and CDK4 in vitro. In contrast to BRAF ( V600E ) or NRAS ( G12D )-expressing melanocytes, melanoma cells have an inherent resistance to suppression of AP-1 activity by BRAF(V600E)- or MEK-inhibitors. Here, CDK2/4 inhibition statistically significantly augmented the effects of BRAF(V600E)- or MEK-inhibitors on melanoma cell viability in vitro and growth in athymic nude Foxn1 ( nu ) mice (P = .03 when mean tumor volume at day 13 was compared for BRAF(V600E) inhibitor vs BRAF(V600E) inhibitor plus CDK2/4 inhibition; P = .02 when mean tumor volume was compared for MEK inhibitor vs MEK inhibitor plus CDK2/4 inhibition; P values were calculated by a two-sided Welch t test; n = 4-8 mice per group).
Asunto(s)
Antineoplásicos/farmacología , Inhibidor p18 de las Quinasas Dependientes de la Ciclina/antagonistas & inhibidores , Inhibidor p18 de las Quinasas Dependientes de la Ciclina/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/antagonistas & inhibidores , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Melanocitos/metabolismo , Melanoma/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas B-raf/metabolismo , Proteínas Proto-Oncogénicas c-jun/metabolismo , Animales , Ácido Aspártico , Benzamidas/farmacología , Western Blotting , Línea Celular Tumoral , Inhibidor p18 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Citometría de Flujo , Regulación Neoplásica de la Expresión Génica , Genes ras , Ácido Glutámico , Glicina , Humanos , Melanocitos/patología , Melanoma/tratamiento farmacológico , Melanoma/enzimología , Ratones , Ratones Desnudos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Proteínas Proto-Oncogénicas B-raf/genética , ARN Interferente Pequeño , Factor de Transcripción AP-1/metabolismo , Transfección , Trasplante Heterólogo , Valina , Vacunas Virales/farmacologíaRESUMEN
We have previously shown that a novel -74 C-to-T mutation in the promoter of the cyclin-dependent kinase inhibitor p18(Ink4c) (p18) gene was associated with a reduced p18 expression in B cells from mice carrying the Sle2c1 lupus susceptibility locus. To determine the function of the -74 C/T single nucleotide polymorphism, we have characterized the proximal promoter of the mouse p18 gene. Functional analysis of the 5' flanking region by sequential deletions revealed crucial elements between -300 and +1, confirming the in silico prediction that the -74 T allele created a novel Yin-Yang 1 (YY-1) binding site adjacent to an existing one common to both alleles. Moreover, we found that YY-1, E2F1, and Sp-1 can synergistically enhance the activity of the p18 promoter. Mutational inactivation revealed that YY-1 binding regulates the p18 activity in an allele-dependent fashion. EMSAs with splenic B cell extracts directly demonstrated that YY-1 binds to the p18 promoter with differences between the C and the T alleles. We also determined in vivo by chromatin immunoprecipitation that the T allele resulted in increased YY-1 and decreased Nrf-2 binding to the p18 promoter as compared with the C allele in B cells. Thus, YY-1 is a direct regulator of p18 gene expression in an allele-dependent fashion that is consistent with the lupus-associated T allele, inducing a lower p18 transcriptional activity by increasing YY-1 binding. These results establish the p18 -74 C/T mutation as the leading causal variant for the B1a cell expansion that characterizes the NZB and NZM2410 lupus-prone strains.
Asunto(s)
Inhibidor p18 de las Quinasas Dependientes de la Ciclina/genética , Variación Genética/inmunología , Lupus Eritematoso Sistémico/genética , Lupus Eritematoso Sistémico/inmunología , Transcripción Genética/inmunología , Factor de Transcripción YY1/genética , Animales , Secuencia de Bases , Inhibidor p18 de las Quinasas Dependientes de la Ciclina/antagonistas & inhibidores , Inhibidor p18 de las Quinasas Dependientes de la Ciclina/biosíntesis , Regulación hacia Abajo/genética , Regulación hacia Abajo/inmunología , Predisposición Genética a la Enfermedad , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NZB , Datos de Secuencia Molecular , Mutación , Células 3T3 NIH , Polimorfismo de Nucleótido Simple/inmunología , Regiones Promotoras Genéticas/genética , Factor de Transcripción YY1/fisiologíaRESUMEN
A combination of extrinsic hematopoietic growth regulators, such as stem cell factor (SCF), interleukin (IL)-3 and IL-6, can induce division of quiescent hematopoietic stem cells (HSCs), but it usually impairs HSCs' self-renewal ability. However, intrinsic negative cell cycle regulators, such as p18INK4C (p18), p27Kip1 (p27) and MAD1, can regulate the self-renewal of HSCs. It is unknown whether the removal of some extrinsic regulators and the knockdown of intrinsic negative cell cycle regulators via RNA interference (RNAi) induce ex vivo expansion of the HSCs. To address this question, a lentiviral vector-based RNAi tool was developed to produce two copies of small RNA that target multiple genes to knockdown the intrinsic negative cell cycle regulators p18, p27 and MAD1. Colony-forming cells, long-term culture-initiating cells (LTC-IC) and engraftment assays were used to evaluate the effects of extrinsic and intrinsic regulators. Results showed that the medium with only SCF, but without IL-3 and IL-6, could maintain the sca-1+c-kit+ bone marrow cells with high LTC-IC frequency and low cell division. However, when the sca-1+c-kit+ bone marrow cells were cultured in a medium with only SCF and simultaneously knocked down the expression of p18, p27 and MAD1 via the lentiviral vector-based RNAi, the cells exhibited both high LTC-IC frequency and high cell division, though engraftment failed. Thus, the simultaneous knockdown of p18, p27 and MAD1 with a medium of only SCF can induce LTC-IC expansion despite the loss of engraftment ability.