RESUMEN
Since the sequencing of the human genome, recent efforts in cancer drug target discovery have focused more on the identification of novel functions of known genes and the development of more appropriate tumor models. In the present study, we investigated in vitro transformed human adult mesenchymal stem cells (MSC) to identify novel candidate cancer drug targets by analyzing the transcriptional profile of known enzymes compared with non-transformed MSC. The identified enzymes were compared with published cancer gene expression data sets. Surprisingly, the majority of up-regulated enzymes are already known cancer drug targets or act within known druggable pathways. Only three enzymes (RNASEH2A, ADARB1, and PPAP2C) are potentially novel targets that are up-regulated in transformed MSC and expressed in numerous carcinomas and sarcomas. We confirmed the overexpression of RNASEH2A, PPAP2C, and ADARB1 in transformed MSC, transformed fibroblasts, and cancer cell lines MCF7, SK-LMS1, MG63, and U2OS. In functional assays, we show that small interfering RNA knockdown of RNASEH2A inhibits anchorage-independent growth but does not alter in vitro proliferation of cancer cell lines, normal MSC, or normal fibroblasts. Knockdown of PPAP2C impaired anchorage-dependent in vitro growth of cancer cell lines and impaired the in vitro growth of primary MSC but not differentiated human fibroblasts. We show that the knockdown of PPAP2C decreases cell proliferation by delaying entry into S phase of the cell cycle and is transcriptionally regulated by p53. These in vitro data validate PPAP2C and RNASEH2A as putative cancer targets and endorse this in silico approach for identifying novel candidates.
Asunto(s)
Adenosina Desaminasa/genética , Antineoplásicos/farmacología , Transformación Celular Neoplásica , Neoplasias/genética , Fosfatidato Fosfatasa/genética , Ribonucleasa H/genética , Células Madre/metabolismo , Adenosina Desaminasa/metabolismo , Células Cultivadas , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Genoma Humano/genética , Genómica , Humanos , Neoplasias/metabolismo , Fosfatidato Fosfatasa/metabolismo , Proteínas de Unión al ARN , Ribonucleasa H/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Regulación hacia Arriba/efectos de los fármacosRESUMEN
The human HDAC (histone deacetylase) family, a well-validated anticancer target, plays a key role in the control of gene expression through regulation of transcription. While HDACs can be subdivided into three main classes, the class I, class II and class III HDACs (sirtuins), it is presently unclear whether inhibiting multiple HDACs using pan-HDAC inhibitors, or targeting specific isoforms that show aberrant levels in tumours, will prove more effective as an anticancer strategy in the clinic. To address the above issues, we have tested a number of clinically relevant HDACis (HDAC inhibitors) against a panel of rhHDAC (recombinant human HDAC) isoforms. Eight rhHDACs were expressed using a baculoviral system, and a Fluor de Lystrade mark (Biomol International) HDAC assay was optimized for each purified isoform. The potency and selectivity of ten HDACs on class I isoforms (rhHDAC1, rhHDAC2, rhHDAC3 and rhHDAC8) and class II HDAC isoforms (rhHDAC4, rhHDAC6, rhHDAC7 and rhHDAC9) was determined. MS-275 was HDAC1-selective, MGCD0103 was HDAC1- and HDAC2-selective, apicidin was HDAC2- and HDAC3-selective and valproic acid was a specific inhibitor of class I HDACs. The hydroxamic acid-derived compounds (trichostatin A, NVP-LAQ824, panobinostat, ITF2357, vorinostat and belinostat) were potent pan-HDAC inhibitors. The growth-inhibitory effect of the HDACis on HeLa cells showed that both pan-HDAC and class-I-specific inhibitors inhibited cell growth. The results also showed that both pan-HDAC and class-I-specific inhibitor treatment resulted in increased acetylation of histones, but only pan-HDAC inhibitor treatment resulted in increased tubulin acetylation, which is in agreement with their activity towards the HDAC6 isoform.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Inhibidores de Histona Desacetilasas , Acetilación , Proliferación Celular , Clonación Molecular , Inhibidores Enzimáticos/metabolismo , Células HeLa , Histona Desacetilasas/clasificación , Histona Desacetilasas/metabolismo , Humanos , Isoformas de Proteínas/antagonistas & inhibidores , Isoformas de Proteínas/metabolismo , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
Reversible histone acetylation is one of the key mechanisms involved in the epigenetic control of gene expression. A variety of recent studies has revealed a role for acetylation in a much broader repertoire of physiological processes, including proliferation control and protein folding, and has highlighted how a variety of non-histone regulatory proteins are influenced by acetylation. Inhibition of histone deacetylase (HDAC) prompts tumour cells to enter apoptosis and, as a consequence, several HDAC inhibitors have entered clinical trials. It is likely that HDAC inhibitor drugs will provide an important class of new mechanism-based therapeutics for cancer.
Asunto(s)
Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/uso terapéutico , Inhibidores de Histona Desacetilasas , Neoplasias/tratamiento farmacológico , Animales , Antineoplásicos/química , Quimioterapia Combinada , Inhibidores Enzimáticos/química , Histona Desacetilasas/metabolismo , Humanos , Neoplasias/enzimologíaRESUMEN
Non-CG methylation is an unexplored epigenetic hallmark of pluripotent stem cells. Here we report that a reduction in non-CG methylation is associated with impaired differentiation capacity into endodermal lineages. Genome-wide analysis of 2,670 non-CG sites in a discovery cohort of 25 phenotyped human induced pluripotent stem cell (hiPSC) lines revealed unidirectional loss (Δß=13%, P<7.4 × 10(-4)) of non-CG methylation that correctly identifies endodermal differentiation capacity in 23 out of 25 (92%) hiPSC lines. Translation into a simplified assay of only nine non-CG sites maintains predictive power in the discovery cohort (Δß=23%, P<9.1 × 10(-6)) and correctly identifies endodermal differentiation capacity in nine out of ten pluripotent stem cell lines in an independent replication cohort consisting of hiPSCs reprogrammed from different cell types and different delivery systems, as well as human embryonic stem cell (hESC) lines. This finding infers non-CG methylation at these sites as a biomarker when assessing endodermal differentiation capacity as a readout.
Asunto(s)
Diferenciación Celular , Metilación de ADN , Endodermo/citología , Células Madre Pluripotentes Inducidas/citología , Biomarcadores/metabolismo , Estudios de Cohortes , Endodermo/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/metabolismoRESUMEN
Primary human endothelial cells have a finite life span in vitro. After 3-4 passages, they tend to de-differentiate and eventually reach senescence. This limits their use in studies of endothelial cell function. To overcome this, we have developed human saphenous vein endothelial cell lines (HSVEC lines). Two cell lines were produced by infection with pZipSVtsA58-U19 which encodes the simian virus 40 large T-antigen, and one cell line was obtained by transfection with pLXSN16E6E7, which encodes the human papillomavirus type 16 E6 and E7 genes. Two of the three HSVEC lines exhibited an extended life span in vitro and retained characteristic endothelial "cobblestone" morphology. These cell lines expressed the known endothelial markers CD31 and vascular endothelial cadherin, and were able to bind Ulex europaeus lectin I, but they did not retain the expression of von Willebrand factor. Furthermore, one cell line was able to functionally up-regulate the expression of intercellular adhesion molecule-1 in response to stimulation with tumor necrosis factor alpha and was also able to incorporate acetylated low-density lipoprotein. Our results suggest that this latter HSVEC line will provide a useful resource to investigate selected responses of the vascular endothelium to physiological and pathological situations.
Asunto(s)
Endotelio Vascular/citología , Endotelio Vascular/metabolismo , Vena Safena/citología , Vena Safena/metabolismo , Anticuerpos/inmunología , Antígenos CD , Antígenos Virales de Tumores/biosíntesis , Antígenos Virales de Tumores/inmunología , Cadherinas , División Celular/efectos de los fármacos , Línea Celular , Relación Dosis-Respuesta a Droga , Humanos , Inmunohistoquímica , Molécula 1 de Adhesión Intercelular/biosíntesis , Molécula 1 de Adhesión Intercelular/efectos de los fármacos , Interleucina-1/farmacología , Lipoproteínas LDL/farmacología , Molécula-1 de Adhesión Celular Endotelial de Plaqueta , Retroviridae/inmunología , Infecciones por Retroviridae/inmunología , Factores de Tiempo , Factor de Necrosis Tumoral alfa/farmacología , Regulación hacia Arriba/efectos de los fármacos , Factor de von Willebrand/biosíntesisRESUMEN
Epigenetic modifications to chromatin are essential for the specification and maintenance of cell fate, enabling the same genome to programme a variety of cellular outcomes. Epigenetic modulation of gene expression is also a critical mechanism by which cells stabilize their responses to environmental stimuli, including both nutritional cues and hormonal signalling. Unsurprisingly, epigenetics is proving to be vitally important in fetal development, and this review addresses our current understanding of the roles of epigenetic regulation in the prenatal phase. It is striking that while there has been a major interest in the intersection of fetal health with epigenetics, there has been relatively little discussion in the literature on epigenetic changes in the pregnant woman, and we attempt to redress this balance, drawing on the fragmented but intriguing experimental literature in this field.
RESUMEN
DNA methylation is one of the most extensively studied, and one of the most stable, of all epigenetic modifications. Two drugs that target DNA methyltransferase enzymes are licensed for clinical use in oncology but relatively little attention has focused on the enzymatic pathways by which DNA methylation can be reversed. Recent breakthroughs have identified at least two classes of enzymes that can achieve functional reversal. This review discusses the significance of DNA demethylation in a range of human diseases, the candidate proteins that mediate the demethylation and the opportunities and challenges in targeting these candidates to develop new therapeutics.
Asunto(s)
Metilación de ADN/efectos de los fármacos , Diseño de Fármacos , Epigénesis Genética , Animales , Antineoplásicos/farmacología , Sistemas de Liberación de Medicamentos , Descubrimiento de Drogas/métodos , HumanosRESUMEN
Chronic and degenerative disorders are a major, and growing, human health burden, and current treatments are in many cases inadequate or very expensive. Epigenetic therapies are attractive options for treating such disorders because they manipulate the processes that maintain cells in an abnormal transcriptional state. The challenges lie in identifying the most appropriate diseases and the enzymes that should be targeted. This review describes the different approaches that can be used to address this problem, focusing particularly on CNS disorders (especially mental retardation, neurodegenerative disease, psychiatric disorders and drug addiction), diabetes and diabetic complications, and autoimmunity and inflammatory diseases.
Asunto(s)
Enfermedades Autoinmunes/terapia , Enfermedades del Sistema Nervioso Central/terapia , Complicaciones de la Diabetes/terapia , Epigenómica , Enfermedades Autoinmunes/genética , Enfermedades del Sistema Nervioso Central/genética , Complicaciones de la Diabetes/genética , HumanosRESUMEN
Epigenetic covalent modifications of DNA and chromatin proteins strongly affect gene expression and cellular activity, and epigenetic misregulation occurs in several diseases, especially cancer. First-generation drugs targeting the relatively promiscuous DNA methylation and histone acetylation modifiers have had successes in the treatment of haematological cancers. Second-generation drug programmes are in the discovery phase, targeting epigenetic enzymes with more tightly defined modes of action. This review highlights some of the challenges in identifying the most appropriate new targets and the issues that need to be addressed to facilitate the successful entry of second-generation epigenetic drugs into the clinic.
Asunto(s)
Antineoplásicos/uso terapéutico , Descubrimiento de Drogas/métodos , Epigénesis Genética/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Antineoplásicos/farmacología , Sistemas de Liberación de Medicamentos/métodos , Humanos , Modelos GenéticosRESUMEN
The potassium channel blocker tetraethylammonium blocks the flow-induced increase in endothelial ICAM-1. We have investigated the subtype of potassium channel that modulates flow-induced increased expression of ICAM-1 on saphenous vein endothelium. Cultured human saphenous vein endothelial cells (HSVECs) or intact saphenous veins were perfused at fixed low and high flows in a laminar shear chamber or flow rig, respectively, in the presence or absence of potassium channel blockers. Expression of K(+) channels and endothelial ICAM-1 was measured by real-time polymerase chain reaction and/or immunoassays. In HSVECs, the application of 0.8 N/m(2) (8 dyn/cm(2)) shear stress resulted in a two- to fourfold increase in cellular ICAM-1 within 6 h (P < 0.001). In intact vein a similar shear stress, with pulsatile arterial pressure, resulted in a twofold increase in endothelial ICAM-1/CD31 staining area within 1.5 h (P < 0.001). Both increases in ICAM-1 were blocked by inclusion of 100 nM apamin in the vein perfusate, whereas other K(+) channel blockers were less effective. Two subtypes of small conductance Ca(2+)-activated K(+) channel (selectively blocked by apamin) were expressed in HSVECs and vein endothelium (SK3>SK2). Apamin blocked the upregulation of ICAM-1 on saphenous vein endothelium in response to increased flow to implicate small conductance Ca(2+)-activated K(+) channels in shear stress/flow-mediated signaling pathways.