RESUMEN
Cells respond to soluble and membrane-bound factors to activate signalling cascades that control cell proliferation and cell death. Vascular endothelial growth factor A (VEGF-A) is a soluble ligand that modulates a variety of cellular responses including cell proliferation and apoptosis. It is not well understood how VEGF-A signalling pathways regulate cell proliferation and cell death. To address this, we examined VEGF-A-regulated signalling pathways in the cytosol and nucleus and functional requirement for such cellular responses. The VEGF-A-regulated transcription factor, ATF-2, is required for cell cycle proteins such as p53, p21 and Cyclin D1. A cytosolic serine/threonine protein kinase (Tpl2) modulates ATF-2-regulated effects on the endothelial cell cycle. Such regulatory effects impact on endothelial cell proliferation, cell viability and apoptosis. These cellular effects influence complex cell-based organisation such as endothelial tubulogenesis. Our study now provides a framework for incorporating VEGF-A-stimulated signalling events from the cytosol to the nucleus which helps to understand how cell proliferation and apoptosis are controlled.
Asunto(s)
Factor de Transcripción Activador 2/metabolismo , Apoptosis , Ciclo Celular , Células Endoteliales de la Vena Umbilical Humana/citología , Quinasas Quinasa Quinasa PAM/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Línea Celular , Proliferación Celular , Humanos , Transducción de SeñalRESUMEN
New blood vessel sprouting (angiogenesis) and vascular physiology are fundamental features of metazoan species but we do not fully understand how signal transduction pathways regulate diverse vascular responses. The vascular endothelial growth factor (VEGF) family bind membrane-bound receptor tyrosine kinases (VEGFRs), which trigger multiple signal transduction pathways and diverse cellular responses. We evaluated whether the MAP3K family member and proto-oncoprotein Tpl2 (MAP3K8) regulates basal and VEGF-A-stimulated signal transduction in endothelial cells. Notably, stimulation with exogenous VEGF-A increased Tpl2 mRNA levels and consequently de novo protein synthesis. Depletion of Tpl2 levels reveals a role in both basal and VEGF-A-stimulated endothelial cell responses, including endothelial-leukocyte interactions, monolayer permeability and new blood vessel formation. Under basal conditions, Tpl2 modulates a signal transduction cascade resulting in phosphorylation of a nuclear transcription factor (ATF-2) and altered endothelial gene expression, a pathway previously identified as crucial in VEGF-dependent vascular responses. Loss of Tpl2 expression or activity impairs signal transduction through Akt, eNOS and ATF-2, broadly impacting on endothelial function. Our study now provides a mechanism for Tpl2 as a central component of signal transduction pathways in the endothelium.
RESUMEN
Humans are frequently exposed to acrylamide, a probable human carcinogen found in commonplace sources such as most heated starchy foods or tobacco smoke. Prior evidence has shown that acrylamide causes cancer in rodents, yet epidemiological studies conducted to date are limited and, thus far, have yielded inconclusive data on association of human cancers with acrylamide exposure. In this study, we experimentally identify a novel and unique mutational signature imprinted by acrylamide through the effects of its reactive metabolite glycidamide. We next show that the glycidamide mutational signature is found in a full one-third of approximately 1600 tumor genomes corresponding to 19 human tumor types from 14 organs. The highest enrichment of the glycidamide signature was observed in the cancers of the lung (88% of the interrogated tumors), liver (73%), kidney (>70%), bile duct (57%), cervix (50%), and, to a lesser extent, additional cancer types. Overall, our study reveals an unexpectedly extensive contribution of acrylamide-associated mutagenesis to human cancers.
Asunto(s)
Acrilamidas/toxicidad , Carcinogénesis/genética , Exposición a Riesgos Ambientales , Mutágenos/toxicidad , Mutación , Neoplasias/genética , Animales , Carcinogénesis/inducido químicamente , Células Cultivadas , Compuestos Epoxi/toxicidad , Genoma Humano , Humanos , Ratones , Neoplasias/inducido químicamente , Proteína p53 Supresora de Tumor/genéticaRESUMEN
Aflatoxin B1 (AFB1) is a mutagen and IARC (International Agency for Research on Cancer) Group 1 carcinogen that causes hepatocellular carcinoma (HCC). Here, we present the first whole-genome data on the mutational signatures of AFB1 exposure from a total of >40,000 mutations in four experimental systems: two different human cell lines, in liver tumors in wild-type mice, and in mice that carried a hepatitis B surface antigen transgene-this to model the multiplicative effects of aflatoxin exposure and hepatitis B in causing HCC. AFB1 mutational signatures from all four experimental systems were remarkably similar. We integrated the experimental mutational signatures with data from newly sequenced HCCs from Qidong County, China, a region of well-studied aflatoxin exposure. This indicated that COSMIC mutational signature 24, previously hypothesized to stem from aflatoxin exposure, indeed likely represents AFB1 exposure, possibly combined with other exposures. Among published somatic mutation data, we found evidence of AFB1 exposure in 0.7% of HCCs treated in North America, 1% of HCCs from Japan, but 16% of HCCs from Hong Kong. Thus, aflatoxin exposure apparently remains a substantial public health issue in some areas. This aspect of our study exemplifies the promise of future widespread resequencing of tumor genomes in providing new insights into the contribution of mutagenic exposures to cancer incidence.
Asunto(s)
Aflatoxina B1/toxicidad , Carcinógenos/toxicidad , Análisis Mutacional de ADN , Mutación/efectos de los fármacos , Animales , Carcinoma Hepatocelular/inducido químicamente , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patología , Línea Celular Tumoral , China , Antígenos de Superficie de la Hepatitis B/genética , Humanos , Neoplasias Hepáticas/inducido químicamente , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patología , Ratones , Mutación/genéticaRESUMEN
TP53 mutations occur in half of all human tumours. Mutagen-induced or spontaneous TP53 mutagenesis can be studied in vitro using the human TP53 knock-in (Hupki) mouse embryo fibroblast (HUF) immortalisation assay (HIMA). TP53 mutations arise in up to 30% of mutagen-treated, immortalised HUFs; however, mutants are not identified until TP53 sequence analysis following immortalisation (2-5 months) and much effort is expended maintaining TP53-WT cultures. In order to improve the selectivity of the HIMA for HUFs harbouring TP53 mutations, we explored the use of Nutlin-3a, an MDM2 inhibitor that leads to stabilisation and activation of wild-type (WT) p53. First, we treated previously established immortal HUF lines carrying WT or mutated TP53 with Nutlin-3a to examine the effect on cell growth and p53 activation. Nutlin-3a induced the p53 pathway in TP53-WT HUFs and inhibited cell growth, whereas most TP53-mutated HUFs were resistant to Nutlin-3a. We then assessed whether Nutlin-3a treatment could discriminate between TP53-WT and TP53-mutated cells during the HIMA (n = 72 cultures). As immortal clones emerged from senescent cultures, each was treated with 10 µM Nutlin-3a for 5 days and observed for sensitivity or resistance. TP53 was subsequently sequenced from all immortalised clones. We found that all Nutlin-3a-resistant clones harboured TP53 mutations, which were diverse in position and functional impact, while all but one of the Nutlin-3a-sensitive clones were TP53-WT. These data suggest that including a Nutlin-3a counter-screen significantly improves the specificity and efficiency of the HIMA, whereby TP53-mutated clones are selected prior to sequencing and TP53-WT clones can be discarded.
Asunto(s)
Genes p53 , Imidazoles/farmacología , Piperazinas/farmacología , Animales , Línea Celular Transformada , Supervivencia Celular , Transformación Celular Neoplásica/efectos de los fármacos , Células Cultivadas , Células Clonales , Resistencia a Medicamentos , Fibroblastos , Técnicas de Sustitución del Gen , Humanos , Ratones , Mutación , Oxígeno/farmacología , Proteínas Proto-Oncogénicas c-mdm2/antagonistas & inhibidores , Selección Genética , Transducción de Señal/efectos de los fármacosRESUMEN
TP53 gene mutations are one of the most frequent somatic events in cancer. The IARC TP53 Database (http://p53.iarc.fr) is a popular resource that compiles occurrence and phenotype data on TP53 germline and somatic variations linked to human cancer. The deluge of data coming from cancer genomic studies generates new data on TP53 variations and attracts a growing number of database users for the interpretation of TP53 variants. Here, we present the current contents and functionalities of the IARC TP53 Database and perform a systematic analysis of TP53 somatic mutation data extracted from this database and from genomic data repositories. This analysis showed that IARC has more TP53 somatic mutation data than genomic repositories (29,000 vs. 4,000). However, the more complete screening achieved by genomic studies highlighted some overlooked facts about TP53 mutations, such as the presence of a significant number of mutations occurring outside the DNA-binding domain in specific cancer types. We also provide an update on TP53 inherited variants including the ones that should be considered as neutral frequent variations. We thus provide an update of current knowledge on TP53 variations in human cancer as well as inform users on the efficient use of the IARC TP53 Database.
Asunto(s)
Bases de Datos Genéticas , Mutación , Neoplasias/genética , Proteína p53 Supresora de Tumor/genética , Curaduría de Datos , Predisposición Genética a la Enfermedad , Genómica , Humanos , Fenotipo , Dominios Proteicos , Programas Informáticos , Proteína p53 Supresora de Tumor/químicaRESUMEN
A nonsynonymous single-nucleotide polymorphism at codon 47 in TP53 exists in African-descent populations (P47S, rs1800371; referred to here as S47). Here we report that, in human cell lines and a mouse model, the S47 variant exhibits a modest decrease in apoptosis in response to most genotoxic stresses compared with wild-type p53 but exhibits a significant defect in cell death induced by cisplatin. We show that, compared with wild-type p53, S47 has nearly indistinguishable transcriptional function but shows impaired ability to transactivate a subset of p53 target genes, including two involved in metabolism:Gls2(glutaminase 2) and Sco2 We also show that human and mouse cells expressing the S47 variant are markedly resistant to cell death by agents that induce ferroptosis (iron-mediated nonapoptotic cell death). We show that mice expressing S47 in homozygous or heterozygous form are susceptible to spontaneous cancers of diverse histological types. Our data suggest that the S47 variant may contribute to increased cancer risk in individuals of African descent, and our findings highlight the need to assess the contribution of this variant to cancer risk in these populations. These data also confirm the potential relevance of metabolism and ferroptosis to tumor suppression by p53.
Asunto(s)
Genes p53/genética , Polimorfismo de Nucleótido Simple , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Animales , Población Negra/genética , Carcinoma Hepatocelular/genética , Muerte Celular/efectos de los fármacos , Muerte Celular/genética , Línea Celular , Cisplatino/farmacología , Codón/química , Codón/genética , Modelos Animales de Enfermedad , Humanos , Ratones , Ratones Endogámicos C57BL , Neoplasias/genética , Unión Proteica/genética , Factores de Riesgo , Activación Transcripcional/efectos de los fármacos , Activación Transcripcional/genéticaRESUMEN
Whole genome sequencing of human tumours has revealed distinct patterns of mutation that hint at the causative origins of cancer. Experimental investigations of the mutations and mutation spectra induced by environmental mutagens have traditionally focused on single genes. With the advent of faster cheaper sequencing platforms, it is now possible to assess mutation spectra in experimental models across the whole genome. As a proof of principle, we have examined the whole genome mutation profiles of mouse embryo fibroblasts immortalised following exposure to benzo[a]pyrene (BaP), ultraviolet light (UV) and aristolochic acid (AA). The results reveal that each mutagen induces a characteristic mutation signature: predominantly GâT mutations for BaP, CâT and CCâTT for UV and AâT for AA. The data are not only consistent with existing knowledge but also provide additional information at higher levels of genomic organisation. The approach holds promise for identifying agents responsible for mutations in human tumours and for shedding light on the aetiology of human cancer.
Asunto(s)
Exposición a Riesgos Ambientales , Genoma , Genómica , Animales , Línea Celular , Transformación Celular Neoplásica , Análisis Mutacional de ADN , Replicación del ADN , Exposición a Riesgos Ambientales/efectos adversos , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Estudio de Asociación del Genoma Completo , Genómica/métodos , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Ratones , Mutagénesis , Mutágenos/efectos adversos , Mutación , Neoplasias/etiología , Transcripción GenéticaRESUMEN
We compared mouse embryonic stem (ES) cells and fibroblasts (MEFs) for their ability to metabolically activate the environmental carcinogens benzo[a]pyrene (BaP), 3-nitrobenzanthrone (3-NBA) and aristolochic acid I (AAI), measuring DNA adduct formation by (32)P-postlabelling and expression of xenobiotic-metabolism genes by quantitative real-time PCR. At 2 µM, BaP induced Cyp1a1 expression in MEFs to a much greater extent than in ES cells and formed 45 times more adducts. Nqo1 mRNA expression was increased by 3-NBA in both cell types but induction was higher in MEFs, as was adduct formation. For AAI, DNA binding was over 450 times higher in MEFs than in ES cells, although Nqo1 and Cyp1a1 transcriptional levels did not explain this difference. We found higher global methylation of DNA in ES cells than in MEFs, which suggests higher chromatin density and lower accessibility of the DNA to DNA damaging agents in ES cells. However, AAI treatment did not alter DNA methylation. Thus mouse ES cells and MEFs have the metabolic competence to activate a number of environmental carcinogens, but MEFs have lower global DNA methylation and higher metabolic capacity than mouse ES cells.
Asunto(s)
Carcinógenos Ambientales/farmacología , Células Madre Embrionarias/efectos de los fármacos , Fibroblastos/efectos de los fármacos , Animales , Ácidos Aristolóquicos/metabolismo , Ácidos Aristolóquicos/farmacología , Benzo(a)Antracenos/metabolismo , Benzo(a)Antracenos/farmacología , Benzo(a)pireno/metabolismo , Benzo(a)pireno/farmacología , Western Blotting , Carcinógenos Ambientales/metabolismo , Aductos de ADN/análisis , Aductos de ADN/metabolismo , Metilación de ADN/efectos de los fármacos , Células Madre Embrionarias/metabolismo , Fibroblastos/metabolismo , Perfilación de la Expresión Génica , Ratones , Ratones Endogámicos C57BL , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Vascular endothelial growth factor A (VEGF-A) regulates many aspects of vascular physiology. VEGF-A stimulates signal transduction pathways that modulate endothelial outputs such as cell migration, proliferation, tubulogenesis, and cell-cell interactions. Multiple VEGF-A isoforms exist, but the biological significance of this is unclear. Here we analyzed VEGF-A isoform-specific stimulation of VCAM-1 gene expression, which controls endothelial-leukocyte interactions, and show that this is dependent on both ERK1/2 and activating transcription factor-2 (ATF-2). VEGF-A isoforms showed differential ERK1/2 and p38 MAPK phosphorylation kinetics. A key feature of VEGF-A isoform-specific ERK1/2 activation and nuclear translocation was increased phosphorylation of ATF-2 on threonine residue 71 (T71). Using reverse genetics, we showed ATF-2 to be functionally required for VEGF-A-stimulated endothelial VCAM-1 gene expression. ATF-2 knockdown blocked VEGF-A-stimulated VCAM-1 expression and endothelial-leukocyte interactions. ATF-2 was also required for other endothelial cell outputs, such as cell migration and tubulogenesis. In contrast, VCAM-1 was essential only for promoting endothelial-leukocyte interactions. This work presents a new paradigm for understanding how soluble growth factor isoforms program complex cellular outputs and responses by modulating signal transduction pathways.
Asunto(s)
Factor de Transcripción Activador 2/metabolismo , Leucocitos/metabolismo , Sistema de Señalización de MAP Quinasas , Molécula 1 de Adhesión Celular Vascular/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Factor de Transcripción Activador 2/genética , Movimiento Celular , Proliferación Celular , Expresión Génica , Humanos , Fosforilación , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Molécula 1 de Adhesión Celular Vascular/genética , Factor A de Crecimiento Endotelial Vascular/genética , Receptor 2 de Factores de Crecimiento Endotelial Vascular/genética , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Experimental models that recapitulate mutational landscapes of human cancers are needed to decipher the rapidly expanding data on human somatic mutations. We demonstrate that mutation patterns in immortalised cell lines derived from primary murine embryonic fibroblasts (MEFs) exposed in vitro to carcinogens recapitulate key features of mutational signatures observed in human cancers. In experiments with several cancer-causing agents we obtained high genome-wide concordance between human tumour mutation data and in vitro data with respect to predominant substitution types, strand bias and sequence context. Moreover, we found signature mutations in well-studied human cancer driver genes. To explore endogenous mutagenesis, we used MEFs ectopically expressing activation-induced cytidine deaminase (AID) and observed an excess of AID signature mutations in immortalised cell lines compared to their non-transgenic counterparts. MEF immortalisation is thus a simple and powerful strategy for modelling cancer mutation landscapes that facilitates the interpretation of human tumour genome-wide sequencing data.
Asunto(s)
Mutación , Neoplasias/genética , Animales , Línea Celular Transformada , Biología Computacional , Análisis Mutacional de ADN/métodos , Exoma , Estudio de Asociación del Genoma Completo , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Técnicas In Vitro , RatonesRESUMEN
Exposure to herbal remedies containing the carcinogen aristolochic acid (AA) has been widespread in some regions of the world. Rare AâT TP53 mutations were recently discovered in AA-associated urothelial cancers. The near absence of these mutations among all other sequenced human tumors suggests that they could be biologically silent. There are no cell banks with established lines derived from human tumors with which to explore the influence of the novel mutants on p53 function and cellular behavior. To investigate their impact, we generated isogenic mutant clones by integrase-mediated cassette exchange at the p53 locus of platform (null) murine embryonic fibroblasts and kidney epithelial cells. Common tumor mutants (R248W, R273C) were compared with the AA-associated mutants N131Y, R249W, and Q104L. Assays of cell proliferation, migration, growth in soft agar, apoptosis, senescence, and gene expression revealed contrasting outcomes on cellular behavior following introduction of N131Y or Q104L. The N131Y mutant demonstrated a phenotype akin to common tumor mutants, whereas Q104L clone behavior resembled that of cells with wild-type p53. Wild-type p53 responses were restored in double-mutant cells harboring N131Y and N239Y, a second-site rescue mutation, suggesting that pharmaceutical reactivation of p53 function in tumors expressing N131Y could have therapeutic benefit. N131Y is likely to contribute directly to tumor phenotype and is a promising candidate biomarker of AA exposure and disease. Rare mutations thus do not necessarily point to sites where amino acid exchanges are phenotypically neutral. Encounter with mutagenic insults targeting cryptic sites can reveal specific signature hotspots.
Asunto(s)
Ácidos Aristolóquicos/efectos adversos , Mutágenos/efectos adversos , Mutación Missense , Preparaciones de Plantas/efectos adversos , Proteína p53 Supresora de Tumor/genética , Neoplasias Uretrales/inducido químicamente , Neoplasias Uretrales/genética , Sustitución de Aminoácidos , Animales , Ácidos Aristolóquicos/farmacología , Biomarcadores de Tumor , Línea Celular Transformada , Humanos , Enfermedad Iatrogénica , Ratones , Mutágenos/farmacología , Preparaciones de Plantas/farmacología , Proteína p53 Supresora de Tumor/metabolismo , Neoplasias Uretrales/metabolismo , Neoplasias Uretrales/patología , Urotelio/metabolismo , Urotelio/patologíaRESUMEN
Progression to malignancy requires that cells overcome senescence and switch to an immortal phenotype. Thus, exploring the genetic and epigenetic changes that occur during senescence/immortalization may help elucidate crucial events that lead to cell transformation. In the present study, we have globally profiled DNA methylation in relation to gene expression in primary, senescent and immortalized mouse embryonic fibroblasts. Using a high-resolution genome-wide mapping technique, followed by extensive locus-specific validation assays, we have identified 24 CpG islands that display significantly higher levels of CpG methylation in immortalized cell lines as compared to primary murine fibroblasts. Several of these hypermethylated CpG islands are associated with genes involved in the MEK-ERK pathway, one of the most frequently disrupted pathways in cancer. Approximately half of the hypermethylated targets are developmental regulators, and bind to the repressive Polycomb group (PcG) proteins, often in the context of bivalent chromatin in mouse embryonic stem cells. Because PcG-associated aberrant DNA methylation is a hallmark of several human malignancies, our methylation data suggest that epigenetic reprogramming of pluripotency genes may initiate cell immortalization. Consistent with methylome alterations, global gene expression analysis reveals that the vast majority of genes dysregulated during cell immortalization belongs to gene families that converge into the MEK-ERK pathway. Additionally, several dysregulated members of the MAP kinase network show concomitant hypermethylation of CpG islands. Unlocking alternative epigenetic routes for cell immortalization will be paramount for understanding crucial events leading to cell transformation. Unlike genetic alterations, epigenetic changes are reversible events, and as such, can be amenable to pharmacological interventions, which makes them appealing targets for cancer therapy when genetic approaches prove inadequate.
Asunto(s)
Proliferación Celular , Metilación de ADN , Epigénesis Genética , Animales , Línea Celular , Transformación Celular Neoplásica , Células Cultivadas , Senescencia Celular/genética , Islas de CpG , ADN/química , Perfilación de la Expresión Génica , Redes Reguladoras de Genes , Humanos , Sistema de Señalización de MAP Quinasas/genética , Ratones , Análisis de Secuencia por Matrices de Oligonucleótidos , Secuencias Repetitivas de Ácidos NucleicosRESUMEN
Despite a ban on the use of plants of the Aristolochia genus in herbal medicine, as they contain known carcinogens, urothelial carcinoma attributable to the use of this plant seems to be more common than previously thought.
Asunto(s)
Aristolochia/envenenamiento , Extractos Vegetales/envenenamiento , Plantas Medicinales/envenenamiento , Neoplasias Ureterales/etiología , Neoplasias Urológicas/etiología , HumanosRESUMEN
Recently, chronic Aristolochia poisoning was found responsible for the aetiology of Balkan endemic nephropathy (BEN) in Croatia, Serbia, and Bosnia, and diet was the likely route of exposure to aristolochic acid (AA). BEN, often associated with an increased incidence of upper urinary tract carcinoma (UUC), also affects residents of certain rural villages in Romania. AA is a nephrotoxin and human carcinogen that forms DNA adducts after metabolic activation, which induce characteristic TP53 mutations in urothelial tumours. Here we present the first evidence linking AA exposure to UUC in residents of an endemic region in the Romanian Mehedinti County. DNA was extracted from kidney and tumour tissue of seven patients who underwent nephroureterectomy for UUC and resided in BEN villages (endemic group). Five patients with UUC from nonendemic villages served as controls. AA-DNA adducts (7-(deoxyadenosin-N(6) -yl)-aristolactam I), established biomarkers of AA exposure, were identified by (32)P-postlabelling in renal DNA of six patients from the endemic group and in one of the nonendemic group (adduct levels ranged from 0.3 to 6.5 adducts per 10(8) nucleotides). Additionally, an A to T transversion in TP53, a base substitution characteristic of AA mutagenic activity was found in urothelial tumour DNA of one patient from the endemic group. Our results provide a molecular link to the cause of urothelial tumours in BEN regions of Romania indicating that AA is the common aetiological agent for BEN across its numerous geographical foci.
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Ácidos Aristolóquicos/toxicidad , Neoplasias de la Vejiga Urinaria/etiología , Anciano , Anciano de 80 o más Años , Nefropatía de los Balcanes , Aductos de ADN/efectos de los fármacos , Aductos de ADN/genética , Femenino , Humanos , Técnicas In Vitro , Riñón/efectos de los fármacos , Riñón/metabolismo , Masculino , Persona de Mediana Edad , Mutación/efectos de los fármacos , Mutación/genética , Rumanía , Proteína p53 Supresora de Tumor/genéticaRESUMEN
This protocol describes a rapid, precise method for generating sets of embryonic stem (ES) cells or mouse embryonic fibroblasts (MEFs) harboring point mutations in the p53 tumor suppressor gene (officially known as Trp53). The strategy uses cells from the Trp53 (p53-null) 'platform' mouse, which allows site-specific integration of plasmid DNA into the Trp53 locus. Simple PCR protocols identify correctly targeted clones and immunoblots verify re-expression of the protein. We also present protocol modifications needed for efficient recovery of MEF clones expressing p53 constructs that retain wild-type function, including growth at low (3%) oxygen and transient downregulation of p53 regulators to forestall cell senescence of primary MEFs. A library of cell lines expressing various p53 mutants derived from the same population of primary fibroblasts or platform ES cells can be acquired and screened in less than 1 month.
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Células Madre Fetales , Fibroblastos , Biología Molecular/métodos , Mutación , Proteína p53 Supresora de Tumor/genética , Animales , Secuencia de Bases , Ratones , Datos de Secuencia Molecular , Plásmidos/genética , Reacción en Cadena de la PolimerasaRESUMEN
Phospholipase A(2) enzymes hydrolyze phospholipids to liberate arachidonic acid for the biosynthesis of prostaglandins and leukotrienes. In the vascular endothelium, group IV phospholipase A(2)α (cPLA(2)α) enzyme activity is regulated by reversible association with the Golgi apparatus. Here we provide evidence for a plasma membrane cell adhesion complex that regulates endothelial cell confluence and simultaneously controls cPLA(2)α localization and enzymatic activity. Confluent endothelial cells display pronounced accumulation of vascular endothelial cadherin (VE-cadherin) at cell-cell junctions, and mechanical wounding of the monolayer stimulates VE-cadherin complex disassembly and cPLA(2)α release from the Golgi apparatus. VE-cadherin depletion inhibits both recruitment of cPLA(2)α to the Golgi and formation of tubules by endothelial cells. Perturbing VE-cadherin and increasing the soluble cPLA(2)α fraction also stimulated arachidonic acid and prostaglandin production. Of importance, reverse genetics shows that α-catenin and δ-catenin, but not ß-catenin, regulates cPLA(2)α Golgi localization linked to cell confluence. Furthermore, cPLA(2)α Golgi localization also required partitioning defective protein 3 (PAR3) and annexin A1. Disruption of F-actin internalizes VE-cadherin and releases cPLA(2)α from the adhesion complex and Golgi apparatus. Finally, depletion of either PAR3 or α-catenin promotes cPLA(2)α-dependent endothelial tubule formation. Thus a VE-cadherin-PAR3-α-catenin adhesion complex regulates cPLA(2)α recruitment to the Golgi apparatus, with functional consequences for vascular physiology.
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Antígenos CD/metabolismo , Cadherinas/metabolismo , Proteínas de Ciclo Celular/metabolismo , Aparato de Golgi/enzimología , Fosfolipasas A2 Grupo IV/metabolismo , Proteínas de la Membrana/metabolismo , alfa Catenina/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Antígenos CD/genética , Cadherinas/genética , Proteínas de Ciclo Celular/genética , Línea Celular , Células Endoteliales/citología , Células Endoteliales/enzimología , Humanos , Proteínas de la Membrana/genética , Genética Inversa , alfa Catenina/genéticaRESUMEN
Currently there are several dozen human polymorphisms that have been loosely associated with cancer risk. Correlating such variants with cancer risk has been challenging, primarily due to factors such as genetic heterogeneity, contributions of diet and environmental factors, and the difficulty in obtaining large sample sizes for analysis. Such difficulties can be circumvented with the establishment of mouse models for human variants. Recently, several groups have modeled human cancer susceptibility polymorphisms in the mouse. Remarkably, in each case these mouse models have accurately reflected human phenotypes, and clarified the contribution of these variants to cancer risk. We recently reported on a mouse model for the codon 72 polymorphism in p53, and found that this polymorphism regulates the ability to cooperate with NF-kB and induce apoptosis. Here-in we present evidence that this polymorphism impacts the apoptotic function of p53 in a tissue-specific manner; such tissue-specific effects of polymorphic variants represent an added challenge to human cancer risk association studies. The data presented here support the premise that modeling human polymorphisms in the mouse represents a powerful tool to assess the impact of these variants on cancer risk, progression and therapy.