RESUMEN
Oxaliplatin is widely used to treat colorectal cancer in both palliative and adjuvant settings. It is also being tested for use in treating hematological, esophageal, biliary tract, pancreatic, gastric, and hepatocellular cancers. Despite its routine clinical use, little is known about the responses it induces in cancer cells. Therefore the whole-cell proteomics study was conducted to characterize the cellular response induced by oxaliplatin. Chemosensitive CCRF-CEM cells were treated with oxaliplatin at 29.3µM (5×IC50) for 240min (half-time to caspase activation). The proteomes of un-/treated cells were then compared by high-resolution mass spectrometry, revealing 4049 proteins expressed over 3 biological replicates. Among these proteins, 76 were significantly downregulated and 31 significantly upregulated in at least two replicates. In agreement with the DNA-damaging effects of platinum drugs, proteins involved in DNA damage responses were present in both the upregulated and downregulated groups. The downregulated proteins were divided into three subgroups; i) centrosomal proteins, ii) RNA processing and iii) ribosomal proteins, which indicates nucleolar and ribosomal stress. In conclusion, our data supported by further validation experiments indicate the initial cellular response to oxaliplatin is the activation of DNA damage response, which in turn or in parallel triggers nucleolar and ribosomal stress. BIOLOGICAL SIGNIFICANCE: We have performed a whole-cell proteomic study of cellular response to oxaliplatin treatment, which is the drug predominantly used in the treatment of colorectal cancer. Compared to its predecessors, cisplatin and carboplatin, there is only a small fraction of studies dedicated to oxaliplatin. From those studies, most of them are focused on modification of treatment regimens or study of oxaliplatin in new cancer diagnoses. Cellular response hasn't been studied deeply and to our best knowledge, this is the first whole-cell proteomics study focused exclusively to this important topic, which can help to understand molecular mechanisms of action.
Asunto(s)
Nucléolo Celular/efectos de los fármacos , Daño del ADN , Neoplasias/tratamiento farmacológico , Compuestos Organoplatinos/farmacología , Proteoma/efectos de los fármacos , Ribosomas/efectos de los fármacos , Antineoplásicos/farmacología , Perfilación de la Expresión Génica , Humanos , Neoplasias/patología , Oxaliplatino , Proteoma/análisis , Proteoma/metabolismo , Proteómica/métodos , Estrés Fisiológico , Células Tumorales CultivadasRESUMEN
Quercetin and phenylpropanoids are well known chemoprotective compounds identified in many plants. This study was aimed at determining their effects on activation of Nuclear factor erythroid 2-related factor 2 (Nrf2) antioxidant response element (Nrf2-ARE) signalling pathway and expression of its important downstream effector phase II detoxification enzyme glutathione-S-transferase P1 (GSTP1) in BJ foreskin fibroblasts and skin HaCaT keratinocytes. Cell lines and their corresponding Nrf2-ARE luciferase reporter cells were treated by ginger phenylpropanoids and quercetin for 10 h and the level of Nrf2 activity was subsequently determined. Both, ginger phenylpropanoids and quercetin, significantly increased the level of Nrf2 activity. Subsequent western blot analyses of proteins showed the increased expression level of glutathione-S-transferase P1 (GSTP1) in BJ cells but not in HaCaT cells. Such phenomenon of unresponsive downstream target expression in HaCaT cells was consistent with previous studies showing a constitutive expression of their GSTP1. Thus, while both ginger phenylpropanoids and quercetin have the property of increasing the level of Nrf2 both in HaCaT and in BJ cells, their effects on its downstream signalling were mediated only in BJ cells.
Asunto(s)
Antioxidantes/administración & dosificación , Gutatión-S-Transferasa pi/biosíntesis , Factor 2 Relacionado con NF-E2/biosíntesis , Quercetina/administración & dosificación , Antioxidantes/química , Línea Celular , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Zingiber officinale/química , Gutatión-S-Transferasa pi/genética , Glutatión Transferasa , Humanos , Queratinocitos/efectos de los fármacos , Queratinocitos/metabolismo , Factor 2 Relacionado con NF-E2/genética , Transducción de Señal/efectos de los fármacosRESUMEN
Eligibility to anti-HER2 therapy for breast tumors strictly depends on demonstrating HER2 overexpression (by immunohistochemistry) or HER2 gene amplification by in situ hybridization (ISH), usually defined by the ratio of HER2 gene to chromosome 17 centromere (CEP17) copies. However, the CEP17 copy number increase (CNI) has been proven responsible for misleading HER2 FISH results and recent small cohort studies suggest that chromosome 17 polysomy is actually very rare. Here we investigated by FISH the frequency of true chromosome 17 polysomy in a consecutive cohort of 5,477 invasive breast cancer patients. We evaluated and selected the LSI 17p11.2 probe for chromosome 17 enumeration on a training cohort of 67 breast cancer samples (CEP17 ≥ 2.5). LSI 17p11.2 was used in the 297/5,477 patients from the validation cohort displaying CEP17 CNI (CEP17 ≥ 3.0). Using HER2/17p11.2 scoring criteria, 37.3%/1.5% patients initially classified as equivocal/non-amplified were reclassified as amplified. For a more accurate assessment of chromosome 17 and ploidy in the samples, we tested six markers located on chromosome 17 and centromeric regions of chromosome 8 (CEP8) and 11 (CEP11) in 67 patients with CEP17 and LSI 17p11.2 CNI. True polysomy (hyperdiploidy) according to these markers was found in 0.48% of cases (24/5,020). CEP8 and CEP11 CNI (≥3.0) was more frequent in the hyperdiploid than CEP17 non-polysomic group (55.6% vs. 6.1% and 25% vs. 2.3%, respectively). Our results suggest that chromosome 17 polysomy is a rare event found in <1% breast cancer cases and that polysomy of other chromosomes frequently occurs with chromosome 17 polysomy.
Asunto(s)
Neoplasias de la Mama/genética , Cromosomas Humanos Par 17 , Variaciones en el Número de Copia de ADN , Estudios de Cohortes , Femenino , Humanos , Hibridación Fluorescente in SituRESUMEN
Target discovery using the molecular approach, as opposed to the more traditional systems approach requires the study of the cellular or biological process underlying a condition or disease. The approaches that are employed by the "bench" scientist may be genetic, genomic or proteomic and each has its rightful place in the drug-target discovery process. Affinity-based proteomic techniques currently used in drug-discovery draw upon several disciplines, synthetic chemistry, cell-biology, biochemistry and mass spectrometry. An important component of such techniques is the probe that is specifically designed to pick out a protein or set of proteins from amongst the varied thousands in a cell lysate. A second component, that is just as important, is liquid-chromatography tandem massspectrometry (LC-MS/MS). LC-MS/MS and the supporting theoretical framework has come of age and is the tool of choice for protein identification and quantification. These proteomic tools are critical to maintaining the drug-candidate supply, in the larger context of drug discovery.
Asunto(s)
Descubrimiento de Drogas/métodos , Proteómica/métodos , Cromatografía Liquida/métodos , Humanos , Proteínas/química , Proteínas/metabolismo , Espectrometría de Masas en Tándem/métodosRESUMEN
Mutant p53 (mtp53) promotes chemotherapy resistance through multiple mechanisms, including disabling proapoptotic proteins and regulating gene expression. Comparison of genome wide analysis of mtp53 binding revealed that the ETS-binding site motif (EBS) is prevalent within predicted mtp53-binding sites. We demonstrate that mtp53 regulates gene expression through EBS in promoters and that ETS2 mediates the interaction with this motif. Importantly, we identified TDP2, a 5'-tyrosyl DNA phosphodiesterase involved in the repair of DNA damage caused by etoposide, as a transcriptional target of mtp53. We demonstrate that suppression of TDP2 sensitizes mtp53-expressing cells to etoposide and that mtp53 and TDP2 are frequently overexpressed in human lung cancer; thus, our analysis identifies a potentially "druggable" component of mtp53's gain-of-function activity.
Asunto(s)
Antineoplásicos Fitogénicos/farmacología , Resistencia a Antineoplásicos , Etopósido/farmacología , Neoplasias Pulmonares/metabolismo , Proteína Proto-Oncogénica c-ets-2/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Línea Celular Tumoral , Proteínas de Unión al ADN , Humanos , Neoplasias Pulmonares/genética , Mutación , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Hidrolasas Diéster Fosfóricas , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteína p53 Supresora de Tumor/genéticaRESUMEN
The E2F family of transcription factors responds to a variety of intracellular and extracellular signals and, as such, are key regulators of cell growth, differentiation and cell death. The cellular response to DNA damage is a multistep process generally involving the initial detection of DNA damage, propagation of signals via posttranslational modifications (e.g., phosphorylation and ubiquitination) and, finally, the implementation of a response. We have previously reported that E2F3 can be induced by DNA damage, and that it plays an important role in DNA damage-induced apoptosis. Here, we demonstrate that E2F3 knockdown compromises two canonical DNA damage modification events, the ubiquitination of H2AX and PCNA. We find that the defect in these posttranscriptional modifications after E2F3 knockdown is due to reduced expression of important DNA damage responsive ubiquitin ligases. We characterized the regulation of one of these ligases, Rad18, and we demonstrated that E2F3 associates with the Rad18 promoter and directly controls its activity. Furthermore, we find that ectopic expression of Rad18 is sufficient to rescue the PCNA ubiquitination defect resulting from E2F3 knockdown. Our study reveals a novel facet of E2F3's control of the DNA damage response.
Asunto(s)
Daño del ADN , Proteínas de Unión al ADN/metabolismo , Factor de Transcripción E2F3/metabolismo , Regulación Neoplásica de la Expresión Génica , Apoptosis , Línea Celular Tumoral , Cisplatino/farmacología , Proteínas de Unión al ADN/genética , Factor de Transcripción E2F1/genética , Factor de Transcripción E2F1/metabolismo , Factor de Transcripción E2F3/genética , Puntos de Control de la Fase G1 del Ciclo Celular , Perfilación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Histonas/genética , Histonas/metabolismo , Humanos , Fosforilación , Antígeno Nuclear de Célula en Proliferación/genética , Antígeno Nuclear de Célula en Proliferación/metabolismo , Regiones Promotoras Genéticas , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Transfección , Ubiquitina-Proteína Ligasas , UbiquitinaciónRESUMEN
We review studies of subunit III-depleted cytochrome c oxidase (CcO III (-)) that elucidate the structural basis of steady-state proton uptake from solvent into an internal proton transfer pathway. The removal of subunit III from R. sphaeroides CcO makes proton uptake into the D pathway a rate-determining step, such that measurements of the pH dependence of steady-state O(2) consumption can be used to compare the rate and functional pK(a) of proton uptake by D pathways containing different initial proton acceptors. The removal of subunit III also promotes spontaneous suicide inactivation by CcO, greatly shortening its catalytic lifespan. Because the probability of suicide inactivation is controlled by the rate at which the D pathway delivers protons to the active site, measurements of catalytic lifespan provide a second method to compare the relative efficacy of proton uptake by engineered CcO III (-) forms. These simple experimental systems have been used to explore general questions of proton uptake by proteins, such as the functional value of an initial proton acceptor, whether an initial acceptor must be surface-exposed, which side chains will function as initial proton acceptors and whether multiple acceptors can speed proton uptake.
Asunto(s)
Proteínas Bacterianas/metabolismo , Complejo IV de Transporte de Electrones/metabolismo , Protones , Rhodobacter sphaeroides/enzimología , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Sitios de Unión/genética , Biocatálisis , Transporte Biológico/genética , Complejo IV de Transporte de Electrones/química , Complejo IV de Transporte de Electrones/genética , Concentración de Iones de Hidrógeno , Mutación , Subunidades de Proteína/química , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Rhodobacter sphaeroides/genética , Rhodobacter sphaeroides/metabolismoRESUMEN
To characterize protein structures that control proton uptake, we assayed forms of cytochrome c oxidase (CcO) containing a carboxyl or a thiol group in line with the initial, internal waters of the D pathway for proton transfer in the presence and absence of subunit III. Subunit III provides approximately half of the protein surrounding the entry region of the D pathway. The N139D/D132N mutant contains a carboxyl group 6 Å within the D pathway and lacks the normal, surface-exposed proton acceptor, Asp-132. With subunit III, the steady-state activity of this mutant is slow, but once subunit III is removed, its activity is the same as that of wild-type CcO lacking subunit III (â¼1800 H+/s). Thus, a carboxyl groupâ¼25% within the pathway enhances proton uptake even though the carboxyl has no direct contact with bulk solvent. Protons from solvent apparently move to internal Asp-139 through a short file of waters, normally blocked by subunit III. Cys-139 also supports rapid steady-state proton uptake, demonstrating that an anion other than a carboxyl can attract and transfer protons into the D pathway. When both Asp-132 and Asp/Cys-139 are present, the removal of subunit III increases CcO activity to rates greater than that of normal CcO because of simultaneous proton uptake by two initial acceptors. The results show how the environment of the initial proton acceptor for the D pathway in these CcO forms dictates the pH range of CcO activity, with implications for the function of Asp-132, the normal proton acceptor.
Asunto(s)
Proteínas Bacterianas/química , Complejo IV de Transporte de Electrones/química , Protones , Rhodobacter sphaeroides/enzimología , Ácido Aspártico/química , Ácido Aspártico/genética , Ácido Aspártico/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sitios de Unión/genética , Biocatálisis , Complejo IV de Transporte de Electrones/genética , Complejo IV de Transporte de Electrones/metabolismo , Concentración de Iones de Hidrógeno , Modelos Moleculares , Mutación , Subunidades de Proteína/química , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Rhodobacter sphaeroides/genéticaRESUMEN
Subunit III of the three-subunit catalytic core of cytochrome c oxidase (CcO) contains no metal centers, but it does bind two lipids, within a deep cleft, in binding sites conserved from bacteria to humans. Subunit III binds to subunit I, where it prevents the spontaneous suicide inactivation of CcO by decreasing the probability of side reactions at the heme-Cu O2 reduction site in subunit I. Subunit III prevents suicide inactivation by (1) maintaining adequate rates of proton delivery to the heme-Cu active site and (2) stabilizing the structure of the active site during turnover [Mills and Hosler (2005) Biochemistry 44, 4656]. Here, we first show that mutating several individual residues of the conserved lipid binding sites in subunit III disturbs the subunit I-III interface. Then, two lipid binding site mutants were constructed with an affinity tag on subunit III such that the mutant CcOs could be isolated with 100% subunit III. R226A eliminates an ion pair to the phosphate of the outermost lipid of the cleft, while W59A-F86A disrupts interactions with the fatty acid tails of both lipids. Once these mutant CcOs are placed into soybean phospholipid vesicles, where extensive exchange of bacterial for soybean lipids takes place, it is shown that altering the lipid binding sites mimics a major loss of subunit III, even though subunit III is completely retained, in that suicide inactivation becomes much more probable. The rate of proton delivery to the active site remains rapid, ruling out slow proton uptake as the primary reason for increased suicide inactivation upon alteration of the lipid binding sites. We conclude that altering the lipid binding sites of subunit III may promote side reactions leading to suicide inactivation by allowing greater movement to occur in and around the O2 reduction site of subunit I during the catalytic cycle.