RESUMEN
BACKGROUND: HER2-targeted therapies have revolutionised the treatment of HER2-positive breast cancer. However, de novo resistance or the emergence of acquired resistance is a persistent clinical problem. Here we report that neratinib, an irreversible pan-HER inhibitor, in combination with the multi-kinase inhibitor dasatinib, currently used to treat certain leukemias, has strong anti-proliferative effects against models of HER2-positive breast cancer that are innately resistant to trastuzumab or have acquired resistance to neratinib. METHODS: Neratinib plus dasatinib was examined in a panel of 20 breast cancer cell lines, including HER2-positive, estrogen-receptor-positive, triple negative, and acquired HER2-targeted therapy resistant models. Drug effects on migration and apoptosis induction was evaluated and signaling alterations were determined by reverse phase protein array (RPPA). In vivo efficacy was examined using orthotopically-implanted HCC1954 cells. RESULTS: Synergy was observed in cell lines innately resistant to trastuzumab, models with acquired resistance to neratinib, and in triple negative breast cancer cell lines. Further investigation showed that neratinib plus dasatinib induced apoptosis and inhibited cell migration to a greater degree than either drug alone. RPPA revealed that the combination caused suppression of key survival signaling through EGFR, Akt, and MAPK inhibition. In vivo, neratinib plus dasatinib was well tolerated and had a prolonged anti-tumor effect against HCC1954 xenografts. CONCLUSIONS: This study provides a strong pre-clinical rationale for the clinical investigation neratinib and dasatinib in HER2+ breast cancer.
RESUMEN
Reliable monitoring of mammalian cells in bioreactors is essential to biopharmaceutical production. Trypan blue exclusion is a method of determining cell density and viability that has been used for over one hundred years to monitor cells in culture and is the current standard method in biomanufacturing. This method has many disadvantages however and there is a growing demand for more detailed and in-line measurements of cell growth in bioreactors. This article assesses a novel dynamic imaging system for single cell analysis. This data shows that comparable total cell density, viable cell density and percentage viability data shown here, generated by the imaging system, aligned well with conventional trypan blue counting methods for an industrially relevant Chinese Hamster Ovary (CHO) cell line. Furthermore, detailed statistical analysis shows that the classification system used by the PharmaFlow system can reveal trends of interest in monitoring the health of mammalian cells over a 6-day bioreactor culture. The system is also capable of sampling at-line, removing the necessity for taking samples off-line and enabling real time monitoring of cells in a bioreactor culture.
RESUMEN
Foam is generated in mammalian cell cultures by excessive agitation or gas sparging. This occurs particularly in cultures that generate recombinant proteins at high cell concentrations. Three antifoam agents were tested for their compatibility with antibody-producing Chinese hamster ovary (CHO) cells. One agent (antifoam 204) was completely inhibitory to growth at a concentration of 10 ppm, one agent (antifoam C) showed partial inhibition and a third (antifoam SE-15) showed no inhibition at this concentration. A novel foam image analyzer (LabCam) was used to evaluate two antifoams (C and SE-15) for their ability to dissipate foam generated in cell culture media by enhanced agitation. The presence of antifoam in the media reduced significantly the foam layer that was generated and this was shown to be rapidly dissipated in the presence of 10 ppm SE-15. The antifoams were also tested for foam dissipation in cultures of CHO cells at >106 cells/mL. Supplementation of the cultures with SE-15 resulted in dissipation of foam generated by excessive gas sparging within 2 min. Under equivalent conditions 75% of foam dissipated in the presence of antifoam C, within 2 min but there was a residual foam layer up to 25 min. This study showed the value of an optical monitoring system (LabCam) for measuring foam generation and dissipation in a bioreactor to assess the efficiency of antifoam agents to reduce foam in a bioreactor. This has the potential for use as a control system that could be designed for continuous monitoring and foam control in a mammalian cell bioprocess.
Asunto(s)
Técnicas de Cultivo de Célula , Cricetulus , Células CHO , Animales , Técnicas de Cultivo de Célula/métodos , Cricetinae , Medios de Cultivo/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/biosíntesisRESUMEN
Introduction Triple negative breast cancer (TNBC) represents a heterogeneous subtype of breast cancer that carries a poorer prognosis. There remains a need to identify novel drivers of TNBC, which may represent targets to treat the disease. c-Met overexpression is linked with decreased survival and is associated with the basal subtype of breast cancer. Cpd A, a kinase inhibitor selective/specific for Met kinase has demonstrated preclinical anti-cancer efficacy in TNBC. We aimed to assess the anti-cancer efficacy of Cpd A when combined with Src kinase, ErbB-family or hepatocyte growth factor (HGF) inhibitors in TNBC cell lines. Methods We determined the anti-proliferative effects of Cpd A, rilotumumab, neratinib and saracatinib tested alone and in combination in a panel of TNBC cells by acid phosphatase assays. We performed reverse phase protein array analysis of c-Met and IGF1Rß expression and phosphorylation of c-Met (Y1234/1235) in TNBC cells and correlated their expression/phosphorylation with Cpd A sensitivity. We examined the impact of Cpd A, neratinib and saracatinib tested alone and in combination on invasive potential and colony formation.Results TNBC cells are not inherently sensitive to Cpd A, and neither c-Met expression nor phosphorylation are biomarkers of sensitivity to Cpd A. Cpd A enhanced the anti-proliferative effects of neratinib in vitro; however, this effect was limited to cell lines with innate sensitivity to Cpd A. Cpd A had limited anti-invasive effects but it reduced colony formation in the TNBC cell line panel.Conclusions Despite Cpd A having a potential role in reducing cancer cell metastasis, identification of strong predictive biomarkers of c-Met sensitivity would be essential to the development of a c-Met targeted treatment for an appropriately selected cohort of TNBC patients.
Asunto(s)
Antineoplásicos/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-met/antagonistas & inhibidores , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Fosfatasa Ácida/metabolismo , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Femenino , Humanos , Proteínas Proto-Oncogénicas c-met/metabolismo , Neoplasias de la Mama Triple Negativas/metabolismoRESUMEN
KH-type splicing regulatory protein (KHSRP) is a multifunctional nucleic acid binding protein implicated in key aspects of cancer cell biology: inflammation and cell-fate determination. However, the role KHSRP plays in colorectal cancer (CRC) tumorigenesis remains largely unknown. Using a combination of in silico analysis of large data sets, ex vivo analysis of protein expression in patients, and mechanistic studies using in vitro models of CRC, we investigated the oncogenic role of KHSRP. We demonstrated KHSRP expression in the epithelial and stromal compartments of both primary and metastatic tumors. Elevated expression was found in tumor versus matched normal tissue, and these findings were validated in larger independent cohorts in silico. KHSRP expression was a prognostic indicator of worse overall survival (hazard ratio, 3.74; 95% CI, 1.43-22.97; P = 0.0138). Mechanistic data in CRC cell line models supported a role of KHSRP in driving epithelial cell proliferation in both a primary and metastatic setting, through control of the G1/S transition. In addition, KHSRP promoted a proangiogenic extracellular environment by regulating the secretion of oncogenic proteins involved in diverse cellular processes, such as migration and response to cellular stress. Our study provides novel mechanistic insight into the tumor-promoting effects of KHSRP in CRC.
Asunto(s)
Biomarcadores de Tumor/metabolismo , Proliferación Celular , Neoplasias Colorrectales/patología , Regulación Neoplásica de la Expresión Génica , Proteínas de Unión al ARN/metabolismo , Transactivadores/metabolismo , Microambiente Tumoral , Adulto , Anciano , Anciano de 80 o más Años , Apoptosis , Biomarcadores de Tumor/genética , Transformación Celular Neoplásica , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/metabolismo , Femenino , Estudios de Seguimiento , Humanos , Masculino , Persona de Mediana Edad , Pronóstico , Proteínas de Unión al ARN/genética , Tasa de Supervivencia , Transactivadores/genética , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
SCOPE: Copper supplementation in nutrition has evolved from using inorganic mineral salts to organically chelated minerals but with limited knowledge of the impact at the cellular level. METHODS: Here, the impact of inorganic and organic nutrient forms (glycinate, organic acid, and proteinate) of copper on the cellular level is investigated on intestinal cell lines, HT29 and Caco-2, after a 2-hr acute exposure to copper compounds and following a 10-hr recovery. RESULTS: Following the 10-hr recovery, increases were observed in proteins involved in metal binding (metallothioneins) and antioxidant response (sulfiredoxin 1 and heme oxygenase 1), and global proteomic analysis suggested recruitment of the unfolded protein response and proteosomal overloading. Copper organic acid chelate, the only treatment to show striking and sustained reactive oxygen species generation, had the greatest impact on ubiquitinated proteins, reduced autophagy, and increased aggresome formation, reducing growth in both cell lines. The least effect was noted in copper proteinate with negligible impact on aggresome formation or extended growth for either cell line. CONCLUSION: The type and source of copper can impact significantly at the cellular level.
RESUMEN
AIM: To identify miRNA-regulated proteins differentially expressed between Caco2 and HT-29: two principal cell line models of the intestine. METHODS: Exponentially growing Caco-2 and HT-29 cells were harvested and prepared for mRNA, miRNA and proteomic profiling. mRNA microarray profiling analysis was carried out using the Affymetrix GeneChip Human Gene 1.0 ST array. miRNA microarray profiling analysis was carried out using the Affymetrix Genechip miRNA 3.0 array. Quantitative Label-free LC-MS/MS proteomic analysis was performed using a Dionex Ultimate 3000 RSLCnano system coupled to a hybrid linear ion trap/Orbitrap mass spectrometer. Peptide identities were validated in Proteome Discoverer 2.1 and were subsequently imported into Progenesis QI software for further analysis. Hierarchical cluster analysis for all three parallel datasets (miRNA, proteomics, mRNA) was conducted in the R software environment using the Euclidean distance measure and Ward's clustering algorithm. The prediction of miRNA and oppositely correlated protein/mRNA interactions was performed using TargetScan 6.1. GO biological process, molecular function and cellular component enrichment analysis was carried out for the DE miRNA, protein and mRNA lists via the Pathway Studio 11.3 Web interface using their Mammalian database. RESULTS: Differential expression (DE) profiling comparing the intestinal cell lines HT-29 and Caco-2 identified 1795 Genes, 168 Proteins and 160 miRNAs as DE between the two cell lines. At the gene level, 1084 genes were upregulated and 711 were downregulated in the Caco-2 cell line relative to the HT-29 cell line. At the protein level, 57 proteins were found to be upregulated and 111 downregulated in the Caco-2 cell line relative to the HT-29 cell line. Finally, at the miRNAs level, 104 were upregulated and 56 downregulated in the Caco-2 cell line relative to the HT-29 cell line. Gene ontology (GO) analysis of the DE mRNA identified cell adhesion, migration and ECM organization, cellular lipid and cholesterol metabolic processes, small molecule transport and a range of responses to external stimuli, while similar analysis of the DE protein list identified gene expression/transcription, epigenetic mechanisms, DNA replication, differentiation and translation ontology categories. The DE protein and gene lists were found to share 15 biological processes including for example epithelial cell differentiation [P value ≤ 1.81613E-08 (protein list); P ≤ 0.000434311 (gene list)] and actin filament bundle assembly [P value ≤ 0.001582797 (protein list); P ≤ 0.002733714 (gene list)]. Analysis was conducted on the three data streams acquired in parallel to identify targets undergoing potential miRNA translational repression identified 34 proteins, whose respective mRNAs were detected but no change in expression was observed. Of these 34 proteins, 27 proteins downregulated in the Caco-2 cell line relative to the HT-29 cell line and predicted to be targeted by 19 unique anti-correlated/upregulated microRNAs and 7 proteins upregulated in the Caco-2 cell line relative to the HT-29 cell line and predicted to be targeted by 15 unique anti-correlated/downregulated microRNAs. CONCLUSION: This first study providing "tri-omics" analysis of the principal intestinal cell line models Caco-2 and HT-29 has identified 34 proteins potentially undergoing miRNA translational repression.
Asunto(s)
Perfilación de la Expresión Génica/métodos , Mucosa Intestinal/metabolismo , Proteómica/métodos , Células CACO-2 , Biología Computacional , Conjuntos de Datos como Asunto , Regulación hacia Abajo , Células HT29 , Humanos , Intestinos/citología , MicroARNs/metabolismo , Análisis por Micromatrices/métodos , Proteoma/genética , Proteoma/metabolismo , ARN Mensajero/metabolismo , Programas Informáticos , Espectrometría de Masas en Tándem , Regulación hacia ArribaRESUMEN
AIMS/HYPOTHESIS: Physical inactivity has broad implications for human disease including insulin resistance, sarcopenia and obesity. The present study tested the hypothesis that (1) impaired mitochondrial respiration is linked with blunted insulin sensitivity and loss of muscle mass in healthy young men, and (2) resistive vibration exercise (RVE) would mitigate the negative metabolic effects of bed rest. METHODS: Participants (n = 9) were maintained in energy balance during 21 days of bed rest with RVE and without (CON) in a crossover study. Mitochondrial respiration was determined by high-resolution respirometry in permeabilised fibre bundles from biopsies of the vastus lateralis. A hyperinsulinaemic-euglycaemic clamp was used to determine insulin sensitivity, and body composition was assessed by dual-energy x-ray absorptiometry (DEXA). RESULTS: Body mass (-3.2 ± 0.5 kg vs -2.8 ± 0.4 kg for CON and RVE, respectively, p < 0.05), fat-free mass (-2.9 ± 0.5 kg vs -2.7 ± 0.5 kg, p < 0.05) and peak oxygen consumption ([Formula: see text]) (10-15%, p < 0.05) were all reduced following bed rest. Bed rest decreased insulin sensitivity in the CON group (0.04 ± 0.002 mg kgFFM-1 [pmol l-1] min-1 vs 0.03 ± 0.002 mg kgFFM-1 [pmol l-1] min-1 for baseline vs post-CON), while RVE mitigated this response (0.04 ± 0.003 mg kgFFM-1 [pmol l-1] min-1). Mitochondrial respiration (oxidative phosphorylation and electron transport system capacity) decreased in the CON group but not in the RVE group when expressed relative to tissue weight but not when normalised for citrate synthase activity. LEAK respiration, indicating a decrease in mitochondrial uncoupling, was the only component to remain significantly lower in the CON group after normalisation for citrate synthase. This was accompanied by a significant decrease in adenine nucleotide translocase protein content. CONCLUSIONS/INTERPRETATION: Reductions in muscle mitochondrial respiration occur concomitantly with insulin resistance and loss of muscle mass during bed rest and may play a role in the adaptations to physical inactivity. Significantly, we show that RVE is an effective strategy to partially prevent some of the deleterious metabolic effects of bed rest.
Asunto(s)
Reposo en Cama , Resistencia a la Insulina/fisiología , Mitocondrias/metabolismo , Músculo Esquelético/fisiología , Absorciometría de Fotón , Adulto , Composición Corporal/fisiología , Estudios Cruzados , Metabolismo Energético/fisiología , Técnica de Clampeo de la Glucosa , Humanos , MasculinoRESUMEN
microRNA engineering of CHO cells has already proved successful in enhancing various industrially relevant phenotypes and producing various recombinant products. A single miRNA's ability to interact with multiple mRNA targets allows their regulatory capacity to extend to processes such as cellular metabolism. Various metabolic states have previously been associated with particular CHO cell phenotypes such as glycolytic or oxidative metabolism accommodating growth and productivity, respectively. miR-23 has previously been demonstrated to play a role in glutamate metabolism resulting in enhanced oxidative phosphorylation through the TCA cycle. Re-programming cellular bioenergetics through miR-23 could tip the balance, forcing mammalian production cells to be more productive by favoring metabolic channelling into oxidative metabolism. CHO clones depleted of miR-23 using a miR-sponge decoy demonstrated an average â¼three-fold enhanced specific productivity with no impact on cell growth. Using a cell respirometer, mitochondrial activity was found to be enhanced by â¼30% at Complex I and II of the electron transport system. Additionally, label-free proteomic analysis uncovered various potential novel targets of miR-23 including LE1 and IDH1, both implicated in oxidative metabolism and mitochondrial activity. These results demonstrate miRNA-based engineering as a route to re-programming cellular metabolism resulting in increased productivity, without affecting growth.
Asunto(s)
Fosfatasa Alcalina/genética , Células CHO , Ingeniería Celular , MicroARNs/metabolismo , Proteómica , Fosfatasa Alcalina/metabolismo , Animales , Proliferación Celular/genética , Cricetinae , Cricetulus , Ácido Glutámico/metabolismo , Humanos , Isocitrato Deshidrogenasa/genética , Isocitrato Deshidrogenasa/metabolismo , MicroARNs/genética , Mitocondrias/genética , Mitocondrias/metabolismo , Fenotipo , Estabilidad del ARN/genética , ARN Mensajero/genéticaRESUMEN
The development of a drug-resistant cell line can take from 3 to 18 months. However, little is published on the methodology of this development process. This article will discuss key decisions to be made prior to starting resistant cell line development; the choice of parent cell line, dose of selecting agent, treatment interval, and optimizing the dose of drug for the parent cell line. Clinically relevant drug-resistant cell lines are developed by mimicking the conditions cancer patients experience during chemotherapy and cell lines display between two- and eight-fold resistance compared to their parental cell line. Doses of drug administered are low, and a pulsed treatment strategy is often used where the cells recover in drug-free media. High-level laboratory models are developed with the aim of understanding potential mechanisms of resistance to chemotherapy agents. Doses of drug are higher and escalated over time. It is common to have difficulty developing stable clinically relevant drug-resistant cell lines. A comparative selection strategy of multiple cell lines or multiple chemotherapeutic agents mitigates this risk and gives insight into which agents or type of cell line develops resistance easily. Successful selection strategies from our research are presented. Pulsed-selection produced platinum or taxane-resistant large cell lung cancer (H1299 and H460) and temozolomide-resistant melanoma (Malme-3M and HT144) cell lines. Continuous selection produced a lapatinib-resistant breast cancer cell line (HCC1954). Techniques for maintaining drug-resistant cell lines are outlined including; maintaining cells with chemotherapy, pulse treating with chemotherapy, or returning to master drug-resistant stocks. The heterogeneity of drug-resistant models produced from the same parent cell line with the same chemotherapy agent is explored with reference to P-glycoprotein. Heterogeneity in drug-resistant cell lines reflects the heterogeneity that can occur in clinical drug resistance.
RESUMEN
The selection of clones displaying a high rate of cell growth is an essential component of Chinese hamster ovary (CHO) cell line development. In recent years various "omics" technologies have been utilised to understand the mechanisms underlying bioprocess phenotypes. In this study, gene expression analysis using a CHO-specific microarray was conducted for a panel of CHO-K1 MAb-secreting cell lines spanning a range of growth rates that were derived from a single cell line development project. In-silico functional analysis of the resulting transcriptomic data revealed the overrepresentation of biological processes such as cell cycle and translation within those genes upregulated during fast growth, while genes associated with cellular homeostasis were downregulated. Using differential expression and correlation analysis we identified a high priority group of 416 transcripts (190 upregulated; 226 downregulated) associated with growth rate. Expression changes of eight of these genes were independently confirmed by qPCR. Finally, we demonstrate the enrichment of predicted mRNA targets of miR17-92, a microRNA (miRNA) cluster known to be upregulated during rapid proliferation, within downregulated transcripts.
Asunto(s)
Células CHO , Ciclo Celular/genética , Proliferación Celular , Perfilación de la Expresión Génica , MicroARNs/genética , Animales , Supervivencia Celular , Cricetinae , Cricetulus , Regulación hacia Abajo , MicroARNs/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Regulación hacia ArribaRESUMEN
BACKGROUND: To study the role of microRNA (miRNA) in the regulation of Chinese hamster ovary (CHO) cell growth, qPCR, microarray and quantitative LC-MS/MS analysis were utilised for simultaneous expression profiling of miRNA, mRNA and protein. The sample set under investigation consisted of clones with variable cellular growth rates derived from the same population. In addition to providing a systems level perspective on cell growth, the integration of multiple profiling datasets can facilitate the identification of non-seed miRNA targets, complement computational prediction tools and reduce false positive and false negative rates. RESULTS: 51 miRNAs were associated with increased growth rate (35 miRNAs upregulated and 16 miRNAs downregulated). Gene ontology (GO) analysis of genes (n=432) and proteins (n=285) found to be differentially expressed (DE) identified biological processes driving proliferation including mRNA processing and translation. To investigate the influence of miRNA on these processes we combined the proteomic and transcriptomic data into two groups. The first set contained candidates where evidence of translational repression was observed (n=158). The second group was a mixture of proteins and mRNAs where evidence of translational repression was less clear (n=515). The TargetScan algorithm was utilised to predict potential targets within these two groups for anti-correlated DE miRNAs. CONCLUSIONS: The evidence presented in this study indicates that biological processes such as mRNA processing and protein synthesis are correlated with growth rate in CHO cells. Through the integration of expression data from multiple levels of the biological system a number of proteins central to these processes including several hnRNPs and components of the ribosome were found to be post-transcriptionally regulated. We utilised the expression data in conjunction with in-silico tools to identify potential miRNA-mediated regulation of mRNA/proteins involved in CHO cell growth rate. These data have allowed us to prioritise candidates for cell engineering and/or biomarkers relevant to industrial cell culture. We also expect the knowledge gained from this study to be applicable to other fields investigating the role of miRNAs in mammalian cell growth.
Asunto(s)
Células CHO/metabolismo , Células CHO/fisiología , Proliferación Celular , MicroARNs/metabolismo , Proteínas/metabolismo , Procesamiento Postranscripcional del ARN/fisiología , ARN Mensajero/metabolismo , Animales , Cromatografía Liquida , Cricetinae , Cricetulus , Análisis por Micromatrices , Reacción en Cadena de la Polimerasa , Proteómica , Espectrometría de Masas en TándemRESUMEN
Clonal variants or subpopulations have been isolated from every major histological type of cancer, and cellular heterogeneity in lung cancer is a common occurrence. These subpopulations may exhibit differences in drug resistance and invasive potential. One therefore needs to consider the subpopulations as well as the tumour to overcome the barriers of drug resistance and metastasis for successful treatment. Isogenic variants of cancer cell lines can be very valuable in providing controlled human experimental systems to study clinically relevant parameters such as drug resistance and invasiveness. These variants can be established by selection based on a characteristic of the subpopulation or by isolating clonal subpopulations from a heterogeneous population. Drug-resistant variants can be generated by pulse selection, which usually generates low-level resistance, which may as well be clinically relevant, or by continuous exposure, which can be used to obtain high-level resistant variants. Clonal subpopulations may also be isolated based on morphological differences using simple cell-culture-based techniques.
Asunto(s)
Antineoplásicos/farmacología , Línea Celular Tumoral/efectos de los fármacos , Línea Celular Tumoral/patología , Separación Celular/métodos , Neoplasias Pulmonares/patología , Células Clonales/efectos de los fármacos , Células Clonales/patología , Relación Dosis-Respuesta a Droga , Resistencia a Antineoplásicos , Humanos , Concentración 50 Inhibidora , Invasividad NeoplásicaRESUMEN
Chemotherapy drug resistance is a major obstacle in the treatment of cancer. It can result from an increase in levels of cellular drug efflux pumps, such as P-glycoprotein (P-gp). Lapatinib, a growth factor receptor tyrosine kinase inhibitor, is currently in clinical trials for treatment of breast cancer. We examined the impact of co-incubation of chemotherapy drugs in combination with lapatinib in P-gp over-expressing drug resistant cells. Unexpectedly, lapatinib treatment, at clinically relevant concentrations, increased levels of the P-gp drug transporter in a dose- and time-responsive manner. Conversely, exposure to the epidermal growth factor (EGF), an endogenous growth factor receptor ligand, resulted in a decrease in P-gp expression. Despite the lapatinib-induced alteration in P-gp expression, use of accumulation, efflux and toxicity assays demonstrated that the induced alteration in P-gp expression by lapatinib had little direct impact on drug resistance.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Quinazolinas/farmacología , Adenocarcinoma/tratamiento farmacológico , Adenocarcinoma/patología , Adenocarcinoma del Pulmón , Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Carcinoma de Células Escamosas/tratamiento farmacológico , Carcinoma de Células Escamosas/patología , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Resistencia a Antineoplásicos/efectos de los fármacos , Factor de Crecimiento Epidérmico/metabolismo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Lapatinib , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/patología , Masculino , Persona de Mediana Edad , Inhibidores de Proteínas Quinasas/administración & dosificación , Quinazolinas/administración & dosificación , Factores de TiempoRESUMEN
Anthrax lethal toxin (LT) is a bipartite protease-containing toxin and a key virulence determinant of Bacillus anthracis. In mice, LT causes the rapid lysis of macrophages isolated from certain inbred strains, but the correlation between murine macrophage sensitivity and mouse strain susceptibility to toxin challenge is poor. In rats, LT induces a rapid death in as little as 37 minutes through unknown mechanisms. We used a recombinant inbred (RI) rat panel of 19 strains generated from LT-sensitive and LT-resistant progenitors to map LT sensitivity in rats to a locus on chromosome 10 that includes the inflammasome NOD-like receptor (NLR) sensor, Nlrp1. This gene is the closest rat homolog of mouse Nlrp1b, which was previously shown to control murine macrophage sensitivity to LT. An absolute correlation between in vitro macrophage sensitivity to LT-induced lysis and animal susceptibility to the toxin was found for the 19 RI strains and 12 additional rat strains. Sequencing Nlrp1 from these strains identified five polymorphic alleles. Polymorphisms within the N-terminal 100 amino acids of the Nlrp1 protein were perfectly correlated with LT sensitivity. These data suggest that toxin-mediated lethality in rats as well as macrophage sensitivity in this animal model are controlled by a single locus on chromosome 10 that is likely to be the inflammasome NLR sensor, Nlrp1.
Asunto(s)
Carbunco/genética , Carbunco/mortalidad , Antígenos Bacterianos/metabolismo , Toxinas Bacterianas/metabolismo , Predisposición Genética a la Enfermedad , Proteínas del Tejido Nervioso/genética , Secuencia de Aminoácidos , Animales , Carbunco/inmunología , Células Cultivadas , Mapeo Cromosómico , Cromosomas de los Mamíferos , Modelos Animales de Enfermedad , Femenino , Fibroblastos/citología , Macrófagos/inmunología , Macrófagos/microbiología , Ratones , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/inmunología , Estructura Terciaria de Proteína , Ratas , Ratas Endogámicas BN , Ratas Endogámicas Dahl , Ratas Endogámicas F344 , Ratas Endogámicas SHR , Ratas Endogámicas WKY , Ratas Sprague-DawleyRESUMEN
Using a selection process designed to reflect clinically relevant conditions, a panel of taxane-selected variants were developed to study further the mechanisms of resistance in lung cancer. Unlike continuous or pulse exposure to high concentrations of chemotherapeutic drugs which yield high resistance and often cross resistance, most variants developed here displayed low level resistance to the selecting drug with slight cross-resistance. Pulsing with taxol resulted in more highly resistant clones (up to 51.4-fold). Analysis of taxol and taxotere in the four major lung cancer cell types showed the taxanes to be more effective against NSCLC (with the exception of SKMES-taxane selected variants) than against the SCLC. Comparison of taxol and taxotere shows that taxol induces higher levels of resistance than taxotere. Further, in taxotere-selected cell lines, the cells are more resistant to taxol than taxotere, suggesting that taxotere may be a superior taxane from a clinical view. Taxol treatment resulted in increased cross-resistance to 5-FU in all classes of lung cancer except DMS-53. The high levels of Pgp in the DMS-53 and selected variant suggests this mechanism is not related to Pgp expression. Analysis of the Pgp and MRP-1 status by combination inhibitory assays and Western blotting showed no consistent relationship between expression of the membrane pumps Pgp or MRP-1 and resistance. However, where high level resistance was seen, the parent cell line expressed Pgp or MRP-1 and was accompanied by increased levels in the variants. Overall we found that the clinically relevant models used here are useful for investigating mechanisms of taxane resistance.
Asunto(s)
Antineoplásicos/farmacología , Hidrocarburos Aromáticos con Puentes/farmacología , Resistencia a Múltiples Medicamentos/efectos de los fármacos , Resistencia a Antineoplásicos/efectos de los fármacos , Neoplasias Pulmonares/tratamiento farmacológico , Taxoides/farmacología , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Adenocarcinoma/tratamiento farmacológico , Adenocarcinoma/metabolismo , Adenocarcinoma/patología , Carcinoma de Células Pequeñas/tratamiento farmacológico , Carcinoma de Células Pequeñas/metabolismo , Carcinoma de Células Pequeñas/patología , Carcinoma de Células Escamosas/tratamiento farmacológico , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patología , Supervivencia Celular/efectos de los fármacos , Docetaxel , Combinación de Medicamentos , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologíaRESUMEN
BACKGROUND: p53 is a tumour suppressor gene, which is mutated in more than half of all tumours. Most chemotherapeutic drugs cause DNA damage, which is sensed by p53; the cell can then try to repair the damage or induce cell suicide. If the p53 machinery is defective, effective chemotherapy is made more difficult. MATERIALS AND METHODS: Wild-type p53 was transfected into lung cancer cell lines with different p53 status. The transfected cells were tested for changes in sensitivity to a range of chemotherapeutic agents. RESULTS: We observed only modest changes in the sensitivity to the chemotherapeutic agents adriamycin, taxol and carboplatin in the transfected cells lines. p53 protein was detected in a transfected clone of the cell line H1299, whose parent cells are p53 null. However, the protein did not accumulate after DNA damage, suggesting that this cell line utilises alternative pathways for responding to stress, and no longer has a functional p53 pathway. CONCLUSION: The results suggest that introduction of wild-type p53 alone is not sufficient to substantially alter the sensitivity of a cell line to a given chemotherapeutic agent.
Asunto(s)
Neoplasias Pulmonares/tratamiento farmacológico , Proteína p53 Supresora de Tumor/fisiología , Carboplatino/farmacología , Línea Celular Tumoral , Doxorrubicina/farmacología , Resistencia a Múltiples Medicamentos , Resistencia a Antineoplásicos , Humanos , Concentración 50 Inhibidora , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Paclitaxel/farmacología , Plásmidos/genética , Transfección , Proteína p53 Supresora de Tumor/biosíntesis , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Gene expression microarray technology has the unique advantage of allowing the study of the expression of thousands of genes simultaneously in cancer cells and tissues. This review provides an overview of how microarrays have been applied to many areas of cancer research, such as biomarker identification, improved diagnostics and targeted therapy, focusing in particular on lung cancer studies. Advances made in the field of microarrays, especially in interpretation of microarray data, have the potential to significantly increase our understanding of the causes and progression of different types of cancer and, hence, to enhance the treatment of patients suffering from these diseases.
RESUMEN
Anxiety disorders are phenotypically complex and may involve multiple genetic influences on many neurotransmitter systems. Rodent tests used to investigate genetic influences on anxiety-like phenotypes have face and predictive validity as models for anxiety in humans. If multiple genes contribute additively to a trait, the trait will be continuously distributed and be amenable to detection of associations between allelic variation at specific chromosomal loci and the phenotypes being studied via quantitative trait loci (QTL) mapping. The elevated plus-maze test provides quantitative measures of both anxiety-like and locomotion phenotypes. Using this test, we assessed four phenotypes in a set of 22 rat recombinant inbred (RI) strains derived from Brown Norway (BN.Lx /Cub) and Spontaneously Hypertensive rat (SHR/Ola) progenitors. QTL analyses were used to determine whether allelic variation at specific chromosomal loci contribute significantly to RI strain-dependent variance in each phenotype. Significant QTL for an anxiety phenotype were found on chromosomes 2, 5, 6, and 7. For a phenotype reflecting both anxiety and locomotion, QTL were found on chromosomes 2, 7, and 8, while for a locomotion phenotype, significant QTL were found on chromosomes 3 and 18.
Asunto(s)
Ansiedad/genética , Nivel de Alerta/genética , Locomoción/genética , Modelos Genéticos , Fenotipo , Sitios de Carácter Cuantitativo/genética , Alelos , Animales , Mapeo Cromosómico , Cruzamientos Genéticos , Femenino , Masculino , Ratas , Ratas Endogámicas BN , Ratas Endogámicas SHR , Recombinación GenéticaRESUMEN
A new contiguous genetic linkage map of the HXB/BXH set of rat recombinant inbred (RI) strains was constructed to enhance QTL mapping power and precision, and thereby make the RI strain set a better genomics resource. The HXB/BXH rat RI strains were developed from a cross between the hypertensive SHR/OlaIpcv and normotensive BN- Lx/Cub rat strains and have been shown useful for identifying quantitative trait loci (QTL) for a variety of cardiovascular, metabolic, and behavioral phenotypes. In the current analysis, the DNAs from 31 existing strains, 1 substrain, and 4 extinct strains were genotyped for a selection of polymorphic microsatellite marker loci, predominantly polymorphic framework markers from high-density integrated rat genome maps. The resulting linkage map consists of 245 microsatellite markers spanning a total length of 1789 cM with an average inter-marker distance of ~8.0 cM. This map covers the rat genome contiguously and completely with the exception of two locations on Chromosomes (Chrs) 11 and 16. The new genotypic information obtained also permitted further genetic characterization of the RI strain set including strain independence, genetic similarity among the individual strains, and non-syntenic associations between loci.