Effect of c-MET inhibitor SU11274 upon sensitive (RPMI8226) or multi-resistant (RPMI8226/R5) Multiple Myeloma cell lines
ABSTRACT: The c-MET signaling axis is increasingly implicated in tumorigenesis and chemioresistance. In this study, we investigated gene expression modifications induced by SU11274 (Selleck Chemicals, Boston, USA), a novel selective c-MET inhibitor, in a pair of isogenic multiple myeloma (MM) cell lines either sensitive (RPMI8226) or multi-resistant, highly c-MET expressing (RPMI8226/R5) cells. On the whole, RPMI8226/R5 cells after SU11274 were characterised by wider and diverse gene expression modifications than RPMI8226, indicating that c-MET over-expression, and its inhibition, is an important aspect of the adaptive response associated to drug resistance. RPMI8226 and RPMI8226/R5 cells are cultured in RPMI 1640 supplemented with10% fetal calf serum (FCS), 100U/ml penicillin and streptomicin (Euroclone UK) and 1mM Hepes buffer at 37 °C in a humified 5% CO2 atmosphere. The effect of SU11274 on gene expression of both cell lines was evaluated in three distinct experiments. 500,000 cells/ml in T25 flasks were treated with SU11274 (10 µM final concentration) or vehicle, for 6 h at 37 °C. At the end of the incubation time, treated and control untreated cells were washed and resuspended for RNA extraction. Linear mRNA amplification and labeling of fluorescent cDNA targets (Cy5, treated sample, Cy3, untreated sample) were performed using the Amino Allyl aRNA Amplification MessageAmp II Kit (Ambion), and examined Phalanx Human OneArray HOA (Phalanx Biotech Group's ) microarrays. Array scanning was carried out using a ChipReader VersArray ® 5 micron dual confocal laser scanner with VersArray ChipReader v3.1 analysis software, while the images were analyzed by scanner row VersArray Analyzer v4.5 software (Bio-Rad Laboratories) with intensity average pixels for each spot. Global Fund has been removed from biquadratic polynomial and the normalization of multiple channels was carried out by local regression (loess), the logarithmic transformation was then performed for each level of expression.
Project description:Fibroblasts are a key cellular component of tumour microenvironment, along with other stromal cells playing a critical role in disease initiation and progression (1). In this study, bone marrow fibroblasts deriving from patients with monoclonal gammopathy of undetermined significance (MGUS) or active multiple myeloma (MM) were subjected to global gene expression analysis, in order to identify progression-associated alterations of gene expression. Through immunoselection procedures, primary coltures of bone marrow fibroblasts were established from bone marrow aspirate of 18 patients fullfilling the International Myeloma Working Group diagnostic criteria for MM (n=10) and MGUS (n=8). Differentially expressed genes were investigated through cDNA microarray (21,329 70-mer oligonucleotides; dual-label competitive hybridization), using the reference design as experimental protocol and t-test statistics for identifying differentially expressed genes. A number of genes functionally involved in survival, proliferation, motility, inflammation and angiogenesis were found to be up-regulated in bone marrow fibroblasts from MM patients with respect to MGUS patients. In parallel, several genes were down-regulated in MM fibroblasts. Overall, bone marrow fibroblasts from MM patients show a distinct gene expression pattern that differentiates them from bone marrow fibroblasts of MGUS. The observation of differentially expressed genes indicates an activation state of fibroblasts in MM, which very likely concur to determine a microenvironment supporting disease progression. 1) 1. Kalluri R, Zeisberg M: Fibroblasts in cancer. Nat Rev Cancer 2006; 6: 392 –401. Bone marrow fibroblasts deriving from patients with monoclonal gammopathy of undetermined significance (MGUS) and active multiple myeloma (MM), were provided by A.Vacca, (Department of Biomedical Sciences and Human Oncology, Università degli Studi di Bari Aldo Moro, Italy). Briefly, primary coltures of bone marrow fibroblasts were established through immunoselection procedures on aspirates of 18 patients fullfilling the IMWG diagnostic criteria for active MM (n=10) and MGUS (n=8). Total RNA was extracted from each primary culture (test samples), and 1 μg-aliquots of RNA from MGUS samples were pooled to obtain the reference RNA. Linear amplification of mRNA and fluorescent labelling of cDNA targets (Cy5, MM and MGUS test samples; Cy3, reference RNA) were performed by using the Amino Allyl MessageAmp II aRNA Amplification Kit (Ambion), and examined on Micro-CRIBI Human Oligo Array (Operon V2.0) microarrays. Array scanning was carried out using a VersArray ChipReader® 5μm dual confocal laser scanner with VersArray ChipReader v3.1 analysis software , while row scanner images were analyzed with VersArray Analyzer Software v4.5 (Bio-Rad Laboratories) using media pixel intensities for each spot. Global background was subtracted by biquadratic polynomial approximation, and cross-channel normalization was performed by local regression (LOESS); logarithmic transformation was then performed for each expression level.
Project description:Effects of 125IUdR-elicited radiation on global gene expression changes in normal human cells. This experiment uses a balanced block design where each array compares a biologically independent sample from each class (treated versus untreated)
Project description:Study of gene expression during Plasmopara viticola infection in the resistant Vitis vinifera cultivar 'Regent'. The oomycete fungus Plasmopara viticola (Berk. et Curt.) Berl. et de Toni is responsible for grapevine downy mildew disease. Most of the cultivated grapevines are sensitive to this pathogen, thus requiring intensive fungicide treatments. The molecular basis of resistance to this pathogen is poorly understood. We have carried out a cDNA microarray transcriptome analysis to identify grapevine genes associated with resistance traits. Early transcriptional changes associated with downy mildew infection in the resistant Vitis vinifera cultivar ‘Regent’, when compared to the susceptible cultivar ‘Trincadeira’, were analyzed. Transcript levels were measured at three time-points: 0, 6 and 12 hours post inoculation (hpi). Our data indicate that resistance in V. vinifera ‘Regent’ is induced after infection. This study provides the identification of several candidate genes that may be related to ‘Regent’ defense mechanisms, allowing a better understanding of this cultivar's resistance traits. 3 time points: 0, 6 and 12 hours post inoculation by P. viticola. Two cultivars: control (Trinacedira) and test (Regent). Two biological replicates were performed at 0 hpi, and 3 biological replicates at 6 and 12hpi. At 12hpi, three technical replicates also were performed.
Project description:Survey of post pollination events in a sexually deceptive orchid (Ophrys fusca): a transcriptional approach Pollination through deception is a widespread phenomenon in angiosperm, and is extremely common in Orchidaceae family. One of the most striking pollination mechanism in orchids is known as sexual deception, in which flowers lure pollinators by foraging chemical (sex pheromones), visual (e.g. labellum colour and/or shape) and tactile (e.g. labellum pilosity) cues of the female insect pollinator. Ophrys has been used as a model genus to study sexual deception mechanism, mainly regarding chemical analysis in plant-insect association. Study was focused on Ophrys fusca, a species widely distributed in Mediterranean Basin. The main objective rely on Ophrys fusca gene expression study after pollination, through a transcriptional approach using cDNA microarrays. In order to evaluate pollination enhanced events, two different time points were selected: 2 days and 4 days after pollination. Ophrys fusca plants were sampled from a Portuguese natural occurring population. Plants were covered with a white and inert net, built specially for preventing pollinator’s visits in both pollinated and unpollinated flowers. Cross- pollination was performed manually with a sterile plastic stick. Five biological replicates (5 plants in each replicate) from each condition (pollinated and unpollinated) were collected in each time-point Flowers that demonstrate strict pollination regulation, as orchids, provide an excellent model system to unravel pollination- elicited mechanisms (i.e. petal senescence, pigmentation changes, ovary growth). Therefore, this study aims to contribute to the overall knowledge on orchid pollination biology, which is still lacking. 2 time points: 2 days and 4 days after pollination.Two-samples accessed: control (nonpollinated labella) and test (pollinated labella). 5 Biological replicates and 2 technical replicates (repeats of labelling and hybridization using randomly chosen biological replicates) in each time point were made.
Project description:Transcriptional profiling of pear tree comparing a resistant/tolerant cultivar with a susceptible cultivar to the Stemphylium vesicarium fungus Rocha' pear is an economically important portuguese Pyrus communis L. cultivar very susceptible to the Stemphylium vesicarium pathogenic fungus, the brown spot agent, causing huge decrease on fruit quality and yield production. Field control of brown spot disease is based in systemic application of antifungal chemicals with high economic costs and dramatic consequences to public health and environmental pollution. Plant-pathogen interactions involve a series of events encompassing constitutive and induced plant defence responses whose dissection has been a research target for control many crop diseases. The biosynthesis of cell wall polymers and antifungal compounds appear to be an efficient physical and chemical barrier to infection.To understand the molecular responses behind defence mechanisms of resistant/tolerant and susceptible cultivars of Pyrus communis L. to the S. vesicarium fungus, cDNA microarray technology was used to identify the genes differentially expressed along a time course leaf inoculation between 'Rocha' pear cultivar (a high susceptible cultivar) and 'Ercolini' pear cultivar (a resistant/tolerant pear cultivar). This study aims to contribute with information on the molecular mechanisms involved in host-pathogen interactions responsible for pear tree brown spot disease and resistance to Stemphylium vesicarium. Experimental condition: 'Ercolini' vs 'Rocha' (each experiment including 5 plants from each cultivar). 3 time-points: water-inoculation (T0h), 6 hours after inoculation with S. vesicarium (T6h) and 24 hours after inoculation with S. vesicarium. Biological replicates: 3 in each time-point. One replicate per array.
Project description:Juvenile rainbow trout were exposed to MWWE in a caging study, with cages held upstream and downstream of a MWWE outfall for two weeks in the fall of 2007. Whole tissues were sampled and analyzed. Microarray analysis was conducted on liver samples. Four condition experiment, Control (0%),10%, 50% and 100% MWWE. Four independent pooled samples for each treatment, hybridized against a reference pool. The reference pool and test sample were randomly assigned for each array.
Project description:Transcriptional profiling of hop samples throughout in vitro culture on medium with sucrose and hormones IAA and BAP Study of gene expression involved in stress response during in vitro culture and in organogenic nodule formation Hop (Humulus lupulus L.) is an economically important plant forming organogenic nodules which can be used for genetic transformation and micropropagation. We are interested in the mechanisms underlying reprogramming of cells through stress and hormone treatments. To investigate the spatio-temporal sequence of events that underlies competence acquisition for organogenesis three main stages were selected: (i) internodes at the time of excision from the parent plant (T0); (ii) internodes grown for 15 days on culture medium in which several prenodular structures are formed inside the calli (T15d); and (iii) nodule forming tissue after 28 days of culture (T28d). Additionally a control was included corresponding to 28 days of culture without hormones, lacking nodule formation and plantlet regeneration abilities, in order to identify genes specifically involved in morphogenesis (T28dWH). Organogenic nodule formation is a morphogenic process that shares features with somatic embryogenesis though in the former no shoot/root pole is established and plantlet regeneration can occur from different peripheral regions of nodules. Previous studies have shown that the organogenesis-determining period (when cells are determined to form nodules) occurs in between 15 and 25 days of culture corresponding to prenodular and first nodular stages. When nodules are fully developed they are surrounded by layers of elongated, highly vacuolated cells that may degenerate when nodules start differentiating plantlets. Plantlet regeneration from organogenic nodules can be observed after 45 days of culture. However, organogenic nodules after 28 days of culture are already determined to undergo plantlet regeneration, since they no longer require exogenous supply of hormones. Explants cultured in medium without hormones can develop incipient prenodular structures with vascular tissue but not nodules, thus lacking morphogenic potential. Keywords: Plant development, Time course 3 time points: 3 Biological replicates and 1 technical replicate (1 dye-swap). 1 control at beginning of culture and 1 control wihout hormones. One replicate per array. Each clone printed twice in the same sub-grid
Project description:Transcriptional profiling of pancreatic tumors comparing to adjacent non-tumoral pancreatic tissue by two strategies. The first strategy implies a traditional microarray assay, while the second involves a previous Suppression Subtractive Hybridization (SSH) and then the traditional microarray assay. Goal was to determine differences in gene expression profile in tumor samples by both strategies. Two-condition experiment, tumoral vs. non-tumoral tissues. Biological replicates: 6 tumoral replicates, 6 non-tumoral replicates. The same replicates for both strategies.