Atra effect on ERBB2 and RARA-coamplified SKBR-3 cells.
ABSTRACT: RNA was extracted with Rneasy Micro Kit (Qiagen, France) from SKBR3 cells treated with 1 uM ATRA. The quantity and purity of the extracted RNA was evaluated using a NanoDropTM spectrophotometer and its integrity measured using an Agilent BioanalyzerTM. For microarray hybridizations, 500 ng of total RNA from each RNA sample was amplified and labeled with two fluorescents dye (Cy5 and Cy3) using the Quick Amplification Labeling kit (Agilent Technologies, Palo Alto, CA, USA) following the manufacturer's protocol. Cy3-labeled and Cy5-labelled cRNA were hybridized to the Agilent Human Whole Genome Oligo Microarray format 4x44K (Agilent Technologies), prior to washing and scanning.
Project description:The mouse aldehyde oxidase, Aox4 (aldehyde oxidase 4), is a molybdo-flavoenzyme. Harderian glands are the richest source of Aox4, although the protein is detectable also in sebaceous glands, epidermis and other keratinized epithelia. We performed whole genome gene expression experiment on Harderian Gland, White Adipose Tissue and Liver of WT and Aox4-/- animals.
Project description:Radiotherapy has a critical role in the treatment of small cell lung cancer (SCLC). Effectiveness of radiation in SCLC remains limited as resistance results from defects in apoptosis. In the current study, we investigated whether using a Bcl-2/Bcl-XL inhibitor S44563 can enhance radiosensitivity of SCLC cells in vitro and in vivo. In vitro studies confirmed that S44563 induce apoptosis in SCLC cells by inducing hallmarks of apoptosis (cleaved caspase-3, sub-G1 fraction induction and induction of the mitochondrial caspase activation pathway). S44563 markedly enhanced sensitivity of H146 cells and H69 cells to radiation in clonogenic assay. In addition, the combination S44563 and cisplatin-based chemo-radiation showed a dramatic tumor growth delay and increased overall survival in mouse xenograft models. In vitro experiments demonstrated a greater induction of apoptosis (sub-G1 fraction and cleaved caspase-3) with the combination as well as in vivo caspase-3 immunostaining. This positive interaction between S44563 and radiation was greater when S44563 was given after the completion of the radiation, which may be explained by the radiation-induced over expression of anti-apoptotic proteins (Bcl-2 and Bcl-XL) through the NF-?B pathway activation. Taken together, these data underline the critical role of sequence administration of targeted therapies with conventional therapies. SCLC cells which survive to IR highly rely on the induction of anti apoptotic proteins in part through NF- ?B activation for their survival yielding to the concept of 'contextual oncogene addiction' to the Bcl-2/Bcl-XL pathway providing rational for targeting survival pathways to potentiate IR. For transcriptome analysis, 106 cells were seeded in T25 flasks, allowed to growth for 24 h, and then left untreated or treated with 2 Gy radiation. After 24 hours, cells were harvested, lysed for the extraction of RNA, and processed to analyze gene expression.The design of this study consists to 6 hybridizations in dual color with Agilent Human Genome 4x44K (design 014850). The reference is always the untreated condition.
Project description:Non-small cell lung cancer (NSCLC), a leading cause of cancer deaths, represents a heterogeneous group of neoplasms, mostly comprising squamous cell carcinoma (SCC), adenocarcinoma (AC) and large-cell carcinoma (LCC). The aim of this study was to gain a systems biology insight into the current clinical classification. Patients and Methods: Comparative genomic hybridization followed by mutational analysis, gene expression and miRNA microarray profiling were performed on 123 paired tumor and non-tumor tissue samples from patients with NSCLC. Using integrated systems biology approaches, we sought to find out if combining data types from different levels of biology would improve clinical assessment of NSCLC. Results: At both DNA, RNA and miRNA levels we could identify molecular markers that discriminated significantly between the various clinicopathological entities of NSCLC. Conclusions: We report proofs of distinct molecular profiles that contribute to distinguishing NSCLC tumor subtypes even in small biopsies. The Gene expression experiments have been made in dual color and dye_swap with Agilent human Human Genome Exon 244K arrays (custom design 14891, from commercial 4x44K (design 014850 plus 195000 oligo - 1 per exon- defined with RefSeq hg18 and 1840 probes from viral transcripts). Note date of surgery is the date of the sample was frozen.
Project description:Previous reports have described the use of microarrays to assess the molecular classification of human breast cancers and defined new subgroups based on gene expression that are relevant to patient management through their ability to predict metastatic relapse and survival relapse. However, different mechanisms may be associated with the development of early and late distant metastases. With the hypothesis that tumors may lead to early or distant metastases based on their intrinsic biological initial features. We aimed at defining molecular profiles for several subgroups of patients based on their outcome over time. Breast primary tumors were selected from retrospective series of patients with frozen material available. These series include patients of all ages, LN- and LN+; Estrogen or Progesteron-receptor positive, Her2-negative, no adjuvant treatment, with a follow-up of more than 10 years (y) for the control group or distant metastatic relapse as first event (DM) for the study group. Patients tumors were classified in 3 groups: no relapse at 10y (M0), DM before 5 y (M0-5), DM between 5 and 15 y (M5-15).Gene expression analysis of breast tumor samples was performed using custom-made Agilent 4x44K high-density microarrays and hybridized against the Mammaprint reference pool (MRP). MRP=MammaReference Pool (The reference sample consisted of pooled and amplified RNA of 105 primary breast tumors, see Glas AM, BMC Genomics, 2006). The design of this study consists to 20 hybridizations in dual color with Agilent Human Genome 4x44K (design 014850) of breast tumors with a MRP reference with an addition as controls of 2 dye swaps and two self-self of the MRP and HMG (Human normal mammary Gland , Clontech).
Project description:Suppressor of cytokine signaling (SOCS1) is an inducible negative regulator of cytokine signaling. SOCS1 is expressed in normal colonic epithelium and colon adenocarcinomas. However, the levels of SOCS1 decreased during progression of colon adenocarcinomas, the lowest level being found in the more aggressive stage and least differentiated carcinomas. The restoration of SOCS1 in a colon cancer cell line induced expression of E-cadherin associated with disappearance of the mesenchymal form of the p120ctn. SOCS1 expression is sufficient to revert many characteristics of EMT. This reversion is dependent on the regulation of expression of the transcription factor ZEB1, both at transcriptional and translational levels. Furthermore, miRNA profiling indicates that SOCS1 also upregulates the expression of the mir-200 family of microRNAs, which can promote the mesenchymal-epithelial transition and reduce tumor cell migration. Expression of SOCS1 within the colon carcinoma cell lines dramatically reduced the invasive properties of the cells in vitro. This property was also observed in mice inoculated intracardially with SW620 cells where SOCS1 expression not only reduced the number of animals bearing metastases. Thus, SOCS1 up-regulates expression of E-cadherin to reduce colon cancer cells invasive properties. This pathway could be associated with colorectal cancer survival by reducing the capacity of generating metastases.This project concern 2 replicates in dye-swap of SW620 cells with and without transfected SOCS1 on a 244K custom human genomic expression microarray.
Project description:VHL is a tumor suppressor gene involved in the oxygen-sensing pathway whose germline mutations predispose to distinct phenotypes. Heterozygous mutations predispose to von Hippel-Lindau disease characterized by the development of multiple tumors (including hemangioblastomas, renal cell carcinomas and pheochromocytomas)1-3. More recently, a specific VHL-R200W mutation was shown to be responsible for Chuvash Polycythemia in homozygous carriers whereas heterozygous individuals have no clinical manifestation4. We report here a family carrying, on the same allele, VHL mutations characteristics of the two types of disease (a Chuvash polycythemia-R200W mutation and a von Hippel-Lindau disease-R161Q mutation). Genotyping, modeling analysis and functional studies, including transcriptomic profile of the distinct mutants validated for the first time on direct HIF target genes, show a gradual capacity of the VHL mutants to regulate the hypoxia responsive pathway that correlate with the severity of the developed phenotype. Our study provide original results that illuminate genotype/phenotype correlations in von Hippel-Lindau disease.
Project description:Docetaxel is used as a standard treatment in patients with metastatic castration-resistant prostate cancer. However, a large subset of patients develops resistance by mechanisms that remain largely unknown. It is thus important to define the relevant pathways implicated in docetaxel-resistance and validate predictive biomarkers that will allow approaches of personalized treatment. In this aim, we established resistant IGR-CaP1 prostate cancer cell lines to different doses of docetaxel (IGR-CaP1-R cell lines) and investigated gene expression profiles by microarray analyses. We generated a signature of 112 genes potentially implicated in docetaxel-resistance whose expression is highly modified (Fold change ≥ 5). Among these genes, significant modification of expression was observed among cell cycle components in the resistant cells. Hence, we focused on the role of the cell cycle regulator LZTS1 located on chromosome 8p which was under-expressed in all our docetaxel-resistant models. LZTS1 extinction was confirmed at the RNA and protein levels. DNA methylation analysis revealed a stretch of 20 highly methylated CpGs in the region encompassing the exon 1 of LZTS1 promoter in the docetaxel-resistant cells suggesting the existence of an epigenetic regulation of LZTS1 expression in the resistant cells. By using siRNA strategy, we found evidence that LZTS1 plays an important role in the acquisition of the resistant phenotype. In addition, immunohistochemical staining showed that LZTS1 protein was absent or down-regulated in 33% of diagnostic biopsies obtained in patients with metastatic castration-resistant prostate cancer. This heterogeneous labeling suggests that LZTS1 might constitute a predictive biomarker of response to docetaxel chemotherapy. Furthermore, as Cdc25C is a LZTS1 partner in the mitosis regulation, we observed that targeting of Cdc25C with the pharmacological Cdc25C inhibitor NSC 663284 specifically killed the docetaxel-resistant cells. These results strongly suggest that Cdc25C plays a role in docetaxel resistance and that Cdc25C might be a therapeutic target to overcome docetaxel resistance. Altogether our findings identify an important role of LZTS1 in developing docetaxel resistance in prostate cancer through its role in regulating phosphatase Cdc25C. The set of gene expression with 4x44K Agilent ( design 014850) correspond to 6 doses of docetaxel 2?5 to 200 ug/ml) in dual color and dye-swap versus the IGR-Cap1 cell line without docetaxel.
Project description:Goal of the experiment Recurrence is a frequent phenomenon in intracranial childhood ependymomas. To understand this process, we investigated whether the gene expression profiling of matched ependymomas at diagnosis and at relapse could reveal key molecular events involved in tumor progression. To gain new insight in this process and identify pathways associated with recurrence, we compared the gene expression profiles of local recurrences with the corresponding initial tumors. Brief description We analyzed 17 tumor samples at diagnosis and a total of 27 paired recurrences. Recurrences analyzed occurred after surgery only in 12 cases, surgery plus chemotherapy only in 9 cases and any treatment plus radiotherapy in 6 cases. We compared the level of gene expression for each tumor at recurrence relative to its expression at diagnosis using Agilent 44K dual color gene expression microarrays.
Project description:Background: Although histology is the corner stone for decision analysis in bladder cancer, it still needs refinement and new tools to better identify tumours with the highest risk of progression or recurrence. Objective: We designed a prospective study to evaluate whether microarray analysis could find a molecular signature for pathologic staging and/or grading. Design, setting and participants: Prospective multicentric study conducted from September 2007 to May 2008 (108 bladder tumours (45 pTa, 35 pT1 and 28> pT1)). Microarray analysis was performed with Agilent Technologies Human Whole Genome 4 x 44K oligonucleotide microarrays and used a method of dual color type versus a reference consists of a pool of tumours. From the lists of genes provided by the BrB Class Comparison analyses, we validated the microarray results of 38 selected differentially expressed genes by RT-QPCR in another bladder tumour cohort (n = 95). Results: The cluster superficial vs invasive stage properly classified 92.9% of invasive stages and 66.3% of superficial stages. Among the superficial tumours, the cluster analysis showed that pT1b tumours were closer to invasive stages than pT1a tumours. We also found molecular differences between low and high-grade superficial tumours. However these differences were less caricatural than the one observed for staging.Conclusions: We confirm that the histopathological classification into subgroups pTa, pT1a and pT1b may have a translation in a molecular signature with a continuous progression of deregulation (overexpression or repression of these genes) from superficial (pTa) to more invasive (pT1a then b stage).
Project description:'Prostate cancer is the second cancer diagnosed among men worldwide. Beside approval of new therapies in the last five years, chemotherapeutic agents, docetaxel and cabazitaxel taxanes remain key treatments for metastatic castration resistant prostate cancers. However, primary and acquired resistance to taxanes still emerged in about half of patients. There is therefore an urgent need to discover and understand the taxane resistance mechanisms in order to identify new therapeutic targets. Indeed, targeted therapies that exploit the signaling pathways involved in prostate cancer are required to overcome chemoresistance and improve treatment outcomes. Molecular chaperones play a key role in the regulation of cellular homeostasis and the development of treatment resistance, and are promising therapeutic targets. Using high throughput siRNA functional screening based on a gene expression signature, we identified FKBP7, involved in acquired resistance to docetaxel and cabazitaxel. FKBP7 is a molecular chaperone that has not been studied in human so far. FKBP7 is overexpressed in prostate tumors and its expression is correlated with recurrence in patients who received docetaxel as neoadjuvant therapy. Moreover, FKBP7 is upregulated in taxane resistant prostate cancer cell lines and its expression sustains their growth in vitro and in a mice model of Docetaxel resistance. Using a high throughput proteomic approach, we identified the signaling pathway regulated by FKBP7 which is responsible for the survival of chemoresistant cells. Finally, we proposed a promising therapeutic strategy to overcome both docetaxel and cabazitaxel chemoresistance by targeting the downstream effector of FKBP7. Gene expression was profiled using a 4×44K Human Whole Genome (G4112F, id : 014850) expression array (Agilent Technologies, Santa Clara, Cal.) with a dual color dye-swap competitive hybridization procedure, according to the manufacturer''s instructions. Total RNA from untreated parental IGR-CaP1 cells was used as the RNA reference (NN1). Total RNA from IGR-CaP1 cells resistant to 5nM (NN2), 11.6nM (NN3), 25nM (NN4), 50nM (NN5), 100nM (NN6), and 200nM (NN7) of Docetaxel respectively, were used as samples. Two independent replicates of each samples (a and b) were used. Image analyses (quantification, normalization) were performed with Feature Extraction software (Agilent Technologies) and gene expression analysis was performed using Bioconductor.'