ABSTRACT: The HERV-K(HML2) family is a group of elements of retroviral origin that are present in the human genome. They are silenced in most normal tissues, but are induced in a number of human cancers. In order to assess the relevance of their expression in these pathologies, the consequences of ectopic expression of the HERV-K(HML2) envelope protein were analysed in human 293T cells. The cells were transfected with a control vector, or an expression vector for the HERV-K(HML2) envelope protein. Total RNA was extracted 24h and 48h post transfection, and duplicate samples were analysed on whole genome transcriptional microarrays.
Project description:Non-small cell lung cancer (NSCLC) with activating mutations in the epidermal growth factor receptor (EGFR) responds to EGFR tyrosine kinase inhibitors such as erlotinib. However, secondary somatic EGFR mutations (e.g. T790M) confer resistance to erlotinib. BMS-690514, a novel panHER/VEGFR inhibitor described here, exerted antiproliferative and pro-apoptotic effects on NSCLC cell lines, with prominent efficacy on H1975 cells expressing the T790M mutation. In this model, BMS-690514 induced a G1 cell cycle arrest, as well as ultrastructural hallmarks of apoptosis, mitochondrial release of cytochrome c, and activation of caspases involved in the intrinsic (e.g. caspase -2, -3, -7 and -9), but not in the extrinsic (e.g. caspase-8) pathway. Caspase inhibition conferred partial protection against BMS-690514 cytotoxicity, pointing to the involvement of both caspase-dependent and -independent effector mechanisms. Transcriptome analyses revealed the upregulation of pro-apoptotic (e.g. Bim, Puma) and cell cycle inhibitory (e.g. p27Kip1, p57Kip2) factors, as well as the downregulation of anti-apoptotic (e.g. Mcl1), heat shock (e.g. HSP40, HSP70, HSP90) and cell cycle promoting (e.g. cyclins B1, D1 and D3, CDK1, MCM family proteins, PCNA) proteins. BMS-690514-induced death of H1975 cells was modified in a unique fashion by a panel of siRNAs targeting apoptosis modulators. Downregulation of components of the NF-kappaB survival pathway (e.g. p65, Nemo/IKK, TAB2) sensitized cells to BMS-690514, whereas knockdown of pro-apoptotic factors (e.g. Puma, Bax, Bak, caspase-2, etc) and DNA damage-related proteins (e.g. ERCC1, hTERT) exerted cytoprotective effects. BMS-690514 is a new pan-HER/VEGFR inhibitor that may become an alternative to erlotinib for the treatment of NSCLC.
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: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:We had previously demonstrated the role of CD103 integrin on lung tumor-infiltrating lymphocyte (TIL) clones in promoting specific TCR-mediated epithelial tumor cell cytotoxicity. However, the contribution of CD103 on intratumoral T-cell distribution and functions, and the prognosis significance of TIL subpopulations in non-small cell lung carcinoma (NSCLC) have thus far not been systematically addressed. Here we investigate the transcriptomic profil of these cell population, using PBMC cells as control.
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:We profiled breast cancer samples with exon arrays (244K Agilent Human exon) in order to identify exons associated with higher risk of relapse. This set contain 178 single dual color of samples (biopsies) versus normal breast reference (Normal breast Clontech) , 5 dye-swap (10 hybridizations) and 5 self self for controls.