Project description:We integrated metabolome and proteome profiles of the parental cell line 143B.TK- versus ρ0, including PTM analyses such as phosphorylation and ubiquitination to characterize the impact of the absence of mtDNA for the entire cell. For quantitative proteome profiling, we used a shotgun LC-MS/MS approach including the classical SILAC labeling. For comprehensive metabolome profiling, we applied a targeted LC-MS approach, based on multiple reaction monitoring (MRM).</br></br>Our study revealed that mtDNA depletion leads to a non-uniform down-regulation of the mitochondrial energy metabolism in ρ0 cells on the proteome level. Metabolites of the TCA cycle were highly dysregulated which in turn had an impact on the amino acid levels, which were up regulated. Perturbation of the mitochondrial energy metabolism could lead to an activation of the retrograde response, indicated by sets of up-regulated signaling pathways in ρ0 cells, further supported by altered phosphorylation in signaling pathways and the cytoskeleton as well as de-ubiquitination of SLC transporters.
Project description:Fibroblasts isolated from human colon submucosal and subperitoneal layer were stimulated by colon cancer cell line (DLD-1) cultured medium. Peritoneal invasion in colon cancer is an important prognostic factor, and the fibrosis with α-SMA was a significant pathological feature of the cancer microenvironment formed by peritoneal invasion (CMPI). The result indicated that the gene expression of subperitoneal fibroblasts showed more various gene modification than submucosal fibroblasts. And ACTA2 expression was higher in fibroblasts from subperitoneal layer than that from submucosal layer. Together with this concordant stromal protein expression in CMPI, this in vitro model is able to reflect the special microenvironment in CMPI. 3 cases of human colon submucosal and subperitoneal fibroblasts were isolated, and these fibroblasts were stimulated with DLD-1 cultured medium. Total RNA were extracted from these samples and hybridized in Affymetrix microarray to compare their gene expression changes through the DLD-1 stimulation
Project description:In order to comprehensively identify genes directly regulated by AP4, a genome-wide chromatin-immunoprecipitation analysis (ChIP) followed by next generation sequencing (ChIP-seq) was performed after activation of a conditional AP4 allele in DLD-1 cells. One DLD-1 Sample was sequenced.
Project description:Colorectal cancer (CRC) is the third most deadly and fourth most diagnosed cancer worldwide. Despite the progress in early diagnosis and advanced therapeutic options, CRC shows a poor prognosis with a 5-year survival rate of ~45%. PRDM2/RIZ, a member of Positive Regulatory Domain (PRDM) gene family, expresses two main molecular variants, the PR-plus isoform (RIZ1) and the PR-minus (RIZ2). The imbalance in their expression levels in favour of RIZ2 is observed in many cancer types. The full length RIZ1 has been extensively investigated in several cancers where it acts as a tumour suppressor, whereas few studies have explored the RIZ2 oncogenic properties. PRDM2 is often target of frameshift mutations and aberrant DNA methylation in CRC. Accordingly, our analysis of Exome- and transcriptome public datasets available at The Cancer Genome Atlas (TCGA) portal revealed that PRDM2 gene is frequently mutated and transcriptionally deregulated in CRC. However, little is known about its role in CRC. In this study, we first evaluated the expression of the different PRDM2 transcripts by in silico analysis on TCGA CRC datasets. Our in-silico analysis on TCGA datasets revealed a highly significant downregulation of RIZ1 in CRC samples whereas a RIZ2 increase was observed in the same samples. Then, we assayed several CRC cell lines by qRT-PCR analysis for the main PRDM2 transcripts and selected DLD-1 cell line, which showed the lowest RIZ2 levels to mimic TCGA datasets analysis results and consequently to assess the PRDM2/RIZ2 role in CRC cells. RNAseq analysis revealed that RIZ2 overexpression induced an EGF overexpression suggesting that RIZ2 could be involved in the EGF autocrine regulation of DLD-1 cell behaviour. Noteworthy, the forced RIZ2 expression increased cell viability, growth, colony formation, migration and organoid formation thus confirming our previous findings on HEK-293 cells. These effects could be mediated by the release of high EGF levels by RIZ2 overexpressing DLD-1 cells. Our findings add novel insights on the putative RIZ2 tumor-promoting functions in CRC, although additional attempts are warranted to depict the underlying molecular mechanism of action
Project description:MicroRNAs (miRNAs) have emerged as important gene regulators and are recognized as key players in tumorigenesis. miR-143 is reported to be down-regulated in several cancers, but knowledge of its targets in colon cancer remains limited. To investigate the role of miR-143 in colon cancer, we have employed a microarray based approach to identify miR-143 targets. Based on seed site enrichment analyses and unbiased word analyses, we found a significant enrichment of miRNA binding sites in the 3M-bM-^@M-^Y-untranslated regions (UTRs) of transcripts down-regulated upon miRNA overexpression. Here we identify Hexokinase 2 (HK2) as a direct target of miR-143 and show that re-introduction of miR-143 in the colon cancer cell line DLD-1 results in a decreased lactate secretion, indicating that miR-143 down-regulation of HK2 affects glucose metabolism in colon cancer cells. DLD-1 cells were transfected with 50 nM miR-143 duplex or mock transfected. Total RNA was harvested 24 hours post-transfection and analyzed on Affymetrix HG-U133 Plus 2.0 human arrays.
Project description:In order to elucidate how FOXOs affect diverse cellular processes such as cell cycle progression, stress response and transformation we made use of an inducible version of the FOXO3a protein fused to the hormone binding domain of the human estrogen receptor (FOXO3a-A3-ER) in which all three Akt phosphorylation sites have been mutated to alanine. FOXO3a.A3-ER was stably expressed in the human colon carcinoma cell line DLD-1 (Kops et al., 2002b). In order to analyse the transcriptional response to FOXO3a activation we generated gene expression profiles from DL23 or parental DLD-1 cells, after 6 or 24 hours of 4-OHT treatment using cDNA microarrays.
Project description:Our goal is to find new genes regulated by p21 in human primary cells . To get it we carried out a gene expression profiling in two different models, human myeloid leukemia K562 cells and human keratinocytes both of them with conditional expression of p21. In order to identify genes specifically modulated by p21 we compared with the cell line with overexpression of p27, because p21 and p27 belong to the same gene family and regulated the same genes specially in cell cycle. So, our intention is to identify only genes regulated by p21 and not p27. In order to confirm these results we studied the p21-dependent repression of mitotic genes in a different cellular system. We chose human primary keratinocytes because they are non-tumorigenic, non-immortalized and epithelial cells, in contrast to human myeloid leukemia K562 cells. Human primary keratinocytes were infected with recombinant adenoviruses expressing the full-length p21 protein. A dramatic increase in p21 in infected keratinocytes was demonstrated by RT-qPCR (as we show in the manuscript). As controls, we also infected the keratinocytes with adenovirus carrying the genes for p27 which overexpression was also confirmed by RT-qPCR (as we show in the manuscript). We prepared RNA 24 h after infection and performed large-scale expression assay using the Afftymetrix platform. The clustering analysis revealed that p21 provoked the down-regulation of a number genes involved in cell cycle control not shared by cells expressing p27 (as we show in the manuscript). Our goal, has been getting genes regulated more strongly by p21 and not by p27 in cell cycle and mitosis. Our result are supported because we have found the same genes in two different models and also we have validated (by RT-qPCR) more than 20 cell cycle and mitotic genes, found in our affymetrix arrays. Also we have found the region of p21 that is sufficient for gene regulation and for one gene we have described as p21 bind to the promoter. Finally, we have discussed in our manuscript how p21 can do this regulation by bioinformatic analysis of p21-target genes. The success of this study is to describe a new role of p21 as a transcriptional co-repressor in some systems.
Project description:To identify genes and molecular pathways directly or indirectly affected by the loss of LAP2? in skeletal muscle, gene expression profiles of primary Lap2?+/+ (n=3) and Lap2?-/- (n=3) neonatal<br><br>myoblasts were compared.
Project description:Phenotypic plasticity, the ability of one genotype to express different phenotypes in response to changing environmental conditions, is one of the most common phenomena characterising the living world and is not only relevant for the ecology but also for the evolution of species. Daphnia, the waterflea, is a textbook example for predator induced phenotypic plastic defences including changes in life-history, behaviour and morphology. However, the analysis of molecular mechanisms underlying these inducible defences is still in its early stages.<br><br>We exposed Daphnia magna to chemical cues of the predator Triops cancriformis to identify key processes underlying plastic defensive trait formation. D. magna is known to develop an array of morphological changes in the presence of T. cancriformis including changes of carapace morphology and cuticle hardening. To get a more comprehensive idea of this phenomenon, we studied four different genotypes originating from habitats with different predation history, reaching from predator-free to temporary habitats containing T. cancriformis.<br><br>We analysed the morphologies as well as proteomes of predator-exposed and control animals. Three genotypes showed morphological changes when the predator was present. Using a high-throughput proteomics approach, we found 294 proteins which were significantly altered in their abundance after predator exposure in a general or genotype dependant manner. Proteins connected to genotype dependant responses were related to the cuticle, protein synthesis and calcium binding whereas the yolk protein vitellogenin increased in abundance in all genotypes, indicating their involvement in a more general response. Furthermore, genotype dependant responses at the proteome level correlated well with local adaptation to Triops predation.<br><br>Altogether, our study provides new insights concerning genotype dependant and general molecular processes involved in predator-induced phenotypic plasticity in D. magna.