DNA microarray experimental analysis

Protein kinase C iota (PKCiota) was implicated in chemoresistance in several types of cancer, particularly in glioblastoma multiforme, a brain cancer. Using profiling of gene expression by Affymetrix DNA microarrays, we show that the specific depletion of the mRNA for that gene with an interference RNA in glioblastoma cells modifies significantly the gene expression of a number of genes [1]. One of those genes was GMFbeta, a p38 MAP kinase enhancer, whose mRNA levels increased with the inhibition of PKCiota. Further experimental verification indicated that PKCiota might confer partial chemoresistance (to cisplatin, an anti-tumoural drug) by suppressing GMFbeta-mediated enhancement of p38 MAP kinase signaling. The p38 MAP kinase pathway had been previously identified as a key mediator of cisplatin cytotoxicity.

Pulmonary arterial hypertension (PAH) is an inherited disease that can result in vasoconstriction in the small pulmonary arteries producing ventricular failure and premature death. We studied a rat model of this phenotype, the Fawn Hooded Rats (FHR), using different techniques including DNA microarray analysis of gene expression [2]. A consomic FHR rat with a chromosome 1 from Brown Norway rat did not develop the phenotype. Our experiments indicate an alteration of the balance of reactive oxygen species (ROS), likely due to a problem related to the mitochondrial-HIF-1a-Kv channel pathway.

We have studied the relation between the elongation factor eEF1A2 and the outcome in breast cancer [3]. DNA microarray analysis indicates that eEF1A2's mRNA is expressed at a low level in normal breast epythelium and at a higher level in 50-60% of primary breast tumours. However, measurement of the levels of eEF1A2 protein in primary breast tumours indicates that high expression increases the probability of survival. Therefore, although the eEF1A2 gene seems to be tumorigenic it may not activate an effective metastatic program.

Dichloroacetate (DCA) is an inhibitor of the mitochondrial pyruvate kinase that has been used in humans (by oral therapy) for the treatment of lactic acidosis. In this study [4], microarray analysis and other experimental evidence indicates that DCA produces a number of modifications in cancer cells but not in normal cells that could be relevant for the use of DCA as pro-apoptopic cancer therapy. This effects include the activation of Kv channels by an NFAT1 dependent mechanism.

We characterized a novel mouse cell type present in small blood vessls in dermal, adipose and skeletal muscle tissue that can be grown as spheres [5]. Cellular and gene expression analyses of these cells suggests that they are endothelial precursors closely associated with small blood vessels.

SNP chips are used to genotype Single Nucleotide Polymorphisms (SNPs). In this work [6] we test their use for the evaluation of the products of an experiment of chromatin immunoprecipitation, in particular, to detect genomic positions of histone H4 hyperacetylation in samples from myoblasts and myotubes. Comparison of the data with gene expression data obtained from the same samples allowed us to see the range of histone H4 hyperacetylation that occurs nearby expressed genes. The computational aspects of this work are further discussed in [7].

To study the epigenetic control of stem cell differentiation we generated mice with a knock down of the Dnmt1 (DNA methyltransferase 1) gene. DNA microarray analysis demonstrated that the gene expression in haematopoietic stem cells of mice with Dnmt1 knock down had similarities to myeloerythroid progenitors, suggesting the importance of DNA methylation on stem cell differentiation [8].

To study the molecular basis of the decrease of muscle regenerative potential associated to ageing, we studied the changes of gene expression in murine skeletal muscle [9]. We compared muscle from 3 week old mice to muscle from 24 month old mice. Many of the functions of the genes down-regulated in the old mice were related to the function of satellite cells and to the Notch and Wnt pathways whose control is associated to adult myogenesis.

Microarray analysis of gene expression can also produce negative results. In this work, we observed a lack of significant changes in gene expression in a mouse model that expressed the intracellular domain (ICD) of megalin [10]. This result argues against the previously proposed hypothesis that the ICD regulates gene expression.

Leukemia hematopoietic Stem Cells (LSCs) are used as a model of induction of leukemia in mouse. Comparison of gene expression between different types of LSCs indicated that higher expression of the reactive oxygen species scavenger glutathione peroxidase 3 (GPx3) correlates with higher proliferation of these cells [11].

Dendrytic cells (DCs) are immune cells with an antigen-presenting function. We found that knock-down of transcription factor Irf8 transforms its progenitor into neutrophils, a type of white blood cells. By comparison of microarray data of gene expression of several samples we observed that a knock-out of Irf8 in progenitors increases the expression of genes characteristic of neutrophils and decreases the expression of genes characteristic of DCs [12]. Using ChIP we then identified a distant enhancer needed for the activation of Irf8 and controlled by transcription factor PU.1. This indicates that the DC progenitors have a default commitment, which requires PU.1 activation of Irf8 for production of DCs.

Colorectal cancer (CRC) is currently one of the most common cancers. Surgical removal of the tumor is used as therapy but 40% of patients die within 5 years due to recurrence or distant metastases. To find markers predicting recurrence we studied gene expression in samples from resections of 45 patients and found a series of genes related to immune response whose upregulation in patients predicted good outcome [13]. Heterogenetity of human samples required using an algorithm based on recursive application of support vector machines (SVMs). Our study suggests that the activation of a network of genes related to the MHC class II complex is important for the survival of CRC patients.

In another approach to the study of gene expression profiles of CRC, we compared gene expression in a CRC cell line (SW480) versus a version ectopically over-expressing MACC1 (known as a progonostic biomarker for metastatic CRC) [14]. One of the strongly up-regulated genes was SPON2. Follow up experiments demonstrated that MACC1 binds to the gene promoter of SPON2 and that SPON2 expression is related to increased cancer proliferation. Together, MACC1 and SPON2 expression allow the identification of CRC patients at risk of shorter survival time.

To investigate the lineage potential of T-cell precursors, we produced mouse DN2 cells (a particular type of T-cell precursors) transduced with a virus expressing the oncogenes Myc and Bcl2 [15]. These transformed cells produced leukemia in vitro and in vivo. Gene expression profiling indicated expression of some of the genes active in the non-transformed cells, including transcription factors Bcl11b and Gata3. Knock-down of either of these genes impaired growth of the DN2 derived leukemias. These DN2 transformed cells might have interest as models for human cancer since they have a counterpart in some samples of human acute myeloid leukemias (AMLs) and are refractory to standard drugs used to treat AML. A screen for alternative therapeutic options indicated Jak2/Stat3 pathway inhibition.

Amyloid deposits in the brain are a feature of several neurodegenerative diseases, but it is unclear whether these aggregates are a cause or only a result of the disease. To find out if treating the aggregates could be of therapeutic use, we first selected compounds that dissociate Abeta42 aggregates (characteristic of Alzheimer's disease, AD) using a fluorescence polarization assay [16]. Disperse Orange 1 (DO1) ([4-((4-nitrophenyl)diazenyl)-N-phenylaniline]) was identified. The compound was tested in vitro and in vivo using 5xFAD transgenic mice, which accumulate deposits of Abeta42 in the brain and are widely used as AD model. Administration of DO1 reduced aggregates, improved behaviour and, generally, made the brain gene expression in the brain of 5xFAD mice more similar to that of normal mice.

References

[1] Baldwin, R.M., M. Garratt-Lalonde, D.A.E. Parolin, P.M. Krzyzanowski, M.A. Andrade, and I.A.J. Lorimer. 2006. Protection of glioblastoma cells from cisplatin cytotoxicity via protein kinase Ciota-mediated attenuation of P38 MAP Kinase signaling. Oncogene. 25, 2909-2919.

[2] Bonnet, S., E.D. Michelakis, C.J. Porter, M.A. Andrade-Navarro, B. Thébaud, S. Bonnet, A. Haromy, G. Harry, R. Moudgil, M.S. McMurtry, E.K. Weir, and S.L. Archer. 2006. An abnormal mitochondrial-HIF-1alpha-Kv channel pathway disrupts oxygen-sensing and triggers pulmonary arterial hypertension (PAH) in fawn-hooded rats: similarities to human PAH. Circulation. 113, 2630-2641.

[3] Kulkarni, G., D.A. Turbin, A. Amiri, S. Jeganathan, M.A. Andrade-Navarro, T.D. Wu, D.G. Huntsman and J.M. Lee. 2007. Expression of protein elongation factor eEF1A2 predicts favorable outcome in breast cancer. Breast Cancer Research and Treatment. 102, 31-41.

[4] Bonnet, S., S.L. Archer, J. Allalunis-Turner, A. Haromy, C. Beaulieu, R. Thompson, C.T. Lee, G.D. Lopaschuck, L. Puttagunta, S. Bonnet, G. Harry, K. Hashimoto, C.J. Porter, M.A. Andrade, B. Thebaud, E.D. Michelakis. 2007. A mitochondria-K+ channel axis is suppressed in cancer and its normalization promotes apoptosis and inhibits cancer growth. Cancer Cell. 11, 37-51.

[5] Grenier, G., A. Scimè, A. Asakura, C. Perez-Iratxeta, M.A. Andrade-Navarro, P.A. Labosky and M.A. Rudnicki. 2007. Resident endothelial precursors in muscle, adipose and dermis contribute to post-natal vasculogenesis. Stem Cells. 25, 3101-3110.

[6] McCann, J.A., E.M. Muro, C. Palmer, G. Palidwor, C.J. Porter, M.A. Andrade-Navarro and M.A. Rudnicki. 2007. ChIP on SNP-chip for genome-wide analysis of human histone H4 hyperacetylation. BMC Genomics. 8, 322.

[7] Muro, E.M., J.A. McCann, M.A. Rudnicki and M.A. Andrade-Navarro. 2009. Use of SNP-Arrays for ChIP assays: computational aspects. Methods Mol Biol. 567, 145-154.

[8] Bröske, A., L. Vockentanz, S. Kharazi, M.R. Huska, E. Mancini, M. Scheller, A. Enns, M. Prinz, R. Jaenisch, C. Nerlov, A. Leutz, M.A. Andrade-Navarro, S.E.W. Jacobsen, F. Rosenbauer. 2009. DNA methylation protects hematopoietic stem cell multipotency from myeloerythroid restriction. Nature Genetics. 41, 207-215.

[9] Scimè, A., J. Desrosiers, F. Trensz, G.A. Palidwor, A.Z. Caron, M.A. Andrade-Navarro, G. Grenier. 2010. Transcriptional profiling of skeletal muscle reveals factors that are necessary to maintain satellite cell integrity during ageing. Mechanisms of Ageing and Development. 131, 9-20.

[10] Christ, A., S. Terryn, E.I. Christensen, M.R. Huska, M.A. Andrade-Navarro, N. Hübner, O. Devuyst, A. Hammes, and T.E. Willnow. 2010. The soluble intracellular domain of megalin does not affect renal proximal tubular function in vivo. Kidney International. 78, 473-477.

[11] Herault,O., K.J. Hope, E. Deneault, N. Mayotte, J. Chagraoui, B.T. Wilhelm, S. Cellot,  A. Faubert, P. Austin, M. Trost, M. Fréchette, L. Voisin, P. Chagnon, E. Soucie, M. Sauvageau, M.A. Andrade-Navarro, G. Sauvageau. 2012. A role for GPx3 in activity of normal and leukemia stem cells. J. Exp. Med. 209, 895-901.

[12] Schönheit, J., C. Kuhl, M.L. Gebhardt, F. Fernández Klett, P. Riemke, M. Scheller, G. Huang, R. Naumann, A. Leutz, C. Stocking, J. Priller, M.A. Andrade-Navarro and F. Rosenbauer. 2013. PU.1 level-directed chromatin structure remodeling at the Irf8 gene drives dendritic cell commitment. Cell Reports3, 1617-1628.

[13] Fehlker, M., M.R. Huska, T. Jöns, M.A. Andrade-Navarro, W. Kemmner. 2014. Concerted down-regulation of immune-system related genes predicts metastasis in colorectal carcinoma. BMC Cancer. 14, 64.

[14] Schmid, F., Q. Wang, M.R. Huska, M.A. Andrade-Navarro, M. Lemm, I. Fichtner, M. Dahlmann, D. Kobelt, W. Walther, J. Smith, P.M. Schlag and U. Stein. 2016. SPON2, a newly identified target gene of MACC1, drives colorectal cancer metastasis in mice and is prognostic for colorectal cancer patient survival. Oncogene. 35, 5942-5952.

[15] Riemke, P., M. Czeh, M. Fischer, C. Walter, S. Ghani, M. Zepper, K. Agelopoulos, S. Lettermann, M.L. Gebhardt, N. Mah, A. Weilemann, M. Grau, V. Gröning, T. Haferlach, D. Lenze, R. Delwel, M. Prinz, M.A. Andrade-Navarro, G. Lenz, M. Dugas, C. Müller-Tidow and F. Rosenbauer. 2016. Myeloid leukemia with transdifferentiation plasticity developing from T-cell progenitors. EMBO Journal. 35, 2399-2416.

[16] Boeddrich A., J.T. Tachu, T. Wiglenda, L. Diez, M. Jacob, W. Nietfeld, M.R. Huska, C. Haenig, N. Groenke, A. Buntru, E. Blanc, J.C. Meier, E. Vannoni, C. Erck, B. Friedrich, H. Martens, N. Neuendorf, S. Schnoegl, D.P. Wolfer, M. Loos, D. Beule, M.A. Andrade-Navarro and E.E. Wanker. 2018. The anti-amyloid compound DO1 decreases plaque pathology and neuroinfammation-related expression changes in 5xFAD transgenic mice. Cell Chem. Biol. 26,109-120.