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Transporter Expression in the Gut and Tumors Pascale Anderle, ISREC Overview Genomic profiling of transporters in Caco-2 cells and human intestine using a custom array Genomic profiling of transporters along the mouse intestine using Affymetrix GeneChips Identification of transporters responsible for chemo-sensitivity and chemo-resistance using a custom array Evaluation of Transporter and Channel Genes in the Intestine Reference: Goals: Caco-2 cells: Differentiated cells vs. undifferentiated cells Small intestinal and colonic tissues vs. Caco-2 cells Differentiation of Caco-2 cells 5 days vs. 3 weeks 5 days vs. 5 days M-values Time 5 days vs. 2 weeks 5 days vs. 1 week 9 11 13 15 7 A-values 7 9 9 11 A-values 13 15 11 A-values M-values M-values 7 7 9 11 A-values 13 15 13 15 Summary Differentiation of Caco-2 cells: During differentiation: Expression pattern changes Up and down regulation usually < 2 fold Significant changes between 5 days to 1 week and 1 week to 2 weeks No significant changes between 2 weeks and 3 weeks Genes in general related to ion household No major differences between flasks and filters (except GLUT3) Typical small intestinal transporters not especially up regulated in differentiated cells Comparison Tissue vs. Caco-2 Cells: Changes more pronounced between tissue and cell line than between undifferentiated and differentiated cells Tissue vs. Caco-2 cells: More ratios > 2 fold No trend observed: undiff. cells to diff. cells = colon-like to small intestinal-like cells Genomic Profiling of Membrane Transporters in the Intestine Objective: Identification of putative segment-specific and non-specific specific drug carriers Study Design: Triplicates → 3 Pools of 10 mice Duodenum Jejunum Ileum 12 x Mu74Av2 12 x Mu74Bv2 12 x Mu74Cv2 Colon Accurate Annotation of Probe Sets A crucial step in classifying genes into different molecular functions is the use of a consistent and universal classification system and a precise annotation of Affymetrix probe sets. Mapping of single probes and attributing of annotation tags to each probe set (quality tags) Assigning of flags to differentially regulated probe sets (F, G, 1) 1 PS per Gene Gene A >2 PS per Gene Gene C Gene B 1=1 >2 PS per Gene G=1 F=1 Isrec Ontologizer Io Selection of hierarchical level Quality filter Classification of probe sets Classification of UniGenes Classification of RefSeqs P-value filter Gene search Export Ambiguity of results P-value Regulation of Transporters along the Intestine Fold Changes along the Intestine rel. expression levels [log2] rel. expression levels [log2] rel. expression levels [log2] Expression along the Crypt-Villus Axis Abca1 Abcc1 Abcc3 Abcg8 Slc10a2 Slc28a2 Slc2a1 Slc34a2 Slc5a8 V C V C V C V C V C V C V C V C V C V C V C V C D J I C D J I C D J I C Summary Majority of transporters not significantly expressed along the intestine Carriers and in particular the classes “symporters” and “antiporters” change significantly Transporter subclasses change along the whole intestine, most between ileum and colon Similar number of transporters over-expressed in small and large intestine, but generally transporters are expressed at higher levels in the small intestine Most differently regulated transporters are similarly expressed along the small intestine, yet various transporters are clearly segment-specific Most transporters similarly expressed in humans and mice Regulation of expression in whole tissue is a good indicator of the combined changed in epithelial crypt and villus cells Identification of various new candidate carriers for drug delivery (mRNA protein) Expression of Transporters in Cancers Reference: Goal: Study the role of transporters in cancer-sensitivity and cancer-resistance NCI Database Scherf et al. 2000, Nature Genetics SLCs: Chemo-sensitivity and -resistance Sorted correlation coefficients SLC29A1 ABCB1 Effects of regulation of transporters on dose-response curves Summary Important role of transporters in chemo-sensitivity and-resistance ABCB1 and ABCB5 identified as resistance genes for various compounds SLC29A1, nucleoside transporter, identified as sensitivity gene Collection of information bits to perform follow-up studies Acknowledgments I UCSF/OSU Xenoport National Cancer Institute Wolfgang Sadée Vera Rakhmanova Ying Huang Shoshana Brown Audrey Papp Zunyan Dai Katie Woodford Noa Zerangue John Weinstein Kimberly Bussey Catalin Barbacioru Uma Shankavaram William C. Reinhold Joe DeRisi Adam Carroll Jingchun Zhu Acknowledgments II ISREC Bioinformatics Core Facility Nestlé Jean-Pierre Kraehenbuhl Martin Rumbo Thierry Sengstag Mauro Delorenzi Muriel Fiaux Robert Mansourian David Mutch Matthew-Alan Roberts Gary Williamson Swiss Institute of Bioinformatics Philipp Bucher Christian Iseli Viviane Praz