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Transporter Expression in the Gut and Tumors

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

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Transporter Expression in the Gut and Tumors

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  1. Transporter Expression in the Gut and Tumors Pascale Anderle, ISREC

  2. 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

  3. 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

  4. -3 -2 -1 0 1 2 3 -3 -2 -1 0 1 2 3 -3 -2 -1 0 1 2 3 -3 -2 -1 0 1 2 3 M-values M-values M-values 5 days vs. 3 weeks 5 days vs. 5 days 7 9 11 13 15 7 9 11 13 15 7 9 11 13 15 7 9 11 13 15 A-values A-values A-values A-values 5 days vs. 2 weeks 5 days vs. 1 week Differentiation of Caco-2 cells Time

  5. 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

  6. Study Design: Triplicates → 3 Pools of 10 mice Ileum Colon Duodenum Jejunum 12 x Mu74Av2 12 x Mu74Bv2 12 x Mu74Cv2 Genomic Profiling of Membrane Transporters in the Intestine Objective: Identification of putative segment-specific and non-specific specific drug carriers

  7. 1 PS per Gene >2 PS per Gene >2 PS per Gene Gene A Gene C Gene B G = 1 1 = 1 F = 1 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)

  8. Selection of hierarchical level Quality filter Classification of probe sets Classification of UniGenes Classification of RefSeqs Export P-value filter Gene search P-value Ambiguity of results Isrec Ontologizer Io

  9. Regulation of Transporters along the Intestine

  10. Fold Changes along the Intestine

  11. Expression along the Crypt-Villus Axis

  12. 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)

  13. Expression of Transporters in Cancers Reference: Goal: Study the role of transporters in cancer-sensitivity and cancer-resistance

  14. NCI Database Scherf et al. 2000, Nature Genetics

  15. SLCs: Chemo-sensitivity and -resistance

  16. SLC29A1 ABCB1 Sorted correlation coefficients

  17. Effects of regulation of transporters on dose-response curves

  18. 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

  19. Acknowledgments I UCSF/OSU Wolfgang Sadée Vera Rakhmanova Ying Huang Shoshana Brown Audrey Papp Zunyan Dai Joe DeRisi Adam Carroll Jingchun Zhu Xenoport Katie Woodford Noa Zerangue National Cancer Institute John Weinstein Kimberly Bussey Catalin Barbacioru Uma Shankavaram William C. Reinhold

  20. Acknowledgments II ISREC Jean-Pierre Kraehenbuhl Martin Rumbo Bioinformatics Core Facility Thierry Sengstag Mauro Delorenzi Nestlé Muriel Fiaux Robert Mansourian David Mutch Matthew-Alan Roberts Gary Williamson Swiss Institute of Bioinformatics Philipp Bucher Christian Iseli Viviane Praz

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