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. Goals: Caco-2 cells: Differentiated cells vs. undifferentiated cells Small intestinal and colonic tissues vs. Caco-2 cells Evaluation of Transporter and Channel Genes in the Intestine Reference:

4. 5 days vs. 3 weeks A-values 7 9 11 13 15 -3 -2 -1 0 1 2 3 Differentiation of Caco-2 cells M-values -3 -2 -1 0 1 2 3 A-values 7 9 11 13 15 5 days vs. 5 days M-values -3 -2 -1 0 1 2 3 A-values 7 9 11 13 15 5 days vs. 1 week 5 days vs. 2 weeks M-values -3 -2 -1 0 1 2 3 A-values 7 9 11 13 15 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. Genomic Profiling of Membrane Transporters in the Intestine Objective: Identification of putative segment-specific and non-specific specific drug carriers Study Design: 12 x Mu74Av2 12 x Mu74Bv2 12 x Mu74Cv2 Duodenum Jejunum Ileum Colon Triplicates → 3 Pools of 10 mice

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

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

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 Goal: Study the role of transporters in cancer-sensitivity and cancer-resistance Reference:

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

15. SLCs: Chemo-sensitivity and -resistance

16. Sorted correlation coefficients ABCB1 SLC29A1

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