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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