transporter expression in the gut and tumors pascale anderle, isrec
TRANSCRIPT
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
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:
5 days vs. 3 weeks
A-values7 9 11 13 15
-3
-
2
-
1
0
1
2
3
Differentiation of Caco-2 cells
M-v
alu
es
- 3
-
2
-
1
0
1
2
3
A-values7 9 11 13 15
5 days vs. 5 days
M-v
alu
es
- 3
-
2
-
1
0
1
2
3
A-values7 9 11 13 15
5 days vs. 1 week 5 days vs. 2 weeks
M-v
alu
es
- 3
-
2
-
1
0
1
2
3
A-values7 9 11 13 15
Time
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:
12 x Mu74Av2
12 x Mu74Bv2
12 x Mu74Cv2
Duodenum Jejunum Ileum Colon
Triplicates → 3 Pools of 10 mice
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>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.
Isrec Ontologizer Io
Selection of hierarchical level
P-value
P-value filterGene search
Classification of probe setsClassification of UniGenesClassification of RefSeqs
Quality filter
Ambiguity of results
Export
Regulation of Transporters along the Intestine
Fold Changes along the Intestine
Expression along the Crypt-Villus Axis
V C V C V C V C
D J I C
V C V C V C V C
D J I C
V C V C V C V C
D J I C
rel.
exp
res
sio
n l
evel
s [l
og
2]re
l. ex
pre
ssi
on
leve
ls [
log
2]re
l. ex
pre
ssio
n l
evel
s [l
og
2] Abca1
Slc34a2Slc2a1
Slc28a2Slc10a2Abcg8
Abcc1 Abcc3
Slc5a8
V C V C V C V C
D J I C
V C V C V C V C
D J I C
V C V C V C V C
D J I C
V C V C V C V C
D J I C
V C V C V C V C
D J I C
V C V C V C V C
D J I C
rel.
exp
res
sio
n l
evel
s [l
og
2]re
l. ex
pre
ssi
on
leve
ls [
log
2]re
l. ex
pre
ssio
n l
evel
s [l
og
2] Abca1
Slc34a2Slc2a1
Slc28a2Slc10a2Abcg8
Abcc1 Abcc3
Slc5a8
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
Goal:
Study the role of transporters in cancer-sensitivity and cancer-resistance
Reference:
NCI Database
Scherf et al. 2000, Nature Genetics
SLCs: Chemo-sensitivity and -resistance
Sorted correlation coefficients
ABCB1
SLC29A1
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
Wolfgang SadéeVera RakhmanovaYing HuangShoshana BrownAudrey PappZunyan Dai
Joe DeRisiAdam CarrollJingchun Zhu
Xenoport
Katie WoodfordNoa Zerangue
National Cancer Institute
John WeinsteinKimberly BusseyCatalin BarbacioruUma ShankavaramWilliam C. Reinhold
Acknowledgments II
ISREC
Jean-Pierre KraehenbuhlMartin Rumbo
Bioinformatics Core Facility
Thierry SengstagMauro Delorenzi
Nestlé
Muriel FiauxRobert MansourianDavid MutchMatthew-Alan RobertsGary Williamson
Swiss Institute of Bioinformatics
Philipp BucherChristian IseliViviane Praz