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Evaluation of MRP1-5 gene expression
in cystic fibrosis patients
homozygous for the DF508 mutation
Cystic fibrosis : variable severity
CFTR : Cl- channel function heterogeneity
Factors CFTR
Structural homology : CFTR / MRPs
out
ABC transporter COOH
NH2
NBD1 NBD2
out
CFTR : Cystic Fibrosis
Transmembrane conductance COOH
Regulator NH2 NH2
R
NBD1 NBD2
C
NH2
out
MRPs : Multidrug
Resistance-associated Proteins
COOH
TMD0
NBD1 NBD2
Functional homology?
MRPs CFTR
inside inside
Organic anions Cl -
conjugated to Organic anions
glutathione Glutathione
glucuronate
sulfate
Detoxification
Inflammation
Oxydative stress
Drug resistance
Clinical status
Age
FEV1
MRP1, -2, -3, -4, -5 gene expression
FVC
RT-PCR Shwachman score
• DF508 homozygous patients (n=19)
• Nasal respiratory epithelial cells Cl - conductances
Relative to controls (n=20)
SPQ assay
Cell type of the analyzed nasal samples Subject Class
1 C 8
2 C 8
3 C 8
4 C 6
5 C 7
Cell type Class 6
7
C
C
8
8
8 C 8
Necrotic cells or absence of respiratory epithelial cells 9 C 8
10 C 8
Necrotic cells 1 11 C 8
Absence of cells 2 12 C 8
13 C 8
Squamous cells or rare bare nuclei 3 14 C 8
15 C 8
16 C 8
17 C 8
Presence of inflammatory (polymorphonuclear) cells 18
19
C
C
8
8
Epithelial cells associated with > 40% polymorphonuclear cells 4 20 C 6
Epithelial cells associated with 20-40% polymorphonuclear cells 5 21 CF 8
22 CF 7
Epithelial cells associated with < 20% polymorphonuclear cells 6 23 CF 8
24 CF 8
25 CF 8
26 CF 4
Absence of inflammatory cells 27 CF nd
28 CF 7
Ciliated cells <50% 7 29 CF 7
30 CF 8
Ciliated cells >50% 8 31 CF 8
32 CF 7
33 CF 8
34 CF 8
35 CF 8
36 CF 6
37 CF 8
38 CF 8
39 CF 8
A B
3 3 p < 0.05 0.75 0,75
0,5
MRP1
MRP3
2 2 0.5
11 0.25 0,25
0
00 0
0 3
C CF 0 C CF 3
(n=20) (n=19) (n=20) (n=19)
E
C D
MDR1
0.50,5
p < 0.01
3
1.51,5 3
2.5 2,5
MRP5
2 2 0 0
1 1
MRP4
0 4
1.5 1,5
C CF
0.50,5 1 1 (n=20) (n=19)
0.5 0,5
0 0
0 0
0 3
0 C CF 3 C CF
(n=20) (n=19) (n=20) (n=19)
Relation of MRP1 and MRP5 levels with clinical status
MRP1 0.15 MRP1 > 0.15 p
(n=5) (n=14)
Age 14 + 2 12 + 2 0.4
FEV1 87 + 8 89 + 4 0.8
FVC 87 + 11 89 + 5 0.8
Shwachman score 74 + 7 88 + 3 0.05
MRP5 0.08 MRP5 > 0.08 p
(n=5) (n=14)
Age 14 + 2 12 + 2 0.6
FEV1 88 + 8 89 + 4 0.8
FVC 84 + 11 90 + 5 0.5
Shwachman score 76 + 7 87 + 3 0.1
Relation of MRP1 and MRP5 levels with Cl- conductances
SPQ (6-methoxy-N-3-sulfopropylquinolinium) fluorescent dye assay
(Cl-)
Cl- conductance (a cell) DF/Dt > 0.1
Results (a subject) % cells with DF/Dt > 0.1
Previous study in controls Lowest % cells with DFcAMP /Dt > 0.1 : 50%
Lowest % cells with DFbasal /Dt > 0.1 : 63%
Relation of MRP1 and MRP5 levels
with cAMP-dependent Cl- conductances
(% cells with DFcAMP /Dt > 0.1 / Lowest value in controls : 50%)
< 50% 50% p
(n=11) (n=8)
MRP1 0.48 + 0.1 0.66 + 0.15 0.34
MRP5 0.69 + 0.15 0.78 + 0.22 0.74
Relation of MRP1 and MRP5 levels with basal Cl- conductances
(% cells with DFbasal /Dt > 0.1 / Lowest value in controls : 63%)
* p < 0.01
1
0.8
0.6
MRP1
0.4
0.2
0
0
< 63% 63%
1
(n=6) (n=13)
< 63% 63%
(n=6) (n=13) p
MRP1 0.23 + 0.12 0.7 + 0.09 < 0.01
MRP5 0.49 + 0.19 0.84 + 0.15 0.19
Conclusion
Lower MRP1 and MRP5 mRNA levels
in DF508 homozygous patients
MRP1 mRNA levels
clinical status
cAMP-independent Cl- conductance
Conclusion
Role in CF pathophysiology?
MRP1
Cl- channel-related function?
Service d’Histologie-Biologie Tumorale, UPRES EA3499,
Hôpital Tenon, 75020 Paris
Ilse Hurbain
Jean-François Bernaudin
Anne Fajac
Inserm U467, Faculté Necker, 75015 Paris
Benoît Vallée
Aleksander Edelman
Service de Pédiatrie, Hôpital Necker, 75015 Paris
Isabelle Sermet-Gaudelus
Gérard Lenoir
Service de Biochimie, Hôpital Necker, 75015 Paris
Marie-Noëlle Feuillet
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