Expression and function of ABC multidrug transport proteins in patients with non-responsive focal epilepsy [Elektronische Ressource] / vorgelegt von: Snieguole Upermonaite

Snieguole Upermonaite

Sujets

• Gesundheit

Informations

• Publié par: ernst-moritz-arndt-universitat_greifswald
• Publié le: 01 janvier 2006
• Nombre de lectures: 26
• Langue: Deutsch
• Poids de l'ouvrage: 1 Mo

Extrait

Aus der Abteilung Klinische Pharmakologie
des Instituts für Pharmakologie
(Leiter Prof. Dr. med. W. Siegmund)
der Medizinischen Fakultät der Ernst-Moritz-Arndt-Universität Greifswald
Expression and function of ABC multidrug
transport proteins in patients with
non-responsive focal epilepsy
Inaugural - Dissertation
zur
Erlangung des akademischen
Grades
Doktor der Medizin
(Dr. med.)
der
Medizinischen Fakultät
der
Ernst-Moritz-Arndt-Universität
Greifswald
2006

vorgelegt von:
Snieguole Upermonaite
geboren am 14.11.1974
in Telsiai, Litauen
















Dekan: Prof. Dr. rer. nat. H. K. Kroemer

1. Gutachter: Prof. Dr. W. Siegmund
2. Gutachter: Prof. Dr. I. Cascorbi

Tag der Promotion: 19. Oktober 2006
2 The important thing in science
is not so much to obtain new facts
as to discover new ways of thinking about them.
Sir W. H. Bragg
TABLE OF CONTENTS
TABLE OF CONTENTS
SUMMARY .....................................................................................................7
1 INTRODUCTION ..........................................................................................10
2 OBJECTS.....................................................................................................14
3 MATERIALS AND METHODS .....................................................................19
3.1 Study population...........................................................................................19
3.1.1 Control..........................................................................................................19
3.1.2 Patients.........................................................................................................19
3.2 Genotyping ...................................................................................................20
3.2.1 DNA isolation................................................................................................21
3.2.2 Polymerase-Chain-Reaction-Restriction-Fragment-Length-Polymorphism
(PCR-RFLP) .................................................................................................21
3.2.3 PCR..............................................................................................................22
3.2.4 Restriction Fragment Length Polymorphism (RFLP) ....................................23
3.2.5 Gel electrophoresis.......................................................................................23
3.3 Isolation of peripheral blood lymphocytes.....................................................23
3.4 Measurement of mRNA expression of MDR1, MRP1 and MRP2 .................24
3.4.1 RNA isolation................................................................................................24
3.4.2 Real-time PCR assay ...................................................................................24
3.5 Measurement of P-glycoprotein and MRP2 by Western Blot........................27
3.5.1 Preparation of samples containing a tagged protein.....................................27
3.5.2 Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)...28
3.5.3 Immunological detection...............................................................................28
3.6 P-glycoprotein functional analysis.................................................................29
3.6.1 Lymphocytes incubation with fluorescent dye...............................................30
3.6.2 Labelling with antibodies...............................................................................30
4 TABLE OF CONTENTS
3.6.3 Flow cytometry..............................................................................................31
3.7 Statistical analysis ........................................................................................32
4 RESULTS.....................................................................................................34
4.1 MDR1 genotype and haplotype distribution..................................................34
4.2 MRP2 genotype and haplotype distribution ..................................................35
4.3 MRP1 expression in lymphocytes.................................................................37
4.3.1 MRP1 expression in healthy controls and patients with epilepsy..................37
4.3.2 MRP1 expression in responders and non-responders
to antiepileptic therapy..................................................................................37
4.4 MRP2 expression in lymphocytes.................................................................38
4.4.1 MRP2 expression in healthy controls and patients with epilepsy..................38
4.4.2 MRP2 expression in responders and non-responders
to antiepileptic therapy..................................................................................38
4.4.3 Correlation between MRP2 mRNA expression and protein content .............39
4.5 MDR1 expression and function of P-glycoprotein in lymphocytes ................40
4.5.1 MDR1 expression in healthy controls and patients with epilepsy .................40
4.5.2 P-glycoprotein function in healthy controls and patients with epilepsy..........40
4.5.3 MDR1 expression in responders and non-responders
to antiepileptic therapy..................................................................................42
4.5.4 P-glycoprotein function in responders and non-responders
to antiepileptic therapy..................................................................................42
4.5.5 Correlation between MDR1 mRNA expression, P-glycoprotein content
and P-glycoprotein function in lymphocytes..................................................44
4.6 Correlation between expression of multidrug transporter proteins
in lymphocytes and brain tissue....................................................................45
4.7 Impact of MDR1 polymorphisms on the MDR1 expression
and function in lymphocytes .........................................................................47
5 TABLE OF CONTENTS
4.8 Impact of MRP2 polymorphisms on the MRP2 expression in lymphocytes ..48
5 DISCUSSION ...............................................................................................49
5.1 Multidrug transporters in the brain ................................................................50
5.2 Multidrug transporters role in epilepsy..........................................................52
5.3 Lymphocytes as a study surrogate ...............................................................54
5.3.1 Multidrug transporters in lymphocytes ..........................................................55
5.3.2 Variability of multidrug transporters in lymphocytes
by drugs, genotype and disease...................................................................57
5.3.3 Expression of multidrug transporters in lymphocytes
of epileptic patients.......................................................................................58
5.3.4 Rhodamine efflux of lymphocytes and natural killer cells..............................60
5.4 Correlation between multidrug transporters expression
in the brain and in lymphocytes ....................................................................62
5.5 Pharmacogenetics in epilepsy ......................................................................63
5.6 Conclusions ..................................................................................................65
6 REFERENCE LIST.......................................................................................66
7 APPENDIX ...................................................................................................81
7.1 Chemicals, enzymes.....................................................................................81
7.2 Instruments, devices.....................................................................................84
7.3 Solutions and buffers....................................................................................85
7.4 Tables...........................................................................................................89

6 SUMMARY
SUMMARY
About 30 % of epileptic patients are non-responsive even to multidrug antiepileptic
therapy. One of non-responsiveness in epilepsy hypothesis claims that non-
responsiveness occurs because of reduced access of antiepileptic drugs to their neu-
ronal targets, as a result of increased efflux or sequestration of antiepileptic drugs
away from these targets. Transporters believed to be involved in non-responsiveness
in epilepsy are mainly but not exclusively the members of the ABC superfamily in-
cluding P-gp (MDR1, ABCB1), MRP1 (ABCC1), MRP2 (ABCC2) and others. These
proteins are normally found in a variety of locations, consistent in each case with their
postulated roles in generating protective barriers such as the blood-brain barrier and
the blood-cerebrospinal fluid barrier. There is emerging evidence that P-gp, MRP1
and MRP2 are up-regulated in epileptogenic brain tissue. The risk of non-
responsiveness could be related also to the MDR1 or MRP2 gene polymorphisms.
We hypothesised that changes in expression and func