Inducible expression of the human TRP3 cation channel in a prostate cancer cell line [Elektronische Ressource] / vorgelegt von Nina Vogel
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2004
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Aus demInstitut für Biochemie und Molekularbiologie _:Zelluläre Signaltransduktiondes Universitätsklinikums Hamburg-EppendorfDirektor Professor G. W. MayrInducible expression of the human TRP3 cation channelin a prostate cancer cell lineDissertationzur Erlangung des Grades eines Doktors der Medizindem Fachbereich Medizin der Universität Hamburg vorgelegt vonNina Vogelaus Bremen/ BremenHamburg 2003iAngenommen vom Fachbereich Medizinder Universität Hamburg am:24. Juni 2004Veröffentlicht mit Genehmigung des Fachbereichs Medizin an der Universität HamburgPrüfungsausschuss, der/die Vorsitzende:Prof. Dr. A. GusePrüfungsausschuss: 2. Gutachter/in:Prof. Dr. W. FiedlerPrüfungsausschuss: 3. Gutachter/in:Prof. Dr. J SchwarziiTable of ContentsTABLE OF CONTENTS...................................................................................... IIITABLE OF FIGURES ..........................................................................................VLIST OF ABBREVIATIONS ...............................................................................VISUMMARY .......................................................................................................VIII1. INTRODUCTION............................................................................................. 11.1 Prostate cancer ............................................................................................................................................. 11.
Voir
Aus dem
Institut für Biochemie und Molekularbiologie _:
Zelluläre Signaltransduktion
des Universitätsklinikums Hamburg-Eppendorf
Direktor Professor G. W. Mayr
Inducible expression of the human TRP3 cation channel
in a prostate cancer cell line
Dissertation
zur Erlangung des Grades eines Doktors der Medizin
dem Fachbereich Medizin der Universität Hamburg vorgelegt von
Nina Vogel
aus Bremen/ Bremen
Hamburg 2003
i
Angenommen vom Fachbereich Medizin
der Universität Hamburg am:
24. Juni 2004
Veröffentlicht mit Genehmigung des Fachbereichs Medizin an der Universität Hamburg
Prüfungsausschuss, der/die Vorsitzende:
Prof. Dr. A. Guse
Prüfungsausschuss: 2. Gutachter/in:
Prof. Dr. W. Fiedler
Prüfungsausschuss: 3. Gutachter/in:
Prof. Dr. J Schwarz
ii
Table of Contents
TABLE OF CONTENTS...................................................................................... III
TABLE OF FIGURES.......................................................................................... V
LIST OF ABBREVIATIONS............................................................................... VI
SUMMARY....................................................................................................... VIII
1. INTRODUCTION............................................................................................. 1 1.1 Prostate cancer ............................................................................................................................................. 1 1.2 Androgen independence in prostate cancer................................................................................................. 2 1.3 Apoptosis and Calcium ................................................................................................................................ 3 1.4 Calcium signalling and calcium channels ................................................................................................... 4 1.5 TRP1............................................................................................................................................................. 6 1.6 TRP2, 4 and 5............................................................................................................................................... 6 1.7 CaT1 and TRP-p8 ........................................................................................................................................ 7 1.8 TRP3, 6 and 7............................................................................................................................................... 7 1.9 TRP3 calcium channel ................................................................................................................................. 8 1.10 Calcium channels and cell death ............................................................................................................... 9 1.11 Inducible hTRP3 expression.................................................................................................................... 10
2. AIMS.............................................................................................................. 11
3. MATERIALS AND METHODS...................................................................... 11
3.1 Materials........................................................................................................................................................ 11
3.2 Cloning of the pBI-EGFP/hTRP3 vector .................................................................................................. 14 3.2.1 Generation of electro competent bacteria .............................................................................................. 14 3.2.2 Transformation of bacteria ..................................................................................................................... 14 3.2.3 Plasmid extraction................................................................................................................................... 14 3.2.4 Restriction enzyme digests ..................................................................................................................... 15 3.2.5 Gel purification of DNA fragments ....................................................................................................... 15 3.2.6 Ligation reactions.................................................................................................................................... 16 3.2.7 Sequencing of the pBI-EGFP/hTRP3 .................................................................................................... 17
3.3 Cell culture of LNCaP prostate cancer cell line ....................................................................................... 17 3.3.1 Cell culture .............................................................................................................................................. 17 3.3.2 Seeding of LNCaP cells for transfection................................................................................................ 17 3.3.3 Growing cells on glass coverslips for calcium measurements .............................................................. 18
3.4 Transfection of LNCaP cells ....................................................................................................................... 18 3.4.1 Transient transfection of LNCaP cells ................................................................................................... 18 3.4.2 Proliferation assay for G418 and hygromycin B ................................................................................... 19 3.4.3 Stable transfection of LNCaP cells with the Tet-on plasmid ................................................................ 19 3.4.4 Preparation of cell extracts for western blot analysis ............................................................................ 20
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3.4.5 Protein estimation ................................................................................................................................... 20
3.5 Western blot analysis ................................................................................................................................... 21 3.5.1 Gel electrophoresis.................................................................................................................................. 21 3.5.2 Protein transfer and hTRP3 detection .................................................................................................... 21
3.6 Calcium measurements ............................................................................................................................... 22 3.6.1 Loading and location of Fura-2/AM in LNCaP cells ............................................................................ 23 3.6.2 Basal and agonist activated calcium inflow........................................................................................... 23
3.7 Viability assay ............................................................................................................................................... 24 3.7.1 Hoechst 33258 staining........................................................................................................................... 25
4. RESULTS....................................................................................................... 25 4.1 Cloning of hTRP3 into the pBI-EGFP vector........................................................................................... 25 4.2 Sequencing of the pBI-EGFP/hTRP3 construct ....................................................................................... 30 4.3 Transient transfection of LNCaP cells ...................................................................................................... 32 4.4 Stable transfection of LNCaP cells ........................................................................................................... 36 4.5 Transfection with the pBI-EGFP/hTRP3 plasmid .................................................................................... 39 4.6 Detection of hTRP3 ................................................................................................................................... 42 4.7 Peak of hTRP3 expression......................................................................................................................... 45 4.8 Localisation of Fura-2/AM ........................................................................................................................ 47 4.9 Thapsigargin stimulated calcium inflow ................................................................................................... 49 4.10 OAG activated calcium inflow ................................................................................................................ 53 4.11 Effect of hTRP3 on cell viability and cell death..................................................................................... 58
5. DISCUSSION.................................................................................................. 60
6. CONCLUSION............................................................................................... 68
BIBLIOGRAPHY............................................................................................... 70
ACKNOWLEDGMENTS................................................................................... 77
LEBENSLAUF................................................................................................... 78
EIDESSTATTLICHE VERSICHERUNG........................................................... 79
iv
Table of Figures
Figure 1:Restriction enzyme digests of pcDNA3/hTRP3 and pGL3-Basic/hTRP3.................................27
Figure 2:Restriction enzyme digest of pBI-EGFP/hTRP3........................................................................29
Figure 3:Sequencing result of the pBI-EGFP/hTRP3 vector.................................................................. .31
Figure 4:Transfection efficiency using different amounts of pBI-EGFP..................................................33
Figure 5:Doxycyclin dependent induction of EGFP after 48 hrs..............................................................35
Figure 6:Proliferation assay for G418 and hygromycin B........................................................................38
Figure 7:Transfection efficiency for LNCaP/Tet-on cells transiently transfected....................................41
Figure 8:blot analysis of hTRP3 expressed in LNCaP/Tet-on cells...........................................43Western
Figure 9:Time course of hTRP3 expression..............................................................................................46
Figure 10: Localisation of Fura-2-AM in LNCaP/Tet-on cells.................4.8...............................................
Figure 11:Calcium inflow in LNCaP/Tet-on............................................................................................50
Figure 12:Thapsigargin stimulated calcium inflow in LNCaP/Tet-on cells.............................................52
Figure 13:Calcium inflow in LNCaP/Tet-on cells................................................................ ...................55
Figure 14:Percentage of cells responding to OAG...................................................................................56
Figure 15:OAG stimulated calcium inflow in LNCaP/Tet-on cells.........................................................57
Figure 16:Doxycyclin dependent induction of EGFP after 48 hrs...........................................................59
v
List of Abbreviations
2 [Ca+]i
CHO
CMV
DAG
DDI- H2O
ER
EGTA
FCS
Fura-2/AM
GFP/EGFP
HA
IP3
kDa
kb
lmp agarose gel
LNCaP cells
OAG
OPA buffer
PLC
RACC
SDS
Concentration of free intracellular calcium ions Chinese hamster ovary
Cytomegalo virus
Diacylglycerol
Double de-ionised water
Endoplasmic reticulum
Ethylene glycerol-bis-(ß-aminoethylether) N, N, N', N'-tetraacetic acid Fetal calf serum
Fura-2 acetoxymethyl ester
Green fluorescence protein/ Enhanced GFP
Haemagglutinin
D-myo-inositol 1,4,5-trisphosphate
Kilo Dalton
Kilo bases
Low melting point agarose gel
Lymphatic nodule cancer of prostate cells
1-oleolyl-2-acetyl-sn-glycerol
One Phor All buffer
Phospholipase C
Receptor activated calcium channel
Sodium dodecyl sulphate
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SERCA
SDS-PAGE
SOCC
TBS-T buffer
TG
TRPC
hTRP
TRPL
VOCC
vii
Sarcoplasmic endoplasmic reticulum calcium ATP-ase Sodium dodecyl sulphate polyacrylamide gel electrophoresis Store operated calcium channel
Tris-buffered saline with Tween-20
Thapsigargin
Mammalian transient receptor potential channel Human transient receptor channel
Transient receptor potential like
Voltage operated calcium channel
Summary
Prostate cancer is the second leading cause of death from cancer in men and there is
currently no curative treatment for the metastatic stage of the disease. Effective
treatments are therefore urgently needed. Inducing apoptotic cell death in prostate cancer
cells is one possible approach. Calcium has been shown to be involved in apoptosis of
prostate cancer cells. Increased calcium levels have been shown to induce cell death in a
number of prostate cancer cell lines suggesting that modulation of intracellular calcium
may be of therapeutic value in prostate cancer cells. Expression of calcium channels in
cancer cell lines is one approach by which intracellular calcium can be increased to
induce cell death.
The aims of this thesis were therefore to inducibly express hTRP3 (the human homologue
of hTRP3 a member of the TRP calcium channel family) in LNCaP prostate cancer cells
and to examine its effects on calcium inflow and cell death. TRP3 was cloned into the
pBI-EGFP expression vector that is part of the Tet-on inducible system and transiently
co-transfected with the Tet-on vector into LNCaP cells.
Western blot analysis and fluorescence revealed that both EGFP and hTRP3 were
inducibly expressed (in the presence of doxycyclin) in LNCaP cells. Calcium
measurements in hTRP3 transfected, doxycyclin treated LNCaP cells suggested that
hTRP3 was activated by thapsigargin induced store depletion and OAG, but was not
constitutively active. Transient transfection of hTRP3 into LNCaP cells also did not
induce apoptosis, which was confirmed by Hoechst 33258 staining. This may have been
viii
due to low levels of hTRP3 expression, which resulted in only small increases in
stimulated calcium inflow. To determine if hTRP3 can be used to induce cell death in
LNCaP cells further experiments optimising expression and activation of hTRP3 need to
be carried out.
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1. Introduction
1.1 Prostate cancer
Prostate cancer is the second leading cause of death in men after lung cancer. The
incidence increases dramatically with age: about 70 % of all men with clinically
diagnosed prostate cancer are 65 years or older. The American Cancer Society estimated
that 189,000 new cases as well as 30,200 deaths will result from prostate cancer in 2002
(www.cancer.org).
The current therapy for early, organ confined stages of prostate cancer, is surgery.
Surgery at these stages of the disease is usually curative. Patients with extra prostatic
disease eventually require systemic androgen ablation therapy. Prostate cancer cells are
androgen dependent and undergo apoptosis when deprived of androgen by surgical or
chemical ablation (Denmeade et al., 1996). However androgen withdrawal is not curative
and androgen independent prostate cancer cells commonly arise. Once this stage of the
disease has been reached prostate cancer is generally lethal and treatment with androgen
ablation is no longer effective. Since less than 5 % of the cancer cells proliferate every
day, chemotherapy and current anti-cancer drugs, which have an effect on fast
proliferating cancer cells have proven to be of limited success. Therefore effective
treatments for the androgen independent stage of prostate cancer are urgently needed.
1
1.2 Androgen independence in prostate cancer
Several mechanisms have been described of how prostate cancer cells survive androgen
withdrawal. Some groups have described up-regulation of the androgen receptor in
prostate cancer cells that increases the sensitivity to androgen so that those cells survive
with very low androgen levels that are present after androgen ablation (Denmeade et al.,
1996, Koivisto et al., 1997). Other groups have described that androgen independence
arises and several mechanisms have been reported. Sadi et al reported a greater androgen
receptor heterogeneity in androgen independent prostate cancer cells (Sadi and Barrack,
1993) and several mutations of the androgen receptor have been described (Taplin et al.,
1995, Elo et al., 1995). Naturally occurring amino acid substitution in the androgen
receptor ligand binding domain has been shown to change its ligand specificity and
affinity. These changes allow the androgen receptors to bind anti-androgens, adrenal
androgens, and other steroids such as estrogen and progesterone allowing the cells to
grow in the presence of these hormones (Hakimi et al., 1996, Veldscholte et al., 1990,
Berrevoets et al., 1993).
Several molecular changes in androgen independent cancer cells have been described that
allow prostate cancer cells to resist apoptosis induced by androgen withdrawal. Mutations
in the p53 tumor suppressor gene and up-regulation of the proto-oncogene bcl-2 are two
key alterations (Bruckheimer et al., 1999). Bcl-2 has been shown to inhibit apoptosis in
androgen independent prostate cancer cells (Furuya et al., 1996). The tumour suppressor
p53 induces apoptosis via up-regulation of the cell death effector of the bcl-2 family, bax
(Soussi and May, 1996, Korsmeyer, 1995). Up-regulation of bcl-2 and mutations in p53
2
