The proliferative and apoptopic in vitro effects of progesterone and seven synthetic progestogens on normal and malignant human breast epithelial cells [Elektronische Ressource] / vorgelegt von Elizabeth Ann Krämer, geb. Austwick

Krämer

Sujets

• Gesundheit

Informations

• Publié par: eberhard_karls_universitat_tubingen
• Publié le: 01 janvier 2007
• Nombre de lectures: 18
• Langue: English

Extrait

Aus der Universitätsfrauenklinik Tübingen
Abteilung Allgemeine Geburtshilfe und Frauenheilkunde

Ärztlicher Direktor: Professor Dr. D. Wallwiener





The proliferative and apoptopic in vitro effects of
progesterone and seven synthetic progestogens
on normal and malignant human breast epithelial cells





Inaugural-Dissertation
zur Erlangung des Doktorgrades
der Humanwissenschaften


der Medizinischen Fakultät
der Eberhard-Karls-Universität
zu Tübingen






vorgelegt von

Elizabeth Ann Krämer, geb. Austwick

aus

Sheffield, UK


2007 2












































Dekan: Professor Dr. I.B. Autenrieth

1. Berichterstatter: Professor Dr. D. Wallwiener
2. Berichterstatter: Professor Dr. H. Planck 3



























To Bernhard,
for making this possible.














4
CONTENTS

Page

1) INTRODUCTION 13

1.1 Hormone replacement therapy studies in women 13

1.2 Breast cancer 23
1.2.1 Epidemiology of breast cancer in women 23
1.2.2 Risk factors for the development of breast cancer 25
1.2.3 Types of breast cancer 27
1.2.4 Relationship between cell proliferation and breast cancer 28

1.3 Estrogens 28
1.3.1 Pharmacology of estrogens 29

1.4 Progestogens 33
1.4.1 Pharmacology of progestogens 33
1.4.2 Progestogens and breast cancer 36

1.5 Growth factors 40

1.6 Apoptosis 41

1.7 Aims of the following work 43

2) MATERIALS AND METHODS 4

2.1 Materials 44
2.1.1 Reagents 44
2.1.2 Cells and cell culture
2.1.3 Media used during working experiments 46

2. Methods 47
2.2.1 Proliferation assays 47
2.2.2 Cell death detection (CDD):proliferation assays 49
2.2.3 Proliferation assays in the presence of proliferation inhibitors 50
2.2.4 Apoptosis markers: cytochrome C, sFasL and p53 51

2.3 Statistics 53






5

3) RESULTS 54
3.1 Suitability of chosen assay media 54

3.2 MCF10A results 57
3.2.1 MCF10A proliferation assays with progestogens in combination
with growth factors 57
3.2.2 MCF10A cell death detection:proliferation assays with
progestogens in combination with growth factors 60
3.2.3 MCF10A proliferation inhibitor assays with progestogens alone
and in combination with growth factors 66
3.2.4 MCF10A apoptosis marker assays (cytochrome C, sFasL
and p53) with progestogens in combination with growth factors 70


3.3 HCC1500 results 73
3.3.1 HCC1500 proliferation assays with progestogens in
combination with estradiol 73
3.3.2 HCC1500 cell death detection:proliferation assays with
progestogens in combination with growth factors and/or estradiol 76
3.3.3 HCC1500 proliferation inhibitor assays with progestogens alone
and in combination with growth factors and/or E2 88
3.3.4 HCC500 apoptosis marker assays (cytochrome C, sFasL and
p53) with progestogens in combination with growth factors
and/or estradiol 92
3.3.5 HCC1500 proliferation assays with progestogens in combination
with estradiol in the presence of tamoxifen or letrozole 103


4) DISCUSSION 105

4.1 MCF10A 107
4.1.1 MCF10A proliferation assays with growth factors 107
4.1.2 MCF10A cell death detection:proliferation assays 112
4.1.3 MCF10A and proliferation inhibitors 113
4.1.4 MCF10A and apoptosis markers (cytochrome C, sFasL and p53) 114

4.2 HCC1500 16
4.2.1 HCC1500 proliferation assays with growth factors and/or
estradiol 116
4.2.2 HCC1500 cell death detection:proliferation assays 120
4.2.3 HCC1500 and proliferation inhibitors 122
4.2.4 HCC1500 and apoptosis markers (cytochrome C, sFasL and
p53) 123
4.2.5 HCC1500 proliferation assays with progestogens in combination
with estradiol in the presence of tamoxifen or letrozole 127
6
4.3 Cell culture 129

4.4 Limitations 13

4.5 Summary 134

5) CONCLUSIONS 135

6) BIBLIOGRAPHY 140

7) ACKNOWLEDGEMENTS 152

8) CURRICULUM VITAE 153


































7
TABLES AND FIGURES

Page
Tables

Table1: Summary of the epidemiological studies of breast cancer
risk under hormone replacement therapy since the
Oxford Reanalysis 19

Table 2: Risk factors for the development of breast cancer 26

Table 3: Classification of and biological properties of progesterone and
synthetic progestogens 39

Table 4: Diary plan of proliferation assays 48

Figures

Figure 1: Percentage of all deaths in women attributable to breast
cancer 23

Figure 2: Standardised mortality for breast cancer in different countries 24

Figure 3: Structure of Estradiol 30

Figure 4: Summary diagram of in vivo estrone, estradiol and estriol
conversion pathways 32

Figure 5: Structure of Progesterone 34

Figure 6: Proliferative responses of MCF10A to growth factors EGF, FGF
-12 -12 -7 -10and IGF-I 10 M (GF 10 M), estradiol (E2) 10 M to 10 M, and
a combination of growth factors and estradiol in AIM-V medium 54

Figure 7: Proliferative responses of HCC1500 to growth factors EGF, FGF
-10 -12 -9 -10and IGF-I (GF) 10 M to 10 M, estradiol (E2) 10 M to 10 M,
and a combination of growth factors and estradiol in AIM-V
medium 55

Figure 8: Proliferative responses of
-10 -12 -9 -10and IGF-I (GF) 10 M to 10 M, estradiol (E2) 10 M to 10 M,
and a combination of growth factors and estradiol in DMEM/FBS-
stripped medium 56




8
Figure 9: Proliferative responses of MCF10A to the various
-6 -10 progestagens in concentrations of 10 M to 10 M, alone and in
-12 combination with growth factors EGF, FGF and IGF-I 10 M
-12 (GF 10 M) in AIM-V medium 58

Figure 10: Proliferative responses
-6 -7 progestagens in concentrations of 10 M to 10 M in
-12 combination with growth factors EGF, FGF and IGF-I 10 M
-12 (GF 10 M) in AIM-V medium 61

Figure 11:Cell death detection measurements of MCF10A after incubation
-6 -7with the various progestagens in concentrations of 10 M to 10 M
-12in combination with growth factors EGF, FGF and IGF-I 10 M
-12(GF 10 M) in AIM-V medium 63

Figure 12: Cell death detection:proliferation measurements of MCF10A after
-6incubation with the various progestagens in concentrations of 10 M
-7to 10 M in combination with growth factors EGF, FGF and IGF-I
-12 -1210 M(GF 10 M) in AIM-V medium 65

Figure 13: Proliferative responses of MCF10A to growth factors EGF, FGF
-12 -12and IGF-I 10 M (GF 10 M) alone and in the presence of the
-6 -6proliferation inhibitors PD98059 10 M and LY294002 10 M
in AIM-V medium 67

Figure 14: Proliferative responses of MCF10A to growth factors EGF, FGF
-12 -12and IGF-I 10 M (GF 10e of the
-5 -5 M and LY294002 10 M in
AIM-V med