Determinants of cell cycle progression in human mammary epithelial MCF12 cells Alexandra

252 Pages·2012·3.21 MB·English

Determinants of cell cycle progression in
human mammary epithelial MCF12 cells

Alexandra Ouertani
Thesis submitted for the degree of
Doctor of Philosophy
UCL School of Pharmacy
University of London
2012
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This thesis describes research conducted in the UCL School of Pharmacy between
October 2008 and September 2011 under the supervision of Prof Andreas Kortenkamp
and Dr Elisabete Silva. I certify that the research described is original and that any parts
of the work that have been conducted by collaboration are clearly indicated. I also certify
that I have written all the text herein and have clearly indicated by suitable citation any
part of this dissertation that has already appeared in publication.

Signature: Date:

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ACKNOWLEDGMENTS
First of all, I would like to thank my supervisor Prof Andreas Kortenkamp for giving me the
opportunity to undertake this graduate work. Thank you for supporting me in many ways
throughout the years, for your time, your positive attitude and the countless discussions we
had about this work!
I am also very grateful to my second supervisor Dr Elisabete Silva for her constant support,
her practical advice and her fresh view on my work.
I would like to thank all members of the Centre for Toxicology, who were great colleagues,
and all of you have contributed in making my PhD the best time of my life! I wish to thank
especially Dr Sibylle Ermler, Dr Richard Evans and Dr Frankie Orton for taking their time to
discuss my work, show me new techniques, or to have a much needed break!
I would like to thank Dr Erika Rosivatz, Dr Ines da Costa Rocha and Dr Sinikka Rahte. You
cheered me up when I doubted myself, and were there for me in many different ways. Thank
you for all of this, but mostly for your friendship!
I am very grateful to my family, especially my parents and my brother, who were so patient
with me during the past years. Finally, I thank my better half Zied, for his encouragement,
support and advice during this time. Thank you for always finding the right words to get me
through the bad times, and for sharing the good times with me!

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ABSTRACT
Cancer of the mammary gland is the most common type of cancer in women worldwide, and
the vast majority of breast cancers originate from a cluster of malignant cells in the epithelial
tissue of the breast, which initially confines the ductal carcinoma in situ. Research has shown
that the signalling pathways that increase differentiation and maintain proliferation in normal
epithelial cells are of utmost importance for sustaining this barrier against malignant cells. As
a model for normal mammary epithelial cells, the MCF-12A cell line was used to determine
factors that are required for cell cycle progression of these cells. A discontinuous treatment
assay was developed in which the MCF-12A cells were treated with epidermal growth factor
(EGF) and insulin at two distinct times to induce cell cycle re-entry. The use of these
chemically defined growth factors enabled us to determine that continuous stimulation with
mitogenic factors is not required for these cells to re-enter the cell cycle. An initial activation
of the MAP kinase pathway and an up-regulation of the transcription factor c-Myc, followed
by activation of the PI3K pathway, resulted in full competence to progress into S phase. The
order in which the growth factors were applied, and thus the sequence in which the
subsequent proteins were triggered, was of great importance for successful S phase entry.
We found that estradiol (E2) was unable to induce the factors necessary for cell cycle
progression. Furthermore, we report for the first time that E2 did not affect estrogen-
regulated genes which normally are under the control of a ligand-bound estrogen receptor
(ER). We suggest that the mechanism by which the ligand-activated ER usually interferes
with the estrogen responsive element in the promoter region of the target genes is defective in
the MCF-12A cell line.
The results presented here may contribute to new approaches in chemotherapy, taking
advantage of the diverse molecular mechanism in place for cell cycle progression and
proliferation in malignant cells compared to normal mammary epithelial cells.

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TABLE OF CONTENTS
Acknowledgments .............................................................................................................. iii
Abstract .............................................................................................................................. iv
List of Figures................................................................................................................... xiii
List of Tables .................................................................................................................... xvi
List of Abbreviations ...................................................................................................... xviii
CHAPTER 1: INTRODUCTION ................................................................... 1
1 GENERAL INTRODUCTION ...................................................................................... 1
2 CANCER OF THE MAMMARY GLAND ................................................................... 1
2.1 Risk factors for breast cancer......................................................................................... 2
2.2 Signalling between normal and malignant cells for the transition from in situ to
invasive carcinoma ...................................................................................................................... 3
3 MAMMARY GLAND EPITHELIUM .......................................................................... 6
3.1 The function of epithelial cells in the mammary gland .................................................. 6
3.2 Signalling for cell cycle progression and proliferation .................................................. 7
4 AIMS ............................................................................................................................ 9
5 THESIS OUTLINE ..................................................................................................... 10
CHAPTER 2: MATERIAL AND METHODS ............................................. 12
1 LIST OF CHEMICALS ............................................................................................... 12
2 ROUTINE CELL CULTURE ...................................................................................... 14
2.1 Routine maintenance of cells ........................................................................................ 14
2.2 Media ............................................................................................................................ 14
2.3 Sub-culturing (passaging) ............................................................................................. 15
2.4 Cryopreservation and resurrection from cryogenic stocks ......................................... 15
2.5 Charcoal-dextran treatment of serum ......................................................................... 16
3 GROWTH FACTORS ................................................................................................. 16
4 INHIBITORS .............................................................................................................. 17
5 FLOW CYTOMETRY ................................................................................................ 17
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5.1 Discontinuous exposure assay ...................................................................................... 17
5.1.1 Seeding of MCF-12A cells ......................................................................................... 17
5.1.2 Serum depletion for induction of quiescence ............................................................... 18
5.1.3 Incubation with growth factors.................................................................................... 18
5.1.4 Incubation with inhibitors ........................................................................................... 18
5.1.4.1 Inhibitors tested during the first pulse ................................................................... 18
5.1.4.2 Inhibitors tested during the second pulse .............................................................. 19
5.2 Sample preparation for flow cytometric analysis ........................................................ 19
5.3 Staining of cell DNA ..................................................................................................... 19
5.4 Analysis on flow cytometer ........................................................................................... 19
5.5 Data analysis with MACSQuantify™ software ........................................................... 20
5.6 Data analysis with FlowJo software ............................................................................. 20
6 IMMUNOBLOTTING ................................................................................................ 20
6.1 Discontinuous exposure assay ...................................................................................... 20
6.1.1 Seeding of MCF-12A cells ......................................................................................... 20
6.1.2 Serum depletion, incubation with growth factors and inhibitors ................................... 20
6.2 Sample preparation for immunoblotting ..................................................................... 21
6.2.1 Determination of protein concentration and protein separation with SDS-PAGE ......... 21
6.2.2 Transfer ...................................................................................................................... 21
6.2.3 Incubation with antibodies .......................................................................................... 21
6.2.4 Protein detection ......................................................................................................... 22
6.2.5 Re-probing of membranes ........................................................................................... 22
7 QUANTITATIVE REAL-TIME PCR ANALYSIS ..................................................... 23
7.1 Discontinuous exposure assay ...................................................................................... 23
7.1.1 Seeding of MCF-12A cells ......................................................................................... 23
7.1.2 Serum depletion for induction of quiescence ............................................................... 23
7.1.3 Incubation with growth factors.................................................................................... 23
7.1.4 Incubation with complete growth medium .................................................................. 23
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7.2 Sample preparation for rt-PCR analysis...................................................................... 23
7.2.1 RNA extraction .......................................................................................................... 24
7.2.2 Determination of RNA concentration .......................................................................... 24
7.2.3 Reverse transcription .................................................................................................. 24
7.2.4 Real-time PCR analysis on iCycler iQ Real-Time PCR detection system .................... 25
7.2.5 Determination of threshold cycles ............................................................................... 25
7.2.6 Calculation of expression levels .................................................................................. 26
CHAPTER 3: METHODOLOGICAL CONSIDERATIONS FOR CELL
CYCLE ANALYSIS BY FLOW CYTOMETRY ........................................ 28
1 PRINCIPLE OF FLOW CYTOMETRY ...................................................................... 28
2 CELL CYCLE ANALYSIS – CHOOSING THE RIGHT METHODS ........................ 37
2.1 Cell cycle phase distributions: automated calculation methods vs. manual gating .... 37
2.1.1 Mathematical models used for cell cycle analysis ........................................................ 37
2.1.2 Variability of cell cycle analysis with the exemplary methods ..................................... 39
2.1.2.1 Analysis applying the algorithm based on Dean-Jett-Fox ...................................... 42
2.1.2.2 Analysis applying the algorithm based on Watson ................................................. 43
2.1.2.3 Analysis with manually set gates ........................................................................... 44
2.1.3 Systematic comparison of results obtained by the exemplary methods ......................... 46
2.1.4 Summary of results obtained by the exemplary methods ............................................. 50
2.1.5 Discussion .................................................................................................................. 50
2.2 Methods for synchronisation of human normal mammary epithelial cells (MCF-12A
cell line) ...................................................................................................................................... 51
2.3 Variability of synchronicity in MCF-12A cells and the need for normalisation ......... 53
3 SUMMARY ................................................................................................................ 60
CHAPTER 4: IMPLEMENTING THE DISCONTINUOUS EXPOSURE
ASSAY WITH MCF-12A CELLS ................................................................ 61
1 INTRODUCTION ....................................................................................................... 61
2 OBJECTIVES ............................................................................................................. 64
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3 METHODOLOGICAL CONSIDERATIONS ............................................................. 64
3.1 Establishing a synchronisation protocol ...................................................................... 64
3.2 Release of cells from G0 block ...................................................................................... 65
3.2.1 Continuous exposure of cells for release from G0 block .............................................. 65
3.2.2 Determination of the intervening time between administration of the two pulses in the
discontinuous exposure assay .................................................................................................. 66
3.2.3 Determination of growth factors to be tested ............................................................... 66
3.2.3.1 Platelet-derived growth factor (PDGF)................................................................. 66
3.2.3.2 Epidermal growth factor (EGF) ............................................................................ 67
3.2.3.3 Insulin and insulin-like growth factor type I (IGF-1) ............................................. 67
4 RESULTS ................................................................................................................... 67
4.1 Cell synchronisation in the G0 phase through serum depletion .................................. 70
4.2 Release of cells from G0 block ...................................................................................... 72
4.2.1 Release with complete growth medium ....................................................................... 72
4.2.2 Replacement of complete medium by starvation medium plus EGF ............................ 73
4.2.3 Other growth factors in starvation medium ................................................................. 75
4.3 Varying the intervening time between administration of the two pulses .................... 76
4.4 Omission of serum from culture media ........................................................................ 77
4.5 Varying the length of the second pulse ......................................................................... 80
4.6 Investigating the influence of omitting the 1st pulse .................................................... 84
4.7 Combinations of growth factors in each pulse ............................................................. 85
4.7.1 EGF as the sole growth factor ..................................................................................... 85
4.7.2 Insulin ........................................................................................................................ 85
4.7.3 PDGF ......................................................................................................................... 88
4.7.4 Estradiol ..................................................................................................................... 89
5 DISCUSSION ............................................................................................................. 90
5.1 Cell synchronisation ..................................................................................................... 90
5.2 Cell cycle re-entry with continuous stimulation ........................................................... 90
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5.3 The discontinuous exposure assay ................................................................................ 91
5.4 Assessment of growth factors ....................................................................................... 92
6 CONCLUSIONS ......................................................................................................... 94
CHAPTER 5: REGULATION OF GENES BY ESTRADIOL
COMPARED TO EGF IN MCF-12A CELLS ............................................. 95
1 INTRODUCTION ....................................................................................................... 95
1.1 Genes coding for nuclear steroid receptors (ESR1 and PGR) ..................................... 97
1.2 Target gene TFF1 (trefoil factor 1) .............................................................................. 97
1.3 The breast cancer susceptibility gene BRCA1 ............................................................. 98
1.4 Target gene PRAD1, coding for cyclin D1 (CCND1) ................................................... 98
1.5 Transcription factor MYC ........................................................................................... 99
2 OBJECTIVES AND METHODOLOGICAL CONSIDERATIONS ............................ 99
3 RESULTS ................................................................................................................. 100
3.1 Effect of E2 on gene expression .................................................................................. 100
3.2 Effect of EGF on gene expression ............................................................................... 101
4 DISCUSSION ........................................................................................................... 103
4.1 The role of E2 for gene expression ............................................................................. 103
4.2 Impact of EGF on gene expression ............................................................................. 106
4.3 E2-triggered signalling in MCF-12A versus MCF-7 cells .......................................... 109
5 CONCLUSIONS ....................................................................................................... 110
CHAPTER 6: SIGNAL TRANSDUCTION FOR CELL CYCLE RE-
ENTRY ......................................................................................................... 111
1 INTRODUCTION ..................................................................................................... 111
1.1 Epidermal growth factor (EGF) and its receptor, EGFR .......................................... 112
1.1.1 Structure of the receptor ........................................................................................... 112
1.1.2 Mode of action of EGF on its receptor (EGFR): ........................................................ 113
1.1.3 Mechanisms of signal transduction by the EGFR ...................................................... 114
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1.2 Insulin and the insulin receptors ................................................................................ 115
1.2.1 Structure of the receptor ........................................................................................... 115
1.2.2 Mode of action of insulin on its receptors .................................................................. 117
1.2.3 Mechanisms of signal transduction by the IR and the IGF-1R ................................... 117
1.3 Signalling pathways triggered through activation of the EGFR and IR ................... 117
1.3.1 The mitogen-activated protein kinase (MAPK) pathway ........................................... 118
1.3.2 The phosphatidyl-inositol-3-kinase (PI3K) pathway .................................................. 118
1.3.3 The phospholipase C (PLC) pathway ........................................................................ 120
1.4 Key mechanisms for the transition from G0/G1 into S phase ................................... 120
1.4.1 The restriction point and its role in controlling proliferation ...................................... 120
1.4.1.1 Elevation of the transcription factor c-Myc ......................................................... 121
1.4.1.2 Elevation of levels of cyclin dependent kinases .................................................... 122
1.4.1.2.1 The MAPK pathway and the cyclin dependent kinases .............................................. 123
1.4.1.2.2 The PI3K pathway and the cyclin dependent kinases ................................................. 123
1.4.1.3 Decrease of p27Kip1 and p21Cip1 ........................................................................... 124
1.4.1.3.1 The MAPK pathway and the inhibitors p21Cip1 and p27Kip1 ........................................ 124
1.4.1.3.2 The PI3K pathway and the inhibitors p21Cip1 and p27Kip1 ........................................... 125
1.4.1.4 Hyperphosphorylation of the retinoblastoma protein........................................... 125
1.4.1.4.1 The MAPK pathway and the Retinoblastoma protein ................................................. 125
1.4.1.4.2 The PI3K pathway and the Retinoblastoma protein .................................................... 126
1.4.2 Phospholipase pathways ........................................................................................... 126
2 OBJECTIVES AND METHODOLOGICAL CONSIDERATIONS .......................... 127
3 RESULTS ................................................................................................................. 129
4 DISCUSSION ........................................................................................................... 134
4.1 First pulse of growth factors (EGF) ........................................................................... 135
4.2 Second pulse of growth factors (EGF and insulin) .................................................... 137
4.2.1 The MAPK and PI3K pathways and mediation of signalling through Src kinase ....... 137
4.2.2 Crosstalk between the pathways that are triggered through EGF and insulin .............. 140
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