M o l e c u l a r M e c h a n is m s U n d e r l y in g

E s t r o g e n-In d u c e d M icronucleus

F o r m a t io n in B r e a st C a n c e r C ells

Submitted by Alena KABIL For the degree of Doctor of Philosophy The School of Pharmacy, University of Lonodon April, 2008 ProQuest Number: 10104729

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ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 Za moje najdraze roditelje ABSTRACT

Molecular mechanisms underlying estrogen-induced micronucleus formation in breast cancer cells

Aneuploidy, or numerical changes of chromosomes, has been documented in

almost all solid tumours and the frequency of micronucleus formation is commonly

taken as a biomarker of aneuploidy. An increasing number of observations suggest

that exposure to physiologically relevant concentrations of steroidal estrogens gives

rise to chromosomal impairments in estrogen receptor competent cells. Although the

action of estrogenic compounds is well explained by their ability to bind and activate

the estrogen receptor, there are still many uncertainties surrounding the mechanism underlying the ability of steroidal compounds to stimulate micronuclei formation.

This has prompted us to investigate whether these effects are linked to

activation of the estrogen receptor alpha. Co-administration of tamoxifen and the pure

estrogen receptor antagonist ICI 182,780 to breast cancer MCF-7 cells with

estrogenic agents did not lead to significant reductions in micronucleus frequencies.

Since these anti-estrogens interfere with the transcriptional activity of the estrogen receptor and block promotion of estrogen receptor-dependent gene expression, it

appears that this process is not involved in micronucleus formation.

In addition to the classical activation of the estrogen receptor at the nuclear

level, it is now accepted that estrogenic compounds can rapidly and transiently trigger

a number of second-messenger signaling pathways, such as the MAPK cascade

Erkl/2 and even their upstream effectors, such as the kinase Src and Raf. Therefore, we wanted to evaluate if the alternative mechanisms of estrogen receptor activation

could be involved in the formation of micronuclei by estrogenic compounds.

When MCF-7 cells were exposed to estrogenic agents in combination with the

specific kinase inhibitors PP2 and PD 98059, reductions in micronucleus frequencies

occurred. These findings suggest that the Src/Raf/Erk pathway plays a role in

micronucleus formation by estrogenic compounds. Enhanced activation of the

Src/Raf/Erk cascade disturbs the localisation of Aurora B kinase to kinetochores,

leading to a defective spindle checkpoint with chromosome malsegregation.

Further on, using anti-kinetochore CREST antibody staining, a high proportion of micronucleus containing kinetochores was observed when the breast

cancer cells were treated with compounds able to activate non-genomic signaling pathways, indicating that such processes are relevant to the induction of micronuclei by estrogens.

Our results suggest that estrogens induce micronuclei by causing improper

chromosome segregation, possibly by interfering with kinase signaling that controls

the spindle checkpoint, or by inducing centrosome amplification. Our findings may have some relevance in explaining the effects of estrogens in the later stages of breast

carcinogenesis. TABLE OF CONTENTS

TITLE PAGE...... 1 DEDICATION...... 2 ABSTRACT...... 3 TABLE OF CONTENTS...... 5 LIST OF ABBREVIATIONS...... 9 LIST OF FIGURES AND TABLES...... 12 ACKNOWLEDGMENTS...... 14 CHAPTER I: INTRODUCTION...... 15 1.1 Breast cancer aetiology and epidemiology ...... 15 1.1.1 Heredity and other risk factors ...... 16 1.2 The role of steroidal estrogens in breast development and breast cancer ...... 18 1.2.1 Estrogens and breast tissue development...... 18 1.2.2 Steroidal estrogens as risk factors for breast cancer ...... 19 1.3 Xenoestrogens and breast cancer risk ...... 21 1.3.1 Pharmaceutical estrogens...... 21 1.3.2 Environmental estrogens...... 23 1.4 Estrogens and cell signaling...... 28 1.4.1 Non-genomic signalling pathways...... 32 1.5 Estrogens as inducers of micronuclei ...... 35 1.6 Analytical Techniques ...... 39 1.6.1 Kinetochore staining with the CREST antibody ...... 41 1.7 Scope of the thesis...... 41 CHAPTER II: IMPORTANCE OF THE SRC/ERK PATHWAY FOR GENOMIC STABILITY...... 44 2.1 Introduction...... 44 2.1.1 Proposed link between the estrogen receptor and micronucleus formation 45 2.2 Materials and methods...... 49 2.2.1 Chemicals, reagents and solutions...... 49 2.2.2 Routine cell culture ...... 50 2.2.2.1 Removal of endogenous E2 from human serum ...... 51 2.2.3 Cytokinases block micronucleus assay ...... 52 2.2.4 Cytotoxicity Assay ...... 54 2.2.5 Kinetochore staining...... 55

5 2.2.6 Statistical analysis ...... 56 2.2.7 Phosphorylation of Erkl/2 by Tamoxifen and EG F ...... 56 2.2.7.1 Cell treatments with Tamoxifen and EGF ...... 56 2.2.12 Cell lysis ...... 57 2.2.7.3 Bradford assay ...... 57 2.2.1.A Western blotting ...... 58 2.3 Results...... 59 2.3.1 Cytotoxicity data ...... 60 2.3.2 Estrogens and MN formation...... 61 2.3.4 Influence of inhibition of genomic and non genomic pathways on micronucleus formation...... 62 2.3.4.1 Inhibition of the ER with ICI 182 780 and Tam ...... 62 2.3.4.2 Influence of inhibition of Src/Erk on MN frequency ...... 64 2.3.5 The effect of EGF on MN frequency ...... 66 2.3.6 Src phosphorylation...... 67 2.3.7 Erk 1/2 phosphorylation ...... 68 2.3.8 MN frequency induced by carcinogen BaP ...... 69 2.3.9 Probing for centromers in MN ...... 71 2.4 Discussion...... 73 CHAPTER III: THE USE OF SMALL INTERFERING RNA (siRNA) TECHNOLOGY FOR THE SILENCING OF TRANSDUCERS OF ESTROGEN SIGNALING AND ITS EFFECTS ON MICRONUCLEUS FREQUENCY...... 81 3.1 The relay of signals from the activated ER to M APK ...... 82 3.1.1 The principles of RNA interference...... 86 3.1.2 Optimisation of the gene delivery...... 89 3.1.2.1 Choice of the transfection method...... 90 3.1.2.2 Concentration of the siRNA/transfection media...... 91 3.1.2.3 Validation of the gene silencing...... 92 3.2 Materials and methods...... 92 3.2.1 siRNA assay optimization...... 92 3.2.1.1 Optimisation of cell density...... 93 3.2.2 Optimisation of transfection conditions...... 93

6 3.2.2.1 Optimisation of transfection media and siRNA ratio...... 94 3.2.3 Validation of silencing...... 95 3.2.3.1 Bradford assay ...... 96 3.2.3.2 Western blot analysis ...... 96 3.2.3.3 Protein detection using monoclonal mouse antibodies ...... 96 3.2.3.4 Protein detection and Western blot analysis ...... 97 3.2.4 CBMN assay following gene silencing ...... 98 3.3 Results...... 98 3.3.1 Validation of ER silencing...... 99 3.3.2 Validation of Grb2 silencing ...... 100 3.3.3 The effects of ER silencing on MN frequency ...... 101 3.3.4 The effects of Grb2 silencing on MN frequency ...... 103 3.4 Discussion...... 104 CHAPTER IV: COSMETIC INGREDIENTS AS ACTIVE MN INDUCERS.109 4.1 Cosmetic ingredients as potential environmental estrogens...... 109 4.1.1 Estrogenic actions of musks and UV filters ...... 110 4.2 Materials and methods...... 113 4.2.1 Chemicals and reagents...... 113 4.2.2 Cell culture and treatments for the MN assay and kinetochore staining ...... 114 4.2.3 Cytotoxicity assay ...... 115 4.3 Results...... 116 4.3.1 Choice of test concentrations...... 116 4.3.2 MN induction by cosmetic compounds ...... 118 4.3.3 The effect of ICI 182 780 on MN frequency ...... 121 4.3.4 The role of the Erk 1/2 signalling pathway in the induction of MN by cosmetic compounds...... 123 4.3.5 Analysis of MN content ...... 124 4.4 Discussion...... 126 CHAPTER V: THE INFLUENCE OF PARACRINE SIGNALLING BETWEEN DIFFERENT TYPES OF BREAST EPITHELIAL CELLS ON THE INDUCTION OF MICRONUCLEI...... 131 5.2 Materials and methods...... 134 5.2.1 Care and passage of MCF-lOA cells...... 134

7 5.2.2 Care and passage of MCF-7 cells...... 136 5.2.3 Optimisation of the media combination for co-culture experiments...... 136 5.2.4 Culture of MCF-7 and MCF-lOA cells in polycarbonated chambers ...... 137 5.3 Results...... 137 5.3.1 MN induction in breast MCF-lOA cells ...... 137 5.3.2 The influence of the presence of MCF-lOA cells on MN induction in MCF-7 cells...... 138 5.4 Discussion...... 140 CHAPTER VI: GENERAL DISCUSSION AND CONCLUSIONS...... 142 6.2 Future work...... 147 REFERENCES...... 149 LIST OF ABBREVIATIONS

3-BC 3-benzylidene camphor 4-MBC 4-methylbenzylidine camphor 4-OH-Tam 4-hydroxy Tamoxifen AF activator function Ago Agonaute proteins Ago Argonaute AHTN And 6-acetyl-1,1,2,4,4,7-hexamethyltetralinem BaP Benzo-alfa- pyrene BFB Breakage-fusion-bridge BN Binucleated cells BP Butylparaben BPA Bisphenol A BSA Bovine serum albumin CBMN Cytokinasis block micronucelus assay calsinosis, Raynoud phenomenon, esophageal dismolity, sclerodaetyly. CREST telangiactasia CytB Cytochalasin B DAPI 4',6-diamidino-2-phenylindole DDE Dichlorodiphenyldichloroethylene DDE dichlorodiphenyldichloroethylene

dH20 Distilled H 2 O DMEM Dulbecco’s Modified Eagle Medium DMF Dimethylformamide DMSO Dimethyl sulphoxide ds Double strand E2 Estradiol E2 Estrone E3 Estriol ECL Chemiluminiscence detection EDTA Ethylenediaminotetraacetic acid EGF Epidermal growth factor EGFR epidermal growth factor receptor EGFR Epidermal growth factor receptor EGTA Ethylene glycol tetraacetic acid ER Estrogen receptor ERE Estrogen responsive element ERK Extra-cellular regulated kinase estren 4-estren-3 a, 17 p-diol EtOH Ethanol FBS Foetal bovine serum FITC Fluorescein isothiocyanate FITC fluorescein isothiocyanate GPR30 G protein-coupled receptor homolog Grb2 Growth factor receptor binding protein 2

H 2O2 Hydrogen peroxide HESS Hank’s buffered salt solution HHCB l,3,4,6,7,8-hexahydro-4,6,6,7,8,8-hexamethyl-cyclopenta-(y)-2-benzopyran HRP Horse redish peroxidase HRP horse redish peroxidase HRT Hormone replacement therapy hsp heat-shock proteins IGF Insuline-like growth factor MAPK Mitogens activated protein kinases MEM-a Alfa minimal essential medium MN Micronuclei MNAR modulator of non-genomic action of estrogen receptor M-phase Metaphase MTT 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide nt Nucleotides OCP organochlorine pesticides CMC Octylmethoxycinnamate OVX Ovariectomized rats PBS Phosphate buffer saline PBS phosphate buffer saline PCB polychlorinated biphenyls PD 98059 2-Amino-3-methoxyflavone PDGFR platelet derived growth factor receptor PDKl phosphoinositide-dependent kinase PDKl phosphoinositide-dependent kinase PI3K Phosphoinositide Kinase-3 PMSF Phenylmethanesulphonylfluoride PP Propylparaben PP2 Pyrazolopyrimidine RISC RNA induced silencing complex RISC RNA induced silencing complex RKIP Raf Kinase Inhibitory Protein RKIP Raf Kinase Inhibitory Protein RKIP Raf Kinase Inhibitory Protein RNAi RNA interference rpm revolutions per minute RT Room temperature RTK Receptor tyrosine kinase

10 SBP steroid-binding protein SDS Sodium dodecyl sulphate She Src homology 2 domain shRNA Shirt hairpin RNA siRNA small interfering RNA Sos Son of Sevenless ss Single strand STAT Signal Transducers and Activators of Transcription Tam Tamoxifen TEMED N,N,N',N'-Tetramethylethylenediamine Tris Trishydroxymethylaminomethane Tween 20 Polyoxyethylene sorbitan monolaurate Tween 20 polyoxyethylene sorbitan monolaurate UHQ ultra-high quality

11 LIST OF TABLES AND FIGURES

Figure 1.1 Structural similarities between estradiol and the xenoestrogens methoxychlor and bisphenol A ...... 23

Figure 1.2 A schematic structural comparison of human ERa and ERp...... 28 Figure 1.3 Activation of estrogen receptor by steroidal estrogens ...... 29 Figure 1.4 Estrogen receptor activation...... 30 Figure 1.5 Schematic representation of non-genomic mode of action of estrogen receptor activation...... 33 Figure 1.6 Micronuclei formation in breast cancer MCF-7 cells ...... 35 Figure 1.7 Micronuclei induced by non-disjunction ...... 36 Figure 2.1 Cytotoxicity of selected test agents at the concentration chosen for assessment of MN...... 58 Figure 2.2 MN induction and frequency in MCF-7 cells ...... 59 Figure 2.3 Effect of the anti-estrogens ICI 182 780 and Tam on the induction of MN by steroidal estrogens ...... 61 Figure 2.4 Influence of inhibition of Src/Erk cell signalling pathway on MN formation...... 62 Figure 2.5 Cytotoxicity of E2 in combination with Src/Erk inhibitors ...... 64 Figure 2.6 Effect of the Src/Erk inhibitors on MN formation when co - treated with EGF...... 65 Figure 2.7 Activation of Src by test agents in the MCF-7 cell line ...... 66 Figure 2.8 Activation of Erk 1/2 by test agents in the MCF-7 cell line ...... 67 Figure 2.9 Decrease in BaP induced MN formation after co-incubation with BaP and inhibitors ...... 68 Figure 2.10 Kinetochore staining of MCF-7 cells with CREST Ab ...... 69 Figure 2.11 Role of RKIP in cell division ...... 77 Figure 3.1 Nongenomic signalling in the wake of activation of the ER ...... 83 Figure 3.2 The siRNA method...... 86 Figure 3.3 ER expression levels...... 98 Figure 3.4 Silencing of Grb2 by using different combinations of siRNA and transfection agent...... 99

12 Figure 3.5 Changes in MCF-7 cell morphology in cells treated for ER silencing, followed by the MN assay ...... 100 Figure 3.6 MN induced in MCF-7 cells following treatment with E2 (1 nM), E3 (10 nM) and BaP (10 pM)...... 101 Figure 3.7 Possible cross-talk between different RTK receptors and ER involved non-genomic activation of Src/Erk signalling ...... 105 Figure 4.1 Concentration-response curves for E2 and the six tested cosmetic compounds...... 114 Figure 4.2 MCF-7 cell viability measured by MTT following treatment with cosmetic compounds...... 116 Figure 4.3 MN induction in MCF-7 cells by cosmetic compounds ...... 117 Figure 4.4 Comparison of MN frequencies and the total number of MN in MCF-7 cells induced by cosmetic compounds ...... 118 Figure 4.5 Effects of the ER antagonist ICI 182 780on MN frequency induced by UV filter substances ...... 119 Figure 4.6 Effects of the ER antagonist ICI 182 780on MN frequency induced by parabens and AHTN...... 120 Figure 4.7 MN frequency following co-treatment of cosmetic compounds with PD 98059...... 121 Figure 5.1 Polycarbonate inserts used for culturing of MCF-7 cells and MCF-lOA...... 132 Figure 5.2 MN frequencies in MCF-7 (clear bars), MCF-lOA (hashed) and MCF- 7/MCF-lOA (crossed) ...... 137

Table 2.1 Induction of CREST-positive/-negative MN in human breast cancer cells Table 4.1 Induction of CREST-positive MN in human breast cancer cell following treatment with cosmetic agent

13 ACKNOWLEDGEMENTS

I am very grateful to Professor Andreas Kortenkamp who gave me the opportunity to work in a stimulating environment and for his continual support, guidance and understanding. I would also like to thank him for his enthusiasm for the project and his patient explanations and for his approachability. I am also indebted to Dr

Elisabete Silva, for her constant availability, willingness to help and her confidence in me through my PhD. She also thought me to “think about it” - something I will never forget. Thank you both for your friendship and for having a faith in me at the times when I had my doubt!

I would also like to thank my second supervisor Dr Rebecka Lever for her invaluable input and all the others in the Tox group for their insight.

I am especially appreciative to my family in Sweden and in London, for beliving in me throughout my research and writing of my thesis. Thank you all for nagging at me until this thesis was finally finished! I am very grateful to my dearest

Peter for his understanding for my need to work irregular hours and my “grumpy” moods when experiments did not work. My final thanks go to my Mrs J. for the morning calls which made me always so happy and for all her motherly care.

14 CHAPTER I

INTRODUCTION

1.1 Breast cancer aetiology and epidemiology

Cancer of the breast is the fifth most common cause of cancer related death in the world and the most common form of cancer in women (WHO, 2006). Women living in western countries have a 1 in 8 chance of developing invasive breast cancer during their lifetime and a 1 in 33 chance of breast cancer causing their death (ACS, 2007).

The incidence and mortality rates from breast cancer are markedly different within various geographic areas throughout the world. For instance, in Europe the highest number of cases can be found in Northern latitudes and the lowest in Central and Eastern areas. Worldwide, the highest incidences are seen in economically developed countries, and the lowest are found in Asia, particularly Japan and India, and Africa. In recent years, marked rises in breast cancer incidence and mortality rates have been noted in many Asian, eastern European, and South American countries, where incidence rates were previously low and it is believed that the increased rate of breast cancer is linked to the adoption of a more Westernised lifestyle and diet (Fang et al. 2005). In women, the risk of developing breast cancer is strongly related to age and according to the ACS more than 80% of all breast cancer cases are in individuals over 50 years old (CRUK, 2007).

The number of breast cancer cases worldwide has increased significantly since the 1970s. In the UK for example, the age-standardised incidence of women

15 diagnosed with breast cancer has risen from 74 cases per 100 000 women in 1975, to

120 cases per 100 000 women in 2004 (CRUK, 2007). The rise in the UK and in

other western countries can be attributed partially to the introduction of nationwide

mammography screening programmes (Feuer and Wun 1992), which meant that more

cancers were detected at an earlier stage. However, the continuing rise of breast

cancer incidence in the UK is not solely explained by screening. The underlying increase in breast cancer predated screening and continues today (CRUK, 2007). The lifetime risk for men of developing breast cancer of about 1 in 1000 has been fairly stable over the last 30 years.

1.1.1 Heredity and other risk factors

Several genetic alterations have been associated with an increased risk of breast cancer. Women with inherited germline alterations of the DNA tumour suppressor genes, breast cancer 1 and 2 (BRCAl and BRCA2), suffer an increased risk of developing these cancers at a younger, premenopausal, age than women not afflicted by this genetic mutation, especially when in combination with other risk factors such as oral contraceptives (Ursin et al. 1998). Interestingly, women born after 1940, with the same mutation, have a much higher risk (67%) of being diagnosed with breast cancer at the same age (King et al. 2003). This observation shows that even in a genetic background that strongly predisposes to breast cancer, other non-genetic factors can dramatically modulate the risk of developing breast cancer. Although these mutations may be responsible for as much as 90% of breast and ovarian cancers in some families, current estimations of the population-wide innate predisposition to

16 cancer associated with inherited mutations show that only 9% of breast cancer cases are attributable to hereditary syndromes (Madigan et al. 1995). The aetiology of the remaining cases cannot be explained by known genetic risk factors.

Recent studies with monozygotic twins brought up in different environments show that factors, such as life style, exercise and diet, also play a vital role in an individual’s risk of developing cancer (Shields and Harris 1991, Lichtenstein et al.

2000, Hemminki et al. 2001). The impact of the environment on breast cancer risk has also been investigated through studies of women emigrating to western countries.

It has been demonstrated that women moving from a country with a low incidence of breast cancer to one where the incidence is high, experience a risk of developing the disease more in line with those women who have grown up in the new host country

(Ziegler et al. 1993).

Findings with monozygotic twins as well as with migrant studies clearly imply that even with a strong genetic predisposition for breast cancer, other external factors can significantly influence one’s risk of developing the disease. This lends further urgency to the question: What external factors play a role in breast cancer incidence and what are the reasons behind the global breast cancer rate increase in women?

17 1.2 The role of steroidal estrogens in breast development and breast cancer

Estrogens play many physiological roles in the body and are necessary for the development and maintenance of healthy reproductive systems in humans as well as wildlife. Paradoxically, steroidal estrogens, key factors in normal female breast development, also play a strong role in the formation of breast cancer.

1.2.1 Estrogens and breast tissue development

Breast tissue differs from that of other organs in that it is structurally dynamic and changes both with age and in response to factors controlled by ovarian steroidal hormones such as the menstrual cycle and the reproductive state.

Mammary glands are composed of a tree-like ductal structure for the release of mothers’ milk. These structures are not fully developed and functional at birth.

Baby girls are bom with a duct structure that extends only a small distance from the nipple. Until puberty, these ducts grow in proportion with the rest of the body, but during puberty they experience a massive growth phase. Essential for this growth are steroidal estrogens. Through estrogen receptors (a and P) that regulate the expression of genes important in growth, estrogens stimulate division of the cells in the blind ends of the ducts, the “end buds”. This process leads to the elongation and branching of the duct system. With every secretion of estrogens during ovulation, the entire structure becomes more elaborate and branched. The final phase of development occurs during pregnancy when there is a further massive branching of ducts and the

18 entire system matures fully. During this state, the breast epithelium experiences its greatest and most rapid phase of proliferation. After breastfeeding and weaning, many of the ducts grown in pregnancy are remodelled to resemble the state before pregnancy (Russo and Russo 1998). The importance of estrogen signalling in mammary gland development has also been shown in work with mice. Borellini and

Oka (1989) showed that castrated immature mice do not have any ductal growth, demonstrating that the mammary ductal development is hormone dependent.

1.2.2. Steroidal estrogens as risk factors for breast cancer

It is thought that in promoting the growth of tissue specific stem cells in the end buds, estrogens may lead to an increase in cells that later in life become prone to cancerous growth (Clarke and Fuller 2006). This idea is supported by the observation that the majority of breast cancers derive from the end buds of the milk ducts, the cells that contain the estrogen receptor (a) and are most responsive to estrogens in breast development (Russo and Russo 1998). Consequently, most breast cancers diagnosed in women are estrogen receptor positive and rely on estrogen for growth (Nandi et al.

1995, Li and Li 1998).

The cyclical secretion of estrogen during a woman’s life is now recognised as a key determinant of breast cancer risk: the more estrogen reaches the sensitive structures in the breast during her lifetime, the higher the overall risk. In general, each year that the onset of regular ovulations is delayed results in an approximate 20% reduction in breast cancer risk ( MacMahon et al. 1973). MacMahon and colleagues also showed that the cumulative number of ovulatory cycles, i.e. cumulative estrogen

19 exposure, leads to an increase in breast cancer risk. A higher number of ovulatory cycles were consistently associated with higher breast cancer risks (MacMahon et al.

1982b). In the same way that early menarche and regular ovulation are related to a greater cumulative lifetime exposure to estrogens and higher risk of developing breast cancer, every year of delay in menopause increases the risk by 3% (Travis and Key

2003).

MacMahon and colleagues observed that there was a decreased risk of breast cancer with increased parity (MacMahon et al. 1982a). They found that nulliparous women, whether single or married, were approximately 1.4 times more at risk of developing breast cancer than parous women. The main finding of this study was that the protective effect of parity was actually due to a protective effect of early age of first birth (MacMahon et al. 1970). Studies by Travis and Key (2003) showed that each child birth is likely to decrease the risk of breast cancer by 7%, and this effect is even more pronounced before the age of 20. The very high levels of estrogen and other hormones that are secreted during pregnancy stimulate the full maturation of the duct system of the breast. It is thought that this leads to a reduction in the number of cells in the end buds that are vulnerable to cancer-causing factors, and thus to a decrease in cancer risk (Russo and Russo 1998).

Further studies exploring the risk of breast cancer have shown a positive relationship between the level of estrogens in serum and breast cancer, as women with breast cancer tend to have higher levels of the steroid hormone than women who did not develop breast cancer (Collaborative group 1997). It is therefore believed that estrogens involved in the critical periods of breast development contribute to an increase in cells that later in life become prone to cancerous growth (Holland and Roy

1995). Importantly for cancer, signalling pathways and their cross-talk serve to

20 amplify cell survival and proliferation signals, and growing evidence suggests that they may also contribute to resistance to various forms of endocrine therapy.

1.3 Xenoestrogens and breast cancer risk

1.3.1. Pharmaceutical estrogens

The cancer-promoting effects of estrogens are not limited to endogenous hormones.

Currently there is overwhelming evidence that synthetic hormones administered as pharmaceuticals in the form of oral contraceptives, anti-miscarriage drugs or hormone replacement therapy (HRT) used for the alleviation of menopausal symptoms are also associated with breast cancer. The use of these therapies has increased enormously during the last decades and following analysis from 51 epidemiological studies it has come to light that for each year of use of postmenopausal hormones, it significantly increases a women’s risk of getting breast cancer (Beral et al. 1997, Colditz 1998,

Travis and Key, 2003).

The potential benefits and harms of HRT were tested in controlled clinical trials. In 2002, one of these trials, the Women’s Health Initiative (WHI) trial, had to be stopped early because estrogen-progesterone HRT led to increased risks of breast cancer among the participating women. These risks were considered to outweigh the benefits of this form of HRT in terms of reduced bone fractures and reduced colon cancer risks (Rossouw et al. 2002).

Coinciding with the completion of the WHI trial, the results of a very large

UK observational study of women receiving mammography screening, the Million

21 Women Study, were published. Over one million women had been examined. The study showed that breast cancer risk is substantially greater for women using HRT therapy that is based on two hormones, estrogen and progesterone, than for estrogen- only HRT. Current users of estrogen-progestagen HRT were at 2-fold increased risk of developing breast cancer while current users of estrogen-only HRT had a 1.3-fold elevated risk (Banks et al. 2003). The Million Women Study authors estimated that the use of HRT by women aged 50-64 in the UK in the decade from 1993-2003 resulted in 20,000 extra breast cancers (Banks et al. 2003).

The case for a role of synthetic estrogens in HRT and breast cancer has received further support with news of a recent decline in breast cancer incidence rates in the USA reversing a steady 20 year upward trend between ca. 1980 and 2000 during which breast cancer incidence in the USA increased by almost 40%. The reported dropping off of between 7 and 14% of breast cancer incidence for women of all ages and tumour types, between 2002 and 2004, coincided with a pronounced national decline in HRT prescriptions (Ravdin et al. 2007, Glass et al. 2007, Warren and Devine, 2007). The decrease occurred only in women over the age of 50 and was more evident in women with cancers that were hormone dependent tumours, lending further support to the idea that HRT may have been involved. A down-tum in breast cancer rates subsequent to reductions in HRT use was also observed in North

Germany (Katalinic and Rawal 2008) and in Canada (Kliewer et al. 2007). However, in The Netherlands, Norway and Sweden declines in HRT use were not accompanied by a drop in breast cancer incidences (Zahl and Maehlen 2007, Soerjomataram et al.

2007). In these countries, HRT use has been less intensive and was of shorter duration than among US women. Under such conditions decreases in breast cancer incidences are not expected to occur upon discontinuation of HRT (Robbins and

22 Clarke 2007). Taken together, the available evidence strongly suggests that the sudden decline in HRT prescriptions may have led to the decrease in breast cancer, but additional, as yet unexplained factors might also have been at play.

Very recent findings on the effects of exposure to the estrogenic anti­ miscarriage drug diethylstilbestrol (DES) have also highlighted the risks associated with pharmaceutical estrogens. DES was prescribed to women with difficult pregnancies during 1938— 1971 to prevent miscarriages or premature delivery. Not only was the drug ineffective for its intended purpose, recent studies have linked the drug to an increased risk of breast cancer among the daughters of women who were prescribed DES while pregnant, and the so called “DES daughters”. Titus-Emstoff and colleagues (2001) calculated that the risk of breast cancer is approximately 30% higher than the risk for non-DES-exposed women; meanwhile Palmer et al. (2006) showed that DES-daughters face twice the normal breast cancer risk. The risk is expected to grow further as these “DES daughters” reach menopausal age.

1.3.2 Environmental estrogens

It has frequently been proposed that exposure to certain environmental chemicals also adds significantly to the body’s estrogenic load and concomitant risk of breast cancer.

The main concern with so called xenoestrogens, foreign estrogens, is their ability to mimic the effects of steroidal hormones once they are taken up, enter systemic circulation and exert direct effects via the estrogen receptor. Recent findings also suggest that xenoestrogenic chemicals may act through multiple mechanisms of action that may induce endocrine disruption. It has also been shown that they can

23 interfere with the actions of endogenous estrogens and/or other reproductive steroids

(Toppari et al. 1996, Bulayeva and Watson 2004), can accumulate in adipose tissue

(Olea et al. 1996, Darbre et al. 2004, Fernandez et al. 2004) and disrupt endocrine homeostasis by inducing up-regulation of circulating plasma levels of steroid-binding protein (SBP) involved in transport and regulation of circulating levels of endogenous estrogens (Tollefsen 2002).

Xenoestrogens are a large and structurally diverse group of compounds, which are widespread in the environment and are common ingredients in many personal care products that people come into contact with on a daily basis. They include certain phthalates (plasticizers), parabens (common ingredients in many cosmetic products), and pesticides such as dieldrin, endosulfan, and methoxychlor. The various compounds do not necessarily exhibit structural similarities with the natural estrogens, however almost all compounds have structural similarities to estrogens in the form of phenolic groups, as presented inFigure 1.1.

OH

CM ■OH HO CCI.

Estradiol Methoxychlor Bisphenol A

Figure 1.1. Structural similarities between estradiol and the xenoestrogens methoxychlor and bisphenol A.

Epidemiological studies carried out to examine whether specific persistent chemicals such as polychlorinated bisphenyls (PCBs), dieldrin and endosulfan are

24 implicated in breast cancer could neither prove nor rule out a possible link. Many scientists believe that it is highly unlikely that the environmental estrogens play a significant role in breast cancer risk in women, as the vast majority of these chemicals are considerably less potent than the steroidal estrogens. Furthermore, they are usually present in human tissues at levels that induce insignificant effects in the laboratory assays, and for this reason it is suspected that they pose neghgible health risks (Safe 1995, reviewed in Aronson et al. 2000, Mendez and Arab 2003,

LopezCarrillo et al. 1997). However, a variety of methodological limitations in these studies mean that it is difficult to conclude there is no relationship. To be identified as a determinant of risk, the effects of a specific chemical have to be quite pronounced.

These difficulties are not limited to studies of the effects of chemicals. Investigations of the role of diet in breast cancer have also failed to show consistent and statistically significant associations between fruit and vegetable intake, or dietary antioxidants and breast cancer (Michels et al. 2007). Furthermore, recent evidence suggests that instead of looking at exposures later in a woman’s life, when the breast tissue is less vulnerable, critical periods of vulnerability during puberty and development in the womb must be considered. Very recent studies demonstrating breast cancer risks from exposure to the pesticide dichloro-diphenyl-trichloroethane (DDT) during puberty, and from exposure to the estrogenic anti-miscarriage drug DES further underline the importance of chemical exposure in breast cancer.

Xenoestrogenic compounds have undergone extensive testing for their ability to induce ER-mediated gene transcription, however most act very weakly (in comparison to pharmaceutical estrogens and natural estrogens, only at 1000- to

10,000-fold higher concentrations than E2), if at all (reviewed in Watson and

Gametchu 1999). Despite their low potency sufficient numbers of weakly estrogenic

25 chemicals can significantly enhance the effects of natural estrogens, even when they are present at levels that individually do not produce measurable effects. To avoid wrongly dismissing a role for xenoestrogenic chemicals in breast cancer risk, it must be noted that the available epidemiological studies have largely focused on single chemicals and have ignored the possibility that large numbers of agents may act in concert (Silva et al. 2002, Rajapakse et al. 2002). Recent evidence from Spain strongly suggest that cumulative exposure to estrogenic chemicals is associated with breast cancer risks (Lopez-Cervantes et al. 2004). There is now good evidence

(reviewed in Kortenkamp 2006) that combined exposure to hormonally active chemicals can produce additive effects at low doses. Whether the individual doses are effective on their own, is not the key determinant. What also drives the likelihood of mixture effects is the sheer number of chemicals present in a “pollution cocktail”.

Thus, in principle, combination effects will result from toxicants at or even below threshold doses, provided sufficiently large numbers of components sum up to a suitably high dose. Whether such combination effects are likely to arise in reality depends on the nature of hormonally active chemicals, and their number. At present, information about these factors is patchy, but indications are that scores of chemicals may be involved (Fernandez et al. 2007). Recent advances in our knowledge about determinants of mixture effects highlight that the focus of the previous human studies of the effects of chemicals on breast cancer was wrong. Instead of concentrating on a few, arbitrarily selected substances, the entirety of hormonally active chemicals should have been considered.

The importance of chemical exposure in breast cancer during periods of heightened sensitivity of the breast has been further underlined following the very recent studies on the importance of exposure to chemicals during puberty such as the

26 pesticide DDT. DDT was widely used as a pesticide and had worldwide use in malaria control starting in the mid-1940s. It was the first man-made chemical identified to possess significant estrogenic activity as it induced an increase in uterine weight in rats and mice (Bitman et al 1968) and in human breast cancer cell lines

(Soto et al. 1995, Andersen et al. 1999). The harmful impact of DDT on wildlife was the subject of "Silent Spring" (1962) by Rachel Carson which caused a ban for almost all uses in the United States and by 1980s DDT was prohibited in most developed countries (Lopez-Cervantez et al 2004). Cohn et al. (2007) looked at the p,p’-DDT levels in blood in women who were mostly under 20 years of age when DDT use in the USA peaked, and observed a five-fold increase in breast cancer risk in women exposed to the high levels of the isotope p,p’-DDT before mid-adolescence. They also assessed the breast cancer risk induced by DDT exposure later in a woman’s life and found no link between breast cancer and p,p-DDE, a compound produced when

DDT breaks down (Cohn et al. 2007).

Both the DES and DDT findings highlight that the risks that stem from exposure to synthetic estrogens at the “wrong” stages of development can play an important role in breast cancer risk. However, to date, studies carried out to examine whether xenoestrogens are implicated in breast cancer could neither prove nor rule out a possible link.

27 1.4 Estrogens and cell signalling

It is often thought that the increase in risk of breast cancer induced by both natural and synthetic estrogens is caused by an increase in the mitotic rate of breast epithelial cells (Cohen and Ellwein 1990, Prestonmartin et al. 1990, Pike et al. 1993). It is also known that the cumulative lifetime exposure to excessive mitogenic stimulation by endogenous or xenoestrogens is a major etiological factor in the development of breast cancer (Pike et al 1993, Russo and Russo 1998). But what is their mode of action in inducing carcinogenesis?

Estrogens are small molecules that can easily diffuse through the plasma membrane of cells. In general, estrogens are able to induce their physiological effects through an interaction with the estrogen receptor (Figure 1.2). The estrogen receptor was first discovered by Jensen and Jacobson and is a member of the nuclear receptor steroid family that exhibit specific structural features, a ligand-binding domain sequence identity and a dimeric structure (Jensen et al. 1972, Beato et al. 1995).

Currently two isoforms of estrogen receptor have been identified- a and p, (Kuiper et al. 1996, Pettersson et al. 1997). The estrogen receptor subtypes share approximately

55% identical amino acid residues in their ligand binding domain, but in spite of this conservation an estrogen receptor a specific ligand has been demonstrated to exhibit a more than 100-fold difference in binding affinity (Sun et al. 1999). This thesis will focus on the estrogen receptor a unless otherwise stated.

28 DNA Hormone bindingI binding ^A/B domain I , D ^ ERa (185),: I domain * domain domain ■COOH - (83)

domain^ C# t,.. - . .. E ERp (10Ô) domain domainH ° ^omaiff : (80) (243) COOH

Figure 1.2. A schematic structural comparison of human ERa and ERp.The two estrogen receptor (ER) isoforms are encoded by two distinct genes having notably high homology between different domains. C domain (DNA binding domain) shows around 96% homology between the two receptors and the E domain (ligand binding domain) shows around 60% homology. A/B domain is knows as transactivation domain, meanwhile D domain represents the hinge region of the receptor.

In the absence of a ligand, the estrogen receptor is inactive and exists as a monomer repressed by heat-shock proteins (hsp) (reviewed in Picard et al. 1990).

However, once a ligand binds to the estrogen receptor, the hsp dissociate from it, changing its configuration in the process. The receptor-ligand complexes then dimerise and translocate to the nucleus, where they bind to specific nuclear binding sites in the genomic DNA called estrogen responsive element (ERE) consisting of inverted palindromes separated by three nucleotides (Beato 1989). This, in association with several transcriptional coactivators, results in the stimulation of gene transcription, as described in Figure 1.3.

29 e

^-Transcription

Figure 1.3. Activation of estrogen receptor by steroidal estrogensThe hormone (E) enters the cell by passive diffusion and binds to the estrogen receptor (ER). Once the ligand binds to the receptor, it dimerises and translocates into the nucleus. The biding triggers estrogen sensitive gene transcription by binding, as a homodimer, to specific estrogen responsive elements (ERE).

The two transcriptional activation functions AF-1 (localised within the C terminal of the ER) and AF-2 (localised in the N-terminal) have considerably different roles in ER activation (Tora et al. 1989, Lees et al. 1989). The presence of both regions is required for the optimal stimulation of the transcriptional activity of the estrogen receptor (Figure 1.4). However, whilst AF-2 requires ligand dependent transcriptional activation, studies with deletion mutants of ERa have shown that AE-

1 mediates ligand independent action and can be modulated by phosphorylation events (IgnarTrowbridge et al. 1993, ElTanani and Green 1997). It also appears that

AF-1 is crucial for the agonist properties of some steroidal antagonists (Graham et al.

2000) even though it is not required for activation by E2.

30 Co-repressors

A/ff-" Co-activators domain domain (AF-1) (AF-2) DNA binding

Figure 1.4. Estrogen receptor activation.The A/B domain of the ER contains hormone-independent transcription activation functions (AF-1). The ligand binding E domain of the receptor is the most complex domain containing the second transcription activation function (AF-2). In classical activation of the estrogen receptor, estradiol (yellow) binds to the hormone binding site and induces conformational changes which facilitate dimérisation of receptor. This is then followed by binding of the receptor dimer to the DNA (red), recruitment of range of different co-activators and co-repressors leading to activation of estrogen receptor transcription.

Estrogens are able to activate the estrogen receptor and a range of different molecular signalling pathways that also communicate with the estrogen receptor and often work together and consequently alter gene transcription and cellular growth

(Preston-Martin et al 1990, Hall and Korach 2003). How exactly this occurs however, is still the subject of a great deal of research. The pathway described in this section is known as genomic or classical mechanism of estrogen receptor activation and gene transcription.

31 1.4.1 Non-genomic signalling pathways

Evidence is accumulating that in addition to the classical genomic pathway of estrogen action, estrogens exert their multiple biological effects by interacting with a multitude of cell cytoplasmic, non-genomic, signalling pathways.

Studies with estrogen-sensitive cells have shown that the activation of rapid signalling pathways involves the phosphorylation of a whole range of different signal transduction molecules. These non-genomic effects are activated quickly (within seconds or minutes) and can regulate different cellular processes such as cell proliferation and survival. The non-genomic responses do not require RNA or protein synthesis and are considered to be mediated by estrogen binding to the plasma membrane (Lieberherr et al. 1993, Aronica et al. 1994, Watson and Gametchu 1999).

Two models are discussed about the nature of the receptor that mediates these rapid responses: According to the first model (Improta-Brears et al. 1999, Razandi et al.

1999) a sub-population of the nuclear estrogen receptor (a) is located near the inside of the cell membrane. Cell membrane effects have been suggested to involve estradiol indirectly activating tyrosine kinase receptors such as epidermal growth factor receptor (EGER) (Nelson et al. 1991, Kato et al. 1995)or c-Neu/erb-B2 (Matsuda et al. 1993). Alternatively, evidence suggests that there is a membrane-bound estrogen receptor (termed mER) (Watson and Gametchu, 1999, Farach-Carson and Davis

2003). Although proof from numerous laboratories supports the existence of such a receptor (Pietras et al. 2001, LeMellay et al. 1997), it is yet to be isolated and structurally and functionally characterised.

32 One of the most reported targets for estrogenic rapid activation are the mitogen activated protein kinases (MAPK) and consequently the pathways that lead to their activation. At the cytoplasmic level, the activated estrogen receptor promotes stimulation of extra-cellular-regulated kinases (Erks) (Migliaccio et al. 2000b) as well as c-Src and c-Ras, which are important up-stream mediators (Castoria et al. 1996,

Migliaccio et al. 1998). The Src/Ras/Erk pathway, described in Figure 1.5, has been repeatedly shown to be involved in the estrogenic activity of steroidal estrogens

(Migliaccio et al 1998, Castoria et al. 1999, Kousteni et al. 2001a), through phosphorylation of the well conserved serine residue Ser*^* of estrogen receptor that is recognised by MAPK and potentiates the AF-1 of estrogen receptor (All et al.

1993, Arnold et al. 1995).

However, it is still not clear how the interaction between the estrogen receptor and the Src/Erk cascade functions in detail. Some researchers believe that both forms of estrogen receptor lack the motifs needed for the initiation of non-genomic effects

(Filardo et al. 2000), while Cheski’s group suggested a scaffolding protein called modulator of non-genomic action of estrogen receptor (MNAR) to be the missing link between the estrogen receptor and Src/Erk activation (Wong et al. 2002, Greger et al.

2007). It has been shown that MNAR can modulate estrogen receptor interaction with members of Src family of tyrosine kinases by forming an estrogen receptor-MNAR-

Src complex which subsequently leads to activation of Erk and a consequent increase in the transcriptional activity of the estrogen receptor, in turn promoting cell proliferation and differentiation.

33 El ER

Gene Src transcription

Ras

Erk ER

cytoplasm I nucleus

Figure 1.5. Schematic representation of non-genomic mode of action of estrogen receptor activation.Estradiol (E2) actions are primarily executed by its binding to nuclear estrogen receptor and ligand inducible transcriptional factors (a). However, apart from genomic signalling it has been proposed that estrogens and estrogen-like compounds are able to trigger nuclear estrogen receptor activation and induce subsequent gene transcription and cell proliferation by activating the Src/Ras/Erk signalling (b).

It has been reported that estradiol can activate the Src/Ras/Erk pathway in human mammary cancer cell lines, MCF-7 and T47D, as well as in the human colon cancer-derived cell line Caco-2 cells (DiDomenico et al. 1996, Migliaccio et al. 1996,

Migliaccio et al. 1998). This activation requires the presence of the estrogen receptor.

The way in which Src contributes to the activation of the Ras/Erk cascade is still not fully resolved. It is believed that the estrogen receptor in the cytoplasm may couple to

Src and provide docking sites for downstream signalling proteins to initiate signalling

(Kato et al. 1995, Sato et al. 1997, Kousteni et al. 2001).

34 It has also been shown that environmental estrogens such as bisphenol A and environmental pollutants that mimic the effects of estrogens such as DDT are able to trigger similar non-genomic actions, such as phosphorylation of Erks.

However, a number of other groups has reported that estrogens have no effect on Erk 1/2 activation (Joel et al. 1998, Lobenhofer et al. 2000, Caristi et al. 2001), and the extent to which estrogens may influence Erk 1/2 activation as well as the potential mechanism by which estrogens could influence cross-talk between different signalling pathways has been the topic of an intensive recent investigation (Brower et al. 2005).

1.5 Estrogens as inducers of micronuclei

Micronuclei are small bodies of nuclear material ejected from the cell nucleus into the cytoplasm of the affected cell. Micronuclei originate mainly from chromosome fragmentation or from lagging chromosomes that failed to engage with the spindle poles during mitosis and therefore provide a convenient and reliable indicator of the degree of both chromosome breakage and loss (Ford et al. 1988, Lindholm et al.

1991, Fenech 1998). Micronuclei are formed at the telophase stage of cell division. A nuclear envelope forms around the lagging chromosomes and fragments, which then uncoil and gradually assume the morphology of an interphase nucleus with the exception that they are smaller than the main nucleus in the cell, hence the term

“micronucleus” (Figure 1.6).

35 y

Figure 1.6. Micronutiei formation in breast cancer MCF-7 cells.Micronuclei are formed during the telophase stage of cell division and originate from acentric fragments or whole chromosomes, which fail to engage with the mitotic spindle and thus are not incorporated into the two daughter nuclei during mitosis. A nuclear envelope then forms around the lagging chromosome, which then assumes the morphology of an interphase nuclei, or micronuclei (Schuler et al. 1997). Micronuclei can be observed in both mononuclated cells (a, purple arrow) and binuclated cells (b, green arrow).

Micronuclei are round or oval in shape and can be easily seen in the cytoplasm of the binucleated cells as shown by the green arrow in Figure 1.6. They are non-refractile and have the same staining intensity as the main nuclei. The size of the micronuclei can vary from 1/16 to 1/3 of the main nuclei. They are completely separated from the main nucleus and the micronuclear boundary is clearly separated from the daughter nuclei as shown.

Micronuclei formed due to chromosomal loss or gain are believed to arise through non-disjunction (Tsutsui et al. 1987), which occurs due to the failure of

36 homologous chromosome pairs to separate properly during the cell division as shown in Figure 1.7, resulting in a cell with an imbalanced chromosome number.

anaphase

Imbalance in chromosomal segregation

Figure 1.7. Micronuclei induced by non-disjunction.Failure of paired chromosomes (red) to separate during anaphase can cause imbalance in chromosomal malsegregation leading to more chromosomes going to one daughter cell and less to the other and subsequently micronuclei formation.

Another important mechanism through which agents are able to induce complex chromosomal rearrangements is through the breakage-fusion-bridge (BFB) described first in the seminal work of McClintock in maize (McClintock 1942). The

BFB cycle usually starts with the breakage of chromosomes followed by replication and fusion of sister chromatids (Cornforth and Goodwin 1991, Saunders et al. 2000).

This creates a dicentric chromosome (a chromosome containing two centromeres) which have two copies of homologous genes positioned between the two centromeres which during anaphase, as they are drawn towards different poles, leads to unequal

37 breakage and subsequently to a range of different chromosomal aberrations such as gene amplification (Fenech and Crott 2002).

The bridge between the centromeres in dicentric chromosomes is often broken during further cell cycles, which can subsequently result in another BFB cycle and thus a continuous loss or amplification of the DNA. In turn, this creates daughter cells with uneven chromosomal number (Fenech 2002b, Reshmi et al. 2004), leading to a hypermutable state which is assumed to be the precursor to cancer (Duesberg et al.

1999, Li et al. 2000).

There is evidence showing that administration of physiologically relevant estrogen levels to estrogen receptor positive breast cancer MCF-7 cells, leads to an upsurge in cell proliferation (Soto and Sonnenschein 1985, Payne et al. 2000). This rise is thought to be associated with increases in micronuclei frequency (Eckert and

Stopper 1996, Schuler et al. 1997, Fischer et al. 2001, Stopper et al. 2003). This idea has been further investigated by analyzing associations between cell proliferation and the extent of micronuclei formation, and by exploring the influence of estrogen receptor antagonists on estradiol-mediated micronuclei induction. Fischer and colleagues (2001) presented evidence that the frequency of micronuclei induction is suppressed by co-incubation with the estrogen receptor antagonist tamoxifen. Stopper et al (2003) showed similar findings in estrogen receptor positive ovarian BG-1 cells.

Observations by Stopper’s group suggest that estrogen receptor dependent cell proliferation induced by steroidal hormones may be accountable for micronuclei induction. However, the proposed association between cell proliferation and MN induction in MCF-7 cells is in conflict with reports by Yared et al. (2002). In their hands, estrone (El), E2 and estriol (E3) all stimulated cell division, but only El and

E2 caused MN.

38 High fidelity maintenance of genomic integrity is ensured by DNA repair and cell cycle checkpoints, surveillance pathways that respond to DNA damage by inhibiting critical cell cycle events (Weinert 1998). Fischer et al (2001) and Stopper et al (2003) hypothesised that impaired fidelity of checkpoint control might give cells the opportunity to proceed through cell division without adequate DNA repair, possibly through overriding the mitotic checkpoints, resulting in elevated genomic damage.

1.6 Analytical Techniques

Micronuclei are only expressed in dividing eukaryotic cells once they have completed nuclear division. The fate of the micronuclei following more than one nuclear division is rather uncertain; hence they are ideally scored when daughter cells have not yet fully separated and are in the binucleated stage (Fenech and Morley 1985).

The current methodology of scoring micronuclei in cells is based on the cytokinesis- block micronucleus assay (Fenech 2000) which is a commonly used method for measuring chromosome breakage, loss, non-disjunction and gene over amplification that can be visualised in form of micronuclei in the cytoplasm of the dividing cells

(Fenech 2002a). By blocking cells from performing cytokinesis it is possible to specifically score micronuclei in cells with completed nuclear division, which are recognized by their appearance as binucleated cells. The most widely used cytokinesis inhibitor is cytochalasin B. It interferes with actin polymerisation which is required for the formation of the microfilament ring that constricts the cytoplasm between the daughter nuclei during cytokinesis.

39 The ability of this assay to detect both structural and numerical chromosome alterations, respectively, is an advantage of this technique (KirschVolders et al. 1997,

Fenech, 2000). The distinction between chromosomal loss/gain and chromosomal fragmentation can be accomplished by identifying whether micronuclei contain entire chromosomes, or whether they are composed of acentric fragments. This is important as it provides an understanding of the mechanisms leading to micronuclei induction

(Thomson and Perry 1988b, Eastmond and Tucker 1989a, Norppa et al. 1993a,

Norppa et al. 1993b).

New developments using molecular probes have allowed identifying micronuclei originating from whole chromosome loss or gain, referred as aneugenic micronuclei. They can be differentiated from those originating from chromosome fragments, also known as clastogenic micronuclei (Fenech 2000, Thomas et al. 2003).

The two main types of micronucleus induction can be distinguished from each other by using molecular probes specific to the presence of centromeric DNA (Ford et al.

1988, Migliore et al. 1993) or to the presence of kinetochore protein (Thomson and

Perry 1988a, Eastmond and Tucker 1989b). In contrast, micronuclei induced via direct DNA fragmentation do not contain centromeric DNA sequences or kinetochore proteins.

Eastmond and Pinkel (1990) were one of the first to show that the irregular chromosomal number of the cell can be assessed by utilizing the florescence in situ hybridization (FISH) method using specific DNA probes to detect lagging of the chromosomes containing centromeres. Zijno and colleagues (1994) combined the use of specific centromere probes with the micronucleus assay method to discriminate between chromosome fragmentation and chromosomal loss occurring through inappropriate chromosome migration or non-disjunction. Their work showed that

40 micronuclei which positively labeled with centromere probes contained whole chromosomes while micronuclei that remained unlabelled in the assay contained acentric chromosome fragments.

1.6.1 Kinetochore staining with the CREST antibody

Another technique for assessing the content of micronuclei formation is based on a bioassay for kinetochores using the CREST (calsinosis, Raynoud phenomenon, esophageal dismolity, sclerodaetyly, telangiactasia) antibody, obtained from the serum of patients suffering from the scleroderma syndrome. This method is now one of the most widely used techniques for distinguishing between chromosomal fragmentation and chromosomal loss, i.e. clastogenic and aneugenic micronuclei formation. Vig and Sweamgin (1986) showed that the anti-kinetochore CREST antibody is able to characterise the origin of micronuclei by directly binding to centromeric kinetochore proteins in micronuclei formed due to chromosomal malsegragation. If agents such as estradiol and some xenoestrogens predominantly induce CREST positive micronuclei it would indicate the presence of kinetochores in micronuclei and the presence of entire chromosomes.

1.7 Scope of the thesis

As highlighted in the introduction above, it appears controversial that genomic signalling where the estrogen receptor functions as a ligand-dependent transcription factor, plays an important role in micronuclei formation, however, relatively little is

41 known about the role of non-genomic signalling pathways in micronucleus formation in breast cancer cells, MCF-7. The first objective of this thesis was to explore the role of the estrogen receptor in micronucleus induction and to test the idea whether

Src/Erk phosphorylation, due to estrogen receptor activation, does play a role in micronuclei formation by estrogenic agents. More in-depth information on the classical and non-genomic effects of estrogens and xenoestrogens is presented in

Chapters 2, 3 and 4. Secondly, it was important to understand if there was a common mode of action through which micronuclei were formed by estrogenic compounds in the breast cancer MCF-7 cells, by looking at the contents of micronuclei using CREST antibody staining. This work has been focused in Chapter

2 and 4.

In Chapter 2 we assessed the effects of genomic and non-genomic signalling in micronuclei formation following the treatment of estrogen receptor ligands as well as epidermal growth factor and for comparison we employed a carcinogen that has no affinity for the receptor benzo[a] pyrene.

Moreover, in Chapter 2 the role of estrogen receptor in micronuclei induction was also assessed by co-treating the breast cancer cells with selection of chemical inhibitors of both genomic and non-genomic signalling pathways.

The work in Chapter 3 follows directly from that carried out in Chapter 2.

The role of estrogen receptor was further investigated, however here we used instead of specific chemical inhibitors small interfering RNA for the silencing of estrogen receptor expression in MCF-7 cells.

In Chapter 4 we further our understanding of the possible way of micronuclei formation by evaluating whether activation of genomic and non-genomic signalling of action is a shared mechanism of micronuclei induction in breast cancer cells for a

42 greater variety of estrogen-like agents. For this purpose we chose a range of cosmetic agents that have recently been established to be able to activate the estrogen receptor.

The work presented in Chapters 2, 3 and 4 have focused around the breast cancer cell line MCF-7 cells and around chemicals that are able to bind to and activate the estrogen receptor. Report by Spink and colleagues (2006) have recently highlighted the role of paracrine interactors between the cells in the breast tissue, suggesting that the presence of inhibitory factors secreted by the non-cancerous neighbouring cells might influence cancer progression. In Chapter 5 we evaluate micronucleus frequency in MCF-lOA cells that are commonly used as representatives of a healthy breast cells, and culturing non-cancerous cells in very close proximity to cancer cells have an effect on micronucleus formation.

43 CHAPTER II

IMPORTANCE OF THE SRC/ERK PATHWAY FOR GENOMIC

STABILITY

2.1 Introduction

The sex hormone estradiol (E2) plays a pivotal role in the control of the development, behavior and reproductive functions of both males and females in humans and many other species. It is generally accepted that cumulative exposure of the mammary gland to E2 is a strong risk factor for the initiation, development and progression of breast cancer in women, most likely through activation of the estrogen receptor (ER)

(Hamelers and Steenbergh 2003). It has been suggested that the ER triggers E2 sensitive gene transcription by binding to the estrogen responsive element (ERE), known as the classical, or genomic, mechanism of activation and was explained in more details in Chapter 1, Section 1.4. Evidence also suggests that steroidal estrogen signalling is required for the transformation of cells from the normal to the malignant phenotype however; exactly what initiates steroidal estrogen dependent carcinogensis is, at present, the subject of a great deal of research.

44 2.1.1 Proposed link between the estrogen receptor and micronucleus formation

The role of E2 in carcinogenesis has traditionally been explained in terms of epigenetic processes, such as increased proliferation of hormone-responsive cells

(Papendorp et al. 1985). With the observation that DNA-reactive intermediates can result from E2 administration to human breast cancer cells in vitro, came the realization that the hormone may also have a genotoxic effect (Roy and Liehr 1999,

Rajapakse et al. 2005). However, steroidal estrogens generally do not induce mutations or chromosomal aberrations and this apparent contradictory behaviour has motivated investigations into the hormone’s ability to induce changes in chromosome numbers (aneuploidy).

Aneuploidy has been observed in cell lines exposed to very high concentrations (> 10 pM) of E2 and which lack the ER (Wheeler et al. 1987, Eckert et al. 1997). At similar, non-physiological levels, the hormone can also produce micronuclei (MN), small bodies of nuclear material ejected from the nucleus into the cytoplasm of the affected cells (Fenech et al. 1999). The frequency of MN formation in cells is commonly taken as an indicator of aneuploidy (Fenech and Crott 2002,

Stopper and Lutz 2002). Nevertheless, the physiological relevance of these observations was in doubt until (Fischer et al. 2001) demonstrated that E2 causes MN at much lower (nanomolar) concentrations in ER-positive human breast cancer

(MCF-7) cells. Similar observations were made with a number of E2 metabolites and derivatives, including El and E3 (Yared et al. 2002). ER-positive human ovary BG-1 cells also show MN formation at nanomolar levels of E2 exposure (Stopper et al.

45 2003). This suggests that the hormone’s ability to induce MN may be related to the presence of the ER.

This idea has been further investigated by analyzing associations between cell proliferation and the extent of MN formation, and by exploring the influence of ER antagonists on E2-mediated MN induction. In MCF-7 cells, administration of E2 led to an upsurge in cell proliferation (Soto and Sonnenschein 1985, Payne et al. 2000), and this rise was associated with increases in MN frequency (Fischer et al. 2001).

Conversely, co-administration of hydroxy-tamoxifen (OH-Tam) led to an almost complete suppression of MN in MCF-7 and human ovarian BG-1 cells. This is again suggestive of the involvement of ER activation in MN induction (Fischer et al. 2001,

Stopper et al. 2003).

Taken together, these observations led Fischer and colleagues to argue that the stimulation of MCF-7 cells by E2 leads to shorter cell cycles with a concomitant overriding of cell cycle checkpoints, with less time for proper DNA repair and a resultant increase in MN. However, the proposed association between cell proliferation and MN induction in MCF-7 cells is in conflict with reports by Yared et al. (2002) who looked at three steroidal estrogens, E2, estrone (El) and estriol (E3).

Their results showed that all three estrogens were able to stimulate cell division, but only El and E2 caused MN formation. The inconsistent results outlined above have prompted further investigation into the possible role of ER activation in MN induction in this thesis.

In addition to the classical genomic pathways, steroids can activate non- genomic, signalling pathways. These non-genomic effects are activated quickly

(within seconds or minutes) and can regulate different cellular processes such as cell proliferation and survival. Studies with E2 sensitive cells have shown that the

46 activation of rapid signalling pathways involve the activation of a whole range of different signal transduction pathways. As mentioned in Chapter 1, Section 1.4.1, these include effects via secondary signalling pathways, for example the stimulation of extra-cellular regulated kinases (Erks) also known as mitogens activated protein kinases (MAPK) (Migliaccio et al. 2000b).

It has been hypothesised that the activated ER relays signals to the MAPK pathway (Reviewed by (Picard et al. 1996), with both c-Src and c-Ras being important down-stream mediators (Castoria et al. 1996). As with genomic pathways, rapid signalling pathways evoked by E2 phosphorylate the ER and stimulate ER dimérisation, nuclear translocation and receptor transcriptional activity. It has also been shown that environmental pollutants that mimic the effects of estrogens, such as dichlorodiphenyldichloroethylene (o,p’-DDE), are able to trigger non-genomic estrogenic actions such as phosphorylation of Erk 1/2 (Bulayeva and Watson 2004).

Further more Saavedra et al. (1999) observed that induced over-expression of

Ras can increase MN frequency in (ER-negative) NIH 3T3 cells. Increased levels of proto-oncogen, v-ras, precipitated the acquisition of one or more centromeres

(centrosome overamplification), leading to the formation of mitotic bridges and malsegregation of chromosomes. All these changes gave rise to increased MN frequencies, but could be suppressed by inhibition of the Erk 1/2 (Saavedra et al.

1999). These results point to the involvement of MAPK in unblocking the mitosis checkpoint, where cell cycle progression is delayed until all chromosomes are correctly aligned along the spindle equator. Regulatory proteins which override this checkpoint, thereby triggering cell cycle progression before correct chromosome alignment is completed, are a phosphorylation target of the Ras/Erk 1,2 pathway

(Bharadwaj and Yu 2004).

47 In the light of these findings it seemed an interesting proposition to test the idea that Erk 1/2 phosphorylation due to ER activation may play a role in MN formation by estrogenic agents.

The hypothesis on whether MN induction by E2 and other estrogenic agents could be suppressed by administration of selective inhibitors of Erk 1/2, as well as of c-Src was investigated here in this chapter using chemical inhibitors. We chose to work with steroidal estrogens E2, El and E3, well known estrogenic chemical bisphenol A (BPA) known to be able to bind to and activate ER (Krishnan and Safe

1993, Olea et al. 1996), epidermal growth factor (EGF) due its ability to activate

Src/Erk signalling pathway. Environmental carcinogen benzo[a]pyrene (BaP) was used as a control as it does not have affinity for ER and is not able to activate the non- genomic signalling in the MCF-7 cells. Furthermore, we used the CREST staining with anti-kinetochore antibodies which allowed us to investigate the mechanism through which MN are formed following the exposure of these compounds and if these compounds can subsequently induce aneuploidy in the breast cancer cells.

On the basis of our findings we argue that it is the overriding of the spindle checkpoint due to MAPK activation that plays a role in the induction of MN by steroidal estrogens and other ER agonists and not a shortening of the cell cycle due to the action of “mitogenic” E2, as was previously suspected (Fischer et al. 2001,

Stopper et al. 2003).

48 2.2 Materials and methods

2.2.1 Chemicals, reagents and solutions

17P-Estradiol (E2, 99% purity), estriol (E3, 98% purity), bisphenol A (BPA, >99% purity) and tamoxifen (Tam, 99% purity) were all purchased from Sigma Aldrich

(Dorset, UK). Benzo[a]pyrene, (BaP, 96.5% purity) was purchased from Acros

Organics. The Erk 1/2 inhibitor, 2'-Amino-3'-methoxyflavone (PD 98059) was obtained from Promega (UK), the c-Src inhibitor pyrazolopyrimidine (PP2) from

Merck Biosciences LTD, (UK) and the ER antagonist ICI 182 780 was a generous gift from Zeneca (Alderly Park, Cheshire, UK). Work was done under COSSH regulations and all the chemicals were desposed appropriately.

All test reagents were dissolved in dimethyl sulphoxide (DMSO) or 100% ethanol (EtOH) and kept as stock solutions. The volume of the dilutions added to the cells was calculated so that the final EtOH concentration did not exceed 0.1% (v/v), since the vehicle has previously been found to have no detrimental effects on MCF-7 cells at this concentration (Payne et al. 2000). The final concentration of DMSO in media for cell treatment did not exceed 0.5% (v/v). A mixture of 0.5% DMSO and

0.1% EtOH was used as a solvent control. Stock solutions and subsequent dilutions were stored at -20 °C.

Primary anti-kinetochore CREST antibody (Antibodies Inc., USA) was utilised for the kinetochore staining, while fluorescein isothiocyanate (FITC) conjugated goat polyvalent anti-human antibody (Sigma, Dorset, UK) was used as a secondary antibody. All antibodies were diluted to the appropriate concentration in

49 phosphate buffer saline (PBS) solution (Sigma, Poole, UK). PBS with 0.1% polyoxyethylene sorbitan monolaurate (Tween 20) (Sigma, Poole, UK) was used as the washing solution in all experiments. All cell culture consumables were obtained from Invitrogen (Paysley, UK), unless otherwise stated.

2.2.2 Routine cell culture

Human mammary carcinoma MCF-7 cells were a generous gift from M. Dufresne of the University of Windsor (Ontario, Canada). Cells were maintained in 75 cm^ cell culture flasks (Greiner, Gloucestershire, UK) in alfa minimal essential medium

(MEM-a) supplemented with heat inactivated 5% foetal bovine serum (FBS). They were kept in a humidified incubator at 37°C/5%C02. This combination will be referred to as "'complete medium'' for the rest of this thesis.

Media were replaced every other day and the cells were sub-cultured once a week, when cells reached approximately 70-80% of confluence. When they were ready to be split, the cells were washed with 10 ml hank’s buffered salt solution

(HBSS, Invitrogen) and disaggregated with a 1.5 min trypsin solution made of 0.05% trypsin and 0.02% ethylenediaminotetraacetic acid (EDTA) to form single cell suspension. The cells were incubated at 37°C until they were fully detached. They were then resuspended in complete media and pipetted up and down several times to form single cell colonies.

The assay medium used contained phenol red-free Dulbecco’s Modified Eagle

Medium (DMEM) supplemented with 5% charcoal/dextran treated FBS. This is because it has been showed that phenol red possesses a weak estrogenic activity

50 (Berthois et al. 1986, Katzenellenbogen et al. 1986) and by removing phenol red, the sensitivity of the cells to the test agents would be increased. Cells were cultured at

37°C in a humidified atmosphere of 5% CO 2 in air, over a maximum of 10 passages.

2.2.2.7 Removal of endogenous E2 from human serum

In order to remove sex steroids present in the serum, this was treated with a charcoal/dextran mixture following a modification of a previously described protocol

(Soto et al 1995). This process is referred to as “stripping”. Briefly, a suspension of

5% charcoal and 0.5% dextran T70 (Amersham Pharmacia Biotech Europe GmbH,

Germany) was prepared in a volume of ultra-high quality (UHQ) water that equalled the volume of serum to be stripped. This was allowed to equilibrate by rolling for

30min (10 cycles/min) at room temperature (RT) and centrifuge for 10 min at 1000 g.

The supernatant was then removed, before adding the serum to the pellet and mixing by rolling (10 cycles/min, RT, 60 min). The mixture was finally centrifuged for

20min at 50000 g at 4°C and the charcoal-dextran stripped serum filter sterilised (0.2 pm, Nalgene, Merck Ltd., Dorset, UK) and was kept at -20°C for up to six months.

As different batches of serum normally vary in terms of fat and serum protein levels, factors that could impact upon work of this kind, a single batch was used throughout this work.

51 2.2.3 Cytokinases block micronucleus assay

Prior to experiments, MCF-7 cells were disaggregated and resuspended in complete medium. Cells were added to 3 ml complete medium and seeded (Ixlri* cells/ml) in

30 mm Petri dishes containing two 18x18 mm glass cover slips (VWR International,

Leicestershire, UK). After 24 hrs this medium was replaced with assay medium containing the test compounds. The cells were typically exposed for 24 hrs, and the test agents and their final concentrations were as follows: E2 1 nM, E3 10 nM, BaP

10 pM and BPA 5 pM, with or without inhibitors and antagonists, including PD

98059 (25 pM), PP2 (25 pM), ICI 182 780 (1 pM) and Tam (10 pM).

Following the 24 hrs treatment period, assay medium containing 3 pg/ml cytokinesis inhibitor cytochalasin B (Cyt B) (Sigma, Poole, UK) was added and left for an additional 18 hrs. After this time the medium was removed and the cells washed briefly with HBSS before being fixed in 70% cold EtOH for 20 min. Cells were then stained in 5% Giemsa at RT. Subsequently, each cover slip was carefully lifted from the Petri dishes with forceps and dipped in distilled H2 O (d H 2O) to wash off all staining compounds.

All cover slips were air dried before being mounted on glass microscopic slides using DPX mounting media (BDH, VWR, Leicestershire, UK). MNs were assessed in binucleated cells (BN) that had completed nuclear division following exposure to the test chemicals. For each slide, 1000 BN, representative of 2000 nuclei, were examined, and the frequency of BN containing MN was determined. In some cases, the number of MN in BN was recorded to obtain information about the frequency of cells containing 1,2 or more MN. All slides were coded to ensure that

52 the researcher scoring the MN frequencies was blind to the treatment regimens. A minimum of three independent experiments were conducted per treatment for the cytokinasis block micronucelus assay (CBMN) following OECD guideline 487.

2.2.4 Cytotoxicity Assay

The MTT assay was performed as described by Mosmann (1983) with few modifications. Briefly, tétrazolium salts 3-(4,5-Dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) were cleaved to form purple formazan precipitates via the succinate-tetrazolium reductase system. This pathway is found in the respiratory chain of mitochondria and is only active in metabolically intact cells.

An increase in the number of living cells results in an increase in total metabolic activity which leads to a stronger colour formation.

MCF-7 cells suspended in complete medium were seeded in flat bottomed 96 well plates (NUNC, VWR International, UK) at a concentration of 5000 cells per well. Cells were left to attach for 24 hrs after which the media was replaced with 100 pi of phenol-red free DMEM medium. The test compounds were applied to the cells dissolved in the assay media at different concentrations. Cells were then incubated for

24 hrs under normal growing conditions. After this period the medium was removed and replaced with a combination of 100 pi DMEM and 20 pi of an MTT solution (5 mg/ml in PBS). Cells were left for 4 hrs in this solution allowing viable cells to reduce the yellow MTT into dark blue crystals of formazan.

Mosmann et al. (1983) used an acidic/alcohol HCl/isopropanol solubilisation solution. However, a more recent development of this assay Hansen and colleagues

53 (1989) used a DMF based solution. Experience in our lab also shows that DMF appears to have a better ability to solubilise the formazan crystals than the HCl/ISo mixture and thus, this modification of the procedure was used in the experiments described here. The crystals were next dissolved in 150 pl/well dimethylformamide

(DMF) based MTT solubilising solution containing: 200 g sodium dodecyl sulphate

SDS, 500 ml DMF, 500 ml d H 2O, 20 ml acetic acid 80%, and 10 ml 2 M HCl overnight. The optical density of solubilised formazan product was photometrically quantitated at a wavelength of 560 nm using a plate reader (Labsystems Multiscan,

UK).

2.2.5 Kinetochore staining

Cells were handled and treated in exactly the same way as for the CBMN assay.

Kinetochore staining was carried out as previously described by Schmitt et al. (2002) and Bonacker et al. (2004), with minor changes. Briefly, cells were fixed in 100% methanol for 1 hr at -20°C, washed three times for 5 min in PBS 0.1% and Tween20 and blocked with goat serum for 1 hr at 37°C / 5% CO 2 . The cells were then incubated with the CREST primary anti-kinetochore antibody (diluted 1:10 in PBS) overnight at 37 °C/5% CO 2 . After washing three times in PBS/0.1% Tween 20, cells were incubated with the secondary antibody (FlTC-conjugated goat polyvalent anti­ human antibody) (1:300 in PBS) for 1 hr at 37°C. Glass slides (VWR International,

Leicestershire, UK) were washed several times with PBS/0.1% Tween 20 and then mounted in a Vectashield anti-fade solution containing 4',6-diamidino-2-phenylindole

(DAPI, Vector Laboratories, Ltd).

54 Using the fluorescence of nuclear material stained with DAPI (excitation at

345 nm) 500 MN per slide were identified and examined for the presence of kinetochores by monitoring CREST-associated FITC fluorescence (excitation 490 nm). Microscopy was carried out with a Leitz (Wetzlar, Germany) Dialux 20 fluorescent microscope equipped with a 63x objective. FITC-stained kinetochores inside the MN revealed the presence of kinetochore positive (CREST positive) and kinetochore negative (CREST negative) MN, indicating aneugenic or clastogenic effects of the tested compounds on the cells.

2.2.6 Statistical analysis

MN frequencies are derived from count values and therefore were not normally distributed which prohibits application of parametric significance tests. Significance was assessed by using the Mann-Whitney test.

2.2.7 Phosphorylation of Erkl/2 by Tamoxifen and EGF

2.2.7.7 Cell treatments with Tamoxifen and EGF

Cells were seeded in 90 mm Petri dishes (Costar, Cambridge, MA) at the density of

5x10^ per dish in 10 ml MEM-a. Following 48 hrs incubation, the cells were washed with 10 ml HBSS and the medium changed to the assay medium containing phenol red free DMEM without serum to reduce the basal levels of phosphorylated Erkl/2.

55 The treatment concentration for Tam was 10 pM. As a positive control, we used EGF (Sigma, stock conc. 100 pg/ml) at a treatment concentration of 100 ng/ml.

As a negative control 0.1% EtOH was used. Phosphorylation of Erkl/2 was studied following the cells treatment with EGF and for 5 min for investigations of Src phosphorylation. For analysis of Erkl/2 phosphorylations by Tam, the cells were first treated with Tam at 37°C for 5 min, 10 min and 20 min. For the investigations of Src, activation at 2 min, 5min, 10 min, and 20 min was used.

2.2.7.2 Cell lysis

After treatment, the MCF-7 cells were placed on ice and the media containing the treatment was removed. Cells were then washed with 10 ml of ice-cold EtOH and lysed with 150 pi of ice-cold lysis buffer containing: 20 mM trishydroxymethylaminomethane (Tris) pH 7.5, 137 mM NaCl, 1 mM

Ethylenediaminotetraacetic acid (EDTA), 1 mM Ethylene glycol tetraacetic acid

(EGTA), 1% Triton X-100, 2.5 mM Sodium pyrophosphate, 1 mM (3-

Glycerophosphate, 1 mM Sodium orthovanadate (Na3V0 4 activated), 1 pg/ml

Leupeptin, 10% Glycerol, 1 mM 2-Mercaptoethanol and 1 mM phenyImethanesulphonyIfluoride (PMS F).

Cells were then scraped off from Petri dishes and left at 4°C and the lysates were centrifuged (1300 rpm for 5-10 min). The supernatants were kept at -20°C until further use.

2.2.7.3 Bradford assay

56 The protein concentrations of the supernatants were determined by using the Bradford assay, a spectroscopic method routinely used to determine the concentration of protein in a solution by measuring the protein's intrinsic UV absorbance. Standard solutions ranging from 0, 2, 4, 6 8 and 10 pg were prepared using a bovine serum albumin (BSA) standard (Sigma, Dorset, UK) mixed with 500 pi of dHzO and 500 pi

Bradford reagents (Bio-Rad, Hertfordshire). An equivalent amount of dH20 and

Bradford reaget was also added to 2 pi of each lysate sample. Following the final addition of 500 pi of dH20 per well, a volume of 500 pi of Bradford reagent (Bio-

Rad, Hertfordshire, UK) was added to all protein standard samples and lysets, and mixed thoroughly. Subsequently, 250 pi of the mixture from each protein standard and lysate sample was transferred into a 96 well multi-titre plate (Nunc, Fischer

Scientific, Leicestershire, UK) in duplicate wells.

Under strongly acid conditions, the Bradford dye is most stable as a doubly- protonated red form. Upon binding to protein, however, it is most stable as an unprotonated, blue form - which is what is measured. The optical density of each sample was measured on a Labsystem Multiskan Multisoft plate reader at a wave length of 570 nm. The concentration of total protein amount for each lysate sample was then calculated from comparison with the BSA standard curve.

2,2.7.4 Western blotting

To make sure there was enough material to work with, three 90 mm Petri dishes were seeded. At the end of the experiment those cells from Petri dishes with identical treatments were pooled together and the samples were submitted directly to SDS- acrylamide (12% acrylamide/Bis) electrophoresis. At the end of the run, the proteins

57 were electrophoretically transferred from the gels onto nitrocellulose membranes for

1 h using a transfer buffer containing the following: 25 mM Tris, 190 mM Glycine,

0.1% SDS and 20% Methanol.

The membranes were soaked in blocking solution containing 5% skimmed milk in TBS/T buffer, and incubated for 1 hr at a RT. Following this the membranes were washed for 3x 5 min, with 15 ml TBS/T buffer made of 20 mM tris-base, 0.1 M

NaCl, and 0.05 % Tween-20. Membranes were then incubated overnight at 4° C. The membranes were then washed for 3 x 5 min with TBS-T buffer. After washing, the membranes were incubated for 1 hr at room temperature with respective secondary antibodies.

For detection of endogenous levels of Erkl/2 we used rabbit polyclonal

Erkl/2 antibody at concentration of 1:1000 made in TBS/T with 5% w/v BSA. As a secondary antibody anti-rabbit IgG horse redish peroxidase (HRP)-conjugated antibody (1:2000) was used. For detection of phosphorylated Erkl/2, phosphor spexific rabbit monoclonal Erkl/2 antibody (1:1000 diluted in TBS/T with 5% w/v

BSA) was used. As a secondary antibody, anti-rabbit HRP linked IgG antibody was used. All antibodies were purchased from Cell signalling, Hitchin, UK.

Detection of phosphorylated Src was accomplished by incubating the membrane with the phospho-Src rabbit polyclonal antibody at concentration of

1:1000, dilution made in TBS/T with 5% w/v BSA, was used. Anti-rabbit HRP- conjugated antibody (1:2000 dilution) was used as a secondary antibody.

For detection of total Src, Src mouse monoclonal antibody was used. Dilution

1:1000 of antibody was made in TBS/T with 5% w/v nonfat dry milk. As a secondary antibody we used anti-mouse HRP-conjugated antibody (1:2000 dilution). All antibodies were purchased from Cell Signalling, Hitchin, UK.

58 Finally membranes were washed again following the procedure describe above. Detection of protein levels for total and phosphorylated Src/Erkl,2 was carried out using the enhanced chemiluminiscence detection (ECL) system according to the manufacturer’s protocol (Cell signalling, Hitchin, UK) and luminescence was captured by using ECL hyperfilms (Amersham).

59 2.3 Results

2.3.1 Cytotoxicity data

The concentrations of the test compounds used in this study were selected on the basis of published results in the literature, as well several years of work with these compounds in our laboratory. The MTT assay was used to check whether these concentrations induced cytotoxic effects on the MCF-7 cells strain used in our lab

(Figure 2.1).

80

Figure 2.1. Cytotoxicity of selected test agents at the concentration chosen for assessment of MN. The assay was run in duplicate on the same plate and the values presented here show the mean of the two duplicates. The dark purple line set at 100% represents the solvent control containing l%DMSO and 0.1% EtOH as this was used as a solvent for the CBMN assay. E2 was run together with all experiments as positive control. The bar is ± SEM from 4 independent experiments.

60 Data shown in Figure 2.1 shows that, at the selected concentrations, test

compounds did not induce significant cytotoxicity of the cells when compared to the

solvent.

2.3.2 Estrogens and MN formation

Exposure of MCF-7 cells to E2 and BPA led to increases in the frequency of MN when compared to solvent control (Figure 2.2).

t

> 4M N

Figure 2.2. MN induction and frequency in MCF-7 cells.Distribution of MN formed by a.) solvent, b.) E2 (InM), c.) BPA (5 pM) and d.) BaP (10 pM). MCF-7 cells were treated with the test compounds for 24 hrs. Following the treatment, the cells were blocked with 3 pg/ml Cyt B for an additional 18 hrs. MNs were scored in 2000 BN cells. Error bars show mean ± SEM from 3 independent experiments.

61 At the concentrations chosen (E2: 1 nM, BPA: 5 pM), the number of cells showing 1 MN was twice as high as in solvent controls. BaP, the well known genotoxic carcinogen and clastogen, used here as a positive control, induced a three­ fold increase of cells with 1 MN relative to solvent controls. The formation of multiple MN in single BN cells was negligible with E2 and BPA, however it occurred more frequently with BaP.

2.3.4 Influence of inhibition of genomic and non genomic pathways on micronucleus formation

23.4.1 Inhibition of the ER with ICI 182 780 and Tam

Fischer et al. (2001) argued that MN formation is linked to ER activation. We therefore reasoned that if ER activation is involved in the events leading to MN, then the treatment of MCF-7 cells with hormones in the presence of ER antagonists should bring about reduced MN frequencies. To test this idea, a series of experiments with the anti-estrogen Tam were conducted. As shown in Figure 2.3, it proved impossible to confirm the observations by Fischer et al. (2001) of an inhibitory effect of Tam in combination with E2. Lack of reduction in MN frequencies was also observed with the steroidal estrogen E3. However, in contrast to Fischer’s findings (but in agreement with the results communicated by Crofton-Sleigh et al. (1993), Tam on its own caused MN frequencies significantly higher than those in solvent controls

(Figure 2.3).

62 300

250 -

200 - T

Z CO I 150 - i i %

100 - i i 50 - i i i Solvent E2 E2 + E2 + E3 E3 + E3 + ICI Tam ICI Tam

Figure 2.3. Effect of the anti-estrogens ICI 182 780 and Tam on the induction of MN by steroidal estrogens.MCF-7 cells were co-treated for 24 hrs with E2 (InM) and E3 (10 nM) individually or in combination with ER antagonists ICI 182 780 (1 pM) and Tam ( 10 pM). Bars are means ± SEM from 3 independent experiments.

In a second series of experiments the effects of the pure ER antagonist ICI

182 780 were explored. On its own, the antagonist induced elevated levels of MN. In combination with the steroidal estrogens E2 and E3, there was very little, if any suppression of MN formation. To ascertain that this lack of effect was not due to inactivity of the antiestrogen, the inhibitors’ ability to block E2 action at the concentration tested was confirmed using the E-Screen assay, conducted as described previously (Silva et al. 2006) (data not shown).

63 2.3.4.2 Influence of inhibition of Src/Erk on MN frequency

Activation of ER by E2 and BPA will lead to rapid phosphorylation of the kinases

Erk 1/2 (Migliaccio et al. 2000b). We investigated whether these phosphorylation events are linked to the induction of MN. Results are shown in Figure 2.4.

250

200 -

100 -

50 -

solvent PP2 PD 98059 BPA BPA + BPA + PP2 PD 98059 PP2 PD 98059

Figure 2.4. Influence of inhibition of Src/Erk cell signalling pathway on MN formation.Cells were treated with test agent (E2 or BPA) in combination with Src inhibitor PP2 (25 pM) or Erk 1/2 inhibitor PD 98059 (25 pM). Error bars show mean ± SEM from 3 independent experiments.

64 Co-incubation of E2-treated cells with the Src inhibitor PP2 led to a significant decrease in MN frequency. A similar effect was observed with the Erkl/2 inhibitor PD 98059. Essentially the same inhibition pattern was seen with BPA.

Administered on their own, PP2 and PD98059 led to slight, but statistically insignificant changes in MN when compared to solvent controls.

The concentrations of inhibitors were chosen on the basis of previously pubhshed reports (Alessi et al. 1995, Bulayeva et al. 2004) and previous personal experience of the activity of these compounds. By using Western blotting, it was possible to ascertain that PD 98059 and PP2, at 25 pM, suppressed the phosphorylation of Erkl/2 and Src, respectively (data not shown). A concern was whether the kinase inhibitors induced cytotoxicity at these chosen concentrations, both on their own, or in combination with E2 or BPA. This was investigated by employing the MTT assay. The Erkl/2 inhibitor PD 98059 (25 pM) caused a reduction in MCF-7 cell numbers to 90% relative to controls, when administered on its own. In combination with E2, cell numbers were 93% of control values. The cytotoxicity seen with the Src inhibitor PP2 was mild, but more pronounced. At 25 pM, PP2 induced a decrease in cell numbers to 68% of controls, and together with

E2, 72% of control values were observed as shown on Figure 2.5.

65 E2+PP2 E2+PD

Figure 2.5. Cytotoxicity of E2 in combination with Src/Erk inhibitors. The MCF-7 cells were treated with inhibitors both singly and in combination with E2 (InM). The values were then compared to the solvent control (1% DMSO and 0.1% EtOH) here seen as a purple line set at 100%. PD 98059 (25 pM) caused a 10% reduction in MCF-7 cell numbers, whilst PP2 (25 pM) showed a more pronounced reduction of around 30% of the total cell number.

2.3.5 The effect of EGF on MN frequency

Our experiments showed that E2 and BPA- mediated induction of MN was sensitive to the disruption of Src/Erk signalling by the chosen inhibitors. This raised the possibility that activation of these pathways may lead to increases in MN frequency.

To test this possibility, the peptide growth factor EGF, a strong activator of the receptor tyrosine kinase EGER, which in turn signals to c-Src and Erkl/2 (Tice et al.

1999) was administered. EGF (100 ng/ml) revealed itself as an inducer of MN, with a

66 frequency similar to that seen with BaP (10 qM) (Figure 2.6), As expected, the formation of MN could be suppressed by co-administration of PP2 and PD 98059.

300

250 -

200 1 Z OQ

150 -

Z

100 4

50 -

% Solvent EGF EGF+ EGF+ EGF-r PP2 PP2 PD 98059 (1 UM) (10 pM) (25 pM) (25 pM)

Figure 2.6. Effect of the Src/Erk inhibitors on MN formation when co - treated with EGF. MN formation decreased when the MCF-7 cells were treated with a combination of EGF (100 ng/ml) and various concentrations of Src inhibitors, PP2 and Erk 1/2 inhibitor PD 98059 (25 pM). MNs were scored in 1000 BN cells.

2.3.6 Src phosphorylation

The observation of MN formation by both ICI 182 780 and Tam led to the investigation of whether the ER antagonist was able to induce Src and Erkl/2 phosphorylation (Figure 2.7).

67 Ethanol 2min 5min 10min 20min EGF

Ÿ > ' Src (ôOKDa) *

Ethanol 5min 10min EGF

Phospho-Src

Figure 2.7. Activation of Src by test agents in the MCF-7 cell line.MCE-7 cells were either treated with solvent control (0. 1% EtOH), or treated with Tam (for the lengths of time indicated) and EGE (positive control). Cells were incubated with the EGE controls for 5 min only. Cells were lysed, protein content measured and probed for phosphorylated Src using the Tyr 416 antibody which recognises total Src. In MCE-7 cells, Tam treatment resulted in rapid activation of Src protein. The elevations in the band of phosphorylated Src could be detected after 5 min. After 10 min of Tam treatment the effect began to diminish.

2.3.7 Erk 1/2 phosphorylation

Following these findings, the effect of Tam on the signalling pathways further down stream of Src was analysed. Phosphorylation of the Erkl/2 from whole cell lysate using an antibody recognizing dual phosphorylated MAPK (anti-phospho-Erkl/2) was also investigated. As shown in Figure 2.8, Tam induced phosphorylation of

Erkl/2 in MCE-7 breast cancer cells.

68 Treatment with Tam and EGF (our positive control) for different time periods enhanced MAPK phosphorylation in MCF-7 cells. Strong activation of these pathways was observed after 10 min incubation with Tam.

Ethanol 5min lOmin 20min EGF a.)

Erk 1 Erk 2 b.) Ethanol 5min lOmin 20min EGF

Phospho-Erk 1 Phospho-Erk 2 #

Figure 2.8. Activation of Erk 1/2 by test agents in tbe MCF-7 cell line.MCF-7 cells were treated with either 10 pM of Tam (for the lengths of time indicated), EGF, or with the solvent control. Cells were then lysed and processed as described and probed for total (a) and phosphorylated (b) Erkl/2. Levels of total MAPK were determined on the blot run in parallel to control for loading variation using the anti- Erkl/2 antibody.

2.3.8 MN frequency induced by carcinogen BaP

Further it was investigated whether the suppression of MN seen with PP2 and PD

98059 was limited to compounds able to bind and activate the ER or if it was a general feature related to MN induction. To this end, it has been tested whether the

69 formation of MN by the genotoxic carcinogen BaP was sensitive to the presence of

PP2 and PD 98059 (BaP itself is unable to bind to and activate the ER). There was a noticeable, albeit statistically insignificant, effect of PP2 and PD 98059 on MN frequency induced by BaP. However, this was less pronounced than the inhibition observed in the presence of E2 and BPA (Figure 2.9).

350

300 -

250 - X.

Z 150 -

100

50 -

Solvent BaP BaP + BaP + BaP + PP2 PD 98059 ICI 182 780

Figure 2.9. Decrease in BaP induced MN formation after co-incubation with BaP and inhibitors. Cells were treated with PD 98059 (25 pM), PP2 (25 pM), ICI 182 780 (1 pM) together with BaP. Error bars show the mean ± SEM from 3 independent experiments.

70 2.3.9 Probing for centromers in MN

If the increased frequency of MN observed in cells treated with estrogenic agents and

EGF is a consequence of mitotic aberrations mediated by Erk 1/2 signalling through overriding of the spindle checkpoint, then the proportion of MN containing entire chromosomes would also increase in cells.

CREST antibody was used for probing of kinetochores. Figure 2.10 shows a typical image of CREST staining for E2 and BaP. MN formed by cells treated with estrogens contained kinetochores more often than those treated with other compounds. In contrast, BaP was found to induce fragmentation of the genomic material and not contain as many MN with kinetochores.

Figure 2.10 Kinetochore staining of MCF-7 cells with CREST Ah.MCF-7 cells were treated with a) E2 and b) BaP in the same way as for the CBMN assay and probed with the CREST antibody. Typically with the clastogenic agents, such as BaP, we observed several MN formations which are indicative of chromosomal fragmentations (see yellow arrows) and are lacking the presence of kinetechores.

71 In general, one MN per BN was seen for slides treated with E2, whilst multiple MN were observed on slides treated with BaP (see the yellow arrows in

Figure 2.10). Anti-centromeric CREST antibody immunofluorescent staining was used to distinguish MN containing whole chromosomes or chromatids (CREST- positive), from MN containing only chromosomal fragments (CREST-negative). As shown in Table 2.1, the fraction of CREST-positive MN was elevated in cells treated with estrogenic chemicals and with EGF, and this effect became more pronounced as the concentration of these agents increased.

Table 2.1. Induction of CREST-positive/ negative MN in buman breast cancer cells. The aneugenic effect of the tested compounds was estimated by measuring the proportion of CREST-positive vs CREST negative MN contents.

Compound N Concentration CREST+ MN(%)

E2 2 1 nM 33.87, 37.97

1 1 pM 54.63

E3 1 10 nM 28.98

1 1 pM 47.30

EGF 1 100 ng/ml 30.00

1 1 pg/ml 46.20

BPA 2 5 pM 39.81,28.99

1 50 pM 39.00

BaP 2 10 pM 24.21,24.59

1 100 pM 22.00

Solvent 2 0.5% DMSO/0.1%EtOH 20.10, 23.00

72 The aneugenic effect of the tested compounds was estimated by measuring the proportion of CREST-positive/negative MN contents. However, the proportion of

CREST-positive MN seen in cells treated with BaP did not differ significantly from that observed in solvent controls. Furthermore, the fraction of CREST-positive MN caused by BaP did not vary in a concentration-dependent manner, as seen with estrogenic agents and EGF.

2.4 Discussion

The experiments detailed here show that the steroidal hormones, E2 and E3, as well as the xenoestrogen BPA, were able to induce MN in MCF-7 cells. This data is in agreement with previously published reports (Yared et al. 2002, Parry et al. 2002, Wu et al. 2003, Stopper et al. 2003). However, the findings do not agree with observations by Fischer et al. (2001), who hypothesized an involvement of the nuclear ER in MN formation by showing that MN induction in response to E2 could be suppressed by an ER antagonist. In the present study, the use of an ER antagonist did not lead to any significant change in MN induction by the test estrogens, E2 and

E3.

The discrepancies between our findings and the results by Fischer (2001) and

Stopper (2002) could be due to differences in technical set up. Following Norppa and

Falck (2003) suggestions regarding accurate estimation of MN, we incubated the

MCF-7 cells with Tam for one cell cycle (24 hrs), facilitating the analysis of MN exclusively in BN cells after one cell division. Fischer and colleagues (2001) treated

73 their cells for 96 hrs, following 120 hrs treatment with Cyt B prior to harvesting.

According to Norppa et al (2003), incubating for longer periods of time could result in loss of MN, which may account for their observed results.

An alternative explanation is that Fischer et al. used 4-OH-Tam, a more potent antagonist than Tam and with elevated binding affinity to the ER (Sarkaria et al.

1994). However, this is unlikely to be the case as, similar to Tam treatment, there was no observable suppression of MN formation with the pure ER antagonist ICI 182 780.

When tested at the same concentration in the E-Screen, both ER antagonists produced a significant reduction in cell proliferation (data not shown).

Data showed here indicate that activation of ER by ligands and subsequent translocation to the nucleus, with binding to ERE, does not mediate MN formation.

Instead the hypothesis is that the formation of MN by estrogens may depend on the induction of non-genomic pathways and may be related to phosphorylation events in the MAPK cascade, which are induced upon ER activation.

It has been previously shown that steroid hormones, such as E2 (Migliaccio et al. 1998, Migliaccio et al. 1996) and environmental estrogens, such as nonylphenol

(Bulayeva and Watson 2004) and BPA (Li et al. 2006) are able to rapidly activate growth factor signalling events, namely the Src/Erk pathway. Activation of this pathway by estrogens can result in more prolonged genomic effects, such as DNA synthesis and gene expression. Here, it has been shown that activation of the Src/Erk pathway by E2, E3 and BPA results in an increase of MN formation in MCF-7 cells.

A suppression of this pathway by specific inhibitors led to a decrease in MN formation. For the first time it has also been show that EGF, an activator of non- genomic pathways, is able to form MN to an even higher extent than steroidal

74 estrogens. Co-treatment with inhibitors of Src/Erk pathway and EOF reduced the number of MN to almost control levels.

The observations presented in this study are supported by previously published studies, showing that the MAPK cascade is involved in genomic instability and MN formation. Saavedra et al. 1999 looked at the levels of MAPK induced by oncogenic transformation of v-ras in a mouse embryonic fibroblast cell line. They observed a significant reduction of MN formation when v-ras overexpressing cells were treated with the MAPK inhibitors PD 98059 and U0126. The results presented by Saavedra and colleagues (1999) also show that the effect of MAPK on MN formation is direct and independent of ER, as the cell line used is ER deficient.

The possibility that non-genomic events, such as activation of the Src/Erk pathway, rather than the direct activation and translocation of ER from cytoplasma to nucleus, can be involved in MN induction is further supported by the clear increase in

MN in the presence of the ER inhibitors Tam and ICI 182 780. Recent literature points towards Tam being able to act in a cytostatic manner by activating non genomic pathways, such as the MAPK cascade, and by inducing GO/Gl arrest by blocking the transcriptional activation of ER responsive genes (Schiff et al. 2004).

Koibuchi and colleagues (1997) observed that following Tam treatment, the tumour size of ovariectomised rats significantly reduced in size. However, the same group also reported that elevated MAPK protein expression following E2 treatment did not decrease with Tam treatment. Similar observations have also been reported by

Fan et al. (2005) who showed that Tam had an antagonistic effect on tumour proliferation, but an agonistic effect on Erkl/2 pathways in MCF-7 cells following long term exposure. This strongly supports the idea presented here, that induction of

MN by Tam could be attributable to activation of MAPK cascade.

75 Direct evidence is presented here for the non genomic pathway activation of the MAPK cascade by Tam in MCF-7 cells. In MCF-7 cells, 10 pM Tam rapidly induces the phosphorylation of the kinases Src, Erkl/2 with a magnitude similar to those of the positive control, 100 ng/ml EOF, suggesting the ability of Tam to activate the Src/Erk cascade. The results clearly demonstrate that Tam is capable of inducing rapid transient phosphorylation of the MAPKs Erkl/2 as well as Src, suggesting a strong involvement of the Src/Erk pathway in the overall action of Tam.

Filardo et al (2000) showed that 10 nM E2 was sufficient to cause phosphorylation on the MAPK in MCF-7 cells, with maximum activity ranging between 0 and 5 minutes of treatment. This confirms that Tam behaves in similar way to E2 in activating these signalling pathways. Data also confirms previously mentioned claims that Tam induces Erkl/2 phosphorylation in MCF-7 cells.

Furthermore, we have shown that the phosphorylation of Erk 1/2 is accompanied by an activation of the upstream kinase Src.

The induction of MN by ICI 182 780 could be explained in a similar way.

However, there are some indications that it might act in a different manner at the cytoplasmic level. For example, Filardo and colleagues (2000) showed that in ER deficient breast carcinoma cells, ICI 182 780 is able to activate Erkl/2 pathways, at a concentration that antagonises nuclear ER.

There are some indications showing that active MAPK are required for regulation of several key events in initiating cell cycle progression by growth factors and steroidal estrogens (Zecevic et al. 1998). Therefore it could be argued, that the observed suppression of MN in combination treatments with PP2 or PD 98059 is a result of the blocking of cell cycle progression by these inhibitors. Observations showed in this chapter indicate that the number of MN was always related to BN, and

76 there was no reduction in the number of BN cells in these experiments. Huang and

Ingber (2002) reported that inhibition of Erkl/2 with PD 98059 does not reduce

cyclin D1 and does not affect down regulation of p27. Thus, it is plausible to explain

the reduction in MN observed in the presence of inhibitors in terms of genomic

instability and not due to a lack of cell cycle progression.

Studies on Xenopus eggs extract have shown that MAPK plays a principal

role in the spindle check point regulation and activation of MAPK establishes a

mitotic checkpoint (Duesbery et al. 1997). The active MAPK pathway is of particular

importance in organising and maintaining of the metaphase spindle check point arrest

phase (Shapiro et al. 1998, Zecevic et al. 1998).

It has been previously reported that over expression of human oncogenic ras

and mos results in the generation of chromosome aberrations (MN) and could lead to

improper segregation of the chromosomes (Saavedra et al 1999). It is interesting to

note that both ras and mos are known to activate the MAPK cascade, leading further

support to the hypothesis that chromosomal instability might be linked to the

activation of these pathways.

Saavedra and colleagues (1999) have shown that active MAPK localizes to

the kinetochores and phosphorylates motor proteins involved in chromosome

movement. If chromosomes fail to attach to the mitotic spindle of the cell, MAPK

serves as a signal to the cell to sense inappropriate chromosomal attachment.

However, instead of degrading the proteins that are involved in the transition,

chromosomes can in some cases enter anaphase and become abnormally segregated,

an event that leads to chromosome gain/loss and ultimately aneuploidy. This might be

also the case for steroidal estrogens and compounds that are able to activate the

MAPK cascade. When the MN contents were evaluated by classification method

77 using anti-kinetochore antibody, CREST, staining to identify the presence or the absence of kinetochore protein in MN, it was clearly illustrated that mitogens overall induced a higher proportion of kinetochore containing MN (K+ MN). This shows that the protein is associated with the centromeres of the chromosome. The high ratio of

K+MN indicates that these MN are formed due to chromosomal gain or loss.

BaP is a classical carcinogen that is known to induce MN (Davis et al. 2002,

Crofton-Sleigh et al. 1993). There are considerable differences in mechanism behind the MN formation between various mitogens and BaP. In the anti-kinetochore

CREST staining assay, MN induced by BaP had lower ratio of kinetochore containing MN in comparison to the remaining test compounds. One possible explanation for how BaP forms MN is through inducing DNA adducts, that ultimately could lead to chromosome breaks that appear as MN (Curry et al. 1996). A number of reports also show that BaP is able to bypass GO/Gl arrest by up-regulating p53 and instigating DNA damaged cells to progress though the cell cycle without being repaired (Davis et al. 2002, Khan and Dipple 2000). BaP is not an ER activator and neither does it directly influence the non-genomic signalling cascade (van Lipzig et al. 2005). Hence the lack of suppression of induced MN in co-treatments with BaP and the inhibitors of Src/Erk pathways was not an unexpected observation. This data lends credit to the hypothesis proposed here, namely that estrogen and estrogen-alike, compounds capable of activating the Src/Ras/Erk pathway, which ultimately leads to ligand independent activation of the ER, are linked to higher occurrences of MN formation.

To summarize, in light of these findings and published reports it has become clear that classical nuclear ER activation does not play a significant role in MN formation. Instead we believe that MN induction by estrogenic chemicals results from

78 activation of the Src/Erk pathways. This effect seems to be linked to the overriding of the spindle checkpoint and consequent numerical changes in chromosomes, rather than chromosome fragmentation. Defects in the spindle check point are crucial for the segregation of the chromosomes during cell division and a growing body of evidence suggests that such defects may promote aneuploidy and induce MN formation

(Solomon et al. 1991, Wassmann and Benezra 2001). Recent evidence has also shown a vital role of Raf Kinase Inhibitory Protein (RKIP) in cancer progression.

RKIP functions as a regulator of spindle assembly and is essential for the fidelity of appropriate cell alignment prior to cell division(Figure 2.11).

Mitogen

RKIP

Ras

Raf

Erk ER

Figure 2.11 Role of RKIP in cell division.High intracellular levels of Raf kinase inhibitor protein suppresses cells from dividing and cancers from growing. Down regulation or loss of RKIP contributes to the invasiveness of the cancer cells.

It has previously been shown by Yeung et al. (2001) and Trakul and Rosner

(2005) that RKIP functions as a negative regulator of Raf and MAPK signalling

79 pathway by disrupting the phosphorylation of the kinases. As shown from Figure

2.11, RKIP’s role in suppression of cancer progression is by inhibition of MAPK via regulation of the activation of Raf.

Depletion and loss of this protein has been observed in several types of cancer

(e.g. breast and prostate). The mechanism of action of this protein is still unknown, however, it is believed that loss of RKIP during metaphase (M-phase) of cell

division, in which chromosomes align in the middle of the cell, just before being

separated, leads to bypass of the spindle assembly check point and the accumulation of chromosomal abnormalities (Rosner, 2007). Incorrect alignment of the chromosomes due to unattached kinetochores has also been shown to induce the inhibitory checkpoint machinery. Future work is essential in order to understand the nature of MN formation, as well as the events involving metaphase checkpoint.

80 CHAPTER III

THE USE OF SMALL INTERFERING RNA (siRNA) TECHNOLOGY

FOR THE SILENCING OF TRANSDUCERS OF ESTROGEN

SIGNALLING AND ITS EFFECTS ON MICRONUCLEUS

FREQUENCY

The data presented in the previous chapter confirms published reports that the

steroidal estrogens (Yared et al. 2002, Fischer et al. 2001), the xenoestrogen BPA

(Lehmann and Metzler 2004, Suarez et al. 2000), and the classical carcinogen BaP

(Simko et al. 2001, Walton et al. 1983), are all able to induce MN formation in MCF-

7 cancer cells. It was shown that MN formation by estrogenic compounds was not

sensitive to inhibition of genomic signaling pathways, where the ER functions as a

ligand-dependent transcription factor. However, this does not mean that ER activation is without significance for these processes. Estrogens and mitogens can rapidly

activate a number of signaling cascades and it is thought that this results from the interaction of a membrane-associated ER with Src and other kinases (Kousteni et al.

2001b, Migliaccio et al. 2000a, Migliaccio et al. 1996). One of the pathways mediated by ER activation via Src is the MAPK cascade with Erk 1/2 as its down­

stream target (Src/Erk pathway). The results presented in Chapter 2 demonstrate that, in the case of ER activating agents, the inhibition of the Src/Erk signalling pathway

using specific chemical inhibitors, leads to the suppression of MN formation. Based

on these observations it is proposed that the activation of the non-genomic signalling

81 cascade by estrogenic chemicals is responsible for elevating MN frequency in MCF-7

cells and that the genomic signalling pathway is of no importance in MN induction.

Although chemicals that function as receptor antagonists or inhibitors of

kinases, such as the ones employed in the experiments in Chapter 2, are frequently

used to probe the importance of the underlying signaling pathways, there are potential

problems and issues with the use of such agents. Chief among these issues are

concerns that chemical inhibitors might not act in the presumed specific ways, but

instead could induce other, poorly defined effects that interfere in unknown ways

with the functional outcomes that are the object of study, in our case MN formation.

One case in point is the action of the anti-estrogens Tam and ICI 182 780. As is well established, both chemicals antagonize the ER in different ways (Wakeling

2000). Tam can occupy the docking site of estradiol, the so-called activator function 2

(AF-2), and will block its function, but leaves the activator function 1 (AF-1) which is a phosphorylation target for certain kinases, unaffected. As a result, the Tam-bound receptor will translocate to the nucleus and bind to the estrogen response element

(ERE) where it shows residual activity because of the intact AF-1. Thus, Tam is a partial ER antagonist. In contrast, ICI 182 780 silences both AF-1 and AF-2 with the result that activation of transcription is totally abrogated. Yet, none of these interactions of Tam and ICI 182 780 with the ER appear to be able to prevent the

triggering of Src/Erk signaling cascades by estrogens. Instead, there is evidence that both Tam and ICI 182 780 can stimulate the Src/Erk pathway (Acconcia and Kumar

2006, Filardo et al. 2000, Zheng et al. 2007). It appears that disruption of nuclear events by ER antagonists is totally divorced from signaling through Src and other kinases, which can proceed even when receptor-mediated promotion of gene expression is blocked by Tam and ICI 182 780. This is further highlighted by Filardo

82 and colleagues (2000) who even argued that estrogen-induced MAPK activation can also arise independently of the presence of the ER. The group proposed that the mechanism responsible for the MAPK-activating properties of ICI 182 780 and Tam is through activation dependent on the presence of epidermal growth factor receptor

(EGFR) and a G protein-coupled receptor homolog (GPR30). The activation of

Src/Erk by the anti-estrogens may be the reason for the surprising finding that both agents induced MN in MCF-7 cells. Thus, it is important to note that there are currently no ways in which the activation of both genomic and non-genomic signalling by the ER can be blocked by chemical means.

In the light of these considerations it became important to consider ways in which ER signaling could be disrupted by alternative means not involving chemical agents. To this end, RNA interference (RNAi), and more specifically, the use of small interfering RNA (siRNA) suggested itself. This technology allows the blocking of biological functions of biomolecules by inactivating the mRNA of transcribed genes before they can be translated into proteins (therefore the name “knocking down” or

“silencing”). Thus, would there be a reduction in MN frequency if certain signaling molecules normally activated in the wake of ER binding were silenced by RNAi?

Successful realisation of this aim depended on a careful choice of the signaling elements to be silenced by RNAi. One obvious candidate target for silencing is the ER receptor itself and it was important to investigate whether removal of the ER by “knock-down”, instead of by using chemical inhibitors, would affect the

MN frequency. The use of siRNA, instead of chemical agents such as Tam and ICI

182 780, might block all subsequent signalling events, including receptor translocation to the nucleus and the rapid phosphorylation events via Src.

83 Apart from the ER, it was considered important to silence an element along

the pathway of rapid Src-mediated signalling. To underpin the choice of a suitable

“knock-down” target in this pathway, it is necessary to briefly look at the details of

ER signaling to Src and other kinases further down-stream.

3.1 The relay of signals from the activated ER to MAPK

The Src/Ras/Erk signalling pathway is implicated in the control of cell growth and differentiation and is a crucial downstream component of the signalling pathways of growth factor receptors with protein tyrosine kinase activity. However, as discussed in Chapter 1, Section 1.4.1, it is still not clear how the interaction between the ER and the Src/Ras/Erk cascade works.

The enhanced total tyrosine kinase activity that has been detected in malignant breast tissue compared to normal or benign tissue controls has been suggested to be largely due to increased c-Src expression and activity (Ottenhoffkalff et al. 1992). It has also been shown that one of the mechanisms for activation of c-Src in breast cancer is through SH2-domain-mediated association with the EGFR and c-Neu/erb-

B2, as both recruit this enzyme and are commonly over-expressed in breast cancers.

In 2002, Song and colleagues showed that MCF-7 cells utilise a classical growth factor signalling pathway, involving direct association of ER in the cytoplasm with tyrosine kinases receptors through the formation of an adaptor protein complex

Grb2/Shc/Sos. These adaptor proteins are also positive Src/Ras/Erk stimulators and mediate the nongenomic signalling pathways of steroidal estrogens. It has been

demonstrated that in response to EGF, Grb2 acts as an adapter molecule by binding to

84 the activated EGFR via its SH2 domain and to a proline-rich guanine nucleotide- releasing factor, Son of Sevenless (Sos). An additional adaptor protein, She (Pawson and Scott 1997) through its SH3 domains, is also involved, leading to activation of the Ras signaling pathway (Lowenstein et al. 1992, Buday and Downward 1993,

Chardin et al. 1993) as shown in Figure 3.1.

EGFR ER

Ras Grb2 " Raf She

MEK

Erkl/2

hicleus ER Cell proliferation

Figure 3.1 Nongenomic signalling in the wake of activation of the ER.Nuclear ER can be activated independent of estrogen presence, through EGF receptor among other tyrosine kinase receptors. Grb2 functions as a link between the RTK and Ras activation, by binding to the activated RTK receptor and forming a complex with Sos and She adaptor proteins.

It has been proposed that estrogens can indirectly activate RTK such as EGFR

(Nelson et al. 1991, Kato et al. 1995) or erb-B2 (Matsuda et al. 1993). Meanwhile,

85 others have proposed that the cytoplasmic ER, localised in the vicinity of the plasma

membrane can directly be linked to the EGFR and Src as shown in Figure 3.1. The

suggested association of ER-EGFR-Src triggers signalling transduction pathways and

facilitates the rapid estrogen stimulated signalling and consequent activation of

Ras/Erkl,2 (Migliaccio et al. 2000a).

Out of the three adaptor proteins involved in complex formation, Grb2 in

particular has been studied as it initiates the association between the ER and the RTK

with downstream regulators (CorbalanGarcia et al. 1996, Vanderkuur et al. 1997).

Grb2 also seems to play an important role in cancer progression and is reported to be

over-expressed relative to normal breast epithelial cells in a subsequent breast cancer

cell lines (Daly et al. 1994).

For this reason and because of its role as an initiator of adaptor complex formation which facilitates the rapid estrogen stimulated Src/Ras/Erk, Grb2 was the obvious candidate for silencing.

3.1.1 The principles of RNA interference

RNAi is a mechanism for RNA-guided regulation of gene expression in which double-stranded ribonucleic acid inhibits the expression of genes with complementary nucleotide sequences. It is a remarkably potent method of knocking down the expression of proteins of interest. Previous knock-down techniques, such as antisense oligonucleotides and ribozymes, have a much lower efficiency and require empirical screening of a number of candidates. RNAi is much more potent and much less

86 complicated. In addition, since the RNA sequence is specific to the gene of interest,

the technique does not disturb the expression of unrelated genes.

The most vital step in siRNA dependent silencing of gene expression involves

targeting and cleaving the intended mRNA which is complementary to the siRNA.

This reaction is catalysed by the multidomain enzyme RNase III Dicer (Bernstein et

al. 2001). The anti sense strand of the siRNA gets incorporated into protein factors

and together they form the multiple-turnover enzyme complex RNA induced

silencing complex (RISC). The role of activated RISC is to form an effector complex with the target mRNA and RISC has been proposed to be an siRNA directed endonuclease, catalysing cleavage of a single phosphodiester bond on the RNA target

(Hammond et al. 2001, Schwarz et al. 2004). It has also been shown that the complex is recycled to cleave additional mRNA targets (Hutvagner and Zamore 2002, Zamore and Haley 2005).

Prior to recognition of the target mRNA and activation of the RISC complex, the siRNA duplex needs to be separated into single strands (ss) and the one selected as the guide strand forms an active RISC complex (Martinez et al. 2002). The guide strand is commonly chosen on the basis of thermodynamical properties. Schwarz and colleagues (2003) analysed functional siRNAs and demonstrated that these molecules have a reduced thermodynamic stability at the 5' end of the anti sense strand relative to the 3' end within the siRNA. This means that the guide strand tends to have a more

stable 5’end. This asymmetry allows for the unwinding of the dsRNA from one end preferentially and ensures that only one, the guide strand, assembles with the RISC to form the active siRISC complex. Once assembled, the central part of RISC-siRNA

activated complex defines the cleavage point of the mRNA, complementary to the

87 incorporated single stranded siRNA, giving 5’-phosphate and 3'-hydroxyl termini at the end of the reaction as shown in Figure 3.2.

siRNA

lllllllllllllll cytoplasm

ATG AAAA

ATG AAAA

Figure 3.2. The siRNA method, a) siRNA is introduced directly to the cytoplasm where it assembles into the RISC, which also includes members of Argonaute protein family. Once the siRNA-RISC complex is activated b) it prevents the expression of mRNA containing the same sequence c) causing cleavage of the targeted mRNA so that the protein the sequence codes for cannot be translated d). The assembly of the siRNA into the RISC induces the activation of Argonaute

(Ago) proteins, which are the catalytically active ‘engine’ of the RISC (Sandy et al.

2005). The Ago proteins are members of the endonuclease family of proteins and are

able to cleave the passenger strand in a manner analogous to cleavage of target

mRNA (Ma et al. 2004). Liu et al. (2004) showed that Ago2 protein is of particular

importance and is responsible for mRNA cleavage as mammalian cells lacking Ago2

were unable to mount an experimental response to siRNA.

For siRNAs, virtually all RISC is assembled through this cleavage-assisted

mechanism. It was initially believed that an ATP-dependent helicase was behind the

unwinding process that separates the siRNA duplex into two siRNA, however

Matranga and colleagues (2005) have proposed that Ago2 instead directly receives

the ds siRNA from the RISC assembly machinery. Ago2 then cleaves the siRNA passenger strand of the siRNA duplex from the RISC, thereby liberating the guide

strand that remains in contact with Ago in the RISC (Maiti et al. 2007).

Once the mRNA target is cleaved, the siRNA-RISC complex dissociates from

the cleaved mRNA and carries on the cleavage cycle. However, the mechanism by which the activated RISC complex locates complementary mRNA within the cell

with the help of Ago proteins is still not understood.

3.1.2 Optimisation of the gene delivery

Both ER and Grb2 are widely studied genes and the siRNA oligonucleotides that are pre-validated, in other words, rigorously tested to give a strong knock-down response,

are commercially available.

89 Transfection of synthetic siRNA oligonucleotides is a good option for gene

knock-down experiments in cells that are readily transfected. However, considering

that mRNA binding proteins may influence the accessibility of siRNA, several

different, randomly chosen siRNAs, from throughout the entire mRNA sequence for

each gene of interest need to be tested to find the most efficient version.

However, there are several elements which might influence the efficiency of

gene silencing in MCF-7 cells. Optimizing the parameters such as cell density at the

time of transfection, the amount of transfection reagent, of siRNA and culture

incubation time before analysis are necessary in order to determine the optimal

conditions for maximum transfection efficiency for a particular cell line.

3.1.2.1 Choice of the transfection method

The recommended density for most cell types at transfection is around 50 and 80%

(Qiagen siRNA manual) and the effect of silencing can usually be observed within 8 hours after transfection. The transfection efficiency of siRNA was monitored using

specially designed transfection controls. Transfection controls are chemically

synthesized fluorescein labeled oligonucleotides with no sequence identity to mammalian sequences of interest. Transfection conditions are usually run during the

start-up experiments and the treatment regimes that produce the greatest percentage

of fluorescent cells are maintained for the in future experiments.

siRNA can be introduced to MCF-7 cells by using a polyamine-lipid

transfection enhancing formulation designed specifically for the efficient transfection

of siRNA in mammalian cell lines. Liposomes, spherical structures consisting of

90 single or multiple concentric bilayers, are composed of synthetic versions of the

cationic phospholipids that comprise cellular membranes.

As with all transfection technologies, the goal of a chemical transfection reagent is to introduce foreign negatively charged molecules (primarily nucleic acids) into eukaryotic cells that have negatively charged membranes. Certain chemicals and non-lipid cationic polymers, as well as cationic lipids, neutralize or impart an overall net positive charge to nucleic acids, allowing them to cross membranes and may, to

some extent, protect them from degradation once inside the cell as they make their way to the nucleus. The positive surface charge of the liposomes mediates the interaction of the nucleic acid and the cell membrane, allowing for fusion of the liposome/nucleic acid, the so called “transfection complex”, with the negatively charged cell membrane by reducing the electrostatic attraction of the nucleic acid to the membrane (Walters et al 2002). The transfection complex is thought to enter the cell through endocytosis and once inside the cell, the complex must escape the endosomal pathway and diffuse through the cytoplasm.

3.1.2.2 Concentration of the siRNA/transfection media

The ratio between the transfection agent and the siRNA oligonucleotides is crucial for maximising gene silencing, whilst also minimising any potential cytotoxicity for the effective knock-down of the gene.

91 3.1.2.3 Validation o f the gene silencing

siRNAs exert their effects at the mRNA level. Numerous techniques can be used to measure the gene silencing in cells which can be monitored at either the protein or the mRNA level.

Monitoring of the gene silencing based on their effect on the endogenous mRNA levels involves methods such as real-time RT-PCR, microarray analysis or northern blotting. However, the reduction of mRNA does not always correspond to a similar lowering of protein levels. It is clear that many proteins are quite stable and their concentrations in cells, therefore, remain relatively constant for a long period of time. Hence monitoring the endogenous level of the protein encoded by the mRNA is usually regarded to be more efficient. Western blotting is usually the recommended and more effective method of choice to monitor knock-down gene expression

(Leirdal and Sioud 2002, Braasch et al. 2003).

3.2 Materials and methods

3.2.1 siRNA assay optimisation

All the optimisation steps outlined below were repeated for each different RNAi ER silencer. However, only the optimised ratios between the transfection reagent and the siRNA were used for the final treatment with test agents in the MN assay.

92 3.2.1.] Optimisation of cell density

MCF-7 cells were maintained following the protocol described in Chapter 2, Section

2.2.2. MCF-7 cells were seeded into 6 well plates (Nunc, Fisher Scientific,

Leicestershire, UK) at various densities. The optimal confluence of cells for the

transfection procedure was found to be between 50 and 80% at the time of

transfection (Qiagen siRNA manual). The individual wells of each 6 well plate were

seeded with lxlO\ 2x10 \ 4x10 \ 6x10"^ and 1x10^ cells per well. The wells were microscopically assessed 24 hrs after seeding and the seeding density that resulted in approximately 70% confluency of the wells was used for all subsequent experiments.

3.2.2 Optimisation of transfection conditions

For the delivery of the synthetic siRNA to the MCF-7 cells we used the lypophilic transfection enhancing agent, TransPass ™R1 following the recommendations of the manufacturer (NEB, Hitchin, UK).

For the silencing of the ER, a mixture of 21-23 nt long siRNAs, Estrogen

Receptor a Shortcut® siRNA Mix was purchased from NEB (Hitchin, UK). The ER siRNA mix is an enzymatically derived heteologous mix of short dsRNA suitable for

RNAi. The mixture is a unique combination of many individual siRNAs that work collectively to knock down the target gene and are thought to minimize the potential for off-target effects.

For the silencing of Grb2, on the other hand, the mixture of siRNA was not commercially available. We used three different siRNAs covering different sections

93 of mRNA and relied on one dsRNA molecule of defined sequence to knock down the

gene expression by RNAi. Grb2 siRNA no.4196 (sense sequence

GGUUUUGAACGAAGAAUGUTT, antisense ACAUUCUUCGUUCAAAA

CCTT) covered exon 2 and exon 3 of the mRNA, whilst Grb2 siRNA no. 106680

(sense sequence GGCCACGUAUAGUCCUAGCTT, anti sense sequence

GCUAGGACUA UACGUGGCCTT and no. 144866 covered exon 5 and exon 6

(sense sequence CGUAUAGUCCUAGCUGACGTT, anti sense sequence

CGUCAGCUAGGACUAU ACGTG). All three Grb2 silencers were purchased from

Ambion (Warrington, UK).

3.2.2.1 Optimisation o f transfection media and siRNA ratio

Optimisation assays were set up in 6 well plates. Once the optimal seeding density was determined, cells were seeded in 6 well plates in a final volume of 2.0 ml of complete medium, containing MEM-a, supplemented with 5% heat-inactivated FBS.

The cells were then allowed to attach and enter the exponential growth phase for 24 hrs. Following 24 hrs incubation at 37°C, the cells were washed with HBSS

containing 10% FBS, to minimise cell stress. For the optimisation of the ratio of

siRNA to transfection media, TransPass™Rl, we used the recommended range for

the experimental set up of transfection media suggested by the manufacturer and

described below.

The siRNA experiment was started by adding the chosen volume of transfection medium, varying from 4 pi to 10 pi of 2.5 mg/ml TransPassRl reagent, to 400 pi serum free medium in separate 1.5 ml Eppendorf tubes. This was then vortex mixed and incubated at RT for 15 min. After incubation, into corresponding

94 tubes, 10 |j M ER and Grb2 siRNA was added at different volumes, starting with a 0.5 p 1/well and proceeding up to highest recommended manufacturer level of 3 p 1/well.

These were then incubated at RT for an additional 15 min.

The transfection reagent/siRNA complex was diluted with 1.5 ml of complete culture medium to the final volume was 1.9 ml and this was then applied to each well containing MCF-7 cells. To reduce the cytotoxic effects, the plates were then incubated in normal cell culture conditions for 12 hrs and assessed in terms of protein expression of the gene of interest.

A negative, untransfected control was always run in parallel with the siRNA experiments so that it was possible to follow the effect of siRNA transfection on cell morphology and cell proliferation.

As a transfection control we used 10 pM Fluorescein-siRNA Transfection

Control purchased from NFB (Hitchin, UK) and used it in the same way as siRNA following the manufacturers’ protocol. Briefly, 1.5 pi of 10 pM Fluorescein-siRNA

Transfection Control was added by gentle pipetting to the diluted transfection reagent and incubated for 10-20 min at RT to form a transfection complex which was then diluted to 1 ml of complete culture media and added evenly to the treatment wells of

6 well plate. The plates were incubated for 12 hrs at 37“C.

3.2.3 Validation of silencing

After completion of the transfection treatment, 6-well plates were placed on ice and the cells were lysed with 20 pi of ice-cold lysis buffer as described in Chapter 2,

Section 2.2.1.2. Cells were scraped off the wells and transferred to 1.5 ml Eppendorf

95 tubes, where the lysates were centrifuged at 1300 rpm for 5-10 min. The supernatant was transferred to fresh tubes and the protein samples were kept at -20°C until further use.

3.23.1 Bradford assay

The concentration of total protein amount for each lysate sample was calculated from comparison with a BSA standard curve using the Bradford assay as described previously in Chapter 2, Section 1.2.13.

3.2.3.2 Western blot analysis

The lysate samples that had a sufficient amount of Grb2 and ERa protein were loaded onto a polyacrylamide gel. The amount of 25 pg of total protein was administered, with the addition of Ix SDS gel-loading buffer (80 mM Tris-HCl (pH 6.8), 2 % SDS,

10% glycerol, 0.1% bromophenol blue and 0.5% 2-mercaptoethanol). A biotinylated ladder (NEB, Hitchin, UK) was always loaded onto the gel to enable the estimation of the molecular weight of the protein band. Technical aspects of Western blotting are described in Chapter 2, Section 2.2.7.4.

3.2.3.3 Protein detection using monoclonal mouse antibodies

For the detection of Grb2 protein, we followed the manufacturer’s suggestions on membrane blocking and antibody incubation (Cell signaling, NEB, Hitchin, UK).

Briefly, once the protein had been transferred from the polyacrylamide gel to the

96 nitrocellulose membrane, the membrane was washed three times with 25 ml 1 x TBS

for 5 min at RT. The membrane was incubated with a blocking solution made up using 5% BSA in TBS-T. Following one hour incubation at RT, the membrane was washed three times for 5 min each with 15 ml TBS/T. The membrane was incubated overnight at 4°C with the primary Grb2 rabbit antibody (1:1000 dilution made in

TBS/T with 5% BSA) purchased from Cell Signalling Technology (NEB, Hitchin,

UK). For the detection of the primary Ab, the membrane was incubated with HRP linked anti-rabbit IgG (made in 1:2000 dilution in TBS/T with 5% BSA) for 1 hr at

RT. The membrane was analysed on the same day.

3.2.3.4 Protein detection and Western blot analysis

Detection of probed ER and Grb2 proteins on the nitrocellulose membranes was achieved using the ECL detection system as previously described in Chapter 2,

Section 2,2.7.4.

Band intensities from each individual experiment were quantified using blot analysis captured by the bio imagining system GeneGenius (Syngene, UK). Results are presented as percentage of band intensity in relation to the corresponding controls.

The most intense band was set as a control (at 100%) using the software provided with GeneGenius.

97 3.2.4 CBMN assay following gene silencing

Investigations of the effects of gene silencing on MN frequency were performed once

all the experimental steps of siRNA were optimised and the gene silencing validated.

The MCF-7 cells were seeded on glass cover slips in 6 well plates at the previously determined optimum density. Following incubation for 12 hrs with the

optimised amounts of corresponding siRNA and transfection media, all wells were gently washed once with serum free media and then treated with the test compounds for the MN analysis as described in Chapter 2, Section 2.2.3.

As test agents we selected two steroidal estrogens E2, E3 and the carcinogen

BaP. Treatment concentrations were the same as those described in the previous chapter. Following 24 hrs incubation with the test compounds, the media was removed and the cells were treated with Cyt B and further treated as described in

Chapter 2, Section 2.2.3.

Statistical analysis was performed as described in Chapter 2, Section 2.2.4.

3.3 Results

MCF-7 cells were grown and transfected with siRNA so that the effects of silencing of the target genes ER and Grb2 on the frequency of MN formation could be assessed. To improve the effectiveness of gene silencing, the cell density and the amount of silencing and transfection media in each well needed to be as favourable as possible.

98 Delivery of siRNA into cells is an essential aspect of transfection efficiency, therefore cell viability in siRNA experiments and the transfection conditions, such as the ratio of RNAi Feet Transfection Reagent to siRNA, were optimised. A new batch of silencers was optimised separately for the siRNA experiment in order to achieve the greatest amount of target-specific knock-down.

3.3.1 Validation of ER silencing

Each volume of ER siRNA was tested with various volumes of transfection reagent

TransPass R1 in order to establish the best possible combination for the silencing.

However, out of 12 possible combinations between the siRNA and transfection reagent TransPass Rl, only one combination was found to give enough protein sample for the assessment of the silencing using Western blotting and this was by combining 0.5 pi of lOpM ER short cut silencing mix with 6 pi of 2.5 mg/ml

TransPass Rl transfection media. All other combinations appeared to be cytotoxic as the cell density in these wells was much lower in comparison to untreated controls. It was then investigated if this combination also induced the desirable silencing of the

ER. The level of ER silencing was measured by Western blotting and the band intensities quantitatively evaluated by densitometry using GeneGenius (Syngene,

UK) (Figure 3.3). The results of ER silencing were expressed as the percentage change in the band density as compared with the control value which is set to 100%.

As shown in Figure 3.3, expression of ER in MCF-7 cells was reduced by over 70% under treatment conditions of 0.5 pi of 10 pM of ER siRNA and 6 pi of 2.5 mg/ml of transfection media.

99 kDa Lane 1 Lane 2 100.00% 28.74%

106.904 93.636

Figure 3.3 ER expression levels. The cell lysates were analysed by Western blotting with antibody specific for ER. The untransfected control (lane 1) shows the presence of ER in MCF-7 cells and was set as a reference value of 100%. Lane 2 shows a much reduced intensity, indicative of over 70% silencing of the ER. The band intensities of all lanes were measured densitometrically and expressed as fractions of the control.

3.3.2 Validation of Grb2 silencing

For the silencing of Grb2 it was necessary to evaluate which of three different silencers gave the best result in MCF-7 cells. First, the ratio of the amount of siRNA and transfection media needed to be optimised.

Grb2 silencer no. 106680 was highly cytotoxic in MCF7 cells at the tested volumes and therefore was disregarded for further work. However, successful silencing of Grb2 expression was been observed with the other two Grb2 silencers.

The results presented in Figure 3.4 show that Grb2 expression was effectively silenced using siRNA. The combination of lOpM of Grb2 silencer no.4196 at a volume of 0.5 pi with 6 pi 25 mg/ml of transfection reagent TransPassRl gave an optimal silencing of the Grb2 expression, reducing the levels of Grb2 protein by more

100 than 90% (lane 1 in Figure 3.4). This combination of silencer and transfection agent was used for an analysis of MN frequencies. At the higher volume of 1.5 pi of the same silencer together with 4 pi of transfection reagent, Grb2 expression was suppressed by approximately 85%. Similar reductions of Grb2 expression were also observed using 1.5 pi of lOpM siRNA no. 144866 with 10 pi of transfection media.

kDa lanel lane2 lane3 Iane4 Grb2 siRNA no.4196 no.4196 N/A no. 144866 9.93% 16.39% 100% 14.79%

37.226

28.244

Figure 3.4 Silencing of Grb2 by using different combinations of siRNA and transfection agent. Cell lysates were separated by SDS-PAGE and probed with anti-Grb2 antibody. Lane 3 corresponds to untransfected controls, with the presence of Grb2 protein clearly visible. This lane also has the highest band intensity, which was set as 100%. The band intensities of all lanes were measured densitometrically and expressed as fractions of the control. Grb2 expression was reduced in lanes 2 and 3 (Grb2 silence no.4196) and in lane 4 (Grb2

silencer no. 144866).

3.3.3 The effects of ER silencing on MN frequency

Cells treated for silencing of the ER were subjected to the cytokinesis block MN assay. In these cells, extensive reductions in proliferation combined with marked

101 morphological changes can be associated with apoptosis which is recognised by the appearance of cell shrinkage, membrane blebbing, chromatin condensation, DNA

(Bardon et al. 1987, Warri et al. 1993) (Figure 3.5).

m «

Figure 3.5. Changes in MCF-7 cell morphology in cells treated for ER silencing, followed by the MN assay. Figure a) shows MCF-7 cells not subjected to siRNA transfection after the MN assay. The cells in Figure b) were treated with ER siRNA and subjected to the procedures of the cytokinesis-block MN assay, which induced notable loss of cell number and morphological changes such as cell shrinkage, change of cell shape and loss of the cell surface structure suggesting that the cells were undergoing apoptosis.

These changes were so extensive that a scoring of MN in these cells was not possible. For this reason, an assessment of MN frequencies could not be carried out.

102 3.3.4 The effects of Grb2 silencing on MN frequency

To investigate the effect of silencing of Grb2 on MN frequencies, the Grb2 protein was first silenced using the experimental condition that induced the most efficient knock-down of the Grb2 protein. For the MN assay, cells were treated with E2, E3 and BaP (data shown in Figure 3.6). Although some reductions in cell numbers were seen, the remaining cells looked healthy overall. 1000 BN cells were, therefore, counted and evaluated following the procedures described in Chapter 2, Section

2.2.3.

300

Blank E2 E3 BaP 250 -

200 — ------Z OQ O 150 -

100 -

50 -

a) b) a) b) a) b) a) b)

Figure 3.6 MN induced in MCF-7 cells following treatment with E2 (1 nM), E3 (10 nM) and BaP (10 pM). Clear bars (a) show the MN frequency in normal MCF-7 cells without Grb2 silencing, while shaded bars (b) show the frequency of MN following silencing of Grb2 protein.

103 The occurrence of MN induced by each compounds was assessed. As previously, all experiments were run in parallel to the controls and the degree of MN formation of both silenced and control cells was compared as shown in the figure above.

Under the experimental conditions described, silencing of Grb2 protein had no significant effect on MN formation in MCF-7 cells exposed to the test compounds.

Treatment of MCF-7 cells with siRNA prior to performing the MN assay generally induced a slight increase of MN formation.

3.4 Discussion

While it was possible to successfully suppress expression of the ER and Grb2 by using siRNA, the conditions of siRNA do not seem to be conducive for subsequent evaluations of MN frequencies. In the case of ER siRNA pre-treatment of MCF-7 cells, insufficient cell proliferation was the limiting factor. One of the major requirements for the reliability of the MN method is that cell number is not affected by compound treatment. With ER silencing however, extensive cell death was observed once the two techniques were combined.

It is conceivable that severe reduction in cell proliferation and cell death have occurred as a direct consequence of silencing the ER. Estrogens, which are key regulators for growth of both the normal breast and malignant breast, mediate most of these effects biological effects of E2 in both normal and cancer cells. The presence of the ER is, therefore, essential for normal MCF-7 cell function and maintenance

104 (Zivadinovic and Watson 2005), and suppression of ER synthesis by siRNA in our cell line appears to have serious down-stream effects which consequently affected the survival of the cell.

It is generally known that the Bax protein is counteracted by Bcl-2, which has been shown to prevent the apoptosis as its overexpression has been reported to inhibit plasma membrane blebbing, nuclear condensation and DNA endonucleolytic cleavage which are all classic features of apoptosis (Hockenbery et al. 1990,

Hockenbery et al. 1993). Estrogens themselves can inhibit apoptosis in the mammary gland, which cyclically undergoes apoptosis at the end of the menstrual cycle

(Ferguson and Anderson 1981). In a recent report by Perillo and colleagues they showed that in human breast cancer cells (MCF-7), estrogens up-regulate transcription of bcl-2 gene via the estrogen responsive element (ERE), thereby preventing the occurrence of apoptosis (Perillo et al. 2000). It is therefore conceivable that the knocking-down of the ER can severely affect MCF-7 cells, leading to apoptosis. For these reasons it was not possible to investigate the effects that ER silencing might have on MN formation.

In contrast to the experiences with silencing ER, suppression of Grb2 expression by siRNA was tolerated better by MCF-7 cells. Although one of the three tested Grb2 siRNA, silencer no. 106680, was also found to have a cytotoxic effect on

MCF-7 cells, the remaining two Grb2 silencers, Grb2 siRNA no.4196 and Grb2 siRNA no. 144866 did not affect cell proliferation to the point where the conduct of the MN assay was compromised. However, Grb2 silencing was without effect on MN formation. Does this observation invalidate our hypothesis proposed in Chapter 2 about the importance of Src/Raf/Erkl, 2 signalling for MN formation?

105 As previously discussed in Section 3.1, the ER is believed to couple to Src to initiate the activation of the signal transducing Src/Ras/Erk pathways in MCF-7 cells.

This signalling pathway, as described in the same section can also be activated by growth factors such as EGF through membrane associated RTKs. Src is believed to contribute to the activation of Ras/Erk signalling pathway by phosphorylating the adaptor proteins She, Grb2 and Sos.

However, the Src signalhng cascade is still not fully resolved. It has been shown that Src can be activated by interaction with a range of different receptors such as platelet derived growth factor receptor (PDGFR) (Twamleystein et al. 1993) and

RTK receptor Erb-B2 (Muthuswamy and Muller 1994). Castoria and colleagues

(2001) have also shown that activation of Src/Erk signalling pathway can also be induced by an involvement of Phosphoinositide Kinase-3 (PI3K) as shown in Figure

3.7.

PI3K is activated immediately after RTK receptor stimulation (at GO/Gl transition) and again in late G1 stage of the cell division and is an early signaling molecule involved in regulation of cell growth and cell cycle entry (Garcia et al.

2006). The activation of non-genomic signalling events by PI3K, as shown in Figure

3.7, is dependent on the presence of adaptor proteins Shc/Grb2/Ras pathway via Src.

However, PI3K has also been shown to be able to activate the Raf/Erkl,2 signalling independently of Src and the adaptor proteins. (Cantrell 2001) among others have shown that Erb-B2 can stimulate PI3K signalling through recruitment and phosphorylation of catalytical subunit of PI3K leading to signal transduction to the key downstream effectors phosphoinositide-dependent kinase (PDKl) and a serine- threonine kinase Akt.

106 Erb-B2

i Grb2

PI3K

Nucleus Cell cycle progression And MN

Figure 3.7. Possible cross-talk between different RTK receptors and ER involved non-genomic activation of Src/Erk signalling. Like other RTKs, Erb-B2 and EGER receptors can phosphorylate each other upon ligand stimulation and serve as a docking site of various adaptor proteins which consequently activate multiple downstream signalling cascades such as Ras/Raf/Erk and PI3K (red arrows) which can stimulate cell cycle progression. In contrast, it has been shown that once activated, PI3K effector can directly activate and phosphorylate number of targets involved in cell cycle progression, one of which MAPK.

In response to PI3K activation, these two kinases play a key role in activating, phosphorylating and regulating the activity of a number of targets including kinases, transcription factors and possibly also MAPK ErkI/2. This suggests that by silencing the Grb2 adaptor protein, the activation of the Ras/Raf/Erk signalling pathway was disrupted. However, Erk 1/2 can still be activated by other signalling pathways, such as PI3K, independent of the presence of Grb2. Thus, the absence of effects on the

107 frequency of MN upon silencing of Grb2 is due to redundancy in signalling, but does not invalidate hypothesis of the relevance of Erk 1/2 for MN formation.

As described in Chapter 2, Section 2.1., steroidal estrogens and the xenoestrogen BPA are full agonists for both classical and non-genomic transcription and simultaneously activate the two. The only way to further test our hypothesis and role of genomic versus non-genomic signal transduction in MN formation would be by testing chemicals that distinguish between different signaling actions of ER, i.e. chemicals that are full agonist for either genomic or the non-genomic signalling pathways. Kousteni and colleagues (2002) for instance described a synthetic estradiol analogue 4-estren-3a, 17(3-diol (estren). They showed that estren is able to induce the non-genomic effects of steroidal estrogens, meanwhile it has very poor agonist potential for the genomic signaling pathways, which requires the presence of ER

(Kousteni et al. 2002). However, many other groups have observed that estren is able to induce both genomic and non-genomic signaling (Bjomstrom and Sjoberg 2005,

Moverare et al. 2003) testing such agents such as estren was initially described to be would be ideal for future research in the subject of relevance of ER presence in MN induction.

108 CHAPTER IV

COSMETIC INGREDIENTS AS ACTIVE MN INDUCERS

4.1 Cosmetic ingredients as potential environmental estrogens

Parabens are a group of alkyl esters of p-hydroxybenzoic acid and typically include methylparaben, ethylparaben, propylparaben, butylparaben, isobutylparaben, isopropylparaben, and benzylparaben. Given that these compounds are thought to have a low toxicity profile and to be stable in relation to pH and temperature, they are considered safe and, therefore, are widely used in both the food and cosmetic industry to preserve fats, proteins and oils. For this reason, they are regarded as an essential constituent in cosmetics, toiletries and pharmaceuticals (Golden et al. 2005), where they can be used in concentrations of up to 1% (Elder 1984b). According to a survey carried out by Rastogi and colleagues (1995), parabens are a common ingredient in

99% of leave-on and in 77 % of rinse-off cosmetics products. Parabens are also permitted as preservatives in food in levels of up to 0.1% (Elder 1984a). These compounds have worldwide legislative acceptance, and their safety was never considered to be an issue, due to their rapid metabolism once entered in systemic circulation.

However, Routledge and colleagues (1998a and 1998b) showed that parabens exhibit estrogenic properties. These compounds were able to interact and activate the

ER in yeast cells and their estrogenic activity was related to the increased length of

109 the alkyl group side chain - methylparaben being less estrogenic when compared with butylparaben.

In the same paper, Routledge and colleagues (1998) also showed that the estrogenicity of parabens is not exclusive to yeast cells. Subcutaneous administration of 1200 mg/day/kg of butylparaben to immature rats induced a 170% increase in the uterus wet weight. The observed increase, they emphasised, was not merely due to water retention because an equivalent increase was seen in uterine dry weight as well.

Similar observations were made with ovariectomized rats. Oishi et al (2001) demonstrated that butylparaben and propylparaben have effects on the male reproductive tract (alterations of reproductive function as well as reductions of sperm counts).

More recently. Byford and colleagues (2002) confirmed the interaction between ER and parabens in breast cancer cells. They showed that parabens increased

ER regulated cell proliferation and growth in MCF-7 breast cancer cells. It has also been demonstrated that the estrogenic effect of propylparaben and butylparaben can be suppressed upon co-administration of the ER antagonist, 4-OH-Tam, demonstrating that these compounds have affinity for ER and can induce ER mediated effects on the MCF-7 cells (Byford et al 2002, Darbre et al. 2002a).

4.1.1 Estrogenic actions of musks and UV filters

As with parabens, UV filter agents and synthetic musks are widely used as ingredients in cosmetic and personal care products. Recently they too have been shown to have estrogenic activity. They can mimic the effects of the natural estrogens

110 by binding to the ER and affecting the expression of E2 dependent genes

(Brzozowski et al. 1997, Schlumpf et al. 2001, Inui et al. 2003, Gomez et al. 2005).

Musk agents are synthetically produced compounds that have been widely used for over 40 years in fragrant formulations in consumer products including perfumes, lotions, detergents as well as food additives, because of their musky scent and their ability to make a fragrance long lasting. Recent reports in the literature have shown alarming increases in the concentrations of these estrogenic compounds in freshwater fish. Eschke et al. (1994) looked at perch and bream and reported high doses of two commonly used musk agents 1,3,4,6,7,8-hexahydro-4,6,6,7,8,8- hexamethyl-cyclopenta-(y)-2-benzopyran, (HHCB, often referred to as galaxolide) and 6-acetyl-1,1,2,4,4,7-hexamethyltetralinem, (AHTN often referred to as tonalide) in these fish from the Ruhr river (Germany). The average concentration of HHCB was 2.9 mg/kg muscle fat of perch and 2.5 mg/kg muscle fat of bream. The mean levels of AHTN were 4.6 mg/kg muscle fat of perches and breams.

Concerns about HHCB and AHTN arose because of reports of their presence in human fat tissue and breast milk (Rimkus and Wolf 1996, Muller et al. 1996).

Eschke et al. (1995) showed the presence of musk compounds in human adipose tissue and breast milk using GC/MS/MS and detected unexpectedly high levels of both HHCB (335 pg/kg milk fat) and AHTN (270 pg/kg milk fat), which is most probably a result of their high lipophilicity and chemical stability.

UV filter substances are organic compounds able to absorb UV-A and/or UV-

B radiation, hence their use in sunscreens for the blocking of sunlight on the skin.

Similar to polycyclic musks and parabens, UV filters have been found in the aquatic environment and in humans. A study of fish from the Meerfelder Maar lake (Eifel,

Germany) in 1991 and 1993 identified UV screens as important environmental

111 contaminants. Nagtegaal et al. (1997) mainly focused on perch and roach and showed that sunscreens are relevant environmental pollutant as both fish species were contaminated with sunscreens, polychlorinated biphenyls and DDT at comparable levels. In 2004, Schlumpf and colleagues showed that 3-benzylidene camphor (3-

BC), octylmethoxycinnamate (OMC) and 4-methylbenzylidine camphor (4-MBC) are weak estrogens. They raised concerns whether they can also be a threat to human health as they are highly lipophilic and therefore can be expected to bioaccumulate in the environment (Schlumpf et al. 2001) and in the adipose tissue, e.g. of the mammary gland. Dabre et al. (2002) even speculated whether deodorants containing parabens, UV filters and musks might contribute to the rising incidence of breast cancer cases in woman. The concentrations of parabens in breast tissue were broadly in the range of the levels of environmental estrogenic chemicals that accumulate in the human breast, such as polychlorinated biphenyls (PCBs) and organochlorine pesticides (OCPs) (Darbre et al. 2002b)

In the light of the reports showing high concentrations of cosmetic compounds in human adipose tissue and mother’s milk and their estrogenic properties, it is vital to investigate if they too are able to induce MN. The issue is not only interesting from a safety evaluation point of view for cosmetic ingredients. It is also relevant in a wider sense, namely whether the ability of steroidal estrogens and bisphenol A to induce MN via fast signalling events in the wake of ER activation is general to chemicals with binding afficity for the ER. Thus, the aim of this chapter is to evaluate the effects of these compounds in terms of MN formation in breast cancer cells and to make comparisons with the potency of BPA and E2 in terms of

• the frequency of MN formed following the exposure with the compound for

one cell cycle,

112 • the effect of inhibition of the Src/Erk pathway on MN induced by these

compounds and

• whether MN are formed due to DNA adducts or by chromosomal loss or gain.

In view of their widespread use in cosmetic and personal care products it was decided to focus on the two more estrogenic alkyl hydroxyl benzoate preservatives, propylparaben and butylparaben, the polycyclic musk fragrance AHTN and three UV filter substances (OMC, 4-MBC and 3-BC).

To further analyse the mode of action of these compounds, we combined the

MN technique with an anti-kinetochore staining method using CREST antibody, directed against the kinetochore complex of chromosomes, to help distinguish if test compounds have an aneugenic (induce MN formation through spindle poisons) or clastogenic effect (acentric fragments induced via DNA adducts) on MCF-7 cells

(KirschVolders et al. 1997).

4.2 Materials and methods

4.2.1 Chemicals and reagents

All stock solutions of test chemicals and subsequent dilution series were prepared in ultrapure HPLC grade ethanol and stored at - 20 °C. Propylparaben (>99% purity) was kept as a stock solution (8.97 mM) and butylparaben as a solution of 7.50 mM.

Both parabens were purchased from Sigma Aldrich (Dorset, UK). The UV filter

113 substances octylmethoxycinnamate (OMC) (98% purity) and 4-methylbenzylidene camphor (4-MBC) (99.7% purity) were purchased from VWR (Merck, West Drayton,

UK) and kept as solutions of 8.26 and 11.4 mM, respectively. 3-benzylidene camphor

(3-BC) was from Induchem (Volketswil, Switzerland) (97% purity) and kept at a concentration of 6.83 mM. AHTN was prepared as a stock solution of 3.3 mM and was purchased from LGC Promochem (Teddington, UK). Given that this is a technical product it had no certificate to state its purity. The Erk 1/2 inhibitor PD

98059 was obtained from Promega (UK) and kept in stock solution made in DMSO.

For each experiment run, the solvent control made of EtOH or DMSO, depending on the treatment, was used. So for instance, for experiments with the

Erk 1/2 inhibitor a combination of DMSO and EtOH was used as a solvent control. To safeguard against cytotoxicity arising from the solvents, the final (v/v) concentrations of DMSO and EtOH did not exceed 0.5% and 0.1%, respectively, at any point during the cell treatment.

4.2.2. Cell culture and treatments for the MN assay and kinetochore staining

Details about the maintenance of MCF-7 cells were as previously described in

Chapter 2, Section 2.2.2. Harvesting, seeding density and treatment of the cells for the micronucleus assay have also been described in Chapter 2, Section 2.2.3.

The cells were exposed to the test compounds for 24 hrs at the following final concentrations: propylparaben 15 pM, butylparaben 15 pM, 4-MBC 40 pM, OMC 20 pM, 3-BC 10 pM and AHTN 3.3 pM. As positive control for MN induction 1 nM E2

114 was chosen. Co-treatment with the cosmetic compounds and the inhibitors was performed in the same way as previously described in Chapter 2, Section 2.2.3.

All slides were coded to ensure that the researcher scoring MN frequencies was blinded towards treatment regimens. A minimum of three independent experiments were conducted, following OECD guideline 487. Statistical analysis was performed as described in Chapter 2, Section 2.2.4.

Kinetochore staining of the MCF-7 cells, following treatment with the cosmetic compounds, was carried out exactly as described in Chapter 2, Section 2.

2.5.

4.2.3 Cytotoxicity assay

Cells were seeded in 96 well Nunc plates (Fisher Scientific, Leicestershire, UK) at a density of 5000 cells per well and left to attach for 24 hrs after which the complete medium was replaced with 100 pi of phenol red-free DMEM medium. The compounds were administered to the cells in DMEM and incubated for an additional

24 hrs, under normal growing conditions. The remainder of the procedure of the MTT assay was described in detail previously in Chapter 2, Section 2.2.4.

115 4.3. Results

4.3.1. Choice of test concentrations

To gain some degree of comparability with the results obtained for E2 (Chapter 2), the concentrations of the cosmetic compounds tested in the MN assay were selected on the basis of their potency in the E-screen estrogenicity assay conducted in our lab by Dr. E. Silva, shown in Figure 4.1.

17P-oestradiol

0.9 Butylparaben PropyiParaben

3.-BC (Unisol S-22)

0.5 4-MBC (Eusolex 6300) 0.4 _— Tonalide (AHTN)

0.3 OMC (Eusolex 2292)

0.1

0.0 0.00001 0.0001 0.001 0.01 100 1000 10000 100000 Concentration [nM]

Figure 4.1 Concentration-response curves for E2 and the six tested cosmetic compounds. The figure is showing the best fitting models for 96 well plate E- SCREEN assay (data produced in our lab by Dr. E. Silva)

16 Similar to the procedure adopted for E2, the concentrations were chosen such that they came near the ieveling-off of the concentration-response relationships shown in Figure 4.1. Thus, treatment concentrations of butyl- and propylparaben selected for the MN assay (15 pM) corresponded to concentrations that produced a

85-90% response in the E-SCREEN (ECgs-go). Because the maximal proliferative response of all other chemicals was considerably lower than for the two parabens, the selected concentrations of the UV filter 3-BC (10 pM) corresponded to a 70% proliferative effect, for 4-MBC (40 pM) to 40% and for OMC (20 pM) to 25%. The treatment concentration of AHTN was based on published literature (Schreurs et al.

2005, Bitsch et al. 2002).

To ascertain that there was no toxicity at the chosen concentrations which might interfere with the induction of MN, it was necessary to carry out cytotoxicity tests. Figure 4.2 shows concentration-response relationships for all chemicals in the

MTT assay. As can be seen, there was minimal or negligible cytotoxicity at the concentrations selected for the MN assay.

At the concentration of 15 pM butylparaben and propylparaben, the number of cells with functional mitochondria was reduced by 10% compared to untreated controls. The test revealed that treatment with 3.3 pM of the musk agent AHTN resulted in a 20% reduction, similar to the values seen with the tested UV filters, 3-

BC at 10 pM, 4-MBC at 40 pM and OMC at 20 pM.

117 AHTN 4-MBC OMC

Figure 4.2. MCF-7 cell viability measured by MTT following treatment witb cosmetic compounds.Individual bars represent percentage of alive cells following treatment with 15 pM butylparaben, 15 pM propylparaben, 3.3 pM AHTN, 10 pM 3- BC, 40 pM 4-MBC and 20 pM OMC. Dark purple line set at 100% of live cells represents solvent control. The values presented here show the average cytotoxity of two duplicates. Abberations are: butylparaben: BP; propylparaben: PP.

4.3.2 MN induction by cosmetic compounds

Exposure of MCF-7 cells to the parabens propylparaben and butylparaben, as well as the musk fragrance AHTN and the UV-filters 4-MBC, OMC and 3-BC led to increases in the frequency of MN, when compared to solvent controls (Figure 4.3).

18 300

250 -

200 -

T 1 — 1— —I— — r OMC 4-MBC 3-BC BP PP AHTN E2 Solvent

Figure 4.3. MN induction in MCF-7 cells by cosmetic compounds.Shown is the number of binucleated MCF-7 cells with micronuclei. Cells were treated with the UV filters OMC (20 pM), 4-MBC (40 pM) and 3-BC (10 pM), the parabens propylparaben and butylparaben (both at 15 pM) and the musk AHTN (3.3 pM). MNs were scored in 1000 BN cells. Bars are means ± SEM from 3 independent experiments. Abberations are: butylparaben: BP; propylparaben: PP.

At a concentration of 15 pM, propylparaben was the most effective MN inducer, causing 2.5 -fold increase in the frequency of MN in comparison to the solvent control, higher than what was observed with 1 nM E2. At the same concentration as propylparaben, butylparaben induced a 2 -fold increase in MN frequency relative to untreated controls. An effect of comparable strength was also observed with 3.3 pM AHTN and 20 pM OMC. Treatment with 40 pM 4-MBC and

19 10 pM 3-BC induced slightly smaller (1.5 -fold) but statistically significant increases in MN formation.

Although the MN frequency (defined as the fraction of binucleated cells containing MN) observed with the cosmetic components differed from E2, the total number of MN per binucleated cells produced by these agents was similar to those produced by the hormone (Figure 4.4). Butylparaben and the UV filter 4-MBC produced somewhat fewer MN per BN cells at the chosen tested concentrations.

500

400 -

o

200 -

100 -

BP PP AHTN4-MBC0MC 3-BC E2 solvent

Figure 4.4. Comparison of MN frequencies and the total number of MN in MCF-7 cells induced by cosmetic compounds.Grey bars show the frequency of binucleated cells with MN when scored in 1000 BN, while the shaded bars represent the total number of MN per 1000 BN cells following treatment with same concentrations. Bars are means ± SEM from 3 independent experiments. Abberations are: butylparaben: BP; propylparaben: PP.

120 4.3.3 The effect of ICI 182 780 on MN frequency

In order to investigate whether the MN induction observed with these compounds is due to the ability to trigger nuclear ER activation, cells were co-treated with the pure

ER antagonist ICl 182 780 (Figures 4.5 and 4.6)

300

250 -

200 - Z OQ

150 -

1 0 0 ------

50 1

OMC 4-MBC 3-BC E2 ICI Solvent

Figure 4.5. Effects of the ER antagonistIC I 182 780 on MN frequency induced by UV filter substances. MCF-7 cells were treated with 20 pM OMC, 40 pM 4- MBC and 10 pM 3-BC individually (grey bars) alone, and in combination with 1 pM of ICl 182 780 (hatched). The values were compared to the solvent control (purple dashed line) and to the positive control (1 nM E2, green dashed line). Data are means

± SEM from 3 different experiments.

121 In the experiments with the UV filter agents OMC, 4-MBC and 3-BC, ICI 182

780 was unable to suppress the MN frequency. Similar to the results obtained with E2

(see Chapter 2, Section 2.3.4), co-treatment with the ER antagonist even led to slightly elevated MN frequencies although this was not statistically significant.

In contrast, co-administration of ICl 182 780 with propylparaben and butylparaben, as well as AHTN, led to slight reductions in MN frequency, however, the effect did not reach statistically significance.

300

250 - 1 I X 200 - Z PQ O o 150

100 %

50 -

BP PP AHTN E2 ICI Solvent

Figure 4.6. Effects of the ER antagonist ICI 182 780 on MN frequency induced by parabens and AHTN. MCF-7 cells were treated with 15 pM BP and PP as well as 3.3 pM AHTN (grey bars) alone and in the presence of 1 pM ICl 182 780 (hatched). The values were compared to the solvent control (purple dashed line) and to the positive control (green dashed line). Bars are means ± SEM from 3 different experiments. Abberations are: butylparaben: BP; propylparaben: PP; AHTN:; E2

122 4.3.4 The role of the Erk 1/2 signalling pathway in the induction of MN by cosmetic compounds

Considering that treatment with ICI 182 780 did not lead to suppression of MN frequency induced by test agents, it was important to test the relevance of the

Src/Erk 1,2 pathway for MN formation and to see whether the induction of MN by cosmetic compounds operated through mechanisms similar to E2 (Figure 4.7).

300

250 -

1 X 200 - I Z OQ X I i o o 150 H

100 -

50 -

OMC 4-MBC 3-BC BP PP AHTN E2

Figure 4.7. MN frequency following co-treatment of cosmetic compounds with PD 98059.MCF-7 cells were treated with 20 pM OMC, 40 pM 4-MBC, 10 pM 3- BC, 15 pM PP and BP and 3.3 pM AHTN on their own (clear grey bars) and together with 25 pM of Erk 1/2 inhibitor PD 98059 (hatched bars). Controls, solvent (dashed line) and 1 nM E2 (green bar) were run in parallel. Bars are means ± SEM from 3 different experiments. Abberations are: butylparaben: BP; propylparaben: PP.

123 Based on the data presented in Figure 4.7, it is clear that the MN frequency induced by the UV filters 4-MBC and 3-BC is sensitive to the disruption of Erk 1/2 signalling pathway with the inhibitor PD 98059. The most effective and statistically significant reduction was observed with 4-MBC. On its own, 4-MBC induced a 2- fold increase over the solvent control. In the presence of PD 98059, MN frequencies similar to the levels of solvent controls were observed.

There was a clear reduction of MN frequencies upon co-treatment of butylparaben and propylparaben with PD 98059. However, only the reductions seen with butylparaben in the presence of PD 98059 reached statistical significance. This trend was not followed by OMC and AHTN, as co-treatment of these compounds with PD 98059 failed to suppress MN formation. With OMC, the presence of PD

98059 even led to a small increase in MN frequency, although this was not statistically significant.

4.3.5 Probing for centromeric sequences in MN

As previously described in Chapter 1, Section 1.6,agents can be divided into two main groups based on their mechanism of MN induction: clastogens and aneugens. In

Chapter 2 it was proposed that the mechanism underlying MN formation by steroidal estrogens and bisphenol A is related to over-stimulation of the Src/Erk 1,2 pathway in the wake of ER activation, with consequent overriding of the mitosis checkpoint. This is likely to result in premature cell cycle progression before all chromatids are correctly aligned along the spindle apparatus mid plane, leading to improper distribution of sister chromatids to daughter cell nuclei. In this way, entire

124 chromosomes might be excluded from the nuclei and will appear as micronuclei in the cytosol. If this mode of action has any relevance for the induction of MN by parabens, UV filter agents and synthetic musks, it would be expected that a significant fraction of the micronuclei identified in MCF-7 cells contain centromeric

DNA sequences. To test this idea, the MN formed by cosmetic ingredients were analysed for the presence of kinetochores by staining the cells with an anti- kinetochore CREST antibody (see Table 4.1).

Table 4.1 Induction of CREST-positive MN in human breast cancer cell following treatment with cosmetic agent.The aneugenic effect of the tested compounds was measured by the number of the CREST- positive MN per slide. The results presented in this table show the percentage of the total number of kinetochore positive MN on the slide, from two individual experiments.

Compound CREST+ MN(%)

15 pM Propylparaben 34.23% , 35.90%

15 pM Butylparaben 35.71%, 37.31%

3.3 pM AHTN 37.66%, 35.19%

10 pM 3-BC 37.36% , 37.62%

40 pM 4-MBC 35.63%, 36.11%

20 pM OMC 26.67%, 32.63%

1 nM E2 33.87% , 37.97%

10 pM BaP 24.21% , 24.59%

Solvent 20.10%, 23.00%

125 All tested compounds produced a higher fraction of CREST-positive MN, in comparison to 10 pM BaP which was employed as a control for MN induction by chromosome breakage (clastogen), and the solvent control. This indicates that these compounds induce chromosome loss, although a degree of chromosome fragmentation also plays a role. The data seen with cosmetic compounds is consistent to data seen with E2, suggesting these compounds have a similar mode of action to steroidal estrogens in forming MN.

4.4 Discussion

In this chapter we set out to investigate whether MN formation was a general feature of estrogen like compounds and if MN induction was a consequence of ER activation.

The results presented here show convincingly that in MCF-7 breast cancer cells, the tested cosmetic ingredients were able to produce increases in MN frequency similar to those induced by steroidal estrogens, which corresponds to an approximately 2- fold increase relative to solvent controls. Our data supports the hypothesis that one consequence of activation of ERa is formation on MN, as all chemicals that were tested here are well documented ER agonist, albeit with varying estrogenic potencies.

However, the formation of MN by the tested cosmetic compounds could not be explained by their ability to bind the ER at the nuclear level, as inhibition of the receptor with the antagonist ICI 182 980 did not reduce the MN frequency in MCF-7 cells. In some cases, ICI 182 980 even increased MN frequencies slightly. These findings clearly show that the genomic pathway following ER activation is not

126 involved in MN formation and are in agreement with our previous observations with steroidal estrogens (Chapter2, Section 2.3.4.I. Nevertheless, the ER appears to play a role in the events leading to MN formation, however involving fast signalling events via the kinases Src and Erk 1/2 (non-genomic signalling).

The nuclear ER functions as a platform for integrating various signalling pathways involved in transformation and cell growth (Alimandi et al. 1995, Roche

1994, Skorski et al. 1995). Steroidal estrogens can concomitantly activate non- genomic signalling pathways as well as the classical signalling pathway through direct ER activation. Our observation that MARK inhibitors were able to reduce the number of MN formed by the estrogenic cosmetics ingredients suggests that activation of the Erk 1/2 cascade and its upstream kinase Src, both mediated by ER in the cytoplasm, are involved in MN formation. Co-administration of specific inhibitors of Src and Erk 1/2 also affected MN induction by a range of different agents that were tested in Chapter 2, including the growth factor EOF, the xenoestrogen BPA and the steroidal estrogen E2.

However, two chemicals deserve further discussion because they did not follow the MN suppression patterns observed with all other estrogernic chemicals.

There was no significant reduction in MN frequency upon co-administration of PD

98059 with OMC and AHTN. Do these findings invalidate the hypothesis that MAPK are relevant for MN formation?

A possible explanation for the lack of MN suppressing ability of PD 98059 with OMC and AHTN could be that the inhibitor was ineffective in blocking the activity of the kinase. If this were indeed the case, reductions of the MN frequencies in the presence of the other tested chemicals, including 4-MBC, 3-BC, propylparaben, butylparaben and E2, should also not have occurred. Furthermore, PD

127 98059 completely inhibits the phosphorylation of Erk 1/2 at the tested concentration of 25 pM (Communal et al. 2000).

An alternative way of interpreting our findings would be to suggest that OMC and AHTN induce MN by other mechanisms, possibly even involving direct breaking of chromosomes. If this idea was correct, then it would be expected that AHTN and

OMC show clastogenicity in cells devoid of the ERa. Literature data show that

AHTN is negative in in vitro mutagenicity assays screening for point mutations, such as the classical Ames assay (Elder 1984a, Kevekordes et al. 1997, Api and San 1999).

Relatively few studies have examined the ability of AHTN to induce chromosome breaks and MN. Kevekordes et al. (1997) assessed MN formation by AHTN in the concentration range between 0.05 pM to 194 pM in cultured human peripheral lymphocytes and human hepatocytes. Hep G2 cells. The treatment did not lead to significant increases in MN relative to spontaneously induced MN in negative controls. However, these results are hard to compare with our findings, because exposure to AHTN was for only 2 hrs, much shorter than the exposure durations employed in our studies,

Api and San (1999) treated ex vivo isolated mouse lymphocytes and bone marrow cells with AHTN (0.05 pM to 194 pM) and did not observe elevated MN frequencies with exposure times of up to 8 hrs. However, there were indications that

AHTN is able to induce chromosome breaks in cells devoid of the ERa. In analyses of chromosomal aberrations in metabolically activated Chinese hamster ovary (CHO) cells, Api and San (1999) observed that AHTN induced structural aberrations of the chromosomes following 4 hrs treatment at concentrations of between 69.7 and 96.7 pM. Although the concentrations of AHTN employed in our study (3.3 pM) were lower than those chosen by Api and San (1999), the considerably longer exposure

128 times (24 hrs) used in our study make it difficult to rule out that AHTN at least partly induced MN by a clastogenic mechanism. Equivalent reports about the genotoxicity of OMC are missing in the published literature, but similar considerations apply.

However, it is unlikely that AHTN or OMC induce MN exclusively via a clastogenic mechanism. Our results combining the MN assay with an anti- kinetochore staining method using CREST antibodies showed that all the tested cosmetic compounds produced elevated fractions of MN showing centromeric DNA sequences. This indicates that the MN induced by the tested agents are likely to originate from the lagging of whole chromosomes, as seen with steroidal estrogens

(Chaper 2, Section 2.3.9),rather than from the lagging of chromosomal fragments, as seen in MN following BaP exposure. This suggests that AHTN and OMC produce

MN by a mixed mechanism, involving both the loss of entire chromosomes as well as chromosomal aberrations. It is therefore reasonable to suggest that administration of

PD 98059 would only block the loss of chromosomes, but because of the simultaneously ongoing fragmentation of chromosomes, administration of the kinase inhibitor very likely did not prevent formation of MN by OMC and AHTN. It is important to stress that this interpretation of our findings relies on only one literature report indicating AHTN clastogencity, and that reports of similar effects of OMC are lacking completely. To strengthen our suggestion it is necessary to conduct further genotoxicity tests with these agents.

In general, the formation of MN by estrogen-like chemicals seems to be dependent on the presence of the ER. The proposed link between the ER and MN is supported by work with the estrogenic isoflavone compound daidzein, a compound which has continuously been shown to be negative in genotoxicity assays. For instance, Schmitt et al. (2003) did not observe MN induction in daidzein treated

129 mouse lymphoma cells. This was corroborated by Di Virgilio and colleagues (2004) who used Chinese hamster V79 cells to study MN induction by daidzein, and also failed to observe MN in these cells. In MDA MB 231 cells, which do not contain

ERa, but do express the ERp, daidzein treatment did not induce MN, however in

MCF-7 cells daidzein induced MN induction (Stopper et al. 2005). Stopper and colleagues (2003) also looked at MN formation in the ovarian cancer cells BG-1 and

UCI 107. They showed that E2 treatment induced MN in the ERa positive BG-1 cell line (Bardin et al. 2004), but not in ER negative, ovarian cancer UCI 107 cells

(Gamboa et al. 1995).

From these reports and our own findings it has become clear that are our hypothesis on relevance of ERa presence in aneuploidy induction and breast tumourigenesis by ER agonist is credible. Our data with cosmetic agents confirms that the activation of MAPK signalling transduction plays an important role in MN induction through chromosomal loss and mitotic spindle poisoning.

We believe that certain agents, such as AHTN and OMC that we showed here, exhibit mixed mechanisms of MN induction in MCF-7 cells and that chromosomal fragmentation occurs concurrently but independently of aneuploidy.

130 C H A PTER V

THE INFLUENCE OF PARACRINE SIGNALLING BETWEEN

DIFFERENT TYPES OF BREAST EPITHELIAL CELLS ON THE

INDUCTION OF MICRONUCLEI

The mammary epithelium is organised into two layers of cells: a luminal epithelium surrounded by a basal layer of myoepithelial cells. The bilayer is surrounded by a basement membrane that separates the epithelium from the connective tissue stroma compartments (Ronnov-Jessen et al. 1996). In the previous chapters we have focused our studies on MCF-7 breast cancer cells, that predominantly express luminal epithelial markers (Gordon et al. 2003), however, it is beginning to be appreciated that the interrelationships between the cancer epithelium and the mammary tissue microenvironment play a critical role in cancer progression. For instance, ER positive cells in mammary tissue do not themselves respond to E2 by proliferation, however they are commonly situated in close proximity to cells able to go through cell division. Clarke et al. (1997), among others, have hypothesised that surrounding neighbouring cells function as so-called ‘effector cells’, stimulated by the paracrine mitotic signals secreted by ER positive cells to induce mitosis following the estrogenic stimulation (Clarke et al. 2003, Anderson et al. 1998).

Guelstein and colleagues suggest that the myoepithelial cell naturally exhibits a tumor suppressor phenotype as they rarely transform, and when they do, transformation generally gives rise to benign neoplasms rather than degrade the

131 extracellular matrix (Guelstein et al. 1993). This anatomical relationship between luminal epithelial cells and those in the myoepithelium suggests that myoepithelial cells may exert important paracrine effects on the epithelium and may influence breast cancer progression. Studies to decipher the interactions among these cell types have employed a number of interesting approaches. Stemlicht and colleagues (1997) for instance were one of the first to use conditioned media of myoepithelial cells showing their ability to inhibit invasion of invasive breast carcinoma and melanoma lines in vitro. Their findings were in agreement with reports using Boy den chambers

(chambers that are separated by a filter through which cells migrate) (Carpenter and

Nguyen 1998).

The MCF-7 cells that we used throughout our work are often employed as representatives of cells that have escaped the paracrine regulatory signalling by neighbouring cells and passed through the basement membrane. In contrast, immortalized, non-cancerous MCF-lOA human breast epithelial cells (Soule et al.

1990) are often used to represent normal breast epithelial cells (Debnath et al. 2002,

Debnath et al. 2003). In standard tissue culture, both cell lines display similar epithelial morphology making it difficult to distinguish the two in co-culture (Spink et al. 1998). Spink and colleagues therefore developed a system to look at the paracrine interactions between these cells by using a co-culture system where MCF-7 cells were tagged with green fluorescent protein (OFF) and grown at the same density on polystyrene tissue culture plates together with MCF-lOA cells (Spink et al. 2006).

They observed that whilst in monoculture and lacking paracrine controls, MCF-7 cells grew autonomously through autocrine stimulation in response to E2 and EOF.

However, whilst the mitogens stimulated cell proliferation in monoculture, once co­ cultured with MCF-lOA they observed inhibition of MCF-7 cell proliferation. They

132 hypothesised that inhibition of MCF-7 cell growth might be induced through the release of inhibitory factors by the MCF-lOA cells in co-culture, such as maspin

(Stemlicht and Barsky 1997, Pemberton et al. 1995), mammastatin (Ervin et al.

1989), matrix metalloproteinase inhibitors (Stemlicht and Barsky 1997, Barsky

2003), among others.

Because potentially important paracrine relationships might exist between myoepithelial cells and epithelial cells in both developmental biology and cancer, we became interested in assessing whether co-culture between MCF-7 cells and MCF-

1OA cells which lack the ER are also able to influence the frequency of MN in MCF-

7 cells. In the previous chapters we showed that in breast cancer MCF-7 cells, treatment with EGF and E2, as well as other ER ligands, yielded high MN frequencies. Spink’s findings prompted us to explore the influence of paracrine signalling between MCF-lOA and MCF-7 cells on the induction of micronuclei. This meant that it was necessary to first evaluate whether MN induction is observed in

MCF-lOA cells lacking ER. Secondly, it was important to assess if the MN frequency in ER-competent MCF-7 cells could be influenced by the presence of paracrine interactions with MCF-lOA by growing them in close proximity.

In the light of their ability to induce MN by very different mechanisms, we chose to study E2 and BaP. However, time constraints meant that labelling the cells with different GFPs was not an option. Instead, we separated the mammary cells by growing MCF-lOA cells on a microporous, polycarbonate membrane placed over

MCF-7 cells grown on a plastic surface (Figure 5.1). This allows the secretion of paracrine factors into the culture medium. The membranes are designed to fit 6 well plates and are held in a well by an insert, leaving a small gap between the bottom of the well glass cover slip and the insert where the MCF-7 cells are seeded.

133 Polycarbonate Membrane 0 24 mm, Pore size: 0.4 pm MCF-lOA

MCF-7 Figure 5.1. Polycarbonate inserts used for culturing of MCF-7 cells and MCF- lOA. Whilst MCF-7 cells were grown on the bottom of the 6 well plate on a 18x18mm glass cover slip, MCF-lOA were grown in specially fitted inserts with a very fine polycarbonated membrane, pore size of 0.4 pm, that allowed paracrine interaction between the cells.

5.2 Materials and methods

5.2.1 Care and passage of MCF-lOA cells

Human mammary MCF-lOA cells were purchased from (ATCC). Cells were maintained in 75 cm^ cell culture flasks (Greiner, Gloucestershire, UK) in a growth medium containing Dulbecco’s modified Eagle’s medium and FI2 medium (DMEM-

F12) (Invitrogen, Paisley, UK) supplemented with 5% horse serum (Invitrogen,

Paisley, UK), hydrocortisone (0.5 pg/ml), insulin (10 pg/ml), EGF (20 ng/ml), penicillin-streptomycin (100 pg/ml) and cholera toxin (lOOng/ml) (Sigma, St. Louis,

MO, USA). The cells were kept in a humidified incubator at 37 °C/5% C 02.

Medium was replaced every other day and the cells were sub-cultured once a week, when cells reached approximately 70-80% of confluence. For cell passage, the growth medium was aspirated and the cells were rinsed with 10.0 ml of BBSS. Then

134 2.0 ml of Ix trypsin solution (0.05% trypsin/0.02% EDTA) was added, aspirated, leaving a thin film behind. This was done as it is not advisable to use high concentrations of trypsin solutions when passaging MCF-10 A cells because they tend to clump if the excess trypsin is not removed, making it difficult to obtain a single-cell suspension. Cells were then incubated in a 5% CO 2 humidified incubator at 37°C for 15-25 min, periodically checking the extent of trypsinisation by gently tapping the plate to dislodge the cells. Cells should be completely dissociated from the plate to avoid clonal selection of adherent cells. Once cells were dislodged, 1.0-

2.0 ml of resuspension medium, containing DMEM/F12 medium supplemented with

20% horse serum and penicillin-streptomycin (100 pg/ml), was added and pipetted to break up the clumps. The cells were then transferred to a 15 ml centrifuge tube and the culture flask rinsed with another 1.0 ml of resuspension medium, to recover remaining cells. An additional 1.0 ml of resuspension medium was added to the centrifuge tube so that ultimately the cells were re-suspended in 3 - 4 ml. Cells were spun down at 150 gin a tissue culture centrifuge for 3-5 min. The medium was aspirated and the cells were re-suspended in 1.0 ml of MCF-lOA growth medium.

Another 4.0-5.0 ml was added to the tube with the re-suspended cells. The cells were seeded at volume of 1.0 ml cells per 10 cm dish in a total of 10 ml of MCF-lOA growth medium.

For the assaying of the MCF-lOA cells for micronuclei, we used again the

DMEM/F12 medium supplemented with the same concentration of the hydrocortisone, cholera toxin, insulin and penicillin-streptomycin as for the growth media, however the horse serum was reduced from 5% to a final concentration to 2%.

This will be referred to as the ''assay medium”.

135 5.2.2 Care and passage of MCF-7 cells

Care and passage of breast cancer MCF-7 cells were described in Chapter 2, Section

2.2.2.

5.2.3 Optimisation of the media combination for co-culture experiments

In terms of culture medium composition, MCF-lOA and MCF-7 cells have different optimal requirements. Because they had to be grown in the same medium for the subsequent experiments with MN frequencies, an optimized medium had to be found that satisfied the growth requirements of both cell lines. This was achieved by assessing cell growth in different combinations of the MCF-7 and MCF-lOA media.

MCF-7 and MCF-lOA cells were seeded at the same density in the 6 well plates together with different compositions of the assay media, at 20:80, 30:70, 50:50, 70:30 and 80:20 MCF-7:MCF-10A medium, for 5 days. Media were replaced every other day. Assay medium containg 80 % of MCF-7 and 20% of MCF-lOA medium was chosen on the basis that it did not have a negative effect on the cell proliferation, which was microscopically assessed and was used for all the experiments with polycarbonate insert chambers. Hereafter, this medium will be referred to as ''80:20 medium”.

136 5.2.4 Culture of MCF-7 and MCF-lOA cells in polycarbonated chambers

MCF-7 cells were added to 1.5 ml of the 80:20 medium and seeded on 18x18 mm glass cover slips placed in a 6 well plate, at a density of 1x10^ cells/ml. MCF-lOA cells were seeded on to the polycarbonate inserts (Costar, VWR International,

Leicestershire, UK) at the same density as the MCF-7 cells.

The 80:20 medium was changed after 24 hrs and was replaced with new medium containing the test compounds. As previously described in Chapter 2,

Section 2.2.3, the cells were exposed for 24 hrs, at a final concentration of 1 nM for

E2 or 10 pM for BaP. After the 24 hrs treatment period,80:20 medium containing 3 pg/ml cytokinesis inhibitor Cyt B (Sigma, Poole, UK) was added and left for an additional 18 hrs. The medium was removed, the cells washed briefly with HBBS and then fixed in 70% cold EtOH for 20 min. Cells were then stained in 5% Giemsa at

RT. The fixing, staining and the counting of the slides were carried out as described in Chapter 2, Section 2.2.3.

5.3 Results

5.3.1 MN induction in breast MCF-lOA cells

In a first series of experiments, the sensitivity of MCF-lOA cells to E2 and BaP, in terms of induction of MN, was investigated. The cells were grown on their own on cover slips and exposed to 1 nM E2 or 10 pM BaP. As shown inFigure 5.2, the MN

137 frequencies in solvent-exposed control cells were somewhat lower than those observed with MCF-7 cells. At the concentrations tested, neither E2, nor BaP were able to induce MN frequencies significantly different from controls. While the lack of effect of E2 can be explained in terms of the absence of the ER in MCF-lOA cells, the inability of BaP to induce MN was surprising.

5.3.2 The influence of the presence of MCF-lOA cells on MN induction in MCF-7 cells

In the second part of the experiment we investigated whether the MN frequency was affected once MCF-7 cells were grown in close proximity to MCF-10 A cells. The assay medium for the MCF-7 and MCF-lOA cells was first optimised and chosen on the basis of the effect it had on cell proliferation. Microscopic examination of the appearance of either cell type, and evaluation of their cell growth revealed that both lines tolerated well a medium composed of 80% of the medium normally used for

MCF-7 cells, and 20% of that for MCF-lOA cells. This agreed well with the results communicated by Spink et al (2006). MCF-7 cells were grown on cover slips, with

MCF-lOA cells cultured on polycarbonate inserts placed above the cover slips in 6- well plates. MN frequencies were examined as described previously. The results were compared with the MN frequencies observed in MCF-7 cells grown under the same conditions, but without the inserts containing MCF-lOA cells. As shown in Figure

5.2, the MN frequencies induced by E2 and BaP agreed very well with the results reported in earlier chapters of this thesis. In both cases, an approximately 3-fold elevated MN frequency relative to solvent controls was found.

138 The close proximity of MCF-lOA cells had a dramatic impact on the ability of

E2 and BaP to produce MN in MCF-7 cells. With E2, the frequency of MN was reduced to control levels (Figure 5.2). In the case of BaP, the presence of MCF-lOA cells exerted a significant protective influence, although the reduction of MN did not quite reach solvent control levels.

300 Solvent 1 nM E2 lOpMBaP 250 -

200 - J- Z OQ o 8 150 H z

100 -

50 -

0 I

Figure 5.2. MN frequencies in MCF-7 (clear bars), MCF-lOA (hatched) and MCF-7/MCF-10A (crossed).As previously reported, treatment with E2 and BaP induced 2 to 3- fold increase in MN frequency compared to the solvent control (purple dashed line) in MCF-7 breast cancer cell. However, the same treatment of MCF-lOA cells as well the co-cultured MCF-7/MCF-10A cells did not induce an increase in the MN frequence and was not statistically different from the controls. Bars refer to ± SEM from 2 independent experiments.

139 5.4 Discussion

The results of our experiments show that the presence of MCF-lOA cells in close proximity to MCF-7 cells protected significantly from the MN-inducing effects of both E2 and BaP. Our observations echo those by Spink et al. (2006) who reported growth-inhibitory effects when MCF-lOA cells were co-cultured with MCF-7 cells.

These findings suggest that MCF-lOA cells secrete factors that protect from the aneugenic effects of E2 and BaP. It is striking that this occurred with two agents that induce MN by very different mechanisms.

Before the implications of these findings are discussed further, it is important to interpret the lack of MN formation with both E2 and BaP in MCF-lOA cells. In the case of E2, our results are readily explained in terms of the lack of ER in MCF-lOA cells. As was shown by Fischer et al. (2001) and Stopper et al. (2003), cells devoid of the ER are essentially insensitive to MN formation by E2.

The mode of action of BaP in inducing MN is unrelated to the presence of the

ER in both cell lines. The formation of MN by BaP is to due to its ability to induce

DNA adducts in the cells during the cell division. Consistent with previous findings,

BaP treatment of MCF-7 cells induced 2-3 fold increases in MN. However, treatment of MCF-lOA cells with did not produce increases in MN over and above background

MN frequencies. This is surprising, considering that BaP causes MN in a wide variety of cell lines (Wu et al. 2005, Styles et al. 1997, Roscher and Wiebel 1988). To our knowledge, BaP has not been tested with MCF-lOA cells, and it is therefore difficult to draw comparisons. It is conceivable that higher BaP levels might yield MN in these cells. However, this question was not pursued further here, considering that the

140 main objective was to investigate the influence of MCF-lOA cells on MN induction in MCF-7 cells. Future studies should address this point by conducting concentration- response studies with BaP in MCF-lOA cells.

The idea that factors secreted by MCF-lOA cells have a growth inhibiting effect on MCF-7 cells was proposed by Spink et al. (2005). By analogy, it seems reasonable to hypothesise that such factors also protect from MN induction by E2 and

BaP, although this requires experimental confirmation. A number of straightforward experiments suggest themselves to clarify the issue. If secreted factors are at play, then medium in which MCF-lOA cells were grown, so-called conditioned medium, should also lead to reductions in MN frequencies in MCF-7 cells, when co­ administered with E2 or BaP. Similarly, serial dilutions of such conditioned medium should restore the ability of E2 and BaP to form MN in MCF-7 cells. Future experiments should address these points.

Spink et al. (2005) have suggested that maspin, mammastatin, matrix metalloproteinase inhibitors, TGF-pi, solubilised CD44, thrombospondin-1 and insulin-like growth factor binding proteins are candidates for the growth inhibition of

MCF-7 cells in the presence of MCF-lOA cells. It is an attractive idea to regard these agents also as candidates responsible for the reduction in MN frequencies observed here. However, this is at present largely speculative and will require further work to resolve.

In general, however, our findings show that tissue context may substantially modulate the ability of chemicals to induce MN in mammary epithelial cells. Further knowledge about such inter-relations will be of great importance in order to come to realistic risk assessment of estrogenic chemicals in the future.

141 C H A PTER VI

GENERAL DISCUSSION AND CONCLUSIONS

Breast cancer incidence continues to rise, and much of the long-term underlying increase in breast cancer incidence is attributed to changes in reproductive patterns, such as delayed childbearing, a recognized risk factor for breast cancer.

Epidemiological evidence indicates that breast cancer risk is linked to prolonged exposure to the steroidal hormone, 17-p estradiol (Pike et al. 1993, Toniolo et al.

1995). Evidence also continues to amass indicating that increases in breast cancer in women are linked to environmental chemicals that are “estrogenic”, i.e. able to interact with the ER by mimicking the actions of endogenous hormone E2.

Research continues based largely around the two hypotheses that estrogens are involved in the development of cancer by either stimulating cellular proliferation or causing DNA damage and thereby mutagenic change. The attention of many cancer researchers has in recent years turned towards the instability induced by alterations of chromosome territories through induction of aneuploidy. Structural and genetic instabilities in chromosomes can negatively affect the genes that regulate the cells’ reproductive cycle, which in turn can further increase the risk of incorrect cell cycle regulation. Rearrangement of the cell’s genetic material induces profound changes of the DNA and is common in majority of solid tumours (Bolt et al. 2004,

Selvarajah et al. 2006).

It was the report by Fischer et al (2001) and Stopper et al (2002) describing that estradiol has the ability to induce micronuclei, via the ER-mediated pathway in

142 breast cancer cells that stimulated our interest to evaluate the ability of xenoestrogens to form micronuclei in a mammalian breast cancer cell line.

In an effort to better understand the role of ER signalling in inducing genomic instability in breast cancer cells, we investigated the role of the classical, so-called genomic pathway of ER activation on micronucleus induction following exposure to steroidal estrogens and the xenoestrogen BPA (Chapter 2). In cells co-exposed to E2 and Tam statistically significant reductions in micronuclei frequencies relative to cells treated with E2 alone were not observed as previously reported by Fischer et al

(2001). Treatment with the ER antagonist Tam also did not lead to diminished micronuclei frequencies in cells incubated with E3, and the occurrence of micronuclei was not reduced by co-administration of the pure ER antagonist ICI 182 780. Instead,

ICI 182 780 even caused slight elevations of micronuclei when combined with steroidal estrogen.

In addition to the classical activation of the ER at the nuclear level, it is now accepted that E2 can rapidly and transiently activate a number of second-messenger signalling pathways, such as the MAPK cascade Erk 1/2 and even their upstream effectors, such as the kinase Src (Migliaccio et al., 1996 and 1998). These cellular signalling pathways are essential for good cell maintenance, and are involved in the regulation of cell proliferation, differentiation and death. An interesting report by

Saavedra et al. (1999) has shown that over-activation of ras in NIH3T3 cells resulted in chromosome aberrations and improper segregation of chromosomes, which, in turn, led to an increase in micronuclei formation. This chromosome instability was demonstrated to be mostly mediated by activation of the MAPK pathway, as the presence of MAPK inhibitors reduced the number of micronuclei formed.

143 Based on these facts, we hypothesised that similar signalling pathways, in particular the Src/Erk pathway, could also be involved in the formation of micronuclei by estrogenic agents. Therefore, we wanted to evaluate wheater the alternative mechanisms of ER activation could be involved in the formation of micronuclei by estrogenic compounds.

In Chapter 2 we further tested this idea by applying the specific chemical inhibitors PD 98059 and PP2 of the Src/Erk signalling pathways. We observed reductions of micronucleus frequencies following co-incubation with PD 98059

(inhibitor of Erk 1/2 activation) and the tested estrogenic chemicals. Similar observations were made with PP2, an inhibitor of Src, suggesting the role for non- genomic ER signalling in micronucleus formation by estrogenic chemicals.

We also showed that E2, E3, EGF and BPA induced a significantly higher proportion of micronuclei which stained positive for kinetochores (CREST-positive micronuclei), when compared to controls or to BaP, a known clastogenic agent. On the other hand, a certain degree of DNA fragmentation, yielding CREST-negative micronuclei also occurred. Our results echo those by Saavedra et al. (1999) who also reported that increased levels of activated Erk led to a disproportionate increase in the frequency of CREST-positive micronuclei. They observed that a small but significant fraction of the micronuclei were CREST-negative, indicative of loss of acentric chromosome fragments from the cell nuclei. Saavedra et al. proposed that two mechanisms may be operating during the Erk-mediated induction of micronuclei.

First, the presence of centromeres and kinetochores in micronuclei (CREST-postive micronuclei) can be attributed to the improper segregation of whole chromosomes during mitosis, very likely through overriding of the spindle checkpoint. Second, the generation of acentric (CREST-negative) fragments is indicative of clastogenic

144 events. Constitutive activation of MAPK can stimulate the amplification of centrosomes, with formation of multi-polar spindles and mitotic bridges which arise from dicentric chromosomes. Alternatively, clastogenicity could also occur through redox-cycling of semiquinones derived from steroidal estrogens, with concomitant release of DNA-reactive intermediates leading to chromosome breaks. There is evidence that steroidal estrogens may induce DNA strand breaks by such a mechanism (Rajapakse et al. 2005), and this may have contributed to the occurrence of CREST-negative micronuclei. However, we believe that centromer amplification and not clastogenicity through DNA-reactive intermediates, is the predominant mode of action operating here, because EGF, an agent essentially unreactive to DNA, also induced acentric micronuclei.

In Chapter 3 we looked into silencing, instead of chemically inhibiting the phosphorylation of the non-genomic signalling pathways out of the concerns that PP2 and PD 98058 might not act in the presumed specific ways, but instead could induce other, poorly defined effects that interfere in unknown ways. We successfully silenced the ER and the adaptor protein Grb2 involved in early activation of the

Src/Erk 1,2 signalling pathway. Once the expression of these proteins has been silenced, the MCF-7 cells were assessed for micronuclei induction. One of the major requirements for the reliability of the micronuclei method is that cell number is not affected by the compound treatment; however with the MCF-7 cells with silenced ER we observed extensive cell death following the micronuclei assay. We hypothesised that this might be due to the relevance of the ER presence for function and maintenance of the MCF-7 cells. Tolerance of the silencing of Grb2 and subsequent early Ras/Src/Erk activation on the other hand was higher and we observed no reduction in cell number. Further on in this chapter, we present the evidence that the

145 silencing of Grb2 protein has no effect in micronuclei frequency induced by the tested agents. However, here we are only looking at one out of many signaling pathways that can lead to Erk activation, so returning to our hypothesis linking ER and micronuclei induction, our Grb2 data can eliminate possibility that there is a very plausible association between the two.

In Chapter 4 we extended the number of tested agents and investigated a range of cosmetic compounds that have recently been shown to exhibit estrogenic properties and are able to activate the ER. In this chapter we showed for the first time that these chemicals are able to induce micronuclei in ways that are mechanistically similar to steroidal estrogens in MCF-7 cells.

We also showed that that xenoestrogenic agents such as AHTN and OMC are able to induce micronuclei by mixed mechanisms in MCF-7 cells as they were unaffected by the inhibition of MAPK signalling cascade. In terms of advancing our understanding of mechanism of micronuclei induction, it would be important to test the agents that are only able to induce non-genomic signalling pathways in the MCF-

7 cells. Considering there are no such agents currently available, until we are able to look at non-genomic and genomic signalling separately, at this stage we are not able to state their exact role in micronuclei induction.

Finally in Chapter 5, we showed that E2 and BaP do not induce significant induction of micronuclei in normal breast MCF-lOA cells. We also showed micronuclei surpression in MCF-7 cells when they are grown in close proximity to

MCF-lOA suggesting that paracrine interaction between the luminal epithelium and myoepithelial cells has an inhibitory effect on chromosomal aberration in cells.

146 6.2 Future work

The possibilities of future work entail a number of different options. The most important issue to explore involves the role of other signalling pathways, such as

PI3K in micronuclei induction. Following from our Chapter 5, where we enlightened the importance of paracrine interactions in cancer progression, it would be therefore of huge significance to investigate paracrine interactors such as maspin, mammastatin and matrix metalloproteinase inhibitors that express a tumour suppressing activity in our micronucleus assay.

Our observations provoke questions as to the relevance and precise role of

MAPK signalling in contributing to genomic instability. In terms of advancing our understanding of the mechanism of micronuclei induction by estrogenic compounds, a multitude of experiments can provide the answers for some questions that resulted from this work. Normally, cell division is halted before all chromatids are properly aligned along the equator of the spindle apparatus, but premature cell cycle progression at this stage may promote micronuclei formation. Key proteins involved in the regulation of mitosis and are also imphcated in chromosomal aligment, cytokinesis and spindle checkpoints are the Aurora kinases (Ducat and Zheng 2004).

There is evidence that active Erk 1/2 can regulate Aurora kinase (Shapiro et al. 1998,

Zecevic et al. 1998), as they monitor the correct attachment of microtubules to kinetochors and are thought to be key players in the spindle checkpoint (Ducat and

Zheng 2004). Interestingly, previous observations have shown that enhanced activation of the Src/Raf/Erk cascade may lead to inhibition of Aurora B kinase by decreasing the localisation of Aurora B to kinetochores with a consequent decrease in

147 mitotic index and a defective spindle checkpoint (Eves et al. 2006, Solomon et al.

1991, Wassmann and Benezra 2001). It would therefore be of extreme importance to analyse whether estrogenic agents are able to amplify the expression of Aurora kinases, which then may promote aneuploidy and induce micronuclei formation

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