RADIATION-INDUCED DNA DAMAGE AND REPAIR IN HORMONE-DEPENDENT BREAST CANCER CELLS Word count: 12275

Anne-Sophie Bom Student number: 01300042

Supervisor(s): Prof. Dr. Ir. Ans Baeyens

A dissertation submitted to Ghent University in partial fulfilment of the requirements for the degree of Master of Science in Biomedical Sciences Academic year: 2018-2019

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PREFACE

First of all, I would like to thank my promotor, Prof. Dr. Ir. Ans Baeyens, for giving me the

opportunity to perform this research and to assist me in completing this master’s thesis.

Many thanks to my mentor, Stephanie Vermeulen, for the huge amount of support she gave

me throughout the year and for the many things she taught me. She made a great effort to

help me whenever needed. She gave me the necessary tools, information and feedback so as

to complete my dissertation successfully.

Thanks to the girls of the flexiroom for the supporting words and relaxing chats between the

numerous lab experiments. In particular to Karlien, whom I spent many lovely moments with

and for her support during tough times.

Lots of love to my best friends who were always there for me and with whom I spent wonderful

moments during my once in a lifetime adventure in Gent.

Lastly, I am grateful to my parents for always believing in me and for supporting me in any way

over the past years.

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TABLE OF CONTENTS

ABSTRACT ........................................................................................................................... 1

1 INTRODUCTION ............................................................................................................ 2

1.1 BREAST CANCER .................................................................................................. 2

1.2 TYPES OF BREAST CANCER ............................................................................... 2

1.3 THE ESTROGEN RECEPTOR ............................................................................... 3

1.3.1 FUNCTIONING OF THE ESTROGEN RECEPTOR ......................................... 3

1.3.2 SUBTYPES OF ER .......................................................................................... 3

1.3.3 CORRELATION BETWEEN ERα/β AND BREAST CANCER .......................... 5

1.4 DNA DAMAGE AND REPAIR ................................................................................. 6

1.4.1 DNA REPAIR ................................................................................................... 7

1.4.2 CELL-CYCLE ARREST ...................................................................................10

1.4.3 SENESCENCE ...............................................................................................11

1.4.4 APOPTOSIS ...................................................................................................11

1.4.5 NECROSIS .....................................................................................................11

1.4.6 P53 AS THE GUARDIAN OF THE GENOME..................................................12

1.4.7 MITOTIC CATASTROPHE ..............................................................................12

1.4.8 RADIOSENSITIVITY .......................................................................................12

1.5 FULVESTRANT .....................................................................................................13

1.6 MICRONUCLEI AS AN INDICATOR OF DNA DAMAGE ........................................13

1.7 RADIOTHERAPY ...................................................................................................14

1.8 RADIOSENSITIVITY IN BREAST CANCER PATIENTS ........................................15

1.9 AIM OF THE STUDY ..............................................................................................16

2 MATERIALS AND METHODS .......................................................................................17

2.1 CHARACTERISTICS OF BREAST CANCER CELL LINES ....................................17

2.2 CELL CULTURE ....................................................................................................17

2.3 CELL PROLIFERATION ASSAY ............................................................................18

2.3.1 PROLIFERATION ...........................................................................................18

2.3.2 MORPHOLOGY ..............................................................................................18

2.4 MICRONUCLEUS ASSAY .....................................................................................18

2.5 Erα/β-STAINING ....................................................................................................20

3 RESULTS......................................................................................................................21

3.1 CELL PROLIFERATION ASSAY ................................................................................21

3.1 ERα/β-STAINING ...................................................................................................23

3.2 MICRONUCLEUS ASSAY .....................................................................................27

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4 DISCUSSION ................................................................................................................31

5 GENERAL CONCLUSION ............................................................................................33

6 REFERENCES ..............................................................................................................34

7 APPENDICES ...............................................................................................................38

APPENDIX 1 Table temperature warmth plate / air humidity .............................................38

APPENDIX 2 Proliferation assay MCF-7 (fulvestrant added after 20h CT) ........................39

APPENDIX 3 Brightfield pictures of the proliferation assays .............................................40

APPENDIX 4 Proliferation assay MCF-7 (fulvestrant added at 0h CT) ..............................55

APPENDIX 5 Proliferation MCF-7 in MN assay (fulvestrant added at 0h CT) ....................56

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LIST OF FIGURES

Figure 1: Functioning of the ER. .......................................................................................... 3 Figure 2: The expression of ERα and ERβ in different tissues. .............................................. 4 Figure 3: Box chart of Allred scores of ERα- and ERβ-staining. ............................................ 5 Figure 4: Different types of DNA damage. ............................................................................ 6 Figure 5: Cellular responses to ionizing radiation. ................................................................ 7 Figure 6: DSB repair pathways: Nonhomologous end-joining (NHEJ) versus homologous

recombination (HR). .............................................................................................................. 9 Figure 7 Different types of chromosome aberrations. ...........................................................10 Figure 8: Cell-cycle checkpoints. ..........................................................................................11 Figure 9: Schematic representation of the functioning of fulvestrant. ....................................13 Figure 10: Various representations of DNA damage due to genotoxic events.. ....................14 Figure 11: Proliferation MCF-7 with fulvestrant added after 20h CT. .....................................21 Figure 12: Proliferation MCF-7 with fulvestrant added at 0h CT.. ..........................................22 Figure 13: Brightfield imaging of MCF-7.. .............................................................................22 Figure 14: Fluorescent imaging of the ERα degradation in MCF-7 following fulvestrant

treatment. .............................................................................................................................23 Figure 15: Intensity of ERα-staining.. ....................................................................................24 Figure 16: Fluorescent imaging of the ERα degradation in MCF-7 following fulvestrant

treatment. . ...........................................................................................................................25 Figure 17: ERβ-staining of MCF-7 and T-47D.. ....................................................................26 Figure 18: MN assay with fulvestrant added 4h before irradiation. ........................................27 Figure 19: MN assay with fulvestrant added 120h before irradiation.. ...................................28 Figure 20: MN assay with fulvestrant added 96h before irradiation.. .....................................29 Figure 21: proliferation MCF-7 in MN assay with fulvestrant. ................................................30

LIST OF TABLES

Table 1: Summary table MCF-7, MDA-MB-231 and T47-D. ..................................................17 Table 2: Dilution series of fulvestrant. ...................................................................................18 Table 3 Different antibodies for ERα/β-staining. ...................................................................20

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LIST OF ABBREVIATIONS

A

AF 1/2 Activation Function 1/2 AI Aromatase Inhibitor AO Acridine Orange

B

BER Base excision Repair BN Binuclear cell BSA Bovine Serum Albumin

C

CBMN Cytokinesis Block Micronucleus CT Culture Time

D

DAPI 4',6-diamidino-2-fenylindool DCIS Ductal Carcinoma In Situ DFS Disease Free Survival DMEM Dulbecco's Modified Eagle Medium DMSO Dimethyl Sulfoxide DNMT DNA Methyltransferase DNA-PKcs DNA-dependent Protein Kinase catalytic subunit DSB Double Strand Break

E

ER Estrogen Receptor EREs Estrogen Response Elements

F

FBS Fetal Bovine Serum

G

GFP Green Fluorescent Protein GY Gray

H

HER2 Human Epidermal growth factor Receptor 2 HR Homologous Recombination

I IDC Invasive Ductal Carcinoma

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L

LCIS Lobular Carcinoma In Situ

M

MDS Multiply damaged site MMR Mismatch Repair MN Micronucleus MNi Micronuclei

N

NBUD Nuclear Bud NDI Nuclear Division Index NER Nucleotide Excision Repair NHEJ Nonhomologous End-Joining NPB Nucleoplasmatic Bridge

O

OS Overall Survival

P

PARP Poly ADP-Ribose Polymerase PBS Phosphate Buffered Saline PFA Paraformaldehyde PR Progesteron Receptor

R

RPM Rounds Per Minute

S

SARRP Small Animal Radiation Research Platform SD Standard Deviation SERD Selective Estrogen Receptor Degrader SERM Selective Estrogen Receptor Modulator SSB Single Strand Break

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ABSTRACT Background: Breast cancer is the most prevalent cancer in women. It can be divided in subtypes depending on which receptors are expressed by the cancer cells. These receptors can be estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2). Cancer cells expressing none of these receptors, called triple negative cells, are insensitive to hormonal therapy. The estrogen receptor-positive (ER+) breast cancers are the most common. Previous studies showed that ER+ cancer cells (e.g. MCF-7) are more radiosensitive than hormone insensitive cancer cells (e.g. MDA-MB-231). A synthetic estrogen antagonist is fulvestrant which causes degradation of the receptor. In this study the impact of fulvestrant on the proliferation and morphology of MCF-7 and a possible link with radiosensitivity was investigated.

Methods: MCF-7 cells were irradiated with X-rays with doses varying from 1 to 4 Gray, in the presence or absence of fulvestrant. An ERα-staining was performed to visualize the presence and degradation of the ERα. The quantity of DNA damage and repair was assessed with the micronucleus (MN) assay.

Results: Results showed a reduced proliferation of MCF-7 cells with increasing concentration of fulvestrant but no changes in morphology. MCF-7 cells treated with fulvestrant showed a lower amount of MN than the untreated MCF-7 cells.

Conclusion: Fulvestrant has an influence on cell proliferation in a dose- and time-dependant manner. There is a strong indication that radiosensitivity increases when ER is present. However, further investigation is needed to confirm these findings.

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1 INTRODUCTION

1.1 BREAST CANCER Breast cancer is the most common cancer in women. Every year over 1 million persons worldwide are diagnosed with breast cancer[1, 2], which is approximately a fourth of all cancer patients. It is the second greatest cause of cancer death in more developed regions after lung cancer[1]. As breast cancer is a heterogeneous disease[3], it is very hard to provide for an appropriate treatment for each patient. For different types of breast cancer, different therapies are needed. So evaluation of the patient’s cancer type at the molecular level is required to define the correct prognosis[4].

1.2 TYPES OF BREAST CANCER Conventionally, breast cancers are divided in subtypes according to their status of three important receptors, namely the estrogen receptor (ER), the progesterone receptor (PR) and the human epidermal growth factor receptor 2 (HER2). Breast cancers can be classified as luminal A, luminal B, HER2 positive or triple negative[5, 6]. In addition, a distinction is made according to which extent they are expressing Ki-67. Ki-67 is a protein acting as a cellular marker for proliferation. Furthermore, Ki-67 would contribute as a prognostic value for survival and tumor recurrence[7, 8].

Luminal breast cancers are characterized by the expression of ER and/or PR[5, 6]. It represents 50%-60% of all breast cancers[9]. Luminal cells are, compared to other cell types, more differentiated and, due to tight intercellular junctions, they have less tendency to migrate[10]. Luminal breast cancers can be further divided into luminal A and B according to their HER2 status[9]. Luminal B cancers show HER2 overexpression, which is associated with ER down-regulation[5]. This results in a decreased luminal cell phenotype and in a more invasive and consequently more aggressive behaviour[11]. Luminal breast cancers have a rather good prognosis with a high survival rate and a low recurrence rate. Comparing the 2 luminal breast cancers, luminal B has a slightly worse prognosis than luminal A[9].

HER2 positive breast cancers are defined by the expression of especially HER2 and no ER and PR[5, 6, 12]. HER2-positive breast cancer accounts for 15-20% of all breast cancers[9]. The HER2 positive group contains cells with luminal features as well as features of the triple negative cells. Over-expression of HER2 causes breakdown of cell-cell junctions and thus more migration[5]. Patients with HER2 positive breast cancer have a relative good prognosis due to good responses to certain drugs, such as Herceptin[13].

Triple negative breast cancers are characterized by low or no expression of all three receptors[3, 14]. It represents 10-20% of all breast cancers[9]. Due to the insensitivity to hormones, more frequent recurrences and worse 5-year survival triple negative breast cancer comes with a poor prognosis[15].

Breast cancers can also be subdivided according to their histological features. The 2 main groups are the in situ carcinomas and the invasive carcinomas. Subsequently they can be sub-classified by lobular or ductal carcinomas. Ductal carcinoma in situ (DCIS) is substantially more common than the lobular carcinoma in situ (LCIS). Of all invasive carcinomas 70-80% are infiltrating ductal carcinomas (IDC)[16].

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1.3 THE ESTROGEN RECEPTOR

1.3.1 FUNCTIONING OF THE ESTROGEN RECEPTOR In ER-positive cells survival and proliferation is driven by estrogen. The ER is a receptor occurring in both cytoplasm and nucleus. Figure 1 shows a schematic illustration of its functioning. It has a transcriptional activity regulated by two activating functional domains (AF): AF1 and AF2[17]. AF1 becomes activated by phosphorylation of various sites by cyclin-dependent kinases Cdk, while the activation AF2, which is located in the ligand-binding domain, requires estrogen binding[18, 19].

Estrogen diffuses into the cell and binds to the ER. Dimerization of the estrogen-ER complexes occurs and the dimer is further translocated to the nucleus where it binds to the estrogen response elements (EREs) in the promoter regions of estrogen-responsive genes. AF1 and AF2

then recruit transcriptional co-activators and co-repressors to the transcriptional complex necessary for the transcription of the concerned genes[17, 20].

Figure 1: Functioning of the ER. Estrogen (E) binds the estrogen receptor (ER) followed by the dimerization of 2 estrogen-ER complexes. Activating functional domain 1 and 2 (AF1/2) recruit transcriptional co-activators and co-repressors which bind the estrogen response element (ERE) together with the dimer. This results in the transcription of the concerned genes. Adapted from[21]

1.3.2 SUBTYPES OF ER The ER appears in two subtypes: ERα and ERβ. They are expressed in various types of cells and tissues and control several physiological functions in different organ systems such as the reproductive, skeletal, cardiovascular, and central nervous system. ERα is known to be present in numerous tissues as illustrated in figure 2[22, 23]. In breast cells ERα is responsible for proliferative actions. It induces cell cycling and stimulates cell growth. This can lead to initiation and development of cancer. In contrast with ERα, ERβ causes a reduction of proliferation in vitro and prevents tumor formation[22, 24, 25].

ERα is expressed in almost 80% of breast tumors, and its presence is the main indicator for antihormonal therapy. But when ERβ is expressed, also patients with ERα-negative early breast cancer can benefit from endocrine therapy[26, 27], such as tamoxifen. Tamoxifen is a ER antagonist and works as a selective ER modulator (SERM), resulting in a decreased growth of breast cancer cells[27]. The ERβ level is an independent marker to predict the response to tamoxifen. In ERα-negative breast cancer, proliferation is not driven by estrogen, so tamoxifen may modulate another growth signaling pathway by binding ERβ[27, 28].

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Figure 2: The expression of ERα and ERβ in different tissues.[23]

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1.3.3 CORRELATION BETWEEN ERα/β AND BREAST CANCER Huang et al. did research on the differential expression of ERα and ERβ in different breast tissues. In normal breast tissue ERα was expressed in approximately 10% of epithelial cells, whereas ERβ was expressed in more than 70% of epithelial cells. In DCIS the percentage of ERα-positive cells was markedly increased, whereas the percentage of ERβ-positive cells was significantly decreased[29]. This is in accordance with other studies which concluded that expression of ERβ markedly decreased in the early stages of mammary carcinogenesis and ERα increased[30, 31]. In the invasive carcinoma, the amount of ERα expression was reduced and ERβ expression was completely lost compared to normal breast tissue(figure 3). Lobular cancers strongly expressed both ERα and ERβ[29].

Figure 3: Box chart of Allred scores of ERα- and ERβ-staining. Compared to normal tissue, ERα expression was increased and ERβ expression was reduced in ductal carcinoma in situ (DCIS) tissue. In invasive ductal carcinoma (IDC) tissue ERβ expression was completely lost. Adapted from [29]

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1.4 DNA DAMAGE AND REPAIR Radiation-induced DNA damage can appear in different forms (figure 4). It includes single strand breaks (SSB), double strand breaks (DSB), base modifications, base losses and crosslinks. When a combination of these damages occur at a close distance in the DNA it is called a multiply damaged sites (MDS)[32, 33]. Most of the time MDS are fatal for the cell. Ionizing radiation causes DNA lesions by direct interaction with DNA or indirectly via radiolysis. X-rays causes mostly DNA damage induced by radiolysis. Radiolysis is caused by interaction of the DNA with reactive oxygen that is generated by the ionization of water[32, 34]..

Figure 4: Different types of DNA damage. Ionizing radiation can induce lesions and modifications in the DNA such as single strand breaks (SSB), double strand breaks (DSB), damage to the base or sugar molecule, crosslinks between DNA molecules or between DNA and a protein.[32]

The most harmful DNA damage caused by ionizing radiation are the DSBs. A DSB occurs when both strands of the double helix are cleaved within one helical turn. The main cellular responses to irradiation are cell cycle arrest, DNA repair, senescence and apoptosis (figure 5). Cell death via mitotic catastrophe and the generation of aneuploid cells are a result of failed activation of p53-dependent DNA damage checkpoints[34, 35]. DNA damage is initially detected by sensor proteins. They are recruited to damaged sites and amplify the damage signal to proteins, called transducers. These transducers relay the signal to downstream effector proteins[36].

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Figure 5: Cellular responses to ionizing radiation. Depending on the severity of the DNA damage one or more of the following cellular responses will be initiated by p53: DNA repair, cell-cycle arrest, senescence and apoptosis. When p53 function is impaired the DNA damage can lead to cell death via mitotic catastrophe or the development of cancer cells due to abnormal division of aneuploid cells.[35]

1.4.1 DNA REPAIR

1.4.1.1 Nucleotide excision repair (NER) NER is responsible for the repair of various DNA lesions, particularly base modifications that distort the normal helical DNA structure. First the damage is recognized by proteins such as XPC-RAD23B. Then ERCC1-XPF and XPG make an incision on both sides of the lesion and the damaged nucleotide is removed. A new nucleotide is synthesized by Pol δ and Pol κ or Pol ε and associated factors. To complete the process the nick is sealed by DNA ligase IIIα-XRCC1 or DNA ligase I. Roughly thirty proteins are involved in NER operating in a coordinated manner[37, 38].

1.4.1.2 Base excision repair (BER) BER repairs common DNA base modifications caused by oxidation, deamination or alkylation. BER starts with the removal of the damaged base by a damage-specific DNA glycosylase, leaving an abasic site (AP-site). An AP endonuclease and lyase cut out the remaining sugar molecule and Poly ADP-ribose polymerase (PARP-1) prevents further degradation of the DNA helix. Then a new nucleotide is synthesized by polymerase β. As last step ligation is performed by XRCC1-DNA ligase 3α. Depending on several conditions BER takes place by short-patch repair or long-patch repair which are similar but largely use different proteins downstream of the base excision[38, 39].

1.4.1.3 Mismatch repair (MMR) This mechanism recognizes and repairs base-base mismatches and insertion–deletion loops that arise during DNA replication and HR (see 1.6.1.4). The mismatch is recognized by MutSα or MutSβ and MutLα resulting in the formation of a ternary complex. MMR recruits proteins

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near the replication fork. Excision is conducted by exonucleases and a RPA-coated single-strand gap is formed. Resynthesis is performed by polymerase δ and the nicks are ligated by a ligase[38, 40].

1.4.1.4 DSB repair

Exposure to ionizing radiation is one of the main DNA damaging events that causes DSB, thus making the DSB repair pathways crucial after irradiation. DSB can be repaired by two pathways: homologous recombination (HR) or nonhomologous end-joining (NHEJ) (figure 6). HR is solely performed during the late S phase and the G2 phase because its mechanism requires the presence of an intact sister chromatid as a template[32]. This repair pathway is relatively error-free. HR is initiated by recognition of the DSB by the MRN complex consisting of the proteins MRE11, RAD50 and NBS1[38]. This complex recruits ATM which phosphorylates the histone variant H2AX[41]. MRE11 then conducts resection of the DNA helix resulting in ssDNA ends. This endonuclease activity is regulated by interaction with CtBP which depends on ATM and BRCA1. After RPA activation a RAD52/BRCA2/RAD51/RAD54 complex is recruited by a BRCA1/PALB2 complex. This facilitates the replacement of RPA with RAD51. RAD51 coordinates invasion into the sister chromatid and formation of a Holliday junction followed by the alignment of the two strands with homologous regions within the sister chromatid[32]. SMC proteins (cohesins) are responsible for holding the homologous regions together as a stable complex[32, 41]. Next DNA synthesis is performed by DNA polymerase δ[32] and the Holliday junction is resolved. Finally, the DNA ends are ligated[38, 41].

NHEJ is more prone to errors but the most important DSB repair method during the G1 phase and the early S phase, due to the fact that there is no sister chromatid available for HR. Initiation of NHEJ is performed by binding of the Ku70/Ku80 heterodimer to the two DSB ends which recruits the DNA-dependent protein kinase catalytic subunit (DNA-PKcs)[32]. The DNA-PKcs complex both stabilizes and aligns the DNA ends. The interaction between the two DNA-PKcs, each at a DSB terminus, activates its autophosphorylation and dissociation[32, 38]. Artemis, an endonuclease, trims the single-stranded overhangs at the DNA ends[32, 38]. After annealing the remaining short nucleotide gaps in the DNA are filled by DNA polymerases. To complete the NHEJ repair the processed ends are ligated by a XRCC4–DNA ligase IV complex[32, 38, 42].

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Figure 6: DSB repair pathways: Nonhomologous end-joining (NHEJ) versus homologous recombination (HR). DSB are repaired by one of two pathways. HR requires a homologous sister chromatid as a template. Strands of the damaged chromatid invade the sister chromatid and form a Holliday junction. The concerning part of the DNA is replicated by a DNA polymerase and ligated into the damaged DNA strand. In the repair process of nonhomologous end-joining ragged ends of DNA are first secured and gaps are filled. Repair is completed by ligating the ends together.[42]

1.4.1.5 Chromosomal aberrations DSB are not always repaired correctly or not repaired leading to chromosomal aberrations[38, 42]. A lot of mutated chromosomes are the result of incorrect repair by NHEJ. There are many different types of chromosomal aberrations depending on the moment of the DSB, the location of the DSB and the rearrangement of the chromatid fragments[33, 43, 44]. If the DSB occurred before DNA replication both sister chromatids will contain the damage. In this case they are called chromosome aberrations. When the DNA was damaged after DNA replication only one sister chromatid will contains the damage and then they are called chromatid aberrations[44]. Intra-chromosomal exchange (figure 7a) involves one chromosome and may lead to pericentric and paracentric inversions, acentric fragments, interstitial deletions and acentric and centric rings. Inter-chromosomal exchange (figure 7b) involves two different chromosomes and may lead to dicentric chromosomes, acentric fragments and translocations. In addition, the exchange can be classified as symmetrical or asymmetrical rearrangement[33, 43, 44]. Symmetrical rearrangements are frequently transmitted to subsequent cell generations because no genetic loss is involved. These mutated chromosomes are considered as ‘normal’ by cell-cycle checkpoints proteins (see 1.6.2) and there are no mechanical separation difficulties at anaphase. This can lead to genetic implications and formation of cancerous cells[44] (e.g. philadelphia chromosome[45]). Asymmetrical exchanges result in more notable chromosome aberrations such as dicentric chromosomes and deletions. These changes in the

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genetic material are easy to detect by cell cycle checkpoints which results in senescence, mitotic catastrophe or apoptosis[44].

Figure 7: Different types of chromosome aberrations. (a) intra-chromosomal aberrations: a centric ring and an acentric fragment as a result of asymmetrical exchange. Pericentric inversion as a result of symmetrical exchange. (b) Inter-chromosomal aberrations: translocation for symmetrical exchange and a dicentric chromosome accompanied by an acentric fragment due to asymmetrical exchange. Adapted from [46]

1.4.2 CELL-CYCLE ARREST Activated effector proteins are responsible for the induction of cell cycle arrest[35]. The cell cycle exists of a series of events that lead to the production of two daughter cells (figure 8). During a cell cycle arrest this series of events is halted[47]. There is the G1 phase where the cells increase in size and the G1 checkpoint that ensures that the DNA is ready for synthesis. In the S phase the DNA is duplicated and the DNA is checked for aberration, followed by a G2 phase where everything is prepared for the cell division. The G2 checkpoint controls whether the cell has reached the necessary state to enter the M phase. In the M phase the actual mitosis takes place. Just before the segregation of the chromosomes another checkpoint controls if the centrosomes and the DNA are in the proper condition. Cells that are not dividing are in the G0 phase[48]. When aberrations or other unusual events are detected during the checkpoints an arrest takes place. This gives the cell more time to restore the ‘normal’ state of the cell, for instance repair damages by repair mechanisms. If the repair was successful the cell will re-enter the cell cycle, if not the cell risks to go into senescence or even apoptosis[34, 35] (see 1.6.3. and 1.6.4.).

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Figure 8: Cell-cycle checkpoints. Nondividing cells remain in the G0 phase. In dividing cells the first checkpoint occurs at the end of the G1 phase and ensures that DNA is ready for synthesis. In the S phase the second checkpoint controls the DNA for aberrations. At the end of the G2 phase the third checkpoint checks if everything is ready to enter the M phase. Lastly the fourth checkpoint ensures that the cell is ready for the segregation of the chromosomes. Adapted from [47]

1.4.3 SENESCENCE Senescence is a term used when cells enter a permanent cell cycle arrest. Even though senescent cells stop dividing, they remain metabolically active. They acquire alterations in gene expression including inhibition of genes involved in cell proliferation and induction of several intracellular and secreted growth inhibitors. Senescence is induced by p53 and p21[34, 35].

1.4.4 APOPTOSIS Apoptosis is considered to be one of the main cell death mechanisms for cells exposed to ionizing radiation. Hallmarks of radiation-induced apoptosis include pyknosis, cell shrinkage, and internucleosomal breakage of chromatin. P53 is crucial for rapid interphase apoptosis. It occurs within a few hours following irradiation exposure as a premitotic event[34, 35].

1.4.5 NECROSIS

Necrosis is a type of cell death independent of DNA damage. As opposed to apoptosis, necrosis is an accidental death of the cell due to exposure to extreme environmental insults. This type of cell death is induced when parts of the cell, such as the cell membrane, are damaged due to high doses of ionizing radiation.( Cellular Pathways in Response to Ionizing Radiation and Their Targetability for Tumor Radiosensitization)

S Checkpoint

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1.4.6 P53 AS THE GUARDIAN OF THE GENOME P53 is a very important tumor suppressor. In more than 50% of all human cancers the p53 gene is mutated, which suggest that the impairment of this protein is favourable for the proliferation of tumor cells[35]. Cells with an impaired activation of p53 will unjust pass the DNA damage checkpoints and can continue through abnormal divisions when exposed to ionizing radiation. This can lead to aneuploidy and the development and progression of tumors[49]. However, tumor cells exposed to ionizing radiation typically undergo mitotic catastrophe.

1.4.7 MITOTIC CATASTROPHE

Mitotic catastrophe is a delayed type of cell death that occurs during or as a result of an aberrant mitosis. In an aberrant mitosis an atypical chromosome segregation and cell division is conducted. This results into the formation of giant cells with aberrant nuclear morphology, multiple nuclei or several micronuclei (MNi)[35, 49].

1.4.8 RADIOSENSITIVITY

Radiosensitivity is the susceptibility of cells to the damaging effect of ionizing radiation. Ionizing radiation are particulate waves or electromagnetic waves with an energy high enough to remove an electron from its orbit resulting in an ionized atom. When these ionizations occur at the level of the genetic material, the DNA can suffer from several types of damage[32, 38, 41, 42]. These damages can lead to different outcomes for the cell and are sometimes fatal[35](see 1.6). The damage in the cell caused by ionizing radiation is mainly dependent on which type of radiation they are exposed to and by which dose[50]. Also some cell types are more sensitive than others[51, 52]. In addition are cells more or less radiosensitive depending on the part of the cell cycle in which they are found. Cells are the most radiosensitive in the G2/M phase, less sensitive in G0/G1, and the least sensitive in the latter part of the S phase[44].

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1.5 FULVESTRANT Fulvestrant is a synthetic ER antagonist. It is a 7α-alkylsulphinyl analogue of 17β-oestradiol. Due to its antagonistic effects, fulvestrant is used as a drug in endocrine therapy[53]. A schematic representation of its functioning is shown in figure 9. Fulvestrant is able to compete with estrogen due to its high binding affinity to ER, which is 89% of estrogen[21]. Once fulvestrant is bound to the ER, dimerization of the receptor is inhibited due to a conformational change[18]. This leads to an unstable complex, which results in degradation of the ER. Thus, fulvestrant acts as a selective ER degrader (SERD) and may therefore be a solution to acquired resistance to selective ER modulators[54]. There is increasing evidence that mutations in ESR1, the gene coding for ERα, ensue the ER to become constitutively active[55]. Whilst AIs and SERMs does not have an effect on mutated ER, fulvestrant maintains the capacity of degrading the mutated ER[56].

Figure 9: Schematic representation of the functioning of fulvestrant. Fulvestrant binds the ER with a higher affinity than estrogen, inhibiting the dimerization of ER. This leads to an unstable complex, which results in degradation of the ER. There is no DNA binding to estrogen response elements (EREs) and no transcription is conducted. [21]

There exists an inconsistency about the effect of fulvestrant on ERβ. In a study using ERβ vectors the findings were that ERβ was degraded in the presence of fulvestrant[57]. Another study on mice showed an up-regulation of ERβ as an effect of fulvestrant addition[58]. An in vitro research on the effect of fulvestrant on ERα and ERβ resulted in an significant decrease in ERα but no altered expression of ERβ[59].

1.6 MICRONUCLEI AS AN INDICATOR OF DNA DAMAGE MNi are small nuclei that are formed whenever a part of the DNA is not incorporated into one of the daughter nuclei during cell division. They can originate from whole chromosomes or acentric chromosomal fragments. The presence of MNi is an indicator for chromosomal instability or genotoxic events. After a genotoxic event, such as exposure to ionizing radiation, changes in the DNA are visible in cultured cells by various representations. Fenech described the cytokinesis block micronucleus (CBMN) assay[60]. The CBMN is a method that can be used to detect DNA damage and misrepair, chromosomal instability, mitotic abnormalities, cell death and cytostasis. In the CBMN assay cytochalasin B is administered to irradiated cells to block the cytokinesis. The DNA in these cells will continue to divide and thus binuclear cells are formed. Observation of the binuclear cells provides information about the amount of incurred DNA damage. Possible representations of DNA damage of irradiated cells are shown in figure 10. MNi can arise when chromosome breaks are not repaired or when DNA is

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misrepaired, resulting in acentric fragments of the chromosome. Also spindle or kinetochore defects and cell-cycle checkpoints malfunctions can lead to MNi due to malsegregation of the chromosomes during anaphase[60, 61].

Figure 10: Various representations of DNA damage due to genotoxic events. After a genotoxic event, such as exposure to ionizing radiation, cells can incur DNA damages. Some cells will undergo cell death (apoptosis, necrosis). Other cells will show aberrations in the genetic material after nuclear division such as nucleoplasmic bridges (NPBs), MNi (chromosome breakage and loss) and nuclear buds (NBUDs). Dicentric chromosomes are a result of the incorrect repair of DNA DSB and telomere end fusion. NPBs are formed by dicentric chromosomes where the 2 centromeres are pulled to opposite sites in the cell during anafase. NBUDs are formed by nuclear elimination of amplified DNA and/or DNA repair complexes[58].

1.7 RADIOTHERAPY Radiotherapy is a treatment frequently used for patients with breast cancer. It uses energy of ionizing radiation to destroy tumor cells[62]. This application is used in early breast cancer after breast conservation surgery and in locally advanced breast cancer patients post mastectomy. Treatment of breast cancer is mostly realised with external X-rays. In a very few rare cases the use of internal radiation through brachytherapy is used[63, 64]. For early-stage breast cancer patients, post-mastectomy radiotherapy reduces locoregional recurrences and improves overall survival in high-risk patients, namely those with pathologically involved axillary nodes or with large tumor size[65]. For triple negative breast cancers radiotherapy is indispensable due to a high recurrence rate and the insensitivity to hormone therapy. Unfortunately, some breast cancers are less radiosensitive than others which means that radiotherapy is not always an option[66].

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1.8 RADIOSENSITIVITY IN BREAST CANCER PATIENTS Previous study showed that different DNA repair mechanisms are responsible for chromosome

damage during different cell cycle phases, leading to the development of breast cancer[67]. γ-

H2AX and RAD51 foci assays demonstrated a higher radiosensitivity in breast cancer patients

when compared to healthy people[68]. Furthermore, the various molecular breast cancer

subtypes show differences as regards radiosensitivity. Moreover, Francies et al. found a

correlation between radiosensitivity and ER+ breast cancers. Here lymphocytes of ER+ breast

cancer patients showed a higher radiosensitivity than lymphocytes of ER- breast cancer

patients. This may suggest that people with ER+ breast cells execute an altered mechanism

of DNA repair which results in more DNA damage[51]. Thus impact of radiation exposure may

have a different biological effect on various breast cancer subtypes and this could influence

the clinical outcome of treatment[69].

.

16

1.9 AIM OF THE STUDY

Previous study[52] suggested that ER+ cancer cells are more radiosensitive than triple negative cancer cells. In this study we aimed to find a possible link between the presence of ER and the radiosensitivity of MCF-7 cells. A wild-type MCF-7 cell line was compared to a with fulvestrant treated MCF-7 cell line, in which receptor activity was blocked. Different doses of radiation were administered and by using the micronucleus assay DNA damage in the treated and untreated MCF7 cells was observed. An immunostaining was performed to visualize the presence and degradation of ER.

17

2 MATERIALS AND METHODS

2.1 CHARACTERISTICS OF BREAST CANCER CELL LINES MCF-7 is a commonly used breast cancer cell line for research[5]. This cell line originates from the pleural effusion of a 69 year-old woman with metastatic breast cancer. It belongs to the luminal A subtype thus it expresses ER and PR[70]. This makes MCF-7 cells adequate for in vitro ER+ breast cancer experiments[71]. MCF-7 is a poorly-aggressive cell line with a very low potency of invasion and development of metastases. Invasiveness is rather dependent on estrogen and other hormones. Another characteristic of MCF-7 cells is that they hold an elevated level of genetic instability[72]. T-47D is an ER-positive luminal A cell line very similar to MCF-7 but ERβ expression is higher than in MCF-7[73] (see 1.4.2). MDA-MB-231 is an epithelial, human breast cancer cell line that was established from a pleural effusion of a woman with a metastatic mammary adenocarcinoma[74]. It is highly aggressive, invasive and poorly differentiated and belongs to the triple-negative breast cancers[75].

Table 1: Summary table MCF-7, MDA-MB-231 and T47-D.

Cell line Luminal A

Triple negative

ER PR HER2 Primary tumor Invasive Origin

MCF-7 ✔ ✘ ✔ ✔ ✘ Invasive ductal carcinoma

✘ Metastasis (pleural effusion)

MDA-MB-231

✘ ✔ ✘ ✘ ✘ Adenocarcinoma ✔ Metastasis (pleural effusion)

T-47D ✔ ✘ ✔ ✔ ✘ Invasive ductal carcinoma

✘ Metastasis (pleural effusion)

2.2 CELL CULTURE MCF-7 breast cancer cells were maintained in Dulbecco's Modified Eagle Medium (DMEM) (Gibco, 61965-026) supplemented with 10% fetal bovine serum (FBS) (Gibco, 10270-106) and 0,1% penicillin/streptomycin solution (Gibco, 15140-122). The cells were grown at 37°C in a humidified 5% CO₂ incubator.

For the defrosting of the cells a cryovial was taken out of a liquid nitrogen tank and thawed at room temperature. The content of the vial was transferred to a 15 ml tube and diluted with cold complete medium (4°C) until 10 ml. After centrifuging (1000 rpm, 5 min) the cells supernatant was removed and the cells were resuspended in 10 ml warm complete medium (37°C). Then they were centrifuged (1000 rpm, 5 min) and supernatant was removed. Lastly 4 ml of warm complete medium (37°C) was added to the cell pellet so the cells could be transferred to a culture flask.

The cells were reseeded every 3 or 4 days (whenever the cells were confluent) into a new culture flask. The cells were washed 3 times with 2 ml phosphate buffer saline (PBS) (1,78 g Na₂HPO₄ / 0,42 g KH₂PO₄ / 7,2 g NaCl / 1 l distilled water) (37°C) and harvested with 1 ml

trypsin (Gibco, Invitrogen, 25300) (5 min, 37°C). After trypsinisation 2 ml complete medium (37°C) was added and the cell suspension was transferred to a 15 ml tube. The cell suspension was centrifuged (1000 rpm, 5 min) and supernatant was removed. Then the cell pellet was washed in 2 ml PBS (37°C). Next the cell pellet was resuspended in 1 complete medium and cells were counted in trypan blue (Gibco, 15250-061) with a Bürker counting chamber.

18

2.3 CELL PROLIFERATION ASSAY

2.3.1 PROLIFERATION MCF-7 cells were seeded at a concentration of 400 000 (fixation after 44h culture time (CT)) cells or 300 000 cells (fixation after 116h CT) per culture flask (T25) in 5 ml complete culture medium. Different concentrations of fulvestrant (Sigma, I4409 ), varying from 10-6 M to 10-9 M, were added immediately or after 20 hours of CT. Fulvestrant was dissolved in dimethyl sulfoxide (DMSO) (Sigma, 101887800) as a stock solution of 10ˉ² M and stored at 4°C in the dark (see table 2). The cells were further grown in the incubator (37°C, 5% CO₂). After exposure to fulvestrant for 48h, 72h, 96h and 116h one culture flask of each concentration of fulvestrant was harvested and counted (see 2.1).

Table 2: Dilution series of fulvestrant.

2.3.2 MORPHOLOGY At 18, 42, 66 and 90 hours of CT imaging of MCF-7 in each culture was performed with a light microscope (Brightfield) to detect any transformations in the morphology of the cells. Every time in three different magnifications: 40x, 100x and 200x. The magnification of 40x provides for the overall picture how the cells attached to the bottom of the flask. At the magnification of 100x any changes in the form of the cells were observed. Detailed changes in the cell were observed with the 200x observation.

2.4 MICRONUCLEUS ASSAY MCF-7 cells were seeded in duplicate at a concentration of 400 000 cells (fixation after 44h CT) or 300 000 cells (fixation after 116h CT) per culture flask in 5 ml complete culture medium and were placed in the incubator (37°C, 5% CO₂).

2,5 µl of fulvestrant working solution (see table 2) was added immediately or at 20 hours CT to the culture flasks intended for the MCF-7 group with blocked ER. 2,5 µl DMSO was added to the untreated cells (=negative control).

After 24 or 96 hours of CT the cells were irradiated. The cells were transported to the small animal radiation research platform (SARRP). For the irradiation an X-ray beam was used with an energy of 220 kV and an intensity of 13 mA. A copper filter was used and a collimator with a fieldsize of 10x10 cm. The cells were irradiated with doses of 0, 1, 2, 3 or 4 Gray (Gy). Immediately after irradiation cytochalasin B (Sigma, C6762) was added with a final concentration of 4,7 . 10ˉ³ M and the cultures were placed back into the incubator.

After 44 or 116 hours of CT the cells were harvested, using trypsin (Gibco, Invitrogen, 25300), and transferred to 15 ml tubes. The cell suspension was centrifuged (5min, 1000rpm, RT) and supernatant was removed. Then the cells were washed with 2 ml PBS (37°C). The cell

Work solution

Concentration work solution

Dilution Final concentration in cell culture (2,5 µl/5 ml medium)

A 2 . 10ˉ³ M 8 µl stock solution (10ˉ² M) + 32 µl DMSO

10ˉ⁶ M

B 2 . 10ˉ⁴ M 4 µl work solution A + 36 µl DMSO 10ˉ⁷ M

C 2 . 10ˉ⁵ M 4 µl work solution B + 36 µl DMSO 10ˉ⁸ M

D 2 . 10ˉ⁶ M 4 µl work solution C + 36 µl DMSO 10ˉ⁹ M

19

suspension is again centrifuged (5min, 1000rpm, RT) and supernatant is removed. Then 7 ml cold KCl (75 mM, 4°C) (VWR) was added to each tube while vortexing. The cell suspension was centrifuged (8 min, 1000 rpm) and supernatant was removed. Followed by drop wise adding, while vortexing, 7 ml of the first fixative, methanol/acetic acid/ringer (3/1/4, 4°C) (ringer = 9g NaCl, 0.42 KCl, 0.24g CaCl2 for 1L, 4°C) and stored overnight at 4°C.

Afterwards 5 ml of the second fixative, methanol/acetic acid (3/1, 4°C) was added drop wise while vortexing. This was repeated 2 times separated by centrifuging the cells (8 min, 1000 rpm) and removing supernatant.

The 15 ml tubes were centrifuged 8 minutes at 1000 rpm. Then the cells were concentrated into a smaller volume of the fixative and resuspended. Clean slides were first brought at the right temperature on a heat plate, depending on the humidity of the air (appendix 1). Then 40 µl of fixated cells were dropped on the slides. Once the slides were dry, they were stained with acridine orange (AO). First the slides were immersed in an AO-work solution for 1 minute. Then they were rinsed in distilled water and immersed in an AO-buffer for 1 minute. Thereafter excess AO-buffer was removed and 20 µl of the AO-buffer was dropped on the slides. A coverslip was put on top and the edges of the coverslip were covered with sealant so no air could get under. The number of MNi in 500 binuclear cells (BN) per slide were counted. In addition, the nuclear division index (NDI) of each slide was calculated using the following formula: NDI = (M1 +2M2 +3M3 +4M4)/N. M1, M2, M3 and M4 represent the number of cells with one, two, three and four nuclei and N the total number of analysed cells (N = 500).

20

2.5 Erα/β-STAINING First 120.000 cells were collected from each concentration of fulvestrant into 1 ml complete culture medium. Using the cytospin (5 min, 500 rpm) 250 µl cell suspension (30.000 cells) were centrifuged onto the slides. Then the cells were fixed 10 minutes with 4% paraformaldehyde (PFA) (VWR, 1040051000). Thereafter the slides were placed on a shaking plate and washed 3 times for 5 minutes with PBS. Permeabilization was done using Triton X-100 (Sigma T8787) for 10 minutes followed by 3 washing steps of 5 minutes with 0,1% Tween 20% PBS (Sigma P1379). Then the blocking serum, bovine serum albumin (0,1g BSA/10ml 0,1% Tween 20% PBS) (Roche, 10 735 086 011), was added for 30 minutes. The first antibody (see table 3) was added for 2 hours followed by washing the cells 3 times for 5 minutes with 0,1% Tween 20% PBS. Afterwards the second antibody, GAM-Dylight-488 (Thermo 35503) (1/1000), was added for 1 hour. The cells were then washed 3 times for 5 minutes with 0,1% Tween 20% PBS and counterstained with DAPI (Sigma D8417) (1/500) for 10 minutes. After DAPI staining, the cells were washed 2 times for 5 minutes with 0,1% Tween 20% PBS, mounted with fluoromount (Sigma) and stored at 4°C until viewing. The intensity of DAPI and GFP (GAM-Dylight-488) were calculated with the software of Image J.

Table 3 Different antibodies for ERα/β-staining.

Staining Antibody Concentration antibody

ERα anti-ERα mouse monoclonal antibody sc-8002 (Santa Cruz)

1/100

ERβ anti-ERβ mouse monoclonal antibody B-3 (sc-373853, Santa Cruz)

1/100 and 1/2000

ERβ anti-ERβ mouse monoclonal antibody B-1 (sc-390243, Santa Cruz)

1/100 and 1/2000

21

3 RESULTS

3.1 CELL PROLIFERATION ASSAY

Figure 11 shows the results of the proliferation assay. Fulvestrant (added after 20h CT) reduced the proliferation of MCF-7 in a dose-dependent manner. The data of the separate experiments can be found in appendix 2.

Figure 11: Proliferation MCF-7 with fulvestrant added after 20h CT. The numbers under the graph

represent the total cell count of one culture flask. Error bars represent the standard deviation (SD)(N=5). After 68h CT a significant difference in proliferation is only observed between 0 M and 10ˉ⁶ M. There is a significant difference in proliferation between 0 M and all the other concentrations of fulvestrant after 92h CT (p<0,05).

When fulvestrant was added at 0h CT, the first 48 hours cells proliferated very little as shown in figure 12. Afterwards cell proliferation was proceeded to an extent dependent on the concentration of fulvestrant. This graph is a representation of the mean. Growth curves of the separate experiments are given in appendix 4.

0h 68h 92h

0M 400 000 1 141 400 1 954 500

10-9M 400 000 1 038 800 1 312 333

10-8M 400 000 992 400 1 140 600

10-7M 400 000 957 100 1 094 333

10-6M 400 000 838 200 1 076 000

0,0

0,5

1,0

1,5

2,0

2,5

Num

ber

of

cells

(x10⁶)

Culture time

22

Figure 12: Proliferation MCF-7 with fulvestrant added at 0h CT. The numbers under the graph represent the total cell count of one culture flask. Error bars represent the SD. A significant difference in growth is observed between 0 M and 10ˉ⁶ M at 116h CT (p<0,05).

As illustrated in figure 13 fulvestrant (added after 20h CT) has no impact on the morphology of MCF-7, neither in a time or dose-dependent manner. Pictures of other magnifications and repeats can be found in appendix 3.

0h 48h (N=1) 72h (N=2) 96h (N=4) 116h (N=4)

0 M 300 000 280 000 747 500 1 041 500 1 283 500

10ˉ⁹ M 300 000 244 750 707 500 776 125 1 079 500

10ˉ⁶ M 300 000 184 250 480 000 613 500 611 375

0,0

0,5

1,0

1,5

2,0

2,5N

um

ber

of

cells

(x10⁶)

Culture time

0 M 10ˉ⁶ M 10ˉ⁷ M 10ˉ⁸ M 10ˉ⁹ M

66h

90h

CT

Concentration fulvestrant

scalebar 100 µm

Figure 13: Brightfield imaging of MCF-7. Fulvestrant was added after 20h CT.

23

3.1 ERα/β-STAINING

Shown in figure 14 and 15 is the demonstration of ERα degradation in time when cells were cultured in 10ˉ⁶ M fulvestrant. After 4h and 72h respectively 23,6% and 34,2% of ERα was degraded. After 96h nearly 65% of ERα was degraded. In figure 14 the presence of ERα is clearly visible in both the nucleus and cytoplasm.

GFP DAPI OVERLAY

MC

F7

M

DA

-

MB

-23

1

0 M

0 M

10

-6

M

4 h

72 h

96 h

96 h

96 h

Figure 14: Fluorescent imaging of the ERα degradation in MCF-7 following fulvestrant treatment. MDA-MB-231 was taken as negative control. DAPI represents blue counterstaining of nuclei, Green fluorescent protein (GFP) represents green staining of the ERα, OVERLAY is the combination of DAPI and GFP images. Scalebar 50 µm.

24

Figure 15: Intensity of ERα-staining. Error bars represent the SD.

In figure 16 the degradation of ERα after treatment with different concentrations of fulvestrant

is shown. After 96 hours and 116 hours of CT with 10ˉ⁶ M fulvestrant an obvious reduction of

ERα is seen. The presence of ERα in cells treated with 10ˉ⁹ M fulvestrant was about the same

as in the untreated cells.

MCF7 0MMCF7 10-

6M 4hMCF7 10-

6M 72hMCF7 10-

6M 96hMDA-MB-231 0M

GFP/DAPI 0,739 0,564 0,486 0,262 0,172

0,0

0,2

0,4

0,6

0,8

1,0

1,2

1,4

Inte

nsity

ratio G

FP

/DA

PI

25

GF

P

DA

PI

OV

ER

LA

Y

MD

A-

MB

-23

1

0 M

10ˉ⁶

M

116 h CT 96 h CT

0 M

10ˉ⁹

M

10ˉ⁶

M

0 M

10ˉ⁹

M

10ˉ⁶

M

0 M

MC

F7

M

DA

-

MB

-23

1

MC

F7

M

CF

7

MD

A-

MB

-23

1

0 M

0 M

10ˉ⁹

M

Figure 16: Fluorescent imaging of the ERα degradation in MCF-7 following fulvestrant treatment. MDA-MB-231 was taken as negative control. DAPI represents blue counterstaining of nuclei, GFP represents green staining of the ERα, OVERLAY is the combination of GFP and DAPI. Fulvestrant was added immediately after seeding. 0 M, 10ˉ⁶ M and 10ˉ⁹ M represent the different concentrations of fulvestrant. Scalebar 50 µm.

26

In figure 17 a ERβ-staining is showed. The B-1 antibody bound nonspecific and resulted in a

staining with high intensity. The B-3 antibody bound specific but very weak. A larger amount

of ERβ was observed in T-47D than in MCF-7. Furthermore, ERβ is present in both cytoplasm

and nucleus.

MC

F7

B-1 B-3

DAPI GFP OVERLAY DAPI GFP OVERLAY

T-4

7D

DIL

UT

ION

1/1

00

D

ILU

TIO

N 1

/20

00

MC

F7

T

-47D

Figure 17: ERβ-staining of MCF-7 and T-47D. 2 different antibodies were administered: B-1 (sc-390243) and B-3 (sc-373853). DAPI represents blue counterstaining of nuclei, GFP represents green staining of the ERα, OVERLAY is the combination of GFP and DAPI. Scalebar 50 µm.

27

3.2 MICRONUCLEUS ASSAY

When 10ˉ⁶ M fulvestrant was added 4h before irradiation the amount of MN in untreated and treated MCF-7 cells were comparable (figure 18). For a dose of 2 Gy the treated cells showed a slightly decreased amount of MN. At doses of 3 Gy and 4 Gy the amount of MN was slightly higher than the untreated cells.

Figure 18: MN assay with fulvestrant added 4h before irradiation. Error bars represent the SD. 0Gy (N=2), 1Gy (N=2), 2Gy (N=2), 3Gy (N=1), 4Gy (N=1).

0

200

400

600

800

1000

0 GY 1 GY 2 GY 3 GY 4 GY

MN

/ 1

000 B

N

Dose

0 M 10ˉ⁶ M

28

As shown in figure 19A and 20A differences in MN amount were observed after 120 hours fulvestrant treatment. At higher concentrations, less MN were detected. Differences in MN amount increased with higher X-rays doses. When cells were not irradiated, the number of MN was comparable between different concentrations of fulvestrant.

Corresponding NDIs are shown in figure 19B and 20B. Higher concentrations of fulvestrant caused lower NDIs. Comparable with the number of MN the difference in NDI increased with higher X-ray doses.

A)

B)

Figure 19: MN assay with fulvestrant added 120h before irradiation. Fulvestrant was added immediately after seeding. A) DNA damage (MN). 0 M (N=4), 10ˉ⁹ M (N=2), 10ˉ⁷ M (N=2), 10ˉ⁶ M (N=1). B) cell culture quality (NDI). Error bars represent the SD.

0

200

400

600

800

1000

0 GY 2 GY

# M

N /

1000 B

N

Dose

0 M 10ˉ⁹ M 10ˉ⁷ M 10ˉ⁶ M

1

1,1

1,2

1,3

1,4

1,5

1,6

0 GY 2 GY

ND

I

Dose

0 M 10ˉ⁹ M 10ˉ⁷ M 10ˉ⁶ M

29

In figure 20A the MN assay with 10ˉ⁶ M fulvestrant was repeated 4 times. Only the data points of 2 experiments are taken into the histogram. This is due to the fact that for the other 2 experiments not enough binuclear cells could be counted for the concentration of 10ˉ⁶ M.

A)

B)

Figure 20: MN assay with fulvestrant added 96h before irradiation. A) DNA damage (MN). 0 M (N=4), 10ˉ⁹ M (N=4), 10ˉ⁶ (N=2). B) cell culture quality (NDI)(N=4). Error bars represent the SD .

0

200

400

600

800

1000

0 GY 1GY

MN

/ 1

000 B

N

Dose

0 M 10ˉ⁹ M 10ˉ⁶ M

1

1,1

1,2

1,3

1,4

1,5

1,6

0 GY 1 GY

ND

I

Dose

0 M 10ˉ⁹ M 10ˉ⁶ M

30

Figure 21 shows the proliferation of MCF-7 within a MN assay with fulvestrant. Fulvestrant was added at 0h CT so the results could be compared to the ERα-staining and the MN assay where cells were irradiated at 96 hours and fixated at 116 hours CT. For the fulvestrant concentration 10ˉ⁶ M the number of cells was the same as at 0h CT. No difference was observed between 0 Gy and 1 Gy within the same concentration. This graph is a representation of the mean. Growth curves of each experiment are shown in appendix 5.

Figure 21: proliferation MCF-7 in MN assay with fulvestrant. Fulvestrant was added at 0h CT. The numbers under the graph represent the total cell count of one culture flask. Error bars represent the SD (N=4). No significant differences are observed between fulvestrant concentrations (p<0,05).

0h 116h (0GY) 116h (1GY)

0 M 300 000 523 750 578 750

10ˉ⁹ M 300 000 492 500 475 000

10ˉ⁶ M 300 000 296 563 304 975

0,0

0,5

1,0

1,5

2,0

2,5

Num

ber

of

cells

(x10⁶)

Culture time

31

4 DISCUSSION

In this study, we aimed to verify the relation between radiosensitivity and ERα expression using fulvestrant to degrade ERα in MCF-7. Radiosensitivity of the untreated and treated MCF-7 cells was compared using the micronucleus assay. Since previous studies suggested that ER+ cancer cells tended to be more radiosensitive[51, 52], we evaluated if radiosensitivity of MCF-7 decreased in correlation to ERα expression.

Cell proliferation experiments showed that the proliferative activity of MCF-7 cells decreased after addition of fulvestrant in a dose-dependent manner (figure 11 and 12). Cells exposed to a concentration of 10ˉ⁶ M survived for several days but lost great proliferation ability, suggesting that 10ˉ⁶ M is the limited concentration for in vitro experiments. Concerning the morphology fulvestrant did not cause any morphological changes. This is in accordance with previously performed proliferation assays conducted by Hutcheson et al. and Wang et al[76, 77].

Hutcheson et al. conducted a proliferation assay on wild type MCF-7 treated with 10ˉ⁷ M fulvestrant. By means of RT-PCR, western blot and immunostaining the presence and degradation of ERα was observed. Fulvestrant decreased growth of MCF-7 cells, with approximately 80–85% inhibition after 7 days. In addition, ERα expression was significantly reduced by fulvestrant, thus confirming the important role of ERα in the proliferation of ER+ cancer cells[76].

Wang et al. combined the treatment of fulvestrant with exposure to ionizing radiation. Cells were treated with 10ˉ⁷ M fulvestrant and irradiated after 4 days with doses of 0, 2, 4 and 6 Gy. The cytotoxic effect of fulvestrant and ionizing radiation was observed with a clonogenic survival assay where both had a cytotoxic effect on MCF-7 cells and showed a synergic effect when combined[77]. When this is compared to the results in figure 21, the cytotoxic effect of fulvestrant is clearly noticeable but a synergic effect between fulvestrant and irradiation is not observed. MCF-7 cells treated with 10ˉ⁶ M fulvestrant proliferated to the same extent for both irradiation doses 0 Gy and 1 Gy. Although this could be due to the low irradiation dose and maybe a synergic effect would be observable with higher doses of ionizing addition.

An ERα-staining confirmed the ERα degradation after treatment with fulvestrant (figure 14, 15 and 16). ERα expression diminished in a time- and dose-dependent manner. Shaw et al. performed a western blot of ERα in MCF-7 cells treated with 10ˉ⁷ M fulvestrant. This resulted in an almost complete disappearance of ERα after 1 year[59]. Khalid et al. treated MCF-7 cells for 48 hours with fulvestrant concentrations varying from 10ˉ⁶ M to 10ˉ⁹ M. In this study a dose-dependent ERα degradation was observed with a western blot[78].

Figure 18 is the representation of the radiosensitivity of MCF-7 cells treated with fulvestrant 4 hours before irradiation. No major differences are observed between the treated and untreated cells. This can be explained by the fact that after 4 hours of 10ˉ⁶ M fulvestrant exposure still 76% of ERα is present in MCF-7 compared to the untreated cells (figure 14 and 15). After 96 hours of 10ˉ⁶ M fulvestrant exposure only 36% of ERα remained in the cells (figure 15). When fulvestrant was added 96 hours (figure 20A) and 120 hours (figure 19A) before irradiation radiosensitivity of the treated cells was lower than the untreated cells depending on the added concentration of fulvestrant. Thus, these results indicate that the radiosensitivity of MCF-7 cells decreased when ERα expression decreased.

As opposed to this study Wang et al. showed an increased radiosensitivity when fulvestrant was used. Cell survival with combined treatment was reduced up to 30% after irradiation suggesting that fulvestrant enhances the effect of irradiation. Furthermore, decreased levels of RAD51 and DNA-PKcs proteins were observed when fulvestrant was administered. Given

32

that both RAD51 and DNA-PKcs have major roles in DSB repair, this could be a cause of the radiosensitizing effect[77].

Previous studies revealed a relation between ERα expression and radiosensitivity. Chen et al. investigated the effects of ER expression on the biological behaviours of cells using gene transfection to introduce ERα in MDA-MB-231 cancer cells. ERα transfection reduced cell proliferation suggesting that ERα cells have a lower proliferative capacity than ER- cells. Secondly ERα transfection increased radiosensitivity of MDA-MB-231 cells. In addition, ERα-transfected cells showed increased double-stranded breaks and delayed repair compared with wild type MDA-MB-231 cells. Furthermore ERα-transfected cells showed a modified cell cycle distribution in which the number of cells in G2/M phase increased and in G1/S phase decreased. Given that cells in the G2/M phase are more radiosensitive, this could explain the difference in radiosensitivity after ERα transfection[52].

A recent study analysed the relation between ER positivity and deficiencies in DNA damage repair capacity in 270 breast cancer patients. Results of a host cell reactivation assay showed that ER+ breast cancer cells are more likely to have deficiencies in DNA damage repair capacity[79]. This is in line with previous studies on altered DNA repair in ER+ cancer cells. Results revealed that ERα transfected MDA-MB-231 cells showed a down-regulation of DNA-PKcs which would lead to a decrease in DNA repair efficiency[80]. Furthermore, ER alters the DNA damage response and DNA repair through the regulation of ATM, ATR, CHK1, BRCA1, and p53. Due to downregulation of ATM and ATR, cell cycle checkpoints are not activated. This results in a continued proliferation after DNA damage and DNA repair is delayed or not engaged. These results suggested that the DNA damage repair ability in ER+ cancer cells differs from ER- cancer cells, resulting in differences in radiosensitivity[81, 82].

In figure 19B and 20B the NDI of different treated cell cultures are demonstrated. NDI is a marker of cell proliferation in cultures and is used to measure the cytotoxicity of fulvestrant. The NDI decreased with both increasing dose of x-rays and increasing concentration of fulvestrant, which is in accordance with the proliferation assays (figure 11, 12 and 21). These low NDIs may confirm that fulvestrant has a radiosensitizing effect on ER+ breast cancer cells[77].

When the ERα-staining is compared with the micronucleus assay, an association is seen between the amount of degraded ERα and the reduced MN. For the fulvestrant concentration 10ˉ⁹ M, only a small number of ERα was degraded. This is in line with the MN amount which does not differ much from the MN amount in the untreated cells. When we look at the cells treated with 10ˉ⁶ M fulvestrant, a greater degradation of ERα is observed and this results in a lower MN number after irradiation. Whether this is the result of a decreased radiosensitivity is not certain because of the fact that the data for 10ˉ⁶ M a collection was of only 2 repeats (figure 20A).

In figure 17 the presence of ERβ is visualized in MCF-7 and T-47D. Antibody B-1 bound nonspecific and antibody B-3 bound specific but weak. Further optimization of ERβ-staining in MCF-7 with B-3 is required. As expected, more ERβ was observed in T-47D than in MCF-7[73].

Looking at both MN and proliferation assays, it is hard to conclude if the decreased amount of MN is due to reduced radiosensitivity or on the contrary due to increased radiosensitivity resulting in less cells dividing, which is necessary for the micronucleus assay.

33

5 GENERAL CONCLUSION

The results of this study showed that number of MN in MCF-7 decreased when ERα expression diminished. Although, further investigation is needed to obtain a clearer picture on this topic.

Given the cytotoxic effect of fulvestrant, a combination with the micronucleus assay is not the ideal method to observe the amount of DNA damage. The reliability of the results is low due to low NDIs. Further optimisation is needed for the combination of a micronucleus assay with fulvestrant.

A foci or comet assay would provide more insight on the incurred DNA damage and DNA repair kinetics. These methods are not depending on the cell proliferation of cancer cells, thus combination with fulvestrant is possible.

34

6 REFERENCES

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71. Sweeney EE, McDaniel RE, Maximov PY, Fan P, Jordan VC (2012) Models and Mechanisms of Acquired Antihormone Resistance in Breast Cancer: Significant Clinical Progress Despite Limitations. Horm Mol Biol Clin Investig 9: 143-163 72. Nugoli M, Chuchana P, Vendrell J, Orsetti B, Ursule L, Nguyen C, Birnbaum D, Douzery EJ, Cohen P, Theillet C (2003) Genetic variability in MCF-7 sublines: evidence of rapid genomic and RNA expression profile modifications. BMC Cancer 3: 13 73. Pons DG, Nadal-Serrano M, Blanquer-Rossello MM, Sastre-Serra J, Oliver J, Roca P (2014) Genistein modulates proliferation and mitochondrial functionality in breast cancer cells depending on ERalpha/ERbeta ratio. J Cell Biochem 115: 949-58 74. Cailleau R, Olive M, Cruciger QV (1978) Long-term human breast carcinoma cell lines of metastatic origin: preliminary characterization. In Vitro 14: 911-5 75. Chavez KJ, Garimella SV, Lipkowitz S (2010) Triple negative breast cancer cell lines: one tool in the search for better treatment of triple negative breast cancer. Breast Dis 32: 35-48 76. Hutcheson IR, Knowlden JM, Madden TA, Barrow D, Gee JM, Wakeling AE, Nicholson RI (2003) Oestrogen receptor-mediated modulation of the EGFR/MAPK pathway in tamoxifen-resistant MCF-7 cells. Breast cancer research and treatment 81: 81-93 77. Wang J, Yang Q, Haffty BG, Li X, Moran MS (2013) Fulvestrant radiosensitizes human estrogen receptor-positive breast cancer cells. Biochem Biophys Res Commun 431: 146-51 78. Khalid S, Hanif R, Jabeen I, Mansoor Q, Ismail M (2018) Pharmacophore modeling for identification of anti-IGF-1R drugs and in-vitro validation of fulvestrant as a potential inhibitor. PloS one 13: e0196312 79. Matta J, Morales L, Ortiz C, Adams D, Vargas W, Casbas P, Dutil J, Echenique M, Suarez E (2016) Estrogen Receptor Expression Is Associated with DNA Repair Capacity in Breast Cancer. PloS one 11: e0152422 80. Licznar A, Caporali S, Lucas A, Weisz A, Vignon F, Lazennec G (2003) Identification of genes involved in growth inhibition of breast cancer cells transduced with estrogen receptor. FEBS Lett 553: 445-50 81. Caldon CE (2014) Estrogen signaling and the DNA damage response in hormone dependent breast cancers. Front Oncol 4: 106 82. Pedram A, Razandi M, Evinger AJ, Lee E, Levin ER (2009) Estrogen inhibits ATR signaling to cell cycle checkpoints and DNA repair. Mol Biol Cell 20: 3374-89

38

7 APPENDICES

APPENDIX 1 Table temperature warmth plate / air humidity

Plate

Humidity 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50

30%

31%

32%

33%

34%

35%

36%

37%

38%

39%

40%

41%

42%

43%

44%

45%

46%

47%

48%

49%

50%

51%

52%

53%

54%

55%

56%

57% too small

58%

59%

60%

61%

62%

63%

explod

ed

cells

partly

explod

ed

partly

explod

ed OK

64%

65%

66%

67% too small

68%

exploded cells

room

temperature,

small but OK

exploded cells

39

APPENDIX 2

Proliferation assay MCF-7 (fulvestrant added after 20h CT)

0,4

0,9

1,4

1,9

2,4

0h 68h 92h

Num

ber

of

cells

(x10⁶)

Culture time

Experiment 1

0,4

0,9

1,4

1,9

2,4

0h 68h 92h

Num

ber

of

cells

(x10⁶)

Culture time

Experiment 2

0,4

0,9

1,4

1,9

2,4

0h 68h 92h

Num

ber

of

cells

(x10⁶)

Culture time

Experiment 3

0,4

0,9

1,4

1,9

2,4

0h 68h 92h

Num

ber

of

cells

(x10⁶)

Culture time

Experiment 4

0,4

0,9

1,4

1,9

2,4

0h 68h 92h

Num

ber

of

cells

(x10⁶)

Culture time

Experiment 5

40

APPENDIX 3

Brightfield pictures of the proliferation assays

Proliferation assay 1 (magnification 40x)

18h

scalebar 500 µm

42h

66h

90h

10-6 M 10-8 M 0 M

Fulvestrant added after 20h CT

CT

Concentration fulvestrant

41

Proliferation assay 1 (magnification 100x)

18h

scalebar 200 µm

42h

66h

90h

10-6M 10-8M 0M

Fulvestrant added after 20h CT

CT

Concentration fulvestrant

42

Proliferation assay 1 (magnification 200x)

scalebar 100 µm

42h

66h

90h

10-6M 10-8M 0M

Fulvestrant addad after 20h CT

CT

Concentration fulvestrant

43

Proliferation assay 2 (magnification 40x)

scalebar 500 µm

66h

18h

90h

0 M 10-9 M 10-8 M 10-7 M 10-6 M

Fulvestrant added after 20h CT

CT

Concentration fulvestrant

44

Proliferation assay 2 (magnification 100x)

scalebar 200 µm

66h

18h

90h

0 M 10-9 M 10-8 M 10-7 M 10-6 M

Fulvestrant added after 20h CT

CT

Concentration fulvestrant

45

Proliferation assay 2 (magnification 200x)

scalebar 100 µm

66h

18h

90h

0 M 10-9 M 10-8 M 10-7 M 10-6 M

Fulvestrant added after 20h CT

CT

Concentration fulvestrant

46

Proliferation assay 3 (magnification 40x)

scalebar 500 µm

42h

18h

66h

0 M 10-9 M 10-8 M 10-7 M 10-6 M

Fulvestrant added after 20h CT

CT

Concentration fulvestrant

90h

47

Proliferation assay 3 (magnification 100x)

scalebar 200 µm

42h

18h

90h

0 M 10-9 M 10-8 M 10-7 M 10-6 M

Fulvestrant added 20h CT

CT

Concentration fulvestrant

66h

48

Proliferation assay 3 (magnification 200x)

scalebar 100 µm

42h

18h

90h

0 M 10-9 M 10-8 M 10-7 M 10-6 M

Fulvestrant added after 20h CT

CT

Concentration fulvestrant

66h

49

Proliferation assay 4 (magnification 40x)

scalebar 500 µm

42h

66h

0 M 10-9 M 10-8 M 10-7 M 10-6 M

Fulvestrant added after 20h CT

CT

Concentration fulvestrant

90h

50

Proliferation assay 4 (magnification 100x)

scalebar 200 µm

42h

90h

0 M 10-9 M 10-8 M 10-7 M 10-6 M

Fulvestrant added after 20h CT

CT

Concentration fulvestrant

66h

51

Proliferation assay 4 (magnification 200x)

scalebar 100 µm

42h

90h

0 M 10-9 M 10-8 M 10-7 M 10-6 M

Fulvestrant added after 20h CT

CT

Concentration fulvestrant

66h

52

Proliferation assay 5 (magnification 40x)

scalebar 500 µm

42h

66h

0 M 10-9 M 10-8 M 10-7 M 10-6 M

Fulvestrant added after 20h CT

CT

Concentration fulvestrant

90h

53

Proliferation assay 5 (magnification100x)

scalebar 200 µm

42h

90h

0 M 10-9 M 10-8 M 10-7 M 10-6 M

Fulvestrant toegevoegd na 20h CT

CT

Concentration fulvestrant

66h

54

Proliferation assay 5 (magnification 200x)

scalebar 100 µm

42h

90h

0 M 10-9 M 10-8 M 10-7 M 10-6 M

Fulvestrant added after 20h CT

CT

Concentration fulvestrant

66h

55

APPENDIX 4

Proliferation assay MCF-7 (fulvestrant added at 0h CT)

0,1

0,3

0,5

0,7

0,9

1,1

1,3

1,5

1,7

1,9

0h 72h 96h 116h

Num

ber

of

cells

(x10⁶)

Culture time

Experiment 1

0,1

0,3

0,5

0,7

0,9

1,1

1,3

1,5

1,7

1,9

0h 48h 96h 116h

Num

ber

of

cells

(x10⁶)

Culture time

Experiment 2

0,1

0,3

0,5

0,7

0,9

1,1

1,3

1,5

1,7

1,9

0h 72h 96h 116h

Num

ber

of

cells

(x10⁶)

Culture time

Experiment 3

0,1

0,3

0,5

0,7

0,9

1,1

1,3

1,5

1,7

1,9

0h 96h 116h

Num

ber

of

cells

(x10⁶)

Culture time

Experiment 4

56

APPENDIX 5

Proliferation MCF-7 in MN assay (fulvestrant added at 0h CT)

0,2

0,3

0,4

0,5

0,6

0,7

0,8

0,9

0h 116h

Num

ber

of

cells

(x10⁶)

Culture time

Experiment 1

0,2

0,3

0,4

0,5

0,6

0,7

0,8

0,9

0h 116h

Num

ber

of

cells

(x10⁶)

Culture time

Experiment 2

0,2

0,3

0,4

0,5

0,6

0,7

0,8

0,9

0h 116h

Num

ber

of

cells

(x10⁶)

Culture time

Experiment 3

0,2

0,3

0,4

0,5

0,6

0,7

0,8

0,9

0h 116h

NN

um

ber

of cells

(x10⁶)

Culture time

Experiment 4