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Cell-cell contact induced resistance to etoposide Oloumi, Arusha 2002

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C E L L - C E L L CONTACT INDUCED RESISTANCE TO ETOPOSIDE by ARUSHA OLOUMI B.Sc. (Chemistry), The University of British Columbia, 1994 A THESIS SUBMITTED IN PARTIAL F U L F I L M E N T OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE F A C U L T Y OF G R A D U A T E STUDIES (Department of Pathology and Laboratory Medicine) We accept this thesis as conforming to the reguirexi standard THE UNIVERSITY OF BRITISH C O L U M B I A February 2002 © Arusha Oloumi, 2002 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department The University of British Columbia Vancouver, Canada DE-6 (2/88) A b s t r a c t Many tumour cell lines grown in close three dimensional cell-cell contact either as multicell spheroids or tumours in mice exhibit a form of multicellular drug and radiation resistance that has been called the "contact effect". This resistance is often associated with agents that produce D N A double-strand breaks such as ionizing radiation and the anticancer drug and topoisomerase II inhibitor, etoposide. The hypothesis was that growth in three dimensional contact results in changes in gene expression that act, directly or indirectly, to increase resistance to etoposide. The objectives were to 1) determine the mechanism for etoposide resistance of spheroids, 2) identify genes that are differentially expressed in monolayers and spheroids, and 3) based on these results, examine the importance of intracellular free calcium levels as mediators of contact resistance. Cycling cells from Chinese hamster V79 spheroids are about 10 times more resistant than monolayers to cell killing by etoposide. Previous results indicated that the outer cells of spheroids and monolayers contained the same total amount and activity of the target enzyme, topo lice, and grew at the same rate. Using immunoblotting and immunohistochemistry, topo Hoc was found to be localized primarily in the cytoplasm of the proliferating outer cells of V79, SiHa and C6 spheroids, while nuclear localization was observed in their corresponding monolayers. Conversely, only WiDr cells, which did not show an increase in resistance to etoposide when grown as spheroids, demonstrated a predominantly nuclear localization of topo Ila. This difference in localization pattern was subsequently explained by the 10-fold decrease in phosphorylation of topo Ila in spheroids relative to monolayers, since phosphorylation is i i apparently required for nuclear translocation of this enzyme. Cells sorted from xenograft tumours grown in immunodeficient mice resembled the spheroid pattern both in terms of sensitivity to etoposide and location of topo Hoc. When the outer cells of V79 spheroids were returned to monolayer growth, the rate of redistribution of topo Hot to the nucleus occurred with the same kinetics as the increase in sensitivity to the cytotoxic effects of etoposide. Thus, close 3-dimensional cell-cell contact can lead to a change in post-translational modification of topo Hoc that results in resistance to etoposide. A more direct approach was taken to identify changes in gene expression that occur when cells are grown as spheroids. Using the technique of differential display, 8 reproducible genes were found to be differentially expressed in the outer layer of V79 spheroids compared to monolayers. Up-regulation of 3 genes (cytochrome oxidase c, mtsl and calretinin) was associated with an increase in calcium binding capacity in outer cycling cell of spheroids, suggesting a possible role for calcium for the development of a contact effect. Consistent with this hypothesis, a 2-fold lower concentration of intracellular calcium was found in spheroids compared to monolayers using fluo-3 as a calcium indicator dye. Exposure of monolayers and outer spheroid cells to non-cytotoxic concentrations of B A P T A - A M , a calcium chelating agent, eliminated the difference in etoposide sensitivity between V79 monolayers and spheroids. Calcium depletion has been previously shown to protect against etoposide-induced damage by affecting both the phosphoryaltion of topo Ila and by stablizing the cleavable complex. To determine whether over-expression of a calcium binding protein would increase resistance to etoposide, V79, SiHa and C6 monolayers were transduced with metastasin (mtsl). Expression of this transgene did not reduce killing by etoposide, however because the in ultimate goal of reduction in intracellular free C a 2 + was also not achieved with this method, the importance of calcium regulation in etoposide resistance cannot be ruled out. In conclusion, growth of cells in 3-dimensional contact as spheroids or as solid tumours induces resistance to etoposide. The basis for this resistance could lie in a change in intracellular C a 2 + that alters cleavable complex formation and affects phosphorylation of topo Ila, both of which can cause resistance to the anti-cancer drug etoposide. iv Table of Contents Page Abstract ii Table of Contents v List of Figures vii List of Tables '• ix Abbreviations x Acknowledgements xi i Dedication '. xiv Chapter 1. Introduction 1 1.1 Tumour Cell Resistance to Therapy 2 1.2 Topoisomerase II and Etoposide 6 1.2.1 Topoisomerases: Essential Roles in Transcription and Replication 6 1.2.2 Inhibitors of Topoisomerase II 9 1.2.3 Etoposide 10 1.2.4 Mechanism of Resistance to Etoposide 11 1.3 Multicell Spheroids 15 1.3.1 Structure and Biology of Spheroids 16 1.3.2 Culture of Spheroids 20 1.3.3 Resistance to Therapy 22 1.4 The "Contact Effect" 23 1.4.1 Ionizing Radiation 23 1.4.2 Other Agents which Show a Contact Effect 24 1.4.3 Proposed Mechanisms for the Contact Effect 25 1.5 Objectives 29 Chapter 2. Materials and Methods 32 2.1 Cell Lines and Culture Conditions 33 2.2 Spheroid Growth 34 2.3 Sequential Trypsinization 35 2.4 Xenograft Cell Sorting 35 2.5 Colony Formation Assay and Relative Resistance 37 2.6 Immunhistochemistry 40 2.7 Topo I la Phosphorylation 41 2.8 Sub-cellular Fractionation 41 2.9 Immunoblotting 42 v 2.10 R N A isolation 43 2.11 Differential Display 45 2.12 Reverse Northern 47 2.13 Sequencing and B L A S T Search 50 2.14 Calcium Measurement 50 2.15 B A P T A - A M Treatment 51 2.16 Mts l Transfection 52 Chapter 3. The Role of Phosphorylation and Localization of Topoisomerase Ila in Resistance to Etoposide 56 3.1 Introduction 57 3.2 Results 61 3.2.1. Response to Etoposide Treatment 61 3.2.2 Localization of Topo Ila by Immunohistochemistry 62 3.2.3 Localization of Topo Ila by Western blot 67 3.2.4 Localization of Topo Ila in Relation to Sensitivity to Etoposide....67 3.2.5 Phosphorylation of Topo Ila 68 3.3 Discussion 74 Chapter 4. Identification of Genes Differentially Expressed in V79 Cells Grown as Multicell Spheroids 77 4.1 Introduction 78 4.2 Results .' 80 4.2.1 Genes Identified with Differential Display 80 4.2.2 Metastasin Northern Blot 82 4.2.3 Metastasin and Calretinin Western Blots 83 4.3 Discussion 88 Chapter 5. The role of Calcium Regulation in Resistance to of spheroids to Etoposide...91 5.1 Introduction 92 5.2 Results 97 5.2.1 Intracellular Free Calcium Measurement 97 5.2.2 Effect of Pre-treatment with B APT A and Etoposide Toxicity 99 5.2.3 Detection and Expression of Transfected Mts l 99 5.2.4 Intracellular Free C a 2 + in Mtsl Overexpressing Cell Lines 100 5.2.5 Effect of mtsl Overexpression on Etoposide-induced Toxicity 101 5.3 Discussion 108 Chapter 6. Summary and Future Work 113 6.1 Summary .114 6.2 Suggestions for Future Work 120 References 125 vi List of Figures Page Fig.l: Topoisomerase Il-targeted drugs 12 Fig. 2: Interaction between topoisomerase II, D N A and etoposide 13 Fig. 3: Schematic representation of spheroid growth in suspension 14 Fig. 4: Xenograft cell sorting 39 Fig. 5: mRNA differential display experiment 48 Fig. 6: Structure of pCR-TRAP vector 49 Fig. 7: Flag-mtsl D N A template 54 Fig. 8: Schematic representation of MIG-flag-mtsl plasmid 55 Fig. 9: Schematic representation of human topoisomerase Hoc 60 Fig. 10: Response of monolayers and outer spheroid cells to etoposide 63 Fig. 11: Response of xenograft tumours to etoposide 64 Fig. 12: Cellular distribution of topo Ila determined by immunohistochemistry 66 Fig. 13: Topoisomerase Hoc in xenograft tumours 69 Fig. 14: Cellular distribution of topo I la determined by Western blot 70 Fig. 15: Anti-topoisomerase Ila antibody staining of outer V79 spheroid cells as a function of time after return to monolayer growth conditions 71 Fig. 16: Change in localization of topoisomerase Ila in comparison with change in sensitivity to etoposide 72 Fig. 17: Phosphorylation of Topo Ila in monolayrs and the outer cells of spheroids 73 Fig. 18: Differential display gel 84 Fig. 19: Reverse northern blot analysis 85 vii Fig. 20: V79 Northern blot with mtsl probe 86 Fig. 21: Western blot with mtsl and calretinin antibodies 87 Fig. 22: Signal transduction by calcium binding proteins 96 Fig. 23: Intracellular free calcium measurement 98 Fig. 24: Effect of B APT A - A M on etoposide induced cell killing 102 Fig. 25: Restriction enzyme digested MIG-mtsl plasmid 103 Fig. 26: Mean GFP fluorescence measured by flow cytometry in virally transfected cell lines 104 Fig. 27: Western blot analysis of mtsl transduced cell lines 105 Fig. 28: Intracellular free calcium measurement in transduced cell lines 106 Fig. 29: Etoposide toxicity in mtsl transduced cell lines 107 Fig. 30: Effect of B A P T A treatment on etoposide induced D N A damage 112 Fig. 31: Possible mechanisms for the contact effect 118 Fig. 32: Model representing the possible link between intracellular calcium and factors that could lead to resistance to etoposide and x-rays 119 Fig. 33: p27 k i p- 1 expression in V79 monolayers and spheroids 122 viii List of Tables Page Table 1: Probable mechanisms associated with tumour resistance to therapy 3 Table 2: Antibodies 44 Table 3: Analysis of 4 cell lines for the presence of a contact effect 65 Table 4: Genes identified using differential display 81 ix List of Abbreviations ATP Adenosine Triphosphate BAPTA-AM [ 1,2-bis-(o-aminophenoxy)ethane-AW N', N'-tetra-acetic-acid tetra(acetoxymethyl) ester] BrdUrd Bromodeoxyuridine BSA Bovine Serum Albumin cAMP Cyclic A M P CKII Casien Kinase II CKI Cyclin-dependent Kinase Inhibitor dATP Deoxyadenosine Triphosphate dCTP Deoxycytidine Triphosphate DD Differential Display dNTP Deoxynucleotide Triphosphate DTT Dithiothreitol EDTA Ethylenediaminetetra acidic acid EGF Epidermal Growth Factor FBS Fetal Bovine Serum GFP Green Fluorescent Protein GST Glutathione S-Transferase InsP3 Inositiol 1,4,5-triphosphate m-AMSA 4'-(9-acridinylamino) methanesulfon-m-anisidide MDR Multi Drug Resistance M E M Minimum Essential Medium MGMT 0(6)-methylguanine D N A methyltransferase MRP Multi-drug Resistance Associated Protein MTT (3-[4,5-dimethythiazol-2-ly]-2,5-diphenyl tetrazolium bromide) NaTT Sodium Tetrathionate PBS Phosphate Buffer Saline PCR Polymerase Chain Reaction PKC Protein Kinase C PTN PBS containing 1 % B S A and 0.1 % Tween 20 R T Reverse Transcriptase TBS Tris-buffered Saline TBS-T TBS containing Tween 20 T E Tris EDTA T G F - a Transforming Growth Factor a TGF-P Transforming Growth Factor P Topo' Topoisomerase x i Acknowledgements First and foremost I would like to thank my supervisor Dr. Peggy L . Olive for giving me the opportunity to work on this project. Her extensive knowledge and relentless curiosity, open door office and sense of humor were key to the completion of this work. Her support and understanding of both my professional and personal life during the course of this research project have been invaluable to me. M y thanks extend to all my colleagues: Susan McPhail, Dr. Judit Banath, Laura Tower, Charline Vikse and Eric Chu for their patience in sharing their expertise with me and supporting me throughout the course of this work particularly during the last few months of my project. Dr. Chenmei Luo and Dr. Peter Johnston also thought me the basics of some molecular biology techniques upon my arrival. M y gratitude goes to the other senior staff scientists of the Medical Biophysics Department at BC Cancer Research Centre particularly Dr. Ralph Durand and Dr. Al ly Karsan for sharing their knowledge and expertise. Also, many thanks to the other members of the department particularly Denise MacDougal and Nancy LePard for running the FACS machine, Chris Hull for his help with the transfection assay and our amazing office administrator Wi l Cottingham. Thanks must also go to Dr. Wan Lam and Dr. Kim Lonergan for their assistance with the differential display experiments. I also want to express my appreciation to the members of my supervisory committee, Dr. Calvin Roskelley, Dr. Shoukat Dedhar, Dr. Don Brunnette, and Dr. Susan Porter. In addition to all the great people at work I have been very fortunate to be surrounded by many wonderful and supportive people in my personal life. Above and beyond everyone I am deeply indebted to my parents. Everything that I have earned in xii life is the result of their love, support and all the sacrifices that they have made in life for the success of their children. They have taught me the importance of education while they have never stopped learning themselves. M y loving husband, Farnoosh, who has believed in my abilitiy, has patiently re-lived the ups and downs of being a Ph.D. student with me. Also, my amazing brother, Aria, has been my best friend who despite his own busy life has never stopped being involved and supportive of mine and my sister-in-law, Sareh, has been a true sister that I have never had. Finally I would like to thank all my other family members and my friends who have always believed in me, and my unborn baby who has been my biggest incentive for completing this work, and has cooperated with me in the past few months. xiii To my parents, to your enormous sacrifices and your hard work. CHAPTER 1 Introduction 1 1.1 Tumour Cell Resistance to Therapy One of the major limitations in the success of cancer therapy is the emergence of tumour cell resistance to treatment. Many tumours often respond to initial treatment, but later start to re-grow. This could be a result of inadequate exposure to the agent or drug, selection of pre-existing resistant cells, or development of acquired resistance. A wide range of metabolic and structural properties of cells may lead to drug resistance. Many different mechanisms have been identified including overexpression of membrane pumps (P-glycoprotein, MRP) and free radical scavengers (GSH, glutathione-S-transferase), inactivation of topoisomerase II, enhancement of D N A repair, alteration in target enzymes, and increase in drug activation or degradation, all of which could explain why some tumour cells become more resistant to either radiation or chemotherapy (summarized in Table 1). Acquired multi-drug resistance (MDR) can occur in several different ways. For instance, reduced intracellular drug accumulation has been associated with overexpression of the multi-drug resistant gene (mdrl) which encodes the ATP-dependent efflux pump P-glycoprotein, a 170 kDa protein. A direct correlation has been found between the content of P-glycoprotein in the cell membrane and the degree of resistance to the selecting drug [22]. This overexpression increases cytotoxic drug efflux, preventing drugs from reaching their cellular target. In some multi-drug resistant cells, there is an increased transcription of mdr mRNA, without amplification of mdr gene [169, 185]. A variety of agents have been identified to inhibit the function of P-glycoprotein and increase the sensitivity of drug resistant cells, some of which act as substrates for P-glycoprotein and competitively inhibit the efflux of anticancer drugs. 2 Table 1: Probable mechanisms associated with tumour resistance to therapy [185]. Mechanisms Treatment Enhancement of D N A repair Alkylating agents, cisplatin, radiation Decrease in cellular uptake or increase in drug efflux Cisplatin, doxorubicin, etoposide, melphalan, 6-mercaptopurine, methotrexate, nitrogen mustard, vinblastine, vincristine Increase in levels of target enzyme Methotrexate Alterations in target enzyme 5-Fluorouracil, 6 mercaptopurine, methotrexate, 6-thioguanine Decrease in drug activation Cytosine arabinoside, doxorubicin, 5-fluorouracil, 6-mercaptopurine, 6-thioguanine Increase in drug degradation Bleomycin, cytosine arabinoside, 6-mercaptopurine Alternative biochemical pathways Cytosine arabinoside Inactivation of active intermediates by binding to sulfhydryl compounds Alkylating agents, cisplatin, doxorubicin, radiation Decrease activity of topoisomerase I I ' " Amsacrine, doxorubicin, etoposide 3 Changes in phosphorylation of P-glycoprotein have also been shown with some of the other inhibitors [185]. In addition, in some cases where resistance to chemotherapeutic agents was not attributable to P-glycoprotein expression, a contribution from multi-drug resistance-associated proteins (MRP) which also belong to ATP-binding cassette transmembrane transporter superfamily was implicated [35]. The function of MRPs is very similar to P-glycoprotein in reducing cellular drug retention. Alteration in drug targets is another category of MDR. This particular mechanism of M D R is best characterized by alteration in the expression of topoisomerase II. Topoisomerase II is a nuclear enzyme that is involved in number of different biological processes (described in Chapter 3) and is the target for several classes of chemo therapeutic agents (doxorubicin, mitoxantrone, and etoposide). Decreased expression of topo II as well as a reduction in the activity of this enzyme possibly due to mutation has been correlated with resistance to these inhibitors [41, 154]. The presence of variant forms of the enzyme whose activity do not facilitate drug-induced D N A damage has also been documented [211]. Enhancement of D N A repair can be considered to be another mechanism of MDR. Since damaging D N A is the mechanism by which many antitumour agents exert their cytotoxic effects, an increase in D N A repair capacity (or at least tolerance of D N A damage) could lead to resistance to therapy. For instance, the D N A repair enzyme O 6 -methylguanine D N A methyltransferase (MGMT) catalyzes the removal of methyl adducts from O 6 methyl guanine resulting from treatment with nitrosoureas [153]. Up-regulation of expression of this gene leads to resistance. Defects in h M L H l expression, a protein involved in D N A mismatch repair, have been correlated with resistance to 4 cisplatin in ovarian cancer [20, 197]. D N A repair capacity can also influence cellular radiosensitivity. A commonly described example is patients with a rare autosomal recessive disorder, Ataxia Telangectasia, that demonstrate a deficiency in the ability to repair D N A damage by radiation. Cells from these patients are approximately 3 times more sensitive to killing by ionizing radiation [166]. Human cancer cells that carry mutations to a breast cancer associated gene, B R C A 1 , may also be more sensitive to radiation therapy [1]. Inhibition of apoptotic signaling pathways is also another type of acquired MDR. Accumulating evidence shows that chemotherapeutic agents often use physiological apoptotic pathways to mediate cell death. Therefore, endogenous apoptotic inhibitors can hinder the effect of these agents [68, 69]. For instance, expression of the Bcl -2 family of proteins that have been demonstrated to play major roles in inhibiting the apoptotic cascade has recently been shown to correlate with acquired M D R [168]. Resistance to chemotherapeutic drugs may also be due to increased expression of anti-apoptotic proteins such as Bcl-2 and B c l - X L , or reduced expression of pro-apoptotic proteins such as B A X and FAS [72, 155]. Deregulation of cell cycle progression, for instance due to p53 mutation, could also result in resistance to radiation and chemotherapy. In cases where cell death after D N A damage is the result of apoptosis, cells that can evade apoptosis due to mutations in p53 generally show resistance to cell killing by D N A damaging agents [19, 144]. Another mechanism of drug and radiation resistance has been attributed to expression of detoxifying agents. In the case of some anticancer drugs such as cisplatin and doxorubicin as well as ionizing radiation, cellular damage is caused by the 5 production of reactive short-lived intermediates. Therefore, cells can adapt and protect themselves from the lethal effects of treatment by synthesizing higher concentrations of sulfhydryl compounds, notably glutathione, which can react with such radicals and reduce their toxicity to vital molecules [185]. Gluthatione in the reduced form can inactivate peroxides and free radicals making them less toxic and more easily excreted. The catalyzing agents for these reactions are glutathione peroxidase and glutathione S-transferase (GST) [105]. Increases in expression of any of these enzymes can promote resistance to some drugs and radiations. However, the resistant mechanisms identified to date do not fully account for all the resistant phenotypes that have been observed. Proposed mechanisms of drug and radiation resistance in cancer are based primarily on studies with resistant variant cell lines isolated from tumour cell lines. Although these studies have helped to characterize a number of important mechanisms involved in tumour resistance to therapy, they have generally emphasized genetic changes occurring in cell monolayers exposed under prolonged exposure to cytotoxic agents, often at doses that are not clinically relevant. A major disadvantage of these models is that they fail to consider the effect of cell-cell interaction at the multicellular level and the critical importance of the tumour microenvironment to response of tumour cells to cytotoxins. 1 . 2 Topoisomerase II and Etoposide 1.2.1 Topoisomerases: Essential Roles in Transcription and Replication The observation that the enzymatic machinery involved in duplication of D N A should ensure faithful replication of genetic material led to the discovery of D N A 6 topoisomerase I from E.coli [201]. There are five distinct topoisomerases identified in mammalian cells with various non-overlapping functions. Topoisomerases I, III and V belong to the type I family of topoisomerases while topoisomerases II and IV belong to the type II family of topoisomerases. These are ubiquitous nuclear enzymes that control the topological interconversions of D N A by transiently breaking and resealing the phosphodiester backbone of one (type I) or both (type II) strands of D N A duplex via a transesterification reaction. The cleavage gives rise to a transient covalent enzyme-DNA intermediate with the tyrosine hydroxyl group of topoisomerase linked to 5' or 3' phosphate. While strand passage by type I topoisomerases is an ATP independent process, type II topoisomerases function at the expense of ATP hydrolysis [9, 64, 137]. Type I topoisomerases function primarily to allow transcription. There are several ways by which one can measure the activity of topoisomerases. A decatenation assay is performed by using trypanosome kinetoplast D N A (kDNA), a mitochondrial D N A of Crithidia fasciculate. This is a catenated network of D N A rings, the majority of which are 2.5 K B monomers. Type II topoisomerases, but not type I, have the ability to decatenate kDNA and generate the monomer D N A which can be separated from the large network of k D N A and detected on an agarose gel after electrophoresis [128]. Detecting the topoisomerase-DNA cleavable complex is another way of measuring the activity of topoisomerases. The cleavable complex which has been stabilized by topo poisons can be resolved from free protein by rapid denaturation with detergents followed by CsCl gradient centrifugation. Immunoblotting allows quantification of both free and D N A bound enzyme. 7 Topoisomerase II is the major non-histone protein present in the nuclear scaffold fractions, and D N A that binds preferentially to the nuclear matrix/chromosomal scaffold contains topoisomerase II cleavage consensus sequences [57]. There are two isoforms of topoisomerase II identified: topo Ila and topo 11(3. Although highly similar, topo Hoc and P are genetically distinct and display different expression patterns. As will be discussed in more detail in Chapter 3 regulation of topo Ila is cell cycle dependent, therefore, it is considered to be a specific marker for cellular proliferation, being required for chromosome condensation and segregation of intertwined daughter chromosomes. In contrast, levels of topo l ip fluctuate less throughout the cell cycle and this isoform is mostly localized in the nucleolus of interphase cells which suggests a role in transcription of ribosomal R N A [114]. Topo II regulation is controlled both at the transcriptional and post-translational levels. In higher eukaryotes the increase of topo I la protein is due to both higher transcriptional activation of the gene and an increased half-life of its mRNA and protein [114]. Topo II can also be modified by ADP-ribosylation which inhibits its catalytic activity in vitro and has been detected in vivo. Phosphorylation is another process by which topoisomerase II is regulated and the effect of phosphorylation on the activity of topo II appears to be species specific. In all organisms, topo II phosphorylation has so far been shown to be primarily on serine/threonine residues in the C-terminal domain [23]. There are several kinases that have been shown to be involved in the phosphorylation of this enzyme. Casein kinase II (CK II) has been identified as the primary candidate involved in phosphorylation of topo II in vivo, controlling its activity [23]. In addition, protein kinase C (PKC), Ca /calmodulm-dependent protein kinase, p34 c d c 2 kinase, and several M A P kinases have been shown to phosphorylate topo II in 8 various systems [3, 23]. The phosphorylation status of some of the sites changes throughout the cell cycle, increasing from Gi /S to G2/M [23]. 1.2.2 Inhibitors of Topoisomerase II Since topo II plays such a vital role in maintaining cell viability, it is not surprising that it is an important target for large number of antitumour agents (Fig. 1). Different mechanisms are involved by which these poisons act as inhibitors of topoisomerase II [64]. Some of these drugs are intercalating agents such as doxorubicin, mitoxantrone and amsacrine which can intercalate between base pairs perpendicular to the long axis of the double helix. Others like etoposide and teniposide are non-intercalators. These agents function by interfering with the D N A cleavage/ religation step of the catalytic cycle, by either enhancing the cleavage reaction or by inhibiting religation. This results in stabilization of the cleavable complex and consequently accumulation of potentially lethal D N A double-strand breaks (Fig. 2). Although the cleavage/ religation step is the main target of topo II inhibitors, other drugs which inhibit the catalytic cycle also exist. These agents deprive the cells of the essential functions of topoisomerase II. Drugs such as staurosporin block topo II catalytic activity without enhancing levels of enzyme-mediated D N A cleavage. Depending on the compound, different steps of the topoisomerase II catalytic cycle such as enzyme-catalyzed ATP hydrolysis and/or strand passage can be inhibited [158]. Also, topoisomerase II-DNA binding can be disrupted by blocking access of topoisomerase II to its substrate. Sometimes D N A binding is allowed while the catalytic role of the enzyme is blocked [64]. 9 It is often debated as to which one of the topoisomerase II isoforms is the more important target for anticancer therapy. It is believed that topo I la is the more significant of the two isoforms because it is most prevalent in rapidly proliferating tissues and is associated with newly replicated D N A [205]. Certainly etoposide killing is dramatically reduced or even eliminated in non-proliferating cells. 1.2.3 Etoposide VP-16 or etoposide is a semisynthetic glycoside derivative of podophyllotoxin which is an antimitotic agent derived from the mandrake plant [185]. VP-16 is currently being used for treatments of many types of hematopoetic and/or solid tumours such as lymphomas, and cancers of the ovary, prostate, testis and lung [34, 86, 94, 112, 150]. Etoposide and teniposide (VM-26) get their family name from their common multi-ringed structure known as epipodophyllotoxin. Despite the binding of podophyllotoxins to tubulin, VP-16 and VM-26 seem to have little effect on microtubule assembly. The major mechanism underlying the cytotoxic effects of VP-16 appears to be interference with the religation step of the catalytic cycle of topoisomerase II leading to stabilization of the cleavable complex (Fig. 2) (reviewed in [9]). Etoposide is administered intravenously in the clinic with an initial half-life for plasma clearance of about 3 hours. About 45% of the administered drug is excreted unchanged in urine while an additional 15% is excreted in the feces. The dose-limiting toxicity of this drug is mainly to bone marrow cells [185]. 10 1.2.4 Mechanism of Resistance to Etoposide Resistance associated with etoposide includes overexpression of the membrane efflux pump, P-glycoprotein [31], or overexpression of MRP protein [35]. In addition, reduction in topoisomerase II levels to the minimal amount required for cell division, or decreased activity of the enzyme mostly due to mutation leads to resistance to etoposide [41, 64, 154]. Changes in topoisomerase II phosphorylation may also affect resistance to the drugs since phosphorylation could affect the activity or location of the enzyme [24]. Improper nuclear translocation of topoisomerase II in human small cell lung cancer cells has been demonstrated to result in resistance to etoposide [202]. As previously discussed, etoposide acts on the actively proliferating cells since topo I la is lacking in non-proliferating cells. Therefore, tumours with high percentages of non-proliferating cells are also resistant to etoposide treatment. An increased expression of genes involved in D N A replication and repair such as P C N A and GADD45 are also possible mechanisms for resistance to etoposide [118]. 11 Fig. 1: Topoisomerase II-targeted drugs. These agents enhance levels of topoisomerase II-mediated D N A cleavage and display antineoplastic activity. 12 DNA Topo II dimer Etoposide Cleavab Mil 34 Binding of topo II homodimer to DNA allows reversible double strand break DNA strand passage and unwinding Binding of etoposide to topo II-DNA complex prevents re-ligation of DNA Fig. 2: Interaction between topoisomerase II, DNA and etoposide. Schematic representation of topoisomerase II with DNA, and the mechanism by which etoposide can interact with the enzyme-DNA complex (cleavable complex) to cause double strand breaks in DNA. 13 Spheroid cells grown for 2 weeks with daily medium replenishment External cells of spheroids are rapidly dividing, well nourished and accessible to drugs. Internal cells of spheroids are quiescent, poorly-nourished, and accessible to drugs. Fig. 3: Schematic representation of spheroid growth in suspension. 14 1.3 Multicell Spheroids When placed in suspension culture many cell lines will aggregate spontaneously and grow to form multicellular structures known as spheroids (Fig. 3). Multicell aggregates were used over fifty years ago to investigate various aspects of normal cell and tumour cell biology. Pioneering work was initially conducted by Holtfreter in the 1940s who used embryo cells cultured on agar to prevent cell adhesion to the bottom of the dish [93]. This was followed by studies by Moscona in 1950's who examined the ability of tumour cells to grow as aggregates and to differentiate [133]. However, utilization of spheroids in a systematic manner did not begin until the early 1970s when Robert Sutherland and colleagues in London, Ontario observed that Chinese hamster V79 cells would not grow in suspension as single cells but invariably clumped in small aggregates. These cell clusters, i f fed daily for several weeks, continued to grow to quite large diameters (3 mm or more) that eventually contained more than 100,000 cells [180, 181]. Rodger Inch, a tumour biologist in London, sectioned these spheroids and noted similarities between spheroids and tumour nodules [96]. This prompted the idea to develop spheroids as an in vitro tumour model. The spherical geometry of spheroids, the ability to eliminate host factors, and their ease of manipulation have all contributed to the popularity of this system for drug testing [25, 175]. Spheroids develop microenvironmental heterogeneity with an intermediate complexity between conventional two-dimensional monolayer culture in vitro and three dimensional tumours in vivo. The external cells of spheroids are well-nourished and have access to growth factors, nutrients and oxygen. Like tumour cells adjacent to blood vessels, they are exposed to the highest drug doses. However, the innermost cells of spheroids are distant 15 from the nutrient supply. These cells often lack oxygen, have low levels of glucose and a a lower extracellular pH which lead to exit from the cell cycle, development of necrosis and eventual cell death. Investigation of growth and cellular characteristic of spheroids is important not only in understanding of the development of cellular and microenvironmental heterogeneity in tumour mircoregions, but also in determining responses to different therapeutic modalities. 1.3.1 Structure and Biology of Spheroids One of the most useful characteristics of tumour spheroids is the range of cellular microenvironments and subsequent development of cellular heterogeneity within the three dimensional structures. As spheroids enlarge, cells located at the periphery continue to divide while cells deeper within the spheroids become quiescent. The quiescent cells which are located more centrally can often be recruited into proliferation i f removed from within the spheroids or i f drug treatment kills the overlying cells. As growth progresses, the proportion of non-proliferating (quiescent) cells increases and cell death and necrosis occur in the centers of spheroids where, in addition to lack of nutrients, toxic metabolic waste products accumulate. The distance from the periphery of spheroids where necrosis occurs, and consequently the thickness of the viable rims of the spheroids, depends on cell type, nutrient consumption rate, cell packing, gradients of different nutrients, oxygen, metabolites, pH, hormones and growth factors in the growth media [2, 87, 175, 176]. Most of the proliferating cells generally reside in the outer two to three cell layers of spheroids. The thickness of viable rims of most spheroids surrounding the necrotic region falls in the range of 100-220 um [175]. Also, similar to 16 the radial distance from small blood vessels in tumours, significant cell heterogeneity develops in spheroids approximately at 5-15 cell diameters. At different depths within spheroids, differences in cellular organization and packing density have been observed. In general, cells near the periphery are more loosely attached to neighboring cells while the intermediate zone in the center of the viable rim is more densely packed, the same as confluent monolayers [177]. However, cells towards the necrotic center have large extracellular spaces surrounding them similar to hypoxic cells which have reached plateau growth [123, 177]. According to Sutherland [175], spheroid growth, like tumour growth, is often described as "Gompertzian" due to the shape of the growth curve that can be described by three phase: 1) exponential phase during which all cells are cycling and ends usually when the spheroid reaches diameter of 50-200 um; 2) second phase, during which the number of quiescent cells increases within the spheroid while the diameter of spheroid increases linearly; and 3) final phase in which spheroid growth is considerably retarded as it reaches 1-4 mm in diameter, depending on cell and culture conditions. After this stage, spheroids can be maintained in culture for several weeks. Enlargement of spheroids can also be accompanied by changes in ploidy [148] a characteristic that is often seen in advanced tumours as well. Different subpopulations of cells either from peripheral or central regions of spheroids can be separated in order to study their biological properties as well as their response to therapy. This means that successive shells of spheroid cells are removed and analyzed independently. Several methods have been used to dissociate spheroids, the most popular ones being controlled exposure to proteolytic enzymes (mostly trypsin) [67] 17 and fluorescence activated cell sorting based on the perfusion of rapidly bound fluorescent (nontoxic) dyes [50, 51]. Using trypsin at lower temperatures (room temperature or lower) and short exposure times, cells that have become detached from spheroids are removed after addition of medium containing serum to stop the action of trypsin. This process can be repeated several times to obtain successive layers from the spheroid. Alternatively, one can use a rapidly bound fluoresecent probe such as Hoechst 33342, which binds to D N A and emits blue fluorescence (470 nm) when excited by ultraviolet light (350 nm). Spheroids incubated for 20 minutes with this stain will show a marked gradient of fluorescence with brightly fluorescent cells on the outside of the spheroid and dimly fluorescent cells in the innermost layers [50]. Fluorescence intensity can vary by a factor of 100 or more. Since this fluorescence is maintained for some time after spheroids are disaggregated with trypsin, it is possible to sort cells from disaggregated spheroids according to their degree of Hoechst 33342 staining (corrected for cell size) and to calculate their depth within the intact spheroids. Concentrations of this dye required for this procedure are generally much lower than those that will cause toxicity and D N A damage [50]. Spheroids have been used extensively to study the role of oxygen and glucose in tumour growth [66, 134, 181]. Oxygen supply to spheroids can be measured both experimentally by microelectrodes and theoretically by diffusion calculations [134]. Oxygen gradients across the viable rim and the necrotic center can be very steep. However, spheroids sometimes adapt to changes in O2 by lowering the rate of oxygen consumption. Although the initial growth of spheroids is independent of oxygen and glucose concentration, the size of spheroid at which growth saturation occurs depends on 18 these factors [175]. It has also been hypothesized that an unexpectedly large decrease in both O2 and glucose consumption during the growth of intact spheroids could be due to an increase in fraction of quiescent cells [66]. Studies of the growth kinetics of spheroids using tritiated thymidine, incorporation of the thymidine analogue BrdUrd, or measurement of proliferation markers (Ki-67) all indicate a decrease in growth fraction associated with the presence of non-cycling cells in the inner layers of spheroids [49, 140]. Therefore, although the doubling time is longer when the tumour cells are grown as spheroids compared to monolayers, it is important to remember that the spheroid doubling time and cell cycle time are not the same. The longer doubling time is due to the presence of non-cycling cells; cells that are in cycle generally cycle at a similar rate as exponentially growing monolayers. Like solid tumours, the majority of the cells of larger spheroids are not in cycle. Some of the signals that regulate the cell cycle have been shown to differ in monolayers versus multicell spheroids, for example, regulation of cyclin dependent kinase inhibitors (CKIs) is different between monolayers and spheroids [115]. Spheroids also represent a useful model for studying intercellular communication. Enlargement of spheroids has been shown to cause changes in extracellular matrix constituents. Presumably a more extensive extracellular matrix is observed in spheroids compared to monolayers [149]. The presence of a small proteoglycan as well as the appearance of fibronectin has been shown to be increased in spheroids relative to monolayers[75]. In addition, evidence for enhanced differentiation determined by morphological and biochemical changes has been demonstrated in spheroids of both malignant and normal cell lines. For instance, spheroids of colon adenocarcinoma cells 19 have been shown to develop pseudoglandular structures [136], and F9 embryonal carcinoma cells in suspension have been shown to differentiate into cells that synthesize and secrete alphafetoprotein [85]. It has been hypothesized that the production of extracellular matrix in close association with the cells during growth is an important environmental factor involved in stimulating differentiation [136]. Inhibition of proliferation and development of hypoxia may also contribute to differentiation and ultimately senescence. 1.3.2 Culture of Spheroids Initiation and growth of multicell spheroids of many different histological types of rodent and human tumours have been accomplished in vitro using several different techniques. It should be noted that previous trypsinization is not believed to affect spheroid formation [87]. The principle method of growing spheroids for investigation of tumour cell biology by Sutherland et al. involves growth of cells in stirred suspension culture in spinner flasks to prevent adhesion (Fig. 3) [181]. Growth of cells as spheroids in suspension culture needs to be optimized for different cell types. For instance, the choice of medium, the frequency of medium replenishment, serum concentration and speed of rotation all influence spheroid growth. Optimum growth conditions can be achieved by assessing consumption of oxygen and glucose as well as changes in pH and reducing cell tendencies to adhere to the glass walls of the flasks. However, three main factors dominate: 1) the initial concentration of cells in the flask; 2) the concentration of fetal calf serum in the growth medium; and 3) the speed of rotation of the spinner in the suspension culture flasks [177]. Generally monolayer cells in exponential growth phase 20 are used for initiation of spheroid culture at a concentration of 10 /ml in 250-ml spinner flask with magnetic spinner rotating at 190 rpm [181]. Enhancement of cell-cell interactions by growth in liquid medium on non-adhesive surfaces such as agar coated dishes, or non-tissue culture plastic dishes has also been used to initiate spheroid growth [178] with subsequent transfer of cell aggregates to suspension cultures. For some cell lines, initial spheroid aggregation is enhanced using this method, and it also shortens the total culture time and reduces the volume of medium required to obtain spheroids. Also, it has been shown with some cell lines such as E M T 6 / R 0 mammary tumour cells that spheroids obtained by using this method are more homogeneous in size because of the more controlled conditions for cell aggregation [177].. In some cases where investigation in unstirred (static) liquid-overlay culture is required, spheroid growth is initiated on the non-stick surface and maintained there without transferring to spinner flask for growth of spheroids to large sizes [87, 208]. Some other investigators have also achieved inhibition of cell adhesion and consequently three-dimensional growth of colonies or aggregates of tumour cells by growth in semisolid agar, agarose or methylcellulose medium [61, 208]. Trypsinized monolayer cultured cells or single cells in suspension culture are seeded in coated dishes. After 2-3 days (if human cell line) or 1-2 days (if rodent cell line) spherical aggregates are formed. In some cases cells remain as single or suspended cells while in other cases they make flat or disk shaped aggregates that are loosely attached to each other. The spherical aggregates can be separated from single cells through repeated medium changes or by sedimentation separation in a pipette [26]. 21 In general, growth of spheroids in static cultures suffers from two important disadvantages: fewer spheroids are produced by this method than by suspension culture and more important, geometry for diffusion is no longer spherical. Regions of hypoxia and nutrient depletion occur at the agar surface rather than in the centre of the spheroid. For these reasons, all experiments in this thesis employed cell lines capable of growing as spheroids in magnetically stirred suspension culture vessels. 1.3.3 Resistance to Therapy Cell culture as spheroids has confirmed the important role of tumour cell microenvironmental factors in cell sensitivity to cytotoxic agents and radiation. Spheroids, even those that contain only 25-50 cells, can develop resistance to radiation and a variety of anti-tumour agents compared to monolayers [175, 179]. Multiple mechanisms could potentially contribute to resistance in large spheroids. In addition to the heterogeneity in spheroid structure, development of hypoxia and lack of glucose could play a role in resistance to therapy [53, 89, 96]. The spheroid model is particularly suitable for examining the response of the chronically hypoxic and quiescent cells to radiation [124]. In fact, radiation-resistant hypoxic cells that develop in spheroids are analogous to the resistant hypoxic population that develops when the same tumour cell line is grown in mice [159]. Different types of resistance develop when cells are cultured as spheroids [53, 104]. Often resistance can be explained because cells in the centre of spheroids are hypoxic, non-cycling, and distant from the supply of drug. However, these conventional explanations fail to account for all forms of resistance. For example, i f spheroids are 22 disaggregated before drug exposure, drug accessibility and availability of nutrients should not limit successful therapy, yet in some situations, resistance is still present. Therefore, a unique form of resistance termed the "contact effect" was first described for multicell spheroids. 1.4 The "contact effect" 1.4.1 Ionizing Radiation One of the initial and unexpected observations with Chinese hamster V79 cells, grown in suspension for only 24 hours, was the fact that they showed higher clonogenic survival after ionizing radiation compared to cells grown as monolayers. This phenomenon called the "contact effect" was first described in 1972 by Durand and Sutherland who observed that spheroids containing only 25-50 cells were more resistant to killing by ionizing radiation than cells grown as monolayers [54]. The increase in cell survival was evident in the shoulder region of the radiation survival curve and the terminal slope was largely unchanged. Differences in this shoulder region are critical since this is the clinically-relevant dose range, and differences in this region will be magnified during a course of fractionated radiotherapy treatments in the clinic. V79 cells develop a contact effect after only 24 hours in suspension culture, corresponding with two cell doublings [145, 178]. Cells dissociated from spheroids immediately prior to irradiation still show the contact effect. However, radioresistance is gradually lost i f spheroid cells are returned to monolayer growth after dissociation; after about 1 cell doubling as monolayers, cells are no longer resistant to radiation [54, 55]. Similar kinetics have been observed for etoposide with loss of resistance after 1-2 cell 23 doublings [142]. A n effect of 3-dimensional contact has also been observed in several studies where murine tumour cells were exposed to ionizing radiation. Tumours irradiated in situ or cells recovered from these tumours shortly after excision survived irradiation better than those allowed to grow as monolayers for several hours before irradiation [90, 117, 145]. It has been estimated that 40% of human and rodent cells display this form of radiation resistance when grown in suspension culture [65]. It should be noted that it is the environment of cells prior to, and not during or after irradiation that is critical for the development of the contact effect. The contact effect is most often studied in very small spheroids before development of hypoxia and cell cycle redistribution that can complicate interpretation of results [55]. However, using only the external cell layer of larger spheroids, representing about 10%> of the total cells, also avoids these complications. Therefore, experiments in this thesis compare the response of cells growing as monolayers with the response of the outer cell layer of larger spheroids. 1.4.2 Other Agents which Show a Contact Effect Since the first description of a contact effect for ionizing radiation, a similar contact resistance has also been demonstrated for variety of cytotoxic agents including hyperthermia, ultrasound, and photodynamic therapy and some topoisomerase II inhibitors. One-day-old V79 Chinese hamster fibroblasts and HT29 human colon adenocarcinoma spheroids showed less cell killing with heat treatment compared to monolayers; the authors eliminated explanations such as heating technique, cell cycle distribution, and nutrient availability [46, 48]. Photodynamic therapy is also twice as effective in killing monolayers compared to small spheroids [203]. Ultrasound treatment 24 produces more toxicity to monolayers than to small spheroids; however, this effect is eliminated i f the cells are disaggregated before treatment [162]. Small V79 spheroids (about 50 cells) show a 30% reduction in D N A damage and cell killing from etoposide compared to monolayers while larger spheroids show a greater resistance. Among other anti-tumour agents, some topoisomerase II inhibitors such as doxorubicin and m-AMSA which also produce D N A double strand breaks, have been shown to be less toxic to spheroids compared to monolayers [142, 145, 204] which can not be explained by the presence of non-cycling cells [142]. Even when the outer cells of V79 spheroids (which have the same cell cycle kinetics as monolayers) are removed from large spheroids before etoposide treatment, 10 times more drug is required to exert the same cytotoxic effect in these cells as in monolayers [145]. 1.4.3 Proposed Mechanisms for the Contact Effect To date, a number of different mechanisms have been proposed to explain the contact effect. These include changes in intercellular communication and signaling, changes in nuclear shape and D N A packaging, and changes in gene expression and apoptosis [52, 145, 165, 175]. A l l of these may ultimately affect the ability of a cell to resist D N A damage or promote repair, and of course, all may contribute to some degree to contact resistance. Resistance of spheroids to radiation and some drugs was initially thought to be the result of increased intercellular communication [54]. Although junctional communication also exists in monolayers, spheroids contain greater cell-cell contact and cellular junctions in 3-dimensions; gap junctions, tight junctions and desmosomes have 25 been observed [175]. At that time, it was believed that gap junctions between spheroids would enhance the ability of cells to resist or recover from radiation damage perhaps by the exchange of molecules involved directly or indirectly in D N A repair [54]. Supportive results for the role of gap junctions in the contact effect is found in the work of Dertinger and colleagues who demonstrated that only those cells that were electrically coupled when grown as spheroids showed a contact effect [43, 44]. However, several studies do not support the role of gap junctional communication in the contact effect. Expression of the major gap junctional protein connexin43 does not appear to influence the formation of spheroids or the development of contact resistance to radiation [126]. Also, the fact that cells only gradually lose their resistance to radiation or etoposide after being dissociated from whole spheroids argues against the requirement for gap junctional communication in the contact effect [90, 145]. The difference in chromatin organization or "packaging" is another mechanism that could potentially explain contact resistance. The role of chromatin structure in contact resistance was first proposed by Olive and colleagues who showed that changes in chromatin organization due to growth as spheroids might enhance repair of radiation-induced damage leading to higher cell survival [146, 147]. These studies showed an inability of D N A from irradiated V79 spheroids to unwind.in an alkali/salt lysis solution. Moreover, the rates of development and loss of ability to unwind correlated with the rate of development and loss of the contact effect [138, 147]. In addition to the alkali unwinding assay, the nucleoid sedimentation method [38] in which exposure to detergent and high salt results in the formation of the "nucleoids" composed of nuclear matrix structures and attached DNA, has confirmed differences in D N A conformation between 26 monolayers and spheroids [77, 146]. Acridine orange and filter elution methods were later used to show the same difference between monolayers and spheroids [143, 145]. Changes in higher order chromatin packing had already been shown to influence D N A repair, thus forming the basis for this hypothesis. Damage to transcribed sequences was shown to be repaired more rapidly than non-transcribed sequences [14], and induction of damage by radiation was thought to be greater in transcriptionally active D N A in close association with the nuclear matrix [33]. Changes in D N A organization that occur as cells move through the cell cycle were also shown to be accompanied by changes to radiosensitivity [187]. In addition, higher order chromatin structure was previously proposed to be important in controlling cellular processes such as differentiation, and it could also play a role in the lethal effect of radiation as the result of introducing an incorrect D N A conformation during repair [37]. Demonstration of the involvement of the nuclear matrix and nuclear matrix proteins in transcription and replication [11, 56] strongly suggests that D N A repair processes are not independent of chromatin organization [121]. Interestingly, topoisomerase II is an important component of the nuclear and chromosome scaffolds and is associated with DNA-matrix attachment regions. Therefore a potential explanation for the greater resistance of spheroids to radiation and etoposide could be a change in nuclear matrix organization that either reduces D N A damage or promotes repair. So far, attempts to identify specific differences in higher order chromatin organization that might be involved in the contact effect have not been successful. Another structural difference that occurs is the change in cell shape from an elongated ellipse when cells are grown as monolayers to a spherical shape in spheroids. 27 Changes in cell shape can have dramatic effects on D N A replication, gene expression and differentiation [62, 79], and there is also a possibility that they can affect fidelity of D N A repair. For example, for a given dose of radiation, small V79 spheroids show fewer radiation-induced gene mutations than monolayers [146]. Expression of several growth factors, including EGF, TGFct and TGF[3 have been examined for their role in the contact effect. In A431 squamous carcinoma spheroid up-regulation of up to 3 fold in both protein and mRNA level of TGFoc has been observed [111]. Addition of EGF dramatically increased spheroid volume in A431 cells, and increased sensitivity to radiation and doxorubicin when added after treatment [110]. An increase in tyrosine phosphatases in the outer cells of spheroids and high cell density monolayers was observed by measuring EGF dependent tyrosine phosphorylation [127]. Also, one of the earlier suggestions for the mechanism of the contact effect involved increases in cyclic A M P (cAMP) levels when cells were grown as spheroids [44, 45]. It has been demonstrated that V79 cells containing high levels of cAMP were also more radioresistant [116]. In addition since one of the earliest events in spheroid growth is exit from the cell cycle, it may not be surprising to find changes in expression of genes involved in growth control, or changes in response to growth factors. With the growing appreciation that D N A repair and the cell cycle are closely linked, and that genes involved in replication or cell cycle progression are also required for effective D N A repair (e.g., p53, PCNA, RPA, topo Ila), potential explanations for the contact effect could lie in changes in cell cycle regulation. 28 Despite many proposed explanations for the contact effect, the exact mechanisms are not yet understood. A goal of this project was to further understand the mechanism behind the resistance of spheroids to one drug known to show a contact effect, etoposide. 1.5 Objectives This work was directed towards examining the mechanisms leading from the changes in the growth environment to changes in the ability of cells to withstand etoposide-induced D N A damage. The differential etoposide sensitivity of monolayers and spheroids in some cell lines but not others provided a unique model system. Studies with regards to cell-cell contact induced resistance to etoposide were originally initiated because toposiomerase II is a nuclear enzyme of fundamental importance for many biological functions in mammalian cells including D N A replication, transcription, recombination and possibly repair [200]. Also, the position of topoisomerase II at the nuclear matrix [57] suggested a critical role for this enzyme in chromatin packaging. Previous studies from our laboratory indicated that 10 times more etoposide was required to produce the same amount of D N A damage and cell ki l l in the outer cells of spheroids relative to monolayers [142], in spite of the fact that cell cycle kinetics were identical. Moreover, disaggregated spheroid cells retained their resistance to etoposide for up to 48 hours after return to monolayer culture conditions even though they were continuously proliferating at the normal rate during this time [142]. Several different mechanisms previously shown to be involved in etoposide resistance were examined in this regard, none of which could explain the reduced sensitivity of outer spheroid cells to etoposide. The growth fraction and cell cycle time 29 were found to be identical for monolayers and outer spheroid cells. The amount of topoisomerase II was examined by immunoblotting and found to be identical for monolayers and outer spheroid cells. The activity of this enzyme, measured by a decatenation reaction using trypanisome DNA, was the same for monolayers and outer spheroid cells. The ability of etoposide to inhibit the decatenation reaction using topo II extracts from monolayers and outer spheroid cells did not differ either. The levels of P-glycoprotein were also found to be the same for cells grown under the two different culture conditions. Two other mechanisms that were ruled out were differences in drug uptake measured using 3H-etoposide, and rate of repair of etoposide-induced D N A breaks which were also quite similar between monolayers and the outer cells of V79 spheroids. Therefore, it was concluded from these earlier studies that the nature of the stable cleavable complex between topoisomerase II, D N A and etoposide must differ for monolayers and spheroids. Although this explanation cannot be ruled out, results from this thesis indicate that there are, in fact, another explanations. Therefore the working hypothesis is that growth in three dimensional contact as spheroids results in changes in gene expression which could directly or indirectly (through post-translational modification of other proteins) make the cells resistant to treatment with etoposide. Three main objectives were pursued in this study in order to identify possible mechanisms regulating the increased resistance to etoposide in the outer cells of spheroids. 1) To study the resistance of outer spheroid cells to etoposide with emphasis on post-translational modifications of the target enzyme, topoisomerase II. 30 2) To compare gene expression patterns between monolayers and outer cells of spheroids. 3) To examine the possible link between one of the differentially expressed genes and etoposide sensitivity in spheroids. There is currently no easy way to determine whether a particular tumour cell or tumour type is likely to display a contact effect, so an understanding of the molecular basis of this phenomenon is essential for identification of this form of resistance prior to treatment. Knowledge of the mechanism of the contact effect could also provide new directions for improving tumour response to treatment. 31 CHAPTER 2 Materials and Methods 32 2.1 Cell Lines and Culture Conditions Four different cell lines were chosen to study the effect of cell-cell contact induced resistance to etoposide (Bristol Laboratories, Montreal, Canada). These cell lines were chosen because all four cell lines have the ability to grow in monolayer culture, in suspension culture as spheroids, and as xenograft tumours. The Chinese hamster V79 lung fibroblast is a well-established mammalian cell line. This cell line has been extensively used in studies on X-ray induced damage and repair processes [141, 178]. V79 cells are immortal, have a high plating efficiency and a rapid doubling time (12-14 hr). Due to mutations in the coding region of p53, these cells do not have a functional p53 protein [30]. The subline maintained in our laboratory, V79-171b, was originally obtained from Dr. Robert Sutherland who had previously obtained it from Dr. Warren H . Sinclair at Argonne National Laboratories. Rat C6 glioma cells, SiHa human squamous cervical carcinoma cells, and WiDr human colorectal adenocarcinoma cell lines were obtained from American Type Culture Collection (ATCC). C6 is a rat glioma cell line that was cloned from a rat glial tumour induced by N-nitrosomethylurea after a series of alternate culture and animal passages. A major product of this line is SI00 proteins, a family of calcium binding proteins (discussed in more detail in chapter 5), the production of which increases as cells grow from low density to confluency. The SiHa cell line came from a cervical squamous cell carcinoma that was established from fragments of primary tissue sample from a Japanese patient. This is an adherent cell line that contains an integrated human papillomavirus type 16 genome (HPV-16) (about 1-2 copies per cell) which results in inhibition of the regulatory functions of pRB and p53 due to the presence of E6 and E7 oncoproteins. 33 This line has the ability to form a poorly differentiated epidermoid carcinoma (grade III) in nude mice. D N A fingerprinting of the WiDr cell line has shown it to be a derivative of the HT-29 line, although it was originally deposited as a colon carcinoma from a 78 year old female. These cells produce p53 antigen with a G to A mutation (resulting in Arg to His at position 273). WiDr cells are tumourigenic in nude mice and express epidermal growth factor (EGF). A l l four cell lines were routinely maintained in exponential monolayer growth by subcultivation twice weekly in Eagle's. M E M (minimum essential medium) (Sigma-Aldrich, Oakville, ON, Canada) containing 10% (v/v) FBS (fetal bovine serum) (Gibco B R L , Grand Island, N Y , USA). The cells were grown as monolayers attached to 100 mm plastic tissue culture plates (Falcon 3003), and were kept at 37°C in a humidified atmosphere of 5% CO2 and 95% air. Cells were typically removed from culture plates by first removing the culture medium and rinsing twice with 0.1% trypsin in a citrated-phosphate saline buffer (Gibco). After the last rinse the plates were incubated for 6 min at 37°C with a film of trypsin remaining on them. The cells were then resuspended in complete medium by vigorous pipetting and were counted using a Coulter counter. 2.2 Spheroid Growth Multicell spheroids represent a three dimensional in vitro model of solid tumours. They are formed when cells in suspension initially aggregate and continue to grow. Spheroids were initiated in suspension culture by seeding 5 x 10^ cells/ml into Bellco glass spinner culture vessels (Vineland, NJ, USA) containing M E M plus 10% FBS. The spinner flasks were initially gassed with 5% CO2 after adding the medium and allowed to 34 equilibrate to 37°C before adding cells. After a few hours the cells aggregate spontaneously, and then divide to form larger spheroids. Spheroids were fed after 3 days and daily thereafter with complete medium supplemented with antibiotics. Spheroids were normally used when they reached a diameter of about 0.6 mm which would typically take about 15 days for the V79 cell line and about 21 days for C6, SiHa and WiDr cell lines. 2.3 Sequential Trypsinization Sequential trypsinization has been used for over 20 years as a method able to provide relatively pure populations of cells from various depths within multicell spheroids [67, 74]. The outer cell layer of spheroids, approximately 0.6 mm in diameter, was removed by agitating spheroids for 5 min in cold 0.1% trypsin in PBS. Outer spheroid cells were released by this process when spheroids were gently transferred to cold medium and allowed to settle to the bottom of a tube. The remaining cores were trypsinized to determine cell recovery and calculate the percentage of cells in the outer layer (generally 7-10% of the spheroid). 2.4 Xenograft Cell Sorting Xenograft tumours were initiated subcutaneously in the dorsum of 8-12 week old female NOD/SCID immune deficient mice by injection of 5 x 105 single cells in 0.05 ml serum free medium. These mice were bred in-house at the B C Cancer Research Centre. They were kept in microisolators, and were checked on a daily basis with their bedding, food and water changed once per week. The protocols for animal maintenance were 35 approved by the U B C Animal Care Committee. To obtain tumour cells closest to the blood vessels, mice bearing approximately 0.5 g tumours were injected intravenously with 4 mg/kg Hoechst 33342 (Sigma-Aldrich). Hoechst 33342 is a DNA-binding fluorescent stain used extensively in flow cytometry studies to quantify D N A content in live cells [7], but it can also be used at lower drug concentration to select cells from different locations within spheroids or experimental solid tumours [29, 51]. Ten minutes later, tumours were excised, and a single cell suspension was prepared by chopping the tumour finely and incubating the suspension with 5 ml of sterile PBS containing 0.3 ml of each of the following: trypsin 25 mg/ml, collagenase 4 mg/ ml and DNase 10 mg/ ml. The mixture was transferred to a 14 ml tube and rotated at 37°C for 30 min [139]. Cells were filtered through 30 micron pore nylon filters and were centrifuged and resuspended in complete medium for incubation with 0 - 1 5 pg/ml etoposide. Etoposide (Bristol Laboratories) was purchased as a 20 mg/ml solution and was diluted in M E M containing 10% (v/v) FBS just before use. As the carrier used for solublizing etoposide in this formulation may have some toxic properties itself, etoposide concentrations were kept below 50 pg/ml. Following a 30 min incubation with etoposide, tumour cells were sorted on the basis of the Hoechst 33342 diffusion gradient using a Becton Dickinson FACS 440 cell sorter with U V excitation [28]. The 10% of cells that contained the highest concentration of Hoechst 33342 (i.e., those closest to the functional tumour blood vessels) were sorted and analysed for protein expression or survival using a colony formation assay. 36 2.5 Colony Formation Assay and Relative Resistance In order to compare etoposide-induced cell kil l in monolayers, outer spheroid cells, or xenograft tumours, a colony formation assay was used. Single cells from each treated population were placed in 100 mm tissue culture dishes containing 10 ml M E M + 10% FBS. After 10 days to 2 weeks, colonies (> 50 cells) were stained with malachite green (Sigma Chemical Co.) and counted. Experiments were repeated at least 3 times to obtain the mean and standard error. For V79 and WiDr cells, survival as a function of etoposide concentration followed a simple exponential curve, so relative resistance was constant at all survival levels. Therefore, etoposide resistance for these cell lines could be expressed as the ratio of the slopes obtained for the monolayer and spheroid cell survival curves. For the SiHa and C6 cell lines, survival was defined by a linear quadratic relationship, and the lethal dose to 90% of the cells (LD90) was calculated from these curves. The in vitro plating assay is one of the most common ways to assess cell survival [156]. Using this assay, treated cells are counted and appropriate dilutions are plated in tissue culture dishes followed by incubation at 37°C. The cells attach to the surface of the tissue culture dish, and those that retain proliferative capacity divide and grow to form discrete colonies of cells (usually defined as >50 cells). After a number of days of incubation, which depends on the growth rate of the cells, the plates are removed from the incubator and the colonies are stained with malachite green (Sigma Chemical Co.) so that they can be easily counted. The plating efficiency of the cells is calculated by dividing the number of colonies formed by the number of cells initially plated. The colony formation assay is a a more reliable method of assessing cell killing after radiation 37 or drug treatment compared to MTT or trypan blue assays. With the latter methods, viability measurement is either achieved by total population staining using M T T (3-[4,5-dimethylthiazol-2-ly]-2,5-diphenyl tetrazolium bromide), or by assessing the extent of cell death from the proportion of cells incorporating a dye excluded by live cells such as trypan blue or propidium iodide at short time (1-4 days) after treatment. This can lead to an underestimation of the overall cell kil l since kinetic differences in the manifestation of cell death is ignored [19]. Cell death does not occur immediately after treatment, and can take hours to days thus reducing the value of assays based on cell metabolism or membrane integrity. 38 Fig. 4: Xenograft cell sorting, (a) Tumours implanted in NOD/SCID mice were labeled with Hoechst 33342 (i.v.) 20 min before sacrifice, (b) A section from the tumour viewed under the microscope reveals that the cells closest to blood vessels show the brightest blue staining for Hoechst. (c) Cells are enzymatically monodispersed and treated with etoposide for 30 min. (d) Hoechst staining allows for sorting on the FACS machine for the cells closest to blood vessels, (e) The sorted cells are then plated in monolayer culture dishes in a colony formation assay. 39 2.6 Immunohistochemistry Cells suspended in ice-cold PBS were deposited by cytospinning (CYTOSPIN II, Shandon, Pittsburg, PA, USA) at 800 rpm for 8 min onto cleaned glass slides at a concentration of 2 x 104 cells in 100 pi. Slides were air-dried for 10 min then immersed in cold 1% paraformaldehyde (Sigma-Aldrich) in PBS for 30 min at 4°C. After rinsing in PBS, slides were immersed in acetone for 30 sec at room temperature, followed by rinsing two times in PBS and immersing in PBS with 1% bovine serum albumin plus 0.1% Tween 20 (PTN) for 20 min at room temperature. Anti-topoisomerase I la antibody (Sigma-Genosys or TopoGEN, Columbus, OH, USA) and p27 monoclonal (BD Biosciences, Mississauga, ON, Canada) were diluted 1:100 in PTN, and 25 pi was deposited on 1cm parafilm strips. The strips were inverted over slides that had been drained and placed in humidified chambers. Chambers were placed at 4°C overnight. Slides were then removed from the chambers and rinsed two times for 5 min in PBS and one time 5 min in PTN. The secondary antibody, Alexa488 goat anti-mouse IgG (H + L) F(ab')2 fragment conjugate (Molecular Probes, Eugene, OR, USA) was diluted 1:150 in PTN and 25 pi was deposited on new Parafilm strips for incubation at room temperature for 2 hr. Rinsed slides were immersed in 0.05 pg/ml DAPI (Sigma) in PBS for 5 min, then rinsed for 5 min in PBS, drained, mounted in Fluorogard mounting medium (BioRAD, Mississauga, ON, Canada), sealed and viewed using a Zeiss Fluorescent Microscope with a Sensicam Camera attached. The images were digitized using Northern Eclipse 5.0 software (Empix, Toronto, ON, Canada). For some images, NIH/Scion image software was used to analyze average nuclear, cytoplasmic and background fluorescence intensities from several cells. 40 2.7 Topo Ila Phosphorylation Cells from V79 monolayers and the outer layer of spheroids (5 x 106) were grown in 5 ml of phosphate free medium containing 10% dialyzed FBS and were labeled with [3 2P] orthophosphoric acid (250 uCi/ml) (Amersham, Oakville, ON, Canada) for 4 hr at 37°C. Cells were lysed after washing in buffer containing phosphatase inhibitors, 2 m M Na3"V04 (Sigma) and 100 mM NaF (Sigma). Cell lysate was pre-cleared with protein A-Sepharose beads (Zymed Laboratories, Inc., San Francisco, CA) and then incubated with 1:80 dilution of Topo Ila monoclonal antibody (TopoGEN Inc.) while rotating overnight at 4°C. Protein A-sepharose beads were washed three times after being incubated with the lysate for 2 hr. Following boiling for 5 min, samples were centrifuged and loaded on a 7.5% SDS-PAGE for electrophoresis. Proteins that did not attach to beads were also analyzed as a loading control. After completion of the run, the gel was fixed, dried and images were taken using a phosphoimager. This experiment was repeated twice. 2.8 Sub-cellular Fractionation Before preparation of nuclei, cells were incubated for 30 min with 10 u.g/ml etoposide at 37°C to stabilize DNA/topo II complexes. To prepare nuclei, approximately 1.5 X 107 outer cells of spheroids or monolayers were rinsed in PBS and resuspended in 100 ul cold buffer A (10 m M HEPES pH 7.9, 1.5 m M M g C l 2 , 10 m M KC1, 300 mM sucrose, 1 m M E D T A pH 8.0, 1 m M DTT, 0.1% NP-40, 1 m M phenylmethyl sulfonyl fluoride, 10 u.g/ml protease inhibitor cocktail and 0.1 m M Na 3V04) (all reagents were obtained from Sigma Chemical Co., Oakville, ON, Canada, unless otherwise indicated). After 10 min of incubation on ice cells were sheared by passing a few times through a 41 Gilson microtip, and were centrifuged for 10 min at 3000 rpm at 4°C. The cytoplasmic supernatant was reserved for analysis by Western blot, and the nuclear pellet was washed two times in buffer A and examined by microscopy for the presence of cytoplasmic tags by staining with nuclear and cytoplasmic fluorescent probes. After the final wash the nuclear pellet was resuspended in 75 pi of buffer B (250 m M Tris-HCl pH 7.9, 5 m M MgS0 4 , 250 m M sucrose, 2 m M NaTT, 1% thiodiglycol, 1% NP-40, 1 m M PMSF, 10 pg/ml protease inhibitor cocktail (Sigma Chemical Co.), 0.1 m M Na3V04). Equal amounts of lysate (same cell number) from the two fractions were used for analysis on a Western blot. 2.9 Immunoblotting Whole cell lysates were prepared by lysing 5.0 x 106 cells in RTPA lysis buffer (50 m M Tris-HCl pH 7.4, 150 m M NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 m M EGTA, and the following inhibitors were added just before use: 1 m M PMSF, 1 pg/ml aprotinin, 1 pg/ml leupeptin, 1 pg/ml pepstatin, 1 m M Na3V04, ImM NaF) (all reagents were obtained from Sigma Chemical Co., Oakville, ON, Canada, unless otherwise indicated). Equal amounts of protein (determined using a Sigma Protein Assay) were denatured in sample loading buffer, and were loaded into the wells of an 8-15% SDS-polyacrylamide gel depending on the size of the protein of interest. After electrophoresis, gels were blotted onto nitrocellulose membrane (BioRad) and blocked with 5% B S A or non-fat dried milk in TBS containing 0.1% Tween-20 (TBS-T) while agitating for 1 hr at room temperature. Blots were further incubated for 1-2 hr with different antibodies diluted in TBS-T: topoisomerase Ilex; monoclonal 1:250 (Sigma-42 Genosys), S100A4 polyclonal 1:2000 (Dako, CA, USA), calretinin polyclonal 1:1500 (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), flag (M5) monoclonal 1:500, p27 monoclonal (BD Biosciences), and control loading markers such as Lamin A/C polyclonal 1:1000 (Santa Cruz Biotechnology Inc.), and a-tubulin monoclonal 1:2000 (Oncogene, Cambridge, M A , USA). After several washes with TBS-T secondary goat anti-mouse antibody or goat anti-rabbit antibody (used against each specific primary antibody) conjugated to horseradish peroxidase (1:5000-1:10,000 dilution) (Sigma) was incubated with blots for 1 hr, and bands were detected using the E C L detection system (Amersham, Oakville, ON, Canada) (summarized in Table 2). 2.10 R N A isolation Total R N A was isolated from V79 monolayers and the outer cells of spheroids using TRIzol™ Reagent (Gibco BRL, Grand Island, N Y , USA). TRIzol™ is a monophasic solution of phenol and guanidine isothiocyanate. During cell lysis it maintains the integrity of the total R N A while disrupting cells and solubilizing cell components. About 1 ml of TRIzol™ Reagent was used to lyse 106 cells by repetitive pipetting followed by incubation at room temperature for 5 min. Vigorous mixing with 0.2 ml of chloroform per 1 ml of TRIzol™ Reagent followed by centrifugation at 12,000g for 15 min at 4°C was done to separate the solution into an upper colorless aqueous phase and a lower red organic phase. Total R N A was precipitated from the aqueous phase by mixing with 0.5 ml of isopropanol per 1 ml of TRIzol™ Reagent and incubating at -20°C for 2 hr. After centrifugation at 12,000g for 15 min at 4°C, the R N A pellet was washed 43 Table 2: Antibodies. Different antibodies used with their sources and concentrations. Antibody Source Immunoblotting concentration Immuno-histochemistry concentration Topoisomerase I la mouse monoclonal TopoGEN (cat#: 2010-1) N / A 1:100 Topoisomerase Ila mouse monoclonal Sigma-Genosys (cat#: OM-11-930) 1:250 1:100 S100A4(mtsl) rabbit polyclonal Dako (cat #: A 5114) 1:2000 N / A Lamin A / C rabbit polyclonal Santa Cruz (cat #: sc-6216) 1:1000 N / A F L A G (M5) mouse monoclonal Sigma-Aldrich (F4042) 1:500 N / A a-tubulin mouse monoclonal Oncogene (cat #: CP06) 1:2000 N / A Calretinin rabbit polyclonal Santa Cruz (cat #: sc-6467) 1:1500 N / A p27 mouse monoclonal BD Biosciences (cat #: K 25020) 1:2000 1:100 44 with 75% ethanol, briefly air-dried and dissolved in 50 ul RNase-free water. R N A concentration and purity was determined by using a U V spectrometer to measure the absorbance at 260 nm (1 A 26o U = 40 pg/ml). Also, because any trace amount of chromosomal contamination in the R N A sample could have been amplified in the subsequent steps, digestion with DNasel was performed. Ten to 100 pg R N A was mixed with 30 pi of DNasel reaction buffer, and 3 pi of DNasel (RNase free) (Gibco BRL) and made up to 300 pi with dTbO, and was incubated at room temperature for 15 min. The reaction was stopped by addition of 30 pi of 50 m M E D T A (pH = 8.0) (Sigma) at 70°C for 10 min. Subsequently, total R N A was recovered by the phenol extraction method [164]. 2.11 Differential Display The method of differential display (DD) originally developed by Liang and Pardee [119] was used to identify differentially expressed genes between V79 monolayers and the outer cells of spheroids. Modifications from the original technique were the use of primers with ten-based nucleotides added at the 5' end, and a low stringency PCR cycle at the beginning [8]. Reverse transcription of mRNA, which was isolated from total R N A using Oligotex mRNA kit (Qiagen, Germany), was achieved by using three different anchored primers in three separate reactions. For 20 pi final volume: 9 pi mRNA (approximately 0.5 pg), 4pl of 5X RT buffer, 2 pi of 100 n M DTT, 2 pi of 200 p M dNTP, and 2 pi of 10 uM H - T U M anchored primer (H = Hindlll , M can be either G, A , or C) (GibcoBRL) were used. The thermocycler program performed as follows: 65°C for 5 min, 37°C for 60 min, 75°C for 5 min, 4°C. Reverse transcriptase 45 enzyme M M L V was added to each tube after 10 min at 37°C. Subsequently, RT-mixes from this step were used in a differential polymerase chain reaction (PCR) along with six different arbitrary primers (GibcoBRL). DD-PCR mixes for each primer combination were set-up to the final volume of 20 ul: 7.3 pi of dH 2 0 , 2 pi of 10X PCR buffer, 2 pi of 25 m M M g C l 2 , 2 pi of 20 p M dNTPs, 2 pi of 4 p M arbitrary 13-mer, 2 p M of the corresponding anchored primer, 0.5 pi of [ct-32P]-dATP (6000 Ci/mmole), 0.2 pi of AmpliTaq (Qiagen, Germany). The primers used in these experiments were made available to us by Dr. Wan Lam in our center, B C C R C . The design of the arbitrary primers were based on the published data by Ayala et al. [8] with slight modification by the presence of EcoRI restriction sites at the 5' ends. The use of longer primers reduces the number of false positive signals associated with the original method, however it also results in a limited repertoire of the tested cDNAs. Therefore, unlike microarrays where thousands of genes can be compared, only a small subset of genes that are differentially expressed will be identified using this approach. PCR cycles using an Eppendorf Mastercycler were performed as follows: 94°C for 2 min, 40°C for 4 min, 72°C for 1 min, 34 cycles of (94°C for 45 s, 60°C for 2 min, 72°C for lmin), 72°C for 5 min, 4°C. Upon completion formamide loading dye was added to each PCR reaction (1:1) and incubated at 80°C for 3 min immediately before adding onto a 6% D N A denaturing gel. Electrophoresis was conducted for 4 hr at 60W constant power. Before exposing to an X -ray film overnight, the gel was dried and oriented with the autoradiogram. Differentially expressed cDNA fragments were excised from the gel and soaked in 200 pi TE buffer for 30 min followed by boiling at 95°C for 10 min. The eluate was transferred to a new tube and D N A was retrieved by ethanol precipitation using 5 pi of glycogen (10 mg/ml) 46 (GibcoBRL) per sample as an oligonucleotide carrier. The cDNA fragments were then re-amplified with the same set of primers used in the initial differential display PCR reaction (Fig. 5). 2.12 Reverse Northern "Reverse Northern" was the preferred method to verify the cDNA fragments tentatively identified by differential display [209]. Because the potential of false positives is a major problem with differential display, reverse northern blotting allows a more efficient way of screening. With this method, many cDNA templates from a differential display gel are dot blotted onto duplicate membranes and probed separately with labeled cDNAs made from the two total R N A samples being compared. After re-amplification, 5 pi of the PCR products were directly ligated with 2 pi pCR-TRAP vector (GeneHunter Corporation, Nashville, TN, USA) (Fig. 6). After transformation by heat shock into G H competent cells and plating on L B plates containing 20 pg/ml tetracycline and overnight incubation at 37°C, at least 4 tetracycline resistant colonies were picked. These colonies were lysed by boiling in 50 pi lysis buffer consisting of 0.1% Tween-20 in TE buffer, pH 8.0, and the cloned cDNA fragments were PCR amplified using primers flanking the cloning site of the vector (Fig. 6). To prepare for blotting, 30 pi of each PCR amplified cDNA insert was mixed with 10 pi 2 N sodium hydroxide (Sigma), denatured by boiling for 5 min and then neutralized with 10 pi 3 M sodium acetate (Sigma), pH 5. After bringing the total volume to 110 pi with dH20, 50 pi of each sample was dot blotted onto duplicate Zeta probe nylon membranes (BioRad) using the Bio-Dot microfiltration system (BioRad). The membranes were washed with 47 I. Reverse Transcription II. PCR Amplification • GAAAAAAAAAAA-An •TAAAAAAAAAAA-An • CAAAAAAAAAAA-An Oligo-dT primer: H - T n G , dNTPs, M M L V reverse transcriptase . GAAAAAAAAAA A-An C T T T T T T T T T T T T T C G A A H-AP1 Arbitrary Primer % H-TnG Anchored Primer * dNTPs ct-[32P-dATP] Amplitaq DNA polymerase III. Denaturing polyacrylamide gel Fig. 5: mRNA differential display experiment. { six different arbitrary primers (H-AP1: C C G T G A A T T C G A C C A G T G , H-AP2: C C G T G A A T T C G C T G G G A T , H-AP3: C C G T G A A T T C G C C G T T G A , H-AP4: C C G T G A A T T C G T G A C G A G , H-AP5: C C G T G A A T T C G G T C A G G T , H-AP6 C C G T G A A T T C G T A G C C T C ) were used in these experiments. * Anchored primers: H - T n M (H = Hindlll , M can be either G, A , or C). Overexpressed or unique cDNAs are retrieved from the polyacrylamide gel and analyzed in the subsequent experiment (cloning and reverse Northern blot). 48 Fig. 6: Structure of p C R - T R A P vector. After re-amplification of the identified differentially expressed cDNAs, PCR products can be cloned into pCR-TRAP vector by blunt ligation at the cloning site (GenHunter Corp., USA). This cloning system utilizes a third generation cloning vector that features positive selection for D N A inserts. Only recombinant vectors that have the PCR product directly inserted into the cl gene lead to the inactivation of the repressor gene and thus the expression of the TetR gene. Vector primers Lgh and Rgh flanking the cloning site are used for checking for plasmids containing a D N A insert. 49 6X SSC and baked at 80°C for 1 hr. The cDNA probes were prepared from 50 pg of each of the two R N A samples by reverse transcription using reverse Northern kit (GenHunter) plus 100 pCi [o>32P]dCTP (3000 Ci/mmol; Amersham). The unincorpor-ated P was removed using spin columns (BioRad). Equal counts (5 -10 x 10 cpm) of the cDNA probes from V79 monolayers and the outer cells of spheroids were heat denatured and used to probe the duplicate blots. 2.13 Sequencing and BLAST Search Sequencing of the cDNA samples for which the differential gene expression was confirmed was performed by the Biotechnology Laboratory at the University of British Columbia (Vancouver, Canada). The obtained sequences were aligned with the database from Genbank using the B L A S T algorithm for nucleic acid sequence analysis [5]. 2.14 Calcium Measurement In order to measure the amount of free intracellular calcium, 106 cells from either monolayers or the outer cells of spheroids were trypsinized and placed in suspension at 37°C for one hour to recover from the effect of trypsinization. Fluo-3-AM (Molecular Probes, Eugene, OR) which was originally developed for use with visible light excitation sources in flow cytometry and confocal microscopy [98, 131] was the fluorescent indicator used for the measurement of Ca 2 + . Cells in suspension were incubated with 1 p M of this dye for 1 hr. After two rinses with indicator-free medium to remove any dye that was non-specifically associated with the cell surface, cells were resuspended in M E M plus 10% FBS for an additional 30 min to permit complete hydrolysis of the 50 acetoxymethyl ester. One of the most important properties of fluo-3 is a very large increase in fluorescence intensity in response to C a 2 + binding. Therefore, using an argon-ion laser source, the mean fluorescence corresponding to the amount of free intracellular C a 2 + was measured by using excitation at 488 nm. In the transfected cell lines expressing green fluorescent protein (GFP) fluorescence could not be measured by fluo-3. Therefore, another C a 2 + indicator, indo-1-A M (Molecular Probes), was used. This is a UV-excitable Ca binding agent that shows a change in emission wavelength from 475 nm to 400 nm upon C a 2 + binding [98]. The ratio of the intensities at the two wavelengths was used, to determine the relative amount of intracellular free C a 2 + . 2.15 B A P T A - A M Treatment The cell permeant buffer B A P T A - A M [l,2-bis-(o-aminophenoxy)ethane-A^A^/V^'.A^'-tetra-acetic-acid tetra(acetoxymethyl) ester] (Molecular Probes) has a high affinity and selectivity for calcium (>10 for Ca over M g ) [99, 192], and can reduce intracellular calcium to very low levels. This drug was used to pre-treat monolayers and spheroids before incubation with etoposide. The effect of intracellular calcium buffering on etoposide toxicity was determined by using different concentrations of B A P A - A M (7.5 p M - 15 pM) for 30 min before and also during the 30 min incubation with etoposide. Spheroids were also incubated with 2 p M of Hoechst 33342 during the last 30 min in order to sort the brightest cells (outer cycling cells) by FACS [50]. Subsequently, treated cells were trypsinized, sorted and plated to measure clonogenic survival. 51 2.16 M t s l Transfection In order to transfect V79 monolayers with mtsl gene, pUC19 plasmid containing mouse cDNA for mtsl (S100A4) (pUC19-mtsl) was kindly provided by Dr. M.S. Grigorian (Danish Cancer Society, Copenhagen, Denmark) [84]. After confirming the sequence of the inserted gene with the database from Genebank, the PCR amplification reaction was set up in order to add a FLAG-tag to the 5' end of mtsl cDNA (Fig. 7 a, b) using a high fidelity (HF) PCR system (Roche Molecular Biochemicals, Mannheim, Germany). The PCR reaction was set up as follows: 2 pi of lOmM dNTP, 3 pi of 10 p M PUC reverse amplification primer (5 ' - A G C G G A T A A C A A T T T C A C A C A G G - 3 ' ) (GibcoBRL), 3 pi of 10 p M flag-mtsl forward primer (5'-C C A G C T A A G C T T C C A C C A T G G A C T A C A A G G A C G A C G A T G A C A A G G C A A G A C CTTGGACGAGG-3 ' ) (UBC Biotechnology Laboratory, Vancouver, B.C. , Canada) 0.5 pg pUC-mtsl plasmid, 10 pi of lOx Expand HF buffer with 15 m M M g C l 2 , 0.75 pi of Expand HF PCR system enzyme mix PCR made to 100 pi with distilled water. The PCR reaction was carried out as follows: 94° C for 3 min, 15 cycles of (94°C for 15 s, 55°C for 30 s, 72° for 45 s) followed by 20 cycles of (94°C for 15 s, 55°C for 30 s, 72°C for 50 s plus cycle elongation of 5 s for each cycle, 72°C for 5 min). After gel purifying this product (flag-mtsl) using a Qiagen extraction kit (Qiagen, Germany) in order to be able to more efficiently sub-clone it into a retroviral vector, it was first cloned into a Qiagen pDrive cloning vector (Qiagen, Germany) specifically designed for PCR products. This vector also contains multiple restriction sites which makes sub-cloning easier. Subsequently, flag-mtsl was cut at its 5' end with Kpnl (GibcoBRL) and blunt ended with 1 unit of Klenow fragment of D N A polymerase I (GibcoBRL), followed by 52 restriction enzyme digestion at its 3' end with Xhol (Gibco BRL) to match Hpal/Xhol sites on MSCV-IRES-GFP (MIG) multiple cloning site (Fig. 8). This vector was kindly provided by Dr. Robert Hawley (American Red Cross Holland Laboratory, Rockville, MD). M I G is a retrovirus based GFP (green fluorescent protein) selectable vector containing ampicilin as its antibiotic resistant gene. By containing 5' long terminal repeat (LTR) from the murine stem cell virus as its promoter and the internal ribosomal entry site (IRES) sequence located between mtsl and GFP, it allows the bicistronic mRNA transcript to be translated simultaneously at the 5' end and at the IRES sequence. Thus retroviral transfected cells containing MIG-flag-mtsl plasmid were selected based on GFP expression. Western blot (as described before) was performed on these cells using both mtsl and flag antibody. 53 pUC19-mtsl LacZ' Kpnl-mtsl-BamHI lad 3' PCR reaction using flag- mtsl as forward primer flag-mtsl forward primer Hindlll recognition start Flag Mtsl * 7-12 sequence codon+ 21-44 " 45-63 * 13-17" * # + a I II I [ 5 ' - C C A G C T A A G C T T C C A C C A I C G A C T A C A A G G A C G A C G A T G A b C A A G G C A A G A C C T T G G A C G A G G - 3 ' Flag-mtsl DNA template Fig. 7: Flag-mtsl DNA Template. Mtsl gene in pUC19 plasmid was amplified in a PCR reaction using high fidelity PCR system (Roche) in order to add a flag-tag to the 5' end of mtsl gene. 54 M I G 6400bp M C S •4--' \ EcoRl 1406 Hpal 1412 flag-mtsl (570 bp) Xhol Fig. 8: Schematic representation of MIG-flag-mtsl. Flag-mtsl was ligated into MIG vector between Hpal and Xhol restriction sites. Permission to use the vector was kindly provided by Dr. Robert Hawley. 5 5 CHAPTER 3 The Role of Phosphorylation and Localization of Topoisomerase Ila in Resistance to Etoposide The data presented in this chapter have been incorporated into the following manuscripts: Luo C, Johnston P.J., MacPhail S.H., Banath J.P., Oloumi A . , and Olive P.L. Cell fusion studies to examine the mechanism for etoposide resistance in Chinese hamster V79 spheroids. Expt Cell Res. 1998 Sep. 15:243(2):282-9. Oloumi A . , MacPhail SH, Johnston PJ, and Olive PL. Changes in subcellular distribution of topoisomerase Ilalpha correlate with etoposide resistance in multicell spheroids and xenograft tumours. Cancer Res. 2000 Oct 15;60(20):5747-53. 56 3.1 Introduction The recognition that the semi-conservative replication of double stranded D N A presents a topological problem resulted in the discovery of D N A topoisomerases by Wang in 1985 [200]. Mammalian cells express two types of topoisomerases. Type I is an ATP-independent enzyme that catalyzes D N A relaxation through a single strand break while type II is an ATP-dependent enzyme that changes the topology of D N A by making a staggered double strand break in D N A [199]. Eukaryotic type II topoisomerases are homodimeric enzymes which contain several functional domains that are extremely conserved from low eukaryotic organisms to humans [9]. Each enzyme monomer can be divided into three distinct domains (Fig. 9). The N-terminal portion, encompassing approximately the first 660 amino acids, contains consensus sequences for ATP binding and hydrolysis. The central or catalytic domain contains the active site tyrosine residue that forms the covalent bond with D N A during breakage and reunion. The C-terminal domain, which is the most variable part from species to species, contains nuclear localization sequences as well as serine and threonine residues that are phosphorylated by a number of different kinases in vivo (reviewed in [9, 137]). Topoisomerase II is an essential nuclear enzyme that exists in two isoforms, a, a 170 kD protein, and [3, a 180 kD protein [114] mapped to chromosomes 17q21-21 and 3p24 respectively [184, 191]. Relationships between the two isoforms are not well defined. However, it is known that topoisomerase Ila is subject to regulation through the cell cycle. Enzyme levels increase throughout S phase and peak at the G2/M boundary [114, 137]. Furthermore, this isoform is found almost exclusively in rapidly proliferating cells [174, 205]. Taken together, these characteristics suggest that topo I la has a major 57 role during D N A metabolic processes including replication, transcription, chromosome segragation, D N A repair, and cell cycle progression [18, 114, 160, 207]. In contrast, the concentration of topo lip is independent of cell cycle stage or growth condition [114, 205]. Moreover, this isoform appears to be present in most cell types regardless of their proliferation status [205]. While this isoform probably functions primarily as a "housekeeping" gene, it is also a target for drugs like etoposide [59], and it may compensate for decreases in nuclear topo Ila levels in some cells [82]. However, as non-cycling cells are very resistant to killing by etoposide, it is unlikely that topo l ip plays a major role as a target for this drug. Topo I la is one of the well-recognized cell proliferation markers for solid tumours [92]. Also, many types of tumours are characterized by an altered pattern of topo II regulation [196] which makes this enzyme an important target for anticancer therapy. Several clinically important anticancer agents including etoposide, doxorubicin and danorubicin have been developed which target topo II. The mechanism by which some of these agents such as etoposide exert their chemotherapeutic effects is by stabilizing the "cleavable complex" between topoisomerase II and D N A [122]. Thus, these drugs are also referred to as topoisomerase II poisons because they convert this essential enzyme to a potent cellular toxin (refer to Chapter I for more detailed explanation). Often, cancer cells develop resistance to topoisomerase II poisons either by drug efflux pumps, p-glycoprotein coded for by the MDR1 gene [31] or the MRP protein [35], or by alterations in the target enzyme [41, 154]. The latter is usually associated with a reduction in the amount of enzyme and/or mutations leading to a decrease in sensitivity of the drug [64]. 58 However, the resistance of the outer cells of spheroids to etoposide compared to monolayers cannot be explained by these mechansims. Both the outer cells of spheroids and monolayers contain similar amounts and activity of the target enzyme topoisomerase II. Also, the uptake and efflux of etoposide appears to be similar for both growth conditions [142]. Moreover, etoposide is equally efficient in inhibiting decatenation reactions using monolayer and spheroid extracts in vitro. Phosphorylation is one of the major post-translational modifications which has been shown to regulate the activity of topo II. For topo Ila, the sites of phosphate incorporation vary in a cell cycle dependent manner [21]. In vitro studies have shown that topo II phosphorylation by serine/threonine protein kinase II and protein kinase C affects its catalytic function by stimulating its ATPase activity [3, 23]. However, in the case of fission yeast, phosphorylation does not seem to affect its catalytic activity but rather enhances its transport into the nucleus [171]. Several studies have also implicated topo II phosphorylation in resistance to topo II poisons [70, 157]. Therefore, in an effort to identify the mechanism underlying the resistance of spheroids to etoposide, the phosphorylation status of this enzyme was examined in V79 monolayers and the outer cells of spheroids. Poor nuclear translocation of topo Ila has been correlated with reduced sensitivity to tumour treatment with topo II inhibitors [60]. A significant difference in intracellular distribution of the target enzyme could potentially affect the sensitivity of the cells to treatment with etoposide. Therefore, experiments were designed to compare the subcellular distribution of topo I la in monolayers and the outer cells of spheroids. In addition to Chinese hamster V79 cells, three other cell lines, which could also be grown 59 Human topoisomerase II ex (1530 a.a.) Fig. 9: Schematic representation of human topoisomerase II « showing the amino-terminal ATPase domain (closed box), breakage/reunion domain (open box), carboxyl terminal domain (grey), and active site tyrosine (Y). The arrows represent sites of phosphoryaltion in vivo. (*) denotes site of mitosis-specific phosphorylation. A large number of kinases, including P K C (A), CKII (#), and proline directed MAP-kinases (@) phosphorylate topo lice in vivo at the sites indicated. N and C represent amino- and carboxyl- terminal ends respectively [9]. 60 as both monolayers and spheroids were examined. Because these three cell lines could be grown as xenograft tumours, the spheroid results were also compared with the response of the more clinically relevant solid tumour models. 3.2 Results 3.2.1. Response to Etoposide Treatment The outer cells of Chinese hamster V79 spheroids were found to be up to 10 times more resistant to etoposide treatment than monolayers (Fig. 10). Resistance to etoposide treatment was also observed in the outer cells of C6 rat glioma and SiHa human cervical carcinoma spheroids. However, for WiDr human colon carcinoma cells, the response to etoposide treatment did not differ between monolayers and the corresponding spheroids. These results are summarized in Table 3 that also shows the response of these same 4 cell lines to ionizing radiation. A question of some importance is whether the response of spheroids to etoposide would mimic the response of solid tumours. To address this question, cell lines were implanted subcutaneously in the flanks of NOD/SCID mice where they formed solid tumours. To obtain tumour cells close to the vasculature that would more closely mimic the external cell layer of spheroids, mice were injected intravenously with Hoechst 33342 that produces a gradient of fluorescence into the tumour. Stained tumour cells were then flow sorted to obtain the brightest 10% cells, and these cells were incubated with etoposide. Figure 11 shows the surviving fraction of xenograft cells treated with etoposide. Results closely resemble those shown for outer spheroid cells, justifying the use of the simpler spheroid model in studying this form of resistance. 61 3.2.2 Localization of Topo Ila by Immunohistochemistry The intracellular distribution of topo Ila was examined in monolayers and cells from the outer layer of spheroids. Representative results given in Fig. 12a showed predominant nuclear localization of topo Ila antibody in V79 monolayers compared to its cytoplasmic localization in the outer cells of V79 spheroids (Fig. 126). Similarly, C6 and SiHa monolayers (Fig. 12c and g) showed more nuclear distribution of topo I la while their corresponding spheroids (Fig. \2d and h) mostly exhibited cytoplasmic staining of the enzyme. Conversely, WiDr spheroids and monolayers showed a similar distribution, with topo II localized primarily in the nucleus (Fig. \2e and f). These observations were confirmed in several separate experiments. It must also be noted that secondary antibody alone, under the same condition of illumination showed no significant fluorescence. The small intracellular variability observed in the fluorescence pattern, especially within the human tumour cell populations is most likely due to the difference in the level of topo Ila through the cell cycle [114]. Brightly fluorescent cells from xenograft tumours sorted based on Hoechst 33342 intensity (cells closest to blood vessels) were also examined for distribution of topo Ila (Fig. 13). Results correlated with the spheroid immunohistochemistry data and showed a predominant cytoplasmic localization of topo Ila in C6 and SiHa tumour cells as opposed to the predominantly nuclear localization in cells from WiDr xenografts. 62 0 10 20 0 10 20 Etoposide (Mg/ml) Etoposide (ug/ml) Fig. 10: Response of monolayers and outer spheroid cells to etoposide. Monolayers and the outer cell layer of spheroids were removed by trypsin and immediately exposed to etoposide for 30 min. Cells were examined for survival using a standard colony formation assay. The means and standard errors for 3 independent experiments are shown. 63 0 10 20 0 10 20 Etoposide (|jg/ml) Etoposide (pg/ml) Fig. 11: Response of xenograft tumours to Etoposide. Cells closest to blood vessels were sorted from xenograft tumours and were treated with etoposide. The resistance observed in spheroids resembles much more closely the data obtained in in vivo models. 64 Cell line Dio Dio Resistance Monolayers Spheroids Factor a) X-ray (Gy) a V79 7.4 ±0.6 9.4 ±0 .6 1.3* C6 5.9 ±0.2 7.9 ±0.6 1.3* SiHa 5.8 ±0.3 7.3 ±0 .4 1.3* WiDr 8.5 ±0.3 8.4 ±0.7 1.0 T. b) Etoposide V79 1.5 ±0.2 12.5 ±2 .0 8.3* C6 3.9 ±0.8 14.5 ± 1.3 3.8* SiHa 6.3 ± 0.9 16.7 ±0.5 2.6* WiDr 35 ±4.0 38 ±5.5 1.1 Table 3: Analysis of 4 cell lines for the presence of a contact effect. a X-ray exposure was performed on 1-2 day old spheroids. Results are from 5-8 experiments determined from complete dose response curves. The dose that results in a surviving fraction of 10% is shown (Oloumi et al., in press) b Etoposide exposure was performed using the outer cell layer of large spheroids (approx. 0.6 mm in diameter). Complete dose response curves were generated using clonogenic assays, and experiments were repeated a minimum of 3 times. 'Significantly different at 95% confidence level using a paired t-test. 65 Figure 12. Cellular distribution of topo Ha determined by immunohistochemistry. Topoisomerase I la distribution in monolayer cells (upper panels) and outer spheroid cells (lower panels) examined using the Sigma-Genosys anti-topoisomerase I la antibody and alexa488 secondary antibody. Cells were allowed to attach to glass coverslips for 2 hours before fixation and staining. Digitized images are shown (magnification x 600). 66 3.2.3 Localization of Topo Ila by Western Blot The predominance of topo I l a in monolayer nuclei was also confirmed with cell fractionation and Western blotting (Fig. 14). Conversely cytoplasmic localization of topo I l a was observed in lysates from the outer cells of V79 spheroids but not in lysates from monolayers. In addition, lamin A , a nuclear protein, confirmed an efficient nuclear and cytoplasmic separation (Fig. 14). 3.2.4 Localization of Topo Ila in Relation to Sensitivity to Etoposide Topo I l a localization was monitored in the outer cells of spheroids upon return to monolayer culture conditions for 48 hr. Nuclear fluorescence increased gradually over time, and by 6-9 h after return to the monolayer growth environment nuclear and cytoplasmic staining for topo I l a antibody was similar (Fig. 15 c and d). By 24-48 In-after return to the monolayer growth condition, the fluorescent staining was primarily nuclear and indistinguishable from the pattern for monolayers (Fig. 15/, g and h). To quantify this observation, the rate of recovery of nuclear fluorescence was obtained by using NEH/Scion image analysis software (Fig. 16a), and was compared to the change in sensitivity of cells to etoposide (Fig. \6b). After return to monolayer culture conditions, the outer cells of spheroids were exposed to etoposide at various times and dose-response curves were generated at each time point. The rate of recovery of nuclear fluorescence correlated with the rate of increase in sensitivity to killing by etoposide (Fig. 16a and b). 67 3.2.5 Phosphorylation of Topo Ila Several studies have shown that phosphorylation of topo I la plays a role in the nuclear localization of the enzyme and possibly resistance to treatment [32, 171]. Therefore, whole cell extracts were prepared from monolayers and outer spheroid cells previously labeled with 32P-orthophosphate. Immunoprecipated topo I la was about 10 times more phosphorylated in monolayers compared to the outer cells of spheroids (Fig. 17a). The phosphoimager analysis of the whole cell lysate is shown in Fig. 176, and independent Western blots confirmed equal loading on the SDS-polyacrylamide gels. 68 Fig. 13: Topoisomerase Ilex: in xenograft tumours. Intracellular distribution of topoisomerase I la in cells from representative WiDr (A) , SiHa (B) and C6 (C) xenograft tumours. Digitized images are shown (magnification x 600). 69 Fig. 14: Cellular distribution of topo Ila determined by Western blot. Western immunoblots of extracts of whole cells, nuclei or cytoplasm from V79 monolayers or the outer cells of spheroids. The Sigma-Genosys anti-topoisomerase Ila antibody was used for detection. Panels a, c and e show protein in the outer cells of spheroids. Panels b, d and f are the monolayer protein levels. This experiment was repeated 3 times with similar results. The nuclear to cytoplasmic ratio was calculated to be 5.1 for monolayers and 0.64 for outer spheroid cells using NIH image software. 70 Fig. 15: Anti-topoisomerase Ila antibody staining of outer V79 spheroid cells as a function of time after return to monolayer growth conditions. Outer spheroid cells were removed using sequential trypsinization and cells were returned to tissue culture dishes. Then at various times after return to monolayer growth, attached cells were fixed and analyzed for topoisomerase I la antibody binding using immunohistochemistry. A=0 hr, B=3 hr, C=6 hr, D=9 hr, E=12 hr, F=24 hr, G=48 hr, H= monolayers. The TopoGEN anti-topoisomerase Ila antibody was used as primary followed by FITC-conjugated anti-mouse IgG as secondary. Photographs were taken using a Zeiss epifluorescence photomicroscope with 488 nm excitation and magnification x 1000. 71 E o 0 10 20 30 40 50 0 10 20 30 40 50 Time after return to monolayers (hr) Fig. 16: Change in localization of topoisomerase Ila in comparison with change in sensitivity to etoposide. Panel a shows the increase in nuclear/cytoplasmic ratio as outer spheroid cells are returned to monolayer growth conditions. Images such as those shown in Fig. 15 were analyzed using NIH/Scion image analysis software to obtain the intensity of nuclear and cytoplasmic fluorescence. The mean and standard deviation for several cells are shown, and the O indicates the monolayer value. Panel b shows the response of outer V79 spheroid cells to etoposide after return to monolayer growth conditions. The degree of resistance to etoposide was determined using a clonogenic assay, and calculating the ratio of the slopes such as those shown in Fig. 11 for each sample time. Results from two series of experiments were combined. 72 b. J 2 P Cell Lysate 1 2 170 K D 170 K D Fig. 17: Phosphorylation of Topo Ila in monolayers and the outer cells of spheroids, (a) Results of two experiments to measure phosphorylation of topo Ila after separation of P-labeled immunoprecipitates on a 7.5% SDS-polyacrylamide gel. (b) The phosphoimager analysis of the whole cell lysate from experiment 1. Lane 1, 3 2 P -labeled monolayer proteins; lane 2, P-labeled spheroid proteins. 73 3.3 Discussion The mechanism underlying resistance to etoposide that results from growth of some cell types in three dimensional contact is still not explained. Mutations in the topo Ila gene which could affect the activity of the enzyme, or changes in the level of topo Ila have been discounted as possible explanations for this type of resistance [142]. Results shown here indicate that return of the outer cells of V79 spheroids to monolayer culture condition reverses this resistance response within a two or so cell doublings (Fig. 16). Moreover, the ability of the enzyme to decatenate trypanosome D N A , an indication of the activity of topo II, has been shown to be unchanged in these outer proliferating cells of spheroids [142]. Cell cycle time and growth fractions have been shown to be identical between monolayers and the outer cells of spheroids [125]. The relationship between D N A damage and survival after etoposide treatment has also been shown to be similar and independent of growth environment [125]. This suggests that the ability to form an effective cleavable complex, not the ability to repair D N A damage is likely to be the primary reason for resistance. Several studies have implicated topo II phosphorylation in resistance to topo II poisons [32, 70, 157], and results shown in Fig. 17 indicate that topo I la from V79 spheroids is about 10 fold less phosphorylated than monolayers topo Ila. Although topo Ila phosphorylation is one of the important post-translational modifications, its role remains controversial. Some reports indicate that phosphorylation regulates the activity of topo I la [36] while others fail to show such an association [171]. Experiments carried out in vitro by Kimura et al. show no apparent effect on decatenation activity of topo I la resulting from changes in phosphorylation [103]. Because phosphorylation of topo Ila 74 occurs at several sites in different domains (Fig. 9) [9] it is not surprising that only phosphorylation of the catalytic domain would regulate the enzyme's activity. The fact that topo I la phosphorylation has little effect on its activity is consistent with previous results showing that topo II decatenation activity was similar for extracts of monolayers and the outer cells of V79 cell spheroids [142]. It appears that changes in phosphorylation of the noncatalytic domain, such as the COOH terminus, does not affect the catalytic activity [171]; however, it can influence intracellular distribution of topo Ila since it has been suggested to be involved in nuclear translocation [60]. In vitro studies, such as those previously performed to measure topo I la activity, failed to appreciate this important form of resistance, that is, resistance due to cytoplasmic localization of the topo Ila. Several studies with human tumour cell lines have associated resistance to topo I la inhibitors with reduced nuclear localization of this isoform [60, 202]. Our immunohistochemistry and Western blot results (Fig. 12 and 14) indicated cytoplasmic localization of a considerable amount of topo Ila in the outer resistant cells of V79, SiHa and C6 spheroids. It should be noted that the level of topo II needs to fall below a critical threshold before cells fail to undergo mitosis [137], and that topo Hp may assume some of the functions of topo Ila. In V79 mutant cells it has been shown that even a drastic reduction in total cellular topo Ila causes minor perturbation of cell growth [88] indicating that only a small portion of topo Ila is required for cell cycle progression. Therefore, it is most likely that changes observed in intracellular distribution of topo I la in outer cells of spheroids are due to the dramatic decrease in the phosphorylation of the enzyme. In fact phosphorylation of the COOH terminus of topo I la has been indicated to 75 play a role in nuclear translocation of topo II without affecting its activity [4, 132]. The long half life of phosphorylated topo II (12-27 hr) [103, 109] may explain in part the requirement for at least 3 days growth in suspension to achieve maximum etoposide resistance in V79 spheroids. Also, when spheroids were returned to monolayer culture conditions, the steady increase in the amount of nuclear topo I la was comparable to the increase in etoposide sensitivity. By 24 h after return to monolayer culture conditions nuclear localization is apparent, and cells are only about 1.6 times less sensitive to etoposide than monolayers (Fig. 15 and 16). Therefore, a significant decrease in the amount of topo I la would be expected to reduce the amount of D N A damage by etoposide and could explain the resistance of as much as 10-fold observed in outer spheroid cells to killing by etoposide (Fig. 10). Previously the resistance of spheroids to etoposide was related to changes in D N A conformation due to the change of the growth environment [142]. The current results do not eliminate that possibility. In fact, it has been suggested that topoisomerase II can even change chromatin architecture through direct binding interactions, apparently independently of the catalytic activity [15]. D N A topoisomerases may also play a role as repair enzymes through both damage recognition and recombinase activities [113] and possibly through misrejoining [188]. Therefore, changes in topo II localization could contribute to changes in chromatin conformation and to D N A repair. This could also partially explain the resistance of these spheroids to radiation which was the original basis of "contact effect" [54]. 76 CHAPTER 4 Identification of Genes Differentially Expressed in V79 Cells Grown as Multicell Spheroids The data presented in this chapter have been incorporated into the following manuscript: Arusha Oloumi, Wan Lam, Judit P. Banath and Peggy L. Olive. Identification of Genes Differentially Expressed in V79 Cells Grown as Multicell Spheroids. Int J Radiat Biol (in press). 77 4.1 Introduction The explanation for the contact effect has been elusive. A direct approach to undercovering the mechanism(s) is to examine differences in gene expression patterns for cells grown as monolayers or spheroids. Presumably differences in gene expression could underlie the ability of spheroids to resist damage by ionizing radiation and etoposide and could therefore indicate likely candidates responsible for this form of resistance. Of course, this is only a first step. The importance to resistance of any gene that is differentially expressed will need to be determined by over-expression, use of gene knockouts, or antisense experiments. Differential gene expression is responsible for cell control over many biological processes such as growth, development and differentiation. Therefore, the molecular approach of analysis of changes in gene expression in cells under different environmental conditions has high biological potential. The method of differential display (DD) originally developed by Liang and Pardee in 1992 [120] is a powerful technique which allows analysis of changes in gene expression. This technique facilitates comparison of the patterns of expressed mRNAs from two or more cell types in parallel; consequently, both qualitative and quantitative changes can be identified. Thus identification of new genes as well as analysis of any changes in gene expression involved in a particular cellular process would be possible. To perform this method, total R N A is fractionated into specific subpopulations of cDNA by reverse transcription followed by PCR amplification using different combinations of primers. Every pair of primers has a certain chance of identifying a limited number of target sequences within the pool of cDNAs. After resolving the PCR 78 products on a denaturing polyacrylamide gel, bands of interest are excised from the gel for further analysis. Even though the strategy seems to be simple and straightforward, difficulties in confirming the differences found with differential display have been reported [172, 209]. The re-amplified cDNA probes from differential display sometimes contain more than one cDNA fragment due to co-migration on the gel making the identification of the truly differentially expressed genes difficult. Also, northern blot confirmation for each identified cDNA fragment is very laborious and difficult particularly when the amount of R N A sample is limited. Some improvements to the method have been made to increase reproducibility and to reduce the number of false positives, and these technical improvements have been incorporated here [8]. The modified differential display [8] was performed as discussed in Chapter 2 (page 44). Briefly, modified long composite primers were developed based on both mRNA differential display and R N A arbitrary primed PCR fingerprinting method. This approach has been shown to increase the reproducibility and sensitivity of the method as well as reducing the false positives while still keeping the characteristics of the original method. Another way of overcoming some of these problems is by cloning the cDNA fragments first and screening them by "reverse northern" to identify the ones that represent the truly differentially expressed mRNAs. Also, in this case, in order to increase the probability of choosing the clone containing the differentially expressed cDNA, several clones are usually picked to be used as probes in the subsequent validation. 79 To uncover differences in gene expression that might contribute to the "contact effect", the technique of differential display was applied to Chinese hamster V79 cells grown either as monolayers or as multicell spheroids. Clearly many changes occur when cells are grown as spheroids that can affect subsequent drug resistance. For example, within a layer or twd of the spheroid surface, cells exit the cycle and enter the resting state called Go. Internal cells of spheroids are also depleted of nutrients such as oxygen and glucose, and the innermost cell layers are subject to acidosis and necrosis. Therefore, to reduce differences in spheroid gene expression that might be associated primarily with cell cycle and nutrient gradients, only the outer (proliferating, well-nourished) cell layer of spheroids was selected for comparison with exponentially growing monolayers. 4.2 Results 4.2.1 Genes Identified with Differential Display Differential display was used to identify altered gene expression between V79 monolayers and the proliferating outer cells of spheroids. At least 2 different sets of mRNA preparations were used for differential display analysis, and only reproducible differences were selected from these experiments. Using six different pairs of primers (as shown in Fig. 5 in Chapter 2), 20 altered bands were selected and excised from the differential display gels. Figure 18 is a representative image of two gels to show differential expression of bands 1, 2 and 6. To confirm differences in expression, the 20 bands were recovered from the DD gel and re-amplified by PCR. 80 Table 4: Genes identified using differential display Band Higher expression shown in...* B L A S T search with D N A sequence** Identities Function 1 Spheroids (>3) R. norvegicus S100A4 (metastasin) 82/92 (89%) Calcium binding protein 2 Spheroids (>3) P. ban deranus Cytochrome c oxidase II 179/209 (85%) Electron transport 3 Spheroids (>4) H. sapiens B-indl 161/193 (83%) Mediator of Rac-1 signaling 4 Spheroids (>3) H.sapiens Translocating chain associating membrane protein (TRAM) 124/136 (91%) Protein translocation 5 Monolayers (>9) Mus musculus Phosphoglycerate kinase 85/94 (90%) Glycolysis 6 Monolayers (>4) R. norvegicus ARL-3;ADP-ribosylation factor 96/108 (88%) Ras-related GTP binding protein 7 Monolayers (>10) Mus musculus MHC class III complement 4A 32/33 (96%) Complement, antigen processing 8 Monolayers (>5) Mus musculus 2,4-dienoyl-CoA 91/95 (95%) Substrate in fatty acid biosynthesis * An estimate of the degree of over-expression from Fig. 19 is shown in brackets. Saturation of images in Fig. 19 limited the accuracy of this estimate. ** Nucleotide sequences were compared with GeneBank, E M B L , and EST databases using the B L A S T algorithm. The species with highest homology is indicated. 81 Subsequently, the PCR amplified products were cloned and at least 4 colonies from each fragment were used in a reverse northern blot. For each fragment, the colony that ultimately showed the strongest positive signal was used for sequencing and further experiments (Fig. 19). At this stage only 8 bands showed positive signals and had a match in a GenBank search, and the remaining ones are not discussed further. A GenBank search demonstrated high sequence homology to known genes in the database; however, due to species variation, some of the matched identities obtained from GenBank appear to be a little low (Table 4). The relative difference in expression of these genes was estimated from the dot blot results using the FluorChem v2.0 Stand Alone software (Alpha Innotech Corp., San Leandro, CA) (Table 4). GenBank accession numbers and their corresponding genes for these clones are 1: metastatin (also known as mtsl or S100A4) (NM012618), 2: cytochrome c oxidase II (U62572.1), 3: B-ind-1 (AJ271091.1), 4: 2,4-dienoyl-CoA (NM026172.1), 5: phosphoglycerate kinase (M23967.1), 6: ADP-ribosylation factor-3 (X76921.1), 7: M H C class III C4A (AF0999334) and 8: translocating chain associating membrane protein (TRAM)(NM014294). 4.2.2 Metastasin Northern Blot Of the eight identified genes, mtsl (S100A4) which had calcium binding properties and was involved in cell proliferation and motility was further examined. Upregulation of this gene was shown by northern blot using a specific probe made with PCR and using total R N A isolated from V79 monolayer cells and the outer cycling cells of spheroids (Fig. 20). Ribosomal R N A stained with ethidium bromide on the denaturing formaldehyde gel was also used as a loading control. About 3.7 times more mts-1 82 message was present in outer spheroid cells than monolayers. This estimate was obtained using the FluorChem v2.0 Stand Alone software (Alpha Innotech Corp.). 4.2.3 Metastasin and Calretinin Western Blot In order to find out whether the difference seen at the cDNA level also existed at the transcriptional level, immunoblotting was performed. Using a polyclonal antibody against S100A4 (mtsl), upregulation of the corresponding protein was seen in the outer cells of V79 and C6 spheroids while no apparent difference was seen for WiDr monolayers and spheroids (Fig. 21). Expression of mtsl could not be detected for SiHa monolayers or spheroids. However, expression of calretinin, another calcium binding protein, was significantly increased in SiHa spheroids relative to monolayers. For this calcium binding protein, little or no expression was observed for the C6 glioma monolayer cells. Again, no difference was observed between WiDr monolayers and spheroids that do not demonstrate a "contact effect". 83 Fig. 18: Differential display gel. Representative differential display gels showing experiments with 2 different primer sets and 2 different preparations of monolayer and spheroid mRNA. Experiments with each primer set were run side by side (m, V79 monolayers; s, outer cells from V79 spheroids). Numbers correspond with the genes identified in Table 4. Thus band number 5 shows upregulation in monolayers relative to outer spheroid cells using primer set 1. Primer set 1 Primer set 2 m s m s msms 84 1 mono sph mono sph • 7^  8 Fig. 19. Reverse northern blot analysis. PCR amplified cDNA fragments obtained from differential display gel were dot blotted on duplicate membranes and hybridized with equal counts of [a- P] labelled total R N A obtained from either V79 monolayers (mono) or the outer cells of spheroids (sph). Bands upregulated in spheroids are 1) S100A4 gene, 2) cytochrome c oxidase II, 3) B-ind-1 and 8) T R A M . Bands down-regulated in spheroids are 4) 2,4-dienoyl-CoA, 5) phosphoglycerate kinase, 6) ADP-ribosylation factor-3, 7) M H C class III C4A. 85 28SrRNA-* 18SrRNA-* Fig. 20: V79 Northern blot with mtsl probe. R N A extracted from V79 monolayers and the outer cells of spheroids was run on a denaturing formaldehyde gel. (a) 28S and 18S ribosomal RNAs were stained with EtBr and used as loading controls. After transferring to a Zeta probe membrane, hybridization was done with a probe specifically made for mtsl gene with RT-PCR and random primer labeling, (b) The autoradiogram shows 3.7 fold upregulation of mtsl in V79 spheroids relative to monolayers. 86 C6 V79 SiHa WiDr sph mono sph mono sph mono sph mono mtsl 10KD a-tubulin 60KD Calretinin 47 KD Fig. 21: Western blot with mtsl and calretinin antibodies. Cell lysates were loaded onto a 12% SDS-PAGE gel, electrophoresed and transferred to nylon membranes. Results from ECL-treated blots were analysed using the FluorChem v2.0 Stand Alone software (Alpha Innotech Corp.). Alpha-tubulin monoclonal antibody is shown as a control. Mts l level was increased 3.7 fold in the outer cells of V79 spheroids and 2.1 fold in C6 spheroids. 87 4.3 Discussion Since the first observation of the "contact effect" in 1972 [54], efforts have been made to identify possible causes for radiation and chemoresistance when cells are grown as spheroids. However, a convincing mechanism for the "contact effect" has not yet emerged. The fact that not all tumour types demonstrate contact resistance should provide an important clue to aid in uncovering the mechanism for this effect. WiDr colon carcinoma cells fail to show a contact effect for ionizing radiation or etoposide. In contrast, human SiHa cervical carcinoma cells, rat C6 glioma and hamster V79 lung fibroblasts show a contact effect for both radiation (Table 3 in Chapter 3) and etoposide (Fig. 10 in Chapter 3). Differential display approaches this question directly since it detects differentially expressed genes unrelated to message abundance and it measures both upregulated and downregulated mRNAs. Differential display revealed changes in expression of several genes involved in a range of cellular functions. B-indl was recently recognized as a mediator of Racl signaling in a transformed mouse cell line. B-indl does not affect cell proliferation but appears to act downstream of the small GTPase, Racl , and collaborates with the activated form of Racl to induce c-Jun N-terminal kinase activity [40]. Racl is a member of the Rho family of GTPases that also regulates actin dynamics and cytoskeletonremodeling. The authors suggest that B-indl may participate in the initial differentiation program. Cytochrome c oxidase II, which is upregulated in spheroids, has a calcium binding site and has been identified as a senescence associated gene [76]. This gene and mtsl, involved in cell differentiation and adhesion, were both found to be overexpressed in 88 lymphoma using subtractive hybridization [186]. T R A M is an abundant endoplasmic reticulum protein required for membrane transfer of secretory proteins [78]. Genes downregulated in spheroids include Arl-3 a member of the ADP-ribosylation factor family of GTP-binding proteins. Several human tumour cell lines express large amounts of Arl-3 but its role in human cell physiology is not known [27]. M H C class III complement 4A plays an important role in processing of immune complexes. 2,4-dienoyl-Co-A reductase is a mitochondrial enzyme involved in the beta-oxidation of unsaturated fatty acids. It also interacts with HSP70 [135]. Phosphoglycerate kinase, an important enzyme involved in glycolysis is significantly downregulated in the outer cells of spheroids. Interestingly, Sutherland and colleagues [66, 176] made a related observation of reduced glucose consumption in spheroids several years ago. The authors suggested that a decrease in cell proliferation contributed to these changes, but also noted that even very small spheroids that show minimal changes in proliferation showed reduced glucose utilization. Due in part to its calcium binding properties, metastatin (mtsl), also known as S100A4, was the most interesting message that was overexpressed in spheroids relative to monolayers. The protein level was increased 3.7 fold in the outer cells of V79 spheroids (Fig. 21). A 2.1 fold increase was also observed in C6 spheroids that demonstrate a contact effect, but no significant increase was observed in the WiDr colon carcinoma cell line that did not develop contact resistance. Mts l is a member of the EF-hand family of calcium binding proteins whose transcription is regulated by the D N A transcription factor, K R C [91]. It is known to be involved in cell cycle progression through links with stathmin and p53, and becomes elevated upon changes in the growth 89 and differentiation of cells [73]. It is associated with actin filaments [73] and enhanced expression of S100A4 affects intercellular adhesion, possibly by remodeling the extracellular matrix [170]. Mts l has also been linked to cell motility and metastasis. In a series of non-small cell lung cancer cells, reduced expression of E-cadherin (a cell-cell adhesion molecule and putative invasion suppression molecule) was correlated with the increase in mtsl and associated with poor prognosis [101, 102]. We have also found upregulation of expression of calretinin (calreticulin) in V79 and SiHa spheroids but not in WiDr spheroids (Fig. 21). This multifunctional calcium binding protein increases the calcium storage capacity of the ER, and plays a role in integrin function [39, 130]. S100C, a closely relative of SI00A, was recently implicated in contact inhibition of cell growth [163], and SI OOP was identified by subtractive hybridization to be overexpressed in doxorubicin-resistant colon cancer cell lines [12]. If increased expression of calcium binding proteins causes a reduction in intracellular calcium, this could also explain resistance to etoposide since calcium depletion protects against etoposide and perhaps X -ray induced damage [6, 13, 152]. The relation between expression of calcium binding proteins, intracellular free calcium levels, and the development of the contact effect is examined further in the following chapter. 90 CHAPTER 5 The role of Calcium Regulation in Resistance of Spheroids to Etoposide The data presented in this chapter have been incorporated into the following manuscripts: Arusha Oloumi, Wan Lam, Judit P. Banath and Peggy L . Olive. Identification of Genes Differentially Expressed in V79 Cells Grown as Multicell Spheroids. Int J Radiat Biol (in press). Oloumi A. , Banath J.P., and Olive P.L. Calcium regulation in spheroids and its correlation with resistance to etoposide (in preparation). 91 5.1 Introduction In the previous chapter, differential display experiments identified 8 genes that were over- or under-expressed in outer spheroid cells versus monolayers. One of these genes was a calcium binding protein called metastasin that was confirmed by immunoblotting to be up-regulated in V79 spheroids. A second gene that has calcium binding properties, cytochrome c oxidase, was also found to be up-regulated in V79 spheroids relative to monolayers. Finally, another C a 2 + binding protein, calreticulin, was also found to be increased in outer cells of V79, C6 and particularly in SiHa spheroids relative to monolayers. These observations led to a study of the potential role of calcium in contact resistance to etoposide. In most cells, C a 2 + has its major signaling function when its concentration is elevated in the cytoplasm from where it can also diffuse into the nucleus or be sequestered by mitochondria. Most cells utilize C a 2 + either from extracellular influx or from intracellular stores to generate intracellular signals (Fig. 22). Multiple mechanisms control the concentration of Ca inside the cell including channels located at the plasma membrane which regulate entry from the extracellular space, and channels inside the cell such as endoplasmic and sarcoplasmic reticulum (ER and SR respectively) (Fig. 22). On the other hand C a 2 + removal from the cytoplasm is achieved by Ca 2 +ATPases on the plasma membrane and ER/SR in addition to other ion exchangers such as Na + / C a 2 + as well as mitochondria. Several different messenger activated channels mediate the release of Ca from the intracellular stores. For instance, the binding of many hormones and growth factors leads to activation of phospholipase C on the plasma membrane leading to cytoplasmic activation of the highly mobile inositol 1,4,5-triphosphate (InsPa). The 92 binding of InsP3 to receptors on ER/SR results in the release of Ca to the cytoplasm from these intracellular stores (Fig. 22) (reviewed in [16, 17]). Calcium impacts on many cellular functions including differentiation and transcription. C a 2 + signals are transduced into intracellular responses via interaction with calcium binding proteins. Some calcium binding proteins act mainly as buffers while others undergo conformational changes upon C a 2 + binding for association with different proteins, therefore, influencing C a 2 + signaling at different points in the pathway [167]. One family of calcium binding proteins is characterized by a common structural motif, known as EF-hand after E- and F-helices of parvalbumin, which binds calcium selectively and with high affinity [106]. Among those is the SI00 family of proteins, one of the largest sub-families of EF-hand proteins, many of which (at least 10) have been found to be clustered on human chromosome lq21 [47] a region that is frequently rearranged in several tumours [71]. These Ca binding proteins are composed of two EF-hands flanked by hydrophobic regions at either terminus and separated by a central hinge region. The carboxyl end terminal EF hand is referred to as canonical Ca2 +-binding loop and encompasses 12 amino acids, whereas the amino terminal has lower affinity for C a 2 + and is formed of 14 amino acids [167]. The biological role of these small (10-12 KDa), acidic proteins is poorly understood; however, binding of C a 2 + has been shown to result in conformational changes in these proteins revealing the hydrophobic patches that enable these proteins to interact with target proteins and transmit biologically important signals. The SI00 family members share about 50% homology in amino acid sequence [210]. They have become of major interest in the past few years due to their differential expression in neoplastic transformation and metastasis and their involvement in a large group of cellular events 93 such as neurite growth, cell-cell communications, motility, intracellular signaling, and cell division [167]. Metastatin (mtsl) also known as S100A4 (synonyms are 18A2, 42A, pE198, p9Ka, and CAPL) belongs to this family of Ca -binding proteins. Jackson-Grusby et al. originally isolated mtsl as an mRNA species that is induced by serum stimulation of growth-arrested fibroblasts and named it 18A2 [97]. Subsequently and independently, mtsl was also cloned by several other groups, from PC 12 cells after nerve growth factor stimulation as 42A [129], from myoepithelial-like cells as p9Ka [10], and from the BALB/c3T3 established cell line as pE198 [80]. Mts l is a 17 kb gene containing three exons and two introns with the first exon representing the non-translated region of the gene [195]. Methylation of the first exon and the first intron of mtsl gene have been shown to serve as an important mechanism of transcriptional control [193, 194]. Expression of mtsl is observed in normal tissues in particular in several embryonic tissues [97]. In adults, predominant expression of mtsl is demonstrated in lympoid cells, such as spleen and lymph nodes [83]. Moreover, detailed studies performed in mouse and human blood cells demonstrated the expression of mtsl in invasive cells such as monocytes, macrophages, neutrophils and T-lymphocytes, and the absence of its expression in B-lymphocytes and erythroid cells [84, 97]. Interestingly the normal tissue types in which mtsl is diffusely distributed in the cytoplasm are all capable of motility. The name "metastasin" derives from results implicating this gene in the regulation of metastasis. Expression of mtsl has been shown to correlate with the metastatic potential of tumour cells [58]. Differential screening of cDNA libraries generated from metastatic and non-metastatic mammary adenocarcinoma cell lines, CSML-0 and C S M L -94 100, respectively, has resulted in cloning of the highly expressed mtsl gene from metastatic cells [58]. It has also been demonstrated that by changing the expression of the mtsl gene, the metastatic phenotype of tumour cells can strongly be influenced. The reduction of expression of mtsl in mammary gland carcinoma cells and Lewis lung carcinoma cells, both with a high metastatic phenotype, by transfection with anti-sense mouse mtsl reverses the metastatic potential [84, 182]. Alternatively, transfection of the non-metastatic tumour cell lines with the sense construct results in induction of metastasis [42, 84]. Mtsl is believed to increase the metastatic potential of tumour cell lines by increasing cell motility [63, 182]. A recent study on expression of mtsl in specimens of primary breast carcinomas by immunohistochemical techniques revealed a significant association with patient deaths due to metastatic disease [161]. The biological function(s) of the mtsl gene product is not fully understood. However, recent efforts to identify proteins which interact with the mtsl gene product have revealed cytoskeletal proteins such as non-muscle tropomyosins [183] and non-muscle myosin (MHC) as the target proteins in a calcium-dependent manner [108]. Interaction of mtsl with M H C results in the inhibition of the protein kinase C-mediated phosphorylation of the M H C molecule [107], which has been proposed to play a role in reorganization of myosin filaments. Therefore, the potential role of calcium in cell-cell contact induced resistance to etoposide was studied by measuring the amount of intracellular free C a 2 + as well as manipulating Ca concentration and examining subsequent changes in etoposide sensitivity. To directly examine the importance of mtsl expression in the contact effect, 95 Extracellular Agonist ^v^. Receptor Ca 2 + • CaBPs C a 2 + .Channels Ptdlns(4,5)P2 Migration ECM component Ins (1,4,5)P, Ca2+ ATPase Na* - Ca2* exchanger C a 2 + Calcium buffering and transport Activation of enzymes Polymerization of cytoskeleton Cell cycle progression Ca2+ ATPase Endoplasmic Reticulum Nucleus Ca 2 + Mitochondria Fig. 22: Signal transduction by calcium binding proteins [167]. Influx of calcium upon stimulation either from intracellular stores or through different C a 2 + channels allows calcium binding proteins to bind Ca 2 + , undergo a conformational change and associate with different target proteins, thereby shaping the biological effects of the C a 2 + signal. 96 cells were transduced with a construct containing mtsl and green fluorescent protein. After selection of GFP positive clones, cell response to etoposide was examined. 5.2 Results 5.2.1 Intracellular Free Calcium Measurement To examine the role of calcium in cell-cell contact induced etoposide resistance, the calcium indicator dye, fluo-3 was used to measure the level of intracellular free C a 2 + in monolayers and the external cells of V79, SiHa and WiDr spheroids. Although there are several calcium indicators available, fluo-3 which shows a large dynamic range and a low compartmentalization (nuclear/cytoplasmic) [189] was chosen for this measurement. V79 spheroid cells showed almost two fold reduction in the amount of free calcium compared to monolayer cells, consistent with the upregulation of calcium binding proteins in V79 spheroids. Also, in SiHa spheroids there was a reduction of more than 60% in the amount of intracellular free calcium relative to monolayers. These increases were shown to be statistically significant using a paired t-test (p = 0.01 for V79 and p = 0.04 for SiHa), while the reduction of about 15% in WiDr spheroids relative to monolayers was not found to be significant (p = 0.28). Figure 23a shows results from 5 independent experiments. Representative bivariate distributions and flow histograms are shown for V79 monolayers (Fig. 23 b,d) and the outer cells of V79 spheroids (Fig. 23 c,e). Intracellular free calcium measurement in C6 cell line using fluo-3 did not give reproducible results possibly due to the fact that SI00 calcium binding proteins are a major protein product of this line. 97 Fig. 23: Intracellular free calcium measurement. The amount of intracellular free calcium measured with fluo-3 in at least 5 independent experiments in V79, SiHa and Widr monolayers and outer cells of spheroids is plotted. * =p value in paired t-test < 0.05. Panel b (V79 monolayer) and c (V79 spheroid) show bivariate distributions of D N A content versus fluo-3 intensity. Panels d (V79 monolayer) and e (V79 spheroid) are representative histograms showing the greater intensity of fluo-3 in V79 monolayers compared to outer spheroid cells. 98 5.2.2 Effect of Pre-treatment with BAPTA on Etoposide Toxicity In order to examine the effect of lowering the levels of intracellular free calcium on etoposide toxicity, V79 monolayers and spheroids were exposed to different concentrations of the calcium chelator, BAPTA, before and during etoposide treatment. B A P T A buffers are highly selective for C a 2 + [99, 192]. Comparative studies with B A P T A have proven useful in demonstrating that the effect of B A P T A on mitotic progression in Swiss 3T3 fibroblasts is due to specific effects on Ca buffering [99]. Toxicity of etoposide was measured using a clonogenic survival assay to detect etoposide-induced cell killing. Monolayers and outer cells of spheroids were exposed to B A P T A - A M , the acetoxy methyl ester of B A P T A that improves cell uptake, for 30 min before and also during the 30 min exposure to etoposide. In order to obtain the comparable amounts of cell ki l l in the absence of B A P T A , V79 monolayers were treated with 4 pg/ml etoposide and spheroids with 30 pg/ml etoposide. As expected etoposide-induced cell ki l l was reduced in both monolayers and spheroids with increasing concentrations of B A P T A - A M . However, the relative effect in monolayers was much greater (Fig. 24). 5.2.3 Detection and Expression of Mtsl Measurement of the effect of mtsl over-expression and its possible role in etoposide-induced cytotoxicity were achieved by transduction of this gene into V79 monolayers using a retroviral construct. Mtsl gene cloned into a pUC19 vector (kindly provided by Dr. M.S. Grigorian) was amplified in a PCR reaction using appropriate oligonucleotides in order to add a FLAG-tag to its 5' end. Furthermore, the PCR 99 amplified flag-mtsl was sub-cloned into a retroviral vector (MIG) containing GFP as a selectable marker. Restriction enzyme digestion followed by sequencing showed the presence of full length flag-mtsl with correct orientation in this plasmid (Fig. 25). V79, C6 and SiHa monolayers were successfully transduced with this vector (MIG-mtsl). Resulting GFP expressing cells were then FACS-sorted based on GFP using a Becton Dickinson FACS 440 cell sorter. The fluorescent signals from GFP in these pooled populations, also determined by flow cytometry, were 18x higher than the background fluorescence in V79 cells, 25x higher in C6 cells, and 13x higher in SiHa cells (Fig. 26). The monolayers of all these three cell lines were also transduced with mock M I G (vector only). To verify expression of mtsl in the clones, immunoblotting was performed for the mts-1 transduced, mock transduced, and parental V79, C6, and SiHa monolayers. As expected, higher mtsl expression at a slightly higher molecular weight was observed due to the presence of flag-tag in all three MIG-mtsl transduced cell lines (Fig. 21a,). In addition, by using an antibody against FLAG-tag, expression of transfected as opposed to the endogenous mtsl was easily distinguishable (Fig. 21b). Immunoblotting with a-tubulin showed equal loading of different cell lysates in these experiments (Fig. 27c). 5.2.4 Intracellular Free Ca2+ in Mtsl Overexpressing Cell Lines Indo-1, a UV-excitable calcium indicator dye, was used to measure the amount of intracellular free C a 2 + in the transduced cell lines. Since these cells express GFP, fluo-3 that is also excited at 488 nm, could not be used. Figure 28 shows flow cytometry measurements of calcium levels in parental, vector only and mtsl expressing cells. The 100 amount of intracellular free C a 2 + measured in mtsl transduced V79, C6 and SiHa monolayers was not significantly different from the parental or mock transfected cell lines. 5.2.5 Effect of Mtsl Overexpression on Etoposide-induced Toxicity In order to further investigate the direct effect of mtsl overexpression on etoposide induced toxicity, the transduced cell lines as well as the parental cell lines were treated with different concentrations of etoposide for 30 min, and cell survival was measured in a colony formation assay. The MIG-mtsl transduced cell lines showed no apparent change in sensitivity to etoposide (Fig. 29). 101 © V79 mono + 4 ug/ml etoposide • V79 sph + 30 ug/ml etoposide 0 2 4 6 8 10 12 14 16 B A P T A - A M jaM Fig. 24: Effect of BAPTA-AM on etoposide induced cell killing. V79 monolayers and outer cells of spheroids were treated with increasing concentration of B A P T A -A M for 30 min before and during the 30 min etoposide treatment. Different concentrations of etoposide were used to introduce the same amount of cell ki l l in monolayer and spheroid cells. The relative effect of B A P T A on etoposide-induced cell kil l was much greater in V79 monolayers compared to spheroids. Two way A N O V A analysis shows that the difference between the curves is significant (p<0.0001). 102 i ! S 13 S Q | 1 M I G vector 6400 bp Flag-mtsl insert 560 bp Fig. 25: Restriction enzyme digested MIG-mtsl plasmid. MIG-mtsl plasmid was digested with Xho I and BamHI restriction enzymes. Flag-mtsl insert is seen on ethidium bromide stained agarose gel at about 560 bp. Restriction enzyme digestion of 3 separate clones is shown in this figure. 103 50 45 • Parental • Vector only • Mtsl V79 C6 SiHa Fig. 26: Mean G F P fluorescence measured by flow cytometry in virally transfected cell lines. GFP fluorescence was compared in non-transduced (parental), transduced with MIG (vector only) and mtsl containing MIG plasmid (mtsl) in V79, C6 and SiHa cells. The significant higher expression of GFP observed in the transduced cell lines compared to background (parental cells) proves successful transduction. 104 Fig. 27: Western blot analysis of mtsl transduced cell lines. Immunobloting of lysates prepared from regular V79, C6, and SiHa monolayers (parental), as well as from M I G containing (vector only) and MIG-mtsl transduced cells (mtsl) with (a) S100A4 (mtsl) antibody shows upregulation of this protein in mtsl cells with a band at a slightly higher molecular weight compared to the controls due to the presence of the attached flag protein. Immunoblotting with flag antibody (b) also distinguishes the expression in the mtsl cells compared to the endogeneous. Also, immunoblotting with cc-tubulin (c) shows similar loading of the cell lysates. 105 2.5 Fig. 28: Intracellular free calcium measurement in transduced cell lines. Using Indo-1 the amount of intracellular free C a 2 + was measured in the transduced V79, C6 and SiHa cells and was compared to their relative amounts in the regular (parental) monolayers. These relative amounts do not appear to be significantly affected by overexpression of mtsl. 106 0 1 2 3 4 5 6 Etoposide |ag/ml 0 5 10 15 Etoposide |ig/ml o.ooi - n — i — i — i Y 1 0 10 20 30 40 50 Etoposide fig/ml • Parental line O Vector only • M t s l Fig. 29: Etoposide toxicity in mtsl transduced cell lines. Cytotoxic effects of etoposide was compared in V79, C6, and SiHa monolayers in parental and transduced cell lines. Overexpression of mtsl had minimal effects on etoposide-induced cell kil l measured by colony formation assay. 107 5.3 Discussion Upregulation of mtsl (S100A4), a small calcium binding protein, was identified by differential display in the outer cells of V79 spheroids. This difference in gene expression was confirmed at the protein level with a polyclonal antibody. The increased expression of this C a 2 + binding protein and calretinin (as shown in the previous chapter) appeared to have a significant effect on the concentration of intracellular free Ca 2 + . The level of calcium measured by fluo-3 was significantly decreased in the outer cells of V79 and SiHa spheroids. Conversely, WiDr spheroids which did not show any significant up-regulation of calcium binding proteins did not show a significant decrease in the amount of intracellular free C a 2 + either (Fig. 23). A reduction in the level of intracellular free C a 2 + could in fact play a role in resistance to etoposide by several different mechanisms. Previous reports have suggested a role for C a 2 + in stabilizing the D N A cleavable complex upon treatment with topo II inhibitors such as etoposide [70, 152]. In these studies buffering of intracellular Ca was also achieved by treatment with B A P T A - A M . HL-60 cells treated with B A P T A - A M exhibited a significant reduction in activity when assayed for VP-16 stabilized topo II-D N A complex formation while decatenation of kinetoplast D N A , reflecting the activity of topo II, was unchanged [6, 70]. Therefore, a reduction in intracellular free calcium in outer cycling cells of spheroids (possibly due to upregulation of calcium binding proteins) would be expected to affect this stabilization by reducing the amount of cleavable complex leading to a decrease in the extent of D N A damage. In fact, previous results from our laboratory also suggest that the ability to form an effective cleavable 108 complex, and not the ability to repair D N A damage would be the primary reason for resistance [125]. Second, calcium levels in cells may directly affect phosphorylation of topo II. Calcium buffering in HL-60 cells has been shown to reduce topo Ila phosphorylation at two specific sites, which have also been shown to be the same hypophosphorylated sites in topo II-poison resistant cells [6, 70]. The two hypophosphorylated sites were not the substrates for casein kinase II [6, 70]. Therefore, the observed hypophosphorylation of topo Ila (Fig. 15 page 70) in the outer cells of spheroids could also be due to the reduced amount of intracellular Ca 2 + , and this could prevent the proper translocation of topo Ila to the nucleus as was previously shown in Chapter 3. However, the fact that the effects of B A P T A were apparent after 30 min treatment suggests that this mechanism is unlikely to explain the results in Fig. 24. Thirdly, changes in C a 2 + signaling may be indirectly responsible for the change in topo Ila phosphorylation. For instance, inhibition of calcium-calmodulin dependent processes has been shown to increase topo II phosphorylation leading to increased sensitivity of the previously resistant cell line to etoposide cytotoxicity [100]. Although the exact mechanism for this enhanced phosphorylation is not known, inhibition of phosphotase 2B such as calcineurin possibly due to inhibition of Ca -calmodulin dependent protein kinase II has been suggested [100]. The presence of intracellular Ca transients has been demonstrated to be required for inhibition of Ca -calmodulin dependent processes [81]. Overexpression of mtsl in transduced V79, SiHa and C6 monolayers failed to directly affect the sensitivity of these cells to etoposide as measured by a cell survival 109 assay. Other results in the laboratory confirm the lack of a difference in etoposide sensitivity when measured using D N A strand breaks as the endpoint. This could be explained by the fact that the degree of mtsl overexpression that was achieved in these transduced cells was not sufficient to reduce the amount of intracellular free calcium. Results shown in Fig. 24 indicate that Ca2+chelation can dramatically reduce etoposide induced cell ki l l in V79 monolayers, although doses required to produce these effects were near-toxic. In addition results from our laboratory using another method to measure cell response to etoposide (the alkaline comet assay) indicate that treatment of V79 monolayers with 10 p M B A P T A reduced D N A damage by more than five fold while it did not significantly affect the outer cells of spheroids (Fig. 30). Under these conditions of reduced intracellular calcium, monolayer and spheroid responses to etoposide are clearly superimposable. Therefore, while overexpression of mtsl did not reduce the sensitivity of monolayers to etoposide, it is possible that upregulation of other calcium binding proteins (such as calretinin) could also be necessary and that relatively small changes in only one calcium binding protein are not sufficient to mimic the effects of BAPTA. In addition since the maintenance of intracellular calcium involves more than just calcium binding proteins (Fig. 22), other molecules involved in different parts of this signal transduction pathway could play a more important role in calcium mediated resistance to etoposide. Some of these molecules such as phosphotidylinositol 3-kinase (PI3-kinase) which works upstream of PLC have already been shown to affect alterations in cell-cell adhesion [190]. Therefore, the picture that is emerging is that growth as spheroids (and presumably xenograft tumours) causes a reduction in intracellular free calcium possibly due to 110 changes in cell-cell contact which has also been shown to play a role in cytoplasmic localization of topo I l a in spheroids [150]. This reduction leads to a decrease in topo I l a phosphorylation that in turn reduces nuclear localization of this enzyme. Less nuclear topo I l a and lower intracellular calcium means less cleavable complex formed in spheroids, and greater resistance of spheroids to killing and D N A damage by etoposide. Ill O monolayer • mono + B A P T A A outer spheroid ^ outer spheroid + B A P T A Etoposide (ug/ml) Fig. 30: Effect of BAPTA treatment on etoposide induced DNA damage. V79 cells were pre-treated for 30 min with 10 p M B A P T A followed by an additional 30 min incubation during etoposide treatment. B A P T A treatment makes V79 monolayers as resistant to D N A damage as the outer cells of spheroids. 112 CHAPTER 6 Summary and Future Directions 113 6.1 Summary The major cause of failure in cancer treatment is the emergence of tumour cell resistance to chemotherapy or radiation therapy. Many different mechanisms have been identified to explain tumour resistance to therapy such as overexpression of the membrane pump P-glycoprotein, expression of sulfhydryl compounds, enhancement of D N A repair, and inactivation of topoisomerase II. However, there may be many more causes for treatment failure. A n in vitro three dimensional tumour model known as multicell spheroids that was established many years ago has been extensively used to address many questions in tumour biology, tumour cell kinetics as well as tumour cell resistance to radiation and chemotherapy. The multicell spheroid is an attractive tumour model with an intermediate complexity between monolayer culture cells and in vivo tumours. While providing heterogeneity similar to that of solid tumours, it avoids the complications due to host factors. A particular area of research where this three dimensional tumour model has been repeatedly used in studies on the mechanism of "contact effect". Almost 30 years ago, Durand and Sutherland demonstrated that many cells show an acquired form of resistance to radiation in a three dimensional construct as spheroids in suspension culture which did not exist when the same cells were grown as monolayers. This form of resistance was therefore referred to as the "contact effect" and has also been observed in cells examined for sensitivity immediately after removal from tumour. Many other agents such as hyperthermia, photodynamic therapy, ultrasound and some topoisomerase II inhibitors like doxorubicin, m-AMSA and etoposide have also demonstrated a contact effect similar to that seen for ionizing radiation. The molecular mechanism behind this difference in sensitivity is not yet adequately understood, but 114 involvement of cell-to-cell communication, cell shape, and chromatin organization have all been proposed (Fig. 31). Therefore, the main objective of this project was to gain a better understanding of how the cellular microenvironment can cause resistance to treatment with the chemotherapeutic agent etoposide, a topoisomerase II inhibitor. Factors such as differences in growth fraction, distribution through the cell cycle, differences in drug uptake and efflux, involvement of gap juctional communication, and differences in the amount and activity of topoisomerase II had already been ruled out. However, in this study it was determined that topoisomerase Ila was about 10 times less phosphorylated in outer cells of V79 spheroids compared to monolayers. Since phosphorylation of topo Ila has been associated with etoposide sensitivity and nuclear translocation, it was not surprising when the immunohistochemistry and immunoblotting experiments revealed that topo Ila is primarily localized in the nucleus of monolayers, but in the cytoplasm of the outer cells of spheroids. The striking change from nuclear to cytoplasmic localization correlated with the increase in resistance to etoposide. More importantly, cells from xenograft tumours grown in SCH) mice confirmed that topo Ila binding patterns in tumours resembled those of spheroids and not monolayers, thus justifying the use of the spheroid as a tumour model. In addition, in an attempt to find out more about the molecular mechanisms involved in the resistance of spheroids to etoposide the method of differential display was employed to examine the changes in gene expression in cells from the outer cell layer of spheroids. Eight genes were identified and confirmed to be differentially expressed with reverse northern blotting. Genes upregulated in the outer cells of spheroids relative to monolayers included: 1) mtsl (metastatin; S100A4), a calcium binding protein 115 implicated in proliferation, metastasis and cell adhesion, 2) cytochrome c oxidase II, which contains a calcium binding site and 3) B-indl , a mediator of Rac signaling. Genes downregulated in spheroids were: 4) 2,4-dienoyl-CoA, a substrate in fatty acid biosynthesis 5) phosphoglycerate kinase, an important gene involved in glycolysis 6) ARL-3 , a ras-related GTP binding protein, 7) M H C class III complement 4A, and 8) T R A M , an endoplasmic reticulum protein. Upregulation of mtsl protein was subsequently confirmed by immunoblotting. In addition, another calcium binding protein calretinin was found to be upregulated in the outer cells of V79, C6 and particularly SiHa spheroids all of which had demonstrated a contact effect for etoposide and radiation. Of note, WiDr spheroid cells that do not show contact resistance, did not show upregulation of mtsl or calretinin. Therefore, these findings led to the study of the potential role of calcium in contact induced resistance to etoposide. Intracellular free calcium levels were found to be significantly lower in V79 and SiHa spheroids compared to their corresponding monolayers. Also, exposure of V79 monolayers and outer spheroid cells to B A P T A - A M , a calcium chelating agent, eliminated the difference in etoposide-induced D N A damage.. Therefore, changes in intracellular calcium regulation could underlie the contact effect. A potential mechanism for controlling phosphorylation of spheroid topo Ila involves the regulation of intracellular free calcium. Topo Ila becomes hypophosphorylated when intracellular calcium is chelated and as shown before, hypophosphorylation is associated with reduction of both cleavable complex formation and cell killing by etoposide. In addition, calcium has been shown to play a role in stabilizing the cleavable complex formed 116 between D N A , etoposide and topo II. Therefore, differences in calcium regulation could directly affect the sensitivity of cells to etoposide (Fig 32). In an effort to determine whether expression of a calcium binding protein would affect response to etoposide, SiHa, V79 and C6 cells were transduced with a mtsl viral construct. Unfortunately overexpression of mtsl had no apparent effect on etoposide sensitivity. However, as mtsl expressing cells also showed no decreases in intracellular free calcium, this result is probably not unexpected. Significant reductions in levels of intracellular free calcium, perhaps by up-regulation of several calcium binding proteins, may be necessary to develop contact resistance. Alternatively, the intracellular location of mtsl may be critical for function, and this was not examined in the transfected cells. Preliminary experiments were performed in our laboratory to determine whether simply adding excess calcium to the medium would affect sensitivity of V79 monolayers to etoposide. Although 10 m M CaCk (equivalent to 40 p M free calcium) more than doubled the amount of D N A damage by etoposide, completely removing calcium with E G T A and an ionophore also enhanced damage, probably by another mechanism (Banath, personal communication). 117 Growth as spheroids or xenographts > monolayers • Change in cell shape and cytoskeleton • Increase in cell-cell contact • Change in cell-ECM relationships • Increase in DNA repair fidelity • Change in regulation of cell cycle molecules Decrease in topo Ha phosphorylation • Change in Ca regulation Increase in calcium binding proteins Decrease in intracellular free Ca Fig 31: Possible mechanisms for the contact effect. Increase in p27 expression Decrease in radiation induced G2 block 118 Growth as Spheroids or Xenograft Tumours E-cadherin (p21 req'd) t other Ca++ binding proteins cyt. c oxidase calreticuli t S100A4 cells prepare to leave the cell cycle •Cleavable complex I formation/ topo II T phosphorylation changes in cytoskeleton t intercellular adhesion II Ca internal stores? C a + + influx? X-ray Toxicity t fidelity of double-strand break repair Fig. 32: Model representing the possible links between intracellular calcium and factors that could lead to resistance to etoposide and x-rays. 119 6.2 Suggestions for Future Work The results from this study have shed light on new molecular mechanisms that can be further pursued for a better understanding of cell-to-cell contact induced resistance to etoposide. In this work it has been shown that despite the same cell cycle kinetics, outer cells of spheroids show a significant amount of cytoplasmic topo Ila compared to nuclear localization of this enzyme in monolayers. Cytoplasmic topo I la has been shown to be more prevalent in late log phase and early plateau phase cultures [198]. Moreover, induction of cell differentiation can also result in a decrease in topo II phosphorylation that precedes the reduction in catalytic activity [36]. While the rate of division and cell cycle distribution in the outer cells of spheroids is the same as monolayers, many of these cells wil l be exiting the cycle as they pass into the interior of the spheroid. Therefore, the presence of cytoplasmic topo Ila in outer spheroid cells could be an early indication of the approaching exit from the cell cycle. We attempted to detect cytoplasmic topo Ila in V79 monolayers as they entered plateau phase but were unsuccessful. Although cells became very resistant to etoposide when approaching confluencing, this was associated with a loss of topo Ila, not with its redistribution. Apparently spheroids behave differently than monolayers in this regard. In another study it has also been demonstrated that resistance of V79 spheroids to another topo II inhibitor, m-AMSA, that acts by intercalation, occurs well before the departure of these cells from the exponential growth [204]. According to our results, changes in topo Ila localization occur only in the cycling cells of spheroids that are in close proximity to the non-cycling cells [151]. When these cells are removed from the outer layer of spheroids and put back in to suspension culture, during the first 24 hours topo Ila moves back to the nucleus possibly 120 due to the fact that neighboring non-cycling cells are not present. Therefore coordinated changes in cell cycle regulation when tumour cells are grown in close 3-dimensional contact could be occurring. Three other genes identified by differential display, T R A M , B-indl and PGK, may also be involved in changes in cell cycle regulation. Our preliminary results also indicate changes in amount and intracellular location of the cyclin dependent kinase inhibitor, p27k i p"1 (Fig. 33) (an opposite pattern to what is shown in this study with topo Ila). Results by Kerbel and colleagues also point to an increase in the level of p27 k i p l in mammary tumour cell spheroids and its link with E-cadherin expression [173]. Further studies should be pursued to examine the role of p27 expression and activity and its binding partners in monolayers, spheroids and xenograft tumours. The integration of these pathways is shown in Fig. 32 where calcium levels are proposed to play a key role in etoposide toxicity, cell cytoskelton, fidelity of D N A repair, and cell cycle regulation. Alterations in signaling pathways responsible for topo Ila phosphorylation could be further investigated. So far, initial experiments indicate differences in patterns of serine, threonine, and tyrosine phosphorylation between monolayers and outer cycling cells of V79 spheroids. Also, treatment with a non-toxic dose of okadaic acid (Oloumi, unpublished results) which is a potent inhibitor of serine/threonine protein phosphatases, PP1 and PP2a, increased the toxicity of etoposide in V79 spheroids, but not monolayers. Therefore, changes in phosphatase activity could be contributing to changes in sensitivity to etoposide. The use of a phosphatase inhibitor could be an effective way to selectively increase damage to tumour cells that exhibit cytoplasmic topo Ila. 121 Mono Outer Mid Inner ^ Spheroids Mono Outer Mid Inner ^ Spheroids Mono Outer Mid Inner I Spheroids Mono Outer Mid Inner SiHa WiDr V79 4 1 l l w Fig. 33. p27 immunohistochemistry and immunobloting. p 2 7 K i p l protein can be found in proliferating as well as non-proliferating cells of V79 and SiHa spheroids. The protein localization is primarily nuclear, although some cytoplasmic p27 is observed in V79 monolayers. 122 Although several genes were identified to be differentially expressed in spheroids compared to monolayers, the possible role of these genes in the contact effect has yet to be determined. As antibodies become available for these proteins, their relevance to the contact effect can be explored. Microarray analysis can now be used to more easily detect differences in gene expression, and based on the importance of topo II and identification of changes in p27 and B-indl , special emphasis should be placed on genes involved in cell cycle regulation. The direct effect of calcium on topo Ila phosphorylation needs further investigation. Monolayers and spheroids radiolabeled with P orthophsphate in the presence or absence of B A P T A - A M can be analyzed with anti-topo I la antibodies. It is also possible to examine the effects of increase in intracellular free calcium on etoposide toxicity. Although preliminary results to raise intracellular calcium were inconclusive, it is also possible to treat cells with thapsigargin to create a transient influx of calcium into the cytosol. The next step will be to determine whether these results can also to some extent explain the contact effect due to ionizing radiation and other agents. One possibility is that less topo I la in the nucleus increases radiosensitivity by altering chromatin conformation to promote more accurate D N A repair. In fact D N A topoisomerases have been suggested to play a role as repair enzymes [113]. More likely, changes in calcium levels can impact on many cellular functions including repair of radiation-induced damage. Calcium binding proteins have been linked with the double-strand break rejoining enzyme D N A - P K [206]. 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