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2001 Characterization of the ING1 candidate tumor suppressor gene in breast cancer cells

Nelson, Rebecca

Nelson, R. (2001). Characterization of the ING1 candidate tumor suppressor gene in breast cancer cells (Unpublished master's thesis). University of Calgary, Calgary, AB. doi:10.11575/PRISM/11964 http://hdl.handle.net/1880/41031 master thesis

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Characterization of the ING1 Candidate Tumor Suppressor Gene in Breast Cancer Cells

by

Rebecca Nelson

A THESIS SUBMITTED TO THE FACULTY OF GRADUATE STUDIES IN

PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF

MASTER OF SCIENCE

DEPARTMENT OF BIOCHEMISTRY AND MOLECULAR BIOLOGY

CALGARY, ALBERTA

AUGUST, 2001

© Rebecca Nelson 2001 Abstract

The nuclear proteins encoded by the ING1 candidate tumor suppressor gene have been suggested to play a role in apoptosis, cellular growth, DNA repair, chromatin remodeling and senescence. Aberrant expression of specific ING1 proteins has been observed in several different cancers and cancer cell lines, including those from breast, blood, and head and neck. Here, the role of the ING1 gene in breast cancer cells has been examined through cDNA microarray experiments. In response to overexpression of ING1b and ING1c proteins, we observed changes in expression of many other genes, including the downregulation of ribosomal proteins and cell structural proteins, and the upregulation of PCNA, a protein known to interact with ING1b under conditions of UV-induced DNA damage. Taken together, these results propose new mechanisms for ING1 involvement in DNA repair and chromatin remodeling, and suggest that ING1 proteins may participate in a new type of cellular stress response.

iii Acknowledgements

The work that went into this thesis would not have been possible without the help and guidance of many people. Firstly, I would like to acknowledge my supervisor, Dr. Karl Riabowol, for his patience, advice and support over the past three years. I would especially like to thank Dr. Doug Demetrick, one of my committee members, who not only provided me with wonderful resources and suggestions, but also with insight and guidance. Additionally, I would like to thank the other members of my committee, Dr. Julie Deans, and Dr. Peter Forsyth, for all of their support, helpful comments and suggestions, as well as Dr. Randy Johnston, who provided generous advice and encouragement. Thank you so much to all of the Riabowol lab members, including Michelle Scott, Paul Bonnefin, Phil Berardi, Diego Vierya, Jason Quarrie, Keith Wheaton, Denise Lawless, Paula Hettiaratchi, Parneet Cheema, Kyle Mackenzie, Yasuo Hara, Donna Boland, Vanessa Berezovski, Brad Unryn, Xiao Lan Feng and Lana Pastyryeva, for everything from work to play. Additionally, I would like to thank Dr. Sabita Murthy for her amazing help and patience with all of the microarray experiments, as well as members of the Demetrick and Forsyth labs, for their kindness and assistance. Outside of the lab, many friends have helped me enjoy my time in the city and in the mountains. A special thanks to my girls - Jen, Sue, Milena, Shanlee, Brenda, Tara and Tracy, for keeping me on track! Thanks also to Scott, Kosta, Greg, Eric, Duncan, JB, Tim, Peter, Heather, Carolyn and Knutforall of the ski trips, hikes, crazy parties and coffee breaks. Don't know what I'd have done without you! Last but not least, I would like to thank those other members of the Cancer Biology Research Group at the University of Calgary, who made my work possible. Many people have given me moral support, guidance and insight over the years, and I am grateful for your kindness.

iv Dedication

This thesis is dedicated to my little sister Amy, and to my mom Lynne and dad Paul, for helping me to climb mountains and reach for the stars. I wouldn't be where I am without you.

V TABLE OF CONTENTS

Approval Page ii

Abstract iii

Acknowledgements iv

Dedication v

Table of Contents vi

List of Tables ix

List of Figures x

List of Abbreviations xi

CHAPTER 1: INTRODUCTION 1

Breast Cancer and Tumor Suppressor Genes 2 Identification of the ING1 Gene 3 ING1 and Cancer 4 Structure of the ING1 Gene 6 ING1 Homologous Gene 6 ING1 Protein Motifs 8 Functions of the ING1 Gene 8 a) Apoptosis and DNA Repair 8 b) Cellular Growth and Senescence 10 c) Chromatin Remodeling 11 d) Cell Cycle Control 13 Research Hypothesis 14

CHAPTER 2: MATERIALS AND METHODS 18

Cell Culture Procedures 19 Animal Procedures 21 Generation of stably integrated inducible cell lines 22 Amplification and Manipulation of DNA 25 Protein analysis by quantitation, gel electrophoresis and western 28 Magnetic cell separation techniques 30 cDNA Microarray Techniques 31

CHAPTER 3: RESULTS 39

vi Part 1: The ecdysone inducible vector system 40 The ecdysone-inducible vector system is appropriate 41 ING1b sense, ING1b antisense, ING1a sense and p53 sense cDNAs can be cloned into the pIND inducible vector 42 Parental breast cancer cell lines are sensitive to antibiotics 42 Transiently transfected breast cancer cells can be induced to overexpress ING1b 43 Increasing levels of gene expression correspond with increasing levels of Muristerone A inducing agent 43 Stably integrated pVgRXR clones can be induced to overexpress pIND/LacZ 44 Stably integrated pVgRXR clones can overexpress plND/ING1b 45 Dual stable integrants are capable of overexpressing high levels of ING1b in initial immunofluorescence assays 45 Dual stable integrants are not capable of expressing high levels of protein upon induction with hormones 46 Single-cell subcloning of initially clonal population does not restore inducibility of the C5 ING1b clone 47 ING1b C5 stable clone is not inducible when either pVgRXR or plND/ING1b vectors alone are transfected in 47 Inducible transgene cannot be amplified in ING1b C5 stable clone, while antibiotic resistance in culture is still retained 48

PART 2: The murine mammary fatpad model is used to study human breast cancer growth and metastasis 49 Equivalent human breast tumors can be established 50 MDA MB 468 mammary fatpad tumors do not metastasize to the lung or contralateral fatpad tissue in a SCID mouse model 51

PART 3: Overexpression of the ING1b and ING1c gene isoforms result in upregulation and downregulation of other genes, as shown by cDNA microarray 52 Transiently transfected breast cancer cells express H-2Kk 54 Transfected cells purified on the MACs column overexpress ING1b and ING1c proteins 55 Total RNA can be isolated from MACs purified cells without significant degradation 55 Overexpression of ING1b and ING1c genes leads to the upregulation and downregulation of various genes in three different breast cancer cell lines 56 a) Fluorescent intensity ratios for each gene on the microarray slide are generated 56 b) Generation of Log-Transformed Intensity Ratios for Genes that are Unidirectionally Expressed 57 c) Cluster and Treeview Analysis of Gene Arrays 58 d) Significance Analysis of Microarrays 60

vii CHAPTER 4: DISCUSSION 101

Part I: The ecdysone-inducible vector system is effective for obtaining high levels of expression in transient assays 102 The Efficacy of Ecdysone-inducible Vector System for Generation of Transient and Stable Cell Lines 102 Cells transfected with ING1 antisense constructs do not consistently show decreases in ING1b levels 103 The ecdysone system is effective for generating stable pVgRXR clones in several breast cancer cell lines 104 Optimization of Conditions for C5 Induction and Expression Experiments are Inconclusive 106 Original C5 clone ceases to overexpress ING1b after several weeks in culture due to loss of transgene 107

PART II: The Murine Mammary Fatpad Model is Effective for generating human breast cancer tumors in SCID mice 110

PART III: The Role of INGIon transcription in breast cancer cells 111 The Origins of the cDNA Microarray 111 The ING1 cDNA Microarray 113 Upregulation of PCNA and MeiS2 115 Upregulation of signaling pathways 116 Upregulation of Receptors and Growth Factors 116 Upregulation of Enzymes and Cytochrome C 117 Downregulation of Proteins involved in RNA and Protein Synthesis 118 Downregulation of cell structural components and related proteins 119 Downregulation of the p53 tumor suppressor gene 121 Downregulation of c-Myb and A-Myb 123 Downregulation of the Rb-family pocket protein p107 125 Other downregulated genes of potential importance 126 Conclusions and Perspectives 127 Do ING1b and ING1c isoforms exert different effects? 127 Limtations of the cDNA Microarray Procedure 128

CHAPTER 5: BIBLIOGRAPHY 131

Appendix A: Buffer Solutions 148

Appendix B: Structures of antibiotics used for selection 151

viii LIST OF TABLES

Table 1. Properties of ATCC breast cancer cell lines selected for study 37

Table 2. Selective concentrations of antibiotic used for isolating stable Clones 62

Table 3. FACs results indicating percentages of cells expressing the H-2Kk molecule before and after separation on the magnetic column 62

Table 4. Summary of overexpressed and underexpressed genes in MCF7, MDA MB 435 and MDA MB 468 cell lines 97

ix LIST OF FIGURES

Figure 1: The different known isoforms of the human ING1 gene 16 Figure 2: The functional motifs of three ING1 isoforms 17 Figure 3: Components of the ecdysone inducible vector system 63 Figure 4: Breast cancer cell lines transiently transfected with various inducible constructs and induced with different concentrations of Ponasterone 64 Figure 5: Breast cancer cell lines transiently transfected with ING1a and p53 inducible constructs and induced with Ponasterone 65 Figure 6. X-gal staining of MDA MB 468 pVgRXR stable integrants 66 Figure 7. pVgRXR clone screening by immunofluorescence 67 Figure 8. MDA MB 468 plND/ING1b sense stable clones screened by immunofluorescence 68 Figure 9. MDA MB 468 plND/ING1b antisense and plND/p53 stable clones screened by immunofluorescent staining 69 Figure 10. Western blots of pVgRXR/pIND stable cell lines containing ING1b antisense and p53 transgenes, induced with Ponasterone A 70 Figure 11. Time courses for induction and dose response of the inducible MDA MB 468 ING1b sense clone 5 (C5) 71 Figure 12. Clonal dilution of the inducible MDA MB 468 ING1b sense clone 5 (C5) 73 Figure 13. Transient transfection of MDA MB 468 ING1b C5 cells with pVgRXR, plND/ING1b and both constructs simultaneously in order to determine if one or both have been lost 73 Figure 14. Verification of the presence of the plND/ING1b inducible construct in the C5 clone which appears to have lost inducibility 74 Figure 15. Mammary Fatpad Tumor Growth Curve of two different volumes of MDA MB 468 breast cancer cells innoculated into SCID mice 75 Figure 16. The bicistronic pKK vector of the magnetic cell separation kit 76 Figure 17. The MACs cell separation technique 76 Figure 18. Verification of ING1b and ING1c overexpression in pKK vectors after magnetic cell separation 77 Figure 19. Verification of RNA integrity by observation of 28s and 18s ribosomal rRNAs during agarose gel electrophoresis 77 Figure 20. cDNA Microarray graphics generated by Scanarray software 78 Figure 21. Composite image generated in Quantarray 78 Figure 22. Scatter plots for each microarray scan 79 Figure 23. Numerical data generated by Quantarray 83 Figure 24. Numerical data generated by Datahandler 84 Figure 25. Data manipulation in Excel 85 Figure 26. Clustered gene tree diagrams 86 Figure 27. Significant gene list (a) and SAM plot (b) for cDNA microarrays 91

X Abbreviations

AML acute myelogenous leukemia APS ammonium persulfate ATP adenosine 5'-triphosphate CBP CREB binding protein CDNA complimentary DNA DAP I 4'-diamidino-2-phenylindole DEPC diethyl pyrocarbonate DMEM Dulbecco's Modified Eagle Medium DMSO dimethylsulfoxide DNA deoxyribonucleic acid dNTP 2'-deoxynucleotide 5'-triphosphate DTT dithiothreitol EDTA ethylynediamine-tetraacetic acid FBS fetal bovine serum FDR false discovery .rate g gram HAT histone acetyltransferase HDAC histone deacetyltransferase HDMEM high glucose DMEM ING1 inhibitor of growth 1 kb kilobase pair kDa kilodalton LB Luria-Bertani bacterial medium LOH loss of heterozygousity m molar mfp mammary fatpad mg milligram mL milliliter mM millimolar

xi mRNA messenger RNA NaCI sodium chloride ng nanogram NLS nuclear localization signal NTS nucleolar targeting sequence PBE phosphate buffered EDTA PBS phosphate buffered saline PCNA proliferating cell nuclear antigen PCR polymerase chain reaction PHD plant homeodomain PIP PCNA-interacting protein Rb retinoblastoma tumor suppressor protein RNA ribonucleic acid RNase ribonucleic acid nuclease rpm revolutions per minute rRNA ribosomal RNA SAM significance analysis of microarrays SCID severe combined immunodeficient SDS sodium dodecyl sulphate SDS-PAGE SDS polyacrylamide gel electorphoresis SSC standard saline citrate SV40 simian virus 40 TAE Tris-acetate EDTA buffer TEMED N,N,N',N'-tetramethylethyldiamine Tris Tris (hydroxymethyl)aminomethane UV ultraviolet X-gal 5-Bromo-4-chloro-3-indolyl-beta-D-galactopyranoside microgram ml microliter HM micromolar °C degree Celsius

xii 1

CHAPTER 1: INTRODUCTION Breast Cancer and Tumor Suppressor Genes Breast cancer is the most common malignancy occurring in women, and is the second most frequent cause of death from cancer [1]. There has been a large and steady increase in the incidence of breast cancer over the past few decades, and the lifetime risk of developing the disease is 12.2% or approximately 1 in 8 women [2]. Multiple risk factors exist which have been shown to increase an individual's chances of developing breast cancer, including genetic and familial factors, hormonal factors, dietary factors, the presence of benign breast disease, and environmental factors [3]. Of major interest are the inherited and acquired genetic alterations, which lead to the malignant phenotype. Tumour suppressor genes, which act as negative growth regulators, are key components in the pathway to malignancy. If they are mutated or become inactivated, a loss of control over cellular growth and/or proliferation can result, and thus a large number of them have been implicated in the development of both sporadic and inherited breast cancer. Mutations in the p53 gene are known to occur in more than 50% of all human tumours; similarly, 50% of breast tumours also harbor p53 mutations [4]. In addition to controlling a number of critical cellular events such as the cell cycle, apoptosis, differentiation and development, p53 also acts as a transcription factor [5, 6]. Other tumour suppressor genes, such as the Rb and p16 cell cycle inhibitors, are found to be inactivated in approximately 20% and 30% of breast tumours, respectively [7]. Also of importance are the BRCA1 and BRCA2 susceptibility genes found mutated in familial breast cancer: germline BRCA1 mutations are associated with a 50% risk of breast cancer by age 45 and a cumulative lifetime risk of 85% [8], while BRCA2 mutations are associated with the development of breast and ovarian cancer at a lower risk than BRCA1 [9]. These proteins have been shown to encode molecules with both transcriptional regulatory and DNA repair functions [7]. Identification of the ING1 Gene Tumour suppressor genes are key protective mechanisms in the negative regulation of cellular growth and proliferation. They serve to maintain a normal growth rate in all tissues and prevent uncontrolled cellular growth and proliferation, leading to tumourigenesis. When wild type function of one or more of these genes is lost, cells gain the ability to multiply repeatedly and can become malignant. Mutations in many tumour suppressor genes, such as p53 and the Retinoblastoma (Rb) genes, have been implicated in a wide range of cancers, such as brain, breast, ovarian, prostate, and numerous others. It is thus important to identify new tumour suppressor genes so that their role in maintaining normal cell growth and in tumourigenesis can be understood. The ING1 gene was first isolated by subtractive hybridization of cDNAs from normal and malignant human breast epithelial cells [10]. The rationale behind cloning genes by subtractive hybridization using normal and malignant cDNAs is that the expression of negative growth regulator or tumor suppressor mRNAs in normal cells may be repressed or aberrant in malignant cells. In order to take advantage of this dissimilarity, several sets of cDNA were prepared; one from normal human breast epithelial cell cDNA and the others from MCF7, Bt-483, BT-474, Hs578T, MDA-MB-468, MDA-MB-435 and BT-20 breast cancer cell lines. Prior to hybridization, cDNAs were restriction digested and ligated to adaptors: "A" adaptors for the normal epithelial cDNAs and "B" adaptors for the breast cancer cell cDNAs. The cDNAs were then combined, hybridized and treated with Mung bean nuclease to eliminate single-stranded A-B adaptor hybrid ends, and homologous A hybrids were amplified by PCR to enrich for sequences expressed preferentially in the normal cDNA pool. This cycle was repeated several times using the recovered cDNAs as tester and the breast cancer cDNAs as driver. After enrichment through several cycles, the remaining cDNA was used to screen a normal senescent cell cDNA library and approximately 200 clones were chosen, fragmented and subcloned into the pLNCX viral vector. The subcloned 4 were packaged into retroviral particles in the BOSC packaging line and recombinant retrovirus was used to infect normal mouse mammary epithelial cells. Sequences which induced tumor formation in a murine tumor model, were rescued by PCR and analyzed. Using this strategy, a cDNA of 182 bp in length and in antisense orientation was isolated, and determined to encode a protein given the name ING1 for "Inhibitor of Growth". Overexpression of ING1 protein in human breast cancer cell lines and normal human fibroblasts was shown to inhibit cell growth and to arrest cells in the G0/G1 phase of the cell cycle. Conversely, overexpression of ING1 antisense mRNA was shown to promote cell transformation in immortalized mouse mammary epithelial cells and in focus formation and colony formation using in vitro assays. Analysis of ING1 expression levels in different transformed cell lines showed that reduced levels of ING1 protein were present in 7 of 7 breast cancer lines when compared with the phenotypically normal breast epithelial cell line MCF10A as a control. From these initial experiments, it was suggested that the ING1 gene encoded a novel growth inhibitor and candidate tumor suppressor protein.

ING1 and Cancer

The p33ING1 protein was shown to localize to the nucleus and its gene was mapped to chromosome 13q33-34 [11, 12]. Interestingly, this juxtaposes ING1 to a chromosomal region that is frequently deleted or translocated in gastric cancers [13], head and neck squamous carcinomas [14], esophageal cancers [15, 16] and lymphoid malignancies [17]. Loss of heterozygosity (LOH) of the 13q region has been reported to occur at high rates in breast cancer [18], and cancers of the ovary [19], kidney [20], lung [21] and head and neck [22]. Recent studies have attempted to identify LOH at the ING1 locus and ING1 gene mutations in colorectal carcinomas [23] lymphoid malignancies [24], and oral squamous cell carcinoma [25]. Neither mutation (as detected by electrophoretic mobility shift assay of single-stranded PCR products) nor LOH were detected in any of the 29 colorectal carcinoma samples tested [24] or in 71 oral squamous cell carcinomas. Of the 16 lymphoid cell lines tested for mutation by the same method, none were found to contain ING1 point mutations or deletions. However, when the same 16 cell lines were assayed by reverse-transcriptase polymerase chain reaction (RT-PCR), 4 of 5 T-cell lines and 5 of 11 B-cell lines exhibited decreased ING1 expression in comparison with normal peripheral blood polymorphonuclear cells [24]. These results suggest that in lymphoid cell lines, decreased ING1 expression may be due to mechanisms occurring at the transcriptional or post-transcriptional level. The most recent mutational analysis on ING1 was performed in human esophageal cancer [26]. Out of 31 cases, allelic loss at 13q33-34 was identified in 58.9% of tumors, and ING1 missense mutations were identified in four of these tumors. All of these mutations were found in the plant homeodomain (PHD) finger and nuclear localization motif of the ING1 gene, two regions that are likely critical for normal ING1 function. In addition, when ING1 protein expression levels were assayed in these tumors by immunohistochemistry, all 31 showed that levels of ING1 protein were undetectable. This study provides significant evidence that ING1 protein expression levels and genetic alterations are correlated with the development of esophageal sqamous cell carcinoma. Importantly, aberrant ING1 gene expression has also been observed in primary tumor tissue from individuals with sporadic breast cancer, as well as several breast cancer cell lines [10, 27]. In a study of 452 primary tumor samples, it was determined that only one germline missense mutation and three germline silent mutations existed in 377 of the samples, however ING1 mRNA levels were markedly decreased in 44% when compared with normal breast tissue and in 100% of breast cancer cell lines examined [27]. Furthermore, it was determined that of the primary tumors with decreased ING1 expression, the majority (58%) had metastasized to regional lymph nodes, whereas only 9% of the tumors with increased ING1 expression (as compared to normal tissue) had metastasized. This observation highly correlates a decrease in ING1 expression with breast cancer metastasis, and is an area requiring further investigation. 6

Structure of the ING1 Gene The ING1 gene structure was initially deduced from both Hs68 and HeLa cDNA library clones [10], and sequence analysis predicted a protein of approximately 33 kilodaltons. The gene was determined to have no significant homology to known coding sequences when compared to available databases, with the exception of limited homology to the retinoblastoma binding protein 2 (RbBP2) and several zinc finger transcription factors. Presently, the ING1 gene is known to comprise at least 3 exons and 2 introns which, when alternately spliced give rise to several mRNA transcripts and protein isoforms (Fig. 1, p. 16) [28-30]. A large number of isoforms are present in different human tissues, and can be observed on western blots using polyclonal antibodies directed against the conserved exon. Two isoforms, ING1a and ING1b, have molecular weights of 47 kDa and 33 kDa respectively, and have been studied in greater detail than the third ING1c isoform of 24 kDa, which was detected and cloned more recently. Both ING1a and ING1b isoforms share an identical 3' region of 742 bp (27 kDa), encoded by a common exon (Fig. 1, p. 16). Although this exon also encodes ING1a, an internal initiation within the common exon results in the truncated 24 kDa protein, lacking 3 kDa encoded by the exon's 5' region. It is suggested in the literature that the ING1c protein is generated from a truncated ING1a mRNA [31], however both ING1b and ING1a could lead to its generation via internal initiation, and its own discrete transcript may encode it. Other studies suggest that additional isoforms exist, yet these await further characterization [32].

ING1 homologous genes ING1 gene homologues have been found in yeast, mouse, rat, Drosophilia, Xenopus and human and their degree of conservation has suggested that their functions may have been conserved evolutionarily. Indeed, studies in Saccharomyces cerevisiae show that when the ING1 homologue YNG2 is knocked out, several mutant phenotypes, such as sensitivity to heat shock and to caffeine are generated that can subsequently be abrogated by expression of either human ING1 or Schizosaccharomyces pombe Png1 [33]. Studies report that the mouse ING1 gene consists of a differentially regulated promoter which produces 3 mRNA transcripts, also containing a shared region encoded by a common 3' exon [34]. Two of these transcripts generate an identical protein, analogous to human ING1c, while the third transcript generates a protein of 37 kDa analogous to human p33ING1b. In addition, several genes encoding proteins related to ING1 have been described. Consistent with a role in regulating cell growth, a gene located on chromosome 4q35.1 is expressed aberrantly in a subset of colon cancers [35]. The gene, named ING1L for "ING1-like," encodes a protein containing a PHD- domain, analogous to ING1. Studies indicated that ING1L encoded a protein of 280 amino acids with a molecular weight of approximately 32.8 kDa with considerably high homology, both at the nucleotide and protein levels, to ING1. The PHD domain was shown to be present in the carboxy regions of both proteins, residues 213-260 in ING1L. A second ING1-homologous gene was cloned in 1999 by Jager et al. During a library screening procedure in an attempt to isolate the original ING1 gene sequence, they also identified a 593-bp novel cDNA with 78% homology to the original ING1 gene. Further analysis indicated that the full length cDNA was 771 bp in length which was most homologous to the original ING1 nucleotide sequence at it's C-terminal end, coincidentally the region of ING1 encoding its PHD motif. The novel cDNA, designated ING2, was assessed for open reading frames (ORF). The longest ORF identified was 129 bp long and encoded a protein of 42 amino acids, which is much smaller than any of the ING1 proteins, and possesses 76% homology to the ING1 protein isoforms. However, this short polypeptide is of unknown function and significance and the term ING2 is now used synonymously with ING1L to differentiate the ING1 and ING2 genes. Thus, we not only see that the ING1 gene is highly conserved between species, but the presence of several other related genes within the same species may suggest that it is one member of a family of genes encoding proteins whose functionalities have yet to be determined.

ING1 protein motifs Several protein motifs and conserved targeting sequences have been identified in the ING1 isoforms. The most conserved sequence motif in ING1 is

the PHD finger, a Cys3-His-Cys3 sequence [33] located within the final 70 amino acids of the C-terminus (Fig. 2, p. 17). In other proteins with PHD finger motifs, such as transcription factors, this sequence is thought to mediate transcriptional regulation and/or chromatin interactions [36]. The ING1 proteins also possess nuclear localization sequences (NLS), encoded in the common exon, two independent nucleolar targeting sequences (NTS) and a PCNA-interacting protein sequence (PIP) [37]. While all ING1 proteins contain PHD, NLS and NTS domains, only ING1b contains a PIP sequence and consequently is the only isoform to bind PCNA in a DNA damage-dependent manner. One additional sequence motif has been identified in the ING1b protein: a partial bromodomain, which is a motif that recognizes and binds certain acetyl-lysine residues [38, 39]. In proteins such as hsGCN5 (GenBank accession number U57136), scGCN5 (Q03330), hs p300 (A54277), hsCBP (S39162), hsP/CAF (U57317), ttP55 (U47321), scBDFI (P3817) and hsCCGI (P21675), the bromodomain motif is very long (more than 100 amino acids) and specifies a four-helix bundle with a left-handed twist [40]. ING1b possesses approximately 40 amino acids of a partial bromodomain with a high degree of conservation of the key residues.

Functions of the ING1 proteins

a) Apoptosis and DNA Repair Given that many tumor suppressors affect apoptosis, it was speculated that

p33ING1b might also play a role in some apoptotic pathway. Consistent with this idea, ING1 protein levels were shown to increase upon induction of apoptosis in serum-starved P19 teratocarcinoma cells [41]. When ING1 and c- Myc were co-overexpressed in P19 cells and rodent fibroblasts, apoptosis was drastically enhanced in a manner consistent with ING1 synergy with c-Myc. This suggested that p33ING1b modulates cell death in a pathway that is linked to Myc- dependent apoptosis. When antisense ING1 mRNA was overexpressed in conjunction with c-Myc, cells were significantly protected against apoptosis,

supporting the idea that loss of function of wild type p33ING1b may contribute to tumorigenesis by enabling cells to evade apoptosis. Reintroduction of wild type p53 function confers sensitivity to apoptosis and growth arrest, which is similar to the increase in apoptotic cells when p33ING1b is overexpressed in c-Myc induced

cells. These observations provide evidence that the p33ING1b protein exhibits characteristics similar to those of the previously defined Type 2 tumor suppressors [42].

The idea that p53 and p33ING1b cooperate to regulate cellular growth was first introduced by Garkavtsev et al. when they observed a physical interaction between the two proteins when they were overexpressed, and determined that

overexpression of p33ING1 corresponded with an upregulation of the p53-

regulated cell-cycle inhibitor p21WAF1 [43]. To further explore this connection, a second group reported that co-infection with adenoviral constructs encoding p53

and p33ING1b dramatically enhanced apoptosis in glioma cell lines, while infection with either construct alone had a more limited effect on apoptosis [44]. In an attempt to further delineate the apoptotic pathway involved, the group did not observe any increases in Bax or Fas expression, nor did they see an upregulation of manganese superoxide dismutase, hsp32, c-Jun or glutathione•

s-transferase II when p53 and p33ING1b were co-infected. However, more recent studies have shown that both p33ING1b and p33ING2 are capable of inducing

p21WAF1 and Bax expression [45]. Thus, the specific mechanism of ING1 involvement in apoptosis remains to be determined. Most recently, the ING1/p53 relationship has been explored using p53 knockout mice, and through experiments on UV-mediated DNA damage in keratinocytes, HeLa cells and the MMRU melanoma cell line [46, 47]. In mouse experiments, ING1b mRNA and protein levels were compared between tissues from p53 knockout mice and normal mice, and no difference was observed. This suggested that ING1b expression was independent of p53. The next set of experiments compared ING1b expression levels in normal and p53-mutated keratinocytes after they had been exposed to UVB radiation. In both cell lines, an elevation in ING1 b protein levels was observed, again suggesting that ING1 b expression is not reliant on p53. When ING1b was cotransfected into melanoma cells with a UV-damaged reporter plasmid, a 2-4 fold increase in repair rate occurred when compared with vector controls. When the same assay was performed in cells with dominant negative p53, no increase in repair rate was observed, suggesting that p53 is required for the repair function of ING1. The p53-responsive GADD45 DNA repair protein was also identified to be weakly associated with ING1b, suggesting that ING1b may play an important role in the GADD45 -mediated nucleotide excision repair pathway. Thus, the relationship between p53 and ING1b does not appear clear-cut, and requires further investigation.

b) Ce llular Growth and Senescence Soon after the discovery of the candidate tumor suppressor, it was studied for involvement in cellular senescence, given that other well-characterized tumor suppressors (e.g. Rb, p53) are known to be involved in this process [48]. It was determined that p33ING1b expression was elevated 8-10 fold in senescent compared to young, dividing Hs68 human fibroblasts [49]. In parallel experiments, suppressing ING1 expression by overexpression of antisense mRNA appeared to extend the normal replicative lifespan of normal fibroblasts by approximately seven population doublings. However, in more recent experiments, upregulation of ING1 gene expression has not been observed in all primary cell strains under different conditions of growth and so the significance of the original observation of upregulation during cell aging is unclear. Additionally, this study established that p33ING1b protein was cell-cycle

regulated: levels of ING1b protein were detectable in G0, decreased to undetectable levels following serum stimulation in early Gi and increased in late

G^ reached a maximum level in S phase, and decreased in G2. Collectively, these observations implicate p33ING1b in cellular growth and senescence pathways, and draw additional similarities between p33ING1b and other classical tumor suppressors.

c) Chromatin Remodeling The first indication that ING1 may play a role in chromatin remodeling came from studies of ING1 homologues in yeast [33]. Using a two-hybrid system, it was demonstrated that Yng2, one of three Saccharomyces cerevisiae proteins showing homology to ING1, interacts with the yeast protein Tra1, a component of histone acetyltransferase complexes (HATs) [50]. The reversible process of histone acetylation is known to serve an important function in the control of gene transcription. It is widely believed that the addition of acetyl groups to conserved lysine residues on core histones neutralizes the positive charge on the histones, increasing their hydrophobicity and thus decreasing their association with DNA [51]. This is thought to alter the conformation of DNA and facilitate the binding of transcription factors and hyperacetylated histones are associated with transcriptionally active promoters. The process of deacetylation removes acetyl groups, re-establishes a highly positive charge on histones, and is thought to be associated with transcriptionally silent DNA [52].

Yeast studies showed that two additional yeast ING1 homologues, as well as human ING1b, could associate in complexes containing HAT activity in yeast. They also identified Esa1, a component of the NuA4 HAT complex, as the Yng2- associated HAT. The human homologue of a second Yng2- associated protein Tra1 is TRRAP, a member of the PI3 kinase/ATM family of proteins. Recently, Scott et al. (2001) demonstrated that human p33ING1b binds TRRAP and other proteins with HAT activity, including CBP and PCAF, suggesting this function has been conserved through evolution [53]. TRRAP is a 434 kDa nuclear protein known to interact with c-Myc, bringing histone acetyltransferase activity to a protein complex containing c-Myc via recruitment of GCN5 [54]. It also binds the E2F -1 transactivation domain and is an essential cofactorfor c- Myc and adenovirus E1 A-mediated oncogenic transformation. CBP and PCAF are both human proteins which possess intrinsic HAT activity [55, 56]. These proteins interact with phosphorylated cyclic AMP (cAMP) and many other transcription factors, including p53 and several nuclear hormone receptors , such as RXR, RAR, and TR. CBP and PCAF preferentially acetylate histones 3 and 4 (H3 and H4), with H2A and H2B being acetylated to a lesser extent, similar to the results obtained when p33ING1b is overexpressed. CBP/p300 have also been implicated in tumor suppression, as certain missense mutations have been found in CBP/p300 in different cancers. [52]. It has also been suggested from independent studies that HAT activity can be a contributing factor in carcinogenesis: a small number of AML patients possess a translocation involving the fusion of two HAT domains [57]. Furthermore, overexpressed p33ING1b increases and p47ING1a decreases acetylation of histones 3 and 4, which are involved in gene expression regulation through local chromatin remodeling. At present, the function of ING1 proteins within these complexes remains to be determined. Additionally, it has recently been established that ING1b also interacts with members of the mSin3a transcriptional corepressor complex, including HDAC1, Sin3, SAP30 and RbAp48 proteins [58]. This further substantiates a role for ING1 proteins in chromatin remodeling, and suggests that the ING1 proteins may be involved in two opposing processes: activating transcription by histone acetylation, and inhibiting gene expression by histone deacetylation. Coincidentally, many well-known tumor suppressor genes are known to associate with proteins involved in chromatin remodeling. Both BRCA1 and BRCA2 are linked with HAT activity: BRCA1 has been shown to interact with CBP, and the BRCA2 molecule possesses intrinsic HAT activity [59, 60]. A classical tumor suppressor molecule known to physically interact with both HDAC1 and HDAC2 is the Retinoblastoma (Rb) protein [61]. When bound on promoters that are active in S-phase, it is thought to recruit these histone deacetylases to the E2F1 transcription factor, constituting one mechanism by which Rb utilizes E2F1 to inhibit cell-cycle progression. Finally, p300 has been shown to interact with the p53 tumor suppressor protein [62], and CBP/p300 is thought to co-activate and enhance p53's transcriptional activity [59].

d) Ce II cycle control

Upon its discovery, the expression of p33ING1b was shown to be regulated through the cell cycle, with maximal protein levels achieved in S-phase [49]. More recently, it has been shown that after treatment of cells with a number of agents known to induce high levels of p53 expression, including y-irradiation, cisplatinum, hydrogen peroxide, and UV light, high levels of p33ING1b expression are induced when keratinocytes but not fibroblasts are treated with UV [47]. Another protein involved in cell cycle regulation which is DNA damage- and serum-inducible in normal human fibroblasts is the proliferating cell nuclear antigen (PCNA) [63]. In a series of immunoprecipitation and colocalization experiments, exposure of fibroblasts to UV light was shown to induce a strong physical association between PCNA and p33ING1b, but not with other ING1 protein isoforms, including p47ING1a and p24ING1c. The site through which p33ING1t interacts with PCNA appears to be one which shares strong homology with other known PCNA-binding proteins, such as p21, the DNA repair protein XPG, GADD45, and the Fen-1 exonuclease protein [64], among others. The short motif, known as the PIP box for PCNA interacting protein sequence appears to

CIP1/WAF1 MTS1 mediate this interaction since overexpression of P21 but not p16 can compete with p33ING1b for PCNA binding [53]. Also, deletion of, or partial mutation within the PIP box abrogates or inhibits p33ING1b binding. This binding suggests a role for ING1 in DNA repair and may link DNA replication/repair directly to chromatin remodeling through modulation of HAT/HDAC activity.

Research Hypothesis The experiments described within this thesis were designed to test two major hypotheses. Since the most recent evidence indicated that the ING1 gene behaved as a growth inhibitor and candidate tumor suppressor in vitro, that p33ING1b expression was reduced in a large number of primary breast tumor samples (when compared with normal contralateral breast tissue), and that levels of p33ING1b expression were highly correlated to metastasis, we speculated that the ING1 gene would act as a bona fide tumor suppressor in an analogous in vivo system. Taking into consideration the identification of novel ING1 protein isoforms, we proposed to test the effects of both ING1a and ING1b expression on breast tumor growth and metastasis. We predicted that the ideal system in which to test this idea was the Ecdysone Inducible Expression System (Invitrogen) because it was reported to tightly regulate gene expression and to avoid potential problems, such as biological selection against expression of a negative growth regulator that appeared to have been encountered by other groups using constituitive expression systems. Additionally, we hypothesized that the optimal model in which to observe tumor growth and metastasis effects was the murine mammary tumor model. This model uses the orthoptic or most anatomically relevant murine site for implantation of human breast tumors and the most effective site for facilitating growth and metastasis is the murine mammary fatpad [65, 66]. Due to its potential role in chromatin remodeling through association with histone acetyltransferase and deacetylase activities, the secondary hypothesis tested was that the ING1 gene may play a role in gene transcription and/or

repression. The aberrant expression patterns of p33ING1b observed in breast cancer cells led us to hypothesize that testing different ING1 isoform expression in this cell system would produce valid and potentially clinically relevant results. To investigate this hypothesis, a cDNA microarray was performed, using control cDNA from human breast cancer cells, and test cDNA from the same cells overexpressing ING1b or ING1c. It was rationalized that this approach would enable us to observe the responses of a wide range of candidate genes, and give some indication as to the role ING1 plays in the various cellular processes with which it is associated. This thesis contains new data on the role of the ING1 gene in human breast cancer cells. It is the first study to identify global differences in functionality of ING1 gene isoforms, and data presented here further solidifies a link between ING1 functionality in apoptosis, cell cycle control, chromatin remodeling and DNA repair. 16 Figure 1: The different known isoforms of the human ING1 gene. The common exon is represented in bold.

INGla MSFVECPYHSPAERLVAEADEGGPSAITGMGLCFRCLLFSFSGRS 45

ING1a GVEGGRVDLNVFGSLGLQPWIGSSRCWGGPCSSALRCGWFSSP 88

INGla PPSKSAIPIGGGSRGAGRVSRWPPPHWLEAWRVSPRPLSPLSPAT 133

INGla FGRGF\AVAV\PGL\N ARGRGCSSDRLPRPAGPARRQFQAASLLTR 178 ING1b MLSPANGEQLHLVNYVEDYLDSIESPFDLQRNV 33

INGla GM/Gf?/AlVPlV/

INGla SQELGDEKIQIVSQMVELVENRTRQVDSHVELFEAQQELGDTAGN 267 ING1b SQELGDEKIQIVSQMVELVENRTRQVDSHVELFEAQQELGDTAGN 122 ING1C SQELGDEKIQIVSQMVELVENRTRQVDSHVELFEAQQELGDTAGN 56

INGla SGKAGADRPKGEAAAQADKPNSKRSRRQRNNENRENASSNHDH 310 ING1b SGKAGADRPKGEAAAQADKPNSKRSRRQRNNENRENASSNHDH 165 ING1C SGKAGADRPKGEAAAQADKPNSKRSRRQRNNENRENASSNHDH 99

INGla DDGASGTPKEKKAKTSKKKKRSKAKAEREASPADLPIDPNEPTYC 355 ING1b DDGASGTPKEKKAKTSKKKKRSKAKAEREASPADLPIDPNEPTYC 210 ING1C DDGASGTPKEKKAKTSKKKKRSKAKAEREASPADLPIDPNEPTYC 144

INGla LCNQVSYGEMIGCDNDECPIEWFHFSCVGLNHKPKGKWYCPKCR 399 ING1b LCNQVSYGEMIGCDNDECPIEWFHFSCVGLNHKPKGKWYCPKCR 254 ING1C LCNQVSYGEMIGCDNDECPIEWFHFSCVGLNHKPKGKWYCPKCR 188

INGla GENEKTMDKALEKSKKERAYNR ING1b GENEKTMDKALEKSKKERAYNR ING1C GENEKTMDKALEKSKKERAYNR Figure 2: The functional motifs of three ING1 isoforms

-common region • N PHD p47 ING1a

B

p N PHD p33ING1b

N PHD p24 ING1c

N nuclear localization signal and nucleolar targeting motifs PHD plant homeodomain motif P PCNA-interacting protein motif B partial bromodomain motif CHAPTER 2: MATERIALS AND METHODS 19 Cell Culture Procedures

Parental Cell Lines Five different ATCC mammary epithelial tumor cell lines were selected for the study. Considerations in selecting cell lines were: a) their tumorigenicity in SCID mice, b) their ability to metastasize in SCID mice, and c) their low levels of endogenous ING1 expression. Parental lines with different mutations were selected in order to provide a diverse genetic environment in which ING1 expression could be assayed. Since a number of different factors are known to contribute to breast cancer development, metastasis, prognosis and serve as indicators for treatment, it is important to analyze the effects of ING1 overexpression and underexpression with some of these factors in mind. The American Type Cell Collection (ATCC) cell lines selected for this study were BT- 20, Hs578T, MCF7, MDA MB 435, and MDA MB 468, and some of their properties are listed in Table 1.

Culture Media Cells were grown in high glucose Dulbecco's modified Eagle's minimal essential media (HDMEM, Gibco BRL). Media was sterilized by filtration and supplemented with 10% fetal bovine serum (FBS, Gibco BRL), as well as penicillin G (75 units/mL) and streptomycin (50 ng/mL). Fortransfection by electroporation, serum- and antibiotic-free HDMEM was used.

Cell Splitting and Plating Cells were maintained as monolayer cultures at 37°C in 5% carbon dioxide. Using trypsin-EDTA (Gibco BRL, 0.05% trypsin (1:250) and 5 mM EDTA), cells were split into new dishes prior to reaching confluence. For immunofluorescence, glass coverslips were bathed in ethanol to sterilize, and dried under UV light for 15 minutes. Dry coverslips were placed into 8-well dishes, and 1.5 ml_ warmed media was added. Cells were aliquoted in 500 jaL volumes into each well, and allowed to adhere for 24 hours prior to use.

Counting Cells Cells were counted by the Trypan Blue Dye-Exclusion method using a hemocytometer. A 10 JJL aliquot of trypan blue dye solution (Sigma, 0.4% trypan blue, in 1x Hank's Balanced Salt Solution, Gibco BRL) was combined with a 10 |aL aliquot of cells, and 10 jaL of this mix was aliquoted onto a hemocytometer slide. Four large squares of viable cells were counted, and the total number of viable cells was calculated:

Total number of viable cells/mL = (cells in four large squares/4) x 2 x 104

Mycoplasma Testing of Cell Lines Cells were tested using the Mycoplasma PCR Plus Test Kit (Stratagene #302007) as recommended by the supplier, using the boiled extract of cell culture media preparation method.

Transfection of Human Breast Cancer Cells by Electroporation Each 15 cm dish was transfected by trypsinizing cells when they had reached 80-95% confluency, resuspending in 450 [xL serum-free high glucose DMEM (GIBCO BRL), and transferring into a 4 mm gap electroporation cuvette. To the cell suspension, 15 |ag plasmid DNA plus 35 ug salmon sperm DNA (Sigma) in a total volume of 50 \A.L was added. For cotransfection experiments with two vectors, this was adjusted to 15 jag of each plasmid plus 20 \ig salmon sperm DNA in a total volume of 50 jaL sterile TE (pH 8.0). The Gene Pulser (Biorad) capacitance was set at 960 fiFD and EXT, and voltage was set at 0.25 kV. Cells in cuvettes were shocked and cells were promptly placed back into a 15 mm dish of HDMEM + 10% FBS media, and returned to the 37° C incubator. Transfection of human breast cancer cell lines with Fuqene 6 Lipid Reagent In other experiments, cells were transfected using Fugene 6 (Roche #1815091) according to the manufacturer's instructions. Per 15 cm dish of cells, 15 jag plasmid DNA was added to serum-free HDMEM media (Gibco BRL), so that volume total was 85 jaL. To this, 15 [iL Fugene 6 transfection reagent was added, and the 100 ^L mixture was incubated for 30 minutes at room temperature. The mixture was then transferred into a 15 cm dish and cells were covered with 20 mL serum-free HDMEM media, and incubated at 37°C for 4 hours. After incubation, 2 mL FBS (Gibco BRL) was added to the serum-free media, and 24 hours later, this was replaced with fresh HDMEM + 10% FBS. Cells were harvested at either 48 or 72 hours post-transfection.

Animal Procedures

Selection of appropriate mouse strains Initially, our study considered using two strains of immunodeficient mice: severe combined immunodeficient (SCID) mice and nude mice. Due to the increased tumorigenicity of the chosen breast cancer cell lines in SCID mice over nude mice [65-67], and the fact that SCID mice are more economical than nude mice (Cross Cancer Institute, Edmonton), SCID mice were purchased from the Cross Cancer Institute and treated according to protocol #821 approved by the University of Calgary, Animal Care Committee.

Mammary Fatpad Tumor Model Based upon the literature, mice have been inoculated in the mfp with numbers of breast epithelial cells ranging from 2.0x106-10.0x106 in 100-200 jiL sterile 1xPBS [65-67]. Actively growing MDA MB 468 cells were harvested, washed twice, resuspended in sterile 1xPBS at a density of 2x107cells/mL or

5x107 cells/mL, and placed on ice. For an initial assay, 16 mice were inoculated with 0.2 mL of 5x107 cells/mL (1x107 cells total) and 16 mice were inoculated with 0.1 mL of 2x107 cells/mL (2x106 cells total) subcutaneously into the mammary fatpad. Tumors were measured (length x width) weekly, over a period of approximately 8 weeks. Mice were sacrificed when they showed morbidity due to excess tumor burden. At the time of sacrifice, mice were necropsied and liver and lungs were examined for the presence of metastases. One set of tissue samples from tumor, contralateral fatpad and lung were snap- frozen in liquid nitrogen and stored at-170°C, and a second set was fixed in 10% formalin and sent to Calgary Laboratory Services at the Foothills Medical Center, Department of Pathology & Lab Medicine for paraffin embedding and hematoxilin and eosin staining (protocol #129).

Generation of stably integrated inducible cell lines

Generation of Antibiotic Kill Curves All five breast cancer cell lines were plated at approximately 25% confluence (2.5 x 105 cells per dish) in 60 mm dishes. Seven dishes per cell line were prepared, with varying concentrations of G418 (Gibco BRL) orZeocin (Invitrogen) (0, 50, 125, 250, 500, 750, 1000 ug/mL) in each dish. Media with antibiotic was replaced every 3-4 days, and the percentage of surviving cells was observed. The number of viable cells was counted by Trypan Blue exclusion method at days 7 and 14, in order to determine the concentration of antibiotic that inhibits growth for each cell line.

Isolation and amplification of stable integrants Stable pVgRXR clones were generated according to the protocol in the Ecdysone Inducible Expression Kit (Invitrogen #K1000-01). Cells from each breast cancer line were transfected with pVgRXR vector, including one plate of cells transfected with pCi.GFP (Clontech) as a positive control and one plate of untransfected cells as a negative control. At 24 hours post-transfection, cells were washed and fresh media was added. At 48 hours post-transfection, fresh medium containing Zeocin was added, at the appropriate selective concentration for each cell line. Also at this time, the GFP positive control plate was observed for transfection efficiency. Cells were selected over a period of 2-3 weeks, changing media every 3-4 days. When colonies were identifiable, sterilized glass cloning rings sealed with vacuum grease at one end were placed on top of 30-50 colonies, 100 trypsin was added, and each colony was transferred to a single well in a 96-well dish. Clones were expanded in selective media and tested for inducible gene expression in transient assays described below. Stable pIND/pVgRXR clones were generated by transfecting the best expressing pVgRXR clone from each breast cell line with the inducible vectors plND/ING1b sense, plND/ING1b antisense, plND/ING1a, plND/p53 or plND(empty vector). The same transfection and selection procedure was repeated as above, however cells were selected in both G418 and Zeocin, and 20-50 colonies were expanded for each inducible line. Clones were tested for inducibility as outlined below.

Generation of dose response curves for Muristerone A and Ponasterone A Muristerone A (Invitrogen, 250 fig) or Ponasterone A (Invitrogen, 250 jag) was resuspended in 500 jxL of 100% ethanol, to a final concentration of 1 mM. Ponasterone A (Invitrogen, 1 mg) was resuspended in filter-sterilized DMSO to a final concentration of 10 mM. For transient transfectants, cells were transfected, and at 24 hours, fresh media was added. Also at this time, Ponasterone or Muristerone of varying concentrations (5-25 fiM) were aliquoted directly into each dish, and media was swirled around to disperse the hormone. Cells were incubated at 37°C for varying lengths of time, (2-72 hours), depending on the assay. For each experiment, a negative control consisting of cells treated with an equivalent volume of ethanol or DMSO alone was included. For stable transfections, cells were cultured in selective media. Hormone was added at the appropriate concentration, for the appropriate length of time, as above. Negative controls in the form of empty vector were also included. pVgRXR clone screening with X-gal (5-Bromo-4-chloro-3-indolyl-beta-D- galactopvranoside) Zeocin-resistant clones were transfected with pIND/LacZ as above, and plated in 6-well dishes, with two wells per clone. At 24 hours post-transfection, media was changed and each clone was induced with 5 uM Ponasterone in DMSO and DMSO alone (negative control) for 24 hours. At 44 hours post- transfection, cells were washed twice with 1xPBS, and all traces of wash buffer were removed. Cells were fixed by adding 1 mL of 0.25% glutaraldehyde (Sigma, diluted in 1xPBS) for 15 minutes at room temperature. During incubation time, X-gal staining solution was prepared (Appendix A). Glutaraldehyde was promptly removed and cells were washed 3x with PBS, again removing all traces of wash buffer. To each well, 1 mL of X-gal solution was added. Cells were incubated 4-24 hours, depending on the cell line, until cells were visibly stained. After removing X-gal, cells were covered with PBS and viewed under the light microscope. Positive-staining clones were photographed for records. pVgRXR and pIND/pVgRXR clone screening by indirect immunofluorescence Transfected pVgRXR clones or stable pIND/pVgRXR clones were plated on coverslips in 8-well dishes and induced with 5 u.M Ponasterone (or an equivalent volume of DMSO for negative controls) for 12-24 hours. For immunofluorescence, cells were analyzed as described previously [64]. Initially, cells were fixed in 0.1% paraformaldehyde in PBS (pH 7.5) for 5 minutes. Next, cells were permeabilized in 0.5% Triton X-100 (Sigma) in PBS for 5 minutes. For ING1 proteins, cells were stained with a mixture of four murine monoclonal antibodies (Cab 1-4, [68]). For p53 staining, cells were stained with a mixture of 25 3 murine monoclonal antibodies (D01, PAb421, PAb240). Cells were incubated with primary antibodies for 30 minutes at room temperature, and then washed in 0.1% Triton X-100 in PBS, followed by PBS alone. Cells were incubated with goat anti-mouse Cy3 (Chemicon) secondary antibody for 30 minutes, followed by washing in 0.1% Triton X-100 in PBS and then in PBS alone. Cells were mounted in 1 mg/mL paraphenylenediamine in PBS/90% glycerol that additionally contained 1 |jg/ml_ DAPI. Digital imaging was performed using a 14-bit cooled CCD camera (Princeton Instruments) mounted on a Leica DMRE immunofluorescence microscope. To remove out-of-focus contributions, VayTek microtome digital deconvolution software was utilized. Images were processed in Adobe Photoshop v5.5 to add false colouring and to adjust levels. False colours were added by changing the image mode to indexed colour, and the colour mode to the desired mixture of RGB (red-green-blue). DAPI was shown as cyan (red = 100, green = 200, blue = 255), ING1 as green (red = 0, green = 255, blue = 0) and p53 as orange (red = 242, green = 170, blue = 14). pVqRXR/pIND clone screening by protein extraction and western blotting Induced cells were harvested at the appropriate times by washing 1x in ice-cold PBS, trypsinizing, and spinning down at 4°C at 800 rpm for 4 minutes. Media was aspirated off, cells were resuspended in protein extraction buffer, and kept on ice. Cell suspensions were sonicated for 10 seconds to shear DNA and stored at -80°C until use.

Amplification and Manipulation of DNA

Agarose gels Agarose gels were prepared with 0.4 g agarose (0.4 g powdered agarose, Gibco BRL) in 50 mL 1xTAE buffer (Tris, acetic acid, EDTA), and boiled for 1 min 30 in a microwave until all agarose had dissolved. To the warm 0.8% (weight/volume) gel, 5 u.L ethidium bromide (Gibco BRL, 5 mg/mL stock) was added, the gel was poured into plastic gel trays and allowed to cool for 20 minutes at room temperature. To permit visualization of DNA samples while running the gel, a 6x DNA loading buffer was added to each. Gels were loaded and run at 100 volts in 1xTAE buffer until the desired separation was achieved when visualizing bands using an ultraviolet light source. Agarose gel purification of DNA After restriction endonuclease-digested DNA fragments were separated on an agarose gel, the desired bands were excised with a clean scalpel. Using the QIAquick gel extraction kit (Qiagen #28704), DNA fragments were purified from the gel.

DNA digestion by restriction endonuclease enzymes Restriction digests were performed with the desired enzyme, using the appropriate buffer (as recommended by the supplier), 1-5 units of enzyme per

microgram of DNA, 1xBSA (if recommended by supplier) and sterile dH20 to optimize the volume. Digests were performed at 37°C in a water bath for 1-3 hours, unless specified otherwise.

T4 DNA Lipase reactions Molar ratios of 3:1 insertvector were used for DNA ligations. Agarose-gel purified inserts (previously digested) were mixed with vector DNA in a minimum volume. Ligase reaction buffer (10x) was added to a final concentration of 1x, along with 1 mM ATP and 1 unit of T4 DNA ligase enzyme (USB #27-0870-04). The reaction was incubated at 16°C for 16-24 hours, after which time it was used for transformation reactions. Transformation of competent bacterial cells Competent DH5a cells were transformed according to the supplier's protocol (Gibco BRL #18265-017). Briefly, 50 |aL cells were thawed on a dry ice/ethanol bath for 5 minutes, and 1-10 ng DNA was added to tubes in a 1-3 (aL volume, tapping eppendorf tube to mix. Cells were incubated on ice for 30 minutes, followed by a heat shock at 37°C for 20 seconds. Cells were placed on ice again for 2 minutes, followed by the addition of 950 |iL sterile LB media. Tubes were placed on a shaker at 37°C for 1 hour, and 200 jiL aliquots were spread on LB plates with selective antibiotic (either ampicillin at 100 ).ig/mL, kanamycin 30 |ag/mL or zeocin 25 ng/mL). Plates were incubated overnight at

Small scale preparation of plasmid DNA (Miniprep) For the preparation of small quantities of plasmid DNA, Qiagen Miniprep Kit (#27106) protocols were used. Initially, single colonies were picked from LB agar plates and inoculated into 3 mL LB media plus antibiotic (ampicillin at 100 ng/mL, kanamycin at 30 jag/mL or zeocin at 25 jag/mL). Cultures were placed on a shaker at 37°C overnight, and the plasmid miniprep was performed according to the company's recommendations. Following miniprep, DNA was restriction- digested and run on an agarose gel for size confirmation.

Large scale preparation of DNA (Maxiprep) For the preparation of several hundred micrograms of plasmid DNA, a Qiagen Maxiprep kit was used (Qiagen #12163), and protocols were followed as stated in the kit manual. Maxiprep cultures were prepared in 100 mL LB + antibiotic (ampicillin at 100 ng/mL, kanamycin at 30 fag/mL or zeocin at 25 jag/mL) with 100 (iL starter culture. After prepping, plasmid DNA was eluted in 0.5-1.0 mL sterile TE (pH 8.0), and a small aliquot of plasmid DNA was digested and run on an agarose gel for size confirmation. 28

Long term storage of bacterial cells Plasmids in bacterial hosts were stored in bacterial medium containing 40% glycerol (V7V) in cryotubes at -80°C.

Protein analysis by quantitation, gel electrophoresis and western blotting

Quantitation of proteins by Biorad Protein Assay

Protein was quantitated as described in the Biorad Protein Assay handbook

(Biorad #500-0001). Disposable glass cuvettes were filled with 795 u.L mqH20,

200 (JL Biorad protein dye concentrate, and 5 |JL protein sample. Standards were prepared with BSA (stock 1 mg/mL) at 0, 1, 2, 3, 5, 10 and 15 ng/mL, and a standard curve of concentration vs. absorbance at 595 nm wavelength was measured on the Beckman UV/visible spectrophotometer. Following standard curve setup, sample absorbances and concentrations were calculated, relative to the standard curve. In some cases, protein sample volumes were adjusted so that measured concentrations remained within the linear range of the standard curve.

SDS-PAGE Sodium dodecyl-sulfate polyacrylamide gel electrophoresis was performed as the standard method for separating proteins based on differences in molecular weights [69]. The Biorad Minigel apparatus was used to assemble all protein gels. The resolving gel was prepared with 12.5% polyacrylamide:bisacrylamide (29:1 ratio), as all proteins of interest were within a similar size range. For each protein sample, 50 u.g was aliquoted into Laemlli sample buffer so that the volume was less than or equal to 30 ul. Samples, in addition to a molecular weight standard (New England Biolabs #7708S) were loaded on the gel and run for 50-100 V for 1-2 hours, until the running dye had reached the bottom of the gel. The protein gels were subsequently transferred to a nitrocellulose membrane as described below.

Protein transfer to nitrocellulose membrane Proteins were transferred from polyacrylamide gels onto nitrocellulose membranes (Schleicher & Schuell #10439396) using the Biorad minigel transfer apparatus. Transfer buffer, chilled to 4°C was used, and the transfer was performed at 50 V and 4°C for one hour at room temperature.

Western Blotting Nitrocellulose membranes with bound proteins were immersed in 5% blocking solution (see Appendix A) and placed on a rocker at 4°C, overnight. Block solution was removed and replaced with primary antibody solution (PBS-Tween with 5% skim milk with antibody, diluted as recommended by the manufacturer), and membranes were incubated for 2 hours at 4°C with gentle rocking. Membranes were then washed twice for 10 minutes each with wash buffer alone, at room temperature. Wash buffer was poured off and membranes were placed in secondary antibody solution (PBS-Tween with 5% skim milk or high detergent buffer + 5% skim milk) for 2 hours at 4°C with gentle rocking. After secondary incubation, membranes were washed twice more, for 10 minutes each, followed by incubation with a 1:1 ratio of chemiluminescence reagents (NEN Life Science Products) for 1 minute. The membranes were placed between clean acetate sheets and exposed to Kodak X-Omat Blue XB-1 film.

Antibodies used for western blotting ING1 proteins were bound with a mixture of four murine monoclonal antibodies, as described (10). The p53 protein was bound with a mixture of 3 antibodies D01, Pab240 and Pab421. Pyruvate kinase antibody, used to blot membranes for a loading control, was obtained from Dr. S. Robbins. 30

Magnetic cell separation techniques

Determination of optimum selection time and enrichment rate

The MACSelect Kk II transfected cell selection kit (Miltenyi Biotech #705-01) was used to enrich for transfected cells overexpressing ING1 gene isoforms. Optimum selection time experiments and others were performed as suggested by the manufacturer in the kit guidelines. Briefly, MDA MB 435, MDA MB 468 and MCF7 cells were transfected with either pKK, pKK/INGIb or pKK/ING1c vectors, and allowed to express for 24, 36, 48 or 72 hours. Cells were then harvested by washing in PBS, trypsinizing, spinning down at 800 rpm for 4 minutes, followed by resuspension in 800 fiL PBE buffer. A 100 aliquot of cells was removed and placed in clean eppendorf tubes. For transfection efficiency, 10 [iLFITC-labeled anti-H-2Kk antibody (provided in kit) was added to the 100 |aL aliquot of cells, and mixed. This sample was incubated in the dark at 4°C for 30 minutes, and subsequently prepared for FACs analysis (see below).

To the 600 (iL aliquot of cells, 80 \xL MACSelect Kk microbeads (provided in kit) was added, and cells were labeled for 15 minutes at room temperature, rocking tube gently to suspend beads every 5 minutes. During incubation time, MS+ columns (Miltenyi Biotech #130-042-201) were placed on the magnet and washed with 500 u,L of PBE. When incubation time had finished, cells were run through the column in 500 u,L aliquots, each followed by a 500 (aL aliquot of PBE buffer to wash the column. After the final aliquot of cells had passed through, the column was rinsed one final time, drained, and then removed from the magnet and placed in a 15 mL conical tube (Falcon). Cells were eiuted into the conical tube by addition of 1 mL PBE and clearing the column with the plunger provided. At this time, a 400 \iL aliquot of separated cells were removed and placed in an eppendorf tube. This aliquot was spun down briefly at 10,500 rpm in a table-top centrifuge at 4°C, and cells were prepared for protein extraction and western blotting as described above. Running this sample on Western blots enabled us to confirm that the desired ING1 proteins, in addition to the H-

2Kk molecule, were being overexpressed. The remaining 600 jaL of cells were spun down and resuspended in 100 jiL PBE. To this sample, 10 (iL of MACSelect control FITC antibody (included in kit) was added, and cells were incubated for 30 minutes in the dark at 4°C, and prepared for FACs analysis. The percentage of cells which stained positive in this fraction represented the enrichment rate.

FACS analysis of FITC-labeled transfected and selected cells After incubation with antibody, 1 mL PBE was added to each cell suspension, and cells were spun down for 4 minutes at 800 rpm, 4°C. PBE was then aspirated off, and cells were resuspended in 1 mL 1% formalin. Cells were transferred to clear 5 mL polystyrene tubes, stored at 4°C in the dark, and sent to the University of Calgary Flow Cytometry Facility within 24 hours of fixing for analysis. Controls for setting gates were included for each cell line, and consisted of parental cell line samples untransfected, both unstained and stained. cDNA Microarray Techniques

Isolation of total RNA and verification of RNA integrity Total RNA from transfected and MACS separated cells was isolated according to protocol, using the Qiagen RNeasy Kit (Qiagen #74104). After isolation, RNA was quantitated on the Pharmacia GeneQuant, in a 500 (iL cuvette, resuspended in RNase-free water. The integrity of RNA samples was tested prior to use by combining 500 ng of each sample with 1 (.iL RNA loading dye and RNase free water to make the total volume 10 (iL. Samples were run on a standard 0.8% agarose gel (prepared as above, not RNase-free) and run at 150 V for 20 minutes. Gel images were captured on a UV light by Kodak digital imaging. 32

Preparation of fluorescentlv-labeled cDNA First strand cDNA synthesis was performed using Superscript II RNase H" Reverse transcriptase enzyme, according to the manufacturer's protocol (Gibco BRL #18064-014), in combination with the protocol included in the Ares DNA Labeling Kits (Molecular Probes, Ares labeling kits, Alexa 546 #A-21667, Alexa 660 #A-21671). For each sample, 20-30 ug RNA was combined with 3 uL oligo dT primer (500 ng/mL stock) and 3 fjL dNTP mix (to a final concentration of 0.5 mM dATP, dCTP, dGTP, 0.15 dTTP and 0.20 aminoallyl-dUTP), and 6 jaL Arabidopsis RNA (20 ng/jaL stock). Volume total was adjusted to 36 jaL with

RNase-free dH20 if necessary. The mixture was heated to 65°C for 5 minutes and then chilled quickly on ice. Tube contents were collected by spinning briefly and the following was added: 12 (aL 5x First Strand Buffer (Gibco BRL), 6 f.iL DTT and 3 {.iL RNase OUT recombinant ribonuclease inhibitor (Gibco BRL, 40 U/|.iL). The contents were mixed and incubated at 42°C for 2 minutes, 3 [.iL Superscript II enzyme was added, and the reaction was mixed by gentle pipetting. The synthesis reaction was then allowed to proceed by incubating at 42°C for 50 minutes.

Hydrolysis of RNA and Purification These procedures were performed as recommended in the manufacturer's protocols (Molecular Probes, Alexa 546 #A-21667, Alexa 660 #A-21671). Briefly, the reverse transcriptase enzyme was inactivated at 95° C for 5 minutes, and directly afterwards samples were snap cooled by placing in an ice bath. To the samples, 1 M NaOH was added to a final concentration of 0.3 M, and contents were mixed and incubated for 15 minutes at 65°C. Samples were neutralized by the addition of 1 M HCI (0.23 M final) and 1 M Tris-HCI (pH 7.0, 0.1 M final), and then passed through Microspin G-50 spin column (Amersham Pharmacia #27- 5330-01) to remove unbound nucleotides and salts. Column eluate was then precipitated by adding 1/10 volume of 3 M sodium acetate (pH 5.2) and 2.5 volumes of 100% ethanol, and samples were frozen at -80°C for 30 minutes. After freezing, samples were spun for 15 minutes at 4°C at 12,000 rpm. Pellets were washed with 70% ethanol and air dried for 5 minutes.

Labeling Reaction Protocols were followed as recommended by the manufacturer (Molecular Probes, Ares Labeling Kits, Alexa 546 #A-21667, Alexa 660 #A-21671), with a

few modifications. cDNA pellets were resuspended in 15 u.L nuclease-free dH20 and warmed at 42°C for 5 minutes to dissolve. To the cDNA, 9 ^L Labeling Buffer (provided in kit) was added, as well as one vial of fluorescent dye which had been resuspended in 10 (aL DMSO. The reaction was vortexed briefly, and then left in the dark for 1 hour at room temperature so that the reaction could

proceed. After incubation, 65 ul_ nuclease-free dH20 was added to the mixture, and at this point, complementarily labeled cDNA from control and test samples were combined into one tube. This mix was purified with the QIAquick PCR purification kit (Qiagen #28104), but substituting 3 washes with 75% ethanol instead of the wash buffer, and three elutions of 5 minutes each, in a total volume of 100 JUL EB buffer. To the purified mixed cDNAs, 5 jiL calf thymus DNA and 5 \xL yeast tRNA were added, and cDNA was precipitated as before. Cell pellets were dried for 5 minutes and resuspended in 100 (aL DIG easy hyb solution.

Array hybridization 1.7k3 Microarray slides were ordered from the Ontario Cancer Institute (http://www.uhnres.utoronto.ca/services/microarray/). For each experiment, hybridization solutions were heated to 65°C for 2 minutes to ensure dissolution of the pellet, followed by the aliquoting of 30 |uL of each cDNA solution onto 3 slides, just above the spotted cDNAs (all experiments were done in triplicate.) 24x60 mm glass coverslips (VWR) were gently placed over the hybridization solution so that the liquid was spread evenly across the slide surface, without introducing air bubbles. Coverslips were sealed around the edges with rubber cement, and slides were promptly placed in moistened hybridization chambers and covered with lids to prevent exposure of the light-sensitive fluors. Chambers were sealed around the outside edges with parafilm and slides were hybridized for 18 hours at 37°C.

Washing array slides After incubation, hybridization chambers were disassembled and slides were immersed in 1xSSC so that the rubber cement could be removed and the coverslip gently slid off. The slide was placed in a staining rack filled with fresh SSC, followed by 3 ten-minute washes at 50°C in 1xSSC, 0.1% SDS with gentle, intermittent agitation. Slides were removed from wash buffer into racks filled with fresh 1xSSC, dipping 4-6 times, and placed in a slide box. Slides were spun dry at 500 rpm for 5 minutes and stored in the dark until scanning.

Array Scanning Arrays were scanned using Scanarray software, on the GSI Lumonics Confocal laser scanner. Lasers 1 and 3 were turned on and allowed to warm up for 15 minutes. Slides were quick-scanned initially, and an array line with dots of medium intensity was chosen for line scanning, so that laser levels could be equilibrated. Once equivalent signals were obtained from both channels, the entire array was rescanned at 10 \im and images for channel 1 (Alexa 546) and channel 2 (Alexa 660) were captured. Images were saved as tiff and bitmap files for importing into quantitation software.

Quantitation of cDNA spot intensities Quantitation was performed as described previously [70]. A pre• programmed 1.7k3 OCI protocol was opened (http://www.uhnres.utoronto.ca/services/microarray/), which consisted of a file containing information on the particular chips being used, the grid pattern of the array and gene identification information. Next, the two complimentary wavelength images (Cy3 and Cy5, saved as bitmap images) for each array were opened together. Under the view menu, composite image was selected so that the different wavelength images were superimposed upon one another. Next, the cDNA spot at the top left-hand corner of the array was located, and the pattern was edited so that each array grid was centered over the spots in that subarray. Then, Quantarray located the spots on the array, and the spot pattern was edited once more to make sure that the center of each spot had been marked properly. Under 'View Reports', scatter plots were viewed and saved for each array, so that the incorporation of the two different dyes could be compared and evaluated at a later stage. Finally, the data consisting of numerical intensity values for each spot was exported and saved in tab-delimited format.

Data Analysis In an Excel macro called Datahandler, channel 1 and 2 intensities were normalized using subarray averages. This involves adjusting channel 1 and channel 2 relative to one another. The intensities for channel 1 and channel 2 were summed on a per sub array basis, so that 16 normalization values were calculated for the 1.7k3 array comprised of 16 subarrays. These normalization values were used for correcting channel 1, or the intensity values of Alexa 546. Numbers were pasted into a new excel spreadsheet, where intensity values for duplicate spots were averaged, and Ch1:Ch2 ratios recalculated. These values were copied into a new master worksheet with single entries for each gene, and including data from each of the triplicate experiments and their reciprocal labels with averaged intensity ratios for each gene. These values were log transformed to base 2, and data was filtered so that only gene which had log intensity values of the same sign in all six experiments were passed. These genes and their logged intensity values were copied onto a third worksheet, and saved in tab delimited format for importing into Cluster (http://rana.lbl.gov/EisenSoftware.htm). Initially, data in cluster was median-centered by array 5 times, and saved, for later use in SAM. Cluster analysis was continued by selecting the hierarchical 36 clustering option, and using the average linkage clustering, with an uncentered correlation coefficient. Clustered data was saved for importing into Treeview (http://rana.lbl.gov/EisenSoftware.htm) later on. Median-centered data was formatted for importation into SAM (http://www- stat.stanford.edu/~tibs/SAM/index.html), where it was analyzed as a one class response with 100 permutations. Significant genes were selected with threshold intensity = 0 and a delta value which gave a false discovery rate 5% or less (genes which were reported as significant had a 95% or greater chance of truly being significant.) Lists of significant genes and line graphs were saved for significant gene analysis. Table 1. Properties of ATCC breast cancer cell lines selected for study

Cell Tumor Type p53 Rb PTEN ER/PR c-erbB2 Miscellaneous Line BT-20 Epithelial + ? ER- ? ? morphology Hs578T Epithelial ? ? ER- ? Mutant H-ras morphology MCF7 Adenocarcinoma + ? ? ER+ but ? Altered length of BRCA2 mutated mRNA (truncated MDA Ductal ? ? ? ER- Moderate mRNA Wild type Ras MB carcinoma levels 435s MDA Adenocarcinoma Point Partial - 44 bp ER- EGFr gene amplified: ?

MB 468 mutation deletion, deletion cells express >106 at 273 inactive EGFR/cell Arg—>His

CO 38 CHAPTER 3: RESULTS 40

Part 1: The ecdysone inducible vector system is useful for obtaining high expression levels in transient assays and in some stably integrated clones In order to test whether the ING1 gene would act as a bona fide tumor suppressor in an in vivo system, we decided to create five inducible breast cancer cell lines, which would later be used to generate mammary fatpad tumors in severe combined immunodeficient (SCID) mice. Prior to using the ecdysone inducible vector system to create our stable cell lines, we first researched the system and verified that it would suit the purposes of our experiments.

The ecdysone-inducible vector system is appropriate for creating stable cell lines that will inducibly express a growth inhibitor gene To test the effect of gene expression in any kind of system, it is advantageous to use an inducible system which gives the researcher tight control over the levels of gene expression during assays. It is also important that the system has low background levels of gene expression (so that the gene is not expressed when undesired), and a high degree of inducibility. The ecdysone vector system, marketed by Invitrogen, was reported to offer tissue-specific targeting and sustained induction, in addition to low basal expression and high inducibility [71, 72]. It is based on the ability of the insect hormone 20-OH ecdysone to activate expression via the ecdysone receptor ligand [72]. Invitrogen has created a modified ecdysone receptor/retinoid hybrid receptor, modified to contain the VP16 transactivation domain, which binds a hybrid ecdysone response element in the presence of the nontoxic hormone ecdysone, or synthetic analogues (Muristerone A or Ponasterone A). Transcription occurs when the heterodimeric receptor binds the response element, and protein induction levels of greater than 200-fold over basal level have been reported in the presence of hormone [72]. This system has many advantages over other inducible systems, such as the Tetracycline On/Off (tTa) systems, which shows mosaic induction, background leakiness, and poor expression of the transactivator [71]. In addition, the steroid hormone ecdysone and its analogues are nontoxic and required in minute amounts to induce gene expression [71]. With tetracycline, robust expression can require the use of toxic levels of tetracycline inducer, which suggests the system may not be appropriate for cancer studies [73]. Thus, we hypothesized that the ecdysone system was best- suited for the objectives of this research thesis.

ING1b sense, ING1b antisense, INGla sense and p53 sense cDNAs can be cloned into the pIND inducible vector All cDNA sequences were subconed into pIND from pCI vectors, with the exception of the p53 inducible vector, which was a generous gift from the Lee lab. Figure 3a (p. 63) represents a vector map of the pIND vector. All ING1 and p53 cDNAs were cloned into restriction enzyme sites within the multiple cloning site. All subcloned sequences were confirmed by restriction digest analysis and by direct sequencing.

Parental breast cancer cell lines are sensitive to low levels of Zeocin and high levels of G418 Antibiotics The first step in preparing stably integrated cell lines was to determine the toxicity of the antibiotics used for selection for each parental cell line. As shown in Figures 3a and 3b (page 63), the pIND vector possesses a G418 or Neomycin- resistance gene, and the pVgRXR vector possesses a Zeocin resistance gene. Zeocin is a glycopeptide antibiotic of the phleomycin family, it binds DNA to generate both single- and double- stranded breaks, and has extremely high toxicity in a broad range of prokaryotic and eukaryotic organisms (APPENDIX B)[74]. Neomycin or G418 is an aminoglycoside antibiotic which causes mistranslation of mRNA in both prokaryotic and eukaryotic ribosomes. The zeocin resistance gene encodes a protein which binds and sequesters the antibiotic, while the neomycin resistance gene encodes a phosphotransferase that confers resistance to neomycin and G418 (http://www.invivogen.com/plasmids/genelist_4.htm). As shown in Table 2, parental cell lines were grown in varying concentrations of Zeocin and G418 over a two-week period, and counted every 3-4 days. The minimum toxic concentrations of drug which killed100% of the cells are indicated in Table 2, and were verified by repeating the experiment a second time. The concentrations of Zeocin used for selection were 250 (ag/mL and 500-750 j.ig/ml_ for G418 antibiotic, for all cell lines.

Transiently transfected breast cancer cells can be induced to overexpress ING1 b after 24 hours of stimulation with Muristerone A In order to assess the inducibility of the prepared vectors and confirm their functionality prior to use in a stable system, it was first necessary to test them in a transient assay, using parental cell lines. Induction of all transiently transfected breast cancer cells showed levels of ING1b similar to endogenous levels when using the pIND empty vector (Fig. 4, p. 64), significant increases in ING1b levels upon transfection with the inducible ING1b vector and addition of 5 or 10 u.M Muristerone, and close to endogenous levels of ING1b with the inducible antisense vector. Little to no background induction was seen in the parental cells transfected with plND/ING1b and induced with ethanol or DMSO alone. We observed similar patterns of induction in all parental cell lines, and using both p53 and INGla inducible vectors (Fig. 5, p. 65). From these experiments, we confirmed that our vectors were inducible in the chosen cell lines, that 5 and 10 u.M concentrations of Muristerone A were effective for inducing high levels of expression of the transgene in the transient system, and that a 24 hour period of incubation with hormone was sufficient to induce high levels of gene expression.

Increasing levels of gene expression correspond with increasing levels of Muristerone A inducing agent Additionally in our transient assay, we used different concentrations of hormone inducer in order to assess the effect on levels of gene expression, as mentioned in the above section. We observed that increasing levels of Muristerone A induced increasing levels of ING1b expression, as seen in Fig. 4 (p. 64). This suggested that once stable clones had been generated, it would be important to optimize gene expression levels by dose-response approaches. Stably integrated pVgRXR clones can be induced to overexpress pIND/LacZ Due to the fact that two vectors are required in the ecdysone system, and that both must be stably incorporated into genomic DNA in order for inducible expression to occur, stable integrants could be generated in either of two ways. Cells could be cotransfected with both vectors and selected in media containing both antibiotics, followed by screening for inducible expression; or, they could be transfected with the pVgRXR vector, screened for expression by transient assay with an inducible vector, and then positive clones would be put through a second transfection and selection process. It was decided that the most effective way to generate stable integrants was to first transfect the pVgRXR vector into all cell lines, select the best expresser by lacZ screening, and use that particular pVgRXR transgenic clone to create all other pIND cell lines. The advantage this confers over cotransfection and dual selection is that ideally fewer total clones have to be screened in order to find a cell line that expresses the transgenes effectively. It also attempts to maintain some consistency between clonal cell lines, and their inducible expression levels. Using the reporter plasmid pIND/lacZ included in the kit (Fig. 3C, p. 63), all clones were tested for expression of the hybrid receptor encoded on pVgRXR. Approximately 50 clones from the MCF7 and MDA MB 468 cell lines were screened by this method, and positive clones stained blue when incubated with X-gal (Fig. 6, p. 66). The absence of staining in the same clones transfected with X-gal but induced with ethanol alone confirmed that there was no leakiness or background levels of gene expression. Clones with the greatest percentage of cells staining blue were then selected for the next transfection and selection process and used to generate stable dual integrants. 45

Stably integrated pVgRXR clones can be used to overexpress plND/ING1b A second screening method was used to confirm inducible expression of the pVgRXR clones. Rather than transfecting with the pIND/LacZ control vector included in the kit, plND/ING1b was transiently transfected into cells, followed by induction with Ponasterone A and immunohistochemical staining with fluorescently labeled antibody to ING1b. As shown in Fig. 7(p. 67), positive clones showed overexpression of ING1b localized in the nucleolus, a pattern characteristically seen when ING1b is overexpressed under constituitive promoters [37]. This screening method also enabled us to confirm once again that our inducible vectors were fully functional, and helped to set up protocols for the screening of stable dual integrants by immunofluorescence assay.

Dual stable integrants are capable of overexpressing high levels of ING1b in initial immunofluorescence assays After the generation of stably integrated pVgRXR clones in all five parental cell lines, a single cell line, MDA MB 468, was chosen to proceed with initially in generating dual integrants. This was simply due to the sheer numbers of clones being generated simultaneously and requiring screening when using 5 different inducible vectors (ING1b sense, ING1 antisense, empty pIND vector, ING1a sense, and p53 sense.) Screening initially by immunofluorescence enabled us to confirm that the clonal cell population was homogenously overexpressing ING1 b, an observation that could not be made by Western blotting. Using this method, one induced clone, C5, was identified which exhibited uniform overexpression of ING1b upon induction in approximately 20-30% of cells. Nucleolar localization of the protein was seen as expected from previous experiments (Fig. 8, p. 68). This clone was then subjected to various induction conditions as described in the next section, in order to optimize expression. Unfortunately, other clones created with inducible ING1 antisense and p53 vectors did not appear to show inducibility of their transgenes by immunofluorescence (Fig 9, p. 69). These clones were retested by Western blotting assays in order to confirm their lack of inducibility.

Dual stable integrants are not capable of expressing high levels of protein upon induction with Muristerone A or Ponasterone hormones All clones tested by immunofluorescence were additionally subjected to induction with 5 JJM Ponasterone, followed by total protein isolation and Western blotting in order to observe the levels of transgene expression. Of 30-50 clones screened for expression of ING1b antisense and of p53 sense insert, and the single inducible ING1b sense clone C5 identified by immunofluorescence, none showed a significant increase in protein levels (or decrease for the antisense clones) after 24 hours of induction (Fig. 10, p. 70). These results indicated that although cells had maintained their antibiotic resistance, for some reason they were not being induced or that expression of the transgene was being hampered. Using C5, different conditions were tested in order to determine if expression could be induced by manipulating the time of incubation with hormone or by changing its concentration. The first assay compared time points between 0 and 48 hours of induction with concentrations of Muristerone A and Ponasterone from 5-25 jaM. As shown in Fig. 11A (p. 71), no significant increases in ING1 b expression were observed. Secondly, time points between 0-12 hours were examined, based on the original positive immunofluorescence screen where induction was observed at 12 hours. Again, Western blots of equal amounts of total cellular protein did not show any increases in ING1b expression at the various time points of induction with 10 (iM Ponasterone (Fig. 11B, p. 72). At this point, it was hypothesized that despite the presence of selective concentrations of Zeocin and G418 in culture media, multiple passages in culture had enabled some (or all) of the cells in the C5 population to lose their transgene. Stably integrated clones have been reported to modify or excise their transgenes in culture, especially when these genes are disadvantageous to growth [75-78]. Due to the possibility that a small percentage of cells within the initially clonal population may still have retained the transgene, the next attempt to restore clonal inducibility involved performing a single-cell dilution series, and growing subclonal populations to be rescreened for inducible expression.

Single-cell subcloning of initially clonal population does not restore inducibility of the C5 ING1 b clone After diluting C5 to single cells, regrowing, and testing by both Western blotting and immunofluorescence, no increases in protein expression were visible when these cell populations were stimulated with Ponasterone A (Fig 12, p. 73). At this point, it was proposed that in spite of the obvious resistance to G418 and Zeocin, likely some part of the inducible construct or heterodimeric receptor construct region had been lost or mutated, which was abrogating gene expression. This idea was tested by transfecting C5 with each construct individually (pVgRXR and plND/ING1 b) and inducing with Ponasterone as an attempt to determine if one or both of the constructs had been lost.

ING1b C5 stable clone is not inducible when either pVgRXR or plND/ING1b vectors alone are transfected in Cells from the original C5 line were transiently transfected with pVgRXR, plND/ING1b and both vectors, and induced with Ponasterone for 24 hours in order to observe if inducible expression could be restored. This would enable us to determine if C5 had lost one or both of the constructs, which may help to provide an explanation for its lack of inducibility. At 24 hours post-transfection, no increase in ING1b protein was visible in the C5 cells transfected with pVgRXR alone, or with plND/ING1b alone (Fig. 13, p. 73). This suggested that critical regions for inducible expression in both constructs had been lost in the C5 clone. In contrast, C5 cells transfected with both constructs and induced with Ponasterone showed a significant increase in ING1b protein, indicating that the system was still inducible in these cells when both vectors were transiently transfected in and had not been subjected to selection for prolonged time periods.

Inducible transgene cannot be amplified in ING1b C5 stable clone, while antibiotic resistance in culture is still retained As a more direct second test, genomic DNA from the original clone, as well as from the parental cell line (as a negative control) was extracted and a PCR performed in order to determine if the plND/ING1b construct was still present. Amplification of a region including the pIND inducible promoter and the first 800 base pairs of the ING1b cDNA was performed on genomic DNA from the parental cell line, from C5, and from an aliquot of plND/ING1 b vector alone as a positive control (Fig. 14A, p. 74). • A very strong band of approximately 1.4 kb was visible in the vector control lane, whereas the same band appears very faintly in the lane containing C5 genomic DNA (Fig. 14B, p. 74). As expected, no band is present in either the MDA MB 468 parental genomic DNA lane, or in the negative control. These results indicate that the C5 clone possesses a very minute amount of construct, suggesting that the majority of cells within the clonal population have lost or modified the transgene. 49

PART 2: The murine mammary fatpad model is used to study human breast cancer growth and metastasis 50

To test the effects of a candidate tumor suppressor on tumor growth rate and metastasis, it is advantageous to use an in vivo model because of its increased physiological relevance over in vitro assays and it's clinical significance. Human tumor cells xenografted into mice maintain their original histological and biological characteristics, and will grow at a rate determined by properties of the particular cells [67]. Studies have shown that the site of administration of human tumor cells can also have an effect on tumor growth rate and metastasis in the mouse model [65, 67]. The most anatomically relevant murine site for implantation of human breast tumors and also the most effective site for facilitating growth and metastasis is the murine mammary fatpad (mfp) [65, 66]. Inoculation of cells into the mfp site is one method of control over the sites at which metastasis occurs, however this varies with each different cell line used [65-67]. In humans, breast cancer most commonly metastasizes to the skin and soft tissues surrounding the primary tumor, nodes and bone [79]. The next most common sites for primary metastasis are the lung and liver, and finally the brain and central nervous system [79]. For human breast epithelial tumors in SCID mice, common sites for metastasis are also nodes, bone, lung and liver [65-67]. In consideration of our goal of exploring the role of ING1 b expression in breast cancer tumor growth and metastasis, we hypothesized that the mammary fatpad model in SCID mice would be the optimal system in which to achieve this.

Equivalent human breast tumors can be established by inoculation of the mammary fatpad with two different volumes of MDA MB 468 breast cancer cells Prior to inoculating mice with transgenic cell lines, it was necessary to perform control tumorigenesis assays using the parental cell lines minus the transgenes. This yielded control curves for tumor growth rate, rates of metastasis and site(s) of metastasis. It would provide tissues for control immunohistochemical staining, FACs analysis and apoptag staining experiments. The MDA MB 468 cell line was injected into 36 mice to test two different inoculation volumes and cell numbers: 2x106 cells in 100 uL and 10.0x106 cells in 200 uL. Both numbers of cells produced tumors: it took slightly less time for tumors to reach the required volume of 0.5 mm2 when more cells were injected, however both curves look quite similar. Therefore, it was concluded that all mice would be injected with 10.0x106 cells in 200 u.L sterile PBS. Tumor growth curves are displayed in Fig. 15 (p. 75).

MDA MB 468 mammary fatpad tumors do not metastasize to the lung or contralateral fatpad tissue in a SCID mouse model In order to look for metastases, paraffin samples of tumor, contralateral tissue and lung were stained with hematoxilin and eosin (H&E). The overall aim of this assay was to observe the number of micrometastases in lungs and compare this between parental and overexpressing cell lines, identifying differences between the two. If ING1 does act as a tumor suppressor and plays a role in breast cancer metastasis, we hypothesized that fewer or smaller metastases might be present in mice with tumors overexpressing ING1b than in control tumors.

In the parental MDA MB 468 cell line, H&E staining showed no lung or contralateral tissue micrometastases. This observation simply provided a baseline with which to compare future MDA MB 468 transgenic cell lines against, and indicated that our method of tissue preparation was effective and successful for generating good quality H&E stains.

Although this tumor model proved to be useful, easy to work with, and appropriate for measuring breast cancer tumor growth, it was not pursued further due to the complications that arose in the creation of stable cell lines. The system may still be useful in the future if stable cell lines are created using a different method, or perhaps using the ecdysone system in other cancer cell lines. In the case of the latter, expression of inducble clones using an appropriate method for drug delivery in the mouse still requires optimization. 52

PART 3: Overexpression of the ING1b and ING1c gene isoforms result in upregulation and downregulation of other genes, as shown by cDNA microarray Due to the lack of success in generating stable ecdysone-inducible breast cancer cell lines, a new approach aimed at identifying genes that are responsive to changes in ING1 gene expression levels was taken. To address the second major hypothesis of this research thesis, we used cDNA from breast cell lines overexpressing ING1b and ING1c isoforms via transient transfection of constituitive ING1 constructs, to perform a cDNA microarray analysis. The use of cDNA microarrays has expanded exponentially in the past several years, as this technology enables the mRNA expression levels of thousands of genes to be quantitatively measured at the same time [80, 81]. We felt that using this technology would enable us to observe the effects of ING1b and ING1c overexpression on a large number of candidate genes, and give us insight into it's involvement in apoptosis, DNA repair, cell cycle control, chromatin remodeling, and other cellular processes in which it is involved.

The basic principle behind this technique involves arraying short cDNA sequences onto a solid support, most commonly a nylon membrane or glass slide [80, 82-84]. Fluorescently or radioactively labeled mRNA from a cellular test population is then hybridized to the support, followed by identification of an expression profile for that mRNA test pool. This technology provides a huge advantage for many biologists and medical scientists, and particularly cancer researchers, because it gives them the opportunity to comprehensively examine expression profiles of different tumors. This is predicted to have great impact on how tumors are classified and treated in the future. For our purposes, comparing expression patterns between control and overexpressing cell lines will tell us which genes are upregulated or downregulated as a result of ING1 gene expression. By comparing expression patterns in metastatic and non-metastatic breast cancer cells, we hoped to further substantiate the correlation between ING1 gene expression and breast cancer metastasis. This data would not only provide insight into the role of ING1 as a modulator of gene expression, but also into its role as a type II tumor suppressor [42]. 54 Transiently transfected breast cancer cells express high levels of cell surface H-2Kk at different time points, and can be enriched with high efficiency on the MACs column In order to prepare cDNA from a population of breast cancer cells that homogeneously overexpress ING1 b, it was first necessary to purify the cells that overexpress ING1b. This is important because when using transient transfection methods, only a small percentage cells uptake the foreign vector DNA [69], and depending on the transfection method used, a large number of cells can die during the process of transfection. The approach taken in order to minimize both of these effects was to use the Magnetic Activated Cell Sorting column, marketed by Miltenyi Biotech. This kit allows for the enrichment of transiently transfected cell populations so that cDNA from cells which homogeneously overexpress the ING1 transgene can be obtained. The kit involves transfection of a bicistronic vector containing a multiple cloning site, into which ING1b or ING1c was cloned

(Fig. 16A, p. 76). Additionally, this vector encodes the cell surface murine H-2Kk molecule, truncated in its cytoplasmic domain to prevent signal transduction (Fig. 16B, p. 76). Both genes are driven by constitutive promoters, resulting in simultaneous expression of the H-2Kk cell surface molecule used for selection and expression of the ING1 gene. After transfection of the vector into breast cancer cells and incubation for 48-72 hours, cells are labeled with paramagnetic microbeads conjugated to a monoclonal antibody directed against the mouse H-

2Kk molecule. Cells are then passed through a magnetized column, where cells expressing H-2Kk are retained in the column by the magnet. After rinsing the column with buffer, it is removed from the magnetic field and the purified population of transfected cells are eluted (Fig. 17, p. 76). From these cells, mRNA can be harvested and transcribed into cDNA, yielding a purified population of transcripts from cells which overexpress ING1. Cells from the 3 different breast cancer cell lines were transfected with pKK vectors containing H-2Kk alone, H-2Kk and ING1b, and H-2KK and ING1c genes. To determine the optimum time for column selection which is ultimately determined by the optimum time for H-2K expression , cells were harvested at different time points, and a small aliquot of these were stained with an FITC- labeled anti-H-2Kk antibody, and sorted by FACs (Table 3). The remainder of these cells were conjugated to magnetic beads, sorted through the column, and then stained with control antibody which binds the covalently linked anti-H-2Kk antibody fixed to the bead. These cells were also analyzed by FACs to determine the enrichment rate (Table 3). From these results, it was determined that the greatest percentages of cells were stained at the optimum harvesting times of 72 hours for MDA MB 468 and MDA MB 435, and at 48 hours for MCF7. These conditions and time points were then used to assess ING1 overexpression.

Transfected cells purified on the MACs column overexpress ING1b and ING1c proteins In order to confirm that ING1b and ING1c were both being expressed from their respective biscistronic pKK vectors, aliquots of transfected and column- purified cells were protein extracted and tested for ING1b and ING 1c expression levels. As shown in Figure 18 (p. 77), these samples were blotted for ING1, in comparison to protein from the same cells transfected with an empty pKK vector

(expressing H-2Kk alone.) These results confirm that column-purified cells overexpress ING1 at the protein level, a direct reflection of corresponding mRNA levels. This enabled us to conclude that our transfection and selection procedure was successful, and that this method would be appropriate for extracting mRNAs from a homogenous population of breast cancer cells overexpressing either ING1borlNG1c.

Total RNA can be isolated from MACs purified cells without significant degradation After MACs column separation, RNA was extracted from positive cells using the Qiagen RNeasy kit, which combines the procedures of guanidine- isothiocyanate lysis with silica-gel-membrane purification (Qiagen #74104). To assess the quality of RNA, an aliquot of each sample was run on an agarose gel. The presence of 28S and 18S ribosomal RNA bands indicated that RNA of good quality had been isolated [69]. Obtaining good quality RNA is important for obtaining a valid expression profile of the breast cancer cells, and for achieving accurate microarray results. Figure 19 (p. 77) indicates a series of RNA samples prepared on the magnetic column whose RNA has remained intact, and that were used to prepare cDNA for the microarray assay.

Overexpression of ING1b and ING1c genes leads to the upregulation and downregulation of various genes in three different breast cancer cell lines

a) Fluorescent intensity ratios for each gene on the microarray slide are generated In order to identify genes that are responsive to changes in ING1 b and ING 1c expression levels, mRNA from breast cancer cell lines overexpressing one of these genes was extracted and transcribed into fluorescently labeled cDNA. This is known as 'test' cDNA. A second population of 'control' mRNA was extracted from breast cells transfected with empty vector alone, which was also transcribed into cDNA and labeled with a different fluorophore. A set of control and test cDNAs were prepared for both ING1b and ING1c-overexpressing cells, in each of the three cell lines. A pair of control and test cDNAs for each cell line, and for each gene isoform was combined and hybridized to glass arrays: each cDNA pool was divided into 3 and aliquoted onto three separate slides, to yield triplicate arrays. The slides were scanned on the confocal laser scanner at wavelengths that excite both dyes (546 and 660 nm), and the resulting graphic depicting dots of different fluorescent intensities was produced by Scanarray software (GSI Lumonics) (Fig. 20, p. 78). This data was imported into the Quantarray software, which generated numerical intensity values for each wavelength and each spot, producing two intensity values which were used to calculate an overall intensity ratio of Alexa660/Alexa546 (Figs. 21,23, pages 78, 83). The software additionally calculates a background fluorescent intensity value for each dot, a normalization factor, and a scatter plot which compares the intensity of the control dye to test dye for each spot on the array (Fig. 22, p. 79- 82). Once these values have been determined (Fig. 23, p. 83), the data is imported into Datahandler, a specialized Excel macro, where it is normalized by subarray averages, followed by recalculation of new intensity ratios for each spot. Due to the representation of each gene in duplicate on the OCI 1.7k3 array slides, the averaged intensities for each dot were then recalculated in Excel, along with a new intensity ratio (Fig. 24, p. 84). It is these ratios that reflect the expression levels of genes in the Alexa660-labeled cDNA relative to the Alexa546- labeled cDNA in the original label, or the reverse ratio for the reciprocal label. In the first or 'original' set of labelings, control cDNA is labeled with Alexa546 and test cDNA is labeled with Alexa660. This signifies that genes upregulated when ING1 is overexpressed have intensity ratios of above 1.0. Genes that are downregulated when ING1 is overexpressed are represented by ratios of less than 1.0. In the experiments with reciprocal labeling, control and test cDNAs are labeled with the opposite Alexas in order to account for differential incorporation between the two dyes, so the intensity ratio is calculated as Alexa546/Alexa660. Internal Arabidopsis cDNA controls on the 1.7k3 slides, spotted at different concentrations, may provide some means of quantifying these expression levels, however we found their spotting to be inconsistent, and as a result they were not used for quantification in our experiments.

b) Generation of Log-Transformed Intensity Ratios for Genes that are Unidirectionally Expressed After calculation of average intensity ratios for each spot, all data from one experiment (including triplicate original labels and triplicate reciprocal labels) was compiled onto one spreadsheet and log-transformed to base 2 (Fig. 25A, p. 85). The use of base 2 enables data points with equivalent inductions or repressions to be treated as numbers equal in magnitude but opposite in sign. This method of analysis is supported by many microarray studies [70, 85]. At this point, the data was filtered so that genes with unidirectional expression across all six experiments were selected for, and all others were eliminated (Fig. 25B, p. 85). A new sublist of genes which passed this screening was then created and saved in tab-delimited format, in preparation for Cluster analysis.

c) Cluster and Treeview Analysis of Gene Arrays Two software programs, created by the Eisen Lab at Stanford, function to identify genes with similar expression patterns and cluster them together [85], and to create a graphical representation of these array patterns. Often, it has been shown that groups of genes that have similarities in function are related, making it possible to interpret expression patterns as cellular processes, and often helping to characterize genes of previously unknown function [85]. Log- transformed gene data was imported into Cluster, where it was then median- centered by array five times. This involved subtracting the column-wise (or array) mean from each value in the column, so that the column median is equal to zero. This 'equalizes' array experiments so that expression levels of individual genes are comparable, and also helps to correct biases due to the different dyes.

The median-centered data was then organized by hierarchical clustering. The basis for this type of clustering is that it organizes genes in a branched-tree structure, so that genes closely related to one another are connected by short branches, and the more distant the relationship between genes, the longer the branches on the tree. Although clustering is most effective when comparing arrays between time points or different treatments, it was used to organize the data in this project so that groups of overexpressed and underexpressed genes could be grouped together and presented in graphical format. The first option in hierarchical clustering is to define similarity, or to what extent two series of numbers are alike. The most common method for defining similarity is by use of the Pearson correlation, and four variations of this equation can be used to relate data in Cluster. In simple terms, the Pearson correlation (r) describes the similarity in shape between two curves X and Y, where each curve is created with the expression data for gene X and gene Y. This correlation coefficient always has a value between 1 and -1, where 1 means the two are identical, 0 means they are independent, and -1 means they are exact opposites. This coefficient does not vary when numbers are transformed by a scalar, for example, if all the Y values are multiplied by 2, the correlation coefficient between X and Y does not change. Thus, two curves with the same shape but different magnitudes will still have a correlation factor of 1. For these experiments, data was correlated using the uncentered correlation, which assumes that the mean is 0, even when it is not. This infers that if two curves (X and Y) have the same shape but are offset from one another by a relative value, the centered correlation coefficient would be 1, while the uncentered coefficient would not be 1. For clustering the arrays in this thesis, the uncentered coefficient was used, however, the use of centered or uncentered coefficients should not be critical in these experiments because we are looking at only one time point, and not necessarily a curve of expression levels over different times. After defining similarity, Average Linkage analysis was performed. This involves assigning a vector to each gene or group of genes, which is then used to determine the distances between that gene and all the other genes or groups of genes in the array. Vectors are determined using the previously calculated similarity coefficients, and consist of the average of all the vectors for that gene (or group of genes.) Agglomerative hierarchical clustering is the method by which Cluster associates items to generate a tree: cycles where the two closest items are joined together by a branch of the tree whose length represents the extent of similarity between the two. The two genes are then removed from the list and replaced by a new 'branch' that represents both of them. Next, the distances between this new branch and all the other items are determined, and the cycle repeated until only one branch (representing the entire array) remains. The computed tree clusters are then exported into Treeview, which transforms the newly ordered genes and tree branches into a graphic. In this setting, genes that are downregulated (or have negative log values) are represented by green colour, and upregulated genes (positive log values) by red colour. The greater the intensity of red or green, the higher or lower the level of expression. The intensity thresholds are determined by the highest and lowest array intensity values. Black represents the absence of expression, or log values close to zero (expression of 1.0). Cluster-analyzed microarray data, graphically formatted in Treeview can be seen in Fig. 26 (p. 86-90).

d) Significance Analysis of Microarrays A third program entitled Significance Analysis of Microarrays (SAM) identified statistically significant genes in the microarray experiments. This program was designed by Tusher, Tibshirani and Chu to allocate scores to each gene in the array, based on changes in gene expression relative to the standard deviation of repeated measurements [86]. It generates a series of t-tests for each gene, and each gene is assigned a score based on how its change in expression compares to the standard deviation of the repeated t-tests. A gene is considered statistically significant if its score is above a given threshold, chosen by the user. The threshold can be increased or decreased to identify smaller or larger sets of significant genes. SAM also calculates a statistic called the False Discovery Rate (FDR) which refers to the percentage of genes identified by chance. In other words, this number represents the number of genes significant by chance alone. Permutations of the intensity measurements are used as 'nonsense' gene measurements to generate the FDR. When the threshold value is changed, the FDR value is also recalculated. SAM provides the user with a 61 means of determining whether the genes identified on the array are there by chance, or because they represent significant results. After data was median-centered in Cluster, files were saved, re-formatted, and imported into SAM (Fig. 27, pgs. 91-96). The type of analysis used in SAM was a one class response, referring to a test where the mean gene expression differs from zero - there are no time points or different treatment groups to compare against. The number of permutations was set at 100, and SAM produced line graphs for each data set (Fig. 27, pgs. 91-96). After a threshold of 0.0 was selected, and an appropriate delta value used so that the maximum number of genes were significant with an FDR of 0.000, a significant gene list was generated for each array (Figure 27). 62

Table 2. Selective concentrations of antibiotic used for isolating stable clones

Cell Line [G418] ng/mL [Zeocin] uxj/mL

BT-20 500 125 Hs578T 500 250 MCF7 750 250 MDA MB 435 750 125 MDA MB 468 750 250

Table 3. Fluorescent activated cell sorter (FACs) results indicating percentages

of cells staining positive for the H-2Kk molecule before and after separation on the magnetic column. Gates were set with both untransfected cells stained with

FITC-labeled anti-H-2Kk antibody, as well as unstained H-2Kk transfected cells.

Time Vector MCF7 (%) MDA MB 435 (%) MDA MB 468 (%)

(h) Before After Before After Before After 48 pKK 13.74 96.46 4.39 83.63 5.27 48.20 ING1b 5.62 79.57 45.01 71.28 2.35 36.79 ING1c 6.76 94.24 50.66 88.44 3.74 28.75 72 pKK 15.49 64.15 45.28 69.84 14.72 76.78 ING1b 16.20 70.95 48.56 97.30 5.36 85.05 ING1c 16.34 76.49 50.29 97.71 3.70 89.72 63 Figure 3. Components of the ecdysone inducible vector system

SV40 pA ColE1 64

Figure 4. Breast cancer cell lines transiently transfected with various inducible constructs and induced with different concentrations of Ponasterone. a) MDA MB 468 cells transiently transfected with pIND empty vector, plND/ING1b sense and plND/ING1b antisense constructs and induced with Ponasterone A. In lanes with 0 |aM Ponasterone A, cells were transfected with vector but induced with 5 fiL volumes of DMSO alone.

pIND pIND Vector pIND INGb INGos v r~ \ i \ [Ponasterone] 5 10 0 5 10 uM 5 10 0

33 — mm «•> aINGIb kDa

aPyruvate Kinase (Loading Control) b) MCF7 cells transiently transfected with pIND empty vector, plND/ING1b sense and plND/ING1b antisense constructs, and induced with various concentrations of Ponasterone A. In lanes with 0 (iM Ponasterone A, cells were transfected with vector but induced with 5 \xL volumes of DMSO alone.

pIND pIND

Vector p|ND INGb INGas

1 * / \t \ [Ponasterone] rj 510 0 510 0 510 UM 33 — MCF7 kDa 65

Figure 5. Breast cancer cell lines transiently transfected with ING1a and p53 inducible constructs and induced with different concentrations of Ponasterone. a) Protein levels of ING1a in MDA MB 468 cells: 1) endogenous; 2) transfected with plND/ING1a but induced with 5 uL DMSO; 3) transfected with plND/ING1a and induced with 5 u.M Ponasterone, and 4) transfected with plND/ING1a and induced with 10 u.M Ponasterone.

ING1a •

aPyruvate Kinase (Loading Control)

b) Protein levels of p53 in MDA MB 468 cells: 1) endogenous; 2) transfected with plND/p53 but induced with 5 uL DMSO; 3) transfected with plND/p53 and induced with 5 uM Ponasterone, and 4) transfected with plND/p53 and induced with 10 u,M Ponasterone.

1 2 3 4 47.5 p53^ 32.5

aPyruvate Kinase (Loading Control) 66 Figure 6. X-gal staining of MDA MB 468 pVgRXR stable integrants.

Cells were transfected with pIND/lacZ at t=0, induced with Ponasterone A at t=24 hrs, and stained at 48 hours. Clone 2 was chosen to proceed with for the creation of dual stable integrants. 67

Figure 7. pVgRXR clone screening by immunofluorescence.

MCF7 and Hs578T pVgRXR clones transiently transfected with plND/ING1b sense, induced with 5 u,M Ponasterone at 24 hours and stained with Cy3- labeled ING1 antibodies at 48 hours. Cells are visualized by fluorescent microscopy. Control cells (- Ponasterone) are induced with 5 uL DMSO to confirm that leakiness is not occurring, and DAPI serves as a control stain for DNA. A) Hs578T cells; B) MCF7 cells.

Ponasterone + Ponasterone Figure 8. MDA MB 468 plND/ING1b sense stable clones screened by immunofluorescence

Cells were induced with 5 u.M Ponasterone (+) or an equivalent volume of DMSO (-) for different time points. All cells were stained with DAPI and a cocktail of four monoclonal ING1 antibodies. Magnification on the upper panel is 20x, while magnification on the lower panel is 63x. DAPI ING1b

0 hrs

+12 hrs

+24 hrs

20x magnification

63x magnification Figure 9. MDA MB 468 plND/ING1b antisense and plND/p53 stable clones screened by immunofluorescent staining.

Cells were induced with 5 uM Ponasterone (+) or an equivalent volume of DMSO (-) for 24 hours. Antisense clones are stained with a cocktail of four monoclonal ING1 antibodies (Cab 1-4). p53 clones are stained with a cocktail of three p53 monoclonal antibodies (D01, PAb421, PAb 240).

C17 C29 C9 C18

ING1b antisense p53 20x magnification 100x magnification 70

Figure 10. Western blots of pVgRXR/pIND stable cell lines containing ING1b antisense and p53 transgenes, induced with Ponasterone A.

A) MDA MB 468 stable clones containing plND/ING1b antisense transgenes, induced with 5 |uM Ponasterone for 24 hours. (+) indicates induced with Ponasterone A, (-) is a negative control, indicating cells induced with an equivalent volume of DMSO, 5 fiL. T he control lane represents endogenous ING1 protein from parental cells without s transgene and without induction. For each lane, 50 fig total protein was loaded.

O L. +-> i + i + i + i + c SNOOOOt-t-MM O i + i + i + i + t-t-vt-CMCMCMCN

47.5— 32.5—* «... — ^ ING1b 25

B) MDA MB 468 stable clones containing plND/p53 transgenes, induced with 5 |iM Ponasterone for 24 hours. (+) indicates induced with Ponasterone A, (-) is a negative control, indicating cells induced with an equivalent volume of DMSO, 5 [iL. For each lane, 50 |ag total protein was loaded.

• + i + CO CD 00 00 CO CO 0)0)(0(ONNOO rrNNrrrrrr rr(M(N(NMrr

^ - —» mmm mm mm mm mm mm mm mmmm mm ^...^M.^ p53 Figure 11. Time courses for induction and dose response of the inducible MDA MB 468 ING1b sense clone 5 (C5).

A) 48 hour time course of induction of MDA MB 468 ING1 b sense C5 with different concentrations of Ponasterone A. For each lane, 50 ug total protein was loaded.

o O 75 > c & 0 5* 5 10 15 25 £ g i 1 i n 1 i 1 i 1 S. UJ 0 12 24 36 48 12 24 24 24 36 48 12 24 24 36 48 12 24 36 48 12 24 36 48 -47.5 32.5 "25 • A XS- (xPK Loading * Muristerone A in EtOH used as inducing agent Control

A Negative control, pIND/empty vector inducible clone, induced with Ponasterone. Negative control, plND/ING1 b C5 induced with an equivalent volume of DMSO. Figure 11. Time courses for induction and dose response of the inducible MDA MB 468 ING1b sense clone 5 (C5).

B) 0-12 hour time course of induction of MDA MB 468 ING1b sense C5 with a 10 uM concentration of Ponasterone A. For each lane, 50 |ag total protein was loaded. The 'Parental' lane represents endogenous ING1b levels in the MDA MB 468 parental cell line.

.2 10 [Ponasterone] a) ______^ uM Ql 0 2 4 6 6 8 10 12 12 Inductiotioin Time (h) ING1b ** «•» mm — 32.5

Loading Control

Negative control, plND/ING1b C5 induced with an equivalent volume of DMSO. 73

Figure 12. Clonal dilution of the inducible MDA MB 468 ING1b sense clone 5 (C5). Cells from the original C5 clone were diluted out to single cells and regrown, to form new subclonal populations A-E. In this assay, subclones were induced with 10 uM Ponasterone for 0-48 hours to test for increases in ING1b protein levels. For each lane, 50 u.g total protein was loaded. The '0' lane represents endogenous ING1b levels in that MDA MB 468 ING1b C5 subclone.

A B C D E Subclone

/ \ t \i \i \i \ Induction Time (n) - 47.5 ING1b • 32.5 • 25

Figure 13. Transient transfection of MDA MB 468 ING1b C5 cells with pVgRXR, plND/ING1b and both constructs simultaneously in order to determine if one or both have been lost. Cells were transfected at t=0, induced with 10 u.M Ponasterone A at 24 hours, and harvested at 48 hours. For each lane, 50 ug total protein was loaded.

n £ cc ^ oc z x 1 X =^ DC _{ CC Q o> 7 cnz > - > ft. Q. a. o. — 47.5 ING1b • W«»fH|-32.5 • — oc

cxPK Loading Control 74

Figure 14. Verification of the presence of the plND/ING1b inducible construct in the C5 clone which appears to have lost inducibility.

To determine if the inducible construct has truly been lost from the previously inducible MDA MB 468 ING1 b clone, a portion of construct DNA spanning the ecdysone response elements and the first 800 bp of the ING1 b gene was amplified, a) PCR primers were designed to amplify a 1.4 kb fragment spanning all five E/GRE response elements, and a large segment of the ING1b gene.

181 5 0 477 588 Reverse primer 3'

E/GRE HSP MCS iNG1b

Forward primer

b) Amplification of genomic DNA from the MDA MB 468 ING1b C5 clone by PCR, using the primers above. Controls include genomic DNA from the parental cell line (negative) and plND/ING1b plasmid DNA (positive). Lanes

are labeled as follows: 1) H20 negative control; 2) MDA MB 468 parental genomic DNA, sample 1; 3) MDA MB 468 parental genomic DNA, sample 2; 4) C5 genomic DNA, sample 1; 5) C5 genomic DNA, sample 2; and 6) C5 plasmid DNA, positive control.

1 2 3 4 5 6 2.0 kb— Figure 15. Mammary Fatpad Tumor Growth Curve of two different volumes of MDA MB 468 breast cancer cells innoculated into SCID mice 76 Figure 16. The bicistronic pKK vector of the magnetic cell separation kit.

This vector (a) encodes the murine cell surface molecule H-2Kk (b) truncated in its cytoplasmic domain so that signalling is impeded. This gene is driven by a mouse immunoglobulin promoter, which yields high levels of expression. Within the multiple cloning site, the ING1b and ING1c genes have been inserted. These are expressed under the control of an SV40 promoter and enhancer.

Figure 17. The MACs cell separation technique. Initially, cells are transfected with pKK vector and express the cell surface H-

2Kk molecule. When magnetic beads are conjugated to the cell surface molecule, cells can be retained in a magnetic column, while cells that do not express the surface molecule (or the gene of interest) pass through. Columns are washed, and then removed from the magnet, and positive cells can be eluted. „ n 77

Figure 18. Verification of ING1b and ING1c overexpression in pKK vectors after magnetic cell separation. Cells from all three breast cancer cell lines were transfected with pKK, pKK/ING1b or pKK/ING1c and separated on the MACs column. After elution, cells were harvested for protein and run on a Western blot probed for ING1 expression. The pyruvate kinase (PK) protein was blotted as a gel loading control.

n o no on

5 5 5 5 o o z z z z z z * ^* £* * £* 5* * £* 2 Q.Q.Q. Q. Q. Q. Q.Q.Q.

ING1b mmmm—*1 ** 'iii1 325

ING1c T mm 25 uPK

MCF7 MDA MB 435 MDA MB 468

Figure 19. Verification of RNA integrity by observation of 28s and 18s ribosomal rRNAs during agarose gel electrophoresis. For each group of cells harvested, RNA integrity was evaluated by running 500 ng total RNA on an agarose gel, and confirming the presence of integral 28s and 18s ribosomal RNAs. MDA MB MDA MB 435 MCF7 468 l 1 l 11 1 no no no (DO O CD O O z z z z z z O.Q.Q.Q.Q. Q.Q.Q.Q.

*** 28s J...J.J.. ^ ...... 18s Figure 20. cDNA Microarray graphics generated by Scanarray software.

Scans of Alexa 546 (equivalent excitation wavelength to Cy3) and Alexa 660 (equivalent excitation wavelength to Cy5) signals. These arrays represent the

MCF7-ING1b array, with control cDNA labeled with Alexa546 and test cDNA labeled with Alexa660

Alexa 546 Alexa 660

Figure 21. Composite image generated in Quantarray.

Superimposied Alexa546 and Alexa660 scans so that spots can be located by the software and numerical values intensity values can be generated. This composite image was generated during the scanning of the MCF7/ING1b array, where control cDNA is labeled with Alexa660 and test cDNA is labeled with

Alexa546 (a reciprocal label experiment). The Arabidopsis control cDNA spots can be seen as the 8 yellow spots in the bottom lefthand corner of the grid. 79

Figure 22. Scatter plots for each microarray scan. Plots depict the ratio of

intensity between Alexa660:Alexa546 (represented on the graphs as a Cy5:Cy3 ratio). Grey dots represent values that are within the intensity ratio range defined by an upper and lower thresholds. In these arrays, the upper ratio threshold is set at 2.0, while the lower ratio threshold is set at 0.5. Red dots represent values brighter than the set thresholds. Triplicate arrays are shown in rows, while reciprocal experiments are found in consecutive rows, a) MDA MB 435 labeled with pKK546/INGb660

b) MDA MB 435 reciprocally labeled with pKK660/INGb546

c) MDA MB 435 labeled with pKK546/INGc660 Figure 22 continued. Scatter plots for each microarray scan, depicting the ratio of intensity between Alexa660:Alexa546 (represented on the graphs as a Cy5:Cy3 ratio). Microarray Scatter plotscontinued.

d) MDA MB 435 reciprocally labeled with pKK660/INGc546

e) MCF7, labeled with pKK546/INGB660

- — T— r^" L __

r'"" """ p_ /

S5 -; —

i ~ _i__-____rn-5?— = y»

f) MCF7, reciprocally labeled with pKK660/INGB546 81

Figure 22 continued. Scatter plots for each microarray scan, depicting the ratio of intensity between Alexa660:Alexa546 (represented on the graphs as a Cy5:Cy3 ratio). Microarray Scatter plotscontinued. g) MCF7 labeled with pKK546/ING1c660

h) MCF7 reciprocally labeled with pKK660/ING1c546

i) MDA MB 468, labeled with pKK546/INGB660 82

Figure 22 continued. Scatter plots for each microarray scan, depicting the ratio of intensity between Alexa660:Alexa546 (represented on the graphs as a Cy5:Cy3 ratio). j) MDA MB 468, reciprocally labeled with pKK660/INGB546

.—III-'

k) MDA MB 468, labeled with pKK546/ING1c660 Figure 23. Numerical data generated by Quantarray. The program assigns numerical intensity values and ratios to all of the spots on the array. This spreadsheet is taken from a single replicate experiment for the MCF7/ING1b array where control cDNA is labeled with Alexa546 and test cDNA is labeled with Alexa660.

User Name nelson Computer ARRAY1 Date Wed May 23 14:00:54 2001 Experimen 8023 Experimen d:\gsi lumonics\nelson\ExperimentSets\8023 Protocol D:\GSI Lumonics\QuantArray\Human 1.7K.pro Version 2

Begin Protocol Info Units Microns Array Row 4 Array Colu 4 Rows 12 Columns 20 Array Row 4000 Array Colu 5000 Spot Rows 200 Spot Colur 200 Spot Diam 100 Interstitial 0 0 is off, 1 is first one missing. 2 is second one missing Spots Per 240 Total Spot! 3840 Data is not crosstalk corrected Data is not background subtracted. Quantificat Histogram End Protocol Info

Begin Image Info Channel Image Fluorophor Barcode Units X Units Per Pixel Y Units PeX Offset Y Offset Status ch1 D:\GSILurCy5 Microns 10 10 0 0 Control Image ch2 D:\GSILurCy3 Microns 10 10 -20 0 End Image Info

Begin Measurements Number Array Row Array Colu Row Column Name ch1 Ratio ch1 Perceich2 Ratio ch2 Percei Ignore Filte 11111 G2/MITOTIC-SPECIFIC CYC 1 55.53304 0.80073 44.46696 1 2 111 2 NOV PROTEIN HOMOLOG 1 57.43429 0.74112 42.56571 1 Figure 24. Numerical data generated by Datahandler.

Datahandler normalizes intensity values of Alexa546 relative to Alexa660 This spreadsheet is taken from a si replicate experiment for the MCF7/ING1b array where control cDNA is labeled with Alexa546 and test cDNA labeled with Alexa660.

Number Array Row Array Colum Row Name X Location Y Location Ch1 Intensity Ch1 Backgrc ch1 Intensity chl Backgrc 1 G2/MrrOTlC-SPECIFIC CYCLIN G1. 2930 4110 3948 68652 193.701492 751.407776 56.479282 2 NOV PROTEIN HOMOLOG PRECUf 3120 4110 3878.0896 225 671646 545 410217 63 78833 3 N-MYC PROTO-ONCOGENE PROTl 3320 4110 3789 92529 232 805969 644 848145 76 170311 4 PROCOLLAGEN-LYSINE,2OX0GL 3510 4110 4206 47754 255.253738 721.592712 78.481483 5 CHLORIDE CHANNEL PROTEIN 6 (( 3710 4110 4467.38818 220.8806 680 160889 73.662521 6 AUTOCRINE MOTILITY FACTOR RE 3920 4120 4535.56738 223 686569 928.263245 73 961334 7 ARGINASE II PRECURSOR (EC 3.5 4120 4130 4481.44775 302 880585 734.059021 109 909676 8 HYPOTHETICAL PROTEIN KIAA029 4310 4120 4560.56738 264,462677 905.920349 85 598839 9 T-LYMPHOCYTE MATURATION-ASI 4520 4130 401564185 248 074631 659.655823 84 986862 10 CHROMATIN ASSEMBLY FACTOR 4720 4120 4408.14941 308.731354 729 388306 103.646042 11 SERUM AMYLOID P-COMPONENT 4920 4120 4223 56738 332.731354 676.052551 118.69883 1? METHIONINE AMINOPEPTIDASE 2 5130 4120 5078 70166 340 910461 746 803101 114 663239 13 NEURONATIN. 5330 4120 5342 05957 514 298523 660.886047 140 307449 14 C-X-C CHEMOKINE RECEPTOR TYI 5510 4120 5133 85059 417 179108 675 04364 119 224251 15 VACUOLAR ASSEMBLY PROTEIN 5730 4130 10394.1941 441 552246 2365 09424 144 747559 16 ITBA4 PROTEIN (FRAGMENT). 5930 4130 9022.76074 428 492523 2041 05774 146 231796 17 MUCIN 1 PRECURSOR (POLYMOR 8120 4130 32133 5215 568.761169 9520 78809 155 680923 18 DIMETHYLANILINE MONOOXYGEN 6320 4130 28465 5215 485 298492 9265.50684 161.325943 19 GLYCEROL-3-PHOSPHATE DEHYC 6530 4130 10647 373 422 686554 3335 62988 165 530304 20 THYROID HORMONE RECEPTOR A 6720 4130 11034.791 400.701508 3237.93677 144 449631 21 PS2 PROTEIN PRECURSOR (HP1./ 2930 4300 3613.56714 203.492538 645.322571 60 610374 22 LUMICAN PRECURSOR (LUM) (KEF 3130 4310 3417.38794 190 820892 549.114319 49 497452 23 IG GAMMA-1 CHAIN C REGION. 3330 4310 3977 43286 222 462692 671.471313 74 68364 24 SH3 DOMAIN-BINDING PROTEIN 3E 3530 4310 3990 26856 195 820892 620 029236 55 680344 25 [3-METHYL-2-OXOBUTANOATE DEI 3750 4320 17589.5977 252.104477 4096.51172 94 438393 ZINC FINGER PROTEIN 35 (ZJNC Fll 3940 4320 16668.0742 328.044769 4102.18164 109 202538 2e PYRUVATE KINASE. ISOZYMES f 4130 4320 413941797 257 567169 822 928162 73 084679 27 SYNTAXIN 3. 4320 4320 4040 43286 263 4776 741.39502 87 927032 28 15-HYDROXYPROSTAGLANDIN DE 4520 4320 4322 225 731339 794 598328 72 073318 29 PLACENTAL THROMBIN INHIBITOR 4720 4310 4267 04492 305.671631 688.612915 94 147919 30 MACROPHAGE COLONY STtMULA 4920 4320 4776 40283 255 746262 715.469666 86 516479 31 PROBABLE TRANS-1,2-DIHYDROI 5130 4320 4726 50732 384.955231 796.190979 121.161476 3? MITOCHONDRIAL 40S RIBOSOMAL 5330 4310 7596 1 3428 494.776123 1486.86316 146 953598 33 C4B-BINDING PROTEIN BETA CHAI 5530 4320 7599.05957 376.2388 1476.55505 153 057236 34 GUANINE NUCLEOTIDE-BINDING 5730 4320 7319 70166 435.343292 1666.98279 165 613968 35 CYTOCHROME C OXIDASE POLYP 5930 4310 8654 6416 590.492554 1846.46338 167 48526 36 CELL-CYCLE NUCLEAR AUTOANTV 6140 4320 6383.62695 426 746277 971,726135 125 141655 37 GUANINE NUCLEOTIDE-BINDING P 6320 4310 6587 13428 514 5224 1080 48315 206 975723 38 PROBABLE LEUCYL-TRNA SYNT1 6520 4310 5937.95508 378 328369 985 90863 124 537254 39 MICROTUBULE-ASSOCIATED PRO 6710 4310 5659.11963 326 417908 763 87146 124 364464 40 RAS-RELATED PROTEIN RAB-13. 2940 4500 6206 49268 269 791046 1927 79517 92 322975 41 P2X PURINOCEPTOR 7 (ATP RECE 3140 4510 6431 43262 265.507477 1768 50024 74 68811 42 6-PH0SPHOGLUC0NATE DEHYD 3340 4510 3913.0896 213.970154 612 39032 79 284866 43 KALLISTATIN PRECURSOR (KALLIf 3540 4510 3813 85083 193.820892 679 655701 56.301117 Figure 25. Data manipulation in Excel.

a) Log transformation of normalized array intensity ratios.

Gene -rj 48_»j 79J 48_»j 48J 74 48^j 79^J 48_»j 48_^J 48_»J 74 48_-J G2/MITOTI jj 1.14348 1.939208 0.917055 0.775825 0.56999 1.106494 0.134076 0.66228 -0.08659 -0.25383 -0.56214 NOV PRO 3.023738 1.124685 2.38548 1.050279 1.067691 0.669553 1.200293 0.117503 0.869401 0.049056 0.065498 -0.40115 N-MYC PF 3.321091 1.12081 0.924111 1.0351 1.286596 0.752654 1.199027 0.114051 -0.07892 0.034498 0.252 -0.28415 PROCOL 3.316889 1.305949 1.119407 0.93933 1.333345 0.639557 0.907286 0.26693 0.112799 -0.06259 0.287691 -0.44698 CHLORIDE 2.47759 1.231991 1.773553 1.199162 1.182438 0.711872 0.964815 0.208632 0.572985 0.181623 0.167578 -0.33986 AUTOCRIf 2.624301 1.199169 1.699886 0.812231 1.131122 0.708872 1.033519 0.181629 0.530561 -0.20797 0.12321 -0.34408 ARGINASi 2.810941 1.030918 0.944176 0.891067 0.640321 0.722649 0.912748 0.03045 -0.05744 -0.11534 -0.44579 -0.32483 HYPOTHE 2.491158 0.883477 0.903842 1.197128 1.396007 1.469891 0.149596 -0.12389 -0.1011 0.179925 0.333616 0.385188 T-LYMPHC 1.161365 0.597462 0.276055 1.110215 1.194929 1.510645 -0.98089 -0.51506 -1.28716 0.104554 0.178087 0.412536 CHROMA! 0.374976 0.928177 0.81212 1.828552 1.769421 0.096006 -0.07453 -0.20811 0.384475 0.603524 0.570653 SERUM A 1.100765 1.291392 1.809212 1.243934 1.199674 0.619892 1.103726 0.255721 0.592891 0.218279 0.18205 -0.47821 METHIONI 3.015381 1.259551 1.478428 0.910562 0.602156 0.694424 0.860228 0.230755 0.39098 -0.09369 -0.50724 -0.36467 NEURON/ 2.3637 0.841945 0.741201 1.066394 1.052299 1.500316 -0.13378 -0.17204 -0.29948 0.064283 0.050977 0.405676 C-X-CCHE 0.874783 1.243526 1.604661 0.889663 0.506185 0.676614 0.990868 0.217951 0.472912 -0.11691 -0.68085 -0.39065 1.235965 -0.7313 VACUO LA 2.693572 1.332669 1.87189 0.895977 0.900124 0.481281 0.287184 0.626949 -0.10984 -0.10522 1.258391 -0.56645 ITBA4 PRC 3.441697 1.126458 1.692944 0.810932 1.140302 0.567534 0.119078 0.526469 -0.20957 0.131293 MUCIN 1 F 3.519752 1.027862 0.655091 1.150185 -0.09725 -0.42298 1.202465 1.361394 0.90733 0.184373 0.308509 0.027481 DIMETHYl 3.158777 1.379207 0.850439 0.962532 -0.00413 -0.162 0.95973 1.715336 0.995876 -0.0411 0.539609 0.321509 GLYCERC 2.618317 1.392613 0 949173 0.427985 -0.05216 0.988668 1.318206 0.86424 -0.0114 0.276272 -0.1459 0.331182 THYRO D 1.534163 1.309464 0.913663 0.804421 -0.09029 1.168981 1.754888 1.012641 0.156133 0.562405 0.012561 0.269618 PS2 PRO"! 2.235401 3.057621 1.967222 -1.65578 0.676623 0.800245 0.231223 1.130088 -0.22284 -1.46437 0.122295 1.117637 LUMICAN 0.190944 0.578307 0.5515 0.7071 -0.59511 1.205803 1.793256 0.951823 0.187145 0.584033 -0.04938 -0.54765 IG GAMM, 2.0281 1.450902 -1.20603 -0.00044 0.372186 0.819473 0.497047 1.070908 0.99956 -0.19909 -0.69907 0.068507 SH3 DOM, 0.299384 0 866449 -0.34566 0.054495 -0.14335 1.017349 0.816642 0.953068 1.056007 0.0172 -0.20255 -0.04807 [3-METHY 0.707751 0.755463 0.691567 0.112348 -0.38274 -0.28042 1.033675 1.118902 0.826953 0.68199 0.033121 -0.19001 ZINC FING 1.996842 1.706251 -0.7739 -0.37593 -0.08836 0.534299 0.763624 0.686648 1.176413 0.915428 -0.26968 0.16247 0.461211 0.619353 1.07401 -O.45023 -0.47908 1.365492 2.406319 0.828095 0.311515 0.878098 -0.18863 PYRUVA 0.637479 1.051322 -0.41444 2.927093 1.422221 0.660709 -0.54974 SYNTAXIN 1.44837 0.783572 0.577103 0.370439 0.35222 -0.24389 2.861431 0.709325 1.108075 -0.57964 -0.34344 1.966323 0.846502 0.560101 0.440185 -0.16664 15-HYDRC 3.028524 1.552995 0.676165

b) Data filtering based on consistency of intensity ratios. Using logged intensity ratios, genes were passed if all six ratios had the same sign, and failed if they did not. In other words, genes that were not consistently upregulated (logged ratio >0) or downregulated (logged ratio <0) were excluded from further analysis.

74, wj 48jrj 79_»J 48^ 48_»J 48_~| 74 48_^j 79_-J 48_»J 48_»J 48jJP/F 0.90693 0.089589 0.982007 -0.02523 0.29784 0.029454 1 1 1 0 1 1 F 0.986804 0.398396 0.991143 0.116888 0.086577 -0.09215 1 1 1 1 1 OF 0.2117 0.246542 0.184013 0.102921 -0.28075 -0.15759 1 1 1.1 0 OF -0.34459 -0.10572 -0.38017 0.411649 0.015407 0.21485 0 0 0 1 1 1 F 0.126348 0.147231 0.000923 0.751199 -0.00348 -0.00742 1 1 1 1 0 OF 0.682773 0.131973 0.982285 0.624339 0.392566 0.138349 1 1 1 1 1 1 P 0.143175 -0.03564 0.391684 0.819064 0.290356 0.433263 1 0 1 1 1 1 F -0.71985 -0.39212 -0.68103 1.551352 0.584885 0.93746 0 0 0 1 1 1 F 0.507577 -0.32583 0.356675 1.162294 0.668585 0.877783 1 0 1 1 1 1 F 0.94024 0.06777 1.168088 -0.94226 -0.00987 -0.46036 1 1 1 0 0 OF 0.748163 -0.06467 0.40144 -O.O6402 0.233465 -0.19348 1 0 1 0 1 OF 0.658814 -0.02731 0.673087 0.197182 0.269504 -0.07503 1 0 1 1 1 OF 0.088752 0.133853 0.272771 0.250647 -0.00224 -0.16897 1 1 1 1 0 OF 0.702583 0.291486 1.318673 -0.1408 -0.44831 -0.38973 1 1 1 0 0 OF 86

Figure 26. Clustered gene tree diagrams. a) Cluster diagram for all passed genes in the MDA MB 435/ING1 b array. Array numbers are depicted vertically at the top, while genes are displayed horizontally.

9) e CM H CO P-

CD82 ANTIGEN (INDUCIBLE MEMBRANE PROTEIN R2) (C33 ANTIGEN) (IA4) HOMEOBOX PROTEIN HOX-B5 fH0X-2At MHO.C101 (HU-11 . ANGIOPOrETIN 1 RECEPTOR PRECURSOR (EC 2.7.1.1121 (TYROSINE-PROTE BETA-ADAPTIN 1 (PLASMA MEMBRANE ADAPTOR HA2/AP2 ADAPTIN BETA SUE SPECTRIN ALPHA CHAIN. BRAIN (SPECTRIN, NDH-ERYTHROID ALPHA CHI IG KAPPA CHAIN C REGION. CATHEPSIN G PRECURSOR (EC 3.4.21.20). ASTROCYTIC PHOSPHOPROTEIN PEA-15. DNA POLYMERASE DELTA SMALL SUBUNIT (EC 2.7.7.71. PLATELET FACTOR 4 PRECURSOR (PF-41 (OHCOSTATIH Al (IROPLACT1 . PROTEIN KINASE C SUBSTRATE. SO KD PROTEIN. HEAVY CHAIN (PKCSH) PEROXISOME PROLITERATOR ACTIVATED RECEPTOR GAMMA (PPAR-GAMMA1. ADP.ATP CARRIER PROTEIN. LIVER ISOFOHM T2 (ADP/ATP TRANSLOCASE TAR RNA BINDING PROTEIN (TRANS-ACTIVATION RESPONSIVE RNA BINDING TRANS ALDOLASE (EC 2.2.1.21. HLA CLASS II HISTOCOMPATIBILITY ANTIGEN. GAMMA CHAIN (HLA-DR I HOMEOBOX PROTEIN MEIS2 (MEIS1-RELATED PROTEIN 11 (FRAGMENT1. PROLIFERATING-CELL NUCLEOLAR ANTIGEN P120

b) Cluster diagrams for significant genes in the MDA MB 435/ING1b array, as determined by SAM.

D\ O CM iH 09 P- nqun^mm —r OB r- O»»B OB *0f8

HOME OB OX PROTEIN MEIS2 (MEIS1-RELATED PROTEIN 11 (FRAGMENT1. PROLIFERATING-CELL NUCLEOLAR ANTIGEN P120 (PROLIFERATION-ASSI Figure 26 continued. Cluster gene tree diagrams,

c) Part of the gene tree from the MDA MB 435/ING1c array.

r- r- H is ON co eo vo in r- r- o si * H r

CYCLIN-DEPENDENT KINASES REGULATORY SUBUHIT 2 (CKS-2). CCAAT BINDING FACTOR (CBFk. CAMP-DEPENDENT PROTEIN KINASE TYPE I-ALPHA REGULATORY CHAIN ( SUPEROXIDE DISHUTASE (CU-ZNk (EC 1.15.1.1). SIGNAL TRANSDUCER CD24 PRECURSOR. SECRETOGRAHIN I PRECURSOR (SGI) (CHROMOGRANXtT Bk. PROTEINASE ACTIVATED RECEPTOR 3 PRECURSOR (PAR-31 (THROMBIN R RAN-SPECIFIC GTPASE-ACTIVATING PROTEIN (RAH BINDING PROTEIN 1 ADENYLATE KINASE ISOENZYME 1 (EC 2.7.4.3) (ATP-AMP TRANSPHOSP TRANSFORMING PROTEIN RHQB (H6k . PEROXISOME ASSEMBLY FACTOR-2 (PAF-2k (PEROXISOMAL-TYPE ATPASE HYPOTHETICAL PROTEIN KEAA0087 (HA1002). KERATIN. TYPE I CYTOSKELETAL 10 (CYTQKERATIH 10k (KlOk (CK FLAVIN REDUCTASE (EC 1.6.99.1k

d) Significant genes from the MDA MB 435/ING1c array.

PROTEINASE ACTIVATED RECEPTOR 3 PRECURSOR (PAR-3k (THROMBIN RECEPTO] RAN-SPECIFIC GTPASE-ACTIVATING PROTEIN (RAN BINDING PROTEIN 1) (RANI ADENYLATE KINASE ISOENZYME 1 (EC 2.7.4.3) (ATP-AMP TRANS PHOSPHORYLft TRANSFORMING PROTEIN RHQB (H6k. PEROXISOME ASSEMBLY FACTOR-2 (PAF-2k (PEROXISOMAL-TYPE ATPASE 1) (PI HYPOTHETICAL PROTEIN KEAA0087 (HA1002k. KERATIN. TYPE I CYTOSKELETAL 10 (CYTQKERATIH 10k (KlOk (CK 10k. FLAVIN REDUCTASE (EC 1.6.99.1) (FR) (NADPH-DEPENDENT DIAPHORASE) (Hi 88

Figure 26 continued. Cluster gene tree diagrams.

e) A sample of significant downregulated data from the MCF7/ING1b array.

H same C*J V0 CO CO TH CM

••••• P PROTEIN (MELANOCYTE-SPECIFIC TRANSPORTER PROTEIN). HYPOTHETICAL PROTEIN KEAA0081 (HA1009) (FRAGMENT). DEATH-ASSOCIATED PROTEIN 1 (DAP-11 . CD82 ANTIGEN (INDUCIBLE MEMBRANE PROTEIN R21 (C33 ANTIGEN! (II AMTLORIDE-SENSITIVE AMINE OXIDASE (COPPER-CONTAINING) PRECURSC CYSTEINE DIQXYGEHASE (EC 1.13.11.201 (CDOl . FMET -LEU-PHE RECEPTOR (FMLP RECEPTOR1 (N-FORMYL PEPTIDE RECEP1 D-ASPARTATE OXIDASE (EC 1.4.3.1) (DASOX) (DDO) . 60S RIBOSOMAL PROTEIN L18. GUANINE NUCLEOTIDE-BINDING PROTEIN G(S1 . ALPHA SUBUNIT (ADES KERATIN. TYPE II CYTOSKELETAL 8 (CYTQKERATIN 8) GLUCOCORTICOID RECEPTOR-AS GUANINE NUCLEOTIDE-BINDING PROTEIN G(Il/G(S1/G(01 GAMMA-4 SUBl INOSIHE-5 MONOPHOSPHATE DEHYDROGENASE 2 (EC 1.1.1.205) (IMP I COLORECTAL MUTANT CANCER PROTEIN (MCC PROTEIN) . ALZHEIMER'S DISEASE AMYLOID A4 PROTEIN PRECURSOR (PROTEASE NE> HLA CLASS I HISTOCOMPATIBILITY ANTIGEN, E E*0101/E*0102 ALPI TRANS ALDOLASE (EC 2.2.1.21. VinWFfTRfTY PUflTFTH VDH /HIHMFIIRnY TOflTPTW HFYl 89

Figure 26 continued. Cluster gene tree diagrams.

f) A sample of significant upregulated data from the MCF7/ING1b array.

SflH GO n CP CM u> CO CO H CM

GAMMA INTERFERON INDUCED MONOKINE PRECURSOR (MEG). Gl/S-SPECIFIC CYCLIN D3. CHOLESTEROL ESTER TRANSFER PROTEIN PRECURSOR (LIPID TRANSFER PROTEIN I) . FOLLISTATIN 1 AND 2 PRECURSOR (FS) (ACTIVIN-BINDING PROTEIN) . CHOLINE KINASE (EC 2.7.1.32). DNA POLYMERASE ALPHA (EC 2.7.7.7)• GLUTBREDOXEH (THIOLTRANSFERASE). 5-AMINOLEVULINIC ACID SYNTHASE MITOCHONDRIAL PRECURSOR. NONSPECIFIC (EC 2. CELL SURFACE GLYCOPROTEIN EMR1 PRECURSOR (EMR1 HORMONE RECEPTOR) . DUAL SPECIFICITY PROTEIN PHOSPHATASE 6 (EC 3.1.3.48) (EC 3.1.3.16) (DUAL SPE EPHRIN TYPE-A RECEPTOR 7 PRECURSOR (EC 2.7.1.112) (TYROSINE-PROTEIN KINASE F CYTOCHROME C. SIGNAL TRANSDUCER AND ACTIVATOR OF TRANSCRIPTION 5A. NEUTROPHIL DEFENSINS 1, 2 AND 3 PRECURSOR (HHP) (DEFENSIN, ALPHA 1). PARVALBUMIH ALPHA. EPHREH-B1 PRECURSOR (EPH-RELATED RECEPTOR TYROSINE KINASE LIGAND 2) CLERK-21 6-PYRUVOYL TETRAHYDROBIOPTERIH SYNTHASE (EC 4.6.1.10) (PTPS) (PTP SYNTHASE). ZINC FINGER PROTEIN 151 (MT.Z-1 PROTEIN) . Bl BRADYKTNIN RECEPTOR (BK-1 RECEPTOR). EOTAXTN PRECURSOR (EOSINOPHIL CHEMOTACTIC PROTEIN) . IRON-RESPONSIVE ELEMENT BINDING PROTEIN 2 (IRE-BP 2) (IRON REGULATORY PROTE]

g) Significant data from the MCF7/ING1c array.

40S44CM CM CM NH m r- r- co co r- r- co

T-LYMPHOCYTE MATURATION-ASSOCIATED PROTEIN. PHE-B CELL ENHANCING FACTOR PRECURSOR. LYSOSOME-ASSOCIATED MEMBRANE GLYCOPROTEIN 1 PRECURSOR (LAMP-1) (CD 107A ANT ZINC FINGER PROTEIN 76. ALKALINE PHOSPHATASE. TISSUE-NONSPECIFIC ISOZYME PRECURSOR (EC 3.1.3.1) MYELIN-ASSOCIATED GLYCOPROTEIN PRECURSOR. MALATE OXXDQREDUCTASE (HADP) . MITOCHONDRIAL PRECURSOR (EC 1.1.1.40) (HAL KERATIN. TYPE I CYTOSKELETAL 18 (CYTQKERATIH 18) (K18) (CK 18). VASCULAR ENDOTHELIAL GROWTH FACTOR B PRECURSOR (VEGF-B) (VEGF RELATED FACT SON PROTEIN (SON3). IDUROHATE 2-SULFATASE PRECURSOR (EC 3.1.6.13). ACUTE MYELOID LEUKEMIA 1 PROTEIN (ONCOGENE AML-1) (CORE-BINDING FACTOR, MUSCARINIC ACETYLCHOLINE RECEPTOR Ml. D-ASPARTATE OXIDASE (EC 1.4.3.1) (DASOX) (DDO) . PEROXISOME PROLITERATOR ACTIVATED RECEPTOR ALPHA (PPAR-ALPHA).

• n* Jiod esi CM CM H 10 r~ V0044

ALCOHOL DEHYDROGENASE CLASS III CHI CHAIN (EC 1.1.1.1) (GLUTATMi HYPOTHETICAL PROTEIN KIAA0136 (FRAGMENT). Figure 26 continued. Cluster gene tree diagrams,

h) Significant genes from the MDA MB 468/ING1b array.

w if » w eo to

OXYTOCIN RECEPTOR (OT-R). GRAVEN (FRAGMENT). MACROPHAGE INFLAMMATORY PROTEIN 3 BETA PRECURSOR (MP-3-BETA) (EBI1- LIG GUANINE NUCLEOTIDE-BINDING PROTEIN G(0), ALPHA SUBUNIT 1. PLECKSTREN (PLATELET P47 PROTEIN). TRAHSLOCON ASSOCIATED PROTEIN, ALPHA SUBUNIT PRECURSOR (TRAP-ALPHA) (S

i) Significant genes from the MDA MB 468/ING1c array.

91 H OS CO OS O CM ai r- -f CM co r- p- oo r- OB

ALDEHYDE DEHYDROGENASE. CYTOSOLIC (EC 1.2.1.3) (CLASS 1) (ALHDII) SIGNAL TRANSDUCER AND ACTIVATOR OF TRANSCRIPTION 1-ALPHA/BETA (TRAD HERA PROTEIN (TUP1 LIKE ENHANCER OF SPLIT PROTEIN 1) . INTERLEUKIN-16 (TL-16) (LYMPHOCYTE CHEMOATTRACTANT FACTOR) (LCF).

MS Tf ON ©> rH p- CM «l 91 P- CM

HISTONE H3.3 (H3.B) (H3.30). [VASCULAR ENDOTHELIAL GROWTH FACTOR B PRECURSOR (VEGF-B) (VEGF RELJ I EPHRTN TYPE-A RECEPTOR 7 PRECURSOR (EC 2.7.1.112) (TYROSINE-PROTE] 1 ARYLACETAMTDE DEACETYLASE (EC 3.1.1.-) (AADAC) . PUTATIVE ATP-DEPENDENT RNA HELICASE KTAA0134. DUAL SPECIFICITY PROTEIN PHOSPHATASE 1 (EC 3.1.3.48) (EC 3.1.3.16) MICROSOMAL STRESS 70 PROTEIN ATPASE CORE PRECURSOR. | DIPEPTIDYL PEPTIDASE IV LIKE PROTEIN (DIPEPTIDYL AMINOPEPTIDASE I 40S RIBOSOMAL PROTEIN S26. GALECTIN-1 (BETA-GALACTOSTDE-BINDING LECTIN L-14-I) (LACTOSE-BIND] POLYADENYL ATE -BINDING PROTEIN 1 (POLY (A) BINDING PROTEIN 1) (PABP CENTROMEREC PROTEIN E (CENP-E PROTEIN) . 70 KD PEROXISOMAL MEMBRANE PROTEIN (PMP70). ANION EXCHANGE PROTEIN 2 (HTJN-EHYTHROID BAND 3-LIKE PROTEIN) (BHD: PROTEINASE ACTIVATED RECEPTOR 3 PRECURSOR (PAR-3) (THROMBIN RECEP] | AMELOREDE-SENSITIVE AMINE OXIDASE (COPPER-CONTAINING) PRECURSOR (E 60S RIBOSOMAL PROTEIN L6 (TAX-RESPONSIVE ENHANCER ELEMENT BINDING I Gl TO S PHASE TRANSITION PROTEIN 1 HOMOLOG (GTP-BINDING PROTEIN GS I LYSOSOME-ASSOCIATED MEMBRANE GLYCOPROTEIN 1 PRECURSOR (LAMP-1) (CI NUCLEOPROTEIN TPR. TRANSCRIPTION FACTOR E2-ALPHA (IMMUNOGLOBULIN ENHANCER BINDING FA( I NEUROENDOCRINE PROTEIN 7B2 PRECURSOR (SECRETORY GRANULE ENDOCRINE Figure 27. Significant gene list (a) and SAM plot (b) for cDNA microarrays.

a) Significant gene list for the MDA MB 435/ING 1b array.

Significant Genes List

Imputation Engine 10-Nearest Neighbor Imputer

Data Type One class Response

Number of Permutations 100 RNGSeed 1234S67

(Delta, Threshold) (048678,0.00000) (Upper Cutoff, Lower Cutoff) (3.27500, --)

Computed Quantities

Computed Exchangeability Factor SO 0.024274998 SO percentile 0 False Significant Number (Median, 90 percentile) (0 00000. 0.00000) False Discovery Rate (Median, 90 percentile) (0.00000,0.00000)

2 Positive Significant Genes

Row Gene Name Gene ID Scoreid) Numerator(f) Denommatoris+s0)

1505262868 0 650616667 0 186611377 l PROLKERA TTVKT CtLL NUaKXAH ANIlGbNP12inW'XHKA 1 Of* ASSOOA ILD MKXL-OLA H PHOTUN W 2(1, 6! HCfcCOeOX PROT3«J r*£JS2 (M3S1-RELATED PROTF3M 1) (FRAG^ONIT) 6 3.274995811 0 54166666" 016539461"

0 Negativ 9 Significant Genes

Row Gene Name Gene ID Scoreid i Numeraior(r) Denominator(s+s0)

Significant: 2 Bella 0.48678 Median * false significant:0:00000 SAM Plot "niresnold 0.00000

b) SAM plot for the MDA MB 435/ING1b array.

Significant: 2 Delta 0.48678

Median tt false significant: 0.00000 SAM Plot Threshold 0.00000

Expected 92

Figure 27. Significant gene list (a) and SAM plot (b) for cDNA microarrays.

c) Significant gene list for MDA MB 435/ING 1c array.

Significant Genes List

Input Parameters Imputation Engine 10-Nearest Neighbor Imputer One cMi Reeponae Number ol Permutation* 100 RNGSeed I234M7 (Dene, Three hold) (0.70784,0.00000) (Upper Cutoff. Lower Cutoff) (3.40416, -»)

Computed Quantities Computed Bech an q • at» lity Feci or So 0.01 187788 SOperoenllle Felae Significant Number (Uedlen, SO percentile) (0.00000, 0 00000) False Discovery Rate (Median, 00 peroentlle) (0.00000, 0 0O00OJ

Significant Genet> Gena Name Sooreidi NumeratoKri UenominntoM KERATN TYf^iC^TOSKELETAl 10 (CYTpXEPATN 10) (KIM tCttJO) 0 IW433333 RAN SPfflFTCGTPASe-AC^AlfJG PPCTEN I[RAN RNOUG PR0THN .. -WNBP-

HVPOTHFTCAL PROTEft KiA A008' •"u*t0Q? &43801a>»3 f*^TEt^SE ACTW HBC^PTQW3 PWCLWSQR IPAR-3> If-ffOMaH WCBPTORg)^ - ; 1 i„J6762493 0 3123 -RA*t5,POf***J f»-OB ih6| 3 «7631_7»b 0*09766667 PEPOXtSOMEASSOwBLV FACTOR-2 (RAF-2) (P9X»tSOMA^ TYPE ATPASE I . 'RFOxl*h

0 Negative Significant Genes

d) SAM plot for the MDA MB 435/INGIc array.

Significant: 7 Delta 0.70754 Median # false significant: 0.00000 SAM Plot Threshold 0.00000

Expected Figure 27. Significant gene list (a) and SAM plot (b) for cDNA microarrays.

e) Some significant genes for the MCF7.ING1b array as calculated by SAM.

Significant Genes List

Input Parameters Imputation Engine 10-Nearest Neighbor Imputer Data Type One class Response Mum bar of Parmutatlons 100 RNQStad 1234567 (Delta, Threshold) (1.03113,0.00000) (Uppar Cutoff, Lower Cutoff) (2.68679, -2.61671)

Computed Quantities Computed Exchangeability Factor SO 0.009654107 SO pe rcentile 0 Falsa Significant Number (Median, 90 percentile) (1.00000,7.20000) False Discovery Rata (Median, 90 percentile) (1.14943. 8.27566)

17 Positive Significant Genes Row Gene Name Score(d) iJumerator(r) Denominator^+i0) 180' SIGNAL TRANSDUCER AND ACTTVATOfi OF TRANSORPTDN 5A 5.922787211; 0162615 0.027455823 57 Ca_L SURFACEGLVCOFBDTBrJ EM=M FfECURSOR (EMR1 HORMONE RECEPTOR). S.007834482 0.14867 0029687483 28 DYSTROPHW. 3.834684669 0.253656667 0.066147986 173 PARVALBLMN ALFHA. 3 528303389: 0.12291; 0.03483544 189 CHOLINE KINASE (EC 2.7.1.32). 3.465133892 0.164456667 0.047460408

164; CYTOCHROME C. 3.428691648; 0.10025 0.029238558 146iEFHFB+B1 PRECURSOR (EPH-RELATED RECEPTOR TYFOStE KINASE LIGAND2) (LERK-2) (ELK LXSAND PRECUR& 3.285018334 0.146626667 0.044634961 187 5-AMNOLEVULINC AOO SYNTHASE MTOCHONDFttAL PRECURSOR NONSPECFCfEC 2.3.1.37} (DELTA - A MNOLE; 3.172761063: 0.13197! 0.041594686 169 B1 BRADYKINW RECEPTOR (BK-1 RECEPTOR) 3.171607738 0.095885 0.030232301 142 NBJTROPHL DEFENSNS 1. 2 AND3 PRECURSOR (HNP) (DEFENSIN. ALPHA 1). 313407187 0.060513333 0.019308215 ia8JRAFrTOTaONCCGEI*SER^aW (C-RAF). 3.107173546; 0.143095! 0.046053108 295JCHOLESTERYL ESTER TRANSFER PROTEIN PRECURSOR (LW) TRANSFER PROTBN I). 3 071776041 0.09643, 0.031392263

70 Negative Signifies nt Genes Row Gene Name Score(d) Numerator(r) Denominator Is *«0) 206 GUANINE NUCLEOTIDE EXCHANGE FACTOR MSS4. -It 75116091 -0.6 0.051058785 35 ZWC FWGER PROTBN 136. -11.20580592 -0662633333 0.059133037 119 MYB FR0TO-ONCOGENE PROTBN(C-lvTYB) -10.36379471 -0.802183333 0.077402472 108 60S RBOSCMAL PROTEIN L1 (L4> -8.537774594 -0 52565 0.061567566; 43 60S RIBOSOMAL FROTE3N L10 (QM PROTBN) (TUMOR SUPRESSOROM) (LAMNW RECEPTOR HOMO LOG). -8.42836486 -0.50895 0.060385376 1 to;WEE1-LIKEPROTEIN KNASE(EC 2.7.1.112). -8.364004159 -0.616883333; 0.073754546 S3 CASPASE-8 PRECURSOR (EC 3.4.22.-) (ICE-LIKE APOPTOTIC PROTEASE 5) (MORT1 -ASSOCIATED CED-3 HOMOLCK -8.302693881 -0.471133333 0056744635 304 KEHATK TYPE ICYTOSKELETAL 18 (CYTOKERATlN 18) (K18) (CK 18) -8.080852057 -0.7323; 0.090621632 92 CYTOCHPOME C OX CASE POLYPEPTIDE VIA-LWER PRECURSOR (EC 1.9.3.1) (FRAGMENT) -7.841976333 •0.42275; 0.053908604 231 ADP-RBOSY LA TON FACTOR 3. -7.726042256 -0.610433333 0.079009836' 54 ENOOTHEL1N-1 PRECURSOR (ET-1). -7,665404895 •0.328033333; 0.042794 117 UBDUfTlN-CONJUGATING ENZYME E2-17 KD(EC6 3.2.19) [UBOUmN-PROTBN LIGASE) (UBOUITIN CARRIER PROT -7.658187287 -0.576116667. 0.075228856! 15|GUANME NUCLEOTIDE-BWDWG PROTBN G(I)-'G(S)/G(T) BETA SUBUMT 2 (TRANSDUCIN BETA CHAN 2) -7.571794147 -0.484133333 0.063939051!

Expected Figure 27. Significant gene list (a) and SAM plot (b) for cDNA microarrays.

g) Some significant genes for the MCF7.ING1c array, as calculated by SAM.

Significant Genes List

Input Parameters Imputation Engine 10-Nearest Ne Data Type One class Ref Number of Permutations 100 RNGSaed 1234567 {Delta, Threshold) (0.59495, 0.00C (Upper Cutoff, Lower Cutoff) (1.95913, -2.95

Computed Quantities Computed fcxchangoability Factor SO 0.013893301 SO percentile 0 False Significant Numbar (Median, 90 percentile) (0.00000, 2.00C False Discovery Rate (Median, 90 percentile) (0.00000, 33.33

4 Positive Significant Genes Row Gene Name Gene ID Score(d) • 28MJSCAPtfvS C ACETYLCHOLINE RECEPTOR Ml 28 2 251173686 24 VASCULAR ENDOTHELIAL GROWTH FACTOR B PRECURS OR (VEGF-B) (VEGF RELATED FACTOR). 24 2.127313853 14 SON PROTEW {SONS). 14 2.010683679 2 T-LYMPHOCYTE MATURATION-ASSOCIATED PROTBN. 2 1.959134101

2 Negative Significant Genes How Gene Name Gene ID Score(d) 33 ALCOHOL DB-T/DrlOCieNlASEC^SS ICHCHAINIK 1 1t.t ) (GLUTATHIONE- DEPENDENT FORMALDEHYDE ClEHYDROGENASE) (EC 1.2.1 1) (FDH). 33 -3329668343 30 HYPOTHETICAL PFOTBN KIAA0136 (FRAGMENT). .50 -2.952970942

f) SAM plot for the MCF7/ING 1c array.

Expected 95

Figure 27. Significant gene list (a) and SAM plot (b) for cDNA microarrays. h) Some significant genes for the MDA MB 468/ING1 b array, as calculated by SAM.

Significant Genes List

Input Parameters Im pu Li l to n Engine ID-Nefires i Neighbor Impute Data Type One elasa Response Number of Permutations 100 RNGSeed 1234567 (Delta. Threshold) (0.37282, 0.00000) (Upper Cutoff. Lower Cutoff) (1.49298, -3.12465)

Computed Quantities Computed Exchangeability Factor SO 0.020041014 0 SO percentile centlle) (o.ooooo, i.ooooo) False SignificanDiscovery t RatNumbee (Medianr (Median. 90 percentile, 90 per ) (0.00000, 33.33333)

2 Positive Significant Genes Row Gene Name Gene ID Score(d) Numerator(r) Denominators+s0) 5: OXYTOON RECEPTOR (OT-R) 5 1 787055276: 0 104453333 0 058452771

3 GRAVN(FRAGMENT) ? 1 492980024: 0 059841667 0 040082026:

1 Negative Significant Genes I Row Gene Name Gene ID Score(d) Numerator(r) Danominator(s+«0) 2 PLECKSTRM (PLATELET P47 PROTBN) 2 -3 12465372V -0.3275 0 104611614.

i) SAM plot for the MDA MB 468/ING1b array.

Significant: 3 Delta 0.37282 Median # false significant: 0.00000 SAM Plot Threshold 0.00000

- ^^^^

.5 -1 ' 0.5 1 1 I n o

.—2 „'.._. ....

Expected Figure 27. Significant gene list (a) and SAM plot (b) for cDNA microarrays. k) Some significant genes for the MDA MB 468/ING1c array, as calculated by SAM.

Significant Genes List

Input Parameters Imputation Engine 10-N*arest Neighbor Impuler Dala Type One class Response Number of Permutations 100 RNGSeed 1234567 (Delta, Threshold) (0.94450, 0.00000) (Upper Cutoff, Lower Culo(f) (3.B6836, -2.10497)

Computed Quantities Computed B< change ability Factor SO 0.016137557 SO percentile 8 False Significant Number (Median, 90 percentile) (1.00000, 15.00000) False Discovery Rale (Median, 90 percentile) (3.70370. 55.55556)

4 Positive Significant Genes Row G^ne Nam a Cans ID Score(d) Numerator(r) DonominaIor(s+s0) 29 PROTEN KNASEPKX1 (EC 2.7.1.-)- 29 4.624942919 0.2266: 0.04899519E 23 ALDEHYDE DEHYDROGENASE CYTOSOLC (EC 1.2 13) (CLASS 1) (ALHDI) (ALDH-E1) 23 2.967695703 0.236525; 0 079699880 150 ETS-RELATED TRANSCRIPTION FACTOR ELF-1. 150 2.893346718 0 162528333. 0.056173127 14i HRA PROTEW (TUPl LKE ENHANCER OF SR.fT PFOTEN 1). 14 2 868355474 0 2947' 0.102741791

23 Negati /e Significant Genes Row Gene Name Gene ID Score(d) Numeretor(i) Senom inator(s+s0) 187 HISTONE H3 3 (H3B) (H3.30) 187 -4 357872152 -0 762816667 0 17963277E 130- VASCULAR ENDOTHELIAL GROWTH FACTOR D PRECURSOR (VEGF-D) (VEGF RELATED FACTOR). 130 -4 31244935 -1 436733333 0 333159464 B2 70 KD PEROXISOMAL MEMBRANE PROTBN(PMP70). 82 -3 862709511 -1 256916607 0 32539766* HI LYSOSOME-ASSOCIATED MEfcBRANE GLYGOFfiOTEN 1 PRECURSOR (LAMP-1) (GDI07A ANTIGEN) 81 -3.731314106 -0 763433333: 0 20460173; B7 PROTENASE ACTIVATED RECEPTOR 3 PRECURSOR (PAR- 3) (THROMBN RECEPTOR 2) 67 -3 69552414 -0 810133333: 0.21922014' 89; CWTO3MERC PFOTEN E (CENP- E PROTT3N). ] 89 -3.605110505 -1 574666667 0 436787351 71, AMLOPJDE-SENSlTTVEAMNEOXIDASE (COPPER-CONTANNG] PRECURSOR (EC 1,4.3,0) [DIAMINEOXIDASE) (DAO 71 -349033455 -0 989016667 0 28335870; 88 ANON EXCHANGE PROTEN 2 (NON-EFYTHROD DA NO 3-LKE PFCTEN) (BND3L) 66 -3 489455262 -1 358016667. 0 38917726f 172: G1 TO S PHASE TRANSfTON PFOTEN 1 HOMOLOG (GTP-DNDNG PROTEN GST1-HS) .72 -3 428794937 •1.011933333 0 29512798; t NUCLEOFROTEN TPR -2 999035501 -1 058316667' 0 35288567: 60 60S RBOSOMAL PROTEN L 6 (TAX-FESPONSIVE ENHANCER ELEMENT BtCtfJG PROTON 107) (TAXREB107) (NEOF.60 -2.B9649246 -0.78685 0 27165615; 66 MCROSOMAL STRESS 70 PROTEN ATPASE CORE PRECURSOR 00 -2 755832149 -1 139666667 0 4I354719( 63 40S RBOSOMAL PROTEN S26 63 -2.723059175 -1.12385 0.41271596V

131:D(P£PTOYLPEPT1DASEIV LKE PROTEN (DIPEPTDYL AMNOPEPTlDASEr RELATED PFOTEK; (DIPEPTDYLPEPT1DAS 131 -2.694448694 •1 198833333 0 444852901

—5-1 Expected Table 4. Summary of Overexpressed and Underexpressed Significant Genes in MCF7(1), MDA MB 435(2) and MDA MB 468(3) Cell Lines. Intensity values in the table are represented by median-transformed, non-logged data. A gene that is equally expressed in control and test cells has a value of 1.0; an upregulated gene has a value greater than 1, while a downregulated gene has a value less than 1.

AVERAGE EXPRESSION LEVEL ACCESSION GENE NAME CHANGE 1-ING1B 1-ING1C 2-ING1B 2-ING1C 3-ING1B 3-ING1C W91932 PROLIFERATING-CELL NUCLEO UP 1.56983907 R35310 HOMEOBOX PROTEIN MEIS2 (M UP 1.45565318 AA130179 SIGNAL TRANSDUCER AND AC1 UP 1.11931415 R08293 CELL SURFACE GLYCOPROTEII UP 1.10854705 W05777 DYSTROPHIN. UP 1.19222511 M010608 PARVALBUMIN ALPHA. UP 1.08892908 W76463 CHOLINE KINASE (EC 2.7.1.32). UP 1.12074392 W03017 CYTOCHROME C. UP 1.0719592 R34203 EPHRIN-B1 PRECURSOR (EPH-F UP 1.10697809 AA098907 5-AMINOLEVULINIC ACID SYN1 UP 1.09578898 W49512 B1 BRADYKININ RECEPTOR (BK UP 1.0687208 T81712 NEUTROPHIL DEFENSINS 1, 2 UP 1.04283675 W73651 RAF PROTO-ONCOGENE SERIN UP 1.10427156 R89213 CHOLESTERYL ESTER TRANSF UP 1.0691246 N53346 ZINC FINGER PROTEIN 151 (MI2 UP 1.09522074 T65624 DUAL SPECIFICITY PROTEIN Ph UP 1.09210697 W52609 GLUTAREDOXIN (THIOLTRANSF UP 1.05457132 AA045904 6-PYRUVOYL TETRAHYDROBIO UP 1.11774803 R15219 EPHRIN TYPE-A RECEPTOR 7 P UP 1.09451494 W52156 OXYTOCIN RECEPTOR (OT-R). UP 1.07509067 N31391 GRAVIN (FRAGMENT). UP 1.04235136 H63198 MSS4 GUANINE NUCLEOTIDE E DOWN 0.65975396 W67485 ZINC FINGER PROTEIN 136. DOWN 0.63172417 N49526 C-MYB PROTO-ONCOGENE PRC DOWN 0.57348063 H69367 60S RIBOSOMAL PROTEIN L1 (L DOWN 0.69464607 W04913 60S RIBOSOMAL PROTEIN L10 ( DOWN 0.7027337 AVERAGE EXPRESSION LEVEL ACCESSION GENE NAME CHANGE 1-ING1B 1-ING1C 2-ING1B 2-ING1C 3-ING1B 3-ING1C AA039639 WEE1-LIKE PROTEIN KINASE DOWN 0.6520781 R07395 CASPASE-8 PRECURSOR DOWN 0.72139767 W79141 KERATIN, TYPE I CYTOSKELET/ DOWN 0.60194351 H47721 CYTOCHROME C OXIDASE PRE DOWN 0.74600127 AA044771 ADP-RIBOSYLATION FACTOR 3. DOWN 0.65499993 R55914 ENDOTHELIN-1 PRECURSOR (E DOWN 0.79662169 AA134026 E2-17 KD UBIQUITIN-CONJUGA1 DOWN 0.67076687 N98261 TRANSDUCIN BETA CHAIN 2 DOWN 0.71492642 N28330 MUC18PRECURSOR DOWN 0.71883539 N42817 CYTOCHROME C OXIDASE POL DOWN 0.72758279 R83533 MACROPHAGE MANNOSE RECE DOWN 0.61973929 T87961 DUAL SPECIFICITY MITOGEN-Ai DOWN 0.66952817 R46284 HORMONE SENSITIVE LIPASE ( DOWN 0.70365167 N31851 HIGH MOBILITY GROUP PROTE DOWN 0.69810535 H60510 KERATIN, TYPE II CYTOSKELE DOWN 0.54822879 AA054303 GLYCERALDEHYDE 3-PHOSPH DOWN 0.64803786 H66535 CENTROMERIC PROTEIN E (CE DOWN 0.6711932 0.33572068 N39691 COFILIN, NON-MUSCLE ISOFO DOWN 0.73053063 H67329 40S RIBOSOMAL PROTEIN S6 (F DOWN 0.82529587 AA019140 ERYTHROCYTE ADDUCIN ALPH DOWN 0.79931348 R01627 HLA CLASS II HISTOCOMPATIE DOWN 0.7389763 H75494 PROTHROMBIN PRECURSOR (E DOWN 0.73410076 R63039 TRICHOHYALIN. DOWN 0.68519348 H01582 PLECKSTRIN (PLATELET P47 P DOWN 0.64144612 0.79691624 H61357 CELLULAR TUMOR ANTIGEN P5 DOWN 0.62118002 H83215 MELANOMA-ASSOCIATED ANTK DOWN 0.66150929 H71793 X BOX BINDING PROTEIN-1 (XBf DOWN 0.67890581 H85763 RETINOBLASTOMA-LIKE PROTE DOWN 0.61135545 T83460 LEUKOCYTE ADHESION GLYO DOWN 0.82403832 H68339 METALLOTHIONEIN-IF (MT-1F). DOWN 0.70220621 R87198 TUBULIN BETA-5 CHAIN. DOWN 0.76029612 N30952 60S RIBOSOMAL PROTEIN L18. DOWN 0.65239456 CO AVERAGE EXPRESSION LEVEL ACCESSION GENE NAME CHANGE 1-ING1B 1-ING1C 2-ING1B 2-ING1C 3-ING1B 3-ING1C AA047157 CD82 ANTIGEN (INDUCIBLE MET DOWN 0.61464132 R19042 D-ASPARTATE OXIDASE (EC 1.4 DOWN 0.69822633 R18479 ALKALINE PHOSPHATASE, TIS DOWN 0.68039762 H13357 MYB-RELATED PROTEIN A (A-M DOWN 0.70910278 R06476 FMET-LEU-PHE RECEPTOR (FM DOWN 0.6753778 N76562 FERRITIN HEAVY CHAIN. DOWN 0.71852819 AA132194 AMILORIDE-SENSITIVE AMINE ( DOWN 0.63442286 0.50382106 N42117 DNA MISMATCH REPAIR PROTE DOWN 0.73407532 W56465 GUANINE NUCLEOTIDE-BINDIh DOWN 0.64058711 N95679 CYSTEINE DIOXYGENASE (EC 1 DOWN 0.71096478 W07452 60S RIBOSOMAL PROTEIN L6 (1 DOWN 0.75248427 0.57960824 H17218 CALMODULIN. DOWN 0.790443 W70135 VITAMIN D3 RECEPTOR (VDR) DOWN 0.77561718 R22872 CATHEPSIN S PRECURSOR (EC DOWN 0.66404366 R20031 DNA-DIRECTED RNA POLYMER/ DOWN 0.71150707 H14843 IDURONATE 2-SULFATASE PRE DOWN 0.65122741 W78758 U1 AND U2 SMALL NUCLEAR Rli DOWN 0.84316292 H09051 GUANINE NUCLEOTIDE-BINDINC DOWN 0.69383603 W33123 ADP-RIBOSYLATION FACTOR-LI DOWN 0.67645537 R11403 40S RIBOSOMAL PROTEIN S7 (S DOWN 0.6697293 R74428 HLA CLASS I HISTOCOMPATIB DOWN 0.64791809 AA127684 INOSINE-5'-MONOPHOSPHATE 1 DOWN 0.71522381 W42848 ALZHEIMER'S DISEASE AMYLOI DOWN 0.77676495 N46036 THYMOSIN BETA-4 (FX). DOWN 0.73579035 N25456 COLORECTAL MUTANT CANCEF DOWN 0.72076457 N40311 TRANSALDOLASE (EC 2.2.1.2). DOWN 0.77799529 T97408 BCL-2 BINDING ATHANOGENE-1 DOWN 0.79493015 AA039258 60S RIBOSOMAL PROTEIN L31. DOWN 0.77147965 W46219 POLYADENYLATE-BINDING PR( DOWN 0.77733048 0.85971279 R86898 DELTA-TYPE OPIOID RECEPTOI DOWN 0.69560972 AA127989 jHOMEOBOX PROTEIN PRH (HOI DOWN 0.77967782 H80498 HYPOTHETICAL PROTEIN KIAAC DOWN 0.9405566 CD AVERAGE EXPRESSION LEVEL ACCESSION GENE NAME CHANGE 1-ING1B 1-ING1C 2-ING1B 2-ING1C 3-ING1B 3-ING1C R87060 VITAMIN K-DEPENDENT GAMM/ DOWN 0.79343466 W79141 KERATIN, TYPE I CYTOSKELE" UP 1.14686851 R68428 RAN-SPECIFIC GTPASE-ACTIVA UP 1.31841083 AA054118 HYPOTHETICAL PROTEIN KIAA( UP 1.10121793 N34619 PROTEINASE ACTIVATED RECE UP 1.23721823 W35103 ADENYLATE KINASE ISOENZYW UP 1.20564068 W67471 TRANSFORMING PROTEIN RHC UP 1.39213585 W56671 PEROXISOME ASSEMBLY FACT UP 1.06403824 T80006 MUSCARINIC ACETYLCHOLINE UP 1.10246111 R90829 VASCULAR ENDOTHELIAL GRO UP 1.12738869 R17312 SON PROTEIN (SON3). UP 1.11172503 R17499 T-LYMPHOCYTE MATURATION-/ UP 1.11155808 N46526 PROTEIN KINASE PKX1 (EC 2.7. UP 1.17007418 H48784 ALDEHYDE DEHYDROGENASE UP 1.17815144 W05657 ETS-RELATED TRANSCRIPTION UP 1.11924691 H23459 HIRA PROTEIN (TUP1 LIKE ENH UP 1.22662988 W70169 ALCOHOL DEHYDROGENASE C DOWN 0.84358577 R80121 HYPOTHETICAL PROTEIN KIAAC DOWN 0.82823764 W31509 HISTONE H3.3 (H3.B) (H3.3Q). DOWN 0.58123091 R90829 VASCULAR ENDOTHELIAL GRO DOWN 0.97800133 R73965 70 KD PEROXISOMAL MEMBRAf DOWN 0.41843729 R89708 LYSOSOME-ASSOCIATED MEMI DOWN 0.58909274 N34619 PROTEINASE ACTIVATED RECE DOWN 0.57032915 R71723 ANION EXCHANGE PROTEIN 2 ( DOWN 0.39011823 H03728 G1 TO S PHASE TRANSITION PF DOWN 0.49588128 R20063 NUCLEOPROTEIN TPR. DOWN 0.48019202 R13040 MICROSOMAL STRESS 70 PRO- DOWN 0.45386443 W32999 40S RIBOSOMAL PROTEIN S26. DOWN 0.45886765 R88469 DIPEPTIDYL PEPTIDASE IV LIKE DOWN 0.43568781 W68192 GALECTIN-1 (BETA-GALACTOSI DOWN 0.51809911 R12985 NEUROENDOCRINE PROTEIN 7 DOWN 0.74937253 H04421 DUAL SPECIFICITY PROTEIN PI- DOWN 0.41751984 CHAPTER 4: DISCUSSION 102

Part I: The ecdysone-inducible vector system is effective for obtaining high levels of expression in transient assays, but not in stable assays.

The Efficacy of Ecdysone-inducible Vector System for Generation of Transient and Stable Cell Lines The ecdysone-inducible vector system, pioneered by No et al in 1996, showed much promise as a transgenic system capable of inducing high levels of expression with low background, low toxicity of the inducer, and rapid induction of responses [72]. One of its main advantages over the Tetracycline-based system, another commonly utilized inducible expression system for both in vitro and in vivo studies, is that the lipophilic characteristic of the steroid hormone inducer allows it to permeate all tissues, including the brain. Due to the short half-life of ecdysone and its synthetic analogues, immediate and robust inductions are possible; it is also cleared quickly and not retained or stored [72]. Again, this is advantageous over tetracycline, which is cleared slowly from bone, impeding rapid and robust inductions [87, 88]. As shown in Figure 4 (p. 63), the ecdysone system was a highly effective for inducing overexpression in all of the breast cancer cell lines used in this project, when introduced by transient transfection. In addition, the inductions seen are those in total protein samples from cells electroporated and cotransfected with both vectors. With only 5-20% transfection efficiency (by electroporation of the different cell lines), this small percentage of cells are roughly overexpressing the transgene, which indicates that levels of induction in those cells are much higher than appear on the Western blot (Fig. 4,5, pgs. 64- 65). As expected, increasing levels of hormone inducer caused increasing levels of transgene expression with ING1b sense, INGla sense and p53 inducible vectors. Although the objective of the experiment in Figure 4 (p. 64) was to verify that the inducible constructs were indeed inducible using the kit manual's recommended levels of hormone, it is likely that even higher levels of expression could have been obtained by increasing Ponasterone levels above 10 u.M. Low levels of background were present in cells transfected with the same vectors, but induced with ethanol or DMSO alone.

Cells transfected with ING1 antisense constructs do not consistently show decreases in ING1b levels In cells transfected with inducible vector containing ING1 antisense, a very modest but inconsistent decrease in ING1b protein levels was visible (Fig. 4, p. 64). This may be explained by the inconsistency of antisense experiments experienced by many researchers [89-96]. Several major obstacles must be overcome when using antisense strategies: antisense RNA must be sequence- specific, hybridize to target RNA quickly (to avoid degradation by nuclease attack) and be relatively non-toxic to the cells into which it is introduced. Most importantly, problems occur when the target RNA is involved in intramolecular pairing, forming secondary structures and preventing antisense RNA from binding [94, 95]. Studies have shown that even structurally similar RNA regions do not necessarily form duplexes, and that prediction of patterns of interaction is extremely complex and challenging [94]. A number of strategies, such as development of antisense oligodeoxynucleotides [92], use of ribozymes [90, 92, 93, 96], and use of 2-unit antisense RNA cassettes [95] have been explored in order to overcome these problems. In spite of these troubles, some antisense methods work, both in in vitro [97] and in vivo [98, 99] systems. From the experiments done in this thesis, it cannot be said conclusively that the ING1 antisense construct efficiently lowered ING1b levels. However, the construct was still used to create stables because the original ING1 paper had originally identified ING1 as a candidate tumor suppressor through antisense studies using a significant part of this sequence [10]. Thus it was reasoned that since some sequence within this construct had been proven to block ING1b expression, it may be effective when expressed at high levels in a stable cell line. However, if antisense ING1 strategies are to be used in the future, it may be appropriate to test several different ING1 RNA fragments, as well as antisense oligodeoxynucleotides, in order to see if a more reproducible reduction in ING1 levels is possible. In addition, the inducible antisense vector was created by cloning full-length ING1 cDNA in reverse orientation, into the multiple cloning site. This means that complementary sequence to all ING1 isoforms was being expressed, and that expression of the antisense mRNA may have blocked one, several or all of these isoforms. In the future, it may also be relevant to test this antisense sequence against individual isoforms to see if it is effective against any of them, or perhaps to generate isoform-specific antisense oligos to block expression of individual isoforms alone.

The ecdysone system is effective for generating stable pVgRXR clones in several breast cancer cell lines, but not stable pIND/pVgRXR clones As seen in Figs. 6 and 7 (pages 66-67), stably-integrated pVgRXR clones were obtained and induced to express either lacZ or ING1 b transgenes when stimulated with Ponasterone for 6-24 hours. These results suggested that the system was indeed effective, and several positive clones were appropriate for the next step of transfection with the pIND constructs and clonal selection. Throughout this entire process, cells were maintained in selective concentrations of Zeocin and G418, in an attempt to maintain the transgenes. Two positive (inducible) clones were selected from each cell line, and used to generate between 25-50 new inducible clones, screened by both immunofluorescence and by Western blotting. The advantage of screening by both of these methods is that immunofluorescent staining enabled us to see what percentage of cells were overexpressing the inducible transgene, and Western blotting enabled us to evaluate the increase in protein levels more quantifiably. As mentioned in the results, since five different parental breast cancer cell lines were being used to create inducible cell lines, all clones were initially transfected with pVgRXR and selected. Since it proved extremely difficult to manage five cell lines simultaneously, each with 25-50 clones, only two MDA MB 468 pVgRXR stable clones were proceeded with initially. These two clones were used to generate a number of ING1 b sense, ING1 b antisense and p53-inducible clones, which were screened by IF and Western blotting. Unfortunately, as indicated in Fig. 8 (p. 68), Western blots did not show any increases in protein levels (or reductions for ING1b antisense) after 24 hours induction with 5 u.M Ponasterone. When the same clones were screened by immunofluorescence, the same results appeared - no changes in protein levels after addition of hormone inducer. The first explanation for this was that perhaps none of the clones selected, in spite of their antibiotic resistance, contained the insert. This seemed unlikely however, because of the large number of clones screened. It was also hypothesized that the time for induction may not have been optimal. For example, the cells had maximally overexpressed the ING1b gene after a very short period of incubation with hormone, and levels had returned to normal by 24 hours when they were screened for expression. On the other hand, it was also possible that the cells were not induced for a sufficient period of time, and their increases in ING1b protein could only be seen after 24 hours. It is unlikely that the short half-life of Ponasterone was a contributing factor since it was used to induce the MDA MB 468 pVgRXR stables for 24 hours, which showed excellent expression of the reporter p-galactosidase gene. In consideration of the first possibility, IF was redone with cells were induced for 0, 12 and 24 hours. This yielded the first positive clone, C5, which showed excellent nucleolar localization of ING1b, but only in approximately 20-30 % of cells (Fig. 9, p. 69). It is also interesting that this localization, characteristic of cells overexpressing ING1b, is absent in the same clone induced for 24 hours. This suggested that optimal time for induction was closer to 12 hours in MDA MB 468, and that testing a large range of time points and hormone inducer concentrations would help to optimize the specific expression pattern for this clone. It was also decided that the best method for screening at this point was to use Western blotting, so that the increase in ING1b protein levels upon induction could be quantified. Optimization of Conditions for C5 Induction and Expression Experiments are Inconclusive In order to optimize C5 expression levels, two time courses were prepared, where cells were induced from 0-12 hours with 10 uM Ponasterone and from 0- 48 hours with various concentrations of both Ponasterone and Muristerone A (Fig. 11, p. 71-72). Based on the results seen with IF, we expected to see an increase in ING1b protein levels in the 0-12 hour time course, especially with the increase in concentration of hormone inducer (from 5 u.M to 10 p.M). Unfortunately, neither 0-12 nor 0-48 hour time courses showed increases in ING1b protein when C5 was induced with different concentrations of Ponasterone. As an attempt to determine if one ecdysone hormone analogue was more effective than the other, the clone was also induced with Muristerone A at a concentration of 5 u.M over different time points (Fig. 11A, p. 71). There appeared to be no difference in induction with either hormone, and no increases in ING1b expression were visible.

From these results it was difficult to make any conclusions, as they were contradictory to the immunofluorescence pictures. In order to explain this inconsistency, we looked back at the percentage of cells in the original C5 clone which had shown nucleolar localization of ING1b (20-30%), and hypothesized that 1) since only 20-30% of cells were overexpressing ING1 b, this increase in protein levels may not have been observable in total protein samples, which were used on the Western blot; 2) if any leakiness occurred in the system, the cells overexpressing ING1b may have died by apoptosis, resulting in a clonal population which does not consist of the best ING1 b expressers; or 3) due to the rapid growth rate of MDA MB 468 cells in culture (doubling time = approximately 24 hours), and significant number of passages they had undergone during the few weeks in culture which it took to prepare these experiments, the cells may have modified their transgenes, resulting in a decrease of its inducibility or expression levels. A considerable amount of evidence for modification of transgenes in mammalian cells is present in the literature [75-78]. Cells can modify genetic inserts commonly by methylation, leading to gene silencing [75] [77]. In addition, unusual DNA structures (such as hairpin DNA formed from triplet repeats and palindromic sequences) can promote genetic instability, resulting in abnormal recombination during mitosis and inversion or truncation of the transgene [100]. Expression of inserts can also be subject to positional effects, in other words genomic sequences flanking either the vector expressing the transactivator gene or response element can dictate the expression level of these genes, and potentially act as silencers [75, 76, 78]. Additionally, a considerable number of mutations exist in the MDA MB 468 cell line (Table 1) which may have influenced the fidelity of DNA replication, increasing the chance that the transgene was altered. It is very likely that other mutations are present in this cell line as well. In consideration of these multiple factors, it was first decided to dilute the clone down to single cells and attempt to isolate a subclone with high inducible levels of expression - in effect, to rescue the original close and ideally increase the percentage of cells which inducibly express the transgene. Unfortunately, after testing the isolated subclones at 12, 24, 36 and 48 hour timepoints of induction, no overexpression was seen in any line (Fig. 12, p. 73). With this lack of success, we decided to return to a frozen stock of the original C5 clone, taken at the original time of isolation and IF experiment, and to try inducing it again.

Original C5 clone ceases to overexpress ING1b after several weeks in culture due to loss of transgene Upon growing a fresh stock of inducible C5 in culture, cells were screened for mycoplasma and tested positive. As a result, C5 cells spent 3 weeks in culture with a course of two antibiotics (BM-cyclin1 and BM-cyclin2), in addition to G418 and Zeocin to maintain selective pressure. Finally, they were once again screened for overexpression by 0-12 hour and 0-48 hour time courses, at which no increase in protein levels could be seen with different times of induction or concentrations of inducer. In order to determine if one or both of the constructs had been lost, cells were transfected with the pVgRXR vector alone, the plND/ING1b vector alone, and with both vectors. Transfectants were induced for 24 hours and total protein was taken. As shown in Fig. 13 (p. 73). neither vector alone resulted in an increase in ING1b protein levels. When both vectors were transfected in, a resulting increase in ING1b protein is quite evident, confirming that the system remains inducible. At this point, it was reasoned that if the cells were to excise, mutate or silence one of the constructs, it would likely be the construct containing the ING1b transgene because this would give cells a growth advantage. Since overexpression of ING1b has been shown inhibit growth and induce apoptosis [10,41, 44], any cells who were not able to overexpress the transgene would ultimately avoid either of these two fates. As a final attempt to conclusively show that one or both of the constructs had been lost from these cells, genomic DNA was extracted from the C5 clone and from MDA MB 468 parental cells, and each were PCR-amplified for a region within the ING1b construct (Fig. 14B, p. 74). From the extremely faint band in the C5 lane after 30 cycles of amplification, it was concluded that the majority of cells within clone C5 did not contain the construct. It is possible that the cells may have mutated, truncated, rearranged or excised this region of the construct, or that cells containing the construct were selected out due to negative selection pressure over the considerable length of time in culture. Regardless of the exact mechanism, we can correlate the lack of inducible expression with the apparent absence of construct in genomic DNA taken from C5 cells. Thus, we conclude that our stable clone had lost expression due to transgene loss, and that the ecdysone system was not successful for generating a stable MDA MB 468 ING1b inducible clone.

One possibility for the lack of expression in the C5 clone is that stable integration of the plasmid actually never occurred, and throughout the screening process, positive results and antibiotic resistance were observed because the genes remained episomal. This idea could have been tested by Southern blotting, using chromosomal DNA isolated from the clonal cell line, and probes complimentary to both pVgRXR and the plND/ING1 b plasmids. This possibility, however, is extremely unlikely due to the absence of a mammalian origin of replication on the either vector, and the likelihood that both vectors would nave oeen diluted out over numerous population doublings which all clones had to undergo, in order to grown up in significant numbers for screening. To date, a number of groups have successfully used the ecdysone system to create stable cell lines which inducibly express tumor suppressor genes [72, 101-103]. The system has also been used for expression of numerous other genes [104] [72], as well as in vivo, to induce expression in murine mammary epithelial glands [71, 72]. Interestingly, the only study which mentions the percentage of stable cells induced to overexpress the transgene is the original paper, where 100% of 293 cells overexpress a stable lacZ gene under the control of the ecdysone-inducible promoter after induction for 24 hours with 1 u.M Muristerone [72]. None of the other studies mentioned if their clonal population was expressing the transgenes homogenously or heterogeneously [101-104], and it is possible that our original 20-30% of overexpressing cells is consistent with studies where heterogeneous expression is observed. Additionally, Invitrogen has recently modified its pIND vector to include 3 SPI sites which increase expression 5-fold (Invitrogen Manual). (As an aside, this is quite interesting, since modification of a vector that is reported to yield inductions of greater than 200-fold [72] seems as though it should not be necessary.) There are also reports indicating that problems have occurred with the ecdysone-expression system. Through personal communication, several labs have noticed leakiness (H. Muzik and D. Demetrick, personal communication.) One study reports that similarly with ecdysone and other inducible systems, genomic sequences which flank either the inducible gene or the transactivator can cause them to become silenced [78]. As a solution, they suggest using a reporter-linked transgene, so that a second set of response elements driving a GFP reporter gene is present behind the original transgene in pIND. By locating cells which express GFP, those cells expressing the transgene of interest in a heterogeneous population can be identified. Most interestingly, a recent study has been published which determined that both muristerone and ponasterone altered IL3-responsive pathways in a pro-B cell line [105]. They showed that introduction of both ecdysone analogues into culture resulted in P13Kinase/AKT activation, which potentially affected the growth and survival of the cells. In spite of the report that the hormone has no effect on mammals and is suggested to be ideal for uses in cultured cells and transgenic animals[72] and D.N, unpublished data, it appears that this is not necessarily the case. For use of the ecdysone-inducible system in the future, it may be useful to test the cells intended for stable integration by performing a cDNA microarray and looking at changes in gene transcription as a result of addition of Ponasterone or Muristerone, at varying concentrations. This may provide some evidence as to whether the cell line is affected or not: it may also help to determine whether different cell lines have different responses to these hormones. It is possible that the MDA MB 468 cell line was not an optimal line for use of this inducible system, and that one or several of the other lines attempted (MCF7, MDA MB 435, etc.) would have been more successful.

PART II: The Murine Mammary Fatpad Model is Effective for generating human breast cancer tumors in SCID mice

Although the results for this tumor model were preliminary, having been performed with only one of the five parental breast cancer cell lines to be used in the study, the tumors grew successfully with both inoculation volumes tested. The mfp model was straightforward to work with: tumors were easily measured with digital calipers and the tumor take rate was quite high, approximately 83% (Fig. 15, p. 75). It has been reported that increased tumor take and metastasis occurs when mice are inoculated in the mfp over subcutaneous injections, with certain cell lines [66]. This model is commonly used for human breast cancer studies in mice [106, 107] because it involves using the 'orthotopic site of injection', or injection and tumor growth in the normal equivalent murine site [65- 67]. As expected, the MDA MB 468 cell line did not metastasize to lung. (This cell line was chosen for our study so that we could observe differences between metastatic and non-metastatic breast cell lines when ING1 expression was induced or repressed in tumor cells.) The lung was the metastatic site chosen for observation because it represents one of the most common site for metastasis from mfp tumors in mice [67]. It is quite possible that other common sites (i.e. lymph nodes, bone, liver) may have been affected, however these organs were not observed during our study. The characteristics we observed for the MDA MB 468 tumors are in agreement with a similar study with the same cells in SCID mice, however a flank model was utilized [67]. In flank tumors, it was reported that tumors had reached a volume of 2.4 cm3 by 2 months, and by week 14, tumors had ceased to grow and became necrotic. At the time of necropsy, tumors were reported to be well-vascularized and encapsulated, which also corresponds with the characteristics of our mfp tumors. In conclusion, the mfp is an easy-to-work-with model, and would still be useful in the future for conducting in vivo studies on ING1 expression levels and breast cancer cell tumor growth and metastasis.

PART 3: The effect of ING1 on expression in breast cancer cells.

The Origins of the cDNA Microarray The cDNA microarray was first conceptualized by Schena, Davis, Brown, and Shalon in 1995 [82]. They identified several key problems in molecular biology: determining what specific sequences are represented in a complex RNA or DNA sample, determining the abundance of different expressed sequences, identifying what genes are expressed in cells under different conditions, and observing which genes are upregulated ordownregulated in a cancer cell. They proposed the array as a method of determining the relative abundance of a large number of expressed gene sequences, simultaneously, in a complex sample. The first array was prepared robotically, by printing spots of Arabidopsis cDNA onto a glass support, and hybridizing to it treated and untreated, two-colour fluorescently labeled cDNA. These experiments were followed by the generation of yeast cDNA arrays [108] and soon afterwards, human cDNA arrays [83]. These original array experiments were conducted with glass slide arrays where each gene cDNA was spotted in duplicate, and accurate expression ratios were calculated by averaging the intensity ratios of the two individual hybridizations. The original pairs of fluorescent probes consisted of lissamine and fluorescein [108], and Cy5 and fluorescein [83]. Arrays were scanned on confocal laser scanners which excited both fluors, and analysis was performed in a variety of manners. The next step in microarray analysis involved diversification in the types of experiments to which it was applied. Herskowitz and colleagues published an article in Science where gene expression of budding yeast at different time points during sporulation was measured by array and compared [109]. The following year, microarray analysis was used to compare expression patterns across a panel of different human breast cancer cells and tumors [110]. As the amount of information derived from array experiments began to accumulate exponentially, the development of data analysis techniques, primarily clustering algorithms, was increasing in importance [85]. The purpose of generating clustering programs was so that genes with similar expression patterns could be grouped together by standard statistical algorithms and graphically output in a format that was intuitive to scientists.

Currently, microarrays are being used for a variety of applications, from comparison of different expression patterns between cell lines [111, 112], primary tumors [81, 110, 113-118], different cell treatments [70, 86], cellular aging [119, 120] and studies in expression of specific genes [121-123]. In addition, a variety of methods for microarray analysis have been proposed, including various clustering algorithms and statistical significance analysis [85, 86, 121, 124, 125]. Since the technology and its analysis techniques are truly in their infancy, there is very little consistency among published microarray studies, both in terms of experimental procedure and data analysis. Thus, for new users, it is a challenge to identify which methods and analysis techniques are optimal and will generate truly valid and reproducible data. In addition, numerous biotech companies offer microarrays printed on a variety of supports: nylon membranes, glass, and even plastic; as well as different types of labels for control and test cDNAs (from different fluorescent dyes to various radioisotopes). Thus, there are many considerations and variables to be conscious of when commencing microarray experiments.

The ING1 cDNA Microarray For our experiments, we chose to use glass cDNA chips produced by the Ontario Cancer Institute (OCI), arrayed with cDNAs representing 1,718 different human genes. These chips were economical, easily obtainable, and were arrayed with a number of potentially interesting candidate genes for studies in ING1 expression. On these chips, each gene is spotted in duplicate, so that an average intensity ratio can be generated from two independently hybridized gene spots. Although there can be inconsistencies between these two ratios, each experiment was performed six times - three times with one set of fluor labelings, and three times where samples were labeled with the reverse fluors. Reciprocal labeling was performed to eliminate any variations due to differential incorporation of the two fluors. When data from all six replicate experiments was compiled, the data was filtered so that only genes which had equivalent ratios (either all 6 upregulated or all 6 downregulated) were analyzed. Finally, genes which passed this stage were median-centered and imported into SAM, which calculated which of these genes showed statistically significant verification. Table 4 shows a summary of the expression levels of all significant genes in three cell lines. Values in the table represent median centered, non-transformed expression levels, so that an expression level of 1.0 represents a gene which is equivalently expressed in control and test cell lines, a gene with a value of 0.5 represents a gene that is expressed at approximately half of control levels, and a an expression level of 1.5 represents a gene which is expressed at one and a half the levels in the control. In order to identify which genes are upregulated or downregulated as a consequence of ING1b or ING1c overexpression, we used three different breast cancer cell lines, MDA MB 435, MCF7 and MDA MB 468. We felt that in choosing three cell lines, we would be able to compare and contrast between the three, and learn different things about ING1 expression from each one. All of these lines have different mutational backgrounds and different metastatic properties (Table 1), which will give us further insight into potential relationships between ING1 and these cellular characteristics. Two important features worth mentioning are that MCF7 cells have wild type p53, while the other two cell lines both express mutant forms. Secondly, MCF7 cells express the estrogen receptor, an important treatment and prognostic indicator for breast tumors, while neither MDA MB 468 nor MDA MB 435 cell lines express this receptor. Since p53 has previously been implicated in the regulation of ING1b function, this may be an important consideration when examining array results.

Due to the identification of a large number of interesting and significant genes, only a small number have been selected for in-depth discussion in this thesis. Genes were chosen for discussion based on their involvement in cellular processes that ING1 is also believed to play a role in, such as apoptosis, cell cycle control, DNA repair and chromatin remodeling. Genes were also chosen for discussion if they were strongly upregulated or downregulated, or if they were part of a large groups of genes with common functions that were all shown to be consistently upreguation or downregulation on the array. Upregulation of PCNA and MeiS2 In the MDA MB 435/ING1b array, two genes were considered statistically significant by SAM; the Proliferating Cell Nuclear Antigen (PCNA), and MeiS2. PCNA is a protein involved in both DNA replication and nucleotide excision repair, and acts as a processivity factor for DNA polymerases 8 and s [126]. It has been shown to interact with ING1b under DNA damage conditions induced by UV [64], and a PCNA-binding motif has been identified in the ING1b protein sequence. There has been no evidence to date that ING1b is involved in the transcriptional regulation of PCNA, although this would be an interesting idea to follow up on. It is an unexpected result because it is a protein involved in DNA synthesis, which seems to be opposite in function to that of overexpressed ING1b, which is to induce growth arrest. More information is needed in order to clarify the relationship between these two proteins, and it is quite possible that MDA MB 435 possesses a mutation in the PCNA promoter, enabling it to become upregulated by ING1b overexpression. The ability of ING1b to induce expression of PCNA should be tested under normal cellular conditions and in cells induced to undergo a UV-damage response. It is interesting to observe this upregulation of PCNA mRNA in a cell line that possesses mutant p53, but not in the MCF7 cell line which has wild type p53. Since PCNA was not upregulated in the MDA MB 468 line, which also possesses a mutant p53, this may represent a cell-line specific effect of ING1b. The Meis2 gene is a transcription factor and homeobox gene that has been implicated in hematological malignancies [127, 128]. As seen in Figure 27 (p. 91-96), it is also upregulated in MDA MB 435 cells upon ING1b overexpression. It is known that PCNA has homeodomain protein binding sites upstream of its promoter [129, 130], although it is unclear as to which homeodomain proteins are able to bind this gene. One explanation for the co- overexpression of ING1b, PCNA and Meis2 could be that ING1b is able to upregulate Meis2, which in turn upregulates PCNA. This relationship should be explored further by verifying the co-overexpression of all genes at the mRNA and protein level, and perhaps determining if the same relationship exists in p53 wild type breast cancer cells.

Upregulation of signaling pathways In the MCF7-ING1b array, we see that STAT5A (signal transducer and activator of transcription), Raf and PYST1 are 3 of the 17 significantly upregulated genes. Since these all participate in the Jak/Stat/Map Kinase pathway, it would be interesting to look further into the role of ING1b in activating or participating in this signaling pathway. Surprisingly, one of the downregulated genes reported in the same array was MAPKK1, another member of this signal transduction chain, suggesting that the role of ING1 b is not by any means obvious, and requires further clarification. In the literature, PYST1 is reportedly activated under celiular stress conditions [131], which may suggest that this pathway links overexpression of ING1b to the growth arrest/apoptotic response that we see in many different cell types. This idea requires further exploration.

Upregulation of Receptors and Growth Factors Several receptor molecules were found to be upregulated by ING1b overexpression. In the MCF7 cell line, these included the Ephrin receptor A7 and B1 precursors, the EMR1 receptor and the Bradykinin B1 receptor. The Ephrin receptors are receptor tyrosine kinases involved in a wide range of processes including embryogenesis, neural development, and vascular development and have been implicated in tumorigenesis [132]. The EMR1 receptor is a G-protein coupled receptor with 7-transmembrane domains and is thought to associate with various hormone ligands, including parathyroid hormone [133]. Both of these receptors would be interesting to follow up on, as they may lead to a better understanding of the signaling pathways in which ING1b is involved. In the MCF7-ING1c array, mRNA expression of the VEGFB growth factor precursor was notably increased, however this same factor was shown to be downregulated in the MDA MB 468 ING1c array. Although the difference in expression may be a result of the different background mutations in each of these cell lines, or due to their different ER status, VEGF is an important inducer of angiogenesis and valuable prognostic indicator in breast cancer [134], and its relationship to ING1b expression in breast cancer cells should be explored further.

Upregulation of Enzymes and Cytochrome C Several enzymes were shown to be upregulated in the MCF7-ING1b microarray. These include choline kinase, gluraredoxin, 5-aminolevulinic acid synthase, and 6-pyuvoyl tetrahydrobiopterin synthase. It may be most interesting to follow up on glutaredoxin, as this enzyme is involved in redox-signaling pathways and plays a role in nucleic acid synthesis [135]. It was also interesting to find that cytochrome C was upregulated in the MCF7-ING1b array, since we know that ING1b co-overexpression with either p53 [44] or c-Myc [41] induces apoptosis, and this protein is released from the mitochondrial membrane during apoptosis. The cytochrome C promoter is reported to be activated by SP1 [136] and surprisingly by E2F [137], however there is not much other literature on its regulation. It is difficult to speculate on a relationship between this protein and ING1b, however it may be useful to pursue the connection between these two proteins, which may further our understanding of the role of ING1b in apoptosis.

Unfortunately, other important apoptotic mediators, such as Bax, Fas, c- Myc, and the caspases were not shown to be upregulated in any of the cell lines overexpressing ING1b. It is still possible that ING1b is involved in the regulation of these or other proteins that promote apoptosis. Perhaps in other cell types or under different conditions these effects would be visible, however within the mutational background of the breast cancer lines tested, these relationships were not evident. Oddly, the caspase 8 precursor protein was downregulated upon ING1b overexpression in MCF7 cells, in addition to the major apoptotic promoter p53. It is possible that these are also indirect effects of ING1 b expression, and that overexpression of ING1 b has caused a change in regulation of a protein which is involved in downregulating either of these. Regardless, all of these potential regulatory relationships await further validation by RT-PCR, Northern and Western analysis.

Downregulation of Proteins involved in RNA and Protein Synthesis In the MCF7-ING1b array, six ribosomal proteins were significantly downregulated. These included the 60s ribosomal proteins: L31, L6, L1, L10 and the 40s ribosomal proteins S7(S8) and S6. Many of these ribosomal proteins have functions in maintaining ribosomal architecture and promoting protein synthesis, although it has been noted that several ribosomal proteins, such as L10, have other functions [138]. Additionally in this array, the DNA- directed RNA polymerase II was downregulated, as well as the U1 and U2 small nuclear ribonucleic proteins, which participate in mRNA processing and splicing [139]. It also may be of importance to note that in the MDA MB 468/ING1c array, the same 60s L6 protein was downregulated, in addition to the 40s protein S26. These collective findings suggest ING1b has some role in downregulating protein synthesis when it is overexpressed. It is possible that ING1b acts as a transcriptional regulator, binding a consensus sequence in several of these genes in order to block their transcription or that its function as a HAT alters chromatin configuration leading to coordinate changes in gene expression. As ING1b possesses a PHD domain, known in other proteins to bind DNA, this is a valid possibility. Perhaps, by decreasing ribosomal protein synthesis, as well as the synthesis of other RNA-processing factors, ING1b slows or inhibits the assembly of RNA-processing machinery, leading to a deceleration of the cell cycle and either growth arrest or apoptosis. Cells initiate a ribotoxic stress response when the 3' end of the large 23s/28s rRNA subunit is damaged by UV or other cytotoxic agents [140]. It is possible that by causing a decrease in transcription of ribosomal proteins and related RNA processing factors, a new kind of stress response could be initiated by the cell. In the literature, numerous other cDNA arrays have probed for changes in expression of ribosomal processing and translational machinery genes [81, 110, 112, 114]. One group reports that expression of these genes was significantly correlated with cell doubling time, suggesting that these genes were regulated with respect to the rate of proliferation of the cell lines studied [112]. It is also possible that ribosomal proteins are downregulated as a secondary effect of INGIb's induction of growth arrest. For example, ING1 b may alter the expression of another gene(s), which is then responsible for downregulating genes involved in RNA processing. To verify either of these cases, the downregulation of ribosomal and related proteins should be tested by RT-PCR and Northern Blot analysis. Coincidentally, we know from previous experiments that ING1b localizes in the nucleolus when overexpressed or cells are UV-irradiated [37], and that the nucleolus is also the site of rRNA synthesis by RNA polymerase I [141]. Although it is possible that ING1b is sequestered in the nucleolus, or that it functions to sequester other protein(s) by residing there, it may actually function in the nucleolus to block rRNA synthesis by inhibiting and/or binding to RNA polymerase I, or by binding up newly synthesized rRNAs. This idea should be tested further, not only by RT-PCR, but also by colocalization experiments, and possibly IP binding assays.

Downregulation of cell structural components and related proteins A number of proteins involved in cell architecture were identified in the MCF7- ING1b array. These included Keratins K18 (type I) and K8 (type II), cofilin (also known as the actin depolymerizing factor [142]), dystrophin, pleckstrin [143], adducin (promotes association of spectrin with actin and caps the growing end of actin filaments; reviewed in [144]), cathepsin S (a matrix-degrading protein [145]) and trichohyalin (a protein that associates with keratin [146]). Keratins are members of the intermediate filament family of structural proteins, and intermediate filaments have been demonstrated to associate directly with the nuclear matrix, connecting cystoskeleton to nucleoskeleton [147]. Disruptions in the nuclear matrix, either directly, or those communicated through the cytoskeleton, are known to influence or regulate which genes are being transcribed. In addition, we know that ING1b and ING1a are both tightly associated with the nuclear matrix [64], and that both protein isoforms interact with proteins involved in gene transcription [58]. The observation that cytokeratins and other structural proteins are downregulated upon ING1b overexpression may suggest another mechanism by which ING1 b can influence the regulation of other genes, either directly or indirectly. If ING1b has a direct effect on downregulating these structural proteins, their decrease will result in a change in cytoskeletal and nucleoskeletal architecture, which will also affect what genes are transcribed. Indirectly, perhaps through INGTs ability to modulate chromosome structure, other genes are activated which have a negative effect on the expression of structural proteins. Upon validation by Northerns or RT-PCR, this would also be a promising finding in consideration of the motile properties of cancer cells. Many of these structural proteins are also involved in cell motility, and may play a role in invasion or metastasis [148,149]. If ING1b expression can decrease the levels of these proteins, resulting in decreased cell motility, perhaps this may provide one explanation for the observation that primary breast tumors with low levels of ING1b protein had a higher rate of metastasis to local nodes [27]. Taken one step further, this may lead to the use of ING1b protein levels as a prognostic indicator for invasive or metastatic tumors. Interestingly, cDNA microarrays in the literature report that untreated MCF7 cells express K8 and K19 [112], and other genes that are 'epithelial' in nature, suggesting that these cells originated from luminal epithelial cells. In addition, the K8, K18 and K19 keratins are estrogen regulated, and drastically downregulated when cells are grown in serum-free media [150]. Perhaps one mechanism by which ING1b downregulates these proteins is by interaction with some part of this steroid hormone pathway. This is another interesting idea that should be looked into in the future. Additionally, it was recently reported that UV-exposure of keratinocytes induced increasing levels of ING1b, in a dose-dependent manner [46]. Thus, there is much evidence suggesting that the role of ING1b expression in relation to keratin, keratinocytes and other proteins associated with cell structure should be studied further.

Downregulation of the p53 tumor suppressor gene Currently, there is much debate in the literature as to whether the expression of ING1b and p53 proteins are interdependent. Evidence in the literature has suggested that the two proteins physically interact when overexpressed [34, 43, 45] and cooperate to augment the apoptotic effect of expression of either protein alone [44]. In primary tumors, both gastric and breast tumors with decreased ING1b expression were also reported to have decreased p53 expression [151, 152]. This relationship was pursued further in p53-knockout experiments, where ING1b protein levels were found to be equivalent in the same murine tissues from both wild type p53 and the p53 knockout mice [46]. This study also showed that similarly increased levels of ING1b were induced by UV-irradiation of keratinocytes with either wild type or mutant p53, and concluded that the expression of ING1b is independent of p53. More recently, the same group identified a weak physical interaction between ING1b and GADD45, a p53- regulated gene known to function in DNA repair, and found that overexpression of ING1b enhances DNA repair in a p53-dependent manner [47].

From the downregulation of p53 observed in MCF7 cells when ING1b is overexpressed, we may have new evidence that it is the ING1b protein which regulates expression of p53. In the literature, it is unanimously suggested that the predominant regulation of p53 occurs at the protein level [153, 154]. Levels of p53 protein remain low and it exists in an inactive form until activated by various cellular conditions by post-translational modifications. The finding that ING1b acts as a negative transcriptional modulator of p53 would not follow along the lines of common thought, with respect to p53 regulation. This relationship appears counterintuitive as well, because it seems as though a protein that induces growth arrest and apoptosis might induce expression of a protein which also has the same effects and could help promote or augment these cellular responses. It is important to note however, that mRNA levels do not necessarily reflect levels in protein, and the status of both of these need to be tested in MCF7 cells in order to verify results. It is also well established that p53 becomes activated under different types of cellular stress conditions, including changes in cellular adhesion [155] and altered ribonucleotide resources [156], which seems a likely response for cells upon sudden overexpression of ING1 b. Most likely, this activation would occur at the protein level, and may not even be detected at the mRNA level. The decrease in p53 expression as a result of ING1b overexpresion may have some relation to other pathways that cause a decrease in p53 at the mRNA level. One protein involved in the UV-mediated DNA repair process, the AP-1 transcription factor family member JDP-2 [157] has been reported to cause decreases in p53 mRNA upon increases in JDP-2 expression levels. JDP-2 protein levels were induced after exposure of serum-starved cells to UV light, and resulted in a decrease of p53 mRNA which was proposed to be regulated by an atypical AP-1 site in the p53 promoter. Perhaps the ING1b gene also induces expression of this transcription factor, resulting in downregulation of p53 mRNA. Since the JDP-2 gene is not present on the array slide, we were not able to observe whether or not this is the case. It is possible that JDP-1 may be involved in same UV-mediated DNA repair processes as ING1b [53]. Additionally, two other factors of interest have been reported to cause decreases in p53 mRNA. In MCF7 cells, Oncostatin M treatment resulted in activation of the ERK kinase pathway and ultimately in a decrease in p53 at the mRNA level [158]. Unfortunately, the ERK pathway member MAPKK1 was found to be significantly downregulated in the MCF7-ING1b array, which opposes this idea. In a second study, phorbol ester treatment of HeLa and A549 cells resulted in activation of the PKC pathway and ultimately decreased p53 mRNA levels [159]. It may be interesting to pursue either the PKC signaling pathway or the expression of AP-1 family transcription factors, especially JDP-1, in the context of ING1b overexpression in order to examine the role of ING1b in p53 transcriptional regulation. Since p53 is known to interact with CBP/p300 and pCAF under different cellular conditions, perhaps the rapid increase in ING1b associates with all of the cellular CBP/p300, preventing its association and modification of p53. Free, unmodified p53 might have some effect on its own production: if the proteins that modify and stabilize it are not present, perhaps this triggers a negative feedback response which ultimately results in the downregulation of p53 at the transcriptional level. It is also known that CBP/p300 can influence p53 transactivation and protein turnover both positively and negatively, and maybe these proteins are also capable of regulating p53 at the transcriptional level. Thus, there may be an indirect effect of ING1b expression on p53 mRNA levels. Unfortunately, neither CBP or PCAF cDNAs are present on the 1.7k3 gene chip used, however the p300 gene is present but was not significantly expressed in any of the arrays performed. Another protein involved in acetylation is the TRRAP protein, which has also been shown to associate with ING1b [33, 64], but is not present on the gene chip either.

Downregulation of c-Myb and A-Myb The defining characteristic of the Myb family of proto-oncogenes is that they all encode nuclear proteins that act as transcriptional coactivators (reviewed in [160]. Although c-myb is normally expressed in immature hematopoetic cells, it is found to be frequently overexpressed in hematopoetic tumors, and in some mammary epithelial cell lines with positive ER status, including MCF7 (1998, Jeng, 4164-74). It is thought to be regulated by Estrogen in these breast cancer cells, and the inability any of these cell types to downregulate c-myb has been suggested to give them oncogenic potential [160]. The consistent downregulation of c-myb and a-myb when ING1b is overexpressed in MCF7 cells is a relevant finding, for many reasons. If this function of ING1b can be validated, an excellent system in which to study ING1 may be in lymphoid cell lines and tumors. Expression of ING1b was already shown to be aberrant in 5/11 B-cell and 4/5 T-cell lines; these included 2 Burkitt's lymphoma, 4 myelomas and 4 ALL cell lines [24]. Perhaps ING1b acts as a direct negative regulator of c-Myb, and possibly a-Myb as well. This relationship may prove extremely valuable later on, for the treatment of any cancers where c-Myb is overexpressed. It is already well established that c-Myb binds both CBP and p300, and that both of these proteins are required for c-Myb transcriptional activity [161]. More recently, c-Myb was shown to be acetylated by CBP at two different lysine residues within its negative regulatory domain (NRD), causing an increase in its transactivation ability by enhancing its interaction with CBP [162]. This is an interesting observation to make, in the context of ING1b overexpression since we know that ING1 b interacts with CBP as well as other proteins which possess histone acetyltransferase activity. Perhaps when ING1b is overexpressed, it outcompetes c-Myb for these proteins, resulting in a destabilization and decrease in its transactivation capabilities, and ultimately leading to downregulation of its transcription. To explore this idea further, the regulation of the Myb gene would need to be examined to determine what other proteins can regulate its promoter. Alternatively, ING1b may interact with and have some negative regulatory effect on, the ER steroid hormone pathway. This may result in downregulation of ER- responsive genes, such as c-myb. This idea could be studied by looking for the downregulation of other estrogen-responsive genes in the MCF7/ING1b array results. In addition, it is likely that the myb proteins are not observed to be underexpressed in the MDA MB 468 and MDA MB 435 arrays because neither of these cells are ER+ nor would express significant levels of myb so that downregulation could be observed. Downregulation of the Rb-family pocket protein p107 The consistent downregulation of p107, a member of the family of pocket proteins which includes pRb and p130 [163] upon ING1b overexpression in MCF7 cells is important to address. It is known that pRb acts as a transcriptional activator and well-known tumor suppressor which functions in cell-cycle progression and its deregulation is a common event in a wide variety of human tumors (reviewed in [164-166]). In addition, biochemical studies have linked pRb and its family members p107 and p130 to both transcriptional regulation and chromatin remodeling [167-170]. In G1 and S phases of the cell cycle, all three family members have been shown to colocalize in perinuclear foci that also contain HDACs and E2F proteins [171]. These foci have been shown to contain DNA replication proteins and are sites where DNA replication is initiated when cells enter S-phase, suggesting that the transcriptional activation by p107 and the other Rb-family proteins occurs in the same vicinity as initial DNA replication events.

Specifically, p107 is a protein structurally similar to pRb, containing a bipartite pocket structure which enables it to associate with members of the E2F transcription factor family, primarily E2F-4 [172]. In contrast to Rb and p130, p107 has not yet been definitively classified as a tumor suppressor [173]. It is not mutated or rearranged in sporadic tumors or their derived cell lines, with the exception of one intragenic deletion found in a B-cell lymphoma line [174]. In addition, elevated levels of p107, although in its inactive hyperphosphorylated state, are present in cycling cells as well as in tumors [163, 175] [176]. How or why would overexpression of ING1b cause a significant decrease in p107 mRNA? Since we know that ING1 b exerts a negative growth effect and induces cell cycle arrest at G0/G1, perhaps it functions to block the DNA replication processes with which p107 is involved as cells enter G1/S phase. By blocking p107 expression, the perinuclear foci may not be properly assembled or functional, contributing to arrest of the cell cycle. It may be interesting to observe these perinuclear foci in cells that overexpress ING1b, as well as to observe the location of histone acetyltransferase proteins involved with both ING1 and Rb/p107/p130 complexes. It is also possible that ING1b and p107 associate with some of the same histone acetyltransferase proteins, and that the cellular excess of ING1b binds up all of these, preventing p107 from forming associations with them. This may have a destabilization effect on p107, leading to its degradation and ultimate transcriptional downregulation: it may also prevent the p107-related replication foci from forming, ultimately contributing to the halting of the cell cycle. This is also a possibility in consideration of the cell structural proteins that ING1b appears to downregulate. Perhaps the scaffolding proteins that anchor these replication foci are downregulated, such that the proteins that function at these sites cannot be anchored, ultimately leading to their instability, and transcriptional downregulation. It is interesting to note that treatment of MCF7 cells with a pure antiestrogen analogue caused a decrease in p107 protein levels such that by 48 hours, it was nearly undetectable [176]. Messenger RNA for cDNA microarrays was also extracted from MCF7 cells at 48 hours. It is possible to speculate that ING1b may be involved in the regulation of the estrogen steroid hormone pathway in breast cancer cells, although currently there is no formal evidence for this. An additional explanation may be that due to the downregulation of a number of ribosomal proteins by ING1b overexpression, a negative effect on protein synthesis occurs, resulting in the non-specific downregulation of p107 and other proteins in the cells. The downregulation of p107 by ING1b overexpression may occur as a result of, or be affected indirectly by the mutant Rb protein in MCF7 cells.

Other downregulated genes of potential importance One guanine-nucleotide exchange factor (MSS4), a guanine-nucleotide binding protein, and both ADP-ribosylation factor 3 and ADP ribosylation factor 3- like protein were all downregulated in MCF7 cells. These proteins may play a role in G-protein mediated signaling pathways that ING1b is able to downregulate. Two chromosomal binding proteins HMG2 (High Mobility Group Protein 2) and CENP-E (Centromeric Protein E) were also downregulated, which may be related to INGIb's role in chromatin remodeling. Two melanoma- associated antigens, MUC18 and MAGE-1 were both downregulated, as well as several immunoglobulin molecules (HLA Class I E, HLA Class II DQp). Several enzymes, including the ubiquitin-conjugating enzyme E2 and cytochrome C oxidase, as well as the signaling molecules Wee1 kinase and calmodulin were also downregulated. Lastly, the metastasis marker CD82, the Bcl-2 binding protein BAG-1, and transcription factors XBP-1 and HEX were also downregulated. Further study of the influence and relationship of ING1 b expression and any number of these genes may be relevant to our understanding of ING1b in breast cancer cells, as well as in a normal cellular environment.

Conclusions and Perspectives

Do ING1b and ING1c isoforms exert different effects? From the differential gene expression patterns of ING1b and ING1c isoforms in all 3 cell lines, it is apparent that the two gene isoforms do affect different target genes in the cell lines tested. In general, fewer significant genes were upregulated and downregulated as a result of ING1c overexpression, in all 3 cell lines, when compared to the total number of significant genes identified in the ING1b arrays. Additionally, there were no consistently regulated proteins in any of the ING1c arrays, neither upregulated nor downregulated. Although this may be a result of INGIc's cell line specificity, it makes any proposed role for ING1c very tenuous. For example, the MCF7-ING1c array shows an upregulation of VEGF-B precursor, while the same mRNA is reported to be downregulated on the MDA MB 468 array. This may be a result of experimental variation, or possibly a cell-line specific effect. Again, there are no precise conclusions that can be drawn from the ING1c arrays, and perhaps further validation of some of these genes will lead to a better-defined role for ING1c. The ING1b arrays were only slightly more consistent than the ING1c arrays, with respect to gene expression. The pleckstrin gene was significantly downregulated by ING1b overexpression in both MCF7 and MDA MB 468 cell lines, indicating that this gene may be a good candidate for regulation by ING1 b. As seen on the Tree diagram (Fig. 26C, p. 87), the MCF7 cell line produced a cluster of strongly downregulated genes, in addition to the greatest number of upregulated genes for any of the arrays. This may have been a result of the positive p53 status of these cells, their positive ER status, or perhaps due to the status of a mutant Rb or other gene. As evidence for a relationship between p53 and ING1 seems to be gathering weight [47], microarray results from the MCF7 cell line may provide the best indication of a relationship between these two proteins in 'normal' (wild type p53) cells. Comparing between ING1b and ING1c arrays, ribosomal proteins are downregulated in both MDA MB 468/ING1c and MCF7/ING1b arrays, as well as the CENP-E protein and cytochrome C oxidase peptide precursors. These genes may also represent good candidates genes related to ING1. It may also be valuable in the future to perform microarrays in the same three cell lines when ING1a is overexpressed, for a more in depth comparison between gene isoforms.

Limitations of the cDNA Microarray Procedure Due to the vast number of variables involved in the microarray procedure, errors can be generated during many steps and all results must be validated by RT-PCR or Northern Blot analysis before absolute conclusions can be proposed. In the methods used for this thesis work, errors may have been generated when intensity ratios were averaged for duplicate spots, as was done in several of the first microarray experiments [82, 108]. It could have been more appropriate to implement a data filter which removed genes that had ratios which significantly differed in value from one another. We rationalized however that with six replicate experiments (3 original label, 3 reverse label), any ratio that was significantly off would be filtered out at the log transformation stage (Fig. 25, p. 85). Additionally, many groups perform pre-array experiments to determine a 'normal' level of expression variation in specific cell types prior to performing their arrays [112]. This enables them to chose significant levels of expression above and below normal for determining cutoff points between significant and insignificant genes. In this thesis, there was no cutoff (threshold = 0, Fig. 27, p. 91) because the 'normal' variation in expression levels was not pre-determined. Additionally, this is not necessarily a valid method for ruling out insignificant genes because some genes can be expressed at very low levels under normal conditions, and very small increases or decreases in expression levels can have substantial overall effects on the cell. It is these small increases and decreases that are deemed insignificant and become excluded from the dataset when threshold values are used, for which reason the data in this thesis was not threshold-limited. Lastly, microarray technology is still in the process of development. Currently there are no standards for microarray chips, reagents, protocols, analysis, controls, or for publishing microarray data. Chips are reportedly printed with significant rates or error [177, 178], and different methods of analysis make it difficult to compare experimental data with that which is published in the literature. Although we used reciprocal labeling methods to help us rule out gene expression results affected by unequal fluor incorporation between test and control cDNAs, this is not common practice in the major microarray experiments published in the literature. This not only makes it difficult for new users to commence microarray experiments, even more importantly it requires that all scientists reading the literature be extremely critical of microarray results, and that expression data generated in array experiments must be confirmed by secondary analysis. 130

In closing, the data in this thesis suggests new relationships between the ING1 proteins and the regulation of genes involved in ribosomal protein synthesis, cell architecture, cell signaling, transcription, DNA repair and many other functions. It proposes a new link between ING1b and p53, and other genes implicated in cancer, including c-Myb and p107. This provides further evidence that studying ING1 gene regulation and function is important for understanding the biology of the cell in both health and disease. CHAPTER 5: Bibliography 132

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Blocking solution for Western Blots 1x PBS 0.5% (VA/) Tween 20 5% non-fat skim milk powder

6x DNA loading buffer 0.25% (WA/) bromophenol blue 0.25% (WA/) xylene cyanol FF 30 % glycerol

2x Laemelli sample buffer 100 mMTris pH 6.8 200 mM dithiothreitol (DTT) 4% SDS 0.2% bromophenol blue 20% glycerol

LB broth 10 g tryptone 5 g yeast extract 10 g NaCI

PBE 1xPBS 0.5% (WA/) bovine serum albumin 5 mM EDTA 1xPBS 8 g NaCI 0.2 g KCI

1.44 g Na2HP04

0.24 g KH2P04

H20 to 1 L, then adjust pH to 7.4

Resolving gel for SDS-Page 12.5 % acrylamide:bisacrylamide (29:1 ratio) 380 mM Tris pH 8.8 0.1 %SDS 0.5 mg/mL APS 0.05% (VA/) TEMED

RNA loading buffer 50% glycerol 1 mM EDTA 0.25% bromophenol blue 0.25% xylene cyanol FF

Running buffer for SDS-PAGE 25 mM Tris pH 8.5 0.2 M glycine 5% (V/V) glycerol 0.1% SDS

1xSSC 3 M NaCI 0.3 M sodium citrate 150 adjust pH to 7.0

Stacking gel for SDS-PAGE 5% acrylamide (W/W) 0.09% acrylamide (W/W) 0.1% SDS 145 mM Tris pH 6.8 1 mg/mL ammonium persulfate (APS) 0.05% (V/V) TEMED

50x TAE 10 mM Tris pH 7.5 0.5% (V/V) Tween 20 150 mM NaCI

Transfer buffer

800 mL distilled H20 3 g Tris base 14.4 g glycine 200 mL methanol

0.2% X-gal staining solution

100 mM K4Fe(CN)6H20

100 mM K3Fe(CN)6

200 mM MgCI2 2% 5-Bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-gal) in dimelthyl formamide (DMF) PBS Filter through 0.2 u.m syringe filter before use APPENDIX B: Structures of antibiotics used for selection 151

A) The structural formula of bleomycin, from the phleomyin family of antibiotics, which also includes Zeocin. Phleomycins promote DNA strand breakage (Gale, 1981).

B) Structural formula of Neomycin. G418, an analogue of this aminoglycoside antibiotic, interferes with translational fidelity, ultimately blocking protein synthesis (Gale, 1981).

Ho ^—7S

R* \S >/ OH