Molecular Characterization of Tea Catechin Treated Human Prostate Cancer Cell Lines Yewseok Suh

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2006 Molecular Characterization of Tea Catechin Treated Human Prostate Cancer Cell Lines Yewseok Suh

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THE FLORIDA STATE UNIVERSITY

COLLEGE OF ARTS AND SCIENCES

MOLECULAR CHARACTERIZATION OF TEA CATECHIN

TREATED HUMAN PROSTATE CANCER CELL LINES

YEWSEOK SUH

A Dissertation submitted to the Department of Chemistry and Biochemistry in partial fulfillment of the requirements for the degree of Doctor of Philosophy

Degree Awarded: Summer Semester, 2006

The members of the Committee approve the dissertation of Yewseok Suh defended on May 12, 2006.

Qing-Xiang Amy Sang Professor Directing Dissertation

Thomas C.S. Keller III Outside Committee Member

Joseph B. Schlenoff Committee Member

Hong Li Committee Member

Approved:

Naresh Dalal, Chair, Department of Chemistry and Biochemistry

Joseph Travis, Dean, College of Arts and Sciences

The Office of Graduate Studies has verified and approved the above named committee members.

This dissertation is dedicated to my parents for their endless love and encouragement, to my lovely wife Inok Park for her support, and to our charming daughter, Tae-won.

iii ACKNOWLEDGEMENTS

I would like to express my gratitude to my major professor for her support throughout the research. Also thanks to all my committee members, Dr. Thomas C. S. Keller III, Dr. Hong Li, and Dr. Joseph B. Schlenoff, for their support, advice, and guidance. Special thanks to our lab members especially, Ziad Sahab and Robert G. Newcomer and former lab member Douglas R. Hurst for their helpful and critical discussions. I would like to thank all my family members including my sister Sun-young, brother Seung-bum, and my uncle Dr. Jeong-hun Suh for their support and encouragement. It has been a pleasure and privilege to work in Dr. Sang’s lab for the past couple of years.

iv TABLE OF CONTENTS

List of Tables viii List of Figures ix List of Abbreviations xi Abstract xv

1. INTRODUCTION 1 Cancer 1 Cancer Statistics 1 Prostate Cancer 2 Apoptosis 3 Green Tea and EGCG 6 EGCG and Cancer 8 Figures and Tables 11

2. MATERIALS AND METHODS 17 Reagents and Cell Culture 17 Tea Polyphenol Treatment and Cell Lysis 17 Nuclear and Cytoplasmic Extraction of Cells 18 Determination of Protein Concentration by BCA Assay 19 SDS-PAGE and Western Blotting 19 Cell Proliferation Assay 20 Cell Death Detection Assay 21 DNA Fragmentation Assay 22 Morphological Analysis of Apoptotic Cell Nucleus 23 Cell Adhesion Assay 23 Anchorage-Independent Growth Assay 24 GeneChip Microarray Analysis 24 Total RNA Preparation 25 Reverse Transcription - Polymerase Chain Reaction 26

v Quantitative Measurements of proMMP1 and MMP3 by ELISA 27 Statistic Analysis of the Data 28 Figures and Tables 30

3. EFFECTS OF EGCG ON THE GROWTH AND APOTOSIS OF PROSTATE CANCER CELL LINES 34 Prostate Cancer and EGCG 34 R1881 Suppresses the Growth of ARCaP Cells but Facilitates LNCaP Cell Growth 36 EGCG Represses the Growth and Induces Detachment and Membrane Blebbing in Cultured LNCaP Cells but Not in ARCaP Cells 36 EGCG Induces Apoptosis in LNCaP Cells but Not in ARCaP Cells 37 EGCG Induces Chromatin Condensation and Nuclei Fragmentation in LNCaP Cells but Not in ARCaP Cells 38 EGCG Induces Caspase 3 Activation, PARP Cleavage and p53 Increase Only in LNCaP Cells Leading to Apoptosis 39 EGCG Triggers Activation of NF-κB in ARCaP Cells but Induces Inactivation in LNCaP Cells 40 EGCG Decreases the Ratio of Bax/Bcl-2 in ARCaP Cells but Increases the Ratio in LNCaP Cells 41 EGCG Does Not Change the Level of p21/CIP1/WAF1 and CDKs in ARCaP Cells but the Level is Affected in LNCaP Cells in Favor of Apoptosis 42 EGCG Affects the Activation of MAPKs towards Cell Survival in ARCaP Cells but Favors Cell Death in LNCaP Cells 44 EGCG Confers Stronger ECM Adhesion via Collagen in ARCaP Cells whereas the Adhesion to ECM Proteins is Significantly Weakened in LNCaP Cells 46 Tea Catechin EGCG Renders ARCaP Cells Capable of Facilitated Anchorage-Independent Growth 49 Figures 51

4. SELECTIVE CHANGES IN GENE EXPRESSION IN EGCG TREATED PROSTATE CANCER CELLS 79 GeneChip Microarray Analysis 79 ADAMs and MMPs 81 Effects of Tea Catechins in Growth of LNCaP and ARCaP Cells 82 Effects of Tea Catechins in MMP1 and MMP3 Expression by RT-PCR 83 Effects of Tea Catechins in proMMP1 and MMP3 Expression by ELISA 84 The Effect of EGCG on the Expression of MMPs 86 The Role of MMPs and ECM in Programmed Cell Death 86 Figures and Tables 90

5. CONCLUSIONS AND FUTURE DIRECTIONS 103 EGCG and Prostate Cancer Chemoprevention 103 EGR1 in Apoptosis 104 Possible Roles of GADD45 and Topoisomerase 105 Current and Future Studies 106 Figures and Tables 108

APPENDIX 110 REFERENCES 157 BIOGRAPHICAL SKETCH 171

vii LIST OF TABLES

1. The composition of tea leaf 15 2. The information of primers used in PCR 32 3. The sequences of primers used in PCR 33 4. Changes of expression levels of adhesion-related proteins in LNCaP cells by GeneChip assay 90 5. Combinations of integrin subunits and their respective ligands 91 6. Changes of expression levels of ADAMs by GeneChip assay in LNCaP cells 92 7. Changes of expression levels of cell cycle-related proteins by GeneChip assay 108 8. The effect of R1881 on the growth of human prostate cancer cell lines 110 9. The effect of EGCG on the growth of LNCaP prostate cancer cell line 111 10. The effect of EGCG on the growth of ARCaP prostate cancer cell line 112 11. The ‘Enrichment Factor’ of ARCaP and LNCaP cells upon EGCG treatment measured by Cell Death Detection Assay PLUS 113 12. The ratio of Bax to Bcl-2 upon EGCG treatment 114 13. The effect of etoposide on the growth of ARCaP cells 115 14. Relative adherence of ARCaP cells to ECM proteins 116 15. Relative adherence of LNCaP cells to ECM proteins 117 16. The changes in relative adherence after EGCG treatment 118 17. The number of colonies formed in agar plate after EGCG treatment 119 18. GeneChip assay of ARCaP cells 120 19. GeneChip assay of LNCaP cells 130 20. The effect of tea catechins on the growth of LNCaP cells 151 21. The effect of tea catechins on the growth of ARCaP cells 152 22. The effect of tea catechins on the production of proMMP1 in LNCaP cells 153 23. The effect of tea catechins on the production of proMMP1 in ARCaP cells 154 24. The effect of tea catechins on the production of total MMP3 in LNCaP cells 155 25. The effect of tea catechins on the production of total MMP3 in ARCaP cells 156

viii LIST OF FIGURES

1. Estimated new cases and deaths by ten leading cancer types, by sex, US, 2006 11 2. Pictures of prostate and the model of prostate cancer progression 12 3. A picture of a cell undergoing apoptosis 13 4. The overview of apoptosis 14 5. Structures of basic flavonoid and major tea catechins 16 6. Schematic representation of Cell Death Detection ELISA PLUS assay 30 7. The schematic diagram of GeneChip assay 31 8. The effect of R1881 on the growth of ARCaP cells 51 9. The effect of green tea polyphenol EGCG on the growth of LNCaP human prostate cancer cells 52 10. The effect of green tea polyphenol EGCG on the growth of ARCaP human prostate cancer cells 53 11. Morphological changes upon EGCG addition in LNCaP cells 54 12. Morphological changes upon EGCG addition in ARCaP cells 55 13. The extent of apoptosis determined by Cell Death Detection Assay PLUS 56 14. The extent of apoptosis determined by DNA fragmentation assay 57 15. The extent of ARCaP cell apoptosis determined by Hoechst staining 58 16. The extent of LNCaP cell apoptosis determined by Hoechst staining 59 17. Immunoblot results with caspase 3 and PARP specific antibodies 60 18. Immunoblot results with p53 specific antibody 61 19. Immunoblot results with p65, phospho-p65, and Iκ-Bα specific antibodies 62 20. The role of Bcl-2 family members in mitochondrial outer membrane permeabilization during apoptosis 63 21. Immunoblot results with Bax, Bcl-2, and Bcl-XL specific antibodies 64 22. The changes of Bax/Bcl-2 ratio upon EGCG treatment 65 23. The role of p21/CIP1/WAF1 in cell cycle progression and apoptosis 66 24. Immunoblot results with p21, CDK2, CDK4, and CDK6 specific antibodies 67 25. The effects of etoposide on growth of ARCaP cells 68 26. The effects of etoposide on apoptosis of ARCaP cells 69

ix 27. Immunoblot results of etoposide treated ARCaP cell lysates with caspase 3, PARP, and p21 specific antibodies 70 28. Immunoblot results with Akt, p38, and JNK specific antibodies 71 29. Cell detachment assay of ARCaP cells 72 30. Cell detachment assay of LNCaP cells 73 31. Extracellular matrix adhesion assay of ARCaP cells 74 32. Extracellular matrix adhesion assay of LNCaP cells 75 33. The anchorage-independent growth assay of LNCaP cells 76 34. The anchorage-independent growth assay of ARCaP cells 77 35. The number of colonies formed in anchorage-independent growth assay 78 36. Changes of expression levels of MMPs by GeneChip assay in LNCaP cells 93 37. The effect of tea catechins on the growth of LNCaP cells 94 38. The effect of tea catechins on the growth of ARCaP cells 95 39. The effect of tea catechins on the expression of MMP1 and MMP3 mRNA in LNCaP cells 96 40. The effect of tea catechins on the expression of MMP1 and MMP3 mRNA in ARCaP cells 97 41. The effect of EGCG on the expression of MMP1 and MMP3 mRNA in DU145, PC3, and HT1080 cells 98 42. The effect of tea catechins on the production of proMMP1 in LNCaP cells 99 43. The effect of tea catechins on the production of proMMP1 in ARCaP cells 100 44. The effect of tea catechins on the production of total MMP3 in LNCaP cells 101 45. The effect of tea catechins on the production of total MMP3 in ARCaP cells 102 46. Changes of expression levels of EGR1 by RT-PCR in ARCaP and LNCaP cells 109

x LIST OF ABBREVIATIONS

ABTS 2,2’-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) ADAM a disintegrin and metalloproteinase ADAM-TS ADAM-thrombospondin motifs ADP adenosine diphosphate ANOVA analysis of variance AP-1 activator protein 1 Apaf-1 apoptotic protease activating protein 1 AR androgen receptor ARCaP androgen repressed carcinoma of prostate ASK1 apoptosis signal-regulating kinase 1 ATP adenosine triphosphate BCA bicinchoninic acid Bcl-2 B cell leukemia / lymphoma 2 BSA bovine serum albumin CAD caspase-activated DNase CAPS N-cyclohexyl-3-aminopropanesulfonic acid CARD caspase recruitment domain Caspases cysteine aspartic acid-specific proteases CD cluster of differentiation CDK cyclin dependent kinase cDNA complementary DNA CIP1 CDK-interaction protein 1 Col collagen cRNA complementary RNA DEPC diethylpyrocarbonate DFF DNA fragmentation factor DHEA dehydroepiandrosterone DHT dihydrotestosterone

xi DISC death inducing signaling complex DMBA 7, 12-dimethyl benzanthracene DMEM Dulbecco’s modified Eagle’s media DMSO dimethyl sulfoxide DNA deoxyribonucleic acid dNTP deoxyribonucleotide triphosphate DRE digital rectal examination DTT dithiothreitol EC (-)-epicatechin ECG (-)-epicatechin gallate ECM extracellular matrix EDTA ethylenediaminetetraaceticacid EGC (-)-epigallocatechin EGCG (-)-epigallocatechin gallate EGR1 early growth response 1 ELISA enzyme linked immunosorbent assay ErbB2 erythroblastosis oncogene B FADD Fas-associated death domain protein FasL Fas ligand FBS fetal bovine serum Fn fibronectin FSH follicle stimulating hormone GADD45 growth arrest and DNA damage inducible gene 45 GAPDH glyceraldehyde-3-phosphate dehydrogenase HEPES N-(2-hydroxyethyl)-piperazine-N’-2-ethanesulfonic acid HER2 human epidermal growth factor receptor 2 HRP horseradish peroxidase hTERT human telomerase reverse transcriptase Iκ-Bα inhibitor of kappa-B alpha IKK I kappa-B kinase IL-1β interleukin-1 beta

xii iPLA2 calcium-independent phospholipase A2

JNK c-jun NH2-terminal kinase LH luteinizing hormone LH-RH luteinizing hormone-releasing hormone Ln laminin LNCaP lymph node carcinoma of prostate LPC lysophosphatidylcholine MAPK mitogen activated protein kinase MDC metalloprotease / disintegrin / cysteine-rich protein MMP matrix metalloproteinase mRNA messenger RNA MT-MMP membrane type - MMP MW molecular weight NAD nicotinamide adenine dinucleotide NF-κB nuclear factor-kappa B NP-40 Nonidet P-40 OD optical density PAGE polyacrylamide gel electrophoresis PARG poly(ADP-ribose) glycohydrolase PARP poly(ADP-ribose) polymerase PBS phosphate buffered saline PCNA proliferating cell nuclear antigen PCR polymerase chain reaction PECAM platelet endothelial cell adhesion molecule PI3-K phosphatidyl-inositol 3-kinase PIA proliferative inflammatory atrophy PIN prostate intraepithelial neoplasia PKB protein kinase B PSA prostate specific antigen PtdSer phosphatidylserine PTEN phosphatase and tensin homologue deleted on

xiii chromosome 10 Rb retinoblastoma RGD Arginine-Glycine-Aspartic acid RIPA radio immunoprecipitation assay RNA ribonucleic acid RT-PCR reverse transcription-polymerase chain reaction SAPK stress activated protein kinase SDS sodium dodecyl sulfate siRNA small interfering RNA Ta annealing temperature TAE Tris-acetate-EDTA TBS Tris buffered saline TE Tris-EDTA Tn tenascin TNF tumor necrosis factor TSP thrombospondin UV ultraviolet Vn vitronectin WAF1 wild-type p53-activated fragment 1

xiv ABSTRACT

Prostate cancer is the most prevalent cancer diagnosed among men in the United States. The major green tea polyphenol epigallocatechin-3 gallate (EGCG) has been shown to exert remarkable preventive effects against various types of cancer including prostate cancer. Recent human clinical study proved that EGCG can prevent progression of high grade prostatic intraepithelial neoplasia (PIN) to prostate cancer. Cellular studies show that EGCG exhibits antiproliferative and apoptotic effects in androgen-responsive LNCaP and androgen- unresponsive DU145, PC3 prostate cancer cell lines. Previously, we have established a new type of prostate cancer line, androgen repressed carcinoma of prostate (ARCaP). ARCaP cells are highly invasive and metastatic and this cell line showed unique response to androgen since the hormone repressed the proliferation. In this study, we show that androgen-repressed ARCaP prostate cancer cell line, which represents more advanced and aggressive type of prostate cancer, is resistant to EGCG treatment. In Western blot analyses, EGCG treated ARCaP cell line showed increase in phosphorylation of NF-κB and decrease in activation of p38 MAPK and Bax/Bcl-2 ratio. The levels of p21/CIP1/WAF1, cyclin-dependent kinases (CDKs) 2, 4, 6, activated forms of Akt and c-Jun NH2-terminal protein kinase (JNK) remain unchanged in EGCG treated ARCaP cells whereas decrease in active Akt, active JNK, and CDKs 2, 4, 6, and increased level of p21/CIP1/WAF1 were observed in LNCaP cells upon EGCG treatment. Moreover, EGCG treatment confers stronger adherence to types I, II, IV collagen extracellular matrix proteins on ARCaP cells. On the contrary, LNCaP cells lost the adhesion significantly to all extracellular matrix proteins tested, including collagens, fibronectin, laminin, vitronectin, and tenascin. Most importantly, ARCaP cells formed more colonies on soft agar in our anchorage-independent assay when treated with EGCG whereas the colony forming ability of LNCaP cells was totally abolished under the same condition. This study suggests that the use of tea catechin EGCG as anticancer agent may not be effective for treating patients with androgen repressed subtype of prostate cancer. This is the first study of apoptosis in ARCaP cell line. The GeneChip microarray analysis revealed several genes that were differentially expressed when treated with EGCG. Among those, matrix metalloproteinases (MMPs) 1 and 3 were significantly up regulated in LNCaP cells upon EGCG treatment. Both RNA transcription

xv and protein secretion/activation of these MMPs were observed by GeneChip assay, reverse transcription-polymerase chain reaction (RT-PCR) and by enzyme linked immunosorbent assay (ELISA) which can detect proMMP1 and total MMP3 in cell culture media. This feature is very unique in that (1) the MMPs are generally known to be involved in tumor invasion and metastasis not the cell death, and (2) the other EGCG sensitive prostate cancer cell lines, DU145 and PC3, did not display such characteristics. EGCG did not affect the expression of these MMPs in ARCaP cells also. Using GeneChip analysis, we found several genes whose expressions were oppositely regulated in LNCaP and ARCaP cells upon EGCG treatment. These include early growth response -1 (EGR1), growth arrest and DNA damage inducible gene 45 (GADD45). The expression level of EGR1 and GADD45 were decreased in ARCaP cells but the level was increased in LNCaP cells after EGCG treatment. These results suggest that the proapoptotic EGR1 and GADD45 may play a role in EGCG induced apoptosis in LNCaP cells and thus may explain, at least in part, the resistance of ARCaP cells against such apoptotic stimuli. The role of these proteins in EGCG induced apoptosis is not known. The decreased level of topoisomerase II in EGCG treated LNCaP cells is also exciting. Topoisomerases are necessary in DNA replication and thus for survival of the organism. Since only LNCaP cells, but not ARCaP cells, displayed reduced expression of topoisomerase II during EGCG induced apoptosis and since ARCaP cells underwent apoptosis when treated with topoisomerase inhibitor etoposide, the function of this enzyme might be involved in life or death decision of ARCaP and LNCaP cells. Elucidating the molecular effects of these proteins and the mechanisms of how these proteins function in ARCaP and LNCaP cell lines would help understanding the prostate cancer and may help with future design of cancer chemopreventive and chemotherapeutic agents.

xvi

CHAPTER 1

INTRODUCTION

Cancer

Cancer is a malignant tumor that can grow potentially unlimited and expands locally by invasion and systemically by metastasis. The oldest description of human cancer was found in Egyptian papyri written between 3000-1500 BC. The father of the modern medicine, Hippocrates, who studied many types of cancer, is credited with naming cancer as karkinos, the Greek name for crab which in English translates to carcinoma, because a cancer looked like such crustacean in that there is a hard central body to a cancer and the claw-like cancer extension appeared as the legs of the crab. When karkinos was translated into Latin in the first Century, the term “cancer”, which means crab in Latin, was used.

Statistics of Cancer

According to the Global Cancer Statistics, 2002 (Parkin et al. 2005), there were 10.9 million new cases of cancer and 6.7 million cancer deaths, and 24.6 million people alive with cancer worldwide. The five most commonly diagnosed cancers in 2002 were lung cancer (1.35 million cases), breast (1.15 million cases), colorectal (1.02 million cases), stomach (0.93 million cases), and prostate cancer (0.68 million cases), whereas in males, the prostate cancer was the second most popular cancer only next to lung cancer (0.97 million cases). In terms of prevalence, breast cancer (17.9 %) was the most common with 4.4 million survivors up to 5 years following diagnosis followed by colorectal (2.83 million survivors, 11.5 %), and prostate cancer (2.37 million survivors, 9.6 %). In the same year, there were 1,240,046 cancer incidence and 557,271 cancer deaths with the cancer incidence rate of 462.6 and cancer death rate of 193.4 per 100,000 in the United States, according to the U.S. Department of Health & Human Services (Centers for

1 Disease Control and Prevention at www.cdc.gov). In the U.S., cancer is the second leading cause of death only next to heart diseases which accounts for 22.7 % and 28.0 % of total death in 2003, respectively. But the cancer becomes the leading cause of the death in both men and women under age 85 when age-adjusted rates are considered (Jemal et al. 2006). The estimated new cases and deaths of ten leading cancer types (Figure 1) suggest that there will be nearly 1.4 million new cancer cases and 564,830 cancer deaths in the U.S., 2006 (Jemal et al. 2006).

Prostate Cancer

There were 679,023 new cases and 221,002 deaths of prostate cancer worldwide (Parkin et al. 2005) and 190,096 incidence and 30,446 deaths in the U.S. in 2002 (www.cdc.gov). Prostate cancer is the most common cancer in men and the second leading cause of cancer- related death among males after lung cancer in the United States. According to the American Cancer Society, it is estimated that 234,460 men in the U.S. will be diagnosed with prostate cancer in 2006 and about 1 man in 6 will develop prostate cancer during his lifetime (Jemal et al. 2006). But for the first time, American Cancer Society estimates that there will be slightly less prostate cancer death than colon and rectum cancer death in 2006 (Figure 1). The prostate is a male reproductive gland about the size and shape of a walnut (Figure 2 A). It is located below the bladder and in front of rectum, and surrounds urethra, the vessel through which urine follows. The main function of prostate is to make and secrete the part of the seminal fluid. The endocrine control of prostate growth is complex. It is regulated by series of action of hormones. Hypothalamus secretes luteinizing hormone-releasing hormone (LH-RH) into the pituitary where it stimulates receptors that lead to the production and release of luteinizing hormone (LH) and follicle stimulating hormone (FSH). While LH and FSH can stimulate testis for testosterone production, LH stimulates adrenal gland to secrete testosterone precursors such as dehydroepiandrosterone (DHEA) into prostate where they are converted to testosterone. In prostate, testosterone is then converted to dihydrotestosterone (DHT) by the enzyme called 5- alpha-reductase. Testosterone and DHT, with stronger affinity than testosterone, bind to the hormone binding domain of the androgen receptor (AR) that resides in cytosol. The hormone- bound AR then translocates to nucleus and bind to target DNA through its DNA binding domain

2 and activates the transcription of the genes that are responsible for growth and function of the prostate via its N terminal transcription activation domain (Brufsky and Kantoff 1998). The risk factors of prostate cancer include age, family history, and race ethnicity. The induction of prostate cancer is a multistage process as depicted (Figure 2 B). Normal prostate can become proliferative inflammatory atrophy (PIA) which may then progress further to prostate intraepithelial neoplasia (PIN), the main precursor of carcinoma of the prostate, and also to prostate cancer. It has been suggested that, while not all PIA lesions are associated with cancer, PIA could progress to cancer directly or indirectly by first developing to PIN (Putzi and De Marzo 2000, Palapattu et al. 2004). Prostate cancer can be categorized in 4 stages according to its progression status. At Stage 1, the cancer is only in one of the lobes of the prostate and cannot be felt by digital rectal examination (DRE) screening. Also, there are no symptoms in this stage of prostate cancer. The cancer becomes more advanced and may involve both of the lobes but still confined to prostate in Stage 2. At this stage, the cancer is detectable by DRE screening. Stage 3 represents the prostate cancer that has spread outside the prostate. The prostate cancer may have spread to seminal vesicles but not to lymph node. Once the cancer has spread to other organs including lymph nodes, it is categorized as Stage 4, a metastatic phase (National Cancer Institue at www.cancer.gov). The symptoms of prostate cancer include urinary problems, erection difficulties, and the existence of blood in semen or urine. The widespread use of blood test for prostate specific antigen (PSA) and DRE screening method have contributed to earlier diagnosis and treatments, such as surgery, radiation therapy and hormone therapy including antiandrogen treatment and orchiectomy have aided in reducing the fatality of this disease but most of the prostate cancer relapses and become refractory which renders prostate cancer a significant public health burden.

Apoptosis

Apoptosis or programmed cell death, also known as cell suicide, is a mode of cell death and is needed for proper embryonic morphogenesis and development such as in the limb formation of the fetus to remove webbing between the digits and also in the resorption of tadpole tail during metamorphosis into frog. Apoptosis is also required to destroy the cells that represent threats to the integrity of the organism, for example, cells infected with viruses or cells bearing

3 severe DNA damage. The programmed cell death also occurs during cell turnover in adults including the maturation of the immune cells. The term apoptosis came from ancient Greek word αποπτωσισ (“apoptwsis”) meaning “falling off of petals from flower” or “falling off of leaves from the tree” and was first introduced by John Kurr in 1972 (Kurr et al. 1972). In a human body about 100,000 cells are produced every second by mitosis and a similar number of cells die by apoptosis (Vaux and Korsmeyer 1999) Apoptosis is characterized by its unique phenotypical changes including cell shrinkage, chromatin condensation, plasma membrane blebbing and the collapse of the cells into smaller membrane intact fragments called apoptotic bodies (Figure 3) (Bohm and Schild 2003). Apoptosis can be induced by two different ways (Figure 4). The intrinsic or mitochondrial pathway can be triggered by the internal damage to the cell, such as DNA damage, which activates cytochrome c release from mitochondria. The released cytochrome c binds to the apoptotic protease activating factor 1 (Apaf-1) using the energy provided by ATP and recruits caspase 9 forming a huge heptameric ‘death machinery’ complex called apoptosome each of which is consisted of Apaf-1/cytochrome c/caspase 9. The binding of caspase 9 to Apaf-1 is achieved through the homophilic CARD-CARD (caspase recruitment domain) interaction and the caspase 9 gets activated via allosteric changes and homodimerization in the apoptosome (Adams and Cory 2002, Lawen 2003). On the other hand, the extrinsic or death receptor pathway can be initiated by binding of the death activator Fas ligand (FasL) or tumor necrosis factor (TNF) to their receptors Fas, also called CD95 and Apo-1, or TNF receptor whose binding recruits Fas-associated death domain protein (FADD). Caspase 8 or caspase 10 then binds to the FADD creating death inducing signaling complex (DISC) where it can be activated through trasactivation. (Lawen 2003, Fadeel and Orrenius 2005) Cysteine aspartic acid-specific proteases (Caspases) are cysteine proteases that cleave after certain aspartic acid residues and are the key enzymes that execute the apoptosis. Caspases can be grouped into initiator caspases and effector caspases. Once the initiator caspases, such as caspases 8, 9, and 10, are activated through intrinsic or extrinsic pathway, they proteolytically cleave and thus activate the effector caspases including caspases 3, 6, and 7 whose functions are known to be responsible for the cleavage of the intracellular substrates that leads to cell death. Caspase 3 is one of the key executioners of apoptosis. Upon activation, caspase 3 can cleave

4 substrates including other effector caspases and fodrin, which forms cytoskeletal network (Slee et al. 2001). DNA fragmentation factor (DFF), also known as caspase-activated DNase (CAD), is one of the enzymes that are responsible for DNA fragmentation during apoptosis and is consisted of latent nuclease DFF40 and the inhibitor DFF45/35. Active caspase 3 cleaves DFF45/35, inactivating its inhibitory function, which in turn makes DFF40 able to dice the genome (Widlak et al. 2003). Lamin is a nuclear protein that lines the inside surface of the inner nuclear membrane and forms nuclear lamina which supports the structure of nucleus. Lamin can be cleaved through caspase 3 activated caspase 6 which leads to rupture of nucleus membrane. Poly(ADP-ribose) polymerase-1 (PARP-1) is also a target of active caspase 3 and is an enzyme that binds to both single and double strand breaks to facilitate DNA repair. PARP-1 resides in the nucleus and is consisted of a DNA binding domain (46 kDa) at the N terminus, an automodification domain (22 kDa), and a C terminal catalytic domain (54 kDa). The DNA binding domain has two consecutive zinc finger motifs for DNA binding and a nuclear localization signal (Kim et al. 2005). In the presence of DNA strand breaks, PARP-1 binds to the damaged site and cleaves its substrate NAD+ to nicotinamide and ADP-ribose, forming and transferring long negatively charged and branched (ADP-ribose) polymers to itself and to other acceptor proteins such as histones (Ivana Scovassi and Diederich 2004). The negative charges of the polymers cause (1) the dissociation of poly(ADP-ribosyl)ated PARP-1 and histones from the DNA through electrostatic repulsion, and (2) the relaxation of local chromatin through destabilization of nucleosomes thus making the site of DNA damage accessible to DNA repair enzymes (Haince et al. 2005). The polymers of poly(ADP-ribose) can be rapidly degraded by the enzyme poly(ADP- ribose) glycohydrolase (PARG) which has both endo- and exo-glycosidic activities. The pool of NAD+ is replenished by both de novo and salvage pathway both of which require ATP (Kim et al. 2005). PARP-1 is thought to be one of the earliest targets for cleavage by caspases during apoptosis. The cleavage occurs between Asp-214 and Gly-215 in the DNA binding domain’s nuclear localization signal of PARP-1 resulting in 24 and 89 kDa fragments which renders PARP-1 inactive. The cleavage of PARP-1 promotes apoptosis by preventing DNA repair- induced survival and by blocking energy depletion-induced necrosis. In general, the cleavage of PARP-1 has been used extensively as a marker of apoptosis (D’Amours et al. 2001).

5 The final phase of apoptosis is phagocytosis of apoptotic bodies that occurs before the loss of membrane integrity to prevent leakage of potentially cytotoxic or antigenic contents. This apoptotic corpse clearance can be mediated by professional phagocytes such as macrophages and dendritic cells and also by any healthy neighboring cells but with much slower kinetics. For the engulfment of apoptotic cells to occur, phagocytes must identify apoptotic cells. This complicated and elaborate process is associated with 3 different types of signals between the dying cells and the phagocytes. The apoptotic cells present “eat-me” signals that serve as identification for phagocytes. One of the best characterized eat-me signals is the translocation of phosphatidylserine (PtdSer) from the inner leaflet to outer leaflet of the plasma membrane lipid bilayer of apoptotic cells. This phenomenon appears to involve the regulation of two enzymes, phospholipid scramblase and flippase. Also, PtdSer binding annexin proteins are known to appear newly on the surface of the plasma membrane on apoptotic cells and colocalize with PtdSer (Fan et al. 2004). These signals can be recognized by PtdSer receptor that is expressed on phagocytes (Savill et al. 1993). Thrombospondin-1 (TSP-1) is an extracelluar matrix glycoprotein that binds to CD36 on the surface of the dying cells. Phagocytes can recognize apoptotic cell bound TSP-1 via cooperation between αvβ3 integrin and CD36 on phagocytes (Hall et al. 1994, Fadok et al. 1998). Several other interactions between apoptotic cells and phagocytes are engaged in the engulfment and serve as eat-me signals (de Almeida and Linden 2005). Nonapoptotic healthy cells prevent themselves from phagocytosis by presenting “don’t- eat-me” signals. Healthy cells signal through CD31 molecules, also known as platelet endothelial cell adhesion molecule-1 (PECAM-1), that are expressed on both healthy cells and phagocytes to repel the phagocytes. In dying cells, this signaling is disabled and thus binding and engulfment by phagocytes are promoted (Brown et al. 2002, Chimini 2002). Apoptotic cells also secrete “come-get-me” signals that can promote the migration of phagocytes to the site of apoptosis. In dying cells, activated caspase 3 cleaves and thus activates calcium-independent phospholipase A2 (iPLA2). Upon activation, iPLA2 mediates hydrolysis of membraneous phosphatidylcholine into lysophosphatidylcholine (LPC) and arachidonic acid. Once released from the apoptotic cells, LPC serves as a chemoattractive signal and thus induces the recruitment of phagocytes and subsequent recognition and engulfment of apoptotic corpse (Lauber et al. 2003).

6 Green Tea and EGCG

Tea is the most popular drink only next to water with the average tea consumption of about 0.12 liter per day per capita worldwide. Tea is derived from the plant Camellia sinensis and has been cultivated and consumed for more than 2000 years. Tea is manufactured in 3 basic forms. Of more than 2.6 million metric tons of tea produced, the majority is black tea whereas only 20 % is green tea and less than 2 % is oolong tea. All 3 forms of tea are derived from the same tea leaf but with different processing (Mukhtar and Ahmad 1999). Flavonoids are 2-phenyl benzopyran based compounds that can be subdivided into 6 different members: flavones, isoflavones, flavanones, flavanols, flavonols, and anthocyanins. Fresh tea leaf is rich in the flavanols and flavonols, especially flavanol group of polyphenols which may constitute up to 25 % of dry weight of fresh leaf (Table 1). Flavanols, more known as catechins, are characterized by the meta-5, 7-dihydroxy substitution of the A ring and di- or trihydroxyl group substitution of the B ring of basic flavonoid structure (Figure 5). Catechins contribute bitterness and astringency to green tea and the 4 major forms of catechins in green tea are (-)-epicatechin (EC), (-)-epigallocatechin (EGC), (-)-epicatechin gallate (ECG), and (-)- epigallocatechin gallate (EGCG) (Figure 5). As depicted in the figure, the catechins are called gallocatechins when the trihydroxyl groups are present at carbons 3’, 4’, and 5’ on the B ring which include EGC and EGCG. If catechin has a gallate moiety esterified at hydroxyl group located at the carbon 3 of the C ring, it is called catechin gallates such as ECG and EGCG. Flavonols are characterized by 4-oxo 3-hydroxy C ring and include quercetin, kaempferol and myricitin (Balentine et al. 1997, Yang et al. 2002). The manufacturing of black tea involves crushing the tea leaves to promote enzymatic oxidation and subsequent condensation of tea catechins. This oxidation process is known as ‘fermentation’ since the process was believed to be a microbial fermentation in the early days. The oxidation of catechins to reactive quinones is initiated by the tea leaf’s own enzyme, polyphenol oxidase, and results in the formation of unique black tea polyphenols including theaflavins and thearubigins. In fresh tea leaf, catechins are confined to vacuoles and thus separated from polyphenol oxidase which can be found in chloroplast. During black tea manufacturing, catechins are decreased by up to 85 % (Balentine et al. 1997). Green tea, consumed primarily in Korea, China, Japan and in some parts in North Africa and the Middle

7 East, is made by steaming or pan-frying the leaves which inactivates polyphenol oxidase thereby preventing the oxidation of catechins. Oolong tea is partially oxidized and mainly produced and consumed in China and Taiwan. While the most of green tea is produced in China and Japan, the majority of the black tea comes from Sri Lanka, India, Kenya, and Indonesia (Graham 1992). When brewed with 2.5 g of tea leaves in 250 ml of hot water for 3 mins, usually 620 ~ 880 mg of solids can be extracted (Yang et al. 2002). It is known that in green tea beverage, catechins constitute 30 ~ 42 %, whereas black tea beverage contains only 3 ~ 10 % catechins and 3 ~ 6 % theaflavins and 12 ~ 18 % thearubigins in wt/wt percentage of extract solids (Graham 1992). Since EGCG is the major catechin in green tea and constitutes 50 ~ 80 % of the total catechin, such brewing will yield 195 mg and 40 mg of EGCG, on average, from green and black tea, respectively.

EGCG and Cancer

A number of biological activities have been ascribed to the green tea polyphenol EGCG. Apoptosis was induced in human epidermoid carcinoma cell line A431 and mouse lymphoma cell line L5178Y upon EGCG treatment but not in normal human epidermal keratinocyte NHEK cells (Ahmad et al. 1997). In another report, it was shown that EGCG induced apoptosis in A431 cells is triggered by G0/G1 phase arrest of cell cycle through up regulation of the expression of cyclin dependent kinase (CDK) inhibitors, down regulation of the expression of cyclins and CDKs, and inhibiting kinase activity of these proteins (Ahmad et al. 2000). Studies with normal human keratinocytes are interesting since it revealed that EGCG induces differentiation but not apoptosis in these cells. EGCG stimulated the expression of type I transglutaminase, an enzyme that is required in keratinocyte differentiation, which is responsible for the assembly of the keratinocyte cornified envelope (Balasubramanian et al. 2005). EGCG also enhanced the expression of involucrin, one of the substrates of transglutaminase, which is also known as a keratinocyte differentiation marker (Balasubramanian et al. 2002). More studies that compared normal cells to their transformed counterparts upon EGCG treatment show that EGCG more likely inhibit the proliferation of the cancer cells rather than their normal counterparts. Chen et al. demonstrated that EGCG displays much more intense growth inhibitory effect on the SV40 virally transformed human fibroblast W138VA than on

8 normal fibroblast W138 cells (Chen et al. 1998). They also observed similar differential growth inhibitory effects between a human colorectal cancer cell line Caco2 and normal colon cell line CCD-33Co. In parallel with these results, Wang and Bachrach compared the effect of EGCG on normal and transformed NIH-3T3 fibroblasts and reported similar results (Wang and Bachrach 2002). Moreover, in the same report, EGCG showed strong inhibition of tyrosine kinase and mitogen activated protein kinase (MAPK) activities in transformed cells but not in normal cells. EGCG also inhibits carcinogenesis in the digestive tract in rodents. In human stomach cancer cell line KATO III, EGCG and other tea polyphenols induced growth inhibition and apoptosis. In addition, it was demonstrated that EGCG not only inhibited gene expression of TNF-α, the endogenous tumor promoter, through inhibiting the tumor promoter okadaic acid- induced activation of AP-1 and NF-κB but also blocked its release from the cells (Okabe et al. 1999). Leukemic cells are also sensitive to EGCG. It was reported that 50 µM of EGCG inhibits proliferation and triggers apoptotic cell death in human leukemic cell lines HL-60, K562, KG1, THP-1 and U937. On the other hand, half the number of normal hematopoietic colonies was still viable even at 100 µM of EGCG (Otsuka et al. 1998). The effects of EGCG in breast cancer are well studied. ErbB2 (HER2/neu) belongs to receptor tyrosine kinase family and its overexpression in breast cancer has been associated with aggressive phenotype and poor prognosis. In ErbB2 overexpressing and constitutively activating breast cancer cell line BT-474 and head and neck squamous cell carcinoma (HNSCC) cell line YCU-H891, treatment of EGCG induced not only growth inhibition but also resulted in marked inhibition of phosphorylation of ErbB2, inhibition of Stat3 activation, inhibition of c-fos and cyclin D1 promoter activity, and decreased cellular levels of the cyclin D1 and Bcl-XL proteins in these cells (Masuda et al. 2003). Another type of breast cancer cell line, MCF-7, known for its elevated telomerase level, is also known to be sensitive to EGCG. Treatment of EGCG inhibited the viability, induced apoptosis, and also repressed the colony forming ability of MCF7 cells. Furthermore, EGCG inhibited both the telomerase activity and the mRNA expression of human telomerase reverse transcriptase (hTERT), a catalytic subunit of telomerase that is known to be expressed in most of malignant tumors but not in most normal tissues, in these cells (Mittal et al. 2004). EGCG is also reported to induce apoptosis in MDA-MB-231 human breast cancer cells (Chisholm et al. 2004) and to show growth inhibitory effects on ErbB2 overexpressing mouse mammary tumor MMTV-Her2/neu NF639 cells (Pianetti et al. 2002).

9 In 1998, Paschka et al. reported that tea polyphenols induce growth inhibition and apoptosis in both androgen-sensitive LNCaP and androgen-insensitive PC3 and DU145 human prostate cancer cells (Paschka et al. 1998). Among the 4 tea catechins tested, EGCG was the most potent inhibitor of cell proliferation and inducer of apoptosis since it exerted its antiproliferative role as low as at 1 µM of concentration. Treatment of prostate cancer cells with EGCG resulted in (1) increase of p53 in LNCaP but not in mutant p53 bearing DU145 cells, and (2) a dose dependent G0/G1 phase arrest of the cell cycle through induction of cyclin dependent kinase (CDK) inhibitor p21/WAF1/CIP1 in both DU145 and LNCaP cell lines which suggests that EGCG induces p21 mediated cell cycle arrest and ultimately, apoptosis, irrespective of p53 status or the androgen association (Gupta et al. 2000). Another study showed that EGCG suppressed growth and induced apoptosis in DU145 cells and this was associated by mitochondrial depolarization. But EGCG did not alter the expression of B cell leukemia 2 (Bcl- 2), Bcl-XL, and Bad proteins in these cells. DU145 cells showed highest sensitivity to tea catechin ECG followed by EGCG and EGC but were resistant to EC (Chung et al. 2001). In LNCaP prostate cancer cells, which possess wild type p53, EGCG triggered apoptosis has been shown to be mediated in part by p53. When treated with EGCG, the expression, stability, and transcriptional activation of p53 have been increased which resulted in its enhanced activation of its transcriptional targets p21/WAF1 and Bax both of which are known to promote apoptosis. The stability of p53 is achieved through phosphorylation and modulation of its regulators p14/ARF and MDM2. On the other hand, another important transcription factor NF- κB, whose transcriptional activity is known to promote cell proliferation, was down regulated upon EGCG treatment and resulted in decreased expression of its target, Bcl-2, a protein that plays a key role in inhibiting apoptosis. Finally, apoptosis was triggered due to the increase in the ratio of Bax/Bcl-2 which is well balanced in normal cells (Hastak et al. 2003). Further study revealed that cell cycle arrest and the following apoptosis in LNCaP and DU145 cells triggered by EGCG treatment were induced via modulating cyclin-cyclin dependent kinase (CDK)-CDK inhibitor machinery through (1) up regulation of CDK inhibitors including p21/WAF1/CIP1, p27/KIP1, p16/INK4a, and p18/INK4c, (2) decrease in cyclins and CDKs including cyclins D1 and E, CDKs 2, 4, 6 but not cyclin D2, and (3) up regulation of cyclinD1-p21/p27 binding (Gupta et al. 2003).

Figure 1. Estimated new cases and deaths by ten leading cancer types, by sex, US, 2006. This figure was adopted from Jemal et al. 2006.

Figure 2. Pictures of prostate and the model of prostate cancer progression. A: The picture of actual prostate with seminal vesicles is shown. The image was obtained from www.malecare.org. B: The diagram of prostate cancer progression model was acquired and modified from Gonzalgo and Isaacs 2003.

Figure 3. A picture of a cell undergoing apoptosis. The collapse of a cell into small intact fragments called apoptotic bodies is clearly shown. The image was obtained from Dr. Gough’s lecture from University of Manchester website available at personalpages.manchester.ac.uk.

Figure 4. The overview of apoptosis. Intrinsic and extrinsic pathway of apoptosis is shown. Note that two pathways converge on the activation of effector caspases. The image was acquired from www.cellsignal.com.

Table 1. The composition of tea leaf. The table was adopted from Balentine et al. 1997.

Components % dry weight Flavanols 25.0 Flavonols and their glycosides 3.0 Other polyphenols 8.0 Caffeine 3.0 Theobromine 0.2 Amino acids 4.0 Organic acids 0.5 Monosaccharides 4.0 Polysaccharides 13.0 Cellulose 7.0 Proteins 15.0 Lignin 6.0 Lipids 3.0 Chlorophyll and other pigments 0.5 Minerals, etc. 5.0

Figure 5. Structures of basic flavonoid and major tea catechins. Only the four major catechins (flavan-3-ols) are shown. The figures of catechin structures were adopted and modified from Yang et al. 2002.

CHAPTER 2

MATERIALS AND METHODS

Reagents and Cell Culture

Human prostate cancer cell lines LNCaP and PC3 were purchased from American Tissue Type Culture Collection (ATCC, Manassas, VA) and ARCaP cell line was established previously. All four prostate cancer cell lines were maintained in Dulbecco’s modified Eagle’s media (DMEM) (Sigma, St. Louis, MO) without phenol red supplemented with 3.7 g/l of

NaHCO3, 10 % fetal bovine serum (FBS) (HyClone, Logan, UT) and 100 U/ml of Penicillin and 100 µg/ml of Streptomycin (Cambrex, Walkersville, MD). The culture media were refreshed every 3 days or whenever necessary. Cells were grown at 37 °C in a humidified atmosphere consisting of 5 % CO2.

Tea Polyphenol Treatment and Cell Lysis

Green tea polyphenols were purchased from Sigma (St. Louis, MO) and dissolved in double distilled water to final concentration of 50 mM. R1881 (methyltrienolone) was obtained from Perkin-Elmer Life Sciences (Boston, MA) and was dissolved in dimethyl sulfoxide (DMSO). Etoposide (9-(4,6-O-ethylidene-β-D-glucopyranoside)), also known as VP16, was purchased from Sigma (St. Louis, MO) and also dissolved in DMSO. Cells were treated with different concentrations of green tea polyphenols when cells were in their exponentially growing phase. Control was treated with the same volume of water. The maximum volume of the added polyphenol solution did not exceed 0.2 % of the volume of culture media. After desired time period, the culture media were removed and briefly centrifuged to collect the dead and dying cells due to apoptosis. The culture plate was then rinsed with phosphate buffered saline (PBS:

137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH7.4) followed by addition

17 of 0.25 % trypsin to detach the cells. The cells from both fractions were combined, spun, and the resulting cell pellet was washed with PBS to remove trypsin solution. After collecting the cells by centrifugation, the cells were resuspended in radio immunoprecipitation assay (RIPA) cell lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.25 % (w/v) sodium deoxycholate, 0.1 % (v/v) NP-40) supplemented with Phosphatase Inhibitor Cocktail II (Sigma, St. Louis, MO) and Halt Protease Inhibitor Cocktail (Pierce, Rockford, IL) and incubated in ice for 30 minutes (mins) for lysis. The crude cell lysates were centrifuged at 12000 g for 15 mins at 4 °C. The supernatant, which is cell lysate, was aliquoted and kept at - 80 °C for further applications including bicinchoninic acid (BCA) assay or kept at - 20 °C after being mixed with 5x sample buffer (250 mM Tris-HCl, pH 6.8, 10 % (w/v) sodium dodecyl sulfate (SDS), 0.5 % (w/v) bromophenol blue, 50 % glycerol, 5 % (v/v) β-mercaptoethanol) and boiling for 7 mins for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting experiments.

Nuclear and Cytoplasmic Extraction of Cells

The subcellular fractionation was performed by using NE-PER Nuclear and Cytoplasmic Extraction Reagents from Pierce (Rockford, IL) according to the manufacturer’s protocol. Briefly, 40 mg of packed cells was obtained from each sample by centrifugation at 500 g for 3 mins at 4 °C. After removing supernatant, 200 µl of cold CER I solution was added, vortexed for resuspension, and incubated in ice for 10 mins. 11 µl of chilled CER II was then added to the tube and vortexed for 5 seconds (secs) and incubated in ice for 1 min. Then another 5 secs of vortex was performed followed by centrifugation of the tube at 16000 g for 5 mins. The resulting supernatant fraction, which represents cytoplasmic extract, was collected and stored at - 80 °C. The remaining pellet was resuspended in 100 µl of cold NER solution by vortexing for 15 secs. The tube was incubated in ice for total of 40 mins with 15 secs of vortex at every 10 mins. Finally, the tube was centrifuged at 16000 g for 10 mins and the resulting supernatant, which represents nuclear extract, was collected and stored at - 80 °C. The protein concentration of both fractions was measured by BCA assay followed by Western blot experiments. Halt Protease Inhibitor Cocktail was added to CER I and NER solutions and when more than 40 mg of packed cells was used, the volume of solutions were adjusted proportionally.

18 Determination of Protein Concentration by BCA Assay

To calculate the concentration of total protein in cell lysate, BCA assay from Pierce (Rockford, IL) was utilized. 100 µl each from the 6 serially diluted bovine serum albumin (BSA) standards in RIPA lysis buffer and the 25x diluted cell lysate samples were mixed with 2 ml of working reagent which contains bicinchoninic acid and cupric acid in 0.1 M sodium hydroxide solution. The mixture was then incubated at 37 °C for 30 mins. During this incubation, Cu2+ reduces to Cu1+ by the protein in the alkaline solution and two molecules of BCA chelates with one cuprous cation (Cu1+) forming a purple-colored, water-soluble reaction product that gives strong absorbance at 562 nm that is nearly linear with increasing protein concentration. After incubation, the reaction mixture was cooled to room temperature and the optical density (OD) was measured at 562 nm. The absorbance of all samples and standards were measured in 10 minutes since the color continues to develop due to the fact that the BCA assay is not an end point method. However, the color development is low enough not to generate significant error if all measurements are made within 10 minutes. The spectrophotometer was calibrated with water filled cuvette and the absorbance of the blank standard (0 µg/ml BSA) was subtracted from that of all the other standards and samples. The formula of the standard curve of the blank corrected absorbance values of BSA standards vs their concentrations was set up using the Linear Square Regression Method. The correlation coefficient was greater than 0.99 and the sample protein concentration was determined using this formula.

SDS-PAGE and Western Blotting

After cell lysis and BCA assay, same amount (25 µg) of protein from each sample was loaded and separated by SDS-PAGE at 25 milliampere (mA) per gel. The composition of the gel running buffer was 25 mM Tris, 250 mM glycine and 0.1 % (w/v) SDS, and was used for both anode and cathode buffer. The proteins on the gel was then transferred onto the nitrocellulose membrane in N-cyclohexyl-3-aminopropanesulfonic acid (CAPS) buffer (10 mM CAPS, 10 % methanol, pH 11) at 50 volt (V), 40 ~ 90 mins depending on the size of the protein to be detected in Western blotting. The blotted membrane was blocked with 5 % (w/v) nonfat dry milk or same concentration of BSA in Tween-20 containing Tris buffered saline (TBS: 10 mM Tris-HCl, pH

19 7.5, 150 mM NaCl, 20 mM KCl, 0.1 % (v/v) Tween-20) solution for 1 hour (hr) at room temperature. Then, the membrane was rinsed briefly with Tween-20 containing TBS and incubated with primary antibody in 5 % (w/v) nonfat dry milk or same concentration of BSA in Tween-20 containing TBS solution for overnight at 4 °C with gentle shaking. Antibodies used in this study were all purchased from Cell Signaling Technology (Beverly, MA) except anti-β-actin antibody which was purchased from Sigma (St. Louis, MO) and were diluted accordingly based on the manufacturer’s manual. ImmunoPure horseradish peroxidase (HRP)-conjugated secondary antibody (Pierce, Rockford, IL) was added at final concentration of 40 ng/ml (1:10,000 dilution) in blocking solution and incubated for 1 hr at room temperature. For washing the membrane, Tween-20 containing TBS was used 5 mins each for three times. The signal was developed on X-Omat film from Eastman Kodak (Rochester, NY) in dark room by using Super Signal West Pico Chemiluminescent Substrate (Pierce, Rockford, IL).

Cell Proliferation Assay

Cells were seeded in 6-well plates with 10 % FBS media. After 24 hrs, media were changed with fresh 10 % FBS media and cells were treated with several different concentrations of tea catechins or solvent, i.e. water. After culturing for a different periods of time, the number of viable cells was counted with hemacytometer using trypan blue at each time point. To validate the effect of androgen in ARCaP cell growth, cells were seeded in 6-well plates and cultured in media with 10 % FBS. After 24 hrs, media were changed with the one that contains 10 % charcoal/dextran stripped FBS and cultured for another 24 hrs. Then, the media were replaced with new media that contains 10 % charcoal/dextran treated FBS (HyClone, Logan, UT) to minimize the effect of steroids that exist in the normal, noncharcoal/dextran treated FBS. Finally 1 nM of R1881 was added to treated cells whereas same volume of DMSO was added to the control cells. Cells were grown and the number of viable cells was counted at each time point using trypan blue exclusion assay. For trypan blue exclusion assay, cell suspension was mixed 1:1 with 0.4 % trypan blue solution and incubated for 2 mins. The viable cells are those that exclude the dye and was counted using hemacytometer.

20 Cell Death Detection Assay

The extent of apoptosis was determined by using Cell Death Detection ELISA PLUS assay kit from Roche Applied Science (Indianapolis, IN). The assay is a quantitative sandwich enzyme linked immunosorbent assay (ELISA) that uses the fact that mono- and oligonucleosomes due to the apoptotic internucleosomal cleavage are released from nucleus to cytosol in apoptotic cells. These can be detected by biotin labeled anti-histone-biotin antibody and peroxidase conjugated anti-DNA antibody. This antibodies-mono (oligo) nucleosome complex will bind to streptavidin coated well plate and gives signal at 405 nm upon the addition of the substrate (Figure 6). After treating the cells for 72 hrs with different doses of apoptotic stimuli, including EGCG and etoposide, the culture media was removed and centrifuged briefly to collect the floated cells. The culture dish was rinsed with PBS and 0.25 % trypsin was added to detach the cells. Once detached, the cell suspension was combined with the cells collected from the media. The resulting mixture of the cells was spun briefly to collect the cells. The supernatant was discarded and the pelleted cells were resuspended in ice cold PBS. The cell suspension was given final centrifugation and the pellet was resuspended in the Roche lysis buffer. After the incubation at room temperature for 30 mins, the reaction mixture was centrifuged for 10 mins at 200 g, 4 °C. The pellet, which contains nucleus, was removed and the supernatant, which represents the cytoplasmic fraction, was aliquoted in new tubes and kept frozen at - 80 °C until usage. This supernatant solution would contain the fragmented nucleosomes if the cells underwent apoptosis. After measuring the protein concentration of the resulting supernatant using BCA assay, 20 µg of total protein in 20 µl was added to the streptavidin coated 96-well plate. 20 µl of each of Incubation buffer and DNA-histone-complex was used as background control and positive control, respectively. Then, 80 µl of Immunoreagent was added to each well and incubated at room temperature for 2 hrs with gentle and continuous shaking. The Immunoreagent contains Incubation buffer and two different antibodies, anti-histone-biotin antibody and anti-DNA-HRP antibody. After the incubation, the solution in wells were thoroughly removed by gentle suction and rinsed with Incubation buffer three times. Finally, 100 µl of 2,2’-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) substrate solution was added to each well and incubated until the color developed to desired strength which took about 5 mins.

21 The multi-well plates were then placed into Bio-Tek µQuant Microplate Spectrophotometer. The absorbance was measured at 405 nm and 495 nm was used as the reference wavelength. The absorbance at 495 nm was deducted from the absorbance at 405 nm for all samples and controls. Then, the OD405 – OD495 value of the background control, which is composed of the incubation buffer and ABTS solution, was subtracted from all OD405 – OD495 values of the samples. The intensity of apoptosis can be expressed as ‘Enrichment Factor’ as shown below where mU is the absorbance x 10-3 after subtracting the reference absorbance and the OD405 – OD495 value of the background control. The Enrichment Factor exhibits the specific enrichment of mono- and oligonucleosomes released into the cytoplasm of dying and dead cells due to apoptosis. Finally, the values were normalized so that the untreated sample could have Enrichment Factor of 1.

(mU of the sample) Enrichment Factor = ĬĬĬĬĬĬĬĬĬĬĬĬĬĬĬĬĬĬĬĬ/ (mU of the corresponding negative control)

DNA Fragmentation Assay

Cells grown in 100 mm dish were rinsed with PBS and were collected by treating with lysis buffer (10 mM Tris-HCl, pH 7.4, 10 mM ethylenediaminetetraacetic acid (EDTA), 0.5 % Triton X-100) for 30 mins in ice. Floated cells in culture media were also collected. Supernatants after the centrifugation were incubated with RNase (0.4 mg/ml) for overnight at room temperature followed by Proteinase K treatment (0.4 mg/ml) for 2 hrs at 37 °C. DNA was extracted with 1x volume of phenol : chloroform : isoamylalcohol (25:24:1) mixture for 10 mins in ice followed by precipitation of DNA by incubating with 2x volume of 100 % ethanol for overnight at - 20 °C. Finally, precipitated DNA was pelleted by centrifugation. The DNA pellet was washed once with 70 % ethanol and then resuspended in 20 µl of Tris-EDTA (TE) buffer (10 mM Tris-HCl, pH 7.5, 1 mM EDTA). The DNA was loaded onto a 1.5 % agarose gel containing 0.5 µg/ml ethidium bromide and electrophoresis was conducted at 100 V in Tris- acetate-EDTA (TAE) buffer (40 mM Tris acetate, 1 mM EDTA, pH 8.3). The gel was visualized and the pictures were taken under UV light.

22 Morphological Analysis of Apoptotic Cell Nucleus

Cells were grown on dishes and different doses of EGCG were treated. After 72 hrs, the cells from dish and media were collected and were fixed with freshly prepared 4 % paraformaldehyde in PBS (pH 7.4) for 10 mins. The fixed cells were then washed briefly with PBS followed by staining with 10 µM Hoechst 33258 (Sigma, St. Louis, MO) in PBS for 30 mins. The number of cells was counted and 20 µl of cell suspension containing 200,000 cells was mounted onto the slide glass, covered with cover glass and analyzed under a Nikon FX fluorescence microscope with excitation at 340 nm.

Cell Adhesion Assay

One of the unique features of ARCaP cells upon EGCG treatment was that the cells show stronger adherence to the culture plate which makes it difficult to detach the cells for further application. To show the images of this unusual characteristic of ARCaP cells, the pictures of cells of EGCG treated ARCaP and LNCaP cells were taken and compared to that of the untreated control cells after the trypsin treatment. ARCaP and LNCaP cells were seeded in 100 mm dishes. After 24 hrs, media were refreshed and cells were cultured with or without 100 µM EGCG for 72 hrs, then, the media were discarded and the dish was rinsed with PBS, three times followed by the addition of 2 ml of 0.25 % trypsin per dish and incubation at 37 °C incubator. At each time point, dishes were pulled out and pictures were taken through the microscope after the trypsin solution that contains detached cells was completely removed. For more scientific analysis and quantification of changes in cell adhesion upon EGCG treatment, Colorimetric Extracellular Matrix Cell Adhesion Array kit from Chemicon (Temecula, CA) was utilized. The assay kit utilizes a colorimetric detection in 96-well microtiter plate. The plate is consisted of 12 x 8-well removable strips. Each well within a strip is precoated with either collagen I, collagen II, collagen IV, fibronectin, laminin, tenascin, vitronectin or BSA. The strip was rehydrated with PBS at room temperature before seeding the cells. When the cells reach exponential growing phase, cells were detached with 0.05 % trypsin in Hank’s balanced salt solution containing 25 mM N-(2-hydroxyethyl)-piperazine-N’-2-ethanesulfonic acid (HEPES). The cells were rinsed with PBS and resuspended in DMEM media containing 10 %

23 FBS and with or without 100 µM of EGCG. The volume was adjusted to have 1.5 x 106 cells/ml of media. After seeding 150,000 cells in 100 µl per well, the strip was incubated for 3 hrs at

37 °C in a CO2 incubator. Then, the media were gently aspirated and the wells were washed three times with the Assay buffer. After washing, 100 µl of Cell staining solution was added to each well and incubated for 5 mins at room temperature. Next, the solution was removed and the wells were gently washed five times with deionized water. After air drying the wells for few minutes, 100 µl of the Extraction buffer was added to each well and incubated for 10 mins at room temperature with gentle rotation on an orbital shaker. When the cell bound stain was completely solubilized, absorbance at 540 nm was measured on a microplate reader.

Anchorage-Independent Growth Assay

Assays of colony formation in soft agar were performed in 6-well plate. Briefly, 1 ml of base agar consisting of 0.6 % Noble agar (Difco, Detroit, MI), 1x DMEM media and 10 % FBS was underlaid in 6-well plates. After cells were trypsinized, 3 x 104 cells were centrifuged and resuspended in 1.5 ml of top agar which is consisted of 0.3 % Noble agar, 1x DMEM media, 10 % FBS and plated onto previously prepared base agar underlayers. Different doses of EGCG were added to both top and bottom agar of the treated samples and same volume of water was added to control. 200 µl of the same media with or without EGCG and without agar was added every week. The plate was incubated at 37 °C in a humidified 5 % CO2 atmosphere for 2 weeks and stained with 1 mg/ml of p-iodonitrotetrazolium violet (Sigma, St. Louis, MO) for overnight in the incubator. Pictures were taken and the number of colonies was counted.

GeneChip® Microarray Analysis

LNCaP and ARCaP prostate cancer cells were grown to subconfluence in DMEM media with 10 % FBS. 100 µM of EGCG or same volume of solvent, water, was added to the culture media. After 24 hrs, the media were removed and the culture plates were rinsed two times with PBS. To each dish, 4 ml of TRIzol reagent (Life Technologies, Gaithersburg, MD) was added and incubated for 5 mins at room temperature. The cells were then collected and kept at - 80 °C until used. These samples were sent to Center for Functional Genomics at University of Albany

24 (Rensselaer, NY) for RNA preparation, complementary DNA (cDNA) synthesis, complementary RNA (cRNA) preparation, and for microarray service. Briefly, total RNA was isolated using RNeasy Mini Kit from Qiagen (Valencia, CA) and the quality of RNA was measured with BioAnalyzer. RNA was reverse transcribed to single strand cDNA. Double strand cDNA was prepared and the antisense cRNA was synthesized using in vitro transcription. The cRNA was labeled with biotin during the synthesis by using biotinylated ribonucleotides as substrates. The labeled cRNAs were then fragmented into small pieces to facilitate the hybridization. The fragmented cRNAs were hybridized to the human genome U133 Plus 2.0 arrays provided by Affymetrix (Figure 7 A) (Santa Clara, CA). Human genome U133 Plus 2.0 array contains a probe set that represents more than 47,000 transcripts. After washing the unbound cRNAs, the chip was stained with streptavidin-phycoerythrin that binds to the biotinylated cRNA-probe complex. The chip was then scanned with scanner and the intensity of the signal in the array was recorded. A brief scheme of the whole procedure is illustrated (Figure 7 B). The data were analyzed by using GeneSpring software.

Total RNA Preparation

For Reverse Transcription - Polymerase Chain Reaction (RT-PCR), total RNA was prepared. Total RNA was extracted from cells using 1 ml of TRIzol reagent per 10 cm2 of culture dish growth area. TRIzol is a mono-phasic solution of phenol and guanidine isothiocyanate that disrupts the cells and dissolves cell components while maintaining the integrity of the RNA. After cell culture with or without the treatments, cells were briefly rinsed with PBS and TRIzol reagent was added for homogenization. After incubating 5 mins at room temperature, the suspended cell solution was transferred to a new tube and 200 µl of chloroform per 1 ml of TRIzol used was added. The tube was vortexed and centrifuged for 15 mins at 12000 g to separate the phases after 5 mins of incubation at room temperature. The RNA containing aqueous phase was collected and transferred to new tube and 500 µl of isopropanol per 1 ml of TRIzol used initially was added to precipitate the RNA. The organic phase which includes protein and DNA was discarded. After a brief spin, the pelleted RNA was rinsed with 75 % ethanol and spun again. The resulting pellet was then dried under hood for several minutes and dissolved in 50 µl of diethylpyrocarbonate (DEPC) treated water for further applications. The

25 purity of the prepared RNA was measured by calculating OD260/OD280. The concentration of the purified RNA was measured by OD260 x 35 µg/ml. On average, the typical yield was 100 µg of RNA per 4 x106 cells.

Reverse Transcription - Polymerase Chain Reaction (RT-PCR)

Total 2 µg of RNA was reverse transcribed to obtain first strand cDNA using random hexamers, as primers, and SuperScript II Reverse Transcriptase (Life Technologies, Gaithersburg, MD). First, DEPC treated water was added to RNA samples to have 2 µg of RNA in 7 µl and mixed with random hexamers and incubated for 10 mins in 70 °C water bath for denaturation. Then, the mixture was supplemented with 7 µl of reaction mixture (see below) and incubated for 5 mins at room temperature. After incubation, 1 µl of SuperScript II Reverse Transcriptase was added and mixed gently. The resulting 20 µl reaction mixture was then incubated for 10 mins at room temperature for annealing followed by 50 mins of incubation at 42 °C for cDNA synthesis by the reverse transcriptase. Termination of the reaction was achieved by inactivation of the reverse transcriptase through incubating the reaction mixture at 70 °C for 15 mins. The constituents of the final reaction mixture were:

purified RNA (2 µg) 7 µl random hexamer 5 µl reaction mixture: 10x PCR buffer 2 µl 25 mM MgCl2 2 µl 10 mM dNTP mix 1 µl 0.1 M DTT 2 µl Reverse Transcriptase (50 units) 1 µl total 20 µl

The synthesized cDNA was stored at - 20 °C until utilized in PCR to amplify the desired gene specific cDNAs. Matrix metalloproteinase 1 (MMP1) and MMP3 cDNAs were amplified using specific primers and the primers for house keeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as control. The information of primers, including the sequences, is summarized (Table 2). The annealing temperature was calculated by Ta = 2(A+T) + 4(G+C) – 4 where each base represents the number of such base in the primer sequence. The primers that were used in this study were synthesized by Integrated DNA Technologies

26 (Coralville, IA) and were solubilized in TE buffer. The condition of the PCR reaction and the constituents of the reaction mixture are listed as follows:

pure water 30 µl 10x PCR buffer 5 µl 2 mM dNTP 5 µl 25 mM MgCl2 3 µl 10 µM 5' primer 2 µl 10 µM 3' primer 2 µl Taq 1 µl cDNA 2 µl total 50 µl

reaction step 1: 94 °C, 2 mins step 2: 94 °C, 45 secs *step 3: annealing, 45 secs step 4: 72 °C, 90 secs **step 5: repeat from step 2 to 4 step 6: 72 °C, 10 mins

NOTE: *In annealing step, different temperatures were used for different templates (Table 3). **The cycle was repeated 31 times for MMPs, 34 times for EGR1, and 26 times for GAPDH.

After the reaction, 10 µl of 6x gel loading buffer (10 mM Tris-HCl, pH 7.5, 50 mM EDTA, pH 8.0, 15 % Ficoll 400, 0.03 % bromophenol blue, 0.03 % xylene cyanol, and 0.4 % orange G) from Promega (Madison, WI) was added and mixed thoroughly. 40 µl of the mixture was loaded onto a 1.5 % agarose gel containing 0.5 µg/ml ethidium bromide and electrophoresis was conducted at 100 V with TAE buffer for 90 mins. The gel was visualized and the pictures were taken under UV light.

Quantitative Measurements of proMMP1 and MMP3 by ELISA

The Quantikine Human proMMP1 and MMP3 Immunoassay Kits are purchased from R&D Systems (Minneapolis, MN). The assay employs the quantitative sandwich immunoassay technique. The microplate is precoated with proMMP1 or MMP3 specific monoclonal antibody. Cells were treated with tea polyphenols for certain period of time and the culture media were

27 collected. The collected media were centrifuged at 4000 g for 5 mins to remove debris and the remaining supernatant was aliquoted and kept at - 80 °C until utilized. 100 µl of Assay diluent was added to each well of the microplate. Then 100 µl of standard, which is provided by the company, and samples, after proper dilution, were added and incubated at room temperature for 2 hrs with gentle horizontal shaking. After incubation, the solution was aspirated and each well was rinsed with 300 µl of Wash buffer for a total of 4 times to remove unbound substances. 200 µl of MMP Conjugate, which is monoclonal antibody against proMMP1 or MMP3 conjugated with HRP, was added to each well and incubated for another 2 hrs at room temperature with horizontal shaking. The solution was removed after the incubation and the microplate was rinsed with Wash buffer as before and 100 µl each of Color reagent A (stabilized hydrogen peroxide) and Color reagent B (stabilized tetramethylbenzidine as chromogen) were added to each well and incubated for 20 mins at room temperature. 50 µl of Stop solution (2 N sulfuric acid) was added to halt the color development and the optical density of each well was determined using Bio-Tek µQuant Microplate Spectrophotometer. The absorbance was measured at 450 nm and 540 nm was used as the reference wavelength. The readings at 540 nm were subtracted from the readings at 450 nm. After the correction, the optical density of zero standard (0 ng/ml) was subtracted from the corrected readings of each standard. The data from standards were linearized by using log scale and Linear Square Regression Analysis was applied to obtain the formula of the standard curve. The correlation coefficient was greater than 0.99 and the concentration of MMP in the cell culture media was finally determined using the formula after the log transformation of the optical density which was corrected by the readings at reference wavelength and subtraction of zero standard absorbance.

Statistic Analysis of the Data

For statistic analysis of the data, Microsoft Excel software was used to calculate the mean and the standard error of the mean (SEM). SEM was used instead of standard deviation (SD) to generate the tables and graphs of the data. To measure the statistical significance of the data, t- test was used for comparing the means of two groups. To compare three or more groups, analysis of variance (ANOVA) was used to avoid inherent flaw that comes from the probability. ANOVA

28 detects the significance between the treatments as a whole. The P values were calculated using student’s t-test or Single Factor ANOVA.

Figure 6. Schematic representation of Cell Death Detection ELISA PLUS assay. The image was adopted from the manufacturer at www.roche-applied-science.com. The cytoplasmic fraction of the cell was utilized to observe the apoptosis-induced translocation of the nucleosomes.

Figure 7. The schematic diagram of GeneChip assay. A: A picture of GeneChip that was used in this microarray analysis is shown. B: The procedure of GeneChip assay is briefly depicted. The alphabet ‘B’ on the bottom panel represents biotin and encircled B after ‘wash and stain’ represents streptavidin-phycoerythrin bound biotin that generates signals. The pictures were acquired from Affymetrix web page available at www.affymetrix.com.

Table 2. The information of primers used in PCR. The GenBank and GenPept ID numbers that were used to design the primers are shown. The ‘amplified area’ next to the GenBank ID column represents the nucleotide region that is amplified during the PCR. The ‘matching region’ of the corresponding protein is shown in the right side of GenPept ID column.

Target Amplified GenBank ID GenPept ID Matching region gene area

MMP1 NM 002421 729 ~ 1478 NP 002412 Leu 220 ~ Asn 469

MMP3 NM 002422 747 ~ 1496 NP 002413 His 228 ~ Cys 477

EGR1 NM 001964 752 ~ 1480 NP 001955 Val 162 ~ Arg 403

GAPDH NM 002046 628 ~ 1079 NP 002037 Thr 176 ~ Val 325

Table 3. The sequences of primers used in PCR. Also, the size of the amplified cDNA product and the annealing temperature used in the PCR are displayed.

Target Product Direction Primer sequence Ta gene size

forward 5’- ctc ggc cat tct ctt gga ctc tc -3’ MMP1 750 bp 60 °C reverse 5’- att ttt cct gca gtt gaa cca gc -3’

forward 5’- cac tca gcc aac act gaa gct ttg -3’ MMP3 750 bp 60 °C reverse 5’- aca att aag cca gct gtt act ctt c -3’

forward 5’- tag tga gca tga cca acc ca -3’ EGR1 729 bp 56 °C reverse 5’- ttc ttc cac aga tgt cgc ag -3’

forward 5’- acc aca gtc cat gcc atc ac -3’ GAPDH 452 bp 58 °C reverse 5’- tcc acc acc ctg ttg ctg ta -3’

CHAPTER 3

EFFECTS OF EGCG ON GROWTH AND APOPTOSIS OF PROSTATE CANCER CELL LINES

Prostate Cancer and EGCG

Prostate cancer is the most common malignancy in men in United States with an estimated lifetime probability of 17.81 % (Jemal et al. 2006). However, the worldwide incidence rate of prostate cancer differs greatly. The prostate cancer rates per 100,000 people in Asian countries, such as Korea, Japan, and China, of which the population consume large amounts of green tea regularly, are much lower than those of Western countries (Cheon et al. 2002, Parkin et al. 2005). The worldwide popular drink, tea, has been receiving great attention during the past decade due to its anticarcinogenic polyphenolic contents. Epidemiologists have revealed the chemopreventive potential of the tea polyphenols against cancer. Imai et al. have demonstrated that consumption of green tea is associated with later onset of cancer in both men and women (Imai et al. 1997). A study employing 7,833 men living in Hawaii with Japanese ancestry demonstrated that there is negative association between tea drinking and prostate cancer incidence (Heilbrun et al. 1986). Another study with Canadian men showed that there is a decrease in prostate cancer risk with more than two cups of tea intake per day (Jain et al. 1998). A recent case control study in Southeast China shows that the risk of prostate cancer decreased with increasing frequency, duration, and quantity of green tea consumption (Jian et al. 2004). Recent studies show promising results on EGCG as anticancer agent in various cancers including prostate cancer (Gupta et al. 1999, Kuroda et al. 1999, Yu et al. 1995, Suganuma et al. 1999, Nakachi et al. 2000). Very recently, a human clinical study on chemopreventive efficacy of the tea catechins for prostate cancer was performed and reported. The study of 60 men bearing high grade prostatic intraepithelial neoplasia (PIN) in Italy demonstrated that only 1 prostate

34 cancer was diagnosed among 30 men who were treated with 600 mg-per-day dosage of green tea catechins, 51.88 % of which was EGCG, for 1 year whereas 9 cancers were diagnosed among 30 men treated with placebo (Bettuzzi et al. 2006). Cellular studies show that EGCG induces cell cycle arrest and apoptosis in prostate cancer cell lines such as androgen-sensitive LNCaP and androgen-insensitive DU145 and PC3 cells (Paschka et al. 1998, Gupta et al. 2000, Chung et al. 2001, Gupta et al. 2003). Moreover, intraperitoneal injection of EGCG to nude mice not only inhibited the growth of human prostate tumors elicited by subcutaneous inoculation of human prostate cancer cells but also reduced the tumor size in some cases. When the EGCG injection was stopped on the 14th day of treatment, tumor growth resumed very rapidly (Liao et al. 1995). In the transgenic adenocarcinoma of the mouse prostate (TRAMP) model, the infusion of green tea polyphenol resulted in apoptosis and almost complete inhibition of metastasis (Gupta et al. 2001). Previously, we have developed a new type of prostate cancer line. In 1996, an androgen repressed prostate cancer cell line (ARCaP) was established from ascites fluid of an 83-year-old Caucasian man whose cancer has metastasized to liver, spine and long bones (Zhau et al. 1996). The growth of this cell line has been shown to be inhibited by the supply of androgen. ARCaP cells are highly invasive and metastatic and thus may represent the advanced form of prostate cancer. The facts that no apoptosis studies of this cell line were performed, to our knowledge, to date and that the EGCG gave promising results as chemopreventive reagent on the subset of human prostate cancer cell lines have prompted us to investigate the effects of EGCG treatment in this unique prostate cancer cell line. In the present study, we used ARCaP cells to test the susceptibility to tea polyphenol EGCG using LNCaP cells as control. Employing different types of assays to detect the apoptosis including DNA fragmentation assay, cell death detection ELISA assay, caspase activation, and Hoechst staining, ARCaP cell line exhibited resistance to such tea catechin treatment. Testing the pathways that are reported to be affected by EGCG in prostate cancer cell lines revealed that the pathways are influenced but in favor of cell survival and proliferation not apoptosis. The characteristics of ARCaP cell line upon EGCG treatment are reported and compared to that of LNCaP cell line herein.

35 R1881 Suppresses the Growth of ARCaP Cells but Facilitates LNCaP Cell Growth

The original article that reported the establishment of the highly metastatic prostate cancer cell line ARCaP revealed that the growth of this cell line is unique since it is suppressed by the addition of androgen by a dose-dependent manner (Zhau et al. 1996). To verify that androgen represses the growth of ARCaP cell line, a set of growth assay was performed in the presence or absence of 1 nM of synthetic androgen R1881. When the media were replaced and R1881 was added after 48 hrs of initial seeding of the cells, this time point was denoted as 0 hr. The number of viable cells was counted at 0 hr, 24 hr, and 48 hr by utilizing trypan blue exclusion assay. As expected, androgen expedited the growth of androgen-responsive LNCaP cells. 1 nM R1881 facilitated the proliferation of LNCaP cells by 15.7 % and 22.7 % at 24 hr and 48 hr period, respectively. In contrast to this observation, the same concentration of synthetic androgen suppressed the proliferation of ARCaP cells by 9.8 % and 22.1 % (P = 0.00033) at the same time period (Figure 8 and Table 8, see appendix) indicating that ARCaP cells are androgen-repressed prostate cancer cell line as reported in the original article. In androgen-unresponsive PC3 cells, the addition of androgen showed slight inhibition of growth but had no statistic significance.

EGCG Represses the Growth and Induces Detachment and Membrane Blebbing in Cultured LNCaP Cells but Not in ARCaP Cells

To assess the effect of EGCG on prostate cancer cells, LNCaP and ARCaP cells were treated with 25, 50, and 100 µM of EGCG for 24, 48, and 72 hrs and the viability of the cells were determined. As shown in the graph, treatment of LNCaP cells with EGCG resulted in inhibition of proliferation (Figure 9 and Table 9, see appendix) which is consistent with and confirming previous observations (Paschka et al. 1998, Gupta et al. 2000). The control LNCaP cells grew quite rapidly, and over the 72 hr period, the number of cells reached over 821,000 on average. Treating LNCaP cells with 25 µM EGCG resulted in slight decrease in growth rate but not the growth itself whereas 50 µM EGCG significantly decreased the growth rate of LNCaP cells over 72 hrs. 100 µM EGCG finally inhibited proliferation of LNCaP cells which resulted in decrease in number of cells over 3 day period. After 72 hrs, only little over 26,000 cells on

36 average were viable which accounts 26.7 % when compared to the number of the cells that were initially seeded. On the other hand, the growth of ARCaP cells was not affected by adding up to 50 µM of EGCG. At 100 µM, EGCG attenuated the rate of ARCaP cell growth but even at this high concentration, EGCG did not stop ARCaP cells from proliferating and ARCaP cells still showed 176.7 %, 285.3 %, and 326.0 % increase in cell number over 24 hr, 48 hr, and 72 hr period, respectively (Figure 10 and Table 10, see appendix). It is important to note that in LNCaP cells, the actual number of cells decreased over time upon the addition of 100 µM EGCG whereas, in ARCaP cells, the number of cells was still increasing at such high concentration of EGCG even though it was not as rapid as control cells. Moreover, the number of trypan blue stained cells increased in both dose- and time-dependent manner in LNCaP cells but in ARCaP cells, the number of dead cells was minimum and stayed at similar level in all ranges tested. We further investigated the possibility whether R1881 might affect the viability of EGCG treated ARCaP cells but the results indicated no correlation. Next, we cultured LNCaP and ARCaP cells and took pictures of cells after 72 hrs of the tea catechin treatment under microscope. In LNCaP cells with EGCG treatment, not only the density of the cells was lower than the control but also showed many floated cells with round shape that represent dead and/or dying cells (Figure 11). Also, EGCG treatment resulted in loose attachment of the cells to the culture plate. Finally, membrane blebbing, which is one of the hallmarks of apoptosis, was observed in cell population when treated with EGCG especially at 100 µM. In ARCaP cells however, the number of the cells decreased in dose dependent manner but the cells exhibited normal morphology and did not show any membrane blebbing as observed in LNCaP cells (Figure 12). Moreover, treatment of EGCG resulted in more robust attachment of the cells which was dose dependent, probably through between cells and also between cells and culture dish (discussed later).

EGCG Induces Apoptosis in LNCaP Cells but Not in ARCaP Cells

In the next figure, we show that EGCG induces intensive apoptosis in LNCaP cells but not in ARCaP cells. To measure the extent of apoptosis, we performed Cell Death Detection Assay PLUS from Roche which detects mono- and oligonucleosomes, the product of internucleosomal cleavage during apoptosis, that are released to cytosol. In LNCaP cells, the

37 color developed in as low as 25 µM of EGCG treated sample. 100 µM of EGCG treated LNCaP sample showed intense dark green color development (Figure 13 A). In ARCaP cells, the extent of color development was minimal demonstrating that EGCG did not induce apoptosis in these cells. The absorbance of each well was then measured with multi-well plate spectrophotometer and the results are also shown (Figure 13 B and Table 11, see appendix). As depicted in graph, EGCG substantially triggered apoptosis dose dependently in LNCaP cells. The average Enrichment Factor increased from 1 to 2.86, 10.63, and 23.61 for 0, 25, 50, and 100 µM of EGCG treated samples, respectively with statistically high significance. In ARCaP cells, the Enrichment Factor increased slightly upon EGCG treatment but there was no statistical significance. DNA fragmentation, which is mediated by DNA fragmentation factor 40 (DFF40) after its release from caspase 3 induced degradation of its inhibitor DFF45, is one of the hallmarks of apoptosis. DNA was purified after 72 hrs of incubation with or without EGCG and was subjected to gel electrophoresis to monitor the DNA fragmentation. The DNA fraction purified from LNCaP cells treated with high concentrations of EGCG showed smearing of its DNA which indicates the multiply fragmented DNA (Figure 14). On the contrary, the fragmentation of DNA was not detected in ARCaP cells.

EGCG Induces Chromatin Condensation and Nuclei Fragmentation in LNCaP Cells but Not in ARCaP Cells

Chromatin condensation within the nuclei is one of the key characteristics of apoptosis and thus used to identify the apoptotic cells. The nuclei of cells under EGCG treatment were analyzed by staining with Hoechst 33258. The nuclei of ARCaP cells treated with EGCG were all stained blue in a similar fashion that stained the nuclei of the control cells which suggests that EGCG did not induce the chromatin condensation in these cells (Figure 15). In LNCaP cells on the other hand, the nuclei of the control cells stained normal, but the nuclei of cells treated with EGCG displayed dose dependent increase of appearance of small bright blue colored areas due to the chromatin condensation and fragmentation of the nuclei (indicated with arrows) that are still confined in the membrane vesicles (Figure 16). These nuclear morphological changes correlated well with the observations that were obtained from the previous assays of apoptosis.

EGCG Induces Caspase 3 Activation, PARP Cleavage and p53 Increase Only in LNCaP Cells Leading to Apoptosis

Apoptosis is mediated through series of actions of proteases called caspases whose activation requires cleavage of proform into active form. Caspase 3 is the key enzyme that executes the apoptosis and once activated, it can cleave various types of substrates including lamin and fodrin leading to the destruction of the cell. DNA repair related protein poly(ADP- ribose) polymerase (PARP) is also one of the substrates of caspase 3 and has been used extensively as a marker of apoptosis. To evaluate the EGCG induced apoptosis by Western blot, we performed the analysis using antibodies against caspase 3 and PARP. Only LNCaP cells showed cleavage and thus activation of caspase 3 upon EGCG treatment (Figure 17). Accordingly, activated caspase 3 cleaved 116 kDa PARP into 24 and 89 kDa fragments in LNCaP cells, but not in ARCaP cells, in EGCG dose dependent manner and the increase in 89 kDa fragment is clearly shown in the same picture. It has been suggested that EGCG induced apoptosis in LNCaP prostate cancer cell line is mediated through the regulation of p53 and NF-κB (Hastak et al. 2003). The authors concluded that EGCG up regulates proapoptotic protein p53 and down regulates prosurvival factor NF-κB in these cells. The tumor suppressor p53 is the most commonly mutated gene in human cancer. p53 regulates transcription of genes that are involved in cell cycle arrest and apoptosis including Bax, Puma, Noxa, and Bid. LNCaP cells possess wild type p53 whereas ARCaP cells are known to be null for p53 due to the nonsense mutation (CGA TGA) at codon 196 (Arg 196 Stop) (van Bokhoven et al. 2003). Our Western blot experiment confirmed the absence of p53 in ARCaP cells (Figure 18 A). The figure shows that LNCaP cells, but not ARCaP cells, have p53 protein. Other prostate cancer cell line DU145 harbors two mutations in p53 gene, CCT CTT (Pro 223 Leu) and GTT TTT (Val 274 Phe), and the resulting full length mutant protein was detected in our Western blot. The p53 protein was not detected in the other control cell line, PC3. This matches the fact that this cell line is reported to have mutation (GCC GC) that results in halt in translation at codon 169 leading to premature protein (Isaacs et al. 1991). Finally, when LNCaP cells were treated with 100 µM of EGCG, not only the expression of p53 protein was increased but also the

39 translocation of p53 from cytosol to place where it functions, nucleus, was augmented (Figure 18 B). Antibody that is specific to β-actin was used as background control.

EGCG Triggers Activation of NF-κκκB in ARCaP Cells but Induces Inactivation in LNCaP Cells

We next examined the changes of NF-κB and its phosphorylated form upon EGCG treatment. NF-κB is a transcription factor that plays versatile roles including cell survival. NF- κB is activated mainly via Iκ-B kinase (IKK) mediated phosphorylation and degradation of inhibitor Iκ-Bα that leads to translocation of NF-κB into the nucleus. NF-κB is consisted of dimer and the phosphorylation of p65 subunit has been suggested for optimal activation (Viatour et al. 2005). Our experimental results indicate that the phosphorylation of p65 subunit of NF-κB increased in ARCaP cells but decreased in LNCaP cells, both dose dependently when treated with EGCG, whereas the total level of p65 NF-κB remained the same (Figure 19). The level of inhibitor Iκ-Bα did not change also as presented in the same figure. Recent studies suggest that aberrant NF-κB regulation is associated with carcinogenesis and this transcription factor is also known to play a key role as a cell survival factor to avoid programmed cell death (Barket and Gilmore 1999, Chen et al. 2001). It has been demonstrated that apoptosis can be facilitated by suppressing the activity of NF-κB in prostate cancer cells (Altuwaijri et al. 2003, Huerta-Yepez et al. 2004). Studies with soy isoflavone genistein revealed that genistein induces apoptosis through inactivation of NF-κB in prostate cancer cells (Davis et al. 1999). Similarly, Hastak et al. have shown that the decrease of translocation and transcriptional activity of NF-κB plays a crucial role in EGCG induced apoptosis in LNCaP cells (Hastak et al. 2003). Thus we examined the level of NF-κB activation which resulted in increase in activation of NF-κB in ARCaP cells but decrease in LNCaP cells upon EGCG treatment. Previously, Andela et al. suggested that NF-κB is a pivotal transcription factor in prostate cancer metastasis to bone by using wild type and dominant negative mutant Iκ-B bearing PC3 prostate cancer cells (Andela et al. 2003). ARCaP cells are originally established from prostate cancer metastasized to bone. Therefore it is highly possible that ARCaP cells might have adapted their physiology to maintain the activity of NF-κB.

40 EGCG Decreases the Ratio of Bax/Bcl-2 in ARCaP Cells but Increases the Ratio in LNCaP Cells

The release of the apoptogenic factors from the mitochondria is regulated by a group of proteins that belongs to Bcl-2 family which includes proapoptotic Bad, Bax and antiapoptotic Bcl-2, Bcl-XL. Bcl-2 was originally discovered from B cell leukemia, hence the name, where Bcl-2 gene locus in chromosome 18 has translocated into a very active enhancer region of antibody heavy chain gene in chromosome 14 (Pegoraro et al. 1984). Therefore B cell leukemias overexpress Bcl-2 and avoid apoptosis. The schematic diagram of the function of Bcl-2 family members is displayed (Figure 20). As depicted in the picture, Bax plays a critical role in apoptosis since its function is involved in channel formation in outer mitochondrial membrane for mitochondrial cytochrome c release into cytoplasm which leads to apoptosome formation and thus apoptosis. Since Bcl-2 can inhibit the function of Bax, inhibiting cytochrome c release, the ratio of the Bax and Bcl-2 is tightly regulated and well balanced in normal healthy cells and either down regulation of Bcl-2 or up regulation of Bax level will break the balance and trigger the cell to undergo programmed cell death. The level of Bcl-2 can be up regulated by NF-κB and can be down regulated by p53 whereas the expression of Bax can be increased by p53. Indeed, Hastak et al. showed that the ratio of the Bax to Bcl-2 increases when EGCG was added in LNCaP cells (Hastak et al. 2003). To investigate the possible changes in expression levels of these pro- and antiapoptotic proteins upon EGCG treatment in ARCaP cells, we performed Western blotting with antibodies against Bax, Bcl-2, and Bcl-XL (Figure 21). In our immunoblot analysis, LNCaP cells exhibited decrease in Bcl-2 expression and increase in Bax level, which correlates well with the changes in NF-κB and p53, resulting in shift of the ratio towards cell death as expected. Surprisingly, in ARCaP cells, the level of Bcl-2 was augmented whereas Bax expression level stayed relatively unchanged. This might be due to the increase in the phosphorylation of p65 subunit of NF-κB since the level of Bcl-2 is positively regulated by NF-κB as shown by Catz and Johnson, that in prostate cancer cells, NF-κB directly binds to the promoter and activates the transcription of Bcl- 2 (Catz and Johnson 2001). The relatively same level of Bax could be explained by the lack of p53 in ARCaP cell line.

41 The density of Bax and Bcl-2 bands in Western blot was measured and the Bax/Bcl-2 ratio was calculated (Figure 22 and Table 12, see appendix). As depicted in graph, the Bax/Bcl- 2 ratio in ARCaP cells decreased from 1 to 0.92, 0.73, and 0.62 upon 0, 25, 50, 100 µM of EGCG treatment, respectively. On the other hand, the ratio increased from 1 to 1.14, 2.05, and 2.29 in LNCaP cells upon EGCG treatment. The immunoblot of Bcl-XL, another prosurvival factor in the Bcl-2 family which has been known to possess an important role in prostate cancer cell survival, is also shown. As shown in the immunoblot, the addition of EGCG did not change the level of Bcl-XL in ARCaP cells but 100 µM of EGCG down regulated this prosurvival factor in LNCaP cells. These results suggest that proapoptotic and antiapoptotic proteins of the Bcl-2 family play an important function in deciding the fate of these prostate cancer cell lines upon EGCG treatment. Several lines of evidence suggest that NF-κB can activate the antiapoptotic proteins such as Bcl-2 and Bcl-XL (Tamatani et al. 1999, Chen et al. 2000) and also that Bcl-2 can activate NF-κB through degradation of the NF-κB inhibitor Iκ-B (de Moissac et al. 1998 and 1999). Bcl- 2 is localized on the outer membrane of mitochondria and blocks the cytochrome c release by preventing Bax activation thereby blocking the channel formation. In fact, several studies show that the ratio of Bax/Bcl-2 is an important factor in determining the cell’s fate whether to survive or undergo apoptosis (Korsmeyer et al. 1993, Raisova et al. 2001) and many studies suggest that Bcl-2 confers resistance to apoptosis on prostate cancer cells (Huang et al. 1998, Mackey et al. 1998). Thus the increase of both the levels of Bcl-2 and NF-κB activation that were observed in ARCaP cells may be responsible for the protection from EGCG.

EGCG Does Not Change the Level of p21/CIP1/WAF1 and CDKs in ARCaP Cells but the Level is Affected in LNCaP Cells in Favor of Apoptosis

The regulation of cell cycle is controlled by a set of cyclin-dependent kinases (CDKs). The activity of CDK-cyclin pairs can be negatively regulated by cyclin-dependent kinase inhibitors (CDKIs) including p21/CIP1/WAF1. p21/CIP1(CDK-interaction protein 1)/WAF1 (wild-type p53-activated fragment 1) is a universal cyclin-dependent kinase inhibitor and can suppress the cyclin-CDK complexes by forming inactive trimeric complexes which block the kinase activity of the CDK. The schematic diagram of the function of p21/CIP1/WAF1 is

42 presented (Figure 23). As shown in the picture, cyclin/CDK complexes phosphorylate retinoblastoma (Rb) proteins in G1 phase of the cell cycle thereby releasing the transcription factor E2F from Rb. The free E2F transcribes proteins that are required for DNA synthesis, which allows the cells to enter the S phase. p21/CIP1/WAF1 can block cell cycle progression through inhibiting the phosphorylation of Rb by cyclin/CDK complex. Since EGCG treated LNCaP cells showed increase in p53 activation and concomitant increased level of p21/CIP1/WAF1 (Hastak et al. 2003) and down regulation of the protein expression of CDK2, CDK4, and CDK6 that cause cell cycle arrest which ultimately leads to apoptotic cell death (Gupta et al. 2003), we hypothesized that p53 null ARCaP cells may show no change in p21/CIP1/WAF1 level conferring escape from cell cycle arrest on ARCaP cells. To test this hypothesis, we treated the two prostate cancer cell lines with different concentrations of EGCG for 72 hrs and employed Western blot analysis with antibodies against p21/CIP1/WAF1 and CDKs 2, 4, 6 (Figure 24). Our results show that wild type p53 bearing LNCaP cells showed marked increase in p21/CIP1/WAF1 level and slight decrease in all three CDKs tested. The densitometry measurements of p21/CIP1/WAF1 level compared to that of β-actin revealed that the level of CDK inhibitor p21/CIP1/WAF1 increased from 1 to 1.13, 2.07 and to 3.37 when treated with 0, 25, 50, 100 µM of EGCG, respectively. In ARCaP cells, the level of p21/CIP1/WAF1 and the CDKs remained unchanged. The level of p21/CIP1/WAF1 is also regulated by p53 but recent studies indicate that p21/CIP1/WAF1 can be regulated by p53-independent pathways (Aliouat-Denis et al. 2005, Agarwal et al. 2002). The p21/CIP1/WAF1 protein plays an important role in growth suppression in prostate cancer cell lines (Sugibayashi et al. 2002) and in apoptosis (Wu et al. 2002). In fact, DU145 prostate cancer cell line carries mutant p53 but the level of p21/CIP1/WAF1 was increased during the EGCG mediated growth inhibition and apoptosis (Gupta et al. 2000). Since the ablation of either p21/CIP1/WAF1 or Bax prevented prostate cancer cells from EGCG induced apoptosis (Hastak et al. 2005), the unaffected level of p21/CIP1/WAF1 in ARCaP cells may explain the resistance of this cell line to EGCG. However, in our studies of apoptosis of ARCaP cells upon topoisomerase II inhibitor etoposide (VP16) treatment, etoposide suppressed the growth of ARCaP cells significantly (Figure 25 and Table 13, see appendix) and induced apoptosis as confirmed by DNA fragmentation assay (Figure 26 A) and Cell Death Detection Assay PLUS (Figure 26 B). In

43 experiment utilizing Cell Death Detection Assay PLUS, the Enrichment Factor increased from 1 to 2.09 and 8.92 when treated with 0, 5, and 50 µM of etoposide, respectively. Western blot experiments of etoposide treated ARCaP cell lysates revealed the cleavage and thus activation of caspase 3, and cleavage and resulting inactivation of PARP showing that the cells underwent apoptosis upon the treatment (Figure 27). Moreover, the level of p21/CIP1/WAF1 was clearly increased dose dependently when treated with etoposide (Figure 27) suggesting that the response of ARCaP cells to these two apoptotic stimuli are mediated via different pathways. Since ARCaP cells don’t possess p53 due the mutation, the p21/CIP1/WAF1 expression level might be regulated by p53-independent pathway. According to Howell’s study, the factors that may contribute to resistance to apoptosis in prostate cancer cells include increased Bcl-2, down regulation of Bax, and the loss of p53 (Howell 2000) all of which were observed in ARCaP cells.

EGCG Affects the Activation of MAPKs Towards Cell Survival in ARCaP Cells but Favors Cell Death in LNCaP Cells

Mitogen activated protein kinase (MAPK) family is composed of several kinases including Akt, p38, c-jun NH2-terminal kinase (JNK) and transduces signals from the cell plasma membrane to the nucleus and thus contribute to a wide spectrum of cellular processes. Akt, also called protein kinase B (PKB), is the most important downstream effector of phosphatidylinositol 3-kinase (PI3-K). Once activated by phosphorylation, Akt can promote cell survival through inhibiting proapoptotic proteins such as Bad and apoptosis signal-regulating kinase 1 (ASK1), and cell cycle inhibitor p21/CIP1/WAF1 (Zhou et al. 2001). Recent research revealed that EGCG decreases the level of phosphorylated form of Akt without affecting the basal level of Akt in both LNCaP and DU145 prostate cell lines (Siddiqui et al. 2004). We tested whether EGCG affects the activation of Akt in ARCaP cells (Figure 28). As shown in Western blots in two top panels, LNCaP and ARCaP cells showed similar levels of total Akt expression whereas phosphorylated, thus activated level of Akt is much higher in control LNCaP cells. This might due the mutation in phosphatase and tensin homologue deleted on chromosome 10 (PTEN) gene in LNCaP cells whose gene product is known to be a negative regulator of Akt activation. But when EGCG was added into the culture, the level of active Akt dropped in LNCaP cells especially when 100 µM of EGCG was treated. In ARCaP cells

44 however, the phosphorylated level of Akt relatively stayed the same with minor fluctuations. These results indicate that the activation of Akt is important in these prostate cancer cell lines and that ARCaP cells might have distinct set of signaling pathway which keeps Akt to its active state and thus avoid from the EGCG induced programmed cell death. Recently, Graff et al. have demonstrated that Akt can contribute to the progression of prostate cancer through dramatically accelerating tumor growth (Graff et al. 2000). Akt can inhibit apoptosis and facilitate the cell survival in many ways including phosphorylation and inactivation of procaspase 9 (Cardone et al. 1998). Akt can also activate the Iκ-B kinase (IKK) by phosphorylation to degrade Iκ-B and thus activate NF-κB (Ozes et al. 1999, Romashkova and Makarov 1999) thereby expressing prosurvival genes. Previously, it was reported that Akt plays a critical role in prostate cancer cell survival upon apoptotic stimuli (Nesterov et al. 2001, Tanaka et al. 2003). While our immunoblot data confirmed previous result that EGCG can inhibit the activation of Akt in LNCaP cells (Siddiqui et al. 2004), the activation of Akt in ARCaP cells was not affected by EGCG. Prostate cancer develops from androgen dependent type to androgen independent type and possibly to hormone repressed type when the tumor is more advanced. A recent study showed that long term androgen ablation induced increased resistance to PI3-K/Akt pathway inhibition in prostate cancer cells (Pfeil et al. 2004) which may explain our data on Akt activation in ARCaP cells. Next, we investigated whether EGCG can affect other MAPKs using the same strategy. The p38 MAPK participates in a signaling pathway controlling cellular responses to stresses including inflammatory cytokines and UV light and its activation can be achieved by phosphorylation. Several studies, including the one in LNCaP prostate cancer cell line, have reported that p38 MAPK signal transduction pathway can be proapoptotic (de Zutter et al. 2001, Tanaka et al. 2003, Porras et al. 2004). Also, the activation of p38 MAPK has been known to induce apoptosis in C4 and C4-2 prostate cancer cells (Murillo et al. 2001). In fact, p38 MAPK has been shown to regulate the translocation of proapoptotic protein Bax. Shou et al. have revealed that the activation of p38 MAPK induces translocation of Bax from cytoplasm to mitochondria which leads to cytochrome c release and caspase activation thereby inducing apoptosis (Shou et al. 2003). Here we show, in two Western blots in the mid panel of the figure, that the treatment of EGCG deactivates p38 MAPK slightly in ARCaP cells but not in LNCaP cells (Figure 28). At 25 µM EGCG treatment, there was no change in the level of phospho-p38

45 MAPK but when treated with 50 and 100 µM of EGCG, phospho-p38 MAPK was down regulated in ARCaP cells whereas the total expression level of basal p38 stayed unchanged.

The c-Jun NH2-terminal protein kinase/stress activated protein kinase (JNK/SAPK) can be activated by stresses such as heat shock, osmotic shock, and UV radiation and has been implicated in numerous functions. Recently, JNK is reported to be essential for the growth of wide variety of prostate cancer cell lines and the growth of LNCaP cells was the most JNK dependent among the cell lines tested (Yang et al. 2003). In our immunoblot experiments as shown in the bottom panel of the same figure (Figure 28), the control ARCaP cells showed higher level of basal JNK expression than the control LNCaP cells whereas the phosphorylated, thus activated level of JNK was much higher in control LNCaP cells than the ARCaP counterpart which exhibited no JNK activation. While the treatment of cells with tea catechin EGCG did not affect either total level of JNK expression or the activation of JNK in ARCaP cells, both of these were affected severely in LNCaP cells. EGCG not only abated the total level of JNK expression but also diminished the level of phosphorylated JNK in LNCaP cells in a dose dependent manner that led to a complete abrogation of JNK activation at 100 µM of EGCG treatment. Taken together, these results suggest that stress activated MAPKs p38 and JNK may play important roles in these cell lines in response to EGCG. The role of JNK is diverse and is cell type specific since it is required for apoptosis in some type of cells, and in some other cells, it is reported to be required for proliferation and in some cases, transformation. JNK is reported to play a crucial role in proliferation in T96G human glioblastoma cells whereas antisense of JNK has been shown to exhibit a marked elevation in the expression of p21/CIP1/WAF1 (Potapova et al. 2000). In prostate cancer cells, treatment of athymic mice bearing established xenografts of PC3 prostate cancer cells with antisense of JNK resulted in significant inhibition of tumor growth (Yang et al. 2003). In DU145 prostate cancer cells, JNK promoted survival of the cells through reducing the interaction between caspase 8 and Fas associated death domain (FADD) in Fas mediated apoptosis (Curtin and Cotter 2004).

EGCG Confers Stronger ECM Adhesion via Collagen in ARCaP Cells whereas the Adhesion to ECM Proteins is Significantly Weakened in LNCaP Cells

46 When performing the cell proliferation assay, we treated the cells with trypsin to detach and thus be able to count the number of cells. During this process, we observed that ARCaP cells became noticeably difficult to detach them from the tissue culture dish when treated with EGCG. This unexpected and extraordinary characteristic of ARCaP cells was EGCG dose dependent. To show this, we performed simple adhesion assay using trypsin. As described in Cell Adhesion Assay in Chapter 2, trypsin was added after the cell culture with or without 100 µΜ of EGCG for 72 hrs. After incubating for the indicated period, all solutions including the fully detached cells were removed carefully and the pictures of cells were taken. As shown, ARCaP control cells detach easily upon trypsin treatment (Figure 29, left panel). In EGCG treated cells, it took approximately 4 times longer incubation time with trypsin to obtain the same degree of detachment (Figure 29, right panel). It took 5 mins in control cells whereas 20 mins were required in treated cells to achieve the same extent of detachment. Similarly, it took 40 mins in treated cells to make the cells become as loose as control cells which were incubated for just 10 mins with the same amount of trypsin. In LNCaP cells, the morphology changed quickly in control cells upon the addition of trypsin and displayed features somewhat similar to floated aggregates (Figure 30, left panel). Since EGCG is an apoptosis inducer in LNCaP cells, it facilitated cells from detaching from each other and from the tissue culture plate as expected (Figure 30, right panel). After incubating EGCG treated LNCaP cells with trypsin, most of the cells detached in 5 mins and at 10 mins, all cells were detached completely. The distinguishing feature of ARCaP cells on adhesion was quite striking since EGCG showed totally the opposite effect in LNCaP cells. For further scientific analysis of this unique feature of ARCaP cells, we employed Extracellular Matrix (ECM) Adhesion assay and the results are presented. Overall, ARCaP cells exhibited endogenously higher attachment to all ECM proteins tested than LNCaP cells except to fibronectin and vitronectin (Figures 31, 32 and Tables 14, 15, see appendix). Control ARCaP cells showed OD540 = 0.904 on fibronectin whereas control LNCaP cells displayed OD540 = 1.459 to the same protein. In case of vitronectin, both ARCaP and LNCaP cell lines recorded similar optical density at 1.608 and 1.661, respectively. ARCaP cells, when treated with 100 µM of EGCG, increased attachment to types I, II, IV collagen, which were 25.1 %, 27.1 %, and 21.7 % up regulation, respectively, with statistic significance when compared to the nontreated cells. On the other hand, the capability of ARCaP cells to adhere onto fibronectin was curtailed

47 to OD540 = 0.418 upon EGCG treatment which was 53.8 % down regulation when compared to their nontreated counterpart. The attachment of EGCG treated ARCaP cells to extracellular matrix proteins laminin, tenascin, and vitronectin changed slightly but was negligible (Figure 31 and Tables 14, 16, see appendix). On the contrary, LNCaP cells showed diminished adhesion capability to all ECM proteins tested upon EGCG treatment. In LNCaP cells, EGCG induced 14.3, 35.0, and 44.8 % detachment to types I, II, and IV collagen, respectively. Adhesion to fibronectin, the only protein that EGCG treated ARCaP cells showed significant loss of adherence to, also decreased in LNCaP cells to a similar degree (41.7 % down regulation) when treated with EGCG. LNCaP cells lost nearly half (46.5 % down regulation) of the attachment to laminin upon EGCG treatment. In case of tenascin, treating LNCaP cells with the tea polyphenol reduced the adhesion from OD540 of 0.674 to 0.167 which is an immense 75.2 % decrease. Attachment to vitronectin also decreased noticeably (30.1 % down regulation) in LNCaP cells when treated with the tea catechin (Figure 32 and Tables 15, 16, see appendix). Taken together, the adherence to types I, II, IV collagen was significantly increased in ARCaP cells but was decreased in LNCaP cells when EGCG was treated. While attachment to fibronectin was down regulated in both cell lines, adhesion to laminin, tenascin, and to vitronectin remained relatively unchanged in ARCaP cells but diminished considerably in LNCaP cells when treated with EGCG. These results indicate that all ECM proteins tested in our experiment except fibronectin confers stronger adherence on ARCaP cells upon the tea catechin EGCG treatment. In our ECM adhesion assay, EGCG treated ARCaP cells gained stronger adherence to collagens whereas the adhesion of LNCaP cells was diminished in all ECM proteins tested including collagens, fibronectin, laminin, tenascin, and vitronectin. In fact, growing evidence supports the role of ECM environment in cell growth (Boudreau et al. 1995 and 1996). Vitronectin has been shown to decrease microvascular endothelial cell apoptosis (Isik et al. 1998) and laminin has been reported to protect Schwann cells from apoptosis which was mediated via PI3-K/Akt pathway (Yu et al. 2005). Since ARCaP cells did not lose adherence to these two adhesion proteins in our assay and LNCaP cells significantly did, our results suggest that laminin and vitronectin might provide environmental cues to these cell lines that may be involved in determining the cell’s fate.

48 Tea Catechin EGCG Renders ARCaP Cells Capable of Facilitated Anchorage-Independent Growth

One of the hallmarks of cancer is that the neoplastic cells lose contact inhibition and gain anchorage independence, allowing the cells to grow and form colonies in semi solid agar media. The anchorage-independent growth assay in soft agar is a valuable in vitro assay to evaluate the cellular transformation which correlates quite well with in vivo carcinogenesis. It has been reported that EGCG suppresses the colony forming ability of NF639 breast cancer cells (Pianetti et al. 2002) and of Hs-294T and A-375 melanoma cells (Nihal et al. 2005) in soft agar media. To assess the effect of EGCG on colony forming ability of prostate cancer cells, we performed anchorage-independent growth assay of LNCaP and ARCaP cells with different doses of EGCG treatment. Here we show that EGCG inhibits the anchorage-independent growth of LNCaP cells completely when treated with 100 µM of EGCG but shockingly enough, the addition of EGCG expedited the colony formation of ARCaP cells on the semi solid media. As expected, the untreated LNCaP cells started forming colonies in the semi solid media couple of days after seeding and formed 4115.0 colonies · 51.07 (SEM) over 14 day period. When 25 µM of EGCG was added, that number decreased to 2988.67 ·G100.98 and the addition of 50 µM EGCG resulted in 334.33 colonies ·G11.46 which is 92 % decrease when compared to the untreated control. One feature to note is that, as the concentration of EGCG got increased, the less number of colonies were formed but the size of each colony grew bigger as can be seen in the picture. Finally, 100 µM of EGCG totally abrogated LNCaP cells from forming colonies in the agar gel since not a single colony was formed at the end of 14 day culture (Figures 33, 35 B and Table 17, see appendix). On the other hand, in ARCaP cells, untreated control cells formed least number of colonies at 673.67 ·G33.42. This is much lower than that of the untreated LNCaP cells and is quite unexpected since ARCaP cell line is known to be more aggressive and metastatic than the LNCaP cell line. Also ARCaP cells formed much smaller colonies when compared to LNCaP cells. The addition of 25 µM of EGCG let ARCaP cells form 2888.0 colonies · 72.47 whereas 50 µM EGCG treatment resulted in 3632.33 colonies ·G184.23. When treated with maximum dose of this green tea catechin, over 4200 colonies were formed in all plates we triplicated with

49 on average 4526.33 colonies ·G218.54 which is an enormous 571.89 % increase when compared to control (Figures 34, 35 B and Table 17, see appendix). The P values for ARCaP and LNCaP cells were 4.92E-7 and 5.02E-11, respectively. These extremely low probability values indicate that there are highly significant differences between the treatments in both cell lines. An enlarged picture of a single colony from LNCaP cells growing in semi solid agar media is presented for reference (Figure 35 A). The most striking result of this study is that the ARCaP cells form more colonies on agar plate when treated with EGCG. As mentioned earlier, EGCG successfully inhibited the formation of colonies in breast cancer and skin cancer system. This is the first colony formation assay that was performed with EGCG treated prostate cancer cells and also the first report that observed the increase in number of colonies in any system when treated with EGCG. The loss of colony forming ability in LNCaP cells when treated with EGCG could be explained by the induction of intensive apoptosis upon EGCG treatment as shown before early in this Chapter. Also, the total abrogation of JNK activation in EGCG treated LNCaP cells might explain, at least in part, the phenomenon, since the activity of JNK has been reported to be involved in colony formation. In A549 human lung cancer cells, colony formation in soft agar was completely inhibited by the antisense of JNK (Bost et al. 1999). In parallel to this observation, studies on NCI-H82 human lung cancer cells revealed that JNK is required not only for transformation but also for anchorage-independent growth (Xiao and Lang 2000). But since ARCaP cells did not show JNK activation whether treated with EGCG or not, this could not explain the observation that ARCaP cells form more colonies when treated with the tea catechin. The mechanism of anchorage-independent growth is not exactly understood. But it has been suggested that PI3-K/Akt pathway plays a crucial role (Wang 2004). Studies with small cell lung cancer cell line and with liver cancer cell line indicate that PI3-K/Akt activation mediates the anchorage-independent growth of these cells (Moore et al. 1998, Nakanishi et al. 2002). Therefore, it is plausible to speculate that the ARCaP cell’s ability to maintain the Akt activity might aid ARCaP cells to form colonies in soft agar despite the presence of EGCG. However, this still cannot explain the actual increase in number of colonies in EGCG dose dependent manner. Identification and direct evidence for this phenomenon awaits further study.

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0hr, - 0hr, + 24hr, - 24hr, + 48hr, - 48hr, + R1881

Figure 8. The effect of R1881 on the growth of ARCaP cells. Initially 50,000 cells were seeded. All samples were triplicated and the data are presented as mean ·Gstandard error of the mean (SEM). Statistical analysis was performed by t-test between the samples with same period of culture with or without R1881 treatment. P values less than 0.05 are considered significantly different. * represents P < 0.05 and ** designates P < 0.01.

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Figure 9. The effect of green tea polyphenol EGCG on the growth of LNCaP human prostate cancer cells. After 24 hrs of initial seeding of 100,000 cells, media were refreshed and different doses of EGCG were added (0 hr) and cultured for 72 hrs. At different time points, the number of viable cells were counted using trypan blue exclusion assay. Data shown are mean ·GSEM of three independent experiments.

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Figure 10. The effect of green tea polyphenol EGCG on the growth of ARCaP human prostate cancer cells. After 24 hrs of initial seeding of 100,000 cells, media were refreshed and different doses of EGCG were added (0 hr) and cultured for 72 hrs. At different time points, the number of viable cells were counted using trypan blue exclusion assay. Data shown are mean ·GSEM of three independent experiments.

Figure 11. Morphological changes upon EGCG addition in LNCaP cells. Cells were seeded in 100 mm dishes and cultured. After 24 hrs, media was discarded and fresh media with indicated doses of EGCG were added and cells were grown for 72 hrs. Cells were visualized under light microscope (100x) and pictures of cells were taken. Note the circular, floating LNCaP cells upon EGCG treatment.

Figure 12. Morphological changes upon EGCG addition in ARCaP cells. Cells were seeded in 100 mm dishes and cultured. After 24 hrs, media was discarded and fresh media with indicated doses of EGCG were added and cells were grown for 72 hrs. Cells were visualized under light microscope (100x) and pictures of cells were taken.

10 Enrichment Factor Enrichment

0 02550100 EGCG (microM)

ARCaP LNCaP

Figure 13. The extent of apoptosis determined by Cell Death Detection Assay PLUS. Cells were incubated with indicated doses of EGCG for 72 hrs. Cell cytoplasm was obtained according to the manufacturer’s protocol and was used for the assay. A: The image of actual color development after adding the substrate is displayed. B: The intensity of color on panel A was measured and shown. The ordinate ‘Enrichment Factor’ represents the enrichment of nucleosomes in the cytoplasm of the apoptotic cells. The data shown are average ·GSEM. ANOVA was used to calculate the P value and to determine the statistical significance. P values were 0.2080 for ARCaP and 6.58E-9 for LNCaP.

Figure 14. The extent of apoptosis determined by DNA fragmentation assay. The DNA was extracted after 72 hrs of culture with varying amounts of the tea catechin and resolved over 1.5 % agarose gel followed by visualization of bands using UV transilluminator. The DNA MW markers are obtained from Promega and the sizes of the markers shown in the picture are 10, 8, 6, 5, 4, 3, 2.5, 2, 1.5, and 1 kb from the top, respectively.

Figure 15. The extent of ARCaP cell apoptosis determined by Hoechst staining. The cells were fixed and stained after 72 hrs of EGCG treatment and same number of cells from each sample was mounted on a slide glass for morphological analysis of nucleus.

Figure 16. The extent of LNCaP cell apoptosis determined by Hoechst staining. The cells were fixed and stained after 72 hrs of EGCG treatment and same number of cells from each sample was mounted on a slide glass for morphological analysis of nucleus. Condensed chromatin and fragmented nuclei due to apoptosis are indicated with arrows.

Figure 17. Immunoblot results with caspase 3 and PARP specific antibodies.

Figure 18. Immunoblot results with p53 specific antibody. A: Western blot of prostate cancer cell lines with p53 specific antibody. 25 µg of total protein was used and the antibody against β- actin was used as loading control. B: Western blot of cytoplasmic and nuclear fraction of LNCaP cell lysates with p53 specific antibody. The subcellular fractionation was performed with NE- PER Nuclear and Cytoplsamic Extraction Reagents. 30 µg of each fraction was used.

Figure 19. Immunoblot results with p65, phospho-p65, and Iκ-Bα specific antibodies. 25 µg of total protein was used and the antibody against β-actin was used as loading control.

Figure 20. The role of Bcl-2 family members in mitochondrial outer membrane permeabilization during apoptosis. The small black dots represent cytochrome C (cyt C). C3; caspase 3, C9; caspase 9, endo G; endonuclease G, IAP; inhibitor of apoptosis protin. This image was adopted and modified from Adams 2003.

Figure 21. Immunoblot results with Bax, Bcl-2, and Bcl-XL specific antibodies. 25 µg of total protein was used and the antibody against β-actin was used as loading control.

2.5

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Ratio of Bax / Bcl-2 / Bax of Ratio 1.0

0.5 ARCaP LNCaP 0 25 50 100 microM EGCG

Figure 22. The changes of Bax/Bcl-2 ratio upon EGCG treatment. To measure the ratio, NIH image analysis program Image J was utilized. The density of Bax and Bcl-2 bands in Western blot were measured and Bax/Bcl-2 ratio was calculated by dividing the density of Bax by the density of Bcl-2. The obtained ratios were normalized to the untreated control of each cell line and plotted in the graph.

Figure 23. The role of p21/CIP1/WAF1 in cell cycle progression and apoptosis. This image was adopted and modified from www.sigma.com.

Figure 24. Immunoblot results with p21, CDK2, CDK4, and CDK6 specific antibodies. 25 µg of total protein was used and the antibody against β-actin was used as loading control.

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Figure 25. The effects of etoposide on growth of ARCaP cells. The number of viable cells were counted using trypan blue exclusion assay after 72 hrs. 50,000 cells were seeded and after 24 hrs, media were refreshed and different doses of EGCG were added (0 hr). Data shown are mean ·G SEM of three independent experiments.

B 10

2 Enrichment Factor

0 0550 Etoposide (microM)

Figure 26. The effects of etoposide on apoptosis of ARCaP cells. A: The extent of apoptosis determined by DNA fragmentation assay. B: The extent of apoptosis determined by Cell Death Detection Assay PLUS.

Figure 27. Immunoblot results of etoposide treated ARCaP cell lysates with caspase 3, PARP, and p21 specific antibodies. 25 µg of total protein was used for blotting.

Figure 28. Immunoblot results with Akt, p38, and JNK specific antibodies. 25 µg of total protein was used for blotting.

Figure 29. Cell detachment assay of ARCaP cells. Cells were cultured with 0 or 100 µΜ οf EGCG for 72 hrs. Cells were then treated with trypsin for indicated period of time. Cells were visualized under light microscope (100x) and pictures of cells were taken.

Figure 30. Cell detachment assay of LNCaP cells. Cells were cultured with 0 or 100 µΜ οf EGCG for 72 hrs. Cells were then treated with trypsin for indicated period of time. Cells were visualized under light microscope (100x) and pictures of cells were taken.

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I V in in in en I n I n c SA g n I s B a ge mi ll la ge na o la La C ol ol Te C C Fibronect Vitronectin ECM Proetins

EGCG - EGCG +

Figure 31. Extracellular matrix adhesion assay of ARCaP cells. Total of 150,000 cells were seeded and 0 or 100 µM of EGCG was added. After incubating the cells for 3 hrs, non-adherent cells were washed out and adherent cells were stained. Finally, the extent of adherence to matrix proteins was measured with microplate reader at 540 nm. All measurements were repeated three times and the mean value with SEM is shown. Statistical analysis was performed by t-test (*P < 0.05 and **P < 0.01).

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II V in in n n c SA s B ge mi la na llagen I La Collagen I Te Col Co Fibronectin Vitronectin ECM Proteins

EGCG - EGCG +

Figure 32. Extracellular matrix adhesion assay of LNCaP cells. Total of 150,000 cells were seeded and 0 or 100 µM of EGCG was added. After incubating the cells for 3 hrs, non-adherent cells were washed out and adherent cells were stained. Finally, the extent of adherence to matrix proteins was measured with microplate reader at 540 nm. Note the scale difference between the ordinates of Figures 31 and 32. All measurements were repeated three times and the mean value with SEM is shown. Statistical analysis was performed by t-test (*P < 0.05 and **P < 0.01).

Figure 33. The anchorage-independent growth assay of LNCaP cells. The assay was performed in soft agar coated 6-well plates with 3x104 cells in each well. Indicated doses of EGCG or same volume of water was added to both top and bottom agar and also to the media and the media was replaced every week with fresh EGCG. Pictures were taken after culturing the cells for two weeks.

Figure 34. The anchorage-independent growth assay of ARCaP cells. The assay was performed in soft agar coated 6-well plates with 3x104 cells in each well. Indicated doses of EGCG or same volume of water was added to both top and bottom agar and also to the media and the media was replaced every week with fresh EGCG. Pictures were taken after culturing the cells for two weeks.

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Figure 35. The number of colonies formed in anchorage-independent growth assay. A: A colony from LNCaP agar plate was chosen and magnified to show that each single colony represents hundreds of cells. B: The number of colonies in the whole well of the all three independent experiments was counted. The data shown are mean values with SEM. ANOVA was used for statistical analysis of EGCG treatment of each cell line as a whole.

CHAPTER 4

SELECTIVE CHANGES IN GENE EXPRESSION IN EGCG TREATED PROSTATE CANCER CELLS

GeneChip Microarray Analysis

To elucidate the genes that are differentially expressed in these two cell lines upon EGCG treatment, we performed GeneChip microarray assay using Affymetrix U133 Plus 2.0 chip which contains a probe set that represents more than 47,000 transcripts that are derived from approximately 39,500 human genes. The procedure is briefly described in a diagram as shown earlier in Chapter 2 (Figure 7). Genes have been filtered to remove any genes that were determined to be absent in both samples by Affymetrix MAS 5 algorithm. The result was expressed as signal intensities and these signal intensities were normalized to the values of the untreated samples of respective cell line to exhibit the ‘fold change’ of expression after the treatment. In ARCaP cells, total of 427 genes were differentially expressed after 100 µM of EGCG treatment (Table 18, see appendix). Among those, 115 genes were up regulated and 312 genes were down regulated. LNCaP cells were more affected by the addition of EGCG since the expression of 4907 genes was influenced (Table 19, see appendix). Of these, total of 1450 genes were up regulated whereas 2457 genes were down regulated by the tea catechin EGCG treatment. In these two tables in the appendix, the fold change of the gene after the treatment with the corresponding GenBank ID number is displayed. Please note that only those of more than 2 fold increase or decrease after EGCG treatment are shown for ARCaP cells whereas for LNCaP cells, only those of more than 3 fold increase or 5 fold decrease are shown. When these genes are categorized according to their function, a series of adhesion related genes are shown to be down regulated in LNCaP cells when treated with EGCG (Table 4). None of these genes’ expression was affected more than 2 fold in ARCaP cells under the same treatment. As shown in the table,

79 the expression levels of several collagen subunits, fibronectin and laminin subunits were reduced.

Also, the expression levels of α2, α4, αv and β1 subunits of the integrin were decreased. The collagen is composed of a triple helix of three α polypeptide chains and each type of collagen is consisted of different α subunits. The basement membrane is mainly composed of type IV collagen and the most abundant form of type IV collagen is [α1(IV)]2 α2(IV), meaning that this collagen has two type IV α1 subunits and a type IV α2 subunit in its triple helix. The other composition includes combination of four different α chains, α3(IV) ~ α6(IV). The expression of

α3(IV) and several α subunits of other collagen types, except α2(VI), were reduced in EGCG treated LNCaP cells. In multicellular organisms, cell to cell and cell to ECM contacts are indispensable for their physiological functions. These interactions are mediated by cell surface proteins including integrins. Integrins are a widely expressed family of cell adhesion receptors through which cells adhere to extracellular matrix or to cells. Integrins are consisted of two noncovalently associated subunits, α and β. With 15 different α subunits and 8 β subunits, integrin can form 23 different heterodimers. The list of integrin heterodimers and their respective ligands are displayed (Table

5). As listed in the table, αv is the most popular α subunit whereas β1 is the most preferred β subunit for the adhesion proteins that were used in our adhesion assay previously.

According to the table, collagen, fibronectin, and laminin prefer β1 as their β subunit of integrin receptor and vitronectin and tenascin usually use αv as their α subunit of integrin receptor. Therefore, it is highly possible that the decrease of EGCG treated LNCaP cells in adhesion to type IV collagen, fibronectin, and laminin, as shown in adhesion assay in Chapter 3, is probably due to the diminished expression of both ligands and the corresponding receptors. Since the expression of the subunits of types I and II collagen was not affected upon EGCG treatment (Table 4), the diminished adherence of LNCaP cells to type I and II collagen that was observed in cell adhesion assay may due to the decreased expression of the corresponding integrin receptors. Similarly, the loss of LNCaP cell adhesion to vitronectin and tenascin in adhesion assay could be explained in the same manner. Taken together, these GeneChip assay results support our data from LNCaP cells that are presented in the previous chapter. However, the increased adhesion of EGCG treated ARCaP cells on types I, II, and IV collagen could not be explained by GeneChip assay results since the expression level of both adhesion related ligands and receptors remain unchained.

80 ADAMs and MMPs

Matrix metalloproteinases (MMPs, also referred to as matrixins) and a disintegrin and metalloproteinases (ADAMs, also referred to as adamalysins and MDCs for metalloprotease / disintegrin / cysteine-rich proteins) are zinc dependent endopeptidases and constitute metzincin superfamily together with astacins and serralysins. Metzincins are characterized by the zinc binding consensus sequence HEBXHXBGBXHZ in the metalloprotease domain. B is a bulky hydrophobic residue and Z is subfamily specific where ADAMs have D, MMPs have S, Serralysins have P, and Astacins have E (Bergers and Coussens 2000). ADAMs are mostly membrane proteins except ADAMTS (ADAM-thrombospondin motifs) proteins and many ADAM family members are known as “sheddases” since ADAM proteins cleave membrane anchored proteins to release a soluble form of the protein. ADAMs have been implicated in a number of cellular processes such as fertilization, myoblast fusion and specific developmental processes. MMPs were first observed in tadpole tail of metamorphosing frogs (Gross and Lapiere 1962) which now constitute a family of at least 25 members from vertebrates and invertebrates. MMPs are mostly secreted proteins except membrane type MMPs (MT-MMPs) and can be subdivided into collagenases, gelatinases, stromelysins, and matrilysins. MMPs contribute to both normal and pathological tissue remodeling by degrading the extracellular matrix. As a consequence, MMPs play important roles in migration, angiogenesis, tumor invasion and metastasis. Next, MMPs and ADAMs from the GeneChip assay result were grouped together. In ADAM proteins, ADAM9, ADAM10, ADAMTS3, and ADAMTS19 showed decreased expression whereas ADAM21 exhibited 2.7 fold increase in EGCG treated LNCaP cells (Table 6). Changes in expression level of ADAM proteins were not detected in ARCaP cells under same condition. Interestingly, the expression level of MMP1 (collagenase 1) and MMP3 (stromelysin 1) were increased in LNCaP cells but not in ARCaP cells when treated with 100 µM of EGCG (Figure 36). As shown in graph, the expression level of MMP1 (GenBank ID: NM_002421) augmented to 1362 % and that of MMP3 (GenBank ID: NM_002422) boosted up to 922 % when treated with EGCG in LNCaP cells.

81 Effects of Tea Catechins in Growth of LNCaP and ARCaP Cells

To test whether other tea polyphenols such as EC, ECG, and EGC have the same effect on MMP1 and MMP3 expression, we treated both LNCaP and ARCaP cells with each polyphenol. Before measuring the expression levels of MMPs, we first performed cell growth assay to compare the growth inhibition effects of all 4 polyphenols in both cell lines and the results are displayed. LNCaP cells were sensitive to all tea catechins tested (Figure 37 and Table 20, see appendix). LNCaP cells were least sensitive to EC followed by ECG which was slightly more toxic to the cells. The statistical significance was measured by Single Factor ANOVA. The calculated P values were 0.0185 and 1.73E-06 for EC and ECG, respectively. The two gallocatechins, EGC (P = 9.36E-08) and EGCG (P = 7.13E-06), were most toxic to LNCaP cells. All measurements displayed very low P values meaning that the results are statistically highly significant. The results presented here indicate that the growth of LNCaP cells is equally highly sensitive to EGC and EGCG. ARCaP cells however, were quite resistant to such catechin treatments except towards ECG treatment (Figure 38 and Table 21, see appendix). Interestingly, the catechin gallate, ECG exhibited high toxicity to these cells whereas the other catechins displayed minor growth inhibitory effects to a similar degree. The calculated P value by Single Factor ANOVA was 0.0343, 0.0037, 0.2799, and 0.2284 for EC, ECG, EGC, and EGCG, respectively. Among the 4 major tea polyphenols, EGCG is shown to be the most potent inhibitor of carcinogenesis and thus is the most studied tea catechin. However, there are some studies that utilized other catechins. In H661 and H1299 human lung cancer cell lines, EGC and EGCG were both effective in inhibiting the growth followed by ECG and then EC (Yang et al. 1998). EGC also has been shown to inhibit cell growth and induce apoptosis through increasing Bax and decreasing Bcl-2 level in human breast cancer cell lines MCF-7 and MDA-MB-231 but not in normal human breast epithelial cells (Vergote et al. 2002). In HCT-116 human colorectal cancer line, ECG was the most effective inducer of apoptosis among the 4 tea catechins (Baek et al. 2004). By contrast, in OECM-1 oral cavity derived cancer cell line, ECG had no effect in inhibiting the cell growth whereas both EGC and EGCG efficiently suppressed the growth of these cancer cells (Lin et al. 2005). In HSC-2 oral cavity derived fibroblast cells, ECG and

82 EGCG were most toxic whereas EGC was moderately toxic and EC being least toxic (Babich et al. 2005) suggesting that the effect of different tea catechins might be cell type specific.

Effects of Tea Catechins in MMP1 and MMP3 Expression by RT-PCR

The semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) assay was performed to measure the changes in mRNA level of MMPs 1 and 3. First, cells were treated with different tea catechins and cultured. Then, total RNA was prepared using TRIzol solution. The RNAs were then reverse transcribed to obtain cDNAs. Finally, PCR was performed with MMP1 and MMP3 specific primers as described in Materials and Methods in Chapter 2. Primers that are specific for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were used for control. As shown in the figure, EC had no effect on the expression of MMPs 1 and 3 in LNCaP cells (Figure 39). ECG induced the increase in expression of MMP1 slightly when treated with 100 µM ECG but the expression of MMP3 was not changed under the same condition. When LNCaP cells were treated with gallocatechins EGC and EGCG, the expression of both MMPs were increased noticeably, especially when treated with EGCG. These results confirm the data that were obtained from GeneChip analysis which was shown previously. The effect of tea catechins on the expression of these MMPs on ARCaP cells was tested. As expected, all 4 catechins did not influence the transcription of MMP1 or MMP3 gene in ARCaP cells (Figure 40). The expression profile of MMPs 1 and 3 in LNCaP cells matches the proliferation assay in that both EGC and EGCG induced suppression of proliferation, and the induction of MMP1 and MMP3 expression. These results suggest that the function of these MMPs might be proapoptotic, at least, in LNCaP cell line. On the other hand, ARCaP cells’ proliferation was most sensitive to ECG treatment as shown in proliferation assay, but ECG treatment did not up regulate the level of MMP1 or MMP3. Together with LNCaP and ARCaP cell lines, the two other popular prostate cancer cell lines, DU145 and PC3, were also tested for any changes in the expression of these MMPs by tea catechins. DU145 and PC3 cell lines are androgen-responsive and are known to be sensitive to EGCG treatment as mentioned earlier. Moreover, HT1080 human fibrosarcoma cell line was also tested. The invasion of HT1080 cells is reported to be strongly suppressed by EGCG treatment

83 (Maeda-Yamamoto et al. 1999) and this cell line is known to produce large amounts of MMPs. RT-PCR of these 3 additional cell lines were performed after different doses of EGCG treatment and the results are displayed (Figure 41). As can be seen from the RT-PCR data, DU145 cells produced more MMP1 mRNA than PC3 whereas PC3 cells expressed more MMP3 than DU145 cells. HT1080 cells expressed ample amount of both MMPs as expected. Overall, ARCaP cells showed highest expression of MMP1 and HT1080 cells ranked the highest in MMP3 expression in all cell lines tested. However, EGCG did not affect the expression of MMP1 or MMP3 in any of these additional cell lines tested. Therefore, it is quite unique that the transcription of MMP1 and MMP3 genes were affected in LNCaP cells by EGC and EGCG.

Effects of Tea Catechins in proMMP1 and MMP3 Expression by ELISA

Since we observed the increase in RNA levels of MMP1 and MMP3 in EGCG treated LNCaP cells by GeneChip analysis and confirmed by RT-PCR, we next measured the MMP proteins that are secreted to the surrounding media. To measure the increase of the secretion of MMPs quantitatively, we performed ELISA assay using Quantikine Human proMMP1 and MMP3 Immunoassay kits as described earlier in Chapter 2. The ELISA kit was available only in forms of proform of MMP1 detection and total MMP3, which includes both proform and active form of MMP3, detection at the time of assay. The culture media were collected after 24 hrs of each tea catechin treatment and were used in the ELISA assay. The average amount of secreted MMPs was calculated from all 12 controls (4 tea catechins x three independent experiments). For both MMPs, ARCaP cells showed higher secretion than the LNCaP cells. LNCaP cells secreted on average of only 0.52 ng of proMMP1 per ml of culture media when not treated with catechins whereas ARCaP cells secreted 1798 ng/ml which is nearly 3500 fold more which confirms our previous RT-PCR data. For MMP3, both cell lines secreted very small amount of total MMP3 when not treated with EGCG. Less than 0.1 ng/ml were detected in media from control LNCaP cells whereas the control ARCaP cells secreted on average of 4.3 ng/ml of active MMP3. This also matches our RT-PCR data since similar degree of intensity was observed in ethidium bromide staining of RNA transcripts of MMPs.

84 Next, the cells were treated with each catechins and the amount of MMPs in the media were measured and displayed as percent of control. As shown, the secretion of proMMP1 did not change significantly when EC or ECG was added into the cell culture. But when LNCaP cells were treated with gallocatechins, EGC and EGCG, the secretion was increased. EGC augmented proMMP1 secretion to 169.93, 261.80, and 337.96 % when treated with 25, 50, and 100 µM of this catechin, respectively, when compared to untreated control cells. EGCG also boosted the secretion of proMMP1 to 178.09, 265.10, and 449.70 % when treated with 25, 50, and 100 µM of EGCG, respectively (Figure 42 and Table 22, see appendix). The statistical significance of the data was calculated with ANOVA. The P values were 0.8952, 0.8055, 0.0007, and 0.0018 for EC, ECG, EGC, and EGCG treatment, respectively, indicating that the increase in proMMP1 secretion in EGC and EGCG treated LNCaP cells is statistically significant. On the contrary, ARCaP cells did not show any significant changes in secretion of proMMP1 (Figure 43 and Table 23, see appendix). The level of total MMP3 in the media when cultured with 4 different tea catechins was also measured. The addition of EC or ECG to LNCaP culture affected the amount of total MMP3 slightly but with no statistical significance (P > 0.91). When LNCaP cells were treated with EGC, the total level of MMP3 in culture media aggrandized hugely. 25 µM of EGC treatment resulted in the increase in MMP3 in LNCaP cell culture media to 134.58 % whereas 50 and 100 µM of EGC accelerated the expression and secretion of MMP3 that reached 337.86 and 785.82 % of control, respectively. The ANOVA analysis showed very low P values (P = 2.72E-07) suggesting that the result is highly significant. EGCG also enhanced the production of MMP3 in LNCaP cells. 25 and 50 µM of EGCG increased the production of MMP3 to 177.09 and 400.39 %, respectively. 100 µM of this tea catechin inflated the total MMP3 production dramatically and reached 871.00 % when compared to the untreated control (Figure 44 and Table 24, see appendix). The P value (P < 0.0004) for EGCG treatment calculated by ANOVA showed that this result is highly significant. ARCaP cells however, did not exhibit any significant changes in production of MMP3 upon the treatment of tea catechins (Figure 45 and Table 25, see appendix). Treating ARCaP cells with ECG increased the MMP3 production slightly but there was no statistical significance (P = 0.1718). These results indicate that the increased expression, secretion and/or activation of MMP1 and MMP3 in LNCaP cells after EGC and EGCG treatment is unique and that it may be

85 related to suppression of proliferation and/or apoptosis of LNCaP cells since the profile of cell growth assay matches to that of expression and secretion/activation of MMPs.

The Effect of EGCG on the Expression of MMPs

While there are large body of evidence that EGCG affects expression and/or activity of gelatinases, i.e. MMP2 and MMP9, there are only few articles that reported the effect of EGCG on the expression of MMP1 and MMP3. In human skin, photodamage is thought to be caused by ultraviolet irradiation via degeneration of collagens in dermis and basement membrane. Recently, it was reported that UV irradiation to normal human skin fibroblast resulted in increase of both mRNA and protein levels of MMP1 and that EGCG decreased this UV induced MMP1 expression (Song et al. 2004). Another study showed that both MMP1 and MMP3 protein levels were decreased when cultured fibroblasts were exposed to EGCG (Lee et al. 2005). Also EGCG suppressed collagen production and collagenase activity, and reduced the transcription of MMP1 in hepatic cells (Nakamuta et al. 2005). In human tendon fibroblasts, interleukin-1 beta (IL-1β) stimulated the expression of MMP1, MMP3 and MMP13 and this induction of MMPs were shown to be suppressed by EGCG (Ahmed S et al. 2004, Corps et al. 2004).

The Role of MMPs and ECM in Programmed Cell Death

Even though the MMPs have long been thought to be associated with tumor invasion and metastasis due to their role in ECM, there are several reports that show the apoptotic ability of MMPs. In fact, the truth that the MMPs were first observed in tadpole tail of metamorphosing frogs already suggests the involvement of MMPs with apoptosis since metamorphosis itself is implicated with severe programmed cell death. MMP1 has been shown to be related to apoptosis. When lung fibroblasts are treated with pulmonary surfactant, both apoptosis and MMP1 expression were induced (Vazquez de Lara et al. 2000). Recently, Saunders and colleagues have revealed that the activation of MMP1 induces type I collagen degradation, capillary tubular network collapse, regression, and endothelial cell apoptosis (Saunders et al. 2005). A possible mechanism of MMP1 induced apoptosis has been suggested. A study revealed that MMP1 can act on cell surface receptors that affect cell survival (Conant et al. 2004). In this

86 study, MMP1 has been shown to interact with integrin α2β1 that is expressed in human neurons which leads to apoptosis. The authors have observed that MMP1 interaction with integrin α2β1 results in dephosphorylation, and thus inactivation, of cell survival protein Akt whose inactivation has been linked to apoptosis and that MMP1 induced dephosphorylation of Akt was inhibited by blocking antibody to integrin α2 subunit. Moreover, MMP1 inactivation of Akt was not inhibited either by MMP1 inhibitor or catalytic mutant form of proMMP1, suggesting that this unique function of MMP1 is proteolytic activity independent. Therefore, it is plausible to speculate that increased MMP1 expression in EGCG treated LNCaP cells might play similar roles as it did in human neurons. However, the expression of both α2 and β1 integrin subunits were diminished more than 2 fold in LNCaP cells when treated with EGCG as shown in our previous GeneChip assay (Table 4). MMP3 also has been reported to be involved in apoptosis. In active MMP3 expressing transgenic mouse model, the introduction of a single MMP gene, MMP3 gene, was insufficient for the progression of mammary tumors induced by carcinogen 7, 12-dimethyl benzanthracene (DMBA). Rather, the expression of MMP3 not only reduced the number of mice developing mammary tumors but also showed 4 fold increase in the number of apoptotic cells in MMP3 transgenic mice (Witty et al. 1995). Similarly, Boudreau and colleagues showed that the programmed cell death can be induced by the overexpression of MMP3 in CID-9 mammary epithelial cells and that the metalloproteinase inhibitor GM6001 can inhibit such cell death suggesting that the apoptosis depends on the proteolytic activity of MMP3 (Boudreau et al. 1995). They also revealed that the expression of MMP3 in transgenic mice resulted in apoptosis in 10 ~ 15 % of CID-9 cells suggesting that ECM degradation by MMP3 leads to apoptosis both in vitro and in vivo. Previously, another stromelysin, MMP11 has been shown to be up regulated with the onset of apoptosis during metamorphosis including tadpole tail resorption in Xenopus laevis (Patterton et al. 1995). MMP2 and MMP3 showed similar pattern. The apoptosis induced by degradation of ECM may be a result of the loss of survival factors such as growth factors and/or the release of apoptotic signals upon the destruction of ECM. However, the exact mechanism of MMP induced apoptosis is not well understood. The potential role of ECM as an actual survival factor has been studied. When human umbilical endothelial cells (HUVEC) were cultured in plates coated with heat denatured BSA

87 and agarose, the attachment was completely blocked and cells underwent apoptosis and displayed 4 fold higher DNA fragmentation than the control cells that were grown in the regular tissue culture plates (Meredith et al. 1993). When the cells were grown in bacteriological plastic dishes coated with BSA, cells underwent apoptosis rapidly and showed nuclear fragmentation in nearly 70 % of the population. But when the cells were cultured in same dishes coated with ECM component fibronectin, the level of programmed cell death decreased to about 5 % suggesting that ECM can function as a survival factor. The requirement of ECM for cell survival was observed not only in endothelial cells but also in Caco-2 human gut epithelial cells. Moreover, the cells incubated in suspension were identical both morphologically and biochemically to cells induced to undergo apoptosis due to serum starvation supporting the idea that ECM can function as a survival factor (Meredith et al. 1993). Similarly, ECM has been shown to be essential for the survival of some types of cells.

For example, inhibition of ECM receptor integrin αvβ3 can induce apoptosis (Brooks et al. 1994).

In parallel to this observation, integrin αv deficient human melanoma cell line M21 progressed rapidly to apoptosis and transfection of these cells with αv integrin gene prevented the cell suicide (Montgomery et al. 1994). Another article showed that endothelial cells treated with RGD peptide underwent apoptosis (Modlich et al. 1996). RGD (Arg-Gly-Asp) is a tripeptide motif found in many ECM proteins including collagen, fibronectin, vitronectin and laminin, and is the site where integrin receptor binds. The idea that ECM can serve as survival factor was proved in another system. In rat mesangial cells, ECM component type IV collagen and laminin protected the cells from serum starvation and etoposide induced apoptosis (Mooney et al. 1999).

In this study, it was also proved that this effect was β1 integrin dependent since blocking antibody to β1 integrin abrogates the protective effect of type IV collagen and laminin. MMPs may contribute to apoptosis by additional route. In fact, MMPs are capable of cleaving membrane anchored tumor necrosis factor-α (TNF-α) precursor thereby releasing the soluble and mature TNF-α which can cause apoptosis in cells expressing TNF-α receptor (McGeehan et al. 1994, Gearing et al. 1994). Also, MMPs are shown to be able to release membrane bound Fas ligand (FasL) which then can bind to its receptor Fas and induce apoptosis (Kayagaki et al. 1995). It was reported that membrane bound FasL can be proteolytically cleaved by MMP7 that results in generation of soluble Fas ligand. When human embryonic kidney 293 epithelial cells were exposed to the conditioned media from FasL expressing splenocytes treated

88 with MMP7, the cells showed 3.7 fold increase in apoptosis compared with control media treated population (Powell et al. 1999). Also, in the same article, MMP7 null mice displayed 67 % reduction in apoptosis supporting the role of MMP7 in apoptosis. These studies indicate that MMPs may contribute to apoptosis at least in two different ways: (1) degradation of ECM and (2) releasing the death ligands such as TNF-α and FasL. Therefore, it is possible that EGCG treated LNCaP cells underwent apoptosis due to (1) up regulated expression of MMPs (Figures 36, 39, 42, 44), (2) down regulation of ECM proteins such as fibronectin, laminin, and collagen whose expression decreased 2.0 ~ 10.4 fold when treated with EGCG (Table 4), (3) down regulation of integrin receptor proteins (Table 4) that transduce extracellular cues into the cells, and (4) decrease in adhesion to ECM proteins (Figure 32, see Chapter 3).

Table 4. Changes of expression levels of adhesion-related proteins in LNCaP cells by GeneChip assay. ARCaP cells did not show any changes in the expression of the listed genes upon EGCG treatment. All the signal intensities were normalized to the values of the control chips so that the gene expression level in control chip is 1 whereas experimental chip gives greater, equal or less than 1. The value of the experimental chip was expressed and displayed as ‘Fold Change’ and shows the level change when compared to control. Only those of more than 2 fold change after EGCG treatment are shown.

Fold Description GenBank ID Change

integrin, α2 N95414 -2.5

integrin, α4 L12002 -4.3

integrin, α4 BG532690 -4.5

integrin, α4 NM_000885 -7.1

integrin, αv AI093579 -5.0

integrin, β1 NM_033669 -2.0

integrin, β1 NM_133376 -2.2 fibronectin W73431 -2.0

laminin, β1 N30158 -10.4

laminin, α4 NM_002290 -2.6

laminin, α4 U77706 -2.7

collagen, type IV, α3 NM_005713 -2.0

collagen, type IV, α3 BC000102 -2.2

collagen, type IV, α3 AA889952 -2.6

collagen, type VI, α2 AL531750 2.0

collagen, type VIII, α1 BE877796 -3.9

collagen, type IX, α1 AI286254 -3.0

collagen, type XII, α1 AA788946 -2.3

collagen, type XXVII, α1 AK021957 -2.1

collagen, type XXVII, α1 AI949136 -10.1

Table 5. Combinations of integrin subunits and their respective ligands. Note that not all subunits are shown. Also, not all ligands are shown. Only the matrix proteins that were used in the Cell Adhesion Assay are shown. The subunits whose expression was decreased upon EGCG treatment in LNCaP cells are marked with an asterisk*. The table was adopted and modified from Eble 1997. Col = collagen, Fn = fibronectin, Ln = laminin, Tn = tenascin, Vn = vitronectin.

* subunits β1 β3 β4 β5 β6 β7 β8

α1 Col, Ln * α2 Col, Ln

α3 Ln, Fn * α4 Fn Fn

α5 Fn

α6 Ln Ln

α7 Ln

α8 Fn, Vn, Tn

αIIb Fn, Vn * αv Fn, Vn Fn, Vn, Tn Vn Fn Vn

Table 6. Changes of expression levels of ADAMs by GeneChip assay in LNCaP cells. ARCaP cells did not show any changes in the expression of the listed genes upon EGCG treatment. All the signal intensities were normalized to the values of the control chips so that the gene expression level in control chip is 1 whereas experimental chip gives greater, equal or less than 1. The value of the experimental chip was expressed and displayed as ‘Fold Change’ and shows the level change when compared to control. Only those of more than 2 fold change after EGCG treatment are shown.

Fold Description GenBank ID Change ADAM9 NM_003816 -3.9 ADAM9 AF495383 -8.6 ADAM10 N51370 -2.8 ADAM10 NM_001110 -3.1 ADAM10 AU135154 -3.7 ADAM21 NM_003813 2.7 ADAMTS3 AB002364 -2.8 ADAMTS19 NM_133638 -12.9

1500 1362

1200 922 900

600

300

Expression (%) Level 100 100 0 MMP1 MMP3

EGCG - EGCG +

Figure 36. Changes of expression levels of MMPs by GeneChip assay in LNCaP cells. The expression level is normalized to control.

120

100

40 Cell (% control)Viability 20

0 EC ECG EGC EGCG

0 25 50 100 microM

Figure 37. The effect of tea catechins on the growth of LNCaP cells. Initially 100,000 cells were seeded in a 6-well plate and cultured. Media were refreshed after 24 hrs and different doses of each catechin were added. After culturing for another 24 hrs, the number of viable cells were counted using trypan blue exclusion assay. Data shown are mean ·GSEM of three independent experiments.

120

100

40 Cell Viability (% control) 20

0 EC ECG EGC EGCG

0 25 50 100 microM

Figure 38. The effect of tea catechins on the growth of ARCaP cells. Initially 100,000 cells were seeded in a 6-well plate and cultured. Media were refreshed after 24 hrs and different doses of each catechin were added. After culturing for another 24 hrs, the number of viable cells were counted using trypan blue exclusion assay. Data shown are mean ·GSEM of three independent experiments.

Figure 39. The effect of tea catechins on the expression of MMP1 and MMP3 mRNA in LNCaP cells. The primers for house keeping gene GAPDH was used as control.

Figure 40. The effect of tea catechins on the expression of MMP1 and MMP3 mRNA in ARCaP cells. The primers for house keeping gene GAPDH was used as control.

Figure 41. The effect of EGCG on the expression of MMP1 and MMP3 mRNA in DU145, PC3, and HT1080 cells. The primers for house keeping gene GAPDH was used as control.

600

500

400

300

200 proMMP1 (% control) 100

0 EC ECG EGC EGCG Tea Catechins

0 25 50 100 microM

Figure 42. The effect of tea catechins on the production of proMMP1 in LNCaP cells. All samples are adjusted to represent 50,000 cells. The data are displayed as % control. Data shown are mean ·GSEM of three independent experiments.

600

500

400

300

200 proMMP1 (% control) 100

0 EC ECG EGC EGCG Tea Catechins

0 25 50 100 microM

Figure 43. The effect of tea catechins on the production of proMMP1 in ARCaP cells. All samples are adjusted to represent 50,000 cells. The data are displayed as % control. Data shown are mean ·GSEM of three independent experiments.

100

1000

800

600

400 MMP3 (%control) 200

0 EC ECG EGC EGCG Tea Catechins

0 25 50 100 microM

Figure 44. The effect of tea catechins on the production of total MMP3 in LNCaP cells. All samples are adjusted to represent 50,000 cells. The data are displayed as % control. Data shown are mean ·GSEM of three independent experiments.

101

1000

800

600

400 MMP3 (%control) 200

0 EC ECG EGC EGCG Tea Catechins

0 25 50 100 microM

Figure 45. The effect of tea catechins on the production of total MMP3 in ARCaP cells. All samples are adjusted to represent 50,000 cells. The data are displayed as % control. Data shown are mean ·GSEM of three independent experiments.

102

CHAPTER 5

CONCLUSIONS AND FUTURE DIRECTIONS

EGCG and Prostate Cancer Chemoprevention

The results from this research indicate that the androgen-repressed subline of prostate cancer cells that represent more aggressive and metastatic tumor is not only resistant to EGCG treatment but also might have acquired unique mechanisms to survive and proliferate upon such apoptotic stimuli. One of the unique characteristics of EGCG in anticancer effects is that EGCG can not only preclude the onset of tumor but also actually reverse the tumor development. Since the ultimate goal of using the chemopreventive agents is to prevent the tumorigenesis, it may be still advantageous to use EGCG as such agent to obviate the onset of cancer in healthy population. The worldwide popular drink, tea, has been receiving great attention during the past decade due to its anticarcinogenic polyphenolic contents. Among various tea polyphenols, EGCG is the one that has been shown to possess the anticancer effects. Prostate cancer has a long dormant period and is typically diagnosed in old male which makes prostate cancer an ideal target for chemoprevention. The accumulating data of many studies on the chemopreventive effects of EGCG indicate that EGCG performs its antitumorigenic effects in prostate cancer cells through shifting the balance between proliferation and apoptosis in favor of apoptosis. Currently, various kinds of agents are being tested to be used as chemopreventive agents to prevent cancer including soy isoflavones and tea polyphenols (Greenwald 2004). Tea has a great potential to be used as chemopreventive agent since it is known to kill the cancer cells without affecting normal counterparts and is generally safe to use. Also tea is inexpensive and can be easily obtained from the local markets. Even though EGCG has such advantages and many investigations reported the signal transduction pathways that are affected by EGCG, there is no clear definitive molecular mechanism of its action that has been reported to date.

103 EGR1 in Apoptosis

We have tested and compared the sensitivity of ARCaP cells to LNCaP cells against EGCG treatment. LNCaP cells were sensitive to EGCG treatment as expected, but surprisingly, ARCaP cells were resistant to such apoptotic stimuli. To find out genes that may be responsible for this characteristic, we performed GeneChip assay. In our GeneChip microarray experiment, total of 427 genes were differentially expressed in ARCaP cells whereas the expression of 4907 genes were affected in LNCaP cells upon EGCG treatment. Of these genes, only 76 genes were common to both cell lines. It would be interesting to test the function and effect of the gene products under EGCG treatment whose expression is shown to be regulated in opposite manner in LNCaP and ARCaP cells when cultured with EGCG. One such example could be early growth response 1 (EGR1). We found that the expression of EGR1 is decreased 3.8 fold after EGCG treatment in ARCaP cell line whereas the expression is increased 4.8 fold in LNCaP cells under the same condition (Table 7). This feature was confirmed by RT-PCR. Using EGR1 specific primers, total of 35 cycles of RT-PCR was performed. The reaction condition is described in Chapter 2 and the information of primers including the sequences is displayed in tables (Tables 2, 3). The result shows that ARCaP cells (ARCaP, 0 µM) have more basal copies of EGR1 than the LNCaP cells (LNCaP, 0 µM) (Figure 46 A). But when treated with 100 µM of EGCG, the expression of EGR1 decreased in ARCaP cells but increased in LNCaP cells. For normalization, the intensity of EGR1 was divided by that of GAPDH. This value from each untreated cell line was used as 100 %. When normalized, there were about 38 % decrease and 150 % increase in EGCG treated ARCaP and LNCaP cells, respectively (Figure 46 B). EGR1, also known as Krox-24, NGFI-A, Zif 268 and TIS 8, is a transcription factor which is consisted of N terminal transcriptional activation domain and C terminal DNA binding domain which harbors zinc finger motifs and a regulatory domain between the two principal domain. EGR1 can specifically recognize the sequence, 5’-GCG(T/G)GGGCG-3’, and can transactivate promoters containing the appropriate cognate sequence. According to Pignatelli and et al., Neuro2A cells exhibited significant increase in apoptosis when EGR1 gene was stably transfected whereas the EGR1 antisense treated cells showed reduction in apoptosis (Pignatelli et al. 2003). Also, the facts that the apoptosis inducing stimuli can up regulate the expression of

104 EGR1 and that the antisense oligomers or dominant negative mutant of EGR1 can prevent cancer cells from apoptosis are well documented (Muthukkumar et al.1995, Ahmed et al. 1996). The proapoptotic EGR1 is involved in apoptosis in three ways: (1) EGR1 can directly activate the transcription of p53 (Nair et al. 1997), (2) EGR1 is also able to induce apoptosis via activating the expression of PTEN (Virolle et al. 2001), which is a negative regulator of Akt, an important cell survival protein, as described earlier in Chapter 3, and (3) recently, the TNF-α and Bax genes are also known to be the targets of EGR1 (Ahmed MM 2004).

Possible Roles of GADD45 and Topoisomerase

Other interesting proteins include the growth arrest and DNA damage inducible gene 45 (GADD45) and type II topoisomerase. As shown in the same table (Table 7), while both α and β types of GADD45 were increased in LNCaP cells, the expression of α form decreased in ARCaP cells upon EGCG treatment. GADD45 is a cell cycle regulator that is involved in G2/M cell cycle checkpoint control (Wang et al. 1999). GADD45 is a nuclear protein and can interact with the cyclin dependent kinase Cdc2 and dissociate Cdc2-cyclin B1 complex which results in G2/M arrest (Zhan et al. 1999). GADD45 encodes a growth inhibitory and in some cell types, proapoptotic protein that is induced by cellular stress including genotoxic stress. Proliferating cell nuclear antigen (PCNA) is a subunit of mammalian DNA polymerase δ and displaces DNA polymerase α with DNA polymerase δ and also enhances the processivity of the latter polymerase during the leading strand synthesis in DNA replication. GADD45 can bind to PCNA and thus is able to inhibit the DNA replication directly (Smith et al. 1994). In MCF-7 breast cancer cell line, troglitazone induced inhibition of proliferation and induction of apoptosis were shown to be directly involved with GADD45 as the expression of GADD45 was increased during the apoptosis and the GADD45 siRNA abrogated troglitazone induced apoptosis (Yin et al. 2004). In fact, the repression of GADD45 has been shown to be essential for cancer cell survival (Zerbini et al. 2004, Zerbini and Libermann 2005). The authors showed that the induction of GADD45 expression in DU145 and PC3 prostate cancer cells resulted in apoptosis whereas inhibition of the gene by siRNA reduced such cell death. These results may help understanding the resistance of ARCaP cells against EGCG treatment since ARCaP cells showed 2.6 fold decrease in GADD45 expression.

105 Topoisomerases are the enzymes that regulate the topology of DNA. Whereas type I topoisomerase relaxes DNA by nicking and ligating one strand of duplex DNA, type II enzyme can cleave and rejoin both strands. These enzymes are essential for cell survival and some studies indicate that EGCG inhibits the activity of topoisomerases (Berger et al. 2001, Suzuki et al. 2001, Sordet et al. 2003). In fact, the study of topoisomerase inhibitors, such as etoposide, as anticancer drugs has become an area of interest over the past few years due to the fact that inhibiting the religation by topoisomerase after the strand break makes topoisomerase a nuclease and blocks further replication of DNA and that this is lethal to the cells (Holden 2001, Topcu 2001). It is interesting to note that LNCaP cells experienced apoptosis and exhibited decreased expression of topoisomerase (Table 7) when treated with EGCG but not in ARCaP cells, whereas the topoisomerase inhibitor etoposide induced apoptosis in ARCaP cells as described in Chapter 3 previously, suggesting a role of this enzyme in prostate cancer cell death. The increased level of CDK inhibitor p21/CIP1/WAF1 confirms our previous data and the other up regulated CDK inhibitors, p15 and p16, may aid in suppressing the proliferation of LNCaP cells during EGCG induced apoptosis (Table 7).

Current and Future Studies

When LNCaP cells were treated with EGC or EGCG, the expression of MMP1 and MMP3 were increased which is a very unique feature. Such phenomena were not observed in the two other EGCG sensitive prostate cancer cell lines, DU145 and PC3, upon tea catechin treatment. Possible roles of MMPs in apoptosis in several systems were proposed. But it is much less investigated than the role of MMPs in invasion and metastasis. Therefore, it would be exciting to explore the role of these two MMPs in LNCaP cells with respect to programmed cell death. Our lab’s current research is focused on identifying proteins that are differentially expressed in ARCaP cells in the absence and presence of EGCG utilizing two dimensional gel electrophoresis and mass spectroscopy. In our preliminary experiment, few new spots appeared in two dimensional gel after treated with EGCG in ARCaP cells whose function of those proteins might confer resistance to the tea catechin EGCG. When these results from proteomics are

106 combined with GeneChip microarray data, it may give clues to understand the resistance of ARCaP cells to EGCG. It would be interesting to elucidate the mechanism of EGCG resistance in ARCaP cells. This will aid in understanding the physiology and pathology of the prostate cancer and could be applied to therapeutic treatments of such malignancy.

107

Table 7. Changes of expression levels of cell cycle-related proteins by GeneChip assay. All the signal intensities were normalized to the values of the control chips so that the gene expression level in control chip is 1 whereas experimental chip gives greater, equal or less than 1. The value of the experimental chip was expressed and displayed as ‘Fold Change’ and shows the level change when compared to control. Only those of more than 2 fold change after EGCG treatment are shown. n/c = no change

Fold Change Description GenBank ID ARCaP LNCaP EGR1 NM_001964 -3.8 4.8 EGR1 AI459194 -7.5 7.9 GADD45, alpha NM_001924 -2.6 3.4 GADD45, beta AF078077 n/c 2.8 GADD45, beta NM_015675 n/c 6.8 topoisomerase II alpha AL561834 n/c -3.7 topoisomerase II alpha AU159942 n/c -3.8 topoisomerase II beta NM_001068 n/c -3.7 CDK inhibitor, p21/CIP1/WAF1 NM_000389 n/c 9.0 CDK inhibitor, p16 U38945 n/c 2.0 CDK inhibitor, p15 AW444761 n/c 2.2

108

B 300 250.5

200

100 100 100 62.4 Expression Level (%) 0 ARCaP LNCaP

EGCG- EGCG +

Figure 46. Changes of expression levels of EGR1 by RT-PCR in ARCaP and LNCaP cells. A: RT-PCR result after 35 cycles is shown. EGR1 specific primer was designed to amplify 729 bp product. MW in first lane is 100 bp DNA ladder and markers from 600 bp to 1 kbp, from bottom, respectively, are shown. GAPDH was amplified for reference and used as control. B: The normalized expression level is shown. See text for details. The relative band density of the treated samples is normalized to the corresponding controls.

109

APPENDIX

TABLES FOR THE RAW DATA

Table 8. The effect of R1881 on the growth of human prostate cancer cell lines.

Number of Cells 0 hr 24 hrs 48 hrs

Batch Cell Lines DMSO R1881 DMSO R1881 DMSO R1881

LNCaP 64000 64000 190000 234000 355000 465000 1 PC3 64000 64000 132000 102000 282000 295000

ARCaP 108000 108000 210000 197000 573000 441000

LNCaP 58000 58000 204000 214000 344000 445000 2 PC3 58000 58000 112000 102000 322000 264000

ARCaP 85000 85000 209000 187000 600000 465000

LNCaP 70000 70000 180000 216000 320000 400000 3 PC3 52000 52000 84000 82000 315000 325000

ARCaP 129000 129000 192000 167000 574500 456000

Average (Number of Cells, x 1000)

LNCaP 64.0000 64.0000 191.3333 221.3333 339.6667 436.6667 PC3 58.0000 58.0000 109.3333 102.8334 306.3333 294.6667

ARCaP 107.3333 107.3333 203.6667 183.6667 582.5000 454.0000

SEM

LNCaP 3.4641 3.4641 6.9602 6.3595 10.3333 19.2209 PC3 3.4641 3.4641 13.9204 6.6666 12.3333 17.6100

ARCaP 12.7060 12.7060 5.8404 8.8191 8.7607 7.0000

110

Table 9. The effect of EGCG on the growth of LNCaP prostate cancer cell line.

Number of Cells EGCG

Batch hrs 0 µM 25 µM 50 µM 100 µM 0 79000 79000 79000 79000 24 191000 162000 79000 49000 1 48 344000 277000 158000 57000 72 710000 556000 166000 20000 0 62000 62000 62000 62000 24 188000 171000 122000 44000 2 48 404000 316000 163000 47000 72 920000 604000 154000 30000 0 86000 86000 86000 86000 24 176000 150000 74000 53000 3 48 358000 295000 162000 45000 72 834000 650000 150000 30000

Average (Number of Cells, x 1000)

0 hr 75.6667 75.6667 75.6667 75.6667 24 hr 185.0000 161.0000 91.6667 48.6667 48 hr 368.6667 296.0000 161.0000 49.6667 72 hr 821.3333 603.3333 156.6667 26.6667

SEM

0 hr 7.1259 7.1259 7.1259 7.1259 24 hr 4.5826 6.0828 15.2352 2.6034 48 hr 18.1230 11.2694 1.5275 3.7118 72 hr 60.9517 27.1375 4.8074 3.3333

111

Table 10. The effect of EGCG on the growth of ARCaP prostate cancer cell line.

Number of Cells EGCG

Batch hrs 0 µM 25 µM 50 µM 100 µM 0 89000 89000 89000 89000 24 231000 249000 234000 178000 1 48 571000 521000 520000 322000 72 668000 572000 586000 342000 0 91000 91000 91000 91000 24 244000 209000 231000 184000 2 48 568000 512000 492000 232000 72 636000 566000 652000 330000 0 106000 106000 106000 106000 24 254000 248000 236000 168000 3 48 510000 500000 510000 302000 72 640000 522000 628000 306000

Average (Number of Cells, x 1000)

0 hr 95.3333 95.3333 95.3333 95.3333 24 hr 243.0000 235.3333 233.6667 176.6667 48 hr 549.6667 511.0000 507.3333 285.3333 72 hr 648.0000 630.3333 622.0000 326.0000

SEM

0 hr 5.3645 5.3645 5.3645 5.3645 24 hr 6.6583 13.1698 1.4530 4.6667 48 hr 19.8522 6.0828 8.1921 27.2845 72 hr 10.0665 15.7621 19.2873 10.5830

112

Table 11. The ‘Enrichment factor’ of ARCaP and LNCaP cells upon EGCG treatment measured by Cell Death Detection Assay PLUS.

ARCaP EGCG

Batch 0 µM 25 µM 50 µM 100 µM

1 1 1.8015 1.5195 1.5326 2 1 1.2826 1.7925 1.1037

3 1 1.1471 1.1472 1.0332

Average 1 1.4104 1.4864 1.2232 SEM 0 0.1994 0.1870 0.1560

LNCaP EGCG

Batch 0 µM 25 µM 50 µM 100 µM

1 1 2.6192 11.5336 23.1482 2 1 2.9736 9.6395 22.3076

3 1 2.9873 10.7141 25.3657

Average 1 2.8600 10.6291 23.6072 SEM 0 0.1205 0.5484 0.9121

113

Table 12. The ratio of Bax to Bcl-2 upon EGCG treatment. The relative intensity of corresponding band was measured and the ratio was calculated. The ratio was normalized to control.

Relative EGCG Intensity

ARCaP 0 µM 25 µM 50 µM 100 µM

Bax 114.24 107.47 110.47 115.00 Bcl-2 39.70 40.60 52.43 65.01

Bax / Bcl-2 2.878 2.65 2.11 1.77

Normalized 1.00 0.92 0.73 0.62

LNCaP 0 µM 25 µM 50 µM 100 µM

Bax 49.22 52.61 86.98 85.16 Bcl-2 200.10 187.58 172.63 151.55

Bax / Bcl-2 0.25 0.28 0.50 0.56

Normalized 1.00 1.14 2.05 2.29

114

Table 13. The effect of etoposide on the growth of ARCaP cells. The average and standard error of mean (SEM) of the cell number were calculated and were used in graph. The average represents number of cells x 1000.

Number Etoposide of Cells

Batch 0 µM 5 µM 10 µM 50 µM 100 µM

1 196000 96000 64000 22000 2000 2 190000 88000 56000 34000 4000

3 202000 82000 52000 16000 4000

Average 196.0000 88.6667 57.3333 24.0000 3.3333 SEM 3.4641 4.0552 3.5277 5.2915 0.6667

115

Table 14. Relative adherence of ARCaP cells to ECM proteins. The average and SEM were calculated and were used in graph.

Relative 0 µM EGCG Adherence

Batch ECM Proteins Average SEM 1 2 3 Collagen I 2.282 2.103 2.181 2.1887 0.0518 Collagen II 1.629 2.079 1.864 1.8573 0.1299

Collagen IV 1.662 1.780 1.647 1.6963 0.0421

Fibronectin 1.025 0.813 0.875 0.9043 0.0629

Laminin 2.419 2.118 2.122 2.2197 0.0997

Tenascin 1.331 1.307 1.306 1.3147 0.0082

Vitronectin 1.610 1.604 1.611 1.6083 0.0022

BSA 0.156 0.155 0.152 0.1543 0.0012

Relative 100 µM EGCG Adherence

Batch ECM Proteins Average SEM 1 2 3

Collagen I 2.893 2.905 2.413 2.7370 0.1620 Collagen II 2.374 2.175 2.531 2.3600 0.1030

Collagen IV 2.036 2.015 2.142 2.0643 0.0393

Fibronectin 0.431 0.456 0.366 0.4177 0.0268

Laminin 2.244 2.157 2.048 2.1497 0.0567

Tenascin 1.337 1.216 1.300 1.2843 0.0358

Vitronectin 1.454 1.771 1.689 1.6380 0.0950

BSA 0.065 0.067 0.063 0.0650 0.0012

116

Table 15. Relative adherence of LNCaP cells to ECM proteins. The average and SEM were calculated and were used in graph.

Relative 0 µM EGCG Adherence

Batch ECM Proteins Average SEM 1 2 3

Collagen I 0.749 1.015 0.869 0.8777 0.0769 Collagen II 0.686 0.699 0.668 0.6843 0.0090

Collagen IV 0.967 1.137 1.045 1.0497 0.0491

Fibronectin 1.255 1.654 1.469 1.4593 0.1153

Laminin 1.311 1.788 1.559 1.5527 0.1377

Tenascin 0.625 0.725 0.672 0.6740 0.0289

Vitronectin 1.594 1.735 1.654 1.6610 0.0409

BSA 0.191 0.140 0.156 0.1623 0.0151

Relative 100 µM EGCG Adherence

Batch ECM Proteins Average SEM 1 2 3

Collagen I 0.758 0.638 0.860 0.7520 0.0642 Collagen II 0.538 0.436 0.360 0.4447 0.0516

Collagen IV 0.677 0.595 0.467 0.5797 0.0611

Fibronectin 0.905 0.863 0.783 0.8503 0.0358

Laminin 0.797 1.027 0.670 0.8313 0.1045

Tenascin 0.146 0.236 0.119 0.1670 0.0354

Vitronectin 1.145 1.385 0.939 1.1563 0.1289

BSA 0.068 0.100 0.072 0.0800 0.0101

117

Table 16. The changes in relative adherence after EGCG treatment. Percentage of changes of relative adherence was calculated as below and the type of regulation is presented.

% change = (a-b) / b x 100 where, a = relative adherence after EGCG treatment b = relative adherence before EGCG treatment

Relative ARCaP LNCaP Adherence

ECM Proteins % change regulation % change regulation

Collagen I 25.1 up 14.3 down Collagen II 27.1 up 35.0 down

Collagen IV 21.7 up 44.8 down

Fibronectin 53.8 down 41.7 down

Laminin 3.2 down 46.5 down

Tenascin 2.3 down 75.2 down

Vitronectin 1.8 up 30.4 down

BSA 57.9 down 50.7 down

118

Table 17. The number of colonies formed in agar plate after EGCG treatment. The average and SEM were calculated and were used in graph.

Number of EGCG Colonies

ARCaP 0 µM 25 µM 50 µM 100 µM

1 666 3012 3641 4962 2 620 2891 3947 4339 Batch 3 735 2761 3309 4278

Average 673.6667 2888.0000 3632.3333 4526.3333

SEM 33.4182 72.4730 184.2257 218.5439

LNCaP 0 µM 25 µM 50 µM 100 µM

1 4195 2835 330 0 2 4130 3179 317 0 Batch 3 4020 2952 356 0

Average 4115.0000 2988.6667 334.3333 0

SEM 51.0718 100.9824 11.4649 0

119

Table 18. GeneChip assay of ARCaP cells.

Fold GenBank ID Description Change 4.4 BG292233 insulin induced gene 1 4.0 NM_000431 mevalonate kinase (mevalonic aciduria) Cluster Incl. AL049226:Homo sapiens mRNA; cDNA DKFZp564M0916 (from clone 3.8 4851682 DKFZp564M0916) /cds=UNKNOWN /gb=AL049226 /gi=4499955 /ug=Hs.126541 /len=1402 3.7 BC035889 hypothetical protein LOC285943 3.6 NM_005542 insulin induced gene 1 gb:AL353580 /DB_XREF=gi:9944152 /FEA=DNA_2 /CNT=1 /TID=Hs.326589.0 /TIER=ConsEnd /STK=0 /UG=Hs.326589 /UG_TITLE=Human DNA sequence from clone RP11-248N6 on chromosome 13 Contains ESTs, STSs and GSSs. Contains two olfactory receptor pseudogenes, an NPM1 (nucleophosmin, nucleolar phosphoprotein B23, numatrin) 3.5 AL353580 pseudogene and a BCR (breakpoint cluster region) pseudogene /DEF=Human DNA sequence from clone RP11-248N6 on chromosome 13 Contains ESTs, STSs and GSSs. Contains two olfactory receptor pseudogenes, an NPM1 (nucleophosmin, nucleolar phosphoprotein B23, numatrin) pseudogene and a BCR (breakpoint cluster region) p... 3.4 BC041472 MDS2 3.1 BC005939 prostaglandin D2 synthase 21kDa (brain) 3.1 D63807 lanosterol synthase (2,3-oxidosqualene-lanosterol cyclase) 3.0 BE300521 insulin induced gene 1 3.0 AK024576 Homo sapiens cDNA: FLJ20923 fis, clone ADSE00893. 3.0 AV699746 solute carrier family 22 (extraneuronal monoamine transporter), member 3 3.0 NM_001891 casein beta 2.9 AK022897 reversion-inducing-cysteine-rich protein with kazal motifs 2.8 AI535835 jun1.P2.A1 conorm Homo sapiens cDNA 3', mRNA sequence. 2.8 AW083054 hypothetical gene supported by BC007804 2.7 AI189359 mevalonate (diphospho) decarboxylase 2.6 W92036 proprotein convertase subtilisin/kexin type 9 2.6 AV704962 sterol-C4-methyl oxidase-like 2.6 AF034102 solute carrier family 29 (nucleoside transporters), member 2 2.5 BC005247 isopentenyl-diphosphate delta isomerase 2.5 BF513410 hypothetical protein FLJ22297 zu57f04.s1 Soares ovary tumor NbHOT Homo sapiens cDNA clone IMAGE:742111 3' similar 2.5 AA405798 to contains Alu repetitive element;, mRNA sequence. 2.5 NM_002130 3-hydroxy-3-methylglutaryl-Coenzyme A synthase 1 (soluble) 2.5 BG035985 3-hydroxy-3-methylglutaryl-Coenzyme A synthase 1 (soluble) 2.5 NM_006992 B7 gene 2.5 BC006005 uncharacterized hematopoietic stem/progenitor cells protein MDS032 2.5 AK074456 Homo sapiens cDNA FLJ23876 fis, clone LNG13582. 2.4 AA678241 stearoyl-CoA desaturase (delta-9-desaturase) 2.4 AA780365 Homo sapiens transcribed sequences 2.4 NM_000363 similar to hypothetical testis protein from macaque 2.4 NM_016125 PTD016 protein 2.4 R59093 Traf2 and NCK interacting kinase 2.4 W74476 Homo sapiens cDNA FLJ10196 fis, clone HEMBA1004776. 2.4 AU144567 ankyrin repeat and SOCS box-containing 3 2.4 BC005016 tripartite motif-containing 2 2.3 D50479 TYRO3 protein tyrosine kinase 2.3 AB029026 transforming, acidic coiled-coil containing protein 1 Human DNA sequence from clone RP5-1118D24 on chromosome 1p36.11-36.33, complete 2.3 AL031276 sequence.

120

Table 18 continued.

Fold GenBank ID Description Change 2.3 NM_005165 aldolase C, fructose-bisphosphate Homo sapiens transcribed sequence with weak similarity to protein ref:NP_055474.1 2.3 AI783767 (H.sapiens) KIAA0377 gene product [Homo sapiens] 2.3 AK026764 hypothetical protein MGC33202 2.3 BC005807 stearoyl-CoA desaturase (delta-9-desaturase) 2.3 BE046983 Homo sapiens transcribed sequences 2.3 AK025759 hypothetical gene supported by AF034176 2.3 AB033044 KIAA1218 protein 601764995F1 NIH_MGC_53 Homo sapiens cDNA clone IMAGE:3997247 5', mRNA 2.3 BF028225 sequence. 2.3 NM_004508 isopentenyl-diphosphate delta isomerase 2.3 AK026466 Homo sapiens cDNA: FLJ22813 fis, clone KAIA2964. 2.3 BC011938 Homo sapiens, clone IMAGE:4541510, mRNA 2.3 AA625847 Homo sapiens transcribed sequences 2.3 NM_001544 intercellular adhesion molecule 4, Landsteiner-Wiener blood group 2.3 NM_000527 low density lipoprotein receptor (familial hypercholesterolemia) 2.3 NM_000609 chemokine (C-X-C motif) ligand 12 (stromal cell-derived factor 1) 2.3 AA224446 adenylate cyclase 2 (brain) 2.2 U87460 G protein-coupled receptor 37 (endothelin receptor type B-like) 2.2 AV728526 macrophage expressed gene 1 2.2 NM_014897 KIAA0924 protein 2.2 S70123 low density lipoprotein receptor (familial hypercholesterolemia) 2.2 AI820964 stromal antigen 1 2.2 AI939442 v-akt murine thymoma viral oncogene homolog 3 (protein kinase B, gamma) 2.2 AF238489 similar to Olfactory receptor 8D1 (Olfactory receptor-like protein JCG9) (OST004) 2.2 NM_000142 fibroblast growth factor receptor 3 (achondroplasia, thanatophoric dwarfism) 2.2 NM_014354 chromosome 6 open reading frame 54 2.2 AI861942 low density lipoprotein receptor (familial hypercholesterolemia) 2.2 BC032952 hypothetical protein LOC51320 2.2 NM_032903 hypothetical protein MGC14425 2.2 AI824013 Homo sapiens transcribed sequences 2.2 AU147405 hypothetical protein FLJ22955 2.2 AK021440 hypothetical protein FLJ10276 2.2 BC002748 hypothetical protein FLJ20533 2.2 AI693193 metaxin 1 2.2 NM_001360 7-dehydrocholesterol reductase 2.2 BM978026 hypothetical protein LOC284356 2.2 AW340139 interleukin 28 receptor, alpha (interferon, lambda receptor) 2.1 AV725947 Splicing factor, arginine/serine-rich, 46kD 2.1 BF032717 KIAA1813 protein 2.1 AB006757 BH-protocadherin (brain-heart) 2.1 AL049245 serine (or cysteine) proteinase inhibitor, clade B (ovalbumin), member 9 2.1 AF037261 vinexin beta (SH3-containing adaptor molecule-1) 2.1 AW237165 zinc finger protein 297B 2.1 D17218 Human HepG2 3' region MboI cDNA, clone hmd3g02m3. 2.1 AI703480 Homo sapiens transcribed sequences 2.1 N38750 LOC339299 2.1 BC038780 four and a half LIM domains 2 2.1 AI357655 similar to HSPC296 2.1 BE551219 claudin 18

121

Table 18 continued.

Fold GenBank ID Description Change 2.1 AW341649 tumor protein p53 inducible nuclear protein 1 2.1 AF217536 mevalonate kinase (mevalonic aciduria) Cluster Incl. N74607:za55a01.s1 Homo sapiens cDNA, 3 end /clone=IMAGE-296424 2.1 4855867_RC /clone_end=3' /gb=N74607 /gi=1231892 /ug=Hs.234642 /len=487 2.1 AA883493 Mid-1-related chloride channel 1 2.1 BC003573 farnesyl-diphosphate farnesyltransferase 1 2.1 BF001156 hypothetical protein MGC10731 2.1 NM_018490 G protein-coupled receptor 48 2.1 AB038783 mucin 3B 2.1 AL136619 hypothetical protein DKFZp564O0523 2.1 R02580 unc-5 homolog C (C. elegans) 2.0 AA705182 Kruppel-like zinc finger protein GLIS2 2.0 AB002386 enhancer of zeste homolog 1 (Drosophila) tg62h06.x1 Soares_NSF_F8_9W_OT_PA_P_S1 Homo sapiens cDNA clone IMAGE:2113403 2.0 AI400209 3', mRNA sequence. 2.0 NM_030971 similar to rat tricarboxylate carrier-like protein 2.0 AL832681 Homo sapiens mRNA; cDNA DKFZp313M0417 (from clone DKFZp313M0417) 2.0 AI635931 Homo sapiens transcribed sequences 2.0 AF116616 predicted protein of HQ0998; Homo sapiens PRO0998 mRNA, complete cds. 2.0 AA809353 similar to RIKEN cDNA 0610009J22 2.0 R08619 PHD finger protein 11 2.0 AW131553 chromosome 21 open reading frame 86 2.0 BE466117 hypothetical protein DKFZp547B0714 2.0 NM_017868 hypothetical protein FLJ20535 2.0 BG396868 hypothetical gene supported by BC033767; BC022830; BC033767 2.0 W93728 guanylate cyclase 1, soluble, beta 3 2.0 AA149594 TGFB inducible early growth response 2 Homo sapiens transcribed sequence with weak similarity to protein ref:NP_060312.1 2.0 AA401963 (H.sapiens) hypothetical protein FLJ20489 [Homo sapiens] 2.0 AI276196 vestigial like 2 (Drosophila) 2.0 U70370 paired-like homeodomain transcription factor 1 -2.0 T90915 splicing factor, arginine/serine-rich 11 -2.0 AA569225 Homo sapiens cDNA FLJ11915 fis, clone HEMBB1000198. -2.0 NM_002463 myxovirus (influenza virus) resistance 2 (mouse) -2.0 AI360167 Homo sapiens transcribed sequences -2.0 AB020716 calmodulin binding transcription activator 2 -2.0 NM_021800 J domain containing protein 1 -2.0 BC039674 Homo sapiens, clone IMAGE:5171352, mRNA -2.0 U37546 baculoviral IAP repeat-containing 3 -2.0 AW976347 strand-exchange protein 1 hv47a05.x1 NCI_CGAP_Lu24 Homo sapiens cDNA clone IMAGE:3176528 3', mRNA -2.0 BE218980 sequence. -2.0 BC041876 tau tubulin kinase 2 -2.0 AA873021 Homo sapiens transcribed sequences -2.0 BF447692 Homo sapiens transcribed sequences -2.0 AW138350 hypothetical protein LOC348938 -2.0 AA417078 Homo sapiens transcribed sequences -2.0 N47725 retinoic acid- and interferon-inducible protein (58kD) Homo sapiens transcribed sequence with moderate similarity to protein sp:Q99687 (H.sapiens) -2.0 H15129 MEI3_HUMAN Homeobox protein Meis3 (Meis1-related protein 2)

122

Table 18 continued.

Fold GenBank ID Description Change Homo sapiens transcribed sequence with weak similarity to protein ref:NP_060312.1 -2.0 BQ446762 (H.sapiens) hypothetical protein FLJ20489 [Homo sapiens] -2.0 AA115127 hypothetical protein LOC148189 -2.0 BG253119 bullous pemphigoid antigen 1, 230/240kDa -2.0 NM_019853 Homo sapiens protein phosphatase 4, regulatory subunit 2 (PPP4R2), mRNA -2.0 BF342851 Melanoma associated gene -2.0 BC041394 Homo sapiens, clone IMAGE:5277116, mRNA -2.0 NM_021163 RB-associated KRAB repressor -2.0 AU148042 FLJ20288 protein -2.1 N29877 taxilin -2.1 AA811657 uncharacterized hypothalamus protein HCDASE Homo sapiens transcribed sequence with moderate similarity to protein pir:I60307 (E. coli) -2.1 AW452971 I60307 beta-galactosidase, alpha peptide - Escherichia coli -2.1 AI523817 KIAA1554 protein -2.1 NM_025047 hypothetical protein FLJ22595 -2.1 BF514864 Homo sapiens cDNA FLJ13825 fis, clone THYRO1000558. Homo sapiens transcribed sequence with weak similarity to protein ref:NP_055301.1 -2.1 AV742010 (H.sapiens) neuronal thread protein [Homo sapiens] -2.1 NM_020667 Homo sapiens COBW-like protein (LOC55871), mRNA. -2.1 AI084610 Homo sapiens transcribed sequences Homo sapiens transcribed sequence with strong similarity to protein ref:NP_009151.1 -2.1 AA521288 (H.sapiens) carbonic anhydrase VB, mitochondrial precursor; carbonic dehydratase [Homo sapiens] -2.1 AA532745 Homo sapiens cDNA FLJ37778 fis, clone BRHIP2026557. -2.1 BF939551 transient receptor potential cation channel, subfamily M, member 7 -2.1 BC002704 signal transducer and activator of transcription 1, 91kDa Homo sapiens transcribed sequence with moderate similarity to protein ref:NP_002361.1 -2.1 AW371924 (H.sapiens) mago-nashi homolog [Homo sapiens] -2.1 NM_006352 zinc finger protein 238 -2.1 AI923090 Homo sapiens transcribed sequences -2.1 NM_144573 likely ortholog of rat F-actin binding protein nexilin -2.1 AL109714 hypothetical protein LOC283687 -2.1 M87507 caspase 1, apoptosis-related cysteine protease (interleukin 1, beta, convertase) -2.1 BC024736 Homo sapiens, clone IMAGE:4915292, mRNA -2.1 BC004918 adrenergic, beta, receptor kinase 2 -2.1 AK000106 Homo sapiens cDNA FLJ20099 fis, clone COL04544. -2.1 AI967971 3'-5' RNA exonuclease -2.1 BC041029 Homo sapiens cDNA FLJ34562 fis, clone KIDNE2002438. -2.1 NM_002535 2'-5'-oligoadenylate synthetase 2, 69/71kDa -2.1 BF509229 Homo sapiens transcribed sequences -2.1 AF225986 sodium channel, voltage-gated, type III, alpha -2.1 BF701384 Homo sapiens transcribed sequences -2.1 NM_012436 sperm associated antigen 8 Homo sapiens transcribed sequence with strong similarity to protein ref:NP_003277.1 -2.1 AW025108 (H.sapiens) DNA topoisomerase I; type I DNA topoisomerase [Homo sapiens] -2.1 U34919 ATP-binding cassette, sub-family G (WHITE), member 1 -2.1 AA018187 chromosome 22 open reading frame 3 -2.1 NM_004723 rho/rac guanine nucleotide exchange factor (GEF) 2 -2.1 AF230409 promyelocytic leukemia -2.1 NM_017912 hypothetical protein FLJ20637

123

Table 18 continued.

Fold GenBank ID Description Change -2.1 AA932964 LOC124491 -2.1 AA863112 Homo sapiens transcribed sequences -2.1 NM_018842 insulin receptor tyrosine kinase substrate -2.1 AF233516 programmed cell death 1 ligand 1 -2.1 AI332638 Homo sapiens transcribed sequences -2.1 NM_003523 histone 1, H2be -2.1 BC008840 hypothetical protein FLJ13710 -2.1 BE259050 zizimin1 -2.1 D25272 Homo sapiens mRNA, clone:RES4-16. Ig kappa chain; Human kappa-immunoglobulin germline pseudogene (cos118) variable region -2.1 M20812 (subgroup V kappa I). -2.1 AK024805 Homo sapiens cDNA: FLJ21152 fis, clone CAS09594. Homo sapiens transcribed sequence with weak similarity to protein ref:NP_060265.1 -2.1 AI084024 (H.sapiens) hypothetical protein FLJ20378 [Homo sapiens] -2.1 NM_022082 chromosome 20 open reading frame 59 -2.1 T78442 Homo sapiens transcribed sequences -2.1 AA599017 dedicator of cyto-kinesis 1 -2.1 AI684643 tumor rejection antigen (gp96) 1 Homo sapiens clone P2-114 anti-oxidized LDL immunoglobulin light chain Fab mRNA, -2.1 X93006 partial cds -2.1 AL049301 Homo sapiens mRNA; cDNA DKFZp564P073 (from clone DKFZp564P073). -2.1 AI087937 hypothetical protein FLJ11036 -2.1 AU146983 Homo sapiens cDNA FLJ12055 fis, clone HEMBB1002049. -2.1 AI884670 LOC90024 -2.1 NM_005238 v-ets erythroblastosis virus E26 oncogene homolog 1 (avian) -2.1 AB001328 solute carrier family 15 (oligopeptide transporter), member 1 -2.1 D42044 KIAA0090 protein -2.1 AL031313 Human DNA sequence from clone RP4-581F12 on chromosome Xq21, complete sequence. -2.1 NM_003224 ADP-ribosylation factor related protein 1 -2.2 BC006291 hypothetical protein FLJ13154 -2.2 AF397731 neuron navigator 3 -2.2 BC025999 Homo sapiens, clone IMAGE:4291396, mRNA -2.2 NM_016582 peptide transporter 3 -2.2 AU158002 prematurely terminated mRNA decay factor-like -2.2 NM_014684 KIAA0373 gene product -2.2 BC001800 orthopedia homolog (Drosophila) -2.2 AI742383 Homo sapiens transcribed sequences -2.2 AA805622 baculoviral IAP repeat-containing 3 -2.2 AA897191 transforming, acidic coiled-coil containing protein 1 -2.2 AU140931 upstream regulatory element binding protein 1 Homo sapiens transcribed sequence with weak similarity to protein ref:NP_060265.1 -2.2 AI333596 (H.sapiens) hypothetical protein FLJ20378 [Homo sapiens] -2.2 AW960454 Homo sapiens transcribed sequences -2.2 AI800470 Homo sapiens transcribed sequences -2.2 NM_018456 ELL-associated factor 2 gb:L23982 /DB_XREF=gi:495865 /FEA=DNA /CNT=1 /TID=Hs.1640.1 /TIER=ConsEnd /STK=0 /UG=Hs.1640 /LL=1294 /UG_GENE=COL7A1 /UG_TITLE=collagen, type VII, -2.2 L23982 alpha 1 (epidermolysis bullosa, dystrophic, dominant and recessive) /DEF=Homo sapiens (clones: CW52-2, CW27-6, CW15-2, CW26-5, 11-67) collagen type VII intergenic region and (COL7A1) gene, complete cds

124

Table 18 continued.

Fold GenBank ID Description Change serine (or cysteine) proteinase inhibitor, clade E (nexin, plasminogen activator inhibitor type -2.2 BC020765 1), member 1 -2.2 NM_024692 hypothetical protein FLJ21069 -2.2 AL080112 Homo sapiens mRNA; cDNA DKFZp586H0722 (from clone DKFZp586H0722). AL526632 Homo sapiens NEUROBLASTOMA COT 25-NORMALIZED Homo sapiens -2.2 AL526632 cDNA clone CS0DC020YC21 3-PRIME, mRNA sequence. -2.2 AA808018 cyclin I -2.2 AL039379 chromosome 6 open reading frame 148 -2.2 AI761561 hexokinase 2 -2.2 AK023743 hypothetical protein FLJ31033 -2.2 AB016517 fibroblast growth factor 5 -2.2 NM_144990 hypothetical protein FLJ23878 -2.2 BC037946 Homo sapiens, clone IMAGE:5285837, mRNA. -2.2 BC000856 Homo sapiens cDNA: FLJ21331 fis, clone COL02520. -2.2 AI539443 Homo sapiens cDNA FLJ12169 fis, clone MAMMA1000643. -2.2 BC003169 calpain 3, (p94) -2.2 AK024889 Homo sapiens cDNA: FLJ21236 fis, clone COL01111. -2.2 AA632147 PRKC, apoptosis, WT1, regulator -2.2 AF258593 hypothetical protein LOC201175 -2.2 T19133 LOC346196 -2.2 BC038556 Homo sapiens, clone IMAGE:3446976, mRNA -2.3 AA132961 phospholipase D1, phophatidylcholine-specific -2.3 AI373166 transcription factor 8 (represses interleukin 2 expression) -2.3 AI873273 solute carrier family 16 (monocarboxylic acid transporters), member 6 -2.3 AW295488 zinc finger protein, multitype 1 Hypothetical 108.52 kDa human protein; Homo sapiens chromosome 19, overlapping cosmids -2.3 AC004144 R28707 and R34001, complete sequence. -2.3 BF515177 pannexin 2 -2.3 NM_001813 centromere protein E, 312kDa -2.3 AI638532 Homo sapiens transcribed sequences -2.3 AJ224167 matrin 3 -2.3 AK002049 ankyrin repeat and SOCS box-containing 2 -2.3 AA460299 hypothetical protein FLJ23468 -2.3 AU144005 Homo sapiens cDNA FLJ11397 fis, clone HEMBA1000622. -2.3 NM_002090 chemokine (C-X-C motif) ligand 3 -2.3 AK095517 hypothetical protein MGC26989 -2.3 AV738585 AV738585 CB Homo sapiens cDNA clone CBFAWD05 5', mRNA sequence. -2.3 BC038556 Homo sapiens, clone IMAGE:3446976, mRNA -2.3 AW962850 EST374923 MAGE resequences, MAGG Homo sapiens cDNA, mRNA sequence. -2.3 BC029456 trinucleotide repeat containing 15 -2.3 AK024851 Homo sapiens cDNA: FLJ21198 fis, clone COL00220. -2.3 NM_014648 zinc finger DAZ interacting protein 3 -2.3 U16125 glutamate receptor, ionotropic, kainate 1 -2.3 BF195207 hypothetical protein LOC339005 -2.3 AA971429 CASP8 and FADD-like apoptosis regulator yr90d10.s1 Soares fetal liver spleen 1NFLS Homo sapiens cDNA clone IMAGE:212563 3', -2.3 H68862 mRNA sequence. -2.3 AI672373 Homo sapiens cDNA FLJ11179 fis, clone PLACE1007450. -2.3 BC018494 hypothetical protein FLJ22527 -2.3 BF513986 LOC341403

125

Table 18 continued.

Fold GenBank ID Description Change Homo sapiens transcribed sequence with weak similarity to protein ref:NP_060312.1 -2.3 AA132172 (H.sapiens) hypothetical protein FLJ20489 [Homo sapiens] -2.3 L36674 synuclein, alpha (non A4 component of amyloid precursor) -2.3 NM_022044 stromal cell-derived factor 2-like 1 gb:NM_018616.1 /DB_XREF=gi:8924113 /GEN=PRO2037 /FEA=FLmRNA /CNT=4 /TID=Hs.283067.0 /TIER=FL /STK=0 /UG=Hs.283067 /LL=55480 /DEF=Homo sapiens -2.3 NM_018616 hypothetical protein PRO2037 (PRO2037), mRNA. /PROD=hypothetical protein PRO2037 /FL=gb:AF116684.1 gb:NM_018616.1 -2.3 AI141861 lumican -2.4 AI765383 KIAA1466 protein -2.4 BE218239 Homo sapiens transcribed sequences -2.4 BC037907 hypothetical gene supported by BC037907 -2.4 NM_006132 bone morphogenetic protein 1 -2.4 AK022215 DKFZP564O092 protein -2.4 AW294215 Homo sapiens transcribed sequences -2.4 W63579 potassium large conductance calcium-activated channel, subfamily M beta member 3 Homo sapiens transcribed sequence with moderate similarity to protein ref:NP_060190.1 -2.4 AA804267 (H.sapiens) hypothetical protein FLJ20234 [Homo sapiens] -2.4 BE220569 Homo sapiens transcribed sequences -2.4 AU153330 RNA helicase family -2.4 AL049988 Homo sapiens mRNA; cDNA DKFZp564F212 (from clone DKFZp564F212). -2.4 BG819763 hypothetical protein LOC147080 -2.4 NM_144975 hypothetical protein MGC19764 -2.4 AA497043 Homo sapiens transcribed sequences Homo sapiens partial mRNA for immunoglobulin heavy chain variable region (IGHV32-D- -2.4 U64494 JH-Cmu gene), clone ET39 serine (or cysteine) proteinase inhibitor, clade A (alpha-1 antiproteinase, antitrypsin), -2.4 AF119873 member 1 -2.4 AI675298 cyclin L1 -2.4 X90579 cytochrome P450, family 3, subfamily A, polypeptide 5 -2.4 BG334930 angiomotin like 1 -2.4 AI138785 Homo sapiens transcribed sequences -2.4 AK094948 hypothetical protein LOC286207 -2.4 NM_020664 2,4-dienoyl CoA reductase 2, peroxisomal -2.4 U79295 Human clone 23961 mRNA sequence -2.4 BC015770 KIAA0062 protein -2.4 AF230411 promyelocytic leukemia J2096F Human fetal heart, Lambda ZAP Express Homo sapiens cDNA clone J2096 5', mRNA -2.4 N55756 sequence. -2.4 AU118165 zinc finger protein 37a (KOX 21) -2.4 NM_000463 UDP glycosyltransferase 1 family, polypeptide A10 -2.4 AI685845 LOC346972 -2.4 AV706343 Ras-associated protein Rap1 -2.4 BC017314 v-ets erythroblastosis virus E26 oncogene homolog 1 (avian) -2.4 NM_001511 chemokine (C-X-C motif) ligand 1 (melanoma growth stimulating activity, alpha) -2.4 AL050106 Homo sapiens mRNA; cDNA DKFZp586I1319 (from clone DKFZp586I1319). -2.4 BF057506 protein tyrosine phosphatase, non-receptor type 23 -2.4 NM_014330 protein phosphatase 1, regulatory (inhibitor) subunit 15A -2.5 BC000658 stanniocalcin 2 -2.5 NM_000594 tumor necrosis factor (TNF superfamily, member 2)

126

Table 18 continued.

Fold GenBank ID Description Change -2.5 BC038672 Homo sapiens, Similar to neuronal thread protein, clone IMAGE:5265833, mRNA. -2.5 BC015435 hypothetical protein LOC149603 gb:BC001957.1 /DB_XREF=gi:12805006 /FEA=FLmRNA /CNT=5 /TID=Hs.306975.0 /TIER=FL /STK=0 /UG=Hs.306975 /DEF=Homo sapiens, Similar to KIAA0144 gene -2.5 BC001957 product, clone MGC:761, mRNA, complete cds. /PROD=Similar to KIAA0144 gene product /FL=gb:BC001957.1 -2.5 NM_016179 transient receptor potential cation channel, subfamily C, member 4 -2.5 BC017587 LOC136263 -2.5 AL080104 neuron navigator 3 -2.5 NM_002462 myxovirus (influenza virus) resistance 1, interferon-inducible protein p78 (mouse) -2.5 AB033832 spinal cord-derived growth factor-B -2.5 BE870625 similar to spermatid WD-repeat protein -2.5 BC015161 Homo sapiens, clone IMAGE:3886015, mRNA -2.5 AU146742 Homo sapiens cDNA FLJ11986 fis, clone HEMBB1001364. -2.5 AK075118 likely ortholog of rat F-actin binding protein nexilin -2.5 BE544375 Rab coupling protein -2.5 M57731 chemokine (C-X-C motif) ligand 2 -2.5 AF070569 hypothetical protein MGC14376 -2.5 M15329 interleukin 1, alpha -2.5 H63394 Homo sapiens transcribed sequences -2.5 NM_016816 2',5'-oligoadenylate synthetase 1, 40/46kDa -2.5 AB020966 RNA (guanine-7-) methyltransferase -2.5 NM_058189 chromosome 21 open reading frame 69 -2.5 AI982694 Homo sapiens transcribed sequences -2.6 NM_172139 interleukin 28B (interferon, lambda 3) -2.6 BC024012 monocyte-to-macrophage differentiation factor 2 -2.6 AA741061 related to the N terminus of tre -2.6 NM_018363 hypothetical protein FLJ11218 -2.6 AF227968 SH2-B homolog -2.6 BC003637 DNA-damage-inducible transcript 3 -2.6 NM_017654 hypothetical protein FLJ20073 -2.6 BF508977 signal transducer and activator of transcription 3 (acute-phase response factor) 601660289R1 NIH_MGC_71 Homo sapiens cDNA clone IMAGE:3905950 3', mRNA -2.6 BE966604 sequence. -2.6 AF302494 potassium voltage-gated channel, Isk-related family, member 3 -2.6 AL050090 myosin VIIA and Rab interacting protein SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily a, -2.6 NM_003070 member 2 -2.6 NM_021127 phorbol-12-myristate-13-acetate-induced protein 1 -2.6 NM_005531 interferon, gamma-inducible protein 16 -2.6 AA083478 tripartite motif-containing 22 -2.6 AU147295 Homo sapiens cDNA FLJ14090 fis, clone MAMMA1000264. -2.6 AW960145 Homo sapiens cDNA FLJ36210 fis, clone THYMU2000155. -2.6 AW295295 solute carrier family 34 (sodium phosphate), member 1 -2.6 AI459157 uncharacterized hematopoietic stem/progenitor cells protein MDS026 -2.6 AW300612 Homo sapiens transcribed sequences -2.6 NM_001924 growth arrest and DNA-damage-inducible, alpha -2.6 BC042988 MUF1 protein leukocyte immunoglobulin-like receptor, subfamily B (with TM and ITIM domains), -2.6 U82979 member 4

127

Table 18 continued.

Fold GenBank ID Description Change -2.7 BE502377 SH3-domain binding protein 2 Cluster Incl. AL021977:bK447C4.1 (novel MAFF (v-maf musculoaponeurotic fibrosarcoma -2.7 4848734_RC (avian) oncogene family, protein F) LIKE protein) /cds=(0,494) /gb=AL021977 /gi=4914526 /ug=Hs.51305 /len=2128 -2.7 BE328402 hypothetical protein KIAA1434 -2.7 AI336836 RAB5A, member RAS oncogene family -2.7 AK054668 hypothetical protein MGC19764 602614662F1 NIH_MGC_76 Homo sapiens cDNA clone IMAGE:4733702 5', mRNA -2.7 BG616498 sequence. -2.7 AB063297 AAT1-alpha -2.7 AV700865 Homo sapiens transcribed sequences -2.7 AF154848 UDP-Gal:betaGlcNAc beta 1,3-galactosyltransferase, polypeptide 3 -2.7 BC022892 Homo sapiens, clone IMAGE:4616140, mRNA -2.7 NM_002534 2',5'-oligoadenylate synthetase 1, 40/46kDa -2.7 AW513672 Homo sapiens clone N11 NTera2D1 teratocarcinoma mRNA -2.7 AW301393 Homo sapiens cDNA FLJ10263 fis, clone HEMBB1000991. -2.7 AA430014 gap junction protein, alpha 7, 45kDa (connexin 45) -2.7 AI656232 chromosome 14 open reading frame 137 -2.8 AW300131 LOC145188 -2.8 NM_000361 thrombomodulin Homo sapiens transcribed sequence with weak similarity to protein ref:NP_060219.1 -2.8 AU121725 (H.sapiens) hypothetical protein FLJ20294 [Homo sapiens] Homo sapiens transcribed sequence with weak similarity to protein ref:NP_060265.1 -2.8 AI393960 (H.sapiens) hypothetical protein FLJ20378 [Homo sapiens] -2.8 AW015417 family with sequence similarity 3, member C -2.8 NM_003447 zinc finger protein 165 -2.8 BC007652 hypothetical protein BC007652 -2.9 AI742057 hypothetical protein LOC129607 -2.9 AI125338 Homo sapiens transcribed sequences -2.9 BE467322 Homo sapiens transcribed sequences -2.9 BC027951 chromosome 20 open reading frame 32 -2.9 AL162054 Homo sapiens mRNA; cDNA DKFZp761J1323 (from clone DKFZp761J1323) -2.9 R11494 Homo sapiens transcribed sequences -2.9 AA142842 XIAP associated factor-1 -2.9 BC008027 LOC343355 -3.0 AI281593 decorin -3.0 BC029545 v-Ki-ras2 Kirsten rat sarcoma 2 viral oncogene homolog -3.0 BC031107 hypothetical protein LOC136288 -3.0 BC039439 Homo sapiens, clone IMAGE:5285600, mRNA -3.1 NM_017523 XIAP associated factor-1 601820836F1 NIH_MGC_58 Homo sapiens cDNA clone IMAGE:4052768 5', mRNA -3.1 BF131886 sequence. -3.1 AU158358 Homo sapiens cDNA FLJ13694 fis, clone PLACE2000115. -3.1 R40058 Homo sapiens transcribed sequences -3.1 AA480392 Homo sapiens mRNA; cDNA DKFZp686H2244 (from clone DKFZp686H2244) Human DNA sequence from clone RP13-178D16 on chromosome Xq11.1-13.1, complete -3.1 AL353587 sequence. yf77c05.r1 Soares infant brain 1NIB Homo sapiens cDNA clone IMAGE:28483 5', mRNA -3.1 R13458 sequence. -3.1 AI242202 hypothetical protein FLJ20574

128

Table 18 continued.

Fold GenBank ID Description Change -3.2 AI972661 tropomyosin 4 -3.2 AK024712 Homo sapiens cDNA: FLJ21059 fis, clone CAS00740. -3.2 BE645480 Homo sapiens transcribed sequences -3.2 AI871619 chromosome 6 open reading frame 60 tz11g09.x1 NCI_CGAP_Ut1 Homo sapiens cDNA clone IMAGE:2288320 3' similar to -3.2 AI699847 contains MSR1.t2 MSR1 repetitive element ;, mRNA sequence. Homo sapiens transcribed sequence with weak similarity to protein ref:NP_060312.1 -3.2 AI732221 (H.sapiens) hypothetical protein FLJ20489 [Homo sapiens] -3.2 AA781795 epithelial stromal interaction 1 (breast) -3.3 AW057518 ELL-related RNA polymerase II, elongation factor -3.3 AI791828 Homo sapiens transcribed sequences -3.3 BC041876 tau tubulin kinase 2 -3.4 AI732587 Homo sapiens EST from clone 60991, 5' end -3.5 AA741307 hypothetical protein FLJ20073 gb:AL031033 /DB_XREF=gi:4958805 /FEA=DNA_5 /CNT=74 /TID=Hs.101742.0 /TIER=Stack /STK=16 /UG=Hs.101742 /UG_TITLE=Human DNA sequence from clone 321D2 on chromosome 16. Contains a gene for a Ribosomal Large Subunit Pseudouridine Synthase (EC 4.2.1.70, Pseudouridylate Synthase, Uracil Hydrolase) LIKE protein, a gene for -3.5 AL031033 a novel protein similar to replication factors, part of a novel gene and ESTs /DEF=Human DNA sequence from clone 321D2 on chromosome 16. Contains a gene for a Ribosomal Large Subunit Pseudouridine Synthase (EC 4.2.1.70, Pseudouridylate Synthase, Uracil Hydrolase) LIKE protein, a gene for a novel protein similar to replication fa... -3.5 AF339764 Homo sapiens clone IMAGE:108721, mRNA sequence -3.5 AL567808 zinc finger protein 23 (KOX 16) -3.5 X68011 H.sapiens ZNF81 gene. -3.7 NM_022767 hypothetical protein FLJ12484 -3.8 BC001298 hypothetical protein 384D8_6 -3.8 NM_001964 early growth response 1 -4.0 AA702930 Homo sapiens transcribed sequences -4.0 AL117523 KIAA1053 protein -4.2 X80821 KIAA0874 protein -4.8 AV704232 AV704232 ADB Homo sapiens cDNA clone ADBBLB11 5', mRNA sequence. -5.1 AB002441 Homo sapiens mRNA from chromosome 5q21-22, clone:LI26. -5.4 N92818 Homo sapiens transcribed sequences -5.4 AA913703 hypoxia-inducible factor 1, alpha subunit (basic helix-loop-helix transcription factor) -5.8 BC041437 hypothetical protein LOC284958 -6.0 M92934 connective tissue growth factor -6.4 AK097943 Homo sapiens, clone IMAGE:5229530, mRNA -7.0 BC006101 POU domain, class 2, transcription factor 2 -7.5 AI459194 early growth response 1 -7.5 AA001021 thyroid hormone receptor interactor 8 -10.4 NM_003604 insulin receptor substrate 4 -12.4 AF118072 Homo sapiens PRO1716 mRNA, complete cds -13.3 NM_021062 histone 1, H2bb -13.7 D26069 centaurin, beta 2

129

Table 19. GeneChip assay of LNCaP cells.

Fold GenBank ID Description Change 31.1 AW071793 Homo sapiens cDNA clone IMAGE:5270189, partial cds 25.9 BF062629 Ras-induced senescence 1 21.3 BC005161 activin beta E 19.0 AK023451 myotubularin related protein 1 13.9 NM_006169 nicotinamide N-methyltransferase 13.6 NM_002421 matrix metalloproteinase 1 (interstitial collagenase) 13.1 AF003934 prostate differentiation factor 11.8 AF180519 GABA(A) receptors associated protein like 3 9.2 NM_002422 matrix metalloproteinase 3 (stromelysin 1, progelatinase) 9.1 NM_017974 hypothetical protein FLJ10035 9.0 NM_000389 cyclin-dependent kinase inhibitor 1A (p21, Cip1) 8.8 AI984061 hypothetical protein LOC90637 8.8 AF135266 p8 protein (candidate of metastasis 1) 8.2 AF279899 proline rich 2 8.2 AI684439 Homo sapiens transcribed sequences 7.9 NM_013246 cardiotrophin-like cytokine 7.9 AI459194 early growth response 1 putative protein; Homo sapiens mRNA; cDNA DKFZp434F1819 (from clone 7.7 AL136790 DKFZp434F1819); complete cds. 7.6 AA524299 low density lipoprotein receptor-related protein 10 Homo sapiens, chromosome 20 open reading frame 169, clone MGC:51951 IMAGE:5212523, 7.5 N30607 mRNA, complete cds 7.2 NM_000584 interleukin 8 7.1 AF087847 GABA(A) receptor-associated protein like 1 yf81g08.s1 Soares infant brain 1NIB Homo sapiens cDNA clone IMAGE:28927 3', mRNA 7.0 R40373 sequence. 6.8 NM_013370 pregnancy-induced growth inhibitor 6.8 NM_015675 growth arrest and DNA-damage-inducible, beta ym24d11.r1 Soares infant brain 1NIB Homo sapiens cDNA clone IMAGE:49103 5', mRNA 6.7 H14782 sequence. 6.5 NM_014330 protein phosphatase 1, regulatory (inhibitor) subunit 15A 6.5 NM_014181 HSPC159 protein 6.5 AA723810 cDNA for differentially expressed CO16 gene td19b05.x1 NCI_CGAP_Co16 Homo sapiens cDNA clone IMAGE:2076081 3', mRNA 6.3 AI825833 sequence. 6.2 AA846863 Homo sapiens transcribed sequences 6.2 BC004490 v-fos FBJ murine osteosarcoma viral oncogene homolog 6.1 X78928 KRAB zinc finger protein KR18 6.1 AF043337 Homo sapiens interleukin 8 C-terminal variant (IL8) mRNA, complete cds. 6.1 AI964053 Homo sapiens, clone MGC:27017 IMAGE:4831023, mRNA, complete cds 6.1 NM_024111 hypothetical protein MGC4504 6.0 AI078167 nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha 5.9 BC002670 CDK4-binding protein p34SEI1 AL531683 Homo sapiens FETAL LIVER Homo sapiens cDNA clone CS0DM008YI01 3- 5.9 AL531683 PRIME, mRNA sequence. 5.8 AF133207 protein kinase H11 5.8 BC012375 KIAA1001 protein 5.7 NM_013323 sorting nexin 11 5.6 NM_006169 nicotinamide N-methyltransferase 5.4 BF125756 GABA(A) receptor-associated protein like 1

130

Table 19 continued.

Fold GenBank ID Description Change 5.4 X56841 major histocompatibility complex, class I, E 5.4 BC031332 NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 10, 42kDa 5.4 NM_024706 hypothetical protein FLJ13479 5.4 BC000669 chromosome 20 open reading frame 16 5.2 NM_014851 KIAA0469 gene product 5.1 NM_002575 serine (or cysteine) proteinase inhibitor, clade B (ovalbumin), member 2 Cluster Incl. U83981:Homo sapiens apoptosis associated protein (GADD34) mRNA, complete 5.1 4901426_RC cds /cds=(222,2246) /gb=U83981 /gi=3258617 /ug=Hs.76556 /len=2331 5.1 NM_001673 asparagines synthetase 5.0 NM_080618 CCCTC-binding factor (zinc finger protein)-like 4.9 BF194773 hypothetical protein FLJ22160 4.9 D17218 Human HepG2 3' region MboI cDNA, clone hmd3g02m3. 4.9 BF514079 Kruppel-like factor 4 (gut) gb:AL354872 /DB_XREF=gi:9717070 /FEA=mRNA /CNT=2 /TID=Hs.19904.1 /TIER=ConsEnd /STK=0 /UG=Hs.19904 /LL=1491 /UG_GENE=CTH /UG_TITLE=cystathionase (cystathionine gamma-lyase) /DEF=Human DNA sequence from 4.8 AL354872 clone RP11-42O15 on chromosome 1. Contains ESTs, STSs, GSSs and a CpG island. Contains the CTH gene for two isoforms of cystathionase (cystathionine gamma-lyase) and a CHORD containing protein 1 (CHP1) pseudogene 4.8 NM_001964 early growth response 1 4.8 AW075105 DNA replication factor 4.7 NM_018310 BRF2, subunit of RNA polymerase III transcription initiation factor, BRF1-like 4.6 AF132818 Kruppel-like factor 5 (intestinal) 4.6 BF314746 TIGA1 4.6 AA767713 Homo sapiens transcribed sequences 4.6 AI344141 RAB40B, member RAS oncogene family 4.6 NM_001674 activating transcription factor 3 602252212F1 NIH_MGC_84 Homo sapiens cDNA clone IMAGE:4344603 5', mRNA 4.6 BF791874 sequence. 4.5 BC030580 HSPC063 protein 4.5 NM_005980 S100 calcium binding protein P 4.5 AF321125 DNA replication factor 4.4 NM_004030 interferon regulatory factor 7 4.3 BG054833 Homo sapiens transcribed sequences Cluster Incl. U88964:Human HEM45 mRNA, complete cds /cds=(37,582) /gb=U88964 4.3 4866504 /gi=2062679 /ug=Hs.183487 /len=701 4.3 NM_002394 solute carrier family 3 (activators of dibasic and neutral amino acid transport), member 2 4.3 NM_007350 pleckstrin homology-like domain, family A, member 1 ze47e01.s1 Soares retina N2b4HR Homo sapiens cDNA clone IMAGE:362136 3', mRNA 4.2 AA001052 sequence. 4.2 NM_016291 inositol hexaphosphate kinase 2 4.2 BC001051 ADP-ribosylation factor-like 7 4.2 NM_152565 ATPase, H+ transporting, lysosomal 38kDa, V0 subunit d isoform 2 4.1 S77154 nuclear receptor subfamily 4, group A, member 2 yr22d05.s1 Soares fetal liver spleen 1NFLS Homo sapiens cDNA clone IMAGE:206025 3' 4.1 H61544 similar to contains Alu repetitive element;contains MER22 repetitive element ;, mRNA sequence. 4.1 NM_001453 forkhead box C1 4.1 AW292273 cysteinyl-tRNA synthetase 4.1 BE542323 vestigial like 1 (Drosophila)

131

Table 19 continued.

Fold GenBank ID Description Change 4.0 AI568533 serine/arginine repetitive matrix 2 4.0 BC004395 apolipoprotein L, 2 4.0 AF298153 BRF2, subunit of RNA polymerase III transcription initiation factor, BRF1-like Cluster Incl. M60278:Human heparin-binding EGF-like growth factor mRNA, complete cds 4.0 4903762_RC /cds=(261,887) /gb=M60278 /gi=183866 /ug=Hs.799 /len=2342 Cluster Incl. AI668643:zb13f10.x5 Homo sapiens cDNA, 3 end /clone=IMAGE-301963 4.0 4864562_RC /clone_end=3' /gb=AI668643 /gi=4827951 /ug=Hs.15827 /len=601 4.0 AW001036 hypothetical gene LOC131963 4.0 NM_000202 iduronate 2-sulfatase (Hunter syndrome) 3.9 AW245401 death effector domain-containing DNA binding protein 2 3.9 NM_000240 monoamine oxidase A 3.9 NM_005978 S100 calcium binding protein A2 gb:AL353580 /DB_XREF=gi:9944152 /FEA=DNA_2 /CNT=1 /TID=Hs.326589.0 /TIER=ConsEnd /STK=0 /UG=Hs.326589 /UG_TITLE=Human DNA sequence from clone RP11-248N6 on chromosome 13 Contains ESTs, STSs and GSSs. Contains two olfactory receptor pseudogenes, an NPM1 (nucleophosmin, nucleolar phosphoprotein B23, numatrin) 3.9 AL353580 pseudogene and a BCR (breakpoint cluster region) pseudogene /DEF=Human DNA sequence from clone RP11-248N6 on chromosome 13 Contains ESTs, STSs and GSSs. Contains two olfactory receptor pseudogenes, an NPM1 (nucleophosmin, nucleolar phosphoprotein B23, numatrin) pseudogene and a BCR (breakpoint cluster region) p... 3.8 NM_002379 matrilin 1, cartilage matrix protein 3.8 M74301 Human immunoglobulin truncated mu-chain mRNA, 5' end. 3.8 BC012504 dynein, cytoplasmic, intermediate polypeptide 1 3.8 NM_002133 heme oxygenase (decycling) 1 3.8 AI459554 Homo sapiens transcribed sequences 3.8 AK000689 hypothetical protein CLONE24945 3.7 NM_006700 FLN29 gene product 3.7 NM_004091 E2F transcription factor 2 3.7 AI291989 glucosidase, beta (bile acid) 2 3.7 NM_006736 DnaJ (Hsp40) homolog, subfamily B, member 2 3.7 X60188 mitogen-activated protein kinase 3 3.7 AK027071 transforming growth factor beta-stimulated protein TSC-22 3.7 NM_006268 requiem, apoptosis response zinc finger gene 3.7 NM_016202 LDL induced EC protein 3.7 BI830259 hypothetical protein MGC26914 3.7 NM_001554 cysteine-rich, angiogenic inducer, 61 Homo sapiens transcribed sequence with weak similarity to protein ref:NP_060265.1 3.6 AU155612 (H.sapiens) hypothetical protein FLJ20378 [Homo sapiens] gb:AF129756 /DB_XREF=gi:4337095 /FEA=DNA_11 /CNT=98 /TID=Hs.247478.0 /TIER=Stack /STK=33 /UG=Hs.247478 /UG_TITLE=Homo sapiens MSH55 gene, partial cds; and CLIC1, DDAH, G6b, G6c, G5b, G6d, G6e, G6f, BAT5, G5b, CSK2B, BAT4, G4, 3.6 AF129756 Apo M, BAT3, BAT2, AIF-1, 1C7, LST-1, LTB, TNF, and LTA genes, complete cds /DEF=Homo sapiens MSH55 gene, partial cds; and CLIC1, DDAH, G6b, G6c, G5b, G6d, G6e, G6f, BAT5, G5b, CSK2B, BAT4, G4, Apo M, BAT3, BAT2, AIF-1, 1C7, LST-1, LTB, TNF, and LTA genes, complete cds 3.6 NM_145811 calcium channel, voltage-dependent, gamma subunit 5 3.6 AA741072 Homo sapiens transcribed sequences 3.6 NM_019058 HIF-1 responsive RTP801 3.6 NM_002640 serine (or cysteine) proteinase inhibitor, clade B (ovalbumin), member 8 3.6 AF250311 chromosome 20 open reading frame 97

132

Table 19 continued.

Fold GenBank ID Description Change 3.6 AI189753 transmembrane 4 superfamily member 1 3.6 AW007238 hypothetical protein FLJ38663 3.6 BF114906 LOC350381 3.6 AA971496 thiopurine S-methyltransferase 3.6 AI685845 LOC346972 gb:AL136306 /DB_XREF=gi:10045289 /FEA=DNA_1 /CNT=1 /TID=Hs.307102.0 /TIER=ConsEnd /STK=0 /UG=Hs.307102 /UG_TITLE=Human DNA sequence from clone RP3-334F4 on chromosome 6 Contains ESTs, STSs and GSSs. Contains a LAMR1 (laminin 3.6 AL136306 receptor 1, ribosomal protein SA) pseudogene and an RPL10 (ribosomal protein L10) pseudogene /DEF=Human DNA sequence from clone RP3-334F4 on chromosome 6 Contains ESTs, STSs and GSSs. Contains a LAMR1 (laminin receptor 1, ribosomal protein SA) pseudogene and an RPL10 (ribosomal protein L10) pseudogene 3.5 NM_017482 adducin 2 (beta) 3.5 AA532807 hypothetical protein FLJ23420 3.5 AI676056 map kinase phosphatase-like protein MK-STYX 3.5 NM_001643 apolipoprotein A-II 3.5 AK094122 Homo sapiens cDNA FLJ36803 fis, clone ADRGL2008966. 3.5 AW139048 ADP-ribosylation factor 1 3.5 AA447560 Homo sapiens, clone IMAGE:5295746, mRNA, partial cds 3.5 NM_002229 jun B proto-oncogene similar to Mycoplasma genitalium. lipoamide dehydrogenase component (E3) of pyruvate 3.5 AB014766 dehydrogenase; Homo sapiens mRNA for DERP12 (dermal papilla derived protein 12), complete cds. 3.5 AI360832 Homo sapiens, clone IMAGE:3660074, mRNA 3.5 NM_017450 BAI1-associated protein 2 3.5 BC016798 hypothetical gene supported by BC039507 3.4 AL533103 hypothetical protein MGC16037 3.4 NM_022152 PP1201 protein 3.4 NM_025182 hypothetical protein FLJ11560 3.4 NM_001902 cystathionase (cystathionine gamma-lyase) 3.4 AI925572 tripartite motif-containing 8 3.4 NM_001924 growth arrest and DNA-damage-inducible, alpha 3.4 NM_014371 neighbor of A-kinase anchoring protein 95 3.4 NM_021724 nuclear receptor subfamily 1, group D, member 1 3.4 AI922797 H2A histone family, member Y2 3.4 NM_003330 thioredoxin reductase 1 au87h07.y1 Schneider fetal brain 00004 Homo sapiens cDNA clone IMAGE:2783293 5' 3.4 AW162846 similar to TR:Q9Z2N6 Q9Z2N6 CAM-KII INHIBITORY PROTEIN. ;, mRNA sequence. 3.4 NM_001661 ADP-ribosylation factor 4-like 3.4 BC005174 activating transcription factor 5 qv38c04.x1 NCI_CGAP_Ov31 Homo sapiens cDNA clone IMAGE:1983846 3', mRNA 3.4 AI251399 sequence. 3.4 NM_004565 peroxisomal biogenesis factor 14 3.3 AB066566 activating transcription factor 3 3.3 AL136606 hypothetical protein FLJ12886 3.3 AL136786 hypothetical protein DKFZp434A1319 3.3 NM_006623 phosphoglycerate dehydrogenase 3.3 AK096324 hypothetical protein FLJ39005 3.3 AA626142 protein kinase C, epsilon 3.3 BF792126 acheron

133

Table 19 continued.

Fold GenBank ID Description Change gb:AC007766 /DB_XREF=gi:5030437 /FEA=DNA_2 /CNT=1 /TID=Hs.128417.1 /TIER=ConsEnd /STK=0 /UG=Hs.128417 /LL=79816 /UG_GENE=FLJ14009 3.3 AC007766 /UG_TITLE=hypothetical protein FLJ14009 /DEF=Homo sapiens chromosome 19, cosmid R26610 3.3 NM_020119 zinc finger antiviral protein 3.3 AA083483 ferritin, heavy polypeptide 1 3.3 AF217514 chromosome 20 open reading frame 111 3.3 BC000487 POM (POM121 homolog, rat) and ZP3 fusion 3.3 AW736788 YC12 gastric carcinoma cell SGC7901 Homo sapiens cDNA, mRNA sequence. 3.3 BG491844 v-jun sarcoma virus 17 oncogene homolog (avian) 3.3 AI740541 Homo sapiens transcribed sequences 3.3 NM_021158 chromosome 20 open reading frame 97 3.3 AI091372 AXIN1 up-regulated 1 3.2 AL537707 putative translation initiation factor 3.2 BE877420 Homo sapiens transcribed sequences 3.2 AF520796 peptidase (mitochondrial processing) beta 3.2 NM_002061 glutamate-cysteine ligase, modifier subunit 3.2 AI023634 similar to junction-mediating and regulatory protein p300 JMY 3.2 BC017122 Homo sapiens, clone IMAGE:3921486, mRNA 3.2 NM_016639 tumor necrosis factor receptor superfamily, member 12A 3.2 AI300077 Homo sapiens transcribed sequences 3.2 NM_019116 similar to ubiquitin binding protein 3.2 NM_153708 hypothetical protein MGC35450 3.2 AF218451 breast cancer anti-estrogen resistance 1 3.2 BC022827 crystallin, zeta (quinone reductase)-like 1 3.2 NM_017853 hypothetical protein FLJ20511 3.2 AI189359 mevalonate (diphospho) decarboxylase 3.2 NM_001421 E74-like factor 4 (ets domain transcription factor) 3.2 AU146742 Homo sapiens cDNA FLJ11986 fis, clone HEMBB1001364. 3.2 AW184034 v-raf murine sarcoma viral oncogene homolog B1 aldo-keto reductase family 1, member C1 (dihydrodiol dehydrogenase 1; 20-alpha (3-alpha)- 3.2 NM_001353 hydroxysteroid dehydrogenase) 3.2 W67644 putative translation initiation factor Cluster Incl. X72631:H.sapiens mRNA encoding Rev-ErbAalpha /cds=UNKNOWN 3.2 4791857 /gb=X72631 /gi=732801 /ug=Hs.211606 /len=2335 3.2 NM_144703 chromosome 20 open reading frame 40 3.2 N39314 hypothetical protein similar to CG7943 3.1 U10473 UDP-Gal:betaGlcNAc beta 1,4- galactosyltransferase, polypeptide 1 3.1 H07100 Homo sapiens transcribed sequences 3.1 AI814405 junction-mediating and regulatory protein 3.1 NM_024620 hypothetical protein FLJ12586 3.1 AF083441 putative translation initiation factor 3.1 NM_000224 keratin 18 3.1 NM_138999 neuropilin (NRP) and tolloid (TLL)-like 1 601658585R1 NIH_MGC_69 Homo sapiens cDNA clone IMAGE:3885812 3', mRNA 3.1 BE964655 sequence. 3.1 AF332238 testis-specific transcript, Y-linked 9 3.1 NM_001706 B-cell CLL/lymphoma 6 (zinc finger protein 51) 3.1 AL561296 E2F transcription factor 2 3.1 BF445047 epithelial membrane protein 1

134

Table 19 continued.

Fold GenBank ID Description Change 3.1 BE300521 insulin induced gene 1 3.1 NM_004417 dual specificity phosphatase 1 3.1 NM_003897 immediate early response 3 3.1 AW190316 NADH:ubiquinone oxidoreductase MLRQ subunit homolog 3.1 NM_024322 hypothetical protein MGC11266 3.1 AK098276 likely ortholog of mouse C114 dsRNA-binding protein 3.1 NM_021070 latent transforming growth factor beta binding protein 3 3.1 BC006151 MGC13170 gene 3.1 H10318 HLA-B associated transcript 4 3.0 BC028196 similar to tre-2/USP6, BUB2, cdc16) domain family, member 1; tbc1 3.0 BC033939 Homo sapiens, clone IMAGE:5271452, mRNA 3.0 NM_003246 thrombospondin 1 3.0 NM_001423 epithelial membrane protein 1 3.0 BG251266 FOS-like antigen 1 3.0 NM_024106 zinc finger protein 426 3.0 AA576961 pleckstrin homology-like domain, family A, member 1 3.0 NM_006186 nuclear receptor subfamily 4, group A, member 2 3.0 R45656 hypothetical protein FLJ30626 3.0 U79283 D site of albumin promoter (albumin D-box) binding protein 3.0 AW135616 Homo sapiens transcribed sequences 3.0 BC000514 ribosomal protein L13a 3.0 AK022050 heterogeneous nuclear ribonucleoprotein M Cluster Incl. AI962879:wt24c06.x1 Homo sapiens cDNA, 3 end /clone=IMAGE-2508394 3.0 4849388_RC /clone_end=3' /gb=AI962879 /gi=5755592 /ug=Hs.81920 /len=490 3.0 NM_003811 tumor necrosis factor (ligand) superfamily, member 9 3.0 NM_015517 MBD2 (methyl-CpG-binding protein)-interacting zinc finger protein 3.0 NM_017848 hypothetical protein FLJ20506 3.0 BC005127 adipose differentiation-related protein 3.0 NM_000713 biliverdin reductase B (flavin reductase (NADPH)) 3.0 W65310 tumor necrosis factor receptor superfamily, member 10a 3.0 NM_003844 tumor necrosis factor receptor superfamily, member 10a 3.0 M90657 Human tumor antigen (L6) mRNA, complete cds. hw08a05.x1 NCI_CGAP_Lu24 Homo sapiens cDNA clone IMAGE:3182288 3' similar to 3.0 BE327172 contains element MSR1 repetitive element ;, mRNA sequence. 3.0 NM_002852 pentaxin-related gene, rapidly induced by IL-1 beta 3.0 NM_003979 retinoic acid induced 3 3.0 NM_006622 serum-inducible kinase 3.0 BC016404 Homo sapiens, clone IMAGE:4183253, mRNA 3.0 AL110298 solute carrier family 2 (facilitated glucose transporter), member 14 3.0 NM_020386 HRAS-like suppressor -5.0 AI745662 KIAA1596 protein -5.0 AA701657 leukemia inhibitory factor receptor -5.0 NM_000052 ATPase, Cu++ transporting, alpha polypeptide (Menkes syndrome) -5.0 AL137364 hypothetical protein MGC24039 -5.0 BE501352 myc-induced nuclear antigen, 53 kDa -5.0 AW665832 hypothetical protein DKFZp434G1415 Homo sapiens, Similar to GLI-Kruppel family member GLI3 (Greig cephalopolysyndactyly -5.0 BC032660 syndrome), clone IMAGE:5532335, mRNA -5.0 AL046419 TIA1 cytotoxic granule-associated RNA binding protein -5.0 AF306508 SUMO-1-specific protease

135

Table 19 continued.

Fold GenBank ID Description Change -5.0 BF110993 translocated promoter region (to activated MET oncogene) -5.0 AI831506 Homo sapiens transcribed sequences -5.0 AL833487 Homo sapiens mRNA; cDNA DKFZp686H1629 (from clone DKFZp686H1629) -5.0 AI201248 zinc finger protein 207 -5.0 NM_014932 neuroligin 1 -5.0 BF439570 optic atrophy 1 (autosomal dominant) -5.0 BF435752 CGI-77 protein -5.0 AI760130 protein phosphatase 2 (formerly 2A), regulatory subunit B'', alpha -5.0 AI252087 PC4 and SFRS1 interacting protein 2 -5.0 AA047225 Homo sapiens transcribed sequences -5.0 AI093579 integrin, alpha V (vitronectin receptor, alpha polypeptide, antigen CD51) -5.0 AW299250 hypothetical protein LOC90624 -5.0 BC013250 PTD002 protein -5.0 NM_153243 hypothetical protein MGC26143 -5.0 NM_017718 hypothetical protein FLJ20220 602362764F1 NIH_MGC_90 Homo sapiens cDNA clone IMAGE:4471166 5', mRNA -5.0 BG250585 sequence. -5.0 AB046813 DKFZP564G092 protein -5.0 AK001728 KIAA1033 protein -5.0 AB037785 flavoprotein oxidoreductase MICAL3 -5.0 BC011266 hypothetical protein BC011266 -5.1 AA204752 syntaxin binding protein 5 (tomosyn) -5.1 AW235355 translocated promoter region (to activated MET oncogene) -5.1 BF590675 hypothetical protein FLJ14281 -5.1 AF360549 BRCA1 interacting protein C-terminal helicase 1 -5.1 AL832759 Homo sapiens mRNA; cDNA DKFZp686E1027 (from clone DKFZp686E1027) Cluster Incl. U90268:Human Krit1 mRNA, complete cds /cds=(25,1614) /gb=U90268 -5.1 4841257 /gi=2149601 /ug=Hs.93810 /len=1986 -5.1 AW157712 hypothetical protein MGC39633 -5.1 AK001672 KIAA1596 protein -5.1 AW962413 EST374486 MAGE resequences, MAGG Homo sapiens cDNA, mRNA sequence. 601435734F1 NIH_MGC_72 Homo sapiens cDNA clone IMAGE:3920600 5', mRNA -5.1 BE892889 sequence. -5.1 BE502765 hypothetical protein LOC144874 -5.1 AK001846 Homo sapiens cDNA FLJ10984 fis, clone PLACE1001810. -5.1 AA029155 mannosidase, alpha, class 2A, member 1 -5.1 AL050065 HMT1 hnRNP methyltransferase-like 1 (S. cerevisiae) -5.1 AW450329 Homo sapiens cDNA FLJ36584 fis, clone TRACH2013450. -5.1 AU150728 zinc finger protein 267 -5.1 AI745136 similar to Chic1 -5.1 AL049265 interleukin 6 signal transducer (gp130, oncostatin M receptor) -5.1 AJ278112 hypothetical protein FLJ20354 AL515269 Homo sapiens NEUROBLASTOMA Homo sapiens cDNA clone CL0BB016ZA10 -5.1 AL515269 3-PRIME, mRNA sequence. -5.1 AV723984 Homo sapiens transcribed sequences -5.1 BF248364 AF15q14 protein -5.1 U49844 ataxia telangiectasia and Rad3 related -5.1 U80082 KIAA0826 protein -5.1 AA479492 Homo sapiens transcribed sequences -5.1 AV727934 phosphoglucomutase 3

136

Table 19 continued.

Fold GenBank ID Description Change -5.1 AL038450 Homo sapiens transcribed sequences -5.1 AI627965 LOC347872 yw73g03.s1 Soares_placenta_8 to 9weeks_2NbHP8to9W Homo sapiens cDNA clone -5.1 N29672 IMAGE:257908 3', mRNA sequence. -5.2 NM_013269 lectin-like NK cell receptor Homo sapiens transcribed sequence with weak similarity to protein ref:NP_060312.1 -5.2 AI560205 (H.sapiens) hypothetical protein FLJ20489 [Homo sapiens] -5.2 BG028770 hypothetical protein DKFZp566D1346 -5.2 BF445343 chronic myelogenous leukemia tumor antigen 66 -5.2 AA464844 Homo sapiens, clone IMAGE:5295779, mRNA -5.2 AF269167 hypothetical protein FLJ20364 -5.2 BF218922 chondroitin sulfate proteoglycan 2 (versican) -5.2 AB020663 rabconnectin-3 -5.2 NM_017577 hypothetical protein DKFZp434C0328 602294256F1 NIH_MGC_86 Homo sapiens cDNA clone IMAGE:4388988 5', mRNA -5.2 BG026789 sequence. -5.2 AF063591 antigen identified by monoclonal antibody MRC OX-2 gb:BC002836.1 /DB_XREF=gi:12803974 /FEA=FLmRNA /CNT=1 /TID=Hs.1422.1 /TIER=FL /STK=0 /UG=Hs.1422 /LL=2268 /UG_GENE=FGR /DEF=Homo sapiens, -5.2 BC002836 Gardner-Rasheed feline sarcoma viral (v-fgr) oncogene homolog, clone MGC:3553, mRNA, complete cds. /PROD=Gardner-Rasheed feline sarcoma viral (v-fgr) oncogene homolog /FL=gb:BC002836.1 -5.2 AL080144 ELYS transcription factor-like protein TMBS62 -5.2 AB017445 X-ray repair complementing defective repair in Chinese hamster cells 4 -5.2 AI694023 thyroid hormone receptor interactor 8 -5.2 NM_001270 chromodomain helicase DNA binding protein 1 -5.2 AA701247 DKFZP564A2416 protein -5.2 AI636080 mucolipin 3 -5.3 AI184562 U2-associated SR140 protein -5.3 W42665 Homo sapiens transcribed sequences -5.3 AW513477 hypothetical protein FLJ14297 -5.3 BC036461 nuclear receptor coactivator 7 -5.3 NM_014783 KIAA0013 gene product -5.3 BG434893 PMS1 postmeiotic segregation increased 1 (S. cerevisiae) -5.3 AI681025 Homo sapiens transcribed sequences -5.3 NM_005760 CCAAT-box-binding transcription factor -5.3 AB023173 ATPase, Class VI, type 11B -5.3 U72937 alpha thalassemia/mental retardation syndrome X-linked (RAD54 homolog, S. cerevisiae) -5.3 BF509391 SEC10-like 1 (S. cerevisiae) -5.3 D32039 chondroitin sulfate proteoglycan 2 (versican) -5.3 AB037795 KIAA1374 protein -5.3 D26069 centaurin, beta 2 -5.3 NM_000110 dihydropyrimidine dehydrogenase -5.3 AA651899 ER-resident protein ERdj5 -5.3 NM_017645 hypothetical protein FLJ20060 -5.4 R20640 Homo sapiens transcribed sequences -5.4 BC029480 Homo sapiens, clone MGC:32876 IMAGE:4734912, mRNA, complete cds -5.4 BC014318 Homo sapiens, clone IMAGE:3684608, mRNA -5.4 AW292751 KIAA0725 protein -5.4 AI700768 Homo sapiens transcribed sequences

137

Table 19 continued.

Fold GenBank ID Description Change -5.4 AK000864 Homo sapiens cDNA FLJ10002 fis, clone HEMBA1000046. -5.4 AK001538 AND-1 protein -5.4 AW299558 Homo sapiens, clone IMAGE:5288833, mRNA continued from dJ881E24.1.1 in Em:AL031683 match: proteins: Sw:P10687 Tr:Q9Z1B3 Sw:P10894; Human DNA sequence from clone RP4-654A7 on chromosome 20 Contains the -5.4 AL049593 3' end of two variants of the PLCB1 gene for phospholipase C beta 1 (phosphoinositide- specific), complete sequence. Homo sapiens transcribed sequence with weak similarity to protein ref:NP_060312.1 -5.4 AW340891 (H.sapiens) hypothetical protein FLJ20489 [Homo sapiens] -5.4 AW003635 Alstrom syndrome 1 Homo sapiens transcribed sequence with weak similarity to protein ref:NP_060265.1 -5.4 AI652899 (H.sapiens) hypothetical protein FLJ20378 [Homo sapiens] -5.4 AL360145 Homo sapiens mRNA full length insert cDNA clone EUROIMAGE 839551. -5.4 NM_014673 KIAA0103 gene product -5.4 AI758317 Homo sapiens transcribed sequences -5.4 AU157224 hypothetical protein DKFZp434D2328 -5.5 AA045174 Homo sapiens mRNA; cDNA DKFZp586B211 (from clone DKFZp586B211) -5.5 AA083478 tripartite motif-containing 22 -5.5 AL049923 oxysterol binding protein-like 8 -5.5 AI680874 hypothetical protein FLJ23861 -5.5 AI810054 hypothetical protein FLJ20354 -5.5 BG484789 programmed cell death 6 interacting protein -5.5 AA045204 mitogen-activated protein kinase kinase kinase 2 -5.5 BF446673 hemicentin -5.5 D83077 tetratricopeptide repeat domain 3 -5.5 NM_024624 SMC6 structural maintenance of chromosomes 6-like 1 (yeast) -5.6 AL559474 hypothetical protein FLJ23047 -5.6 AA758751 hypothetical protein FLJ32949 -5.6 AF047033 solute carrier family 4, sodium bicarbonate cotransporter, member 7 -5.6 AA747287 Homo sapiens transcribed sequences -5.6 AF008915 Homo sapiens EVI5 homolog mRNA, complete cds -5.6 NM_005570 lectin, mannose-binding, 1 -5.6 U12170 transcription factor 8 (represses interleukin 2 expression) -5.6 NM_003316 tetratricopeptide repeat domain 3 -5.6 NM_005767 purinergic receptor P2Y, G-protein coupled, 5 -5.6 NM_005739 RAS guanyl releasing protein 1 (calcium and DAG-regulated) -5.6 AU145365 Homo sapiens cDNA FLJ11662 fis, clone HEMBA1004629. -5.6 AA521504 Homo sapiens transcribed sequences -5.6 AI828221 SNF2 histone linker PHD RING helicase -5.6 AI807408 transcription factor MLR1 -5.6 BF979668 hypothetical protein FLJ25795 -5.6 AI743588 ankyrin 3, node of Ranvier (ankyrin G) -5.6 AL136821 KIAA0701 protein -5.6 BF940270 KIAA1999 protein -5.7 BE503286 hypothetical protein FLJ20793 -5.7 AV699744 KIAA0650 protein -5.7 BF435809 cullin 5 -5.7 AI028528 Homo sapiens transcribed sequences unnamed protein product; Homo sapiens cDNA FLJ13765 fis, clone PLACE4000128, weakly -5.7 AK023827 similar to Mus musculus putative transcription factor mRNA.

138

Table 19 continued.

Fold GenBank ID Description Change -5.7 AW192700 LOC346584 -5.7 AW117264 Homo sapiens transcribed sequences -5.7 AW451086 Homo sapiens transcribed sequences -5.7 AW081113 SR rich protein -5.7 AL136588 hypothetical protein DKFZp761D112 -5.7 NM_002645 phosphoinositide-3-kinase, class 2, alpha polypeptide -5.7 AW264125 FLJ10378 protein -5.7 BG403671 THO complex 2 -5.7 AW157501 SR rich protein -5.7 BF223237 zinc finger protein 292 -5.7 AL049311 Homo sapiens mRNA; cDNA DKFZp564B226 (from clone DKFZp564B226). -5.7 BF508634 HSPB (heat shock 27kDa) associated protein 1 -5.7 AW044631 Rho GTPase activating protein 5 -5.8 NM_030631 solute carrier family 25 (mitochondrial oxodicarboxylate carrier), member 21 -5.8 BF062244 lin-7 homolog A (C. elegans) -5.8 AI908188 TRK-fused gene -5.8 NM_005732 RAD50 homolog (S. cerevisiae) -5.8 AF260333 hypothetical protein DKFZp434L142 -5.8 NM_018098 epithelial cell transforming sequence 2 oncogene -5.8 NM_016248 A kinase (PRKA) anchor protein 11 -5.8 NM_015384 IDN3 protein -5.8 NM_019012 phosphoinositol 3-phosphate-binding protein-2 -5.8 AI885338 tetratricopeptide repeat domain 3 -5.8 AI823905 hypothetical protein FLJ20333 -5.8 AW592266 v-myb myeloblastosis viral oncogene homolog (avian)-like 1 -5.8 AI732587 Homo sapiens EST from clone 60991, 5' end -5.9 AI983837 protein phosphatase 4, regulatory subunit 2 -5.9 AW591660 TATA element modulatory factor 1 -5.9 BC042069 Homo sapiens mRNA; cDNA DKFZp686N23117 (from clone DKFZp686N23117) -5.9 AA608855 down-regulated by Ctnnb1 -5.9 AI814728 RecQ protein-like (DNA helicase Q1-like) -5.9 AI373676 chondroitin sulfate proteoglycan 6 (bamacan) -5.9 AI656807 PRO2000 protein -5.9 NM_006444 SMC2 structural maintenance of chromosomes 2-like 1 (yeast) -5.9 BF939551 transient receptor potential cation channel, subfamily M, member 7 -5.9 AW965339 tripin -5.9 AA960844 Homo sapiens, clone IMAGE:4081483, mRNA -5.9 AI953360 lysophospholipase-like 1 -5.9 BE350312 Homo sapiens transcribed sequences -5.9 AA501453 hippocampus abundant gene transcript 1 -5.9 AW071458 Homo sapiens cDNA FLJ36663 fis, clone UTERU2002826. -5.9 BG261090 hypothetical protein FLJ22728 -5.9 BF056095 hypothetical protein MGC29956 -6.0 AI685944 RecQ protein-like (DNA helicase Q1-like) -6.0 BF062175 chromosome 14 open reading frame 106 -6.0 AI686936 Homo sapiens cDNA FLJ39245 fis, clone OCBBF2008366. -6.0 BF062139 polymerase (RNA) III (DNA directed) (32kD) Homo sapiens transcribed sequence with weak similarity to protein pir:T02670 (H.sapiens) -6.0 AU147903 T02670 probable thromboxane A2 receptor isoform beta - human -6.0 AU152088 U2-associated SR140 protein

139

Table 19 continued.

Fold GenBank ID Description Change -6.0 NM_005496 SMC4 structural maintenance of chromosomes 4-like 1 (yeast) -6.0 AW979271 Homo sapiens transcribed sequences -6.0 AI935415 Tax1 (human T-cell leukemia virus type I) binding protein 1 -6.0 AI038615 Homo sapiens transcribed sequences -6.0 BF577193 similar to F10G7.10.p -6.0 AB011108 PRP4 pre-mRNA processing factor 4 homolog B (yeast) -6.0 NM_014810 centrosome-associated protein 350 -6.0 BC031044 DnaJ (Hsp40) homolog, subfamily A, member 4 -6.0 BF513121 Homo sapiens, clone IMAGE:4794726, mRNA -6.0 AA769995 Homo sapiens transcribed sequences -6.0 AI275605 phosphatidylinositol glycan, class K Homo sapiens transcribed sequence with weak similarity to protein ref:NP_060312.1 -6.0 R16784 (H.sapiens) hypothetical protein FLJ20489 [Homo sapiens] -6.1 AB002391 D15F37 (pseudogene) -6.1 BF056459 Human retinal pigment epithelium mRNA. -6.1 NM_024696 hypothetical protein FLJ23058 -6.1 AK025191 Homo sapiens cDNA: FLJ21538 fis, clone COL06151. -6.1 AA843541 Homo sapiens transcribed sequences Homo sapiens transcribed sequence with strong similarity to protein pdb:1BGM (E. coli) O -6.1 AI097095 Chain O, Beta-Galactosidase (Chains I-P) -6.1 D42063 RAN binding protein 2 -6.1 AK023668 PTB domain adaptor protein CED-6 -6.1 AK024910 Homo sapiens cDNA: FLJ21257 fis, clone COL01407. -6.1 AA160181 HDCMA18P protein -6.1 AI763431 Homo sapiens transcribed sequences -6.1 AB051495 kinesin family member 21A -6.1 BF688144 alkylglycerone phosphate synthase -6.1 BF224151 interleukin-1 receptor-associated kinase 1 binding protein 1 -6.1 NM_015153 PHD finger protein 3 -6.1 AI953914 Homo sapiens transcribed sequences -6.1 BG287503 ADMP -6.1 BF055107 S164 protein -6.2 NM_016195 M-phase phosphoprotein 1 -6.2 AK054730 KIAA0107 gene product -6.2 AW501507 KIAA1911 protein Homo sapiens transcribed sequence with weak similarity to protein ref:NP_009032.1 -6.2 AI452469 (H.sapiens) sarcosine dehydrogenase; dimethylglycine dehydrogenase-like 1 [Homo sapiens] -6.2 BF218804 hypothetical protein FLJ20986 -6.2 NM_020890 KIAA1524 protein -6.2 BF673049 restin (Reed-Steinberg cell-expressed intermediate filament-associated protein) -6.2 BG109597 PRKC, apoptosis, WT1, regulator -6.2 AB020631 PCF11p homolog -6.2 AV683221 hypothetical protein BC014148 -6.2 AB014578 KIAA0678 protein -6.2 AI983886 zinc finger protein 254 -6.2 BC040740 Munc13-3 -6.2 BF435259 KIAA1349 protein -6.2 BF031819 hypothetical protein FLJ25795 -6.2 U09820 alpha thalassemia/mental retardation syndrome X-linked (RAD54 homolog, S. cerevisiae) -6.2 AL117598 Homo sapiens, clone IMAGE:5312754, mRNA

140

Table 19 continued.

Fold GenBank ID Description Change UI-H-BW0-ajs-b-03-0-UI.s1 NCI_CGAP_Sub6 Homo sapiens cDNA clone IMAGE:2732860 -6.3 AW298092 3', mRNA sequence. Homo sapiens transcribed sequence with weak similarity to protein ref:NP_009056.1 -6.3 AU146949 (H.sapiens) ubiquitously transcribed tetratricopeptide repeat gene, Y chromosome; Ubiquitously transcribed TPR gene on Y chromosome [Homo sapiens] cloned using degenerate PCR primers representing conserved protein kinase subdomains VII -6.3 Z25435 and IX; encodes an open reading frame between subdomains VII and IX of protein kinase catalytic domain; H.sapiens protein-serine/threonine kinase gene, complete CDS. -6.3 BF244214 Homo sapiens transcribed sequences -6.3 AL359571 ninein (GSK3B interacting protein) -6.3 AI950314 HIV-1 rev binding protein 2 Homo sapiens transcribed sequence with strong similarity to protein ref:NP_060840.1 -6.3 AA724995 (H.sapiens) hypothetical protein FLJ11259 [Homo sapiens] -6.3 NM_003693 scavenger receptor class F, member 1 -6.3 AF245436 hypothetical protein FLJ23518 -6.3 AL110163 Homo sapiens, clone IMAGE:5259584, mRNA -6.3 BF207870 Homo sapiens transcribed sequences -6.4 AW235548 hypothetical gene supported by AK090859 -6.4 NM_001812 centromere protein C 1 -6.4 AW629014 chorea acanthocytosis -6.4 AI197932 Homo sapiens transcribed sequences -6.4 AW294894 hypothetical protein FLJ21924 -6.4 BC030757 Homo sapiens, clone IMAGE:4797534, mRNA, partial cds -6.4 AL832339 hypothetical protein MGC50559 -6.4 AW296050 hypothetical protein FLJ10808 -6.4 BC012936 Homo sapiens, clone IMAGE:4454258, mRNA -6.4 AI821935 Homo sapiens transcribed sequences -6.4 W87688 putative dimethyladenosine transferase -6.4 AL162039 Mob4A protein -6.4 BF056048 similar to CG3073 gene product -6.4 NM_018844 B-cell receptor-associated protein BAP29 -6.4 AA873021 Homo sapiens transcribed sequences -6.5 BC005400 leucine zipper protein FKSG14 -6.5 NM_002312 ligase IV, DNA, ATP-dependent -6.5 N21475 putative dimethyladenosine transferase amylo-1, 6-glucosidase, 4-alpha-glucanotransferase (glycogen debranching enzyme, glycogen -6.5 NM_000645 storage disease type III) -6.5 AF346629 transient receptor potential cation channel, subfamily M, member 7 zl50d02.s1 Soares_pregnant_uterus_NbHPU Homo sapiens cDNA clone IMAGE:505347 3', -6.5 AA147933 mRNA sequence. -6.5 AI669022 Homo sapiens transcribed sequences -6.6 NM_006346 progesterone-induced blocking factor 1 -6.6 BF475862 hypothetical protein FLJ34233 -6.6 BC027179 tyrosinase (oculocutaneous albinism IA) -6.6 NM_024920 hypothetical protein FLJ14281 gb:BC030966.1 /DB_XREF=gi:21410194 /TID=Hs2.170098.2 /CNT=9 /FEA=FLmRNA /TIER=FL /STK=6 /LL=9652 /UG_GENE=KIAA0372 /UG=Hs.170098 /DEF=Homo sapiens, -6.6 BC030966 Similar to KIAA0372 gene product, clone MGC:32587 IMAGE:4108519, mRNA, complete cds. /PROD=Similar to KIAA0372 gene product /FL=gb:BC030966.1 -6.6 BE466077 Homo sapiens cDNA FLJ12786 fis, clone NT2RP2001936.

141

Table 19 continued.

Fold GenBank ID Description Change -6.6 AA489100 Homo sapiens cDNA FLJ34866 fis, clone NT2NE2014113. -6.6 AI798790 bullous pemphigoid antigen 1, 230/240kDa -6.6 NM_016122 NY-REN-58 antigen Homo sapiens transcribed sequence with moderate similarity to protein ref:NP_060265.1 -6.6 AI651255 (H.sapiens) hypothetical protein FLJ20378 [Homo sapiens] -6.6 AB046824 KIAA1604 protein -6.6 AK000323 DKFZP434D193 protein Homo sapiens transcribed sequence with weak similarity to protein ref:NP_071431.1 -6.6 BI713506 (H.sapiens) cytokine receptor-like factor 2; cytokine receptor CRL2 precusor [Homo sapiens] -6.6 AI334015 Homo sapiens transcribed sequences -6.7 AK095307 F-box only protein 9 Homo sapiens transcribed sequence with weak similarity to protein ref:NP_060312.1 -6.7 N51102 (H.sapiens) hypothetical protein FLJ20489 [Homo sapiens] -6.7 NM_014648 zinc finger DAZ interacting protein 3 -6.7 AB023203 chorea acanthocytosis -6.7 AI076172 Homo sapiens transcribed sequences -6.7 AI760464 CDA14 -6.7 BG538482 hypothetical protein BC022889 -6.7 BF977231 hypothetical protein LOC283464 -6.7 U79271 serologically defined colon cancer antigen 8 -6.7 AU153330 RNA helicase family Human DNA sequence from clone RP3-393D12 on chromosome 6q16.1-16.3, complete -6.7 AL132776 sequence. -6.7 NM_002912 REV3-like, catalytic subunit of DNA polymerase zeta (yeast) -6.7 R41296 DKFZP434D193 protein -6.7 BF109303 hypothetical protein LOC283508 -6.8 AI002966 hypothetical protein LOC57821 -6.8 AI138934 ELYS transcription factor-like protein TMBS62 -6.8 AF323119 nuclear factor (erythroid-derived 2)-like 2 -6.8 AL119491 hypothetical gene supported by AF086452 -6.8 AL049325 cerebral cavernous malformations 1 -6.8 BC041348 hypothetical protein FLJ35954 -6.8 NM_031217 kinesin family member 18A -6.8 BC041921 Homo sapiens, clone IMAGE:5300163, mRNA -6.8 AI978754 musashi homolog 2 (Drosophila) -6.9 BC036049 chromosome 6 open reading frame 157 -6.9 AI561173 interleukin-1 receptor-associated kinase 1 binding protein 1 -6.9 AW024890 Homo sapiens transcribed sequences -6.9 BE048628 ubiquitin ligase mind bomb -6.9 AB028966 KIAA1043 protein -6.9 AL043646 serine/threonine kinase 18 -6.9 NM_002345 lumican -6.9 AI962943 hypothetical protein FLJ22028 -6.9 AA969238 hypothetical protein LOC158257 -6.9 AL136820 KIAA1411 protein -6.9 AI130715 Homo sapiens transcribed sequences -6.9 N63551 hypothetical protein FLJ22728 -7.0 AI912618 hypothetical protein LOC286170 -7.0 AI587307 mannosidase, endo-alpha -7.0 AW004016 beta-galactoside alpha-2,6-sialyltransferase II

142

Table 19 continued.

Fold GenBank ID Description Change -7.0 AB046794 hypothetical protein FLJ20060 -7.0 AA227879 Homo sapiens transcribed sequences -7.0 AL565362 solute carrier family 2 (facilitated glucose transporter), member 13 -7.1 NM_152460 hypothetical protein FLJ31882 -7.1 BE543064 mannosidase, alpha, class 1A, member 2 -7.1 AL042660 Homo sapiens transcribed sequences -7.1 AI435036 ubiquitin specific protease 16 -7.1 AI683802 Bardet-Biedl syndrome 7 -7.1 BF590263 chondroitin sulfate proteoglycan 2 (versican) -7.1 AI302244 KIAA1915 protein -7.1 NM_000885 integrin, alpha 4 (antigen CD49D, alpha 4 subunit of VLA-4 receptor) -7.1 BF111821 SOCS box-containing WD protein SWiP-1 -7.1 AI829314 ligase IV, DNA, ATP-dependent -7.1 AB037736 CASP8 associated protein 2 -7.1 AK091575 hypothetical protein FLJ13305 -7.1 AW450549 Homo sapiens transcribed sequences -7.1 BU619319 Homo sapiens transcribed sequences -7.2 NM_016649 chromosome 20 open reading frame 6 -7.2 AA834576 Homo sapiens TIPR mRNA for inositol 1,4,5-trisphosphate receptor type 2, complete cds. -7.2 AI401379 phosphoinositide-3-kinase, class 2, alpha polypeptide Homo sapiens transcribed sequence with weak similarity to protein pir:I60307 (E. coli) -7.2 BF512183 I60307 beta-galactosidase, alpha peptide - Escherichia coli -7.2 AU155234 AU155234 OVARC1 Homo sapiens cDNA clone OVARC1001344 3', mRNA sequence. -7.2 AL565516 pantothenate kinase 3 gb:U82828 /DB_XREF=gi:2304970 /FEA=mRNA /CNT=113 /TID=Hs.194382.2 /TIER=Stack /STK=42 /UG=Hs.194382 /LL=472 /UG_GENE=ATM /UG_TITLE=ataxia -7.2 U82828 telangiectasia mutated (includes complementation groups A, C and D) /DEF=Homo sapiens ataxia telangiectasia (ATM) gene, complete cds -7.2 AW183154 kinesin family member 14 -7.3 BE326569 Homo sapiens, clone IMAGE:5259584, mRNA -7.3 BE566023 KIAA0372 gene product -7.3 BF221673 IDN3 protein -7.3 AU146384 Homo sapiens cDNA FLJ14056 fis, clone HEMBB1000335. -7.3 AW074143 Homo sapiens cDNA FLJ36189 fis, clone TESTI2027238. -7.3 BC040287 Homo sapiens, clone IMAGE:4819775, mRNA -7.3 AI963028 hypothetical protein FLJ90430 UI-H-BI2-age-h-01-0-UI.s1 NCI_CGAP_Sub4 Homo sapiens cDNA clone IMAGE:2724312 -7.4 AW207734 3', mRNA sequence. -7.4 NM_025191 chromosome 1 open reading frame 22 -7.4 AI082004 Homo sapiens transcribed sequences -7.4 AI478771 zinc finger protein 148 (pHZ-52) -7.4 AL133663 hypothetical protein from EUROIMAGE 2005326 -7.4 AI472310 potassium channel regulator -7.4 BF224436 Homo sapiens transcribed sequences -7.4 BG168666 ER-resident protein ERdj5 -7.4 X95152 H.sapiens brca2 gene exon 2 (and joined coding region). Homo sapiens transcribed sequence with weak similarity to protein ref:NP_071431.1 -7.4 AI436356 (H.sapiens) cytokine receptor-like factor 2; cytokine receptor CRL2 precusor [Homo sapiens] -7.4 AI742358 similar to hypothetical protein -7.5 AL136877 SMC4 structural maintenance of chromosomes 4-like 1 (yeast)

143

Table 19 continued.

Fold GenBank ID Description Change -7.5 NM_019002 ETAA16 protein -7.5 AA252512 hypothetical protein FLJ23861 bx18f09.x1 Human Iris cDNA (Un-normalized, unamplified): BX Homo sapiens cDNA clone -7.5 BF725688 bx18f09 3', mRNA sequence. -7.5 NM_006447 ubiquitin specific protease 16 -7.5 AL133101 Homo sapiens, clone IMAGE:5270591, mRNA -7.5 NM_016613 hypothetical protein DKFZp434L142 -7.5 U79286 HMT1 hnRNP methyltransferase-like 1 (S. cerevisiae) -7.5 AL833240 Homo sapiens mRNA; cDNA DKFZp761P2319 (from clone DKFZp761P2319) -7.5 NM_004986 kinectin 1 (kinesin receptor) -7.5 AW293174 Homo sapiens transcribed sequences -7.5 AU131711 tetratricopeptide repeat domain 3 -7.6 NM_020121 UDP-glucose ceramide glucosyltransferase-like 2 -7.6 BE500942 Homo sapiens mRNA; cDNA DKFZp761M0111 (from clone DKFZp761M0111) -7.6 AL080057 Homo sapiens mRNA; cDNA DKFZp564D032 (from clone DKFZp564D032) -7.6 AB040957 KIAA1524 protein -7.6 AA678564 Homo sapiens transcribed sequences Homo sapiens transcribed sequence with strong similarity to protein pdb:1BGM (E. coli) O -7.6 AW170044 Chain O, Beta-Galactosidase (Chains I-P) xr78h03.x2 NCI_CGAP_Lu26 Homo sapiens cDNA clone IMAGE:2766293 3' similar to -7.6 BE138647 TR:O13561 O13561 YLR169WP. ;, mRNA sequence. -7.6 AL390143 Homo sapiens mRNA; cDNA DKFZp547N074 (from clone DKFZp547N074) -7.7 NM_006614 cell adhesion molecule with homology to L1CAM (close homolog of L1) -7.7 AV729462 chromosome 20 open reading frame 80 -7.7 NM_018365 meiosis-specific nuclear structural protein 1 -7.7 AU155298 chromodomain helicase DNA binding protein 1 gb:NM_005196.1 /DB_XREF=gi:4885132 /GEN=CENPF /FEA=FLmRNA /CNT=3 /TID=Hs.77204.0 /TIER=FL /STK=0 /UG=Hs.77204 /LL=1063 /DEF=Homo sapiens -7.7 NM_005196 centromere protein F (350400kD, mitosin) (CENPF), mRNA. /PROD=centromere protein F (350400kD, mitosin) /FL=gb:NM_005196.1 gb:U19769.1 -7.7 AI148006 Homo sapiens transcribed sequences -7.8 AI393725 Homo sapiens transcribed sequences -7.8 AI634543 hypothetical protein LOC256112 -7.8 BE551138 type 1 tumor necrosis factor receptor shedding aminopeptidase regulator -7.8 NM_015032 androgen-induced proliferation inhibitor -7.8 AA398321 hypothetical protein FLJ36754 -7.8 BG532121 KIAA1911 protein -7.8 BE504653 DKFZP434D193 protein -7.9 R94644 chondroitin sulfate proteoglycan 2 (versican) -7.9 NM_015216 KIAA0433 protein -7.9 BC020926 Homo sapiens, clone IMAGE:4249347, mRNA gb:AL163248 /DB_XREF=gi:7717304 /FEA=mRNA /CNT=4 /TID=Hs.288773.2 /TIER=ConsEnd /STK=0 /UG=Hs.288773 /LL=26046 /UG_GENE=ZNF294 -8.0 AL163248 /UG_TITLE=zinc finger protein 294 /DEF=Homo sapiens chromosome 21 segment HS21C048 -8.0 NM_024641 mannosidase, endo-alpha -8.0 W93847 mucin 15 -8.0 AJ293392 opsin 3 (encephalopsin, panopsin) -8.1 N22548 Rho-associated, coiled-coil containing protein kinase 1 -8.1 AF247167 AD031 protein

144

Table 19 continued.

Fold GenBank ID Description Change -8.1 AB002330 U2-associated SR140 protein -8.2 AL137753 KIAA1033 protein -8.2 AA126789 KARP-1-binding protein -8.2 AU146081 Homo sapiens cDNA FLJ11854 fis, clone HEMBA1006767. -8.2 NM_014639 KIAA0372 gene product -8.2 AI535737 cong2.P5.a4 conorm Homo sapiens cDNA 3', mRNA sequence. -8.2 AK025247 KIAA0912 protein -8.2 BF970829 oxysterol binding protein-like 8 -8.3 AF017061 cullin 5 -8.3 AI277617 solute carrier family 25 (mitochondrial carrier; dicarboxylate transporter), member 10 -8.3 N38985 retinoblastoma-associated protein 140 -8.3 AW082208 hypothetical protein FLJ20618 -8.3 U80774 Human EST clone 53125 mariner transposon Hsmar1 sequence -8.3 AB014550 KIAA0650 protein -8.4 AW137073 Homo sapiens mRNA; cDNA DKFZp451M139 (from clone DKFZp451M139) -8.4 AF086473 Homo sapiens full length insert cDNA clone ZD88D08 -8.4 NM_022782 M-phase phosphoprotein 9 -8.4 AI767750 TATA element modulatory factor 1 -8.4 NM_014953 mitotic control protein dis3 homolog -8.4 AA639220 Homo sapiens cDNA FLJ33199 fis, clone ADRGL2006377. -8.5 AB011166 SMC5 structural maintenance of chromosomes 5-like 1 (yeast) -8.5 AI709335 solute carrier family 18 (vesicular monoamine), member 2 -8.5 BC040287 Homo sapiens, clone IMAGE:4819775, mRNA -8.5 AV721958 Homo sapiens, clone IMAGE:5259584, mRNA -8.6 BF513060 hypothetical protein FLJ11273 -8.6 AU146655 Sjogren syndrome antigen A2 (60kDa, ribonucleoprotein autoantigen SS-A/Ro) -8.6 AU155565 opsin 3 (encephalopsin, panopsin) Homo sapiens transcribed sequence with weak similarity to protein prf:2109260A (H.sapiens) -8.6 AW193531 2109260A B cell growth factor [Homo sapiens] -8.6 AI742305 hypothetical protein FLJ12178 -8.6 BF692592 Homo sapiens, clone IMAGE:4043849, mRNA -8.6 AI066599 small nuclear RNA activating complex, polypeptide 3, 50kDa -8.6 AV724508 serologically defined colon cancer antigen 1 -8.6 AF495383 a disintegrin and metalloproteinase domain 9 (meltrin gamma) -8.6 BC029545 v-Ki-ras2 Kirsten rat sarcoma 2 viral oncogene homolog th57e01.x1 NCI_CGAP_Kid11 Homo sapiens cDNA clone IMAGE:2122392 3', mRNA -8.7 AI473255 sequence. -8.7 AI122787 hypothetical protein FLJ10706 -8.7 NM_033029 leishmanolysin-like (metallopeptidase M8 family) -8.7 AA744613 hypothetical protein FLJ10392 -8.8 NM_012120 CD2-associated protein -8.8 AA209332 optic atrophy 1 (autosomal dominant) -8.8 AF326731 cell division cycle associated 1 -8.9 AK095276 hypothetical protein LOC283588 -8.9 NM_014684 KIAA0373 gene product -8.9 BG259856 THO complex 2 -8.9 AI912122 neuroligin 1 -9.0 AI445833 Homo sapiens transcribed sequences -9.1 NM_006139 CD28 antigen (Tp44) -9.1 BF670827 Homo sapiens mRNA; cDNA DKFZp586N2424 (from clone DKFZp586N2424)

145

Table 19 continued.

Fold GenBank ID Description Change -9.1 AF288405 Homo sapiens G protein interaction factor 1-like mRNA sequence. -9.1 NM_012485 hyaluronan-mediated motility receptor (RHAMM) -9.1 M74088 adenomatosis polyposis coli yi89e12.s1 Soares placenta Nb2HP Homo sapiens cDNA clone IMAGE:146446 3', mRNA -9.1 R79759 sequence. -9.2 AW003119 WW domain-containing adapter with a coiled-coil region -9.2 AA287223 Homo sapiens transcribed sequences -9.2 NM_014875 kinesin family member 14 -9.3 BE670797 Homo sapiens mRNA; cDNA DKFZp686J23256 (from clone DKFZp686J23256) -9.3 AV692425 Homo sapiens, clone IMAGE:4798349, mRNA -9.3 AW051349 LOC346584 -9.3 AK027340 hypothetical protein LOC222159 -9.3 AI041204 centrosome-associated protein 350 -9.4 NM_018353 chromosome 14 open reading frame 106 -9.4 NM_014720 Ste20-related serine/threonine kinase -9.5 AI375486 adenomatosis polyposis coli -9.5 BF114870 Homo sapiens clone DKFZp762N022 mRNA, complete sequence -9.5 BC043430 Homo sapiens, clone IMAGE:5294683, mRNA -9.6 AB040891 KIAA1458 protein -9.6 AL157478 Homo sapiens mRNA; cDNA DKFZp761H032 (from clone DKFZp761H032) -9.7 AK023613 chromosome 6 open reading frame 84 -9.7 AW051603 B double prime 1, subunit of RNA polymerase III transcription initiation factor IIIB -9.8 AL832095 similar to Chic1 -9.8 AI923944 Homo sapiens transcribed sequences -9.8 BF965546 hypothetical protein MGC10067 -9.8 AA355403 ubiquinol-cytochrome c reductase core protein II alpha-N-acetylneuraminyl 2,3-betagalactosyl-1,3)-N-acetyl galactosaminide alpha-2,6- -9.9 NM_030965 sialyltransferase E Homo sapiens transcribed sequence with weak similarity to protein ref:NP_038602.1 -10.0 AV700086 (M.musculus) L1 repeat, Tf subfamily, member 18 [Mus musculus] -10.0 AA418403 hypothetical protein FLJ90492 -10.0 NM_017651 hypothetical protein FLJ20069 -10.0 NM_016181 melanoma antigen -10.0 AW576457 ATPase, Ca++ transporting, plasma membrane 1 -10.1 AI949136 collagen, type XXVII, alpha 1 -10.1 U49844 ataxia telangiectasia and Rad3 related -10.2 NM_003566 early endosome antigen 1, 162kD -10.2 AF194973 polymerase (DNA directed) kappa -10.3 AL049383 Rho-associated, coiled-coil containing protein kinase 2 -10.4 AI701480 hypothetical protein LOC153577 -10.4 NM_020654 sentrin/SUMO-specific protease -10.4 NM_000081 Chediak-Higashi syndrome 1 -10.4 N30158 laminin, beta 1 -10.4 BC036345 Homo sapiens, clone IMAGE:5172449, mRNA -10.4 U30872 centromere protein F, 350/400ka (mitosin) -10.4 BC005342 nucleosomal binding protein 1 -10.5 AI832363 golgi autoantigen, golgin subfamily a, 1 -10.5 NM_004318 aspartate beta-hydroxylase -10.5 AB029032 hypothetical protein KIAA1109 -10.5 AK027226 Homo sapiens cDNA: FLJ23573 fis, clone LNG12520.

146

Table 19 continued.

Fold GenBank ID Description Change -10.6 NM_017684 hypothetical protein FLJ20136 Homo sapiens transcribed sequence with strong similarity to protein pir:T12479 (H.sapiens) -10.6 AW029169 T12479 hypothetical protein DKFZp564N1362.1 - human (fragment) -10.7 AW084937 Homo sapiens transcribed sequences -10.7 AU154486 SMC2 structural maintenance of chromosomes 2-like 1 (yeast) -10.7 AU148006 Homo sapiens cDNA FLJ12360 fis, clone MAMMA1002356. -10.7 AV709727 Homo sapiens mRNA; cDNA DKFZp586P1124 (from clone DKFZp586P1124) -10.8 AF020043 chondroitin sulfate proteoglycan 6 (bamacan) -10.8 BE670036 Homo sapiens, clone IMAGE:3352913, mRNA -11.0 AI743753 Homo sapiens transcribed sequences -11.1 AV682436 phosphoinositide-3-kinase, class 2, alpha polypeptide -11.2 BG391951 similar to F-box only protein 5 (Early mitotic inhibitor 1) -11.2 AK021887 Homo sapiens cDNA FLJ11825 fis, clone HEMBA1006494. -11.2 AI088609 Homo sapiens transcribed sequences -11.3 BE858593 Homo sapiens transcribed sequences -11.4 AK023111 translocated promoter region (to activated MET oncogene) -11.4 AL121021 hypothetical protein FLJ14281 Homo sapiens transcribed sequence with strong similarity to protein pdb:1BGM (E. coli) O -11.4 N72610 Chain O, Beta-Galactosidase (Chains I-P) -11.5 AW148862 LOC346617 602148771F2 NIH_MGC_62 Homo sapiens cDNA clone IMAGE:4307896 5', mRNA -11.6 BF978541 sequence. -11.7 AI828648 sodium channel, voltage-gated, type VII, alpha -11.9 NM_013256 zinc finger protein 180 (HHZ168) -11.9 AI932370 spastic ataxia of Charlevoix-Saguenay (sacsin) -11.9 BC039509 hypothetical protein LOC340109 -12.1 AA947873 Homo sapiens transcribed sequences -12.2 BF058559 purine-rich element binding protein B -12.2 AW069285 SMC6 structural maintenance of chromosomes 6-like 1 (yeast) -12.2 AU158022 Rho GTPase activating protein 18 -12.3 AF304443 Homo sapiens B lymphocyte activation-related protein BC-2048 mRNA, complete cds. -12.6 BF197009 AUT-like 1, cysteine endopeptidase (S. cerevisiae) Homo sapiens transcribed sequence with strong similarity to protein ref:NP_006000.2 -12.6 AI223870 (H.sapiens) tubulin, alpha 3; tubulin, alpha, brain-specific; hum-a-tub1; hum-a-tub2 [Homo sapiens] Homo sapiens transcribed sequence with strong similarity to protein ref:NP_006700.2 -12.6 AW512988 (H.sapiens) ankyrin repeat-containing protein isoform a; NG36 protein [Homo sapiens] -12.8 BF962082 Homo sapiens mRNA full length insert cDNA clone EUROIMAGE 138904. -12.9 AA456099 Homo sapiens transcribed sequences -12.9 NM_133638 a disintegrin-like and metalloprotease (reprolysin type) with thrombospondin type 1 motif, 19 -13.0 AI590207 similar to hypothetical protein MGC38936 -13.1 BG545769 interleukin-1 receptor-associated kinase 1 binding protein 1 -13.1 AW779917 Homo sapiens transcribed sequences -13.3 BC008384 Homo sapiens, clone IMAGE:3949969, mRNA -13.3 AI991033 heparan sulfate proteoglycan 2 (perlecan) -13.5 NM_002078 golgi autoantigen, golgin subfamily a, 4 -13.6 BC022892 Homo sapiens, clone IMAGE:4616140, mRNA -13.7 BC029474 Homo sapiens, clone IMAGE:4723738, mRNA -13.7 AL136599 sentrin/SUMO-specific protease -13.7 BC000973 hypothetical protein FLJ20333

147

Table 19 continued.

Fold GenBank ID Description Change -13.8 BC039537 Homo sapiens, clone IMAGE:5744875, mRNA Homo sapiens transcribed sequence with weak similarity to protein ref:NP_060265.1 -13.9 AA769450 (H.sapiens) hypothetical protein FLJ20378 [Homo sapiens] -14.0 Z83800 dynein, cytoplasmic, heavy polypeptide 2 -14.0 AL050039 Homo sapiens hypothetical gene supported by AL050039 (LOC349279), mRNA -14.1 AI457436 bone morphogenetic protein receptor, type II (serine/threonine kinase) -14.2 Z22551 kinectin 1 (kinesin receptor) -14.3 AW172431 Homo sapiens, clone IMAGE:4698512, mRNA, partial cds -14.5 AI680541 leukemia inhibitory factor receptor -14.6 AF086073 Homo sapiens full length insert cDNA clone YZ55H04 -14.6 AU156209 small fragment nuclease -14.7 AB037781 hypothetical protein FLJ10074 -14.8 AI609043 similar to hypothetical protein DKFZp761J107.1 - human (fragment) -15.2 AY137776 strand-exchange protein 1 -15.3 AA573449 mitochondrial translational release factor 1 -15.4 H18004 RP42 homolog -15.4 N31731 tripin -15.5 NM_018084 hypothetical protein FLJ10392 -16.1 BG548427 hypothetical protein from BCRA2 region -16.2 R56794 YY1 associated factor 2 -16.5 BG573839 hypothetical gene supported by BC036583 -16.5 NM_014733 zinc finger, FYVE domain containing 16 -16.6 AI922972 Homo sapiens transcribed sequences -16.6 AF090925 c-myc promoter-binding protein -16.8 NM_017661 hypothetical protein FLJ20086 -16.8 AW195579 Msx-interacting-zinc finger -16.9 AW572909 KIAA0874 protein -16.9 AV700621 Homo sapiens transcribed sequences gb:AL161659 /DB_XREF=gi:9581594 /FEA=FLmRNA /CNT=72 /TID=Hs.88820.0 /TIER=Stack /STK=18 /UG=Hs.88820 /LL=51575 /UG_GENE=HDCMC28P -17.0 AL161659 /UG_TITLE=HDCMC28P protein /DEF=Human DNA sequence from clone RP11-526K24 on chromosome 20 Contains two novel genes, two CpG islands, ESTs, GSSs and STSs /FL=gb:NM_016649.1 gb:AF068285.1 -17.0 NM_145312 Zinc finger protein 93 (Zinc finger protein HTF34) -17.4 AU144136 Homo sapiens cDNA FLJ11418 fis, clone HEMBA1000972. -17.7 AI286012 hypothetical protein FLJ23518 -17.8 NM_000059 breast cancer 2, early onset -17.9 AA020920 similar to testis expressed gene 9 -18.0 NM_006838 methionyl aminopeptidase 2 -18.9 AI623184 Homo sapiens transcribed sequences Homo sapiens transcribed sequence with weak similarity to protein sp:P11369 (M.musculus) -19.0 AW182938 POL2_MOUSE Retrovirus-related POL polyprotein [Contains: Reverse transcriptase ; Endonuclease] -19.1 NM_138731 mirror-image polydactyly gene 1 -19.3 AI143879 Homo sapiens cDNA FLJ25677 fis, clone TST04054. Cluster Incl. AI610355:tp18g08.x1 Homo sapiens cDNA, 3 end /clone=IMAGE-2188190 -19.4 4857864 /clone_end=3' /gb=AI610355 /gi=4619522 /ug=Hs.39328 /len=463 Homo sapiens transcribed sequence with weak similarity to protein ref:NP_060265.1 -20.3 N30071 (H.sapiens) hypothetical protein FLJ20378 [Homo sapiens] -20.8 AB033055 KIAA1229 protein

148

Table 19 continued.

Fold GenBank ID Description Change -21.3 NM_016220 zinc finger protein (ZFD25) -22.6 AW190593 interleukin 7 -22.7 AI368859 hypothetical protein LOC144874 -23.3 AA922154 Homo sapiens transcribed sequences -23.4 AI916242 early endosome antigen 1, 162kD -23.4 BF939071 cerebral cavernous malformations 1 -23.4 AL050097 DKFZP586B0319 protein -23.6 NM_017653 dymeclin -23.9 BF592957 chromosome 13 open reading frame 10 -23.9 AW779983 Homo sapiens transcribed sequences -23.9 AK095101 KIAA1726 protein -23.9 NM_030795 stathmin-like 4 -24.4 AW275016 hypothetical protein MGC27466 -25.0 BC037932 Homo sapiens, clone IMAGE:5285165, mRNA -25.2 NM_001813 centromere protein E, 312kDa -25.3 AF322916 uveal autoantigen with coiled-coil domains and ankyrin repeats -25.8 AW129145 Homo sapiens transcribed sequences Homo sapiens transcribed sequence with moderate similarity to protein ref:NP_060190.1 -26.7 BF447323 (H.sapiens) hypothetical protein FLJ20234 [Homo sapiens] Homo sapiens transcribed sequence with moderate similarity to protein sp:P26639 (H.sapiens) -27.1 AA020784 SYTC_HUMAN Threonyl-tRNA synthetase, cytoplasmic (Threonine-tRNA ligase) (ThrRS) -28.1 NM_005509 Dmx-like 1 -28.4 AF317887 hypothetical protein FLJ13615 -31.4 X98258 M-phase phosphoprotein 9 yh75g06.s1 Soares placenta Nb2HP Homo sapiens cDNA clone IMAGE:135610 3', mRNA -31.4 R32893 sequence. -31.4 AK021888 Homo sapiens cDNA FLJ11826 fis, clone HEMBA1006497. -31.9 NM_004775 UDP-Gal:betaGlcNAc beta 1,4- galactosyltransferase, polypeptide 6 -32.8 AL359583 cell adhesion molecule with homology to L1CAM (close homolog of L1) -33.7 AW268886 lectin-like NK cell receptor similar to Gamma-interferon-inducible protein Ifi-16 (Interferon-inducible myeloid -34.3 AI827431 differentiation transcriptional activator) (IFI 16) -34.3 BG413612 Homo sapiens, clone IMAGE:5288750, mRNA -35.8 AW665138 rhotekin 2 -38.2 U83410 cullin 2 synonyms: MCPH5, FLJ10517, FLJ10549; microcephaly, primary autosomal recessive 5; -38.5 NM_018123 Homo sapiens asp (abnormal spindle)-like, microcephaly associated (Drosophila) (ASPM), mRNA. -39.2 AI652848 tetratricopeptide repeat domain 3 -39.9 AI052103 Homo sapiens transcribed sequences -40.4 AA765470 thyroid hormone receptor interactor 11 xr78h03.x2 NCI_CGAP_Lu26 Homo sapiens cDNA clone IMAGE:2766293 3' similar to -41.8 BE138647 TR:O13561 O13561 YLR169WP. ;, mRNA sequence. -42.0 AK001380 asp (abnormal spindle)-like, microcephaly associated (Drosophila) ow12a02.s1 Soares_parathyroid_tumor_NbHPA Homo sapiens cDNA clone IMAGE:1646570 -42.4 AI025829 3', mRNA sequence. -43.6 AI336848 early endosome antigen 1, 162kD -44.5 NM_152355 zinc finger protein 441 -46.4 X80821 KIAA0874 protein -50.4 NM_153039 hypothetical protein FLJ32803

149

Table 19 continued.

Fold GenBank ID Description Change -52.7 AI640594 retinoblastoma binding protein 1 -54.0 NM_025103 capillary morphogenesis protein 1 -54.1 BF438106 SGT1, suppressor of G2 allele of SKP1 (S. cerevisiae) -57.9 AB033038 hypothetical protein FLJ10392 -66.1 AW502434 KIAA0570 gene product -91.1 AW295395 Homo sapiens transcribed sequences -97.2 AI868167 hypothetical protein LOC286097 af48a03.s1 Soares_total_fetus_Nb2HF8_9w Homo sapiens cDNA clone IMAGE:1034860 3' -100.0 AA621615 similar to contains L1.b2 L1 repetitive element ;, mRNA sequence.

150

Table 20. The effect of tea catechins on the growth of LNCaP cells.

Cell Viability EC (% control) 0 µM 25 µM 50 µM 100 µM 1 104.48 95.52 88.06 82.09 2 100.00 108.91 99.5. 88.06 3 95.52 103.48 94.03 87.56 Average 100.00 102.64 93.86 85.90 SEM 2.5865 3.8883 3.3035 1.9121

Cell Viability ECG (% control) 0 µM 25 µM 50 µM 100 µM 1 104.48 87.06 77.11 56.72 2 100.00 97.01 74.13 52.74 3 95.52 94.03 73.13 51.74 Average 100.00 92.70 74.79 53.73 SEM 2.5865 2.9483 1.1954 1.5210

Cell Viability EGC (% control) 0 µM 25 µM 50 µM 100 µM 1 101.98 98.02 43.08 23.32 2 102.37 84.19 44.27 24.22 3 95.65 87.35 50.20 25.30 Average 100.00 89.85 45.85 24.28 SEM 2.1779 4.1840 2.2020 0.5724

Cell Viability EGCG (% control) 0 µM 25 µM 50 µM 100 µM 1 104.48 77.61 40.80 23.86 2 100.00 81.59 45.77 22.89 3 95.52 103.48 51.74 24.88 Average 100.00 87.56 46.10 23.88 SEM 2.5865 8.0425 3.1625 0.5745

151

Table 21. The effect of tea catechins on the growth of ARCaP cells.

Cell Viability EC (% control) 0 µM 25 µM 50 µM 100 µM 1 100.00 102.04 98.98 87.76 2 106.12 98.98 100.00 90.82 3 93.88 94.9 104.08 91.84 Average 100.00 98.64 101.02 90.14 SEM 3.5333 2.0681 1.5581 1.2259

Cell Viability ECG (% control) 0 µM 25 µM 50 µM 100 µM 1 100.00 101.02 90.82 80.00 2 106.12 95.10 94.90 71.79 3 93.88 109.18 82.65 79.30 Average 100.00 101.77 89.46 77.03 SEM 3.5334 4.0817 3.6014 2.6278

Cell Viability EGC (% control) 0 µM 25 µM 50 µM 100 µM 1 100.00 102.04 94.90 97.96 2 106.12 101.33 100.00 97.96 3 93.88 104.29 102.04 86.73 Average 100.00 102.55 98.98 94.22 SEM 3.5334 0.8922 2.1233 3.7433

Cell Viability EGCG (% control) 0 µM 25 µM 50 µM 100 µM 1 100.00 107.20 101.14 89.96 2 106.12 99.20 105.10 91.96 3 93.88 92.98 89.12 87.04 Average 100.00 99.79 98.45 89.65 SEM 3.5334 4.1157 4.8046 1.4285

152

Table 22. The effect of tea catechins on the secretion of proMMP1 in LNCaP cells.

Secretion EC (% control) 0 µM 25 µM 50 µM 100 µM 1 100 111.50 113.79 108.00 2 100 69.61 110.55 102.64 3 100 131.43 101.50 86.50 Average 100 104.18 108.61 99.06 SEM 0 18.2173 3.6785 6.4725

Secretion ECG (% control) 0 µM 25 µM 50 µM 100 µM 1 100 101.67 106.66 120.00 2 100 81.89 81.89 97.13 3 100 83.91 104.04 74.75 Average 100 89.16 97.53 97.30 SEM 0 6.2815 7.8561 13.0644

Secretion EGC (% control) 0 µM 25 µM 50 µM 100 µM 1 100 197.38 312.23 373.28 2 100 183.71 241.50 377.29 3 100 128.69 231.69 263.30 Average 100 169.93 261.80 337.96 SEM 0 20.9911 25.3665 37.3452

Secretion EGCG (% control) 0 µM 25 µM 50 µM 100 µM 1 100 205.96 333.98 591.20 2 100 140.89 207.44 384.24 3 100 187.43 253.87 373.64 Average 100 178.09 265.10 449.70 SEM 0 19.3561 36.9570 70.8194

153

Table 23. The effect of tea catechins on the secretion of proMMP1 in ARCaP cells.

Secretion EC (% control) 0 µM 25 µM 50 µM 100 µM 1 100 99.55 99.03 97.86 2 100 104.66 100.48 93.45 3 100 91.25 98.12 94.08 Average 100 98.49 99.21 95.13 SEM 0 3.9074 0.6879 1.3779

Secretion ECG (% control) 0 µM 25 µM 50 µM 100 µM 1 100 91.36 99.08 98.62 2 100 100.93 97.44 97.66 3 100 94.98 94.47 111.52 Average 100 95.76 97.00 102.60 SEM 0 2.7917 1.3504 4.4684

Secretion EGC (% control) 0 µM 25 µM 50 µM 100 µM 1 100 93.52 98.49 101.04 2 100 106.43 99.74 99.97 3 100 97.85 99.05 101.62 Average 100 99.27 99.09 100.88 SEM 0 3.7931 0.3601 0.4831

Secretion EGCG (% control) 0 µM 25 µM 50 µM 100 µM 1 100 102.70 97.93 93.45 2 100 107.52 94.90 96.84 3 100 101.46 91.59 91.41 Average 100 103.89 94.81 93.90 SEM 0 1.8487 1.8314 1.5845

154

Table 24. The effect of tea catechins on the secretion of MMP3 in LNCaP cells.

Secretion EC (% control) 0 µM 25 µM 50 µM 100 µM 1 100 100.00 79.91 79.91 2 100 109.56 128.89 104.77 3 100 95.80 95.80 100.00 Average 100 101.79 101.53 94.89 SEM 0 4.0723 14.4283 7.6168

Secretion ECG (% control) 0 µM 25 µM 50 µM 100 µM 1 100 106.10 112.23 124.57 2 100 61.64 65.40 69.18 3 100 105.49 105.49 116.54 Average 100 91.08 94.37 103.43 SEM 0 14.7179 14.6153 17.2814

Secretion EGC (% control) 0 µM 25 µM 50 µM 100 µM 1 100 155.61 396.85 728.95 2 100 131.57 260.67 839.43 3 100 116.57 356.04 789.08 Average 100 134.58 337.86 785.82 SEM 0 11.3698 40.3489 31.9339

Secretion EGCG (% control) 0 µM 25 µM 50 µM 100 µM 1 100 149.46 291.63 882.57 2 100 262.71 658.76 751.27 3 100 119.09 250.77 979.16 Average 100 177.09 400.39 871.00 SEM 0 43.6999 129.7223 66.0397

155

Table 25. The effect of tea catechins on the secretion of MMP3 in ARCaP cells.

Secretion EC (% control) 0 µM 25 µM 50 µM 100 µM 1 100 87.72 109.31 72.70 2 100 104.22 86.27 95.13 3 100 97.72 102.49 125.51 Average 100 96.55 99.36 97.78 SEM 0 4.7985 6.8348 15.3000

Secretion ECG (% control) 0 µM 25 µM 50 µM 100 µM 1 100 142.96 139.00 111.37 2 100 111.20 122.93 161.63 3 100 126.59 115.40 120.28 Average 100 126.92 125.77 131.09 SEM 0 9.1699 6.9597 15.4821

Secretion EGC (% control) 0 µM 25 µM 50 µM 100 µM 1 100 89.81 85.66 81.15 2 100 89.81 91.36 86.93 3 100 126.78 108.54 117.03 Average 100 102.14 95.19 95.04 SEM 0 12.3207 6.8771 11.1214

Secretion EGCG (% control) 0 µM 25 µM 50 µM 100 µM 1 100 100.27 103.46 94.62 2 100 100.27 98.03 88.32 3 100 105.83 116.46 112.41 Average 100 102.13 105.98 98.45 SEM 0 1.8518 5.4698 7.2139

156

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BIOGRAPHICAL SKETCH

The author was born in Jeonju, Republic of Korea on June 19, 1970. He entered Chonbuk National University and after one year, he served in the R.O.K. Army (March 1992 - June 1994) as a military police officer. He graduated summa cum laude from CNU in 7 semesters with a Bachelor’s degree in molecular biology in February, 1997. He joined the graduate program in the same university and graduated in February, 1999 with Master’s degree in molecular biology. During his time for graduate studies at CNU, he worked on a project under guidance of Dr. Chung-ung Park. The title of the thesis was ‘Purification and characterization of N terminal calmodulin binding site in beta subunit of cGMP gated ion channel in bovine retina’. He started his PhD program in Biochemistry at the Dept. of Chemistry and Biochemistry, Florida State University, in Fall 2000. He met a lovely woman Inok Park in summer of 1997 and they were married on June 18, 2000. They currently have one gorgeous kid, Tae-won Minji Suh who was born in November 30, 2001.

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