GLUTATHIONE TRANSFERASE N STRUCTURE and GENE REGULATION

GLUTATHIONE TRANSFERASE n STRUCTURE AND GENE REGULATION

Chu-Lin Xia

This report is in part-fulfillment of the requirements for the degree of doctor of philosophy in the University of London

University College, University of London

January 1992 ProQuest Number: 10609162

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

In the early stage of this research, amino acid residues that are essential for the activity of human pi class glutathione transferase (GST n) were identified using chemical modification with group-specific reagents. Protection from inactivation by substrates, substrate analogues and inhibitors was used as the criterion for active site specificity. The results suggested the apparent involvement of one cysteine (Cys), one lysine (Lys), one arginine (Arg), one histidine (His) or tyrosine (Tyr) or both, one aspartate (Asp) or glutamate (Glu) and tryptophan (Trp) residues in the glutathione binding site (G-site) of GSTtc. It was concluded that without the knowledge of the three-dimensional structure of GST 7i, further work in this area would not be profitable and therefore, attention was turned to the regulation of GST n gene expression. Recently, the three-dimensional structure of porcine G ST pi was solved and interpretation of these results in terms of the structure of the porcine GST pi by computerized modelling revealed that Tyr7, Arg13, Trp38, Lys44 and Asp98 were associated with the G-site. The roles of Cys47 and His71 in the active site are not clear.

Studies of the regulation of GST n gene involved the fusion of fragments of both the GST n gene and 13.5 kb of its 5’ flanking region to the chloramphenicol acetyl transferase (CAT) reporter gene. Transfection into a number of human cell lines (Hela, HepG2, EJ and MCF7) demonstrated that the consensus AP1 binding site, located between nucleotides -58 to -65, was essential for the basal level promoter activity of the gene. A positive cis- acting DNA element between nticleotides +8 to +72 was also identified which could be bound by protein(s) both in vivo and in vitro. This element is part of the promoter since it functions in an orientation and position dependent manner. No other regulatory activity was identified within the 17 kb sequence analyzed. Regulation by both retinoic acid and insulin was observed, but the mechanisms of the regulation remain to be elucidated.

2 ACKNOWLEDGEMENTS

I would like to thank my supervisor Professor Brian Ketterer, my advisor Dr. Elizabeth Shephard and Professor Peter Campbell for their support and

encouragement in this project. I am very grateful to my fellow members of the Cancer Research Campaign Molecular Toxicology Research Group: Drs. John Taylor, David Meyer, Ian Cowell, Sally Pemble, Kathy Dixon, Sharon Spencer and Brian Coles, Misses Kim Gilmore and Gillian Fraser and Mr. Jonathan Harris, for their friendship, advice and support. In particular, I would like to thank Dr. John Taylor and Dr. David Meyer for their expert guidance on my work on the gene regulation of GST n and the structure of the active site of GST n respectively. I would also like to thank Miss Lucia Christodoulides, the laboratory administrator, for many kindnesses and acts of help. Finally, I thank my husband, Dr. Jiahua Wang, for his unfailing encouragement and support and for his help in preparation of this manuscript.

The studies reported here were supported by the Frances and Augustus Newman Foundation, the Cancer Research Campaign, the Sino-British Friendship Foundation in collaboration with the British Council and the European Molecular Biology Organization.

3 LIST OF CONTENTS

ABSTRACT 2 ACKNOWLEDGEMENTS 3 LIST OF CONTENTS 4 LIST OF FIGURES 11 LIST OF TABLES 14 LIST OF ABBREVIATIONS 15

PREFACE 18

CHAPTER I INTRODUCTION 20

1.1 Drug metabolism, glutathione, glutathione 21 conjugation and glutathione transferase 1.1.1 Drug metabolism 21 1.1.2 Biosynthesis and metabolism of glutathione and 23 glutathione conjugates 1.1.3 Multifunctional nature of GSTs 25 1.2 Classification of GSTs 29 1.2.1 Purification and identification of GSTs 29 1.2.2 Classification and nomenclature of GSTs 30 1.3 Protein structure and evolutionary relationships 31 of GSTs 1.3.1 Primary structure of GSTs and evolutionary 31 relationships 1.3.2 Secondary, tertiary and quaternary structure 38 1.4 Structure, organization and chromosomal 40 location of GST genes 1.5 Catalytic mechanism and structure of the 42 active site 1.5.1 The kinetic mechanism of GSTs 42 1.5.2 The catalytic mechanism of GSTs 42 1.5.3 The active site of GSTs 1.6 GSTs expression in rat and human 45 1.6.1 Tissue distribution of GSTs in rat and man 45 1.6.2 Developmental change of GSTs expression 47

4 Content

1.7 Modulation of GST expression 47 1.7.1 Post-translational modification 47 1.7.2 Hormonal regulation of GST expression 48 1.7.3 Chemical induction of GSTs 49 1.8 Expression of Pi class GSTs in carcinogenesis 49 and drug resistance 1.8.1 GST 7-7 and carcinogenesis 49 1.8.2 GST n and human tumours 51 1.8.3 GST n and drug resistance 53 1.9 Control of the expression of GST genes 56 1.9.1 Control of the expression of mouse GST 56 subunit Ya gene 1.9.2 Control of the expression of rat GST 57 subunit 1b gene 1.9.3 Control of the expression of rat GST 60 subunit 7 gene 1.9.4 Control of the expression of human GST n gene 63 1.10 Aim of this thesis 63

CHAPTER II MATERIALS AND METHODS 65

Foreword 66 PART I 66 2.1.1 Chemicals 66 2.1.2 Protein determination 67 2.1.3 Enzyme activity assay 67 2.1.4 Purification of GST n from human placenta 67 2.1.5 Modification by DTNB 68 2.1.5.1 Reaction of GST n with DTNB 68 2.1.5.2 Determination of the rate constant of 68 DTNB modification 2.1.5.3 Effect of GS-OCT on DTNB modification 68 2.1.6 Modification by NEMI 69 2.1.6.1 Kinetics of NEMI inactivation of GST n 69 2.1.6.2 Effects of GS-OCT, CDNB and bilirubin 69

5 on NEMI inactivation of GST n Modification of GST n by TNBS 69 Modification by TNBS 69 Effects of GS-OCT, bilirubin and CDNB 70 on TNBS inactivation of GST n Modification of GST n by DiAC 70 Kinetics of DiAC modification 70 Effects of GSH, GS-OCT, GS-HEX, bilirubin 70 and CDNB on DiAC modification of GST n The modification of GST n by EDAC 70 Kinetics of EDAC modification 70 Effects of GSH, GS-OCT, GS-HEX, bilirubin 71 and CDNB on EDAC inactivation of GST n Modification of GST n by DEPC 71 Detection of the reaction of GST n with DEPC 71 Determination of the numbers of histidine and 71 tyrosine residues of GST n modified by DEPC Modification by DEPC and enzyme activity 72 Effects of GSH or CDNB on DEPC modification of GST n 72 Peptide analysis of DEPC modification of GST n 72 N-terminal sequencing of GST n and 73 DEPC modified GST n Modification of GST n by NBS 73

74 Chemicals 74 Basic chemicals 74 Bacterial and cell culture reagents and media 74 Enzymes 74 Enzyme substrates and inhibitors 75 Hormones and vitamins 75 Oligonucleotides and nucleic acids 75 Radiochemicals 75 Reagent Kits 76 Other materials 76

6 Commonly used solutions 76 Bacterial cell culture 76 Bacterial strains 76 Bacterial growth media 77 Plasmids and cosmids 77 Isolation of plasmid DNA 77 Mini-scale isolation of plasmid DNA 77 Large scale isolation of plasmid DNA 78 Enzymatic modification of DNA 79 Restriction digestion 79 Exonuclease digestion 80 Treatment of DNA fragment with alkaline 80 phosphatase Isolation of restriction fragments 80 Agarose gel electrophoresis of DNA fragments 80 Isolation of restriction fragments from 81 agarose gels Southern blot analysis 81 Transfer of DNA fragments to solid support 81 Oligo-labelling of DNA fragment by 82 hexadeoxynucleotide primers Hybridization 82 Polymerase chain reaction (PCR) 83 Plasmid cloning of DNA fragments 83 Ligation 83 Preparation of competent cells 84 Transformation of E.coli JM83 84 Colony hybridization 84 DNA sequencing 85 Annealing template and primer 85 Labelling reactions 86 Termination reactions 86 Denaturing gel electrophoresis 86 Eukaryotic cell culture 87 Eukaryotic cell lines 87

7 Content

2.2.12.2 Routine cell culture 88 2.2.13 Transfection of plasmid DNA into eukaryotic 88 cell lines 2.2.13.1 Transfection 88 2.2.13.2 Chloramphenicol acetyl transferase (CAT) assay 89 2.2.13.3 Assay of 3-galactosidase activity 90 2.2.13.4 Determination of protein concentration 90 2.2.14 Slot blotting 91 2.2.14.1 RNA preparation from cultured cells 91 2.2.14.2 Blotting 91 2.2.14.3 RNA blot hybridization 92 2.2.15 Gel retardation 92 2.2.15.1 Preparation of cell nuclear extract 92 2.2.15.2 preparation of gel 93 2.2.15.3 End labelling of oligonucleotides 93 2.2.15.4 Formation of the DNA-protein complex 93 2.2.15.5 Electrophoresis 94 2.2.15.6 Autoradiography 94 2.2.16 Quantification of GST n in cultured cells 94 2.2.16.1 Preparation of cytosol from cultured cell 94

2.2.16.2 Analysis of GSTk subunit by reverse phase HPLC 95

CHAPTER III THE G-SITE OF GLUTATHIONE TRANSFERASE n 96

Forward 97 3.1 Cysteine residues 97 3.1.1 Modification of GST n by DTNB 97 3.1.2 Effect of GS-OCT on DTNB modification of GST n 99 3.1.3 Kinetics of NEMI inactivation of GST n 101 3.1.4 Effects of GS-OCT, bilirubin and CDNB on 102 NEMI modification of GST n 3.1.5 The involvement of cysteine residue in the G-site 104 3.2 Lysine residues 106 3.2.1 Modification by TNBS 106 3.2.2 Effects of GS-OCT, bilirubin and CDNB on 107

8 Content

TNBS modification of GST n 3.3 Arginine residues 110 3.3.1 Modification of GST n by DiAC 110 3.3.2 Effects of GSH, GS-OCT, GS-HEX, bilirubin 110 and CDNB on DiAC modification of GST n 3.3.3 The involvement of arginine residue in the G-site 112 3.4 Carboxyl groups 114 3.4.1 Modification of GST n by EDAC 114 3.4.2 The Effects of GSH, GS-OCT, GS-HEX, bilirubin 114 and CDNB on DEAC modification of GST n 3.5 Histidine and tyrosine residues 115 3.5.1 Modification of GST n by DEPC 115

3.5.2 Modification of GST k by DEPC and 117 enzyme activity 3.5.3 The effects of GSH and CDNB on 117 DEPC modification 3.5.4 Localization of DEPC-modified 120 histidine of GST n 3.5.5 Localization of DEPC-modified 122 tyrosine of GST n 3.6 Tryptophan residues 123 3.7 Summary 124

CHAPTER IV THREE-DIMENSIONAL STRUCTURE OF GST tc 126

Foreword 127 4.1 The three-dimensional structure ofporcine 127 GST n in complex with glutathione sulfonate 4.2 His71 and Tyr7 130 4.3 Lys44 132 4.4 Arg13 132 4.5 BAsp98 and Glu97 135 4.6 Trp38 135 4.7 Cys47 135 4.8 Summary 136

9 Content

CHAPTER V REGULATION OF GST n GENE EXPRESSION 140

Foreword 141 5.1 A function of the consensus AP1 binding 141 sequence 5.1.1 Construction of pBS**CAT plasmids 141 5.1.2 Transfection of pBS**CATs into 142 cultured human cells 5.2 Analysis of upstream and downstream regions of GST n 146 gene for functionally active c/s-acting sequence 5.2.1 Restriction mapping of the fragment -3.0 146 to -13.5 5.2.2 Repetitive sequence present in the 5’ flanking 146 region of the GST n gene 5.2.3 Construction of plasmidspUH3.5CAT, pUP5.3CAT, 148 pD SH 1.8C A Tand pDB2.3CAT 5.2.4 The CAT activities 151 5.3 Identification of the downstream c/s-acting 151 element of the GST n gene 5.3.1 A c/s-acting regulatory element located in the 154 sequence +8 to +72, downstream of the tsp 5.3.2 The downstream c/s-acting element binds 157 protein in vivo and in vitro 5.3.3 The Function of the downstream c/s-acting 158 element is position dependent 5.3.4 Effect of the downstream c/s-acting element 161 on mRNA stability 5.4 Negative regulation of the GST % promoter 164 activity by retinoic acid (RA) 5.5 Insulin induction of the promoter activity 167 of GST 7i gene

CHAPTER VI DISCUSSION TO CHAPTER V 171

6.1 The consensus AP1 binding site of the GST n 172

10 Content

promoter 6.2 Regulation of the GST n promoter by retinoic acid 176 6.3 Intron 1 and GST n promoter activity 178 6.4 Regulation of the GST n promoter by insulin 181

APPENDIX 184 REFERENCES 188 LIST OF PUBLICATIONS AND PRESENTATIONS 213

LIST OF FIGURES

Fig.1.1 Xenobiotic metabolising system 22 Fig.1.2 The major path way of metabolism of benzo[a]pyrene 22 Fig. 1.3 The structure of glutathione 23 Fig. 1.4 Glutathione metabolism 24 Fig.1.5 Glutathione conjugate metabolism 25 Fig. 1.6 Examples of the reactions catalyzed by GST 27 Fig.1.7 Alignment of the N-terminal sequences 35 of GST subunits from different classes Fig. 1.8 Sequence alignment of the second highly 36 conserved region of GST subunits from different classes Fig.1.9 Sequence alignment of the third highly 37 conserved region of GST subunit from different classes Fig.1.10 Structure of GST genes 41 Fig. 1.11 Schematic representation of the regulatory 59 elements of the rat subunit 1bgene Fig.1.12 Schematic Representation of the regulatory 61 elements of the rat subunit 7 gene Fig. 1.13 Sequence of the promoter region of GST n gene 64

Fig.3.1 The reaction of cysteine residue with DTNB 97

Fig.3.2 DTNB modification of GST k 98

11 Content

Fig.3.3 Determination of the rate constants of 100 GST n cysteine residue modification by DTNB Fig.3.4 The reaction of cysteine residue with NEMI 101 Fig.3.5 Kinetics of NEMI modification of GST n 103 Fig.3.6 Effects of GSH, GS-OCT, GS-HEX, bilirubin 105 and CDNB on NEMI modification of GST n Fig.3.7 The reaction of lysine residue with TNBS 106 Fig.3.8 TNBS modification of GST n 108 Fig.3.9 Effects of GS-OCT, bilirubin and CDNB on 109 TNBS modification of GST n Fig.3.10 The reaction of arginine residue with DiAC 110 Fig.3.11 Kinetics of DiAC modification of GST n 111 Fig.3.12 Effects of GSH, GS-OCT, GS-HEX, CDNB and 113

bilirubin on DiAC modification of GST k Fig.3.13 The reaction of carboxyl group with EDAC 114 Fig.3.14 Effects of GSH, GS-OCT, GS-HEX, CDNB and 116 bilirubin on EDAC modification of GST n Fig.3.15 The reaction of histidine residue with DEPC 115 Fig.3.16 The reaction of tyrosine residue with DEPC 117 Fig.3.17 Modification of GST n by DEPC 118 Fig.3.18 DEPC modification of the GST n and 119 the enzyme activity Fig.3.19 Peptide analysis of DEPC-GST n 121

Fig.4.1 The three-dimensional structure of porcine GST pi 128 Fig.4.2 Stereo drawing of the C“ positions of the 131 environment of DEPC modified His71 Fig.4.3 Stereo drawing of the Ca positions of the 133 environment of DEPC modified Tyr7 Fig.4.4 Stereo drawing of the C“ positions of 134 a-helix A with Tyr7 and GSA Fig.4.5 Stereo drawing of the C“ positions of the 137 environment of DTNB-modified Cys47 Fig.4.6 Stereo drawing of the Ca position of the 138 environment of NEMI-modified Cys47

12 Transfection of pBS**CATs into EJ, MCF7 143 and HepG2 cells pB S**C A Ttransfection of HepG2, EJ and 145 MCF7 cells Restriction map of the 5’ flanking region 147

of GST k Detection of Alu sequence in the 149 5’ flanking region of GST n gene Detection of repetitive sequence in 150 5’ flanking region of GST n gene Scheme for generation of GST rc-CAT 152 constructs containing fragments from 5’ flanking region of GST n gene Scheme for generation of GST rc-CAT 153 constructs containing fragments from the downstream region of GST n Transfection of pUH3.5CAT, pUP5.2CAT, 154 pD SH 1.8C A Tand pD B 2.4C A Tinto EJ cells Chimeric constructs of GST n promoter 155 CAT assay with HepG2, Hale, EJ and MCF7 cells 158 transfection with GST n promoter-CAT constructs The effect of co-transfection with 160 p S S O .IC A Ton pS S 0.2C A Tand pCR0.15CAT Gel shift assay using nucleotides -8 to +72 162 of GST n gene as probe CAT assay of HepG2 cells transfected 163 with GST n promoter-CAT chimeric constructs Slot blot analysis of the mRNA stability 165 F?/4F?8-mediated RA repression of GST n promoter 166 activity in EJ cells Analysis of RA repression of GST n expression 168 by HPLC Induction of GST n promoter activity by insulin 169 Northern blot analysis of the induction of GST n 170 mRNA by insulin

13 Comparison of the promoter region, exon 1 180 and intron 1 of the human GST n and the rat GST subunit 7 genes

The primary sequence of GST n subunit 185 The structure of pSV2CAT 186 The structure of pICCAT 187

LIST OF TABLES

Nomenclature and physical characteristics 32 of rat GSTs Nomenclature and physical characteristics 33 of human GSTs Comparison of GST activity in human tumour 52 tissues with their counterparts GST activity and molecular form in drug-resistance 55 cancer cell lines

Buffers for chemical modification reactions 66

The effect of GS-OCT on DTNB modification 101 of GST n Modification of GST n by EDAC 115 Modification of Tyr7 by DEPC 123 Summary of the chemical modification of GST n 125

Secondary structural components of porcine GST pi 129 Summary of the computerized modelling of 139 chemical modification of GST n

Relative CAT activities generated by GST n 157 chimeric gene constructs

14 LIST OF ABBREVIATIONS

AAF N-acetyl-2-aminofluorene ABP Amino biophenyl Ah-R Aryl hydrocarbon receptor ARE Antioxidant response element Amp Ampicillin AP1 Activator protein 1 BCA Bicinchonicic acid BCNU Bis-chloroethyl nitroso urea BHA Butylated hydroxyanisole t-BHQ t-Butylhydroquinone B[d]P Benzo[a]pyrene BPDE Benzo[d]pyrene 7,8-diol-9,10-oxide BSA Bovine serum albumin BSP Bromosulfophthalein CAT Chloramphenicol acetyl transferase cDNA Complementary DNA CDNB 1 -chloro-2,4-dinitrobenzene CREB cAMP response element binding protein DAB 3-Methyl diaminoazo benzene DCNB 1,2-dichloro-4-nitrobenzene DEN Diethylnitrosamine DEPC Diethyl pyrocarbonate DiAC Diacetyl dldC Poly(dl-dC) -poly(dl-dC) DMN Dimethylnitrosamine DMSO Dimethyl sulphoxide DNA Deoxyribonucleic acid DTNB 5,5’-Dithio-bis(2-nitrobenzoic acid) DTT Dithiothreitol EDAC 1-Ethyl-3-(3-dimethylaminopropyl)- carbodiimide EDB Ethyl dibromide EDTA Ethylendiaminetetraacetic acid EPNP 1,2-Epoxy-3-(nitrophenxy)propane

15 Abbreviation

ERE Electrophile response element FPLC Fast protein liquid chromotography GPEI GST-P enhancer I GPE1I GST-P enhancer II GSA Glutathione sulfonate GSH Glutathione GS-HEX S-n-Hexylglutathione G-site Glutathione binding site GS-OCT S-n-Octylglutathione GSSG Glutathione disulfide GST Glutathione transferase T-GT T-glutamyl transferase HEPES N-(2-Hydroxyethyl)piperazine- ethanesulfonic acid) hMTIIA Human metallothione<>? II A HNE Hydroxynonenal HNF Hepatocyte nuclear factor HPLC High performance liquid chromatography H-site Hydrophobic substrate binding site IGF II Insulin like growth factor II IL6-DBP IL6 dependent DNA binding protein L-1 Long interspersed element 1 (also LINE-1) LAP Liver-enriched transcriptional activator-protein LINE-1 Long interspersed element 1 (also L-1) MAB N-methyl-4-aminoazobenzene 3MC 3-Methyl-cholanthrene MDR Multidrug resistance MOAB 3-Methyl diaminoazobenzene mRNA Messenger RNA NABQI N-acetyl-p-benzoquinonimine NADPH Nicotinamide adenine dinuceotide phosphate (reduced) NBS N-bromosuccinimide NEMI N-ethj^lmaleimide 6-NF B-napthoflavone

16 Abbreviation

NP Nitropyrene ONPG o-nitrophenyl-B-galactopyranoside PAGE Polyacrylamide gel electrophoresis PB Phenobarbital PCR Polymerase chain reaction PDGF Platelet derived growth factor PG Prostaglandin PMSF Phenylmethyl sulfonyffluoride RA Retinoic acid RAR Retinoic acid receptor RNA Ribonucleic acid RXR Retinoid X receptor SP1 Stimulatory protein 1 SV40 Simian virus 40 TCDD 2,3,7,8-tetrachlorodibenzo-p-dioxin TCL Thin layer chromatography TEMED N,N,N\N’-tetramethylethylenediamine TFA Trifluoroacetic acid TLCK N-a-P-Tosyl-L-lysine chloromethyl ketone TNBS 2,4,6-Trinitrobenzene sulfonic acid TPA 12-o-tetradecanoyl phorbol-13-acetate TPBO frans-4-phenyl-3-buten-2-one TPCK N-Tosyl-L-phenylalanine chloromethyl ketone TRE TPA responsive element tsp Transcription start point UDP Uridine diphosphate UDP GA UDP-glucuronic acid XRE xenobiotic responsive element

Additional Abbreviations

ACTH Adrenocorticotrophic hormone

DEAE- Diethylaminoethyl-

SDS Sodium dodecyl sulphate

SSC Saline sodium citrate

SSPE Saline sodium phosphate EDTA PREFACE

Glutathione transferases (GSTs) (EC 2.5.1.18) are a group of multifunctional dimeric enzymes involved in the detoxication of numerous carcinogenic, toxic and pharmacologically active electrophiles through the catalysis of their conjugation with glutathione (GSH, 7-glutamyl-cysteinyl-glycine) (Jakoby, 1978; Mannervik, 1985; Coles & Ketterer, 1990). GSTs can also sequester toxins through high affinity binding, functioning as carrier proteins, and remove toxic peroxides through their intrinsic organic GSH peroxidase activity (Mannervik, 1985; Mannervik & Danielson, 1988; Ketterer e ta/., 1988). In addition, GSTs are able to catalyze reactions in the biosynthetic pathways of physiologically active compounds such as leukotriene C4 (Tsuchida et a!., 1987), and prostaglandins E2 (PGE2), prostaglandin F2a (PGF2a) (Ketterer, 1986; Meyer & Ketterer, 1987).

GSTs have been found in bacteria, lower eukaryotes and all members from both the plant and the animal kingdoms examined so far (Ketterer et a!., 1988). GSTs from human, rat and mouse are the most extensively studied. The cytosolic forms of GST subunits can be grouped into four evolutionary classes, namely alpha, mu, pi and theta, based on their primary structures, kinetic and immunological properties (Mannervik, 1985; Meyer e ta l., 1991).

The pi class forms, namely GST n in human and GST 7-7 in rat, possess broad substrate specificity, glutathione peroxidase activity toward lipid hydroperoxides, low sensitivity to organic anion inhibitors and high sensitivity to active oxygen species (Sato, 1989; Tamai et al., 1990). They are particularly interesting since they are associated with malignancy (Sato etal., 1984a; Pemble et a/,, 1986a) and also cancer chemotherapeutic drug- resistance (Hayes & Wolf, 1988; Batist et al., 1986).

18 This project initially investigates the enzyme active-site, especially the glutathione binding site (G-site) of GST rc, which apart from its intrinsic importance may provide information for the development of powerful GST inhibitors. Such inhibitors could be used to further evaluate the role of GST in detoxification of xenobiotics especially in carcinogenesis and drug- resistance. This project also studies the factors regulating the expression of the GST n gene. Both approaches are of great interest with respect to the understanding of the development and possibly the treatment of cancer.

19 CHAPTER I

INTRODUCTION

20 Introduction

1.1 Drug Metabolism, Glutathione, Glutathione Conjugation and Glutathione Transferase

1.1.1 Drug Metabolism:

Living organisms ingest, inhale or absorb many compounds of no nutritional value which are referred to as xenobiotics. They are potentially toxic and

present a constant challenge. To eliminate xenobiotics, each organism is equipped with its own disposal system including a range of xenobiotic metabolizing enzymes, which facilitate their detoxification and excretion (Jakoby & Ziegler, 1990).

Xenobiotic metabolising reactions can be divided into two phases, namely phase I and phase II reactions (Fig. 1.1). Sipes & Gandolfi (1991) regard the production of -OH, -SH, -NH2, and -COOH as phase I reactions. However to this list, must also be added arene oxides, which can be hydrated to diols by epoxide hydrolase. The most important enzymes involved in phase I reactions are the cytochrome P450 containing monooxygenases which hydroxylate carbon and nitrogen in many xenobiotics and also produce arene oxides. In phase II reactions, xenobiotics or their phase I derived metabolites are covalently linked to endogenous hydrophilic molecules. The major phase II enzymes are those which catalyze conjugations with glucuronic acid ([UDP]- glucuronosyltransferase), sulphate (sulphotransferase) and glutathione (glutathione transferase). Glutathione does not conjugate with -OH, -SH, -NH2 and -COOH groups, but requires compounds containing electrophilic centres such as arene oxides, a,B-unsaturated ketones, alkyl and arene halides.

An extensively studied example of xenobiotic biotransformation is that of benzo[a]pyrene (BP) (Fig. 1.2). A P450 isoenzyme transforms BP to its 7,8 epoxide which is then hydrolysed by epoxide hydrolase to give BP-7,8-diol.

21 Introduction

PHASE I PHASE II

Nucleophilic ____ Glucuronides metabolites Sulfates

Xenobiotics

Electrophilic GSH conjugates metabolites

DNA RNA Protein

Fig. 1.1 Xenobiotic Metabolising System

P450

Epoxide v hydrolase

P450

OH OH

GST

Fig. 1.2 The Major Pathway of Metabolism of Benzo[a]pyrene

22 Introduction

Subsequently .another P450-dependent epoxidation at position 9,10, results in the reactive electrophile BP-7,8-diol-9,10-epoxide (BPDE). These

enzymatic reactions involved in the activation of BP to BPDE are

stereospecific, as shown in Fig. 1.2, with the major product being (+) anti BPDE. This stereoisomer, which reacts with DNA, is not a substrate for

epoxide hydrolase, but is a substrate for GST. Therefore its genotoxicity can be prevented by GST (Ketterer etal., 1988).

1.1.2 Biosynthesis and Metabolism of Glutathione and Glutathione Conjugates

Glutathione, 7 -glutamyl-L-cysteinyl-glycine (Fig. 1.3) is widely distributed in animal tissues, plants and microorganisms. It is an essential co-factor for various enzymes such as GST, GSH peroxidase and GSSG reductase (Meister, 1988). The principal organ of its biosynthesis is the liver. Two ATP-

dependent synthetases are involved (Fig. 1.4).7-Glutamyl cysteine synthetase

condenses L-glutamic acid and L-cysteine to form dipeptide with a 7 -peptide bond and glutathione synthetase elongates the dipeptide at the C-terminal by coupling it to glycine (Meister & Anderson, 1983).

Fig. 1.3 The Structure of Glutathione

GSH turnover is initiated by 7 -glutamyl transpeptidase (7 -G T ), which is located at the outer cell membrane of various types of ceil. This enzyme removes the 7 -glutamyl group of GSH, and the resulting cysteinyl-glycine is then hydrolysed by dipeptidases to give the corresponding amino acids (Fig. 1.4) (Meister et al., 1981).

23 Introduction

The GSH S-conjugates are typically converted to mercapturic acid derivatives (Chasseaud, 1976). The first step is mediated by 7 -G T , resulting in a cysteinyl glycine conjugate which is hydrolyzed at the cysteinyl glycine peptide

bond. The resulting cysteine conjugate is usually N-actetylated by N- acetyltransferase in either liver or kidney to give an N-acetyl cysteine conjugate, which is excreted mainly in the urine (Fig. 1.5) (Boyland & Chasseaud, 1969). Less frequently, enzymes like 6-lyase in kidney or C-S

lyase in the intestinal microflora are capable of cleavage of the C-S bond in the cysteine moiety, giving rise to a toxic thiol derivative. Subsequently S- methylation and oxidation result in the formation of the S-methyl sulfoxy conjugate (Dekant etal., 1988).

GSSG h 2o NADPH + HT H20 2 z / OXIDA TION Glutathione peroxidase \ GSSGG freductase REDUCTION

H ,0 ; K NADP*

Glutathione ------GLUTATHIONE /DP + P, transferase A A X . T [GSH] TGlutathione synthetase 7-Glu-Cys-Gly r I ^ ^ A T P X 7 -Glutamyl transpeptidase 7-Glu-Cys -ADP + P CON JUG A TION V 7-Glu-Cys synthetase Cys-Giy- Dipeptidase j y v . r ^ a tp 7-Glu-AA /u

^ + P; 7-Glutamyl cyclotransferase 5-Oxoprolinase

] ^^5-Oxoproline ATP AA '

7 -GLUTAMYL CYCLE

Fig. 1.4 Glutathione Metabolism Introduction

r-Glytamyl Cys-Gly- N-Acetyl R transpeptidase R dipeptidase R transferase R 7-Glu-Cys-Gly ^ C y s - G l y \ ► Cys N-Ac-Cys

Glu Gly S-Lyase

C-S Lyase

R-SH

S-methyl transferase 0 m Oxidase Oxidase R-S-CH3^ ------R-S-CH,-* R-S-CHo

Fig. 1.5 Metabolism of Glutathione Conjugate

1.1.3 Multifunctional Nature of GSTs:

Most of the enzymatic activities of GST fall into three categories: firstly, the conjugation of glutathione to hydrophobic electrophiles (Jakoby, 1978; Chasseaud, 1979; Coles & Ketterer, 1990); secondly, the reduction of organic hydroperoxides, for example, cumene hydroperoxide and free fatty acid hydroperoxide (Prohaska & Ganther, 1977); thirdly, the catalysis of certain GSH-dependent isomerizations, for example A 3-3-ketosteroid isomerase activity (Benson et al., 1977). Some of the reactions catalyzed by GSTs are shown in Fig. 1.6. Apart from an electrophilic carbon atom, nitrogen (nitrate

esters), sulphur (thiocyanates, disulphides) and oxygen (organic hydroperoxides) atoms can also be attacked by GSH (Jakoby, 1978).

25 Introduction

a. Nucleophilic displacement from saturated carbon

SG Cl NO, / \ HCI NO-

p-nitrobenzyl chloride

N HCl OH 'OH GSH GS NH. NH2

melphalan b. Nucleophilic displacement from aromatic carbon

GSH

NO- — s q + hci V

n o 2 no2

1 -chloro-2,4-dinitrobenzene c. Michael addition

X A„

GSH

N-acetyl-p-benzoquinone imine

GSH

4-hydroxy-non-2-enal • OH

26 Introduction d. Strained oxirane ring opening

GSH SG HO

OH OH OH OH

anti-benzo[a]pyrene-7,8 diol-9,10-oxide

(7R,8S,9S,10R) e. Isomerase activity

GSH O' O

GS^

androst-5ene-3,17-dione f. Peroxidase activity (Se-independent)

GSH + GSSG + HoO C/\ = S 4 OH- OOH GSH

cumene hydroperoxide

OOH COOH GSH COOH GSH + GSSG + H:0

Fatty acid hydroperoxide

Fig. 1.6 Examples of the Reactions Catalyzed by GST

27 Introduction

Most model substrates used for GST conjugation, such as 1-chloro-2,4- dinitrobenzene (CDNB), 1,2-dichloro-4-nitrobenzene (DCNB), trans-4-phenyl-3- buten-2-one (TPBO) and 1,2-epoxy-3-(p-nitrophenxy)propane (EPNP), have no biological relevance but have been chosen because their conjugation reactions can be followed spectrophotometrically and some of them are enzyme selective (Habig et al.,1974).

Many xenobiotics which either are intrinsically electrophilic or can be

metabolized to give electrophiles in vivo, can be substrates for GSTs, and many are human hazards. For example, pollutants from the petrochemical industry, the internal combustion engine, coking ovens and tobacco smoke. These include benzo[a]pyrene (BP) (Jernstrom et al., 1985; Robertson & Jernstrom, 1986), nitropyrene (NP) (Djuric et al., 1987), amino biphenyl (ABP) and dimethylnitrosamine (DMN) (Hemminki, 1982). In addition, there are many products from the chemical industry, including acrylonitrile, ethylene dibromide (EDB), methylene chloride, styrene oxide and vinyl chloride (Peterson & Guengerich, 1988). There are food contaminants, such as aflatoxin B, (Degan & Neumann, 1978; Moss et al., 1985; Coles et al., 1985) and heterocyclic amine carcinogens in cooked meat (Sugimura et

al., 1986). Pharmacologically active compounds, such as paracetamol, may also give rise to toxic electrophiles (Coles et al., 1988), and cancer chemotherapeutic agents, for example, the nitrogen mustards, melphalan (Dulik et al., 1986; Dulik & Fenselau, 1987) and chlorambucil (Dulik et al.,

1990), which frequently owe their pharmacological activity to their electrophilic nature (Coles & Ketterer, 1990).

Peroxidation has been implicated in carcinogenesis (Rushmore et al., 1984; 1986; Frenkel & Chrzan, 1987). Lipid hydroperoxides have proved to be substrates for GSTs (Prohaska & Ganther, 1977). Decomposition of lipid peroxides yields free radicals which can initiate further lipid damage and also generate genotoxic hydroxyalkenals (Esterbauer, 1985; Eckl & Esterbauer,

28 Introduction

1989). Hydroxyalkenals are among the best substrates for GST catalyzed conjugation (Alin et al., 1985; Jensson et al., 1986).

In addition, GSTs are important for the biosynthesis of some physiologically active compounds such as leukotriene C4 (Mannervik et al., 1984; Tsuchida et al., 1987), Prostaglandin E2 (PGE2) and prostaglandin F2a (PGF2a) (Meyer & Ketterer, 1987). Furthermore, apart from their enzymatic functions, GSTs

also serve as binding or carrier proteins for bilirubin and heme (Ketley et al., 1975; Tipping et al., 1976; Senjo et al., 1985; Xia & Chen, 1989c; Boyer & Olsen, 1991); steroid and thyroid hormones (Maruyama & Listowsky, 1984; Homma e ta l, 1986); carcinogens such as azodyes and 3-methylcholanthrene (3-MC) (Listowskyet al., 1988); leukotriene C4 (Sun et al., 1986; Falk et al., 1989) and several anionic dyes such as indocyanine green and bromosulfophthalein.

1.2 Classification of GSTs:

1.2.1 Purification and Identification of GSTs:

Many molecular forms of GSTs have been identified from microorganisms and various organs in a variety of species of both plant and animal kingdoms (Jakoby, 1978; Ketterer et al., 1986; Mannervik & Danielson, 1988). Among them, GSTs from rat, man and mouse are the most extensively studied.

The purification of GSTs is greatly assisted by the use of GSH (Simons & Vander Jagt, 1977), S-hexyl-glutathione (GS-HEX) (Guthenberg & Mannervik, 1979) or other GSH conjugate (Clark etal., 1977; Ishigaki etal., 1989) affinity chromatography. These affinity matrices allow most GSTs to be separated from other proteins of the cytosolic fraction. GSTs are then separated by various established techniques of protein isolation including chromatography and chromatofocusing. The GSTs are identified by their catalytic activities towards 1-chloro-2,4-dinitrobenzene (CDNB) and other model substrates for

29 Introduction

GST (Habig, 1974). However, some GSTs do not bind to GSH or GSH conjugate affinity matrices and have very low catalytic activity towards CDNB. Rat GST 5-5 and human 0 are such examples. GST 5-5 is purified from the rat liver cytosol by collecting the flow-through from GSH-affinity columns and separating the fraction by an Orange A column, then a hydroxyapatite column followed by Mono Q anion exchange chromatography (Meyer et al., 1991).

Substrate specificity, isoelectric separating, SDS-PAGE, immunochemical techniques, HPLC and protein sequence analysis all provide characteristics which can be used to identify GSTs.

1.2.2 Classification and Nomenclature of GSTs:

The isoenzymes of GST from rat, man and mouse can be grouped into three classes namely alpha, mu and pi with respect to their N-terminal amino acid sequences, substrate specificity, sensitivity to inhibitors and immunological cross-re actions (Mannervik, 1985; Mannervik & Danielson, 1988). A new class theta, which includes rat GST 5-5, 12-12 and human GST 0, has been added recently (Meyer etal., 1991). Cytosolic GSTs are homo- or hetero-dimers, which form between subunits belonging to the same class.

A number of nomenclature systems have been used for rat GSTs. Jakoby et al. (1976) introduced a system whereby the GSTs of rat liver were named as AA, A, B, C, D & E, in the reverse order of their elution from the carboxymethyl-cellulose. Bass et al. (1977) described rat GST subunits by their mobility in SDS-PAGE (Ya, Yb, Yc). In the system used here, the subunits are named as 1, 2, 3 .., in the chronological order of their characterization (Table 1.1) (Jakoby et al., 1984). Human GSTs have been named by Greek letters such as a, 5 and e (Kamisaka et al., 1975), and ^ (Awasthi etal., 1980), p. (Warholm et al., 1981), n (Guthenberg et al., 1979) and 0 (Meyer etal., 1991). The human liver alpha class subunits have been

30 Introduction

described by Stockman et al. (1985) as B1 and B2 and by Ostlund-Farrants et al. (1987) as a* and Oy (Table 1.2).

As more novel human GSTs are discovered, a new unifying nomenclature for human GSTs, which is similar to that of the cytochrome P450 supergene family (Nebert et al., 1987), has been published recently by Mannervik etal. (1992). In this system, the subunits of GST are numbered within the class

using Arabic numbers. Thus respective molecular forms are named by.capital a letter to indicate the class they belong to, followed byxcombination of numbers to indicate the subunit composition (Table 1.2).

1.3 Protein Structure and Evolutionary interrelationships of GSTs

1.3.1 Primary Structure of GST and Evolutionary Relationships

Full length cDNA sequences of two rat subunit 1 (Ya) (Pickett etal., 1984; Lai et al., 1984; Taylor et al., 1984), 2 (Yc) (Telakowski-Hopkins et al., 1985), 3 (YbJ (Ding et al., 1985; Lai etal., 1986), 4 (Yb2) (Lai & Tu, 1986; Ding etal., 1986), 5 (Pemble etal., 1992), 6 (Alin et al.f 1986), 7 (Yp) (Suguoka etal., 1985; Pemble et al., 1986b), 8 (Stenberg etal., 1992) are known. Likewise, the sequence of human GST n (Kano etal., 1987), two human hepatic alpha family subunits Ha1 and Ha2 (Tu & Quian, 1986; Rhoads et a/.,1987) and four mu class subunits (DeJongetal., 1988a; Seideg&rd etal., 1988; Campbell et al., 1990; Vorachek et al., 1991) have been deduced from cloned cDNAs. The N-terminal amino acid sequences are known for many others. Sequence comparisons of GSTs indicate that protein sequences have 70-90% identity within a class, but only 20-30% between classes (Mannervik & Danielson,

1988).

Alignment of the amino acid sequences of GSTs from alpha, mu and pi classes reveals three highly conserved regions between different classes: the

31 Introduction

Table 1.1 Nomenclature and Physical Characteristics of Rat GSTs

Class Enzymes Mr (K) Pi

alpha 1-1 Ya-Ya B 25 10

1-2 Ya-Yc B 25/28

2-2 Yc-Yc AA 28 9.8

2-10 Yc-YI

8-8 Yk-Yk K 24.5 6.0

10-10 YI-YI 25.5 9.8

mu 3-3 Y ^ -Y b , A 26.5 8.5

3-4 Ybr Yb2 C 26.5 7.7

4-4 Yb2-Yb2 D 26.5 6.9

4-6 Yb2-Yn1

6-6 Yn1-Yn1 Mt 26 5.8

3-6 Y ^ -Y n ,

6-9 Ynr Yn2 Mt 26 5.8

9-9 Yn2-Yn2 m t 26 5.8

11-11 Yo-Yo 26.8 5.2

Pi 7-7 p 23 7.0

theta 5-5 E 26.5

12-12 Yrs-Yrs M 26.3

13-13 Mito 27.5 9.3 chondria

Micro Micro 14 somal somal

32 Introduction

Table 1.2 Nomenclature and Physical Characteristics of Human GSTs

Class GST new other Mr (K) Pi name name

alpha B A A1-1 e 25 8.9 0*0* BA A1-2 8 25 8.75 OCxOCy

B2B2 A2-2 a - 7 25 8.4 OyOy

9.9 A3-3 27 9.9

mu P M1a-1a m , m 3 26.7 6.0 NA

* M1b-1b 26.7 5.5 4 M2-2 N2N2 25.5 5.0

5 M3-3 4.6

Pi 71 P1-1 23 theta e T1-1 26

Micro Micro MIC 15.4 somal somal

33 Introduction

N-terminal domain (Fig. 1.7), the residues from positions 60 to 80 (Fig. 1.8)

and those between 160 and 180 (Fig 1.9). By alignment to GSTtc, it is shown that residues at several positions contain the same amino acids throughout all GSTs. For example, Tyrosine 7 (5 to 9) and Isoleucine 70 (68-75) are absolutely conserved. The roles of such conserved regions or domains are not clear, although either an involvement in the GSH binding site (G-site) or subunit-subunit interaction is possible candidate. In an evolutionary

perspective, it could be inferred that the three GST classes, alpha, mu and pi, have arisen by divergent evolution from a common ancestor (Mannervik 1985; Mannervik & Danielson, 1988). The separation into distinct classes would appear to have taken place before the divergence of mammalian species, since similarities of the GST structures within the same class from different species are significantly greater than those from different classes of the same species.

The N-terminal sequences of GSTs from the newly discovered theta class show little similarity to those of the other classes (apart from the conservation of Tyr5), although it is highly conserved within the class (Meyer et al., 1991; Pemble et al., 1992). On the other hand, the amino acid sequence of rat subunit 5 between position 60 and 80 shows significant homology with those of alpha, mu and pi classes (Fig. 1.8). The evolutionary relationship between the theta class and the other three classes of cytosolic GST is yet to be establised.

The evolutionary origin of the rat liver microsomal GST is thought to be distinct from those of the cytosolic GSTs since it shows no homology with any of the cytosolic enzymes (DeJong etal., 1988b). Recently Harris etal. (1991) purified GST 13-13 from rat liver mitochondria, the N-terminal sequence of which showed some similarity to GSTs from theta class, particularly over

34 Introduction

Fig 1.7 Alignment of the N-terminal Sequences of GST Subunits from Different Classes

Alpha class Ref. Rat subunit 1 (a&b) SGKP-VLHYFNARGRMECIRWLLAAAGVEFDEK (a) Rat Subunit 2 PGKP-VLHYFDGRGRMEPIRWLLAAAGVEFEEQ (b) Rat subunit 8 EVKP-KLYYFQGRGRME SIRWLLATAGVEFEEE (c) Human Hax AEKP-KLHYSNIRGRMESIRWLLAAAGVEFEEK (d) Human Ha2 AEKP-KLHYFNARGRME SIRWLLAAAGVEFEEK (e)

Mu class Rat subunit PMILGYWNVRGLTHPIRLLLEYTDSSYEEK (f) Rat subunit PMTLGYWDIRGLAHAIRLFLEYTDTSYEDK (g) Rat subunit PMTLGYWDIRGLAHAIRLLLEYTDSSYEEK (h) Rat subunit AMTLGYWNVRGLTHPIRLLLEYTDSNTEEK (i) Human fi & \p PMILGYWDIRGLAHAIRLLLEYTDSSYEEK (j) Human GST 4 PMTLGYWNIRGLAHSIFLLLEYTDSSYEEK (k) Human GST 5 CESSMVLGYWDIRGLAHAIRLLLEFTDTSSEEK (1)

Pi class Rat subunit 7 PPYTIVYFPVRGRCEATRMLLADQGQSWKEE (m) Human ti P P Y T WY F P VRGRC AALRMLL AD Q G Q S WKEE (n)

Theta class Rat subunit 5 VLELYLDLLSQPCRAIYIFAKKNNIPFQM (o) Rat subunit 12 GLELYLDLLSQPCRAVYIFAKKNGIPFQL (o) Human 6 GLELYLDLLSQPCRAVYIFAKKNDIPFEL (o) Rat subunit 13 GPAPRVLELFY-DVLS-P (p)

Others Maize GST III P LKL-YGMPLSPNV-VRVATVLNEKGLDFE (q) Maize GST I PMLK-YGAVMSWNL-TRCATALEEAGSDYE (q)

a) Pickett et a l, 1984; Lai et a l, 1984; Taylor et al, 1984. b) Telakowski-Hopkins e ta l, 1985; c) Stenberg e ta l, 1992. d) Tu & Quian, 1986; Rhoads e ta l, 1987. e) Board & Webb 1987. f) Ding e ta l, 1985; Lai et a l, 1 9 86 .’ g) Lai & Tu, 1986; h) Alin et a l, 1986. i) Abramovitz & Listowsky, 1987. j) DeJong e t a l 1988a. k) Vorachek et a l, 1991. I) Campbell e ta l, 1990. m) Pemble e ta l, 1986b; Suguoka et al., 1985. n) Kano et a l, 1987; Cowell et al, 1988. o) Meyer et a l, 1991. p) Harris et a l, 1991. q) Grove e ta l, 1988.

35 Introduction

Fig. 1.8 Sequence Alignment of the Second Highly Conserved Region of GSTs from Different Classes

Alpha class Ref. Rat subunit 1 (a&b) DG-MKLAQTRAILNYIAT (a) Rat subunit 2 DG-MKLVQTRAILNYIAT (b) Rat subunit 8 DG-MLLTQTRAILSYLAA (c) Rabbit alpha 1 DG-MKLVQTRAILNYVAN (d) Rabbit alpha 2 DG-MKLVQTRAIFNYIAD (d) Mouse Ya DG-MKLAQTRAILNYIAT (e) Human ax and ay DG-MKLVQTRAILNYIAS (f)

Mu class

Rat subunit 3 DGSRKITQSNAIMRYLAR (g) Rat subunit 4 and 6 DGSHKITQSNAILRYLGR (h) Human |! and DGAHKITQSNAILCYIAR (i) Human GST 4 DGTHKITQSNAILRYIAR (j) Human GST 5 DGKNKITQSNAILRYIAR (k)

Pi Class

Rat subunit 7 DGDLTLYQSNAILRHLGR (1) Human n DGDLTLYQSNTILRHLGR (m)

Theta class

Rat subunit 5 DGGFTLCESVAILLYLAH (n)

Others

Maize 1 DGDLYLFESRAICKYAAR (o) Maize 3 DGDEVLFESRAINRYIAS (o)

The sequences shown are from a region equivalent to amino acid residues 57 to 74 of human GST n. a) Lai etal., 1984; Pickett etal., 1984. b) Telakowski-Hopkins etal., 1985. c) Stenberg et al., 1992. d) Gardlik et al., 1991. e) Daniel et al., 1987. f) f) Board & Webb 1987; Tu & Quian, 1986; Rhoads et al., 1987. g) Ding et al., 1985; Lai et al., 1986. h) Lai & Tu*1986. i) DeJong et al., 1988a.j) Vorachek et al., 1991. k) Campbell etal., 1990. I) Suguoka et al., 1985; Pemble etal., 1986b. m) Kano etal., 1987; Cowell etal., 1988. n) Meyer et al., 1991, Pemble etal., 1992. o) Grove etal., 1988.

36 Introduction

Fig. 1.9 Sequence Alignment of the Third Highly Conserved Region of GSTs from Different Classes

Alpha class Ref

Rat subunit 1 (a&b) LLYVEEFDASLLTSFPLLKAFKSRI (a) Rat subunit 2 LYHVEELDP SALANFPLLKALRTRV (b) Rat subunit 8 L-MVEEVSAPVLSDFPLLQAFKTRI (c) Human av & a„ LYYVEELDSSLISSFPLLKALKTRI (d)

Mu class

Rat subunit 3 LDQYHIFEPKCLDAFPNLKDFLARF (e) Rat subunit 4 LDQHRIFEPKCLDAFPNLKDFVARF (f) Rat subunit 6 LERNQVFEATCLDAFPNLKDFIARF (g) Human fi LDLHRIFEPNCLDAFPNLKDFISRF (h) Human \j/ LDLHRIFEPKCLDAFPNLKDFISRF (i) Human GST 4 LERNQVFEP SCLDAFPNLKDFISRF (j) Human GST 5 LDQNRIFDPKCLDEFPNLKDFIARF (k)

Pi class

Rat subunit 7 LLVHQVLAP GCLDNFPLLSAYVARL (1) Human n LLIHEVLAP GCLDAFPLLSAYVGRL (m)

Theta class

Rat subunit 5 FLVGPHISLADWAITECMHPVGGG (n)

The sequences shown are from a region equivalent to residues 159- 182 of human GST k. a) Lai et al., 1984; Pickett et al., 1984; Taylor et al., 1984. b) Telakowski- Hopkins et al., 1985. c) Stenberg et al., 1992. d) Board & Webb,1987; Tu & Qian,1986; Rhoads etal., 1987. e) Ding et al., 1985; Lai et al., 1986. f) Lai & Tu, 1986. g) Alin et al., 1986; Abramovitz & Listowsky, 1987. h) DeJong et al., 1988a.i) Seideg&rd et al., 1988. j) Vorachek et al., 1991. k) Campbell e ta l., 1990. I) Suguoka et al., 1985; Pemble et al., 1986b. m) Kano et al., 1987; Cowell et al., 1988. n) Meyer et al., 1991; Pemble et al., 1992.

37 Introduction

residues 6-16 (Fig. 1.7). Furthermore, like the theta class enzymes, it fails to bind to the GSH affinity matrix and has a Km for GSH above 1 mM.

1.3.2 Secondary, Tertiary and Quaternary Structure.

Until recently, relatively little was known about the folding of the polypeptide chain of GSTs. In the absence of definitive data, most structural information

came from indirect studies. Circular dichroism spectra indicated that both a- helix and 3-sheet contributed to the native conformation of rat GST subunit 1 (Ketterer, et al., 1974), and that 23 % a-helix and 25 % 3-sheet structures for human GST p (Warholm et al., 1983). Five rat GST subunits, 1, 2, 3, 4 and 7, have been subjected to an analysis which predicts the secondary structure from amino acid sequence deduced from cDNA clones (Persson et al., 1988). The prediction of a-helix and 3-sheet was similar for all sequences and was not significantly different from the estimates obtained by circular dichroism spectroscopy. A recent study by Ahmad et al. (1990) using Chou and Fasman's method of analysis of polypeptide conformation of human GSTti predicted that the polypeptide chain was significantly a helical with predominant helices represented by residues 13-22, 27-34, 81-93, 155-165 and 188-195. The polypeptide chain possessed significant 3-sheet between residues 2-8, 31-35, 99-111, 175-183 and 204-207. Considerable 3-turn potential was predicted throughout the molecule with major turn potential at residues 23-26, 114-117, 136-139 and 166-169. These calculations indicate that the a-helix, 3-sheet and 3-turn constituted about 37%, 16% and 47% of the polypeptide chain respectively. Like the rat GSTs, GST n structure was predicted to have alternated a-helices and 3-pleated sheets.

Crystals suitable for X-ray diffraction analyses have been obtained from classes alpha, mu and pi (Sesayetal., 1987; Schaffer et al., 1988; Cowan et al., 1989; Parker et al., 1990; Dirr et al., 1991). However, only the three-

38 Introduction

dimensional structure of GST pi from pig lung has been resolved recently in a complex with glutathione sulfonate (GSA) at 2.3 A resolution (Reinemer et al., 1991). This structure will be discussed extensively in Chapter IV.

All soluble GSTs appear to be dimers of identical or similar subunits from the same class. Interspecies hybrid forms can be formed in vitro between subunits belonging to the same class (Mannervik, 1985). The dimeric

structure is required for the enzyme activity of GSTs.Studies have suggested that each subunit exhibits catalytic activity independent of the other subunit. A hetero-dimeric form shows activities towards all substrates for the respective

homodimers. Sensitivities of the heterodimers towards many inhibitors are

also additive (Mannervik & Danielson, 1988).

There is evidence that GSTs may undergo conformational changes in response to substrate, product or ligand binding. Vander Jagt et al. (1983) have reported time-dependent alterations of the kinetic properties caused by bilirubin or protein binding. The order of the addition of substrates (enzyme- memory) and pre-incubations have been reported to influence the kinetics of the enzyme. For example, GST was able to use B-mercaptoethanol as the nucleophilic co-substrate only after the enzyme was pre-incubated with S- methylglutathione, although the activity was much lower than that obtained with GSH (Principato et al., 1988). Xia & Chen (1989c) have demonstrated that bilirubin has an allosteric effect on the affinity of GSTtc with GSH. In a recent study using computer program (EKPLOT) to elucidate the mechanism by which ethacrynic acid inhibits the mouse GST YfYf (a member of pi class), Phillips & Mantle (1991) showed that ethacrynic acid can compete with CDNB at the active site but simultaneously bindsto an allosteric site on the enzyme, causing a conformational change which results in the elevation of the Vmax for the conjugation of CDNB and GSH. However, the nature of these implied conformational changes and the structural level at which they operate are not known.

39 Introduction

1.4 Structure, Organization and Chromosomal Location of GST Genes

As stated above, the GSTs can be grouped into four families or classes. The alpha and mu families are complex. In rat, both families encode at least 5 subunits (Pickett & Lu, 1988): 1a, 1b, 2, 8 and 10 in alpha class and 3, 4, 6, 9 and 11 in mu class. On the other hand, the pi class does not share the diversity. Only one subunit form of pi family is evident in both rat and man.

Genomic clones of rat subunits 1b (Telakowski-Hopkins et al., 1986), 3 (Morton et al., 1990), 4 (Lai et al., 1988) and 7 (Okuda et al., 1987), mouse Ya (Daniel etal., 1987) and of human GST n (Cowell et al., 1988; Morrow et al., 1990) have been described. The genes range in size from 3 kb for rat GST 7-7 and human GST n to approximately 10 kb for alpha family genes. As shown in Fig. 1.10, the intron/exon arrangement of GSTs is conserved for rat and mouse alpha family genes, rat mu family genes and rat and human pi family genes. However, no conservation of gene structure between families is apparent (Fig. 1..1Q).

In situ hybridization studies have suggested that within each family, genes are clustered, but that each cluster is located on a different chromosome. Using a human hepatic alpha family GST cDNA probe, a single hybridizing region, which contained sequences coding for at least two related genes, was localized to chromosome 6p12 (Board & Webb, 1987; Chow et al., 1988). Similarly, a cDNA probe for a human mu GST gene detected only a single clusteraX chromosome 1p31 (DeJong et al., 1988a). However, there is evidence that a second locus for human mu family gene occurs on chromosome 3 (Islam et al., 1989). The GST n gene has been mapped to chromosome 11 q13 (Moscow et al., 1988) and the rat subunit 7 gene was assigned to chromosome 1q43 (Masuda et al., 1986). The gene coding human microsomal GST has been localized to chromosome 12 (DeJong etal., 1990).

40 Introduction

1Kb

Rat subunit 1 (a)------^------j—j ------j ----- 1 — |-

1 2 3 4 5 6 7

Mouse Va (b) -j------1 ------j - | | | |"

1 2 3 4 5 6 7

Rat subunit 3 (c) -|| j—^-j— jj| '' —y

1 2 3 4 5 6 7 8

Rat subunit 4 (d) Hill II r I

1 23 45 67 8 !

Rat subunit 7 (e] l l l l 1 II

1 2 3 4 5 6 7

Human GST n (f)

1 2 3 4 5 6 7

Fig. 1.10 Structure of GST Genes a) Telakwski-Hopkins etal., 1986. b) Daniel etal., 1987. c) Morton etal., 1990. d) Lai et al., 1988. e) Okuda et al., 1987. f) Cowell et al., 1988.

41 Introduction

1.5 Catalytic Mechanism and Structure of the Active Site

1.5.1 The Kinetic Mechanism of GSTs

Most kinetic studies have been restricted to the members of mu class GST. It was shown that the initial rate pattern for the rat GST 3-3 changed from a sequential mechanism at high GSH concentrations to a ping-pong mechanism at low GSH concentrations (Pabst et al., 1974). However, studies incorporating the effect of product inhibitions suggested that the complex initial rate pattern was better explained by a steady-state random mechanism (Jakobson et al., 1979). Danielson & Mannervik (1988) have proposed a similar mechanism for GST 4-4. More recent studies on GST homodimers 3-3 and 4-4, with two inhibitory GSH analogues have concluded that the kinetic mechanisms of both enzymes are either random sequential or ordered sequential with GSH added first (Chen et al., 1985). In the case of human GST n, a rapid-equilibrium random bi-bi mechanism has been proposed (Ivanetich & Goold, 1989) and more recently studies by Phillips & Mantle (1991) of the mouse orthologue of GSTrc namely GST YfYf also suggested a rapid-equilibrium random mechanism occurred during the conjugation.

1.5.2 The Catalytic Mechanism of GSTs

To investigate the catalytic mechanism, the effect of the electrophilicity of the non-thiol substrate on the rate of enzymatic conjugation with GSH has been evaluated and compared with its spontaneous reaction rate (Keen et al., 1976). Using a series of 1-chloro-2-nitrobenzene (CDNB) analogues bearing substituents at position 4, Hammettplots were obtained with GST 1-2, 3-4.

The second order rate constants for both the spontaneous and the enzyme catalyzed reactions showed good correlation with the resonance d-values (electronegativity of the substituent at position 4). This indicates that within a series of closely related compounds, the rates of both spontaneous and

42 Introduction

enzymatic conjugation reactions depend principally upon the electrophilicity of the substrates. The rate-determining step therefore, is likely to be the bond alteration in the ternary complex, for it is unlikely that a conformational change or slow release of Cl' or GSH conjugate, would show strict dependence on the substrate electrophilicity (Keen et al., 1976). On the other hand, in a similar but more recent study using GST 4-4, a much lower dependence of the enzymatic reaction on the electrophilicity of the acceptor substrate was observed.

Both spectroscopic evidence and the binding of anionic analogues of GSH strongly suggest that GS' is the predominant species of GSH bound at the active site of GST at neutral pH. Titration at pH 5 to 8 indicates that the pKa of enzyme bound GSH is approximately 2.5 units lower than that in aqueous solution (Graminski et al., 1987; Graminski et al., 1989; Chen et al., 1988). It is proposed that the enzyme utilizes part of the intrinsic binding energy of GSH to destabiize the thiol by placing it in a positively charged electrostatic field (Graminski et al., 1989), and that a basic amino acid residue could form a hydrogen bond with the thiol proton, thus facilitating the deprotonation of GSH at physiological pH (Chen et al., 1988). Together with the propinquity of the enzyme bound substrate with an electrophilic centre, this ensures the enhancement of the reaction rate. In addition, catalysis may be further assisted by polarization of the electrophile.

1.5.3 The Active Site of GSTs:

The existing evidence indicates that each subunit of the enzyme possesses an active site, which is thought to consist of two sub-sites: G-site and H-site (Mannervik & Danielson, 1988). Each GST has characteristic activities towards a wide range of hydrophobic electrophiles. The H-site is therefore a characteristic of each respective subunit. Nevertheless, there is considerable overlapping, for example BPDE is utilized by GSTs of both mu and pi classes

43 Introduction

and NABQI is utilized by GSTs of both alpha and pi classes (Coles et al., 1988). On the other hand, GSTs show a high specificity towards GSH, thus G-site may be common to all GST subunits. However, the fact that the Km values for GSH are different for each subunit, and that GSTs of different classes show activity toward GSH analogues to different degrees with mu class enzymes being less selective than alpha and pi class enzymes (Adang et a l.,\988; 1989), indicates that the G-site may vary between different classes.

Using GSH analogues, Adang etal. (1990) have suggested a model of the G- site of GST which consists of two positively charged residues and two negatively charged residues. Enzyme modification studies have indicated the involvement of a histidine residue in the catalytic mechanism of the human GSTiA (Awasthi etal., 1987). A cysteine, a histidine and a tryptophan residue were associated with the active site of rat GST 4-4 (van Ommen etal., 1989). Chimeric enzymes have been constructed by substitution of small pieces (8 or 9 amino acid residues) of N- or C-terminai regions from subunit 4 into corresponding regions of subunit 3 and it has been found that the kinetic properties of the chimeric enzymes are significantly different from those of the native enzyme, suggesting that the active site of the enzyme responds to the modifications of both the N- and the C-terminal regions of the polypeptide (Zhang & Armstrong, 1990).

Using site-directed mutagenesis, Stenberg etal. (1991c) have revealed that the replacement of arginine residues with alanine at position 13, 20, 69 and 187 of human GST B1B1 resulted in significantly altered Km values for GSH and decreased specific activity towards CDNB. Similar studies have revealed that replacement of the His159 of rat GST 1-1 by lysine or tyrosine resulted in decreased enzyme activity. However, replacement of His159 with asparagine does not affect the activity, suggesting that His159 in 1-1 is not essential for the catalytic activity (Wang et al., 1991). Photoaffinity labelling studies using

44 Introduction s-(p-azidophenacyl)[3H]glutathione, which binds to the G-site, revealed that the C-terminal sequence of rat subunit 1 and 2 incorporated the ligand, suggesting that C-terminal regions (212-218 of subunit 1 and 206-218 of subunit 2) may form a portion of the active site (Hoesch & Boyer, 1989).

Interestingly, a second region (91-110) of subunit 1 but not subunit 2 also appeared to form a portion of the active site. Studies on a truncated form of GST B ^ , showed that the C-terminal segment might be a component of the

H-site but appeared not to be directly involved in the G-site (Board & Mannervik, 1991).

Pi class GSTs have in common a sensitivity to thiol modification reagents and active oxygen (Tamai et al., 1990). One cysteine residue has proved to be essential for the enzyme activity of rat GST 7-7 (Tamai et al., 1990). Modification of Cys47 of rat GST 7-7 by NEMI abolishes both the enzyme activity and the affinity towards S-hexylglutathione-Sepharose (Tamai etal., 1990), suggesting that Cys47 may be part of G-site of GST 7-7. However, a mutant enzyme in which Cys47 was replaced by alanine retained enzyme activity (Tamai et al., 1991).

1.6 GSTs Expression in Rat and Man:

1.6.1 Tissue Distribution of GSTs in Rat and Man:

The tissue specificity of GST expression suggests that GST enzymes, in spite of their overlapping activities, may have specialized functions. The tissue distribution of mRNAs of GST subunits was detected by Northern blotting and immunoprecipitation of in vitro translated polypeptides (Pemble et al.,

1986a;b). Quantitative analysis of subunit composition of a given organ was achieved by reverse phase HPLC analysis of GST fractions obtained by GSH affinity chromatography (Ostlund-Farrantsetai., 1987; Ketterer et al., 1986).

45 Introduction

Alpha family subunit 1 mRNA was largely limited to liver and kidney, while subunit 2 mRNA was detected in epididymis, kidney, adrenal, liver and to a lesser extent, testis and lung (Pemble et al., 1986a; Ostlund-Farrants et al., 1987). In agreement with this, GST subunit 1 has so far been detected with certainty in only two organs, namely liver and kidney. This is in contrast to subunit 2, which has been seen in most rat tissues analyzed and rat heart is the only organ where subunit 2 has not been detected. Subunit 8 has been

found to be predominant in erythrocytes (Ketterer et al., 1986; Ostlund- Farrants et al., 1987; Meyer et al., 1989).

Rat mu class GST subunits show a similarly complex pattern of tissue distribution. mRNA of subunit 3 and 4 were detected in brain, heart, kidney, liver, lung, spleen and testis with the strongest signal obtained from liver. It has been shown that the major forms in liver are subunit 3 and 4, whereas in brain, subunit 6 and in testis, subunits 6 and 9 are predominant. (Ketterer et al., 1986; Li etal., 1986; Ishikawa etal., 1987; Ostlund-Farrants etal., 1987). Tissue distribution of rat subunit 7 of the pi class is very broad, its mRNA is detectable in a wide range of tissues, although its expression is very low in testis and liver (Satoh et al., 1985; Pemble et al., 1986b).

Immunohistochemistry has shown that the expression of GSTs is cell-type specific. In normal liver, subunit 7 is present in all bile duct cells and occasionally hepatocytes (Tatematsu etal., 1985), and that, although subunits

1, 2, 3 and 4 are present in all hepatocytes, they are more abundant in the area around the central vein than in the periportal region (Tatematsu et al., 1985). A similar, study of the kidney has shown that alpha class GSTs were located in the proximal tubules of the renal cortex (Boyce et al., 1987).

The tissue distribution of GSTs in man is similar to that of rat. GSTs of alpha family are expressed abundantly in liver, kidney, with moderate levels in intestine, prostate, adrenal, skin and uterus. GST p or GST^are expressed

46 Introduction

in the liver in only about 50% of individuals. GST k is expressed as a major form in many organs other than liver. The expression of GST n, like rat subunit 7, is very broad although its level of expression can be very low. It pr&eht In has been reported placenta (Guthenberg & Mannervik, 1979), spleen

(Koskelo, 1983), lung (Dao etal., 1984; Di llio etal., 1988), heart (Singh etal., 1985), foetal liver (Guthenberg etal., 1986), kidney (Singh etal., 1987), uterus (Di llio et al., 1988), prostate (Tew et al., 1987) and skin (Konohana et al.,

1990).

1.6.2 Developmental Change in GSTs Expression:

Enzymic activity, HPLC and Northern blotting show that the expression of rat foetal liver GSTs can be detected at day 12. Mu family GSTs are expressed at a level lower than that of alpha, and within the alpha family, subunit 2 is the major form in the foetus, whereassubunit 1 is more dominant in adult liver. Subunit 7 is prominent in foetal liver, but present at very low levels post partum. Subunit 10 is expressed perinatally but falls to low level several weeks after birth (Tee et al., 1992).

In humans, GST n expression can be detected as early as 10 weeks in embryonic liver, kidney, spleen, heart, brain and skeletal muscle (Strange et al., 1985; Faulder et al., 1987) and GST n is apparently the only GST expressed at this stage except in liver, where an alpha class GST is also expressed (Guthenberg et al., 1986). From 10 to 40 weeks of development, expression of mu and alpha class enzymes begins and GSTs start to resemble the patterns seen in adult tissue.

1.7 Modulation of GST Expression:

1.7.1 Post-Translational Modification:

47 Introduction

It is not yet known whether GST activity is modulated by post- translational modification. Taniguchi & Pyerin (1989) observed that rat GSTs 1-1, 1-2 and 2-2 could be phosphorylated in vitro by protein kinase C and the phosphorylation of GST 1 -1 exhibited a decreased affinity for bilirubin. Siegel & Wright (1985) observed both in vitro and in vivo methylation of rat liver GST. Using reverse phase HPLC and chromatofocusing, Johnson et al. (1990) demonstrated that rat GST 3-3, 3-4 and 4-4 were the primary GSTs methylated and the reaction can be stimulated with calmodulin. However, the

biological significance of these modifications has yet to be understood. Recent studies suggest that rat GST 7-7 and human GST n are glycosylated and the glycosylation may be involved partly in the microheterogeneity of these subunits observed on isoelectric focusing (Kuzmich et al., 1991).

1.7.2 Hormonal Regulation of GST Expression:

Some mammalian GSTs are induced or repressed in response to hormones. Sex difference in the subunit composition in the liver has been studied (Igarasfri et al., 1985). In males, subunits 3 and 4 of the mu class are more abundant than those in females, whereas the levels of the alpha class GSTs are higher in females than in males. A mouse pi class GST is expressed at a higher level in the livers of male mice than those of female mice (McClellan & Hayes, 1987). Castration of male mice reduces the level of the hepatic pi class GST and this is restored by administration of testosterone (Hatayama etal., 1986). Ovariectomy followed by diethylstilbestrol administration resulted in decrease of mRNA of subunits 6, 2 and 7 in rat seminal vesicles and pituitary gland (Li & Tu, 1986). Studies have also shown that rat subunit 3 is repressed by androgen (Chang et al., 1987). Hypophysectomized rats a demonstrated^large increase in the level of subunit 4 in the adrenal gland, and the lack of ACTH was apparently related to this phenomenon (Mankowitz et al., 1990). Subunit 1 mRNA was reduced in the liver but not in the kidney of adrenalectomized rat (Listowsky et al., 1990). Studies have suggested that

48 Introduction the expression of subunit 7 is enhanced by insulin and/or epidermal growth factor (Hatayama et al., 1991) ; but depressed by dexamethasone

(Abramovitz etal., 1989).

1.7.3 Chemical Induction of GSTs:

Evidence that GSTs mediate xenobiotic detoxification lies in their ability to be induced by xenobiotics in co-ordination with other drug metabolizing enzymes such as certain cytochrome P450 isoenzymes and UDP glucuronyl transferases. Rat and mouse GSTs, mainly alpha and mu class: enzymes, are selectively induced by various chemicals or drugs such as 3- methylcholanthrene (3-MC), phenobarbital (PB) (Igarashi etal., 1987), oltipraz (Davidson et al., 1990), ethoxyquin (Kensler et al., 1986), butylated hydroxyanisole (BHA) (Sato et al., 1984b), 3-methyl diaminoazobenzene (MDAB) and 2-acetylaminofluorene (AAF) (Kitahara et al., 1984). The mechanism of the induction of rat liver GST by 3-MC has been studied. The gene encoding rat subunit 1bhas been cloned and its 5’ flanking sequence has been shown to confer 3-MC inducibility (Telakowjztski-Hopkins et al., 1986; 1988). This gene and its regulation are discussed extensively in section 1.9.2. Less is known about the mechanism of PB induction. The gene encoding mouse subunit Ya and its 5’ flanking region have also been cloned and the mechanisms of its induction by 3-MC and 6NF are discussed in section 1.9.1. The induction of GST subunit 7 by TPA is discussed in section 1.9.3.

1.8 Expression of Pi Class GSTs in Carcinogenesis and Drug Resistance:

1.8.1 GST 7-7 and Carcinogenesis

Although the patterns of expression of alpha and mu class GSTs were noted to be altered during rat chemical hepatocarcinogenesis, attention has

49 Introduction

been focused on the pi class GST, ie. GST 7-7. During the application of the Solt-Farber regimen, the expression of rat GST 7-7, which is barely detectable in normal liver, is induced de novo to become an abundant GST in

preneoplastic foci, hyperplastic nodules and hepatocellular carcinoma

(Kitahara etal., 1984). Hepatomas induced by other carcinogens, for instance diethylnitrosamine (DEN), dimethylnitrosamine (DMN), aflatoxin B1 and heterocyclic amines, also strongly express GST 7-7 (Sato, 1989). Studies have shown that rat GST 7-7 is one of the best markers for pre-neoplastic cells in rat model systems of chemically induced hepatic cancers (Sato etal., 1984a; Meyer et al., 1985; Satoh et al., 1985; Tatematsu et at., 1985). Increased expression of GST 7-7 has also been observed in chemically

induced rat lung pre-neoplastic foci (Yamamoto et al., 1988).

Immunohistochemical staining revealed that very small GST 7-7 positive foci or single cells appear in rat liver 1 or 2 weeks after administration of a single dose of carcinogen and in one case even as early as 48 hours (Moore et al., 1987). This indicates that GST 7-7 is induced at a very early stage following the carcinogen insult to the liver. The multistage nature of carcinogenesis has been extensively studied in rat liver chemical hepatocarcinogenesis (Farber & Cameron, 1980; Farber, 1984). Activation or overexpression of ras and myc genes were detected in liver tumours induced in rats by aflatoxin (Makino et al., 1984; McMahon et al., 1986). GST 7-7 has also been demonstrated to be highly expressed with malignant transformation in vitro of

primary liver epithelial cell culture by transfection with ras oncogene or by treatment with activated aflatoxin B , (Power et al., 1987). Transfection of rat liver epithelial cells with v-H-ras or v-raf also caused expression of GST 7-7 (Burt et al., 1988). Therefore, it is possible that the induction of GST 7-7 at on early stage after treatment with carcinogen is due to the increased activity of ras oncogene.

It is postulated that the foci and pre-neoplastic nodules are the manifestation

50 Introduction

of the adaptive response of the liver to carcinogen insult and are more resistant to cytotoxic agents including hepatocarcinogens (Farber, 1984). GST 7-7 possesses conjugation activity toward benzo[a]pyrene metabolites, however, its actual contribution is not clear. GST 7-7 also demonstrates

selenium-independent glutathione peroxidase activity toward lipid hydroperoxides such as arachidonate and linoleate (Meyer etal., 1985). This may help to prevent lipid peroxidation which is considered to play an important role during tumour promotion.

1.8.2 GST 71 and Human Tumours

Unlike rat, human hepatocellular carcinomas do not express GST n. However, significant induction of GST n was observed in metastatic liver tumours originating from other tissues such as gall bladder, stomach and colon (Soma et al., 1986). Examination of tumours from a range of human tissues other than liver has revealed the elevated level of GST n in melanoma (Shea & Henner, 1987), cervical carcinoma (Shiratori et al., 1987), stomach cancer (Peters et al., 1990; Howie et al., 1990), some bladder carcinomas (McQuaid & Humphries, 1988) and some carcinomas of lung (Di llio et al., 1988) (Table 1.3). It has been reported that GST n may prove a useful marker for human colon, stomach, cervix and oesophageal cancer (Kodate et al., 1986; Tsutsumi et al., 1987; Campbell et al., 1991; Ishioka et al., 1991).

A recent study by Kantor et al. (1991) has demonstrated that GST k is present in almost all primary human tumours including tumours of kidney, ovary, lung, stomach, urinary tract, bladder, colon, breast, melanocytes, skin, brain, endometrium, prostate, testis, cervix and soft tissues. In addition, the expression of GST n has also been found to be increased in preneoplastic tissues such as colon adenoma (Kodate et al., 1986), dysplasia of uterine cervix (Shiratori et al., 1987) and other tissues compared with their normal counterparts (Kantor eta!., 1991). Furthermore, investigation of GST k levels in sera of patients with a malignant gastrointestinal tumour has revealed a

51 Introduction

Table 1.3 Comparison of GST Activity in Human Cancer Tissues with Their Normal Counterparts

Tissue Activity* nmol/min/mg Ref.

Tumour Normal

Colon 130±30 30±10 (a)

239±76 174±44 (b)

350 180 (c)

Stomach 272±24 200±18 (d)

500 300 (c)

Bladder 150±130 29±20 (e)

Cervix 260±190 40±10 (f)

Oesophagus 230±160 40±20 (a)

Lung 205±131 112±70 (g)

500 190 (c)

*GST activity toward CDNB, mean±S.D. The predominant GST isoenzyme is GST % in all cases.

(a) Tsuchida etal., 1989. (b) Moorghen etal., 1991. (c) Howie etal., 1990. (d) Peters etal., 1990. (e) Lafuente etal., 1990. (f) Shiratori etal., 1987. (g) Di llio etal., 1988.

52 Introduction

significant elevation (Niitsu etal., 1989; Tsuchida et al., 1989) with the levels decreasing to the normal range after surgical removal of the tumour.

1.8.3 GST k and Drug-Resistance

Drug-resistance can be divided into two broad categories: intrinsic and acquired resistance. Intrinsic resistance describes a cell or organism that possesses an inherent ability to tolerate a drug. In contrast, acquired resistance refers to the instance, where a cell that was initially sensitive to a drug develops the ability to resist the drug through prolonged exposure. Often, these cells become resistant not only to the original or related drug, but also to a whole range of other agents with no similarity in structure or mechanism of action. This phenomenon is calMmulti-drug resistance (mdt) (Moscow & Cowan, 1988; Hayes etal., 1990).

Overexpression of P-glycoprotein, a 170 kilodaltons plasma membrane protein, has been associated with mdr. P-glycoprotein is thought to decrease the intracellular drug concentration by acting as a drug-pump, therefore increasing the drug efflux (Juliano & Ling, 1976). The involvement of P- glycoprotein in mdr has been demonstrated by the introduction of P- glycoprotein cDNA into drug-sensitive cell lines, resulting in their acquisition of mdr phenotype (Gross et al., 1986; Ueda et al., 1987; Fojo etal., 1987).

Drug-resistance in tumours can be achieved by a variety of mechanisms other than their transport. Many other proteins have been implicated in drug resistance. Elevated GST expression has been reported in a number of mdr cell lines compared with the drug-sensitive parental cell from which they were derived (Batist et al., 1986; Hayes & Wolf, 1988; Kramer, 1988; Moscow et al., 1989; Townsend & Cowan, 1989) (Table 1.4). o

53 Introduction

Transfection studies with cDNAs encoding GSTs and studies using inhibitors for GST have suggested the direct involvement of GST in mdr. Several cell

lines transfected with cDNAs for GST k, rat subunit 1 and 3 demonstrated

increased resistance to a particular drug (Miyazaki etal., 1990; Nakagawa et al., 1990; Puchalski & Fahl, 1990). Treatment with an inhibitor of GST such as ethacrynic acid (Tew etal., 1988; Hansson etal., 1991) and indomethacin (Hall et al., 1989) increased the sensitivity of the cells to alkylating agents.

However, transfection of MCF7 cells with GST n cDNA did not result in resistance to adriamycin although the level of expression of GST n was comparable with that in the adriamycin resistant MCF7 cells (Moscow et al., 1989). MCF7 cells transfected with cDNA encoding human GST subunit B1

also failed to demonstrate resistance to either adriamycin or chlorambucil (Leyland-Jones et al., 1991). Therefore, the expression of GST may not be involved in drug resistance in these circumstances.

It is evident that GSTs have the capability to detoxify certain anti-cancer drugs especially alkylating agents such as melphalan and chlorambucil (Dulik etal., 1986; 1990). However, their role, if any, in the resistance to drugs such as adriamycin, which are not GST substrates, is not clear. Studies have shown that GST 7i can be reversibly inactivated by active oxygen species, which suggests that GSTn may function like a scavenger to remove active oxygen metabolites (Murata et al., 1990; Tamai et al., 1990). As the cytotoxicity of adriamycin has been suggested to be dependent on the formation of free radicals, this possible scavenger function as well as the glutathione peroxidase activity of GST n towards lipid hydroperoxides may contribute to the adriamycin resistance. However, the mechanism of regulation of GST n expression in mdr remains to be elucidated.

54 .o •♦5 o o c

Table 1.4 GST Activity and Molecular Form in Drug-Resistant Cancer Cell Lines o o CO tr < © ' T3 < X £ > t © © w O) c © 03 D) o ! >» CO C k_ c > © 3

o E C - i -C -iZ -C -C © -Q is ooi --D^ foin ifio o o t ^ o ^w o io o i-i-(D c s ^ r in o o o )o O O D c cm t- NfCNjcocooocor^r^ D O CD < < O "O "D t _Q "O O© E E © E E © E © © r = © © o c © c c c c E 2 ^5 o O) - I E o c c © © © © © © . > s a.’cocMi^'*-© K K K K tsK K K K — 0 0 C C N CO CO 00 00 O MOMM-M- LOt— CMLOf^OOCD CMCOCMCMt-CM^- o © c o © E o >> O X "©"© CO > > tO QC * > • ■*—> to E E c c ) D)D) © © c © o o E © E ° © E ©

’■S © 0 0 g J3 c cz c o ©

sz c ZD —N X . 0 Z S Z S J c/) UJ < o o E © c © o s _o © E c E E E © E © c © c c c E o g ££ £ -g E g ° © E c o E © Z J © 3 ZJ 3 3 © ^ > * © g . .E .E c ©>- X c © ZJ

3 JC

M O O O N 00 i— CD 00 - T CM < < < ■D "D E E

0. CO CO co © * - ^ © O F © © 2 E II

O -O zz CO CM CD 3 03 q 03 2 2 E 1 E c o E © o o o © © J3 ZJ

\ N n N C\1

O _Q t _c E o 0 0 CM 00 00 — *2 03 o © E E © © © O) © o © 2 c CO E o> 3 E — X CO o z _ O o O X zz sz o 3 J sz o to c © © © © E o © 2 © © o E o > o 3 U0 m Introduction

1Resistant drug, Anticancer drug to which resistance has been developed. 2Activity of GST toward CDNB, n mol/min/mg protein. 3R, resistant cells. P, parental cells. a) Baptist etal., 1986; b) Cole etal., 1990. c) Ali-Osman et al., 1990. d) Wang etal., 1989. e) Lewis etal., 1988a. f) Gupta etal., 1989. g) Kuroda etal., 1991. h) Deffie etal., 1988. i) Wang & Tew, 1985. j) Buller et al., 1987. k) Evans et al., 1987. I) Smith et al., 1989. m) Lewis etal., 1988b.

1.9 Control of the Expression of GST Genes:

1.9.1 Control of the Expression of Mouse GST Subunit Ya Gene:

The studies of mouse GST subunit Ya have revealed that two cis- acting regulatory elements are located in the 5’ flanking region of subunit Ya gene. One is responsible for the basal level of expression and the other is responsible for the inducible expression by planar aromatic compounds such as 6-NF and 3-MC and is functional only in cells withAh receptors (Daniel et al., 1989; Friling et al., 1990).

Using murine hepatoma cell mutants which are defective in either the Ah receptor or cytochrome P450IA1 genes, it was found that the induction by planar aromatic compounds but not by electrophilic inducers requires a functional Ah receptor and cytochrome P450 activity. It is concluded that the activation of mouse subunit Ya gene by planar aromatic compounds involves metabolism of these inducers by the phase I enzymes into electrophilic compounds. Therefore this sequence is named as an electrophile-responsive element (ERE) (Friling etal., 1990).

56 Introduction

1.9.2 Control of the Expression of Rat GST Subunit 1b Gene:

Rat GST subunit 1b is transcriptionally up-regulated by 3-MC and PB (Ding &

Pickett, 1985). An upstream region comprising nucleotides -1651 to -663 of

GST subunit 1b gene has been shown to contain multiple regulatory elements required for both maximal basal expression and induction by planar aromatic compounds, such as 3-NF (Telakow^ski-Hopkinsetal., 1988). Nucleotides -

867 to -857, which contain a sequence with identity to the hepatocyte nuclear factor 1 recognition motif found in several liver specific genes (Courtois etal., 1987-, 1988), contribute to the maximum basal level expression of subunit 1b gene (Rushmore etal., 1990) (Fig. 1.11).

Sequence homology search revealed the presence of a xenobiotic regulatory element, located between nucleotides -908 and -899 (Fig. 1.11), which is similar to the xenobiotic responsive element (XRE) sequence found as a cluster of six in the planar aromatic hydrocarbon responsive cytochrome P450IA1 gene (Denison et al., 1989). This XRE contributes to part of the responsiveness of subunit 1b to 3-MC and B-NF induction (Paulson et al., 1990; Rushmore et al., 1990). DNA-binding studies have shown that this site (-908 to -889) was bound by a 3-MC-inducible protein similar to the Ah receptor, which has been postulated to be a member of the steroid hormone receptor superfamily (Hapgood etal., 1989).

Another element, located between nucleotides -722 and -682 (Fig. 1.11), has been shown to respond to B-NF and has been assigned as a B-NF responsive element (Rushmore et al., 1990). Rushmore & Pickett (1990a) have demonstrated that this element is responsible for part of the transcriptional activation of the subunit 1b gene by planar aromatic compounds. However, the induction does not require the Ah receptor. Recently, it has been demonstrated that this B-NF responsive element is required for the transcriptional activation of the subunit 1b gene by phenolic

57 Introduction

antioxidants such as t-butylhydroquinone (t-BHQ) through a mechanism which also does not require a functional Ah receptor, and has thus been renamed as antioxidant-response element (ARE) (Rushmore & Pickett, 1990b). When

linked to a heterologous promoter, this ARE confers responsiveness to both t-BHQ and (3-NF but not to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). An ARE has also been identified in the 5’ flanking region (-434 to -404) of the rat NAD(P)H quinone reductase gene. The core sequence of the ARE, puGTGACNNNGC (where N is any nucleotide), has no resemblance to the XRE core sequence TNGCGTG in the 5’ region of the P450IA1 gene, the GST subunit 1b gene or the NAD(P)H quinone reductase gene. On the other hand, this ARE has homology with the ERE which confers electrophile responsiveness in the 5’ region of mouse GST Ya subunit gene (Rushmore et al.f 1991) (See section 1.9.1).

The results of induction of the GST subunit 1bgene by t-BHQ, 3MC, B-NF and TCDD are consistent with the model proposed by Talalay et al. (Prochaska & Talalay, 1988; Talalay etal., 1988) who have suggested that the mechanisms of the induction of phase II drug metabolizing enzymes by monofunctional and bifunctional inducers are distinct. Monofunctional inducers, such as t-BHQ, activate gene expression by means of an electrophilic signal which operates independently of the Ah receptor and the ARE mediates such induction. However bifunctional inducers such as B-NF and 3-MC, can transcriptionally activate gene expression directly through an Ah receptor dependent

mechanism which is mediated by the XRE. Furthermore, inducers, such as B-NF can also act through the ARE once they are metabolized to compounds resembling monofunctional inducers, whereas poorly metabolizable inducers such as TCDD can transcriptionally activate gene expression only through the XRE-Ah receptor-dependent mechanism.

In addition, Rushmore et al. (1991) have found that phenolic antioxidants, which undergo one- or two-electron reductions as well as redox cycling to

58 Introduction

-908 -899 -867 -857 -722 -682 tsp

XRE Basal ARE expression

Ah receptor Ah receptor Ah receptor

TCDD t-BHQ.

Fig. 1.11 Schematic Representation of Regulatory Elements of the Rat Subunit 1b Gene

XRE, xenobiotic response element; ARE, antioxidant response element. TCDD, 2,3,7,8-tetrachlorodibenzo-/>dioxin.

form reactive oxygen species and oxidants such as hydrogen peroxide, activate both the GST subunit 1b and the NAD(P)H quinone reductase genes through AREs. This may suggest that the ARE is a common cis -acting element in the 5’ flanking region of genes that are transcriptionally activated

by oxidants and hence the ARE and the proteins that bind to it are part of a signal transduction pathway that senses and responds to oxidative stress (Rushmore etal., 1991).

59 Introduction

1.9.3 Control of the Expression of the Rat Subunit 7 gene:

The gene encoding GST subunit 7 or GST-P, together with its 5’ flanking region has been characterized by Muramatsu and colleagues who identified multiple regulatory elements. The promoter region of subunit 7 (Fig. 1.12) contains a TATA box, a G/C box as well as a consensus phorbol 12-0- tetradecanoyl 13-acetate (TPA) responsive element (TRE) sequence which is identical to the TRE sequence found in the human metallothionein IIA gene (Lee et al., 1987a). Two enhancer elements, termed GPE I and GPE II, are located about 2.5 kb upstream of the gene (Fig. 1.12) (Sakai et al., 1988a). GPE I is composed of two imperfect TREs whereas GPE II contains two enhancer sequences similar to those of Simian virus 40 and one sequence similar to that of polyoma virus. The region of GPE I responsible for its enhancer activity has been honed down to 27 bp which contain the consensus TRE or AP1 binding motif TGATTCAG in a region of partial diad symmetry, GTCAGTCA.CAT. TGATTCA (Sakai et al., 1988a-, Okuda et al., 1989; Muramatsu etal., 1989) thus implicating the possible role of AP1 in the activity of the enhancer. Deletion analysis demonstrated that this region, but not the consensus TRE sequence adjacent to the promoter, was responsible for the TPA induction of the gene. However, studies have suggested that the TRE adjacent to the promoter, as well as the G/C box, are functional cis -acting DNA elements (Okuda etal., 1987; Sakai etal., 1988a).

Further investigation has revealed that each of the two imperfect TREs of GPE I has no activity per se, but acts synergistically to form a strong enhancer which is active even in F9 cells, in which the activity of AP1 is very low (Okuda etal., 1990). In addition, it has been demonstrated that the activity of GPE I is greatly influenced by the relative orientation and the distance between the two TREs (Okuda et al., 1990).

TPA acts through the protein kinase C signal transduction pathway

60 Introduction

-2.5 kb -1 bp

GPE I GPE II S tsp

^ \ / \ S \ / ^

XGTCAGTCACTATGATTCAG^ / ^ \

— ~ GPEI *■— ' ^ \ \

TTGACTCAGCATCCGGGGCGGGGCGCAATGCCCCTTATAAG

API G/C TATA

pPromoter l r

Fig. 1.12 Schematic Representation of Regulatory Elements of Rat GST Subunit 7 Gene

The promoter and the core sequence of the enhancer element GPE I are shown enlarged, and enhancer element GPE II and silencer are indicated. In the promoter region, consensus AP1 binding site and SP1 (the G/C box) and TATA box are marked. In the GPE I region, the boxes and arrows emphasize the symmetry of the two imperfect TRE sequences.

61 Introduction

(Rozengurt, 1986) and thus mimics stimulation with serum growth factors such as PDGF. The target for TPA is protein kinase C itself and its activation is believed to result in the alteration of transcription factor complexes such as

AP1 (Lee et al., 1987b) which modulate transcription of genes with the appropriate transcription factor binding sites. AP1 appears to be a mixture of proteins including the nuclear oncogene c-jun (Angel et al., 1988; Curran & Fran^za, 1988; Bos et al., 1988) or other members of jun family (Hirai et al.,

1989), and c-fos (Nakabeppu etal., 1988). The AP1 binding site defined by Angel et al. (1988) is a binding site for jun/jun homodimeric and jun/fos heterodimeric complexes, but most transcription activations are thought to be mediated preferentially by the fos/jun heterodimer (Chiu etal., 1988; Sassone-

Corsi et al., 1988; Schonthal et al., 1988; Zerial et al., 1989).

Acting at another level in the protein kinase C signal transduction pathway are the products of the ras protooncogenes (Fleischman et al., 1986; Imler etal., 1988). It has been reported that GST subunit 7 mRNA levels in rat liver epithelial cells were induced by transformation of activated N-ras or H-ras (Power et al., 1987; Li et al., 1988; Burt et al., 1988).

A silencer has been identified in the region 400 bp upstream of the tsp site of GST subunit 7 (Sakai et al., 1988a). Using DNA-binding and transient expression assays, it has been found that this silencer is active not only in rat non-hepatoma and hepatoma cell lines but also in human and mouse cell lines, suggesting that it functions as a general silencer (Imagawa et al., 1991a). This silencer consists of multiple negative elements to which nuclear factors bind and function in a cooperative manner. At least three trans-acting factors (A, B and C) have been found to bind to this silencer. Among them, SF-B has been cloned and the deduced amino acid sequence reveals that SF- B is almost identical to an IL-6 inducible trans-activator LAP/IL6-DBP. LAP and IL6-DBP were identified as a liver-enriched transcriptional activator protein (Descombes etal., 1990) and as an IL-6 dependent DNA binding protein (Poli

62 Introduction

et al., 1990) respectively. It seems that SF-B/LAP/IL6-DBP functions as a dual positive and negative regulator (Imagawa et al., 1991b).

1.9.4 Control of the Expression of Human GST n gene:

The gene encoding GST n has also been sequenced (Cowell et al., 1988; Morrow et al., 1989), and it spans approximately 3 kb, interrupted by six introns. Transient expression assays have revealed that a fragment from +8 to +99 bp upstream of the tsp site promotes transcription, but there is no evidence for any enhancer activity in a further 6 kb of 5’ flanking sequence. Analysis of the sequence by reference to a primate sequence database revealed the presence of an Alu repeat, occupying positions -520 to -820, which was inserted into the 3’ end of a LINE 1 repetitive element. The LINE 1 repeat extended as far as the available sequence data. The immediate promoter of GST n contains similar motifs to the promoter of the rat subunit 7 gene, namely a TATA box and two G/C boxes as well as a consensus TRE or AP 1 binding site (Fig.1.13) (Cowell et al., 1988; Morrow et al., 1989). Studies have shown that the consensus AP1 binding site failed to respond to TPA (Dixon et al., 1989; Morrow et al., 1990) and that there was no correlation between expression of activated ras and GST n mRNA (Dixon et al., 1989).

1.10 Aim of This Thesis

As summarised in the Preface, this thesis describes studies on the G-site of GST n and also studies on factors which regulate the expression of the GSTrc gene. Both approaches are of great interest with respect to the understanding of the development and possibly the treatment of cancer.

63 Introduction

-80 -60

CCGCGGGACCCTCCAGAAGAGCGGCCGGCGCCGTGACTCAGCACTGGGGCGGA Sst II AP1 G/C -40 -2 0 + 1

GCGGGGCGGGACCACCCTT&TAAGGCTCGGAGGCCGCGAGGCCTTCGCTGGAG G/C TATA Stu I + 20 + 4 0 r TTTCGCCGCCGCAGTCTTCGCCACCAGTGAGTACGCGCGGCCCGCGTCCCCGG t + 60 Intron 1

GGATGGGGCTCAGAGCTCCCAGC Sst I

Fig. 1.13 Sequence of the Promoter Region of GST n Gene

The position of the TATA box, G/C box and the consensus AP1 binding site are indicated. In addition, restriction sites Sst I, Sst II and Stu I are underlined.

64 CHAPTER II

MATERIALS AND METHODS

65 Materials and Methods

Foreword

This Chapter is divided into two parts, Part I describes the procedures that were used to investigate the active site of GST n. Part II describes the methods employed to study the control of the expression of GST n gene.

PARTI

2.1.1 Chemicals:

All basic chemicals were of analytical grade and were obtained from BDH

Limited, Fluka Chemical Limited, Sigma Chemical Company and Pharmacia Limited. Buffers used for modification reactions are listed in Table 2.1.

Table 2.1 Buffers for Modification Reactions*

Buffers Reagents

A 0.1 M sodium phosphate pH 7.4 DTNB, NEMI

B 0.1 M sodium phosphate pH 8.0 TNBS

C 0.05 M sodium borate pH 8.5 DiAC

D 0.01 M diaminopropane/HCI pH 4.8 ED AC

E 0.1 M sodium phosphate pH 7.0 DEPC

F 0.1 M sodium phosphate pH 5.8 NBS

*AII buffers contain 2 mM EDTA and buffers B-F also contain 2 mM 2- mercaptoethanol.

66 Materials and Methods

2.1.2 Protein Determination:

The protein concentration of GST n was determined by the method of

Bradford (1976) using ovalbumin as standard.

2.1.3 Enzyme Activity Assay:

The standard assay for GST n activity was based on the reaction of CDNB with GSH (Habig et al., 1974). A stock solution of CDNB was prepared in

40% ethanol/water (v/v). The reaction mixture contained 1 mM GSH, 1 mM CDNB (final concentration of ethanol was 4%) and 0.1 M sodium phosphate, pH6.5. The reaction was initiated by addition of enzyme and was monitored spectrophotometrically at 340 nm (e=9600 M'1crrf1). 1 unit of enzyme activity is defined as 1 pmol product formed/min/mg GSTtc.

2.1.4 Purification of GST n from Human Placenta:

GST n was purified from human placenta as follows: All procedures were carried out at 4°C unless otherwise stated. A homogenate was prepared from 150 g human placenta, using a Waring blender, in 2 volumes (v/w) of homogenization buffer (buffer 1) containing 10 mM sodium phosphate, pH 7.0, 160 mM KCI, 2 mM DTT and 25 pM PMSF. The soluble supernatant obtained by centrifugation at 105,000xg for 1 hour was passed through a GSH-agarose affinity column (Pharmacia). After washing with 10 volume of buffer 1, the GST fraction was eluted with 50 mM Tris/HCI, pH 9.1 containing 5 mM HEX- GSH and 5 mM GSH (buffer 2). The active fractions were pooled, concentrated to 2 ml using Centriprep concentrators (Amicon) and transferred into buffer 3, which contained 10 mM Bis-tris/HCI at pH 6.1, 0.5 mM DTT and 1 mM GSH, by rapid gel filtration on a PD-10 column (Pharmacia) loaded with buffer 3. The total elutenf(2 ml) was applied to FPLC Mono Q HR5/5 anion- exchange column (Pharmacia) equilibrated with 0.2 ji nitrocellulose filtered

67 Materials and Methods

buffer 3. The column was washed with 4 ml of buffer 3 followed by a gradient

of 0 - 0.3 M KCI in buffer 3 at a flow rate of 0.4 ml / min. GST n was eluted

at approximately 0.2 M KCI and had a specific activity of 130 units/mg protein. The enzyme was dialyzed against 0.1 M potassium phosphate, 2 mM EDTA with or without 1 mM 2-mercaptoethanol, pH7.0 before being subjected to modification. AM n e ^ X e d Mere ris e *.

2.1.5 Modification by DTNB:

2.1.5.1 Reaction of GST n with DTNB:

GST k (6 pM) in buffer A (Table 2.1) or 6 M urea was incubated with 0.5 mM DTNB at 12°C for 10 min and the temperature was raised to 37°C for a further 20 min. The reaction was monitored at 412 nm. Aliquots were removed to measure enzyme activity at various times and the enzyme activities were determined. The number of cysteine residues modified were calculated from A412 with 0.5 mM DTNB in buffer A or 6 M urea as reference (e=14129 M'1 cm' 1 in buffer A or 14290 M'1cm'1 in 6 M urea) (Ellamn, 1959).

2.1.5.2 Determination of the Rate Constant of DTNB Modification:

7 pM GST n was incubated with 0.14 mM DTNB at 12°C in buffer A (Table 2.1). The modification reaction was monitored at 412 nm with 0.14 mM DTNB in buffer A as reference.

2.1.5.3 Effect of GS-OCT on DTNB modification:

7 pM GST n in buffer A (Table 2.1) was incubated with 0.14 mM DTNB at 12°C in the absence or presence of GS-OCT at 1.4 pM, 14 pM and 140 pM. The product of DTNB modification was monitored at 412 nm with 0.14 mM

68 Materials and Methods

DTNB in buffer A as reference.

2.1.6 Modification of GST n by NEMI:

2.1.6.1 Kinetics of NEMI Modification of GST n :

0.23 pM enzyme was incubated with various concentrations of NEMI in buffer A (Table 2.1) at 18°C. Aliquots were removed at 4 min intervals and enzyme

activity was measured.

2.1.6.2 Effects of GS-HEX, GS-OCT, CDNB and Bilirubin on NEMI modification of GST n :

0.46 jiM enzyme was pre-incubated alone, with 1 pM GS-OCT, 5 pM GS- HEX, 100 pM bilirubin or 500 pM CDNB at 25°C in buffer A (Table 2.1) for 5 min. 50 pM NEMI was added to each reaction mixture and an equal volume of buffer A was added to each control. Aliquots were removed at 5 min intervals and assayed for the remaining enzyme activities.

2.1.7 Modification of GST n by TNBS:

2.1.7.1 Modification by TNBS:

In a 2.0 ml reaction mixture, 6 pM GST n was incubated in buffer B (Table 2.1) at 22°C, in the dark, for 30 min with freshly made TNBS at various concentrations. 0.1 ml of each reaction mixture was removed after 30 min incubation and assayed for the remaining enzyme activity. Formation of the product TNB-GST n was measured at A420 (e=19200 M*1cm"1; Hollenberg, 1971).

69 Materials and Methods

2.1.7.2 Effects of GS-OCT, Bilirubin and CDNB on TNBS Modification:

0.5 pM enzyme was preincubated alone, with 1 pM GS-OCT, 100 pM bilirubin

or with 500 pM CDNB in buffer B (Table 2.1) for 5 min. 50 pM TNBS was

added to each reaction mixture and an equal volume of buffer B was added to each control. Aliquots were removed at 5 min intervals and assayed for the enzyme activities.

2.1.8 Modification of GST n by DiAC:

2.1.8.1 Kinetics of DiAC Modification:

0.14 pM enzyme was incubated with DiAC at various concentrations in buffer C (Table 2.1) at 25°C. Aliquots were removed at 5 min intervals and assayed for the remaining enzyme activities.

2.1.8.2 Effects of GSH, GS-HEX, GS-OCT, Bilirubin and CDNB on

DiAC Modification of GST tc:

0.14 pM enzyme was pre-incubated alone, with 100 pM GSH, 1 pM GS-OCT, 10 pM GS-HEX, 100 pM bilirubin or 500 pM CDNB in buffer C (Table 2.1) at 25°C for 5 min. 50 mM DiAC was then added to each reaction mixture and an equal volume of buffer D was added to each control. Aliquots were removed at 5 min intervals and the enzyme activities were measured.

2.1.9 The Modification of GST n by EDAC:

2.1.9.1 Kinetics of Modification by EDAC:

0.27 pM enzyme was incubated with EDAC (dissolved in ethanol), at various

70 Materials and Methods

concentrations, at 25°C in buffer D (Table 2.1). The final concentration of ethanol was 4% (v/v). Aliquots were taken and the enzyme activity was measured.

2.1.9.2 Effects of GSH, GS-OCT, GS-HEX, Bilirubin and CDNB on EDAC Modification of GST n:

0.27 pM enzyme preincubated alone, with 500 pM GSH, 1 pM GS-HEX, 1 pM GS-OCT, 100 pM bilirubin or 500 pM CDNB in buffer D (Table 2.1) for 5 min at 25°C. EDAC (dissolved in ethanol) was added to each reaction mixture to a final concentration of 50 mM and an equal volume of ethanol was added to

each control. Aliquots were removed for enzyme activity assay at 5 min intervals.

2.1.10 Modification by DEPC: C A toastin' e ta l.,>937)

2.1.10.1 Detection of Modification of GST n by DEPC:

16 pM GST 7i in 400 pi buffer E (Table 2.1) was placed in a cuvette and 8 pi of 250 mM DEPC (freshly made in dry ethanol) was added (final concentration of DEPC was 5 mM). In the reference cuvette, 16 pM enzyme in 400 pi buffer E was mixed with 8 pi ethanol. The sample was scanned from 310 nm to 210 nm at 4°C, at 0, 2, 4, 6, 8 min intervals and finally at 40, 45, 55 and 60 min to monitor the completion of the reaction of DEPC with histidine and tyrosine, and to monitor the hydrolysis of DEPC.

2.1.10.2 Determination of the Numbers of Histidine and Tyrosine Residues of GST n Modified by DEPC:

12 pM of GST 71 in 400 pi buffer E (Table 2.1) was mixed with 8 pi 250 mM

71 Materials and Methods

DEPC freshly made in ethanol (final concentration of DEPC was 5 mM) and a reference blank was made by mixing 12 pM GST % in 400 pi of buffer E with 8 jil ethanol. The control was prepared by mixing 400 pi buffer E with 8 pi 250 mM DEPC and a reference blank was made by mixing 8 pi ethanol with 400 pi buffer E [\final concentration of ethanol was 2% (v/v)]. After 1 hour at n 4°C, both the sample and the control were scanned from 310 to 210 nm. The number of modified histidine residues was calculated from A246 (e=3200 M'1 min'1) and that of the tyrosine residues was calculated from A^g (e=1300 M'1min'1).

2.1.10.3 Modification^ DEPC and Enzyme Activity:

10 pM enzyme was pre-incubated in buffer E (Table 2.1) at 4°C and the reaction was started by addition of 0, 0.5, 1, 2 and 5 mM of DEPC (final concentration). Final concentration of ethanol was 2% (v/v). After 30 min, 0.1 ml reaction mixture was withdrawn and the enzyme activity was assayed. The extent of carbethoxylation of enzyme was q u a n tify by recording the absorbance increase at 246 nm between the reaction mixture and a control containing an equal amount of DEPC in buffer B (e=3200 M'1cm'1).

2.1.10.4 Effects of GSH or CDNB on DEPC Modification of GST n:

12.5 pM enzyme was preincubated alone, with 500 pM GSH or 500 pM CDNB at 4°C in buffer E (Table 2.1) for 5 min and 50 mM DEPC (final concentration) was added and incubated for 5 min. The enzyme activity was assayed.

2.1.10.5 Peptide Analysis of DEPC Modification of GST n:

Chymotrypsin was prepared at 3 mg/ml in 0.1 M NH4H C 0 3. To 400 pi DEPC- modified GST n (75 pg) dissolved in buffer E (in the control, 75 pg non

72 Materials and Methods

modified GST n was used), 1.25 pi chymotrypsin (containing 3.75 pg chymotrypsin) was added. After a 4 hour incubation at room temperature, another 1.25 pi chymotrypsin was added and the mixture was incubated at room temperature overnight. A chymotrypsin blank was also prepared.

To separate the resulting peptides, 200 pi of the mixture was subjected to reverse-phase HPLC on a Brownlee Aquapore RP 300 (30 mm x 2.1 mm) C8 column (Anachem, luton, Beds., U.K.). The column was pre-washed with 0.06% (v/v) trifluoroacetic acid (TFA). A solvent gradient of 2-45 % (v/v) acetonitrile in water (both solvents containing 0.06% (v/v) TFA) was developed over 60 min, at a flow rate of 0.13 ml/min. The elutentwas monitored at 214

and 240 nm. Fractions which had an increased absorbance at 240 nm, compared with 214 nm, were collected and subjected to automated Edman degradation in an Applied Biosystems 470A gas-phase sequencer with a 120A on-line PTH analyser (Applied Biosystems).

2.1.10.6 N-terminal Sequencing of GST n and DEPC Modified GST n:

10 pg of GST 7t or DEPC modified GST 7r obtained in section 2.1.10.1 was subjected to automated Edman degradation in an Applied Biosystems 470A gas-phase sequencer with a 120A on-line PTH analyser (Applied Biosystems).

2.1.11 Modification of GST n by NBS:

6 pM enzyme was incubated with 10 pM NBS in buffer F (Table 2.1) at 25°C for 25 min and the enzyme activity was measured (Spands, 1967).

73 Materials and Methods

PART II

Ml m etficvts described betovo frere. -fake* M an^ils et

2.2.1 Chemicals:

2.2.1.1 Basic Chemicals:

All common chemicals were of Analar Grade and were obtained from BDH Limited, Fluka Chemical Limited, Sigma Chemical Company, Gibco BRL Limited and Pharmacia Limited.

2.2.1.2 Bacterial and Cell Culture Reagents and Media:

Bacto tryptone, yeast extract and Bacto agar were supplied by Difco Laboratories Limited. The cell culture media Dulbecco’s Modified Eagles Medium (DMEM), Modified Eagles Medium (NEM), RPMI 1640 and OPTIMEN 1 as well as foetal calf serum and trypsin-EDTA we/e the products of Gibco BRL limited. Dimethyl sulphoxide (DMSO) w as obtained from Sigma Chemical Company. Ampicillin and gentamycin were purchased from Sigma Chemical Company. Streptomycin and penicillin were obtained from Gibco BRL Limited.

2.2.1.3 Enzymes

All restriction endonucleases, T4 DNA ligase, T4 polynucleotide kinase and Bal 31 exonuclease were supplied by Gibco BRL Limited. Calf intestinal alkaline phosphatase was purchased from Boehringer Mannheim Biochemical Limited. Large fragment DNA Polymerase I (Klenow) was obtained from Pharmacia Limited and Ribonuclease (RNase) A from Sigma Chemical Company.

74 Materials and Methods

2.2.1.4 Enzyme Substrate and Inhibitors

Acetyl CoA was supplied by Boehringer Mannheim Biochemicals Limited. O- nitrophenyl-6-galactopyranoside (ONPG) was obtained from Sigma Chemical Company. Phenylmethylsulfonyfluoride (PMSF), N-a-tosyl-L-lysine chloromethyl ketone (TLCK), N-tosyl-L-phenylalanine chloromethyl ketone (TPCK), 6-glycerol phosphate, levamisole, antipain, leupeptin, aprotinin and pepstatin A were the products of Sigma Chemical Company.

2.2.1.5 Hormones and Vitamins

Insulin, hydrocortisone and retinoic acid were purchased from Sigma Chemical Company. Insulin-like growth factor II (IGFII) was obtained from Intergen Company.

2.2.1.6 Oligonucleotides and Nucleic Acids

Plasmid vector pUC13,1 kilobase and 300 basepair molecular weight markers were supplied by Gibco BRL Limited. The primensfor sequencing and the polymerase chain reactions were synthesised by Enzymatic Company. Hexadeoxynucleotides were purchased form Pharmacia Limited.

2.2.1.7 Radiochemicals:

Adenosine 5’ triphosphate [r -32P] (r-32ATP, 5000 Ci/mmol), deoxyadenosine 5’-(a-thio) triphosphate [a-35S] (a-35S dATP, 500 Ci/mmol), deoxycytidine 5’- triphosphate [a-32P] (a-32P dCTP, 600 Ci/mmol) were purchased from

Amersham International Limited. [14C] chloramphenicol (50 Ci/mmol) was obtained from DuPont (U.K.) Limited.

75 Materials and Methods

2.2.1.8 Reagent Kits

SequenaseR Version 2.0 kit was purchased from United States Biochemical Corporation. GeneAmp™ DNA Amplification Reagent kit was obtained from Perkin-Elmer Corporation. Pierce BCA* Protein Assay Reagents kit was supplied by Pierce Corporation.

2.2.1.9 Other Materials

X-ray films, X-Omat S and X-Omat AR, were supplied by Kodak Limited. 3MM and 1 MM Whatman Filters were purchased from Whatman Lab Sales Limited. Sephadex G-50 was obtained from Pharmacia Limited. Hybond N was the product of Amersham International Limited. Silica thin layer chromatography (TLC) plates were obtained from Merck Limited.

2.2.2 Commonly Used Solutions:

TE: 10 mM Tris/HCI pH 8.0, 1 mM EDTA; TBE: 89 mM Tris/HCI 89 mM boric acid pH 8.3, 2 mM EDTA. SSC: 150 mM NaCI, 15 mM trisodium citrate, pH 7.0. Phenol/Chloroform: phenol/chloroform/isoamyl alcohol : 25/24/1, equilibrated with TE (pH 8.0). PBS: 140 mM NaCI, 27 mM KCI, 10.6 mM Na2HP04, 15 mM KH2P04. 100 x Denhardt’s Solution: 2 % (w/v) Ficol(,2 % (w/v) polvinylpyrrolidone, 2 % (w/v) BSA.

2.2.3 Bacterial Cell Culture:

2.2.3.1 Bacterial Strains

E.coli JM83 (Vieira & Messing, 1982) (lac proA,B) ara, rpsl, (>80 lacZ M15

(!■ « *. m K*)- E.coli JM109 was obtained from Promega.

76 Materials and Methods

2.2.3.2 Bacterial Growth Media:

LB: 1 % (w/v) Bacto tryptone, 1 % (w/v) NaCI, 0.5 % (w/v) yeast extract; 2 x TY: 1.6 % (w/v) Bacto tryptone, 1 % (w/v) NaCI, 1 % (w/v) yeast extract, 0.2 % (w/v) glucose. Agar-LB: 1.4 % agar/LB (w/v). All selective media contained 100 pg/ml ampicillin.

2.2.4 Plasmids and Cosmids:

Cosmid 3.5.1 (Cowell, 1989, Ph.D. Dissertation, UCL); pSV2CAT (Gorman e ta., l 1982); pICCAT (Dixon etal., 1989); pSS0.2CAT (Dixon etal., 1989); pSSO.ICAT (Dixon etal., 1989); pCH110, Product of Promega; hRARB, A gift from Dr. P. Brickell, Department of Biochemistry, UCL.

2.2.5 Isolation of Plasmid DNA:

Cosmid and plasmid DNAs were maintained in the host cells ED8767 and JM 83 or JM 109 respectively/ Comii. i?s<-ij.

2.2.5.1 Mini-Scale Isolation of Plasmid DNA:

10 ml LB medium containing ampicillin was incubated with a single bacterial colony, from a fresh plate, overnight at 37°C. The culture was centrifuged at 3000 rpm for 10 min at 4°C. The pellet was resuspended in 1 ml of 50 mM glucose, 25 mM Tris/HCI, 10 mM EDTA, pH 8.0 and after 5 min at room temperature, 2 ml of 200 mM NaOH, 1 % SDS was added. The suspension was thoroughly mixed at room temperature for 5 min. 1.5 ml of 3 M potassium acetate, pH 4.8 was added, and the suspension was incubated on ice for 10 min. The precipitate was pelleted by centrifugation at 3000 rpm for 5 min and then the supernatant was removed and further centrifuged in 1.5 ml microfuge tubes at 12,000 rpm for 10 min. The supernatant was pooled,

77 Materials and Methods

RNase was added to 1 pg/ml and incubated at 37°C for 30 min. 2.4 ml isopropanol was added and the plasmid DNA precipitate was collected by centrifugation at 3000 rpm for 10 min at 4°C. The pellet was resuspended in (2 060*9) 400 pi TE and deproteinized by phenol/chloroform and chloroform extractions. The aqueous phase was removed and DNA was precipitated by the addition of NaCI to a final concentration of 100 mM, and 2 volumes ethanol. After incubating at -70°C for 30 min, the DNA was recovered by centrifugation at 12000 rpm for 10 min. The pellet was washed with 70 % ethanol, dried and (tZ400*$f taken up in 20 pi TE and stored at -20°C.

2.2.5.2 Large Scale Isolation of Plasmid DNA:

A small culture (10 to 15 ml) was initially grown for 6 to 8 hours from a single colony. 1 ml of the culture was used to inoculate 500 ml of 2 x TY medium. The culture was grown at 37°C overnight. Cells were pelleted by centrifugation at 5000 rpm (2600 x g) for 5 min. The pellet was resuspended in 50 ml of ice cold 50 mM glucose, 25 mM Tris/HCI, pH 8.0, 10 mM EDTA and incubated at 4°C for 10 min. 100 ml of 0.2 M NaOH, 0.1 % SDS was added to the suspension and the mixture was allowed to stand at room temperature for 10 min. Chromosomal DNA was precipitated by adding 75 ml of 3 M potassium acetate, pH 4.8, incubated at 4°C for 10 min and centrifuged at 8000 rpm (7000 x fi) for 20 min at 4°C. Remnants of the precipitate were removed by filtration through nylon mesh. Isopropanol (0.6 volumes) was added to the filtrate and precipitated material was pelleted by centrifugation at 8000 rpm (7000 x g) for 10 min at 4°C. The pellet was resuspended in 10 ml TE. Caesium chloride was added to a final concentration of 1.0 g/ml and ethidium bromide was added to a final concentration of 1 mg/ml. The solution was subjected to centrifugation at 55000 rpm (230000 x g) for 20 hours at 22°C. At the end of the centrifugation run the rotor was allowed to coast to a halt. A hole was pierced in the top of the tube and the banded DNA was removed from the side of the tubes in a volume of 1 to 2 ml using a 5 ml

78 Materials and Methods

syringe and a 19G needle. Ethidium bromide was removed by extraction with TE-saturated butanol. After dialysis against TE, 2 volumes ethanol and 0.1 M NaCI (final concentration) was added to the solution and left at -20°C for a minimum of 2 hours. DNA was collected by centrifugation and washed with 70 % ethanol, dried, taken up in 0.5 to 2 ml TE and stored at -20°C. For transfection into mammalian cells, plasmid DNA was subjected to two rounds of caesium chloride density gradient centrifugation.

2.2.6 Enzyme Modification of DNA:

2.2.6.1 Restriction Digestion:

Restriction digestions were performed using the buffers provided by manufacturers as follows: For digestion with Acc I and Apa I, the reaction buffer contained 6 mM Tris/HCI pH 7.6, 6 mM MgCI2, 6 mM NaCI, 1 mM DTT and 100 pg/ml BSA. For digestion with Hind III, Sst I and Sst II, the reaction buffer contained 50 mM Tris/HCI pH 7.9, 10 mM MgCI2, 50 mM NaCI and 100 fig/ml BSA.

For digestion with Eco Rl and Hinc II, the reaction buffer contained 50 mM Tris/HCI pH 7.6, 6 mM MgCI2, 100 mM NaCI and 100 pg/ml BSA. For digestion with Bgl II, Ned I and Pst I, 10 mM Tris/HCI pH 7.6, 10 2- mM MgCI2, 100 mM NaCI, 6 mM^mercaptoethanol and 100 jig/ml BSA. For digestion with Tth111 I and Xba I, the reaction buffer contains 10 mM Tris/HCI pH 7.4, 10 mM MgCI2, 50 mM NaCI, 10 mM 2-mercaptoethanol and 100 jig/ml BSA. For digestion with Sma I, the reaction buffer contained 20 mM Tris/HCI pH 7.6, 5 mM MgCI2, 50 mM KCI. An excess of enzyme (usually 2 to 5 fold) was used and the digestions were carried out for 1 to 3 hours at 37°C, except for Sma I which was performed at 30°C.

79 Materials and Methods

2.2.6.2 Exonuclease Digestion:

For digestion with exonuclease Bal 31, 100 pi of the reaction contained 20

mM Tris/HCI pH 8.0,12 mM MgCI2, 600 mM NaCI, 12 mM CaCI2, 1 mM EDTA and 5 fig DNA. 0.2 U of Bal 31, which can digest double-stranded DNA at 10 bp/min/end, was used. The reaction was carried out at 30°C. 10 pi aliquots of the reaction mixture were removed at 1, 2, 3, 4, 5, 6, 8 ,10,12 and 15 min and mixed with 3 pi 0.5 M EDTA (pH 8.0) to stop the reaction. DNA fragments were purified by agarose gel electrophoresis.

2.2.6.3 Treatment of DNA Fragments with Alkaline Phosphatase:

Treatment of DNA fragments with 1 U/pl alkaline phosphatase was carried out in buffer TE, or any restriction enzyme buffers, at 37°C or room temperature for 15 to 30 min. The reaction was stopped by heating the mixture to 65°C for 5 min or by mixing with the sample buffer for agarose gel electrophoresis.

2.2.7 Isolation of Restriction Fragments:

Restriction fragments were separated by agarose gel electrophoresis.

2.2.7.1 Agarose Gel Electrophoresis of DNA Fragments:

The appropriate quantity (0.6 to 2 %) of agarose was melted into 1 X TBE. The slurry was cooled to about 60°C, ethidium bromide was added to a final concentration about 0.5 pg/ml and the resulting mixture was poured into a perspex gel support tray with a comb in position to form sample wells. The gel was allowed to set for at least one hour. Samples for electrophoresis contained up to 2 pg of DNA, 10 % (v/v) glycerol, 0.05 % (w/v) bromophenol blue and 1 x TBE. Electrophoresis was carried out in 1 x TBE containing 0.5 pg/ml ethidium bromide at 5 V/cm.

80 Materials and Methods

2.2.7.2 Isolation of Restriction Fragments from Agarose Gels:

Restriction fragments were separated by agarose gel electrophoresis. After

electrophoresis, the desired bands were made to migrate onto a piece of NA45 DEAE cellulose membrane placed immediately in front of the band. DNA was eluted from the membrane into 400 pi 1 M NaCI for 1 hour at 70°C. Two volumes of ethanol were added to the eluate and DNA was precipitated at -20°C for 2 hours.

2.2.8 Southern Blot Analysis:

DNA fragments were subjected to (0.8 to 1.5 %) agarose gel electrophoresis in 1 X TBE containing 0.5 pg/ml ethidium bromide. Prior to blotting onto nylon membranes, the gel was soaked in a denaturing solution (1.5 M NaCI, 0.5 M NaOH) for 30 min with gentle shaking. Then the gel was soaked in a neutralizing buffer (1.5 M N aC I, 0.5 M Tris/HCI pH 7.2, 1 mM EDTA) for 30 min.

2.2.8.1 Transfer of DNA Fragments to Solid Support:

DNA fragments were transferred to the membrane (Hybond-N) by capillary action (Southern, 1975). A perspex "bridge" covered with Whatman 3MM paper was arranged over a reservoir of 10 x SSC such that the ends of the paper acted as wicks in the reservoir. The filter paper covering the bridge was thoroughly wetted with 10 x SSC and the gel was placed carefully onto its surface. The membrane was placed on the gel and then two pieces of Whatman 3MM paper were placed on top of the membrane followed by a thick layer of paper tissues as the absorbent material. Transfer was allowed to proceed overnight and the membrane was exposed to U.V. irridiation for 10 min to fix the transferred DNA.

81 Materials and Methods

2.2.8.2 Oligo-Labelling of DNA Fragments Using Hexadeoxynucleotide Primers:

Double-stranded DNA labelling was carried out using the random primer labelling method. DNA was denatured by heating, and annealed to random hexadeoxynucleotide primers from which DNA synthesis was performed in the presence of labelled deoxynucleotide triphosphate, using the Klenow fragment of E. coli DNA polymerase I.

DNA to be labelled (25 to 50 ng) was heated to 100°C for 3 min, briefly cooled on ice before^ added to 50 pi of standard reaction mixture which contained 50 mM Tris/HCI pH 6.6, 200 mM HEPES, 25 mM MgCI2, 10 mM 2- mercaptoethanol, 0.5 mM of dATP, dTTP and dGTP, 30 pg/ml random hexadeoxynucleotides, 0.5 mg/ml BSA, and 0.1 mCi/ml/c*-*7^dCTP. 2 to 4 units of Klenow polymerase I was added to initiate the reaction. The reaction was performed at room temperature for 5 hours or more and was stopped by addition of 100 pi stop solution which contained 20 mM NaCI, 10 mM Tris/HCI pH7.5, 2 mM EDTA, 0.25 % SDS and 1 mM dCTP. The labelled DNA was purified by filtration through Sephadex G-50.

2.2.8.3 Hybridization:

Before prehybridization, the membranes were wetted in 2 x SSC, 0.1 x SDS. Prehybridization was carried out for 2 hours at 65°C in sealed plastic bags containing 6 X SSC, 20 mM sodium phosphate pH 7.0, 5 x Denhardt's solution, 7 % SDS and 100 pg/ml heat-denatured salmon sperm DNA. Hybridization was carried out in the above solution following the addition of 5-

10 ng/ml (final concentration) of 32P-labelled oligonucleotide probe which was heated in a boiling water bath for 5 min before being added to the hybridization solution. The hybridization was carried out at 65°C overnight.

82 Materials and Methods

After hybridization, the membranes were washed twice in 2 X SSC, 0.1 % SDS at room temperature, and then twice in 0.2 x SSC, 0.1 % SDS for 1 hour at 60°C. Autoradiography was performed at -70°C using Kodak X-omat S X- ray film.

2.2.9 Polymerase Chain Reaction (PCR)

PCRs were performed by using GeneAmp™ DNA Amplification Reagent Kit. The standard reaction mixture contained 50 mM KCI, 10 mM Tris/HCI pH 8.3, 1.5 mM MgCI2, 0.01 % (w/v) gelatin, 200 pM of each dATP, dGTP, dCTP and dTTP, 5 to 20 pmoles of primers 1 and 2 and 1 pg template DNA. The reaction was carried out in a Perkin Elmer Cetus DNA Thermal Cycler. A standard PCR operation included the following steps: Firstly, the reaction mixture was incubated at 94°C for 4 min allowing the template to be fully denatured before entering the second cycling stage. Each cycle began with a 1 min incubation at 94°C, followed by 2 min at 37 to 70°C (depending on the length and GC content of the primers), allowing the primers anneal to the template and then 3 min at 72°C to enable the polymerase to elongate the DNA. This was repeated for 20 to 30 cycles. The reaction mixture was then incubated at 72°C for 10 min to allow full extension of the DNA. Finally, the reaction mixture was kept at 4°C for up to 48 hours. The fragment generated by PCR was purified by agarose gel electrophoresisr/«*/s &to/ j <990).

2.2.10 Plasmid Cloning of DNA Fragments:

2.2.10.1 Ligation

Fragments of interest, generated by either restriction digestion or PCR, were purified from agarose gels and ligated into a suitably linearised vector. A standard 10 pi ligation reaction contained 10 ng linearised vector and an excess amount of the DNA fragment to be cloned (usually two to three fold

83 Materials and Methods

molar excess), in 6.6 mM MgCI2> 10 mM DTT, 0.4 mM ATP, 66 mM Tris/HCI pH 7.6 and 10 units T4 DNA ligase.

2.2.10.2 Preparation of Competent Cells:

A single colony of E.coli JM83 from a fresh streaked LB-agar plate was used to start a 10 ml LB culture at 37°C overnight. 0.25 ml of this was used to inoculate 40 ml of LB. This was incubated at 37°C for about 2 hours until the culture reached an optical density at 600 nm of 0.3. Cells were pelleted by centrifugation at 3000 rpm in a bench top centrifuge at 4°C for 3 min. The ( 2.060i9) pellet was resuspended in 20 ml ice cold FB [25 mM Tris/HCI pH 8.0, 50 mM CaCI2, 10% (v/v) glycerol]. After incubating on ice for 20 min, the cells were sedimented as above and resuspended in 2 ml FB. The cell suspension was either used immediately or plunged into liquid nitrogen and stored for up to half a year at -70°C.

2.2.10.3 Transformation of E.coli JM83:

Ligated plasmid DNA (1-10 jil) was mixed with 0.2 ml competent cells and left on ice for 1 -2 hours, followed by heat shock for 3 min at 42°C. The cells were returned to ice for 3 min before being mixed with 0.8 ml LB. The cells were then incubated at 37°C for 1 hour and spread on to plates containing an appropriate selective medium.

2.2.10.4 Colony Hybridization:

Two sets of 9 cm LB-agar plates, one with Hybond-N membrane and both containing 100 |ig/ml ampicillin, were each labelled at the back with the numbers 1 to 40. Each bacterial colony, containing a recombinant plasmid from the transformation experiment, was transferred to one of each set of the LB-agar plates at the same position. The plates were incubated at 37°C until

84 Materials and Methods colonies were fully grown. Plates without Hybond-N Membrane were kept at 4°C for future use.

iphat The colonies^grew on the Hybond-N membrane were lysed by placing the membranes on sheets of Whatman 3MM paper saturated in 0.5 M NaOH for 10 min and then transferred to 1 M Tris/HCI pH 7.4 for 10 min and finally to 0.5 M Tris/HCI pH7.4, 1.5 M NaCI for 10 min. Debris was removed by wiping the membranes with tissue paper. The membranes were then washed in 2 x SSC and allowed to dry. DNA was fixed to the membranes by exposing them to U.V. irridiation for 10 min.

The hybridization was carried out as described in 2.2.11.3.

2.2.11 DNA Sequencing:

The DNA sequence of the cloned restriction fragments was determined by the dideoxy chain termination method of Sanger et al. (1977), using double stranded DNA as template. The sequence reactions were carried out using a SequenaseR 2.0 sequencing kit.

2.2.11.1 Annealing Template and Primer:

2 pg of purified and RNA-free plasmid DNA was denatured by incubation in 0.2 M NaOH, 0.2 mM EDTA at 37°C for 30 min. The mixture was then neutralized by adding 0.1 volume of 3 M sodium acetate (pH 5.0) and the DNA precipitated with 2 volumes of ethanol at -70°C for 15 min. After washing with 70 % ethanol and drying in vacuo, the DNA was redissolved in 7 pi distilled water. 2 pi of 5 x Sequenase reaction buffer (200 mM Tris/HCI pH 7.5, 100 mM MgCI2, 250 mM NaCI) and 1 pi primer (2-3 pmoles) were added. Annealing was carried out by warming to 65°C followed by cooling down slowly to room temperature over a period of about 30 min.

85 Materials and Methods

2.2.11.2 Labelling Reactions:

To 10 pi annealed template-primer, the following reagents were added: 1 pi 0.1 M DTT, 2 pi labelling mixture (1.5 pM dGTP, 1.5 pi dCTP, 1.5 pi dTTP), 0.5 pi [a-32S]dATP (1000-1500 Ci/mmol) and finally 2.0 pi Sequenase* 2.0 in 10 mM Tris/HCI pH 7.5, 5 mM DTT, 0.5 mg/ml BSA. The sample was mixed thoroughly and incubated for 2-5 min at room temperature.

2.2.11.3 Termination Reactions:

The termination solutions used were as follows: ddA termination mix: 80 pM dGTP, 80 pM dATP, 80 pM dCTP, 80 pM dTTP, 8 pM ddATP, 50 mM NaCI. ddG termination mix: 80 pM dGTP, 80 pM dATP, 80 pM dCTP, 80 pM dTTP, 8 pM ddGTP, 50 mM NaCI. ddC termination mix: 80 pM dGTP, 80 pM dATP, 80 pM dCTP, 80 pM dTTP, 8 pM ddCTP, 50 mM NaCI. ddT termination mix: 80 pM dGTP, 80 pM dATP, 80 pM dCTP, 80 pM dTTP, 8 pM ddTTP, 50 mM NaCI. 2.5 pi of each termination mix was placed in tube with cap and pre warmed to 37°C. When the labelling incubation was complete, 3.5 pi aliquot was removed and added to each termination mix and incubated for a further 3-10 min at 37°C. 4 pi stop solution (95 % formamide, 20 mM EDTA, 0.0 5

% bromophenol blue, 0.05 % xylene cyanol FF) were added to each of the termination reactions and mixed thoroughly. The samples were heated to 95°C for 2 min prior to being loaded onto the denaturing gel.

2.2.11.4 Denaturing Gel Electrophoresis:

Electrophoresis was carried out using 40 cm x 20 cm plates held apart by spacers of 0.4 mm thickness. Gels were 6 % acrylamide containing 1 x TBE

86 Materials and Methods and 8.3 M urea. Polymerization was achieved by the addition of 20 pi TEMED (N,N,N\N’-tetrairfhyl-ethylenediamine) and 150 pi freshly prepared 10 % (w/v) ammonium persulphate to 36 mis of gel mixture. The gel was allowed to set for 1 hour at room temperature and was used within 2 to 20 hours. The gel was prerun for 10 to 15 min at 25 mA before samples were loaded. Electrophoresis was carried out at 25 mA in 1 x TBE. After electrophoresis, the gel was soaked in 10 % (v/v) acetic acid and 12 % (v/v) methanol in water for 10 min to remove the urea and dried at 80°C using a vacuum gel-drier.

Autoradiography was carried at room temperature for 2 to 3 days using Kodak X-omat S film.

2.2.12 Eukaryotic Cell Culture:

2.2.12.1 Eukaryotic Cell Lines:

Hela: A human cervical carcinoma cell line (Scherer etal., 1953). Hela cells were maintained in Modified Eagle’s Medium (MEM) supplemented with 5 % (v/v) foetal calf serum (FCS), 5 % (v/v) nonessential amino acid mix and gentamycin (20 pg/ml).

HepG2: A human hepatocellular carcinoma cell line (Knowles e ta l, 1980). This was maintained in Dulbecco’s Modified Eagle's Medium (DMEM), supplemented with 10 % (v/v) FCS and gentamycin (20 pg/ml).

EJ: A human bladder carcinoma cell line (Tabin et a l, 1982). This was maintained in DMEM and supplemented with 10 % (v/v) FCS and gentamycin (20 pg/ml).

MCF7: A human breast carcinoma cell line (a gift from Dr. P. Beverley, ICRF,

87 Materials and Methods

London). Maintained in RPMI 1640 supplemented with 10 % (v/v) FCS and gentamycin (20 pg/ml). For the purpose of transfection, the medium was changed to DMEM during the transfection and changed back to RPMI 1640 four hours after transfection.

SVK14: A human keratinocyte cell line (a gift from Dr. T. Kamalaki, Department of Biochemistry, Charing Cross Hospital, London). This was maintained in RPM11640 supplemented with 10 % FCS (v/v), gentamycin (20 pg/ml) and 5 pg/ml hydrocortisone.

2.2.12.2 Routine Cell Culture:

Cell culture was carried out in a humid atmosphere containing 5 % C 0 2 at 37°C. During routine culture, cells were grown in 25 ml flasks and were passaged at a dilution from 1:5 to 1:20 depending on the cell line. Trypsinization was carried out as described below. Medium was removed from the flasks by aspiration and the monolayers of cells were treated with 3 ml of trypsin/EDTA solution (0.05 % trypsin, 0.5 mM EDTA in PBS) for about 30 seconds. The trypsin/EDTA solution was removed by aspiration (some cell ed lines need to be trypinizied twice) and the flasks were returr^ to an incubator at 37°C for 2 to 15 min. Trypsinization was stopped by addition of 10 ml medium and a portion of the cell suspension was pipetted in to a new flask containing 7 ml of the appropriate medium. Flasks were replaced in the incubatorf Freshn&ij. t 988).

2.2.13 Transfection of Plasmid DNA into Eukaryotic Cell Lines:

2.2.13.1 Transfection C fo rm ** '***•>

Cells were seeded into 90 mm plates 24 to 30 hours prior to transfection. Cell densities per plate were: He[a: 2 x 105; HepG2: 5 x 105; MCF7: 5 x 1 05;

88 Materials and Methods

SVK14: 1 x 106; EJ: 5 x 105. The medium was replaced approximately 4 hours before transfection. Transfection was achieved by using the calcium phosphate co-precipitation technique (Gorman et al.t 1982). For a standard

transfection, 15 to 30 pmoles plasmid DNA (purified by two successive CsCI

density gradient centrifugations) was used in each transfection together with pUC13 DNA to make the total amount of DNA up to 10 pg. The DNA/CaCI2 precipitate was formed by addition of 250 pi 2 x HBS (50 mM Hepes, 280 mM NaCI, 1.5 mM Na^PC^ pH 7.12). For even precipitate formation, the DNA/CaCI2 was added into 2 x HBS and mixed thoroughly by vortexing for 20 sec. The precipitate was left at room temperature for 15 to 20 min before being pipetted onto the cultured cells.

The medium with its added DNA precipitate was removed after 4 to 18 hours by aspiration. Cells were washed with serum free medium and the complete medium was then replaced. Cells were harvested 40 to 60 hours after transfection. Medium was washed from the plates with PBS containing 1 mM EDTA. Cells were removed by scraping off with a "rubber policeman" and were pelleted in 1.5 ml microfuge tubes, by centrifugation for 2 min at low speed, using MSE microcentaur.

Cell extracts were prepared by freeze-thawing the cells in 100 pi 0.25 M Tris/HCI pH 7.8 three times, by alternation between liquid nitrogen and a 37°C water bath, followed by 10 min high speed centrifugation using MSE

microcentraur. The supernatant, referred to as cell extract, was either used for CAT assay immediately or stored at -20°C.

2.2.13.2 Chloramphenicol Acetyl Transferase (CAT) Assay:

Each CAT assay contained 10 to 50 pi cell extract, 0.2 pCi [14C] chloramphenicol (50 Ci/mmole), 0.4 mM acetyl CoA and 220 mM Tris/HCI pH 7.8 in a final volume of 150 pi. All components of the reaction except acetyl

89 Materials and Methods

CoA were mixed and preincubated at 37°C. The reaction was started by addition of 20 |il 4 mM acetyl CoA and left for 30 min at 37°C. The reaction was stopped by addition of 0.5 ml ethyl acetate followed by vortexing for 5 sec. The organic phase was removed and evaporated under vacuum. The

residue was taken up in 5 pi ethyl acetate and loaded onto a plastic-backed 0.2 mm silica thin layer chromatography (TLC) plate. Chloramphenicol and its acetylated forms were separated by ascending TLC in solvent chloroform/methanol :95/5. The separation was visualized by autoradiography. The amounts of acetylated and nonacetylated chloramphenicol were determined by excising pieces of the TLC plate, which corresponded to the radio-image on the autoradiogram, and quantitating them by scintillation counting. CAT activity was calculated as a percentage of chloramphenicol acetylation, corrected for protein concentration. In later experiments, pCH110, which contains the 6-galactosidase encoding gene, was used as internal control for transfection efficiency.

2.2.13.3 Assay of B-Galactosidase Activity:

The B-galactosidase activity, used to monitor transfection efficiency for each dish, was assayed as described by Herbomel et al. (1984). 1 ml of 60 mM Na2HP 0 4, 40 mM NaH2P 0 4, 10 mM KCI, 1 mM MgCI2, 50 mM 2 - mercaptoethanol and 0.2 ml of a solution of ONPG (2 mg/ml in 60 mM Na2HP 0 4, 40 mM NaH2P 0 4) were added to 20 to 40 pi of cell extract. The reaction was carried out at 37°C for 30 to 60 min and was stopped by addition of 0.5 ml 1 M NajjCOa. The 6-galactosidase activity was measured at 420 nm and expressed as A420 x 100, extrapolated to 1 hour of reaction and for total 150 pi cell extract.

2.2.13.4 Determination of Protein Concentration:

The protein concentration of cell extracts was measured using the Pierce BCA

90 Materials and Methods protein assay reagent which combines the reaction of protein with Cu2+ in an alkaline medium (biuret reaction) together with a selective detection reagent for Cu1+, namely bicinchoninic acid (BCA). The purple reaction product, formed by interaction of two molecules of BCA with one cuprous ion (Cu1+), is water soluble and exhibits a strong absorbance at 562 nm. BSA was used as standardsds eUsoribed m jyp'focot <*>•'&, to. k -f ) .

2.2.14 Slot Blotting:

2.2.14.1 RNA Purification from Cultured Cells:

About 108 cells were harvested and washed with ice-cold autoclaved PBS. The cell pellet (up to 107 cells) was resuspended in 400 pi of ice-cold RNA lysis buffer(150 mM NaCI, 10 mM Tris/HCI, pH 7.4, 1 mM MgCI2, 0.5% NP40, autoclaved prior to the addition of NP40), left on ice for 5 minutes and spun in a microfuge for 5 minutes. The supernatant was vortexed in a microfuge tube with 200 pi Tris/HCI( pH 7.8hbuffered phenol and 50 pi 10% SDS and spun in a microfuge for 5 minutes. The aqueous phase was transferred to a fresh microfuge tube and extracted with 200 pi Tris/HCI( pH 7.8)-buffered phenol. The aqueous phase was mixed with 40 pi autoclaved 2 M NaAc, pH 5.2 and 1 ml absolute ethanol was added and left at -20°C for 2 hours. The RNA precipitate was spun down and washed with 70% ethanol and vacuum dried. RNA was resuspended in DEPC-treated water and stored at -70°C.

2.2.14.2 Blotting:

Hybond N filters were soaked for 5 min in double-distilled water and then for 30 min in 20 x SSPE. RNA samples were dissolved in 200 pi of 6 x SSPE and 8% (v/v) formaldehyde and were heated at 65°C for 5 min. The RNA solutions were then chilled on ice, and were applied to a Hybond N filter with the aid of a slot blot apparatus and a water pump.

91 Materials and Methods

2.2.14.3 RNA Blot Hybridization:

The blot was washed at 65°C with 5 x SSPE containing 0.1% (w/v) SDS for 30 min and then pre-hybridized for 2 hours at 45°C in sealed plastic bags containing 10 ml of 5 x SSPE, 50% (v/v) formamide, 5 x Denhardt’s solution, 0.5% (w/v) SDS and 100 pg/ml heat-denatured salmon sperm DNA. Hybridization was carried out at 45°C overnight in the above solution following the addition of 5-10 ng/ml (final concentration) 32P-labelled oligonucleotide probe (for the method of DNA labelling, see section 2.2.8.2) which was heated in a boiling water bath for 5 min before being added to the hybridization solution. After hybridization, the membranes were washed twice in 50 ml of 2 x SSPE, 0.1% SDS (w/v) at 55°C for 20 min and in 50 ml of 1 x SSPE, 0.1% SDS (w/v) at 55°C for 30 min and then twice in 0.1 x SSPE, 0.15 (w/v) SDS at room temperature for 15 min. The blot was sealed in a plastic bag and autoradiography was performed at -70°C using Kodak X-omat S X-ray film.

2.2.15 Gel Retardation: ( PerbtU>fi&)

All work ms carried out at 4°C unless specified.

2.2.14.1 Preparation of Cell Nuclear Extract:

5 x 107 to 1 x 10s cells were harvested by trypsinization and washed twice with PBS. The cell pellet was resuspended in 5 ml of buffer A (10 mM HEPES pH 7.9, 1.5 mM MgCI2, 10 mM KCI, 0.5 mM DTT, 0.5 mM PMSF, 10 pg/ml TLCK, 10 pg/ml TPCK, 10 mM 8-glycerol phosphate, 10 mM levamisole, 1 pg/ml antipain, 1 pg/ml leupeptin, 1 pg/ml aprotinin and 1 pg/ml pepstatin A) and incubated at 4°C for 10 min. The cells were pelleted by centrifugation at 2000 rpm for 10 min and resuspended in 3 ml buffer A with 0.05 % NP40 and homogenized with 20 passes by hand in a tightly fitting

92 Materials and Methods

homogenizer. Cell lysis was checked under a phase contrast microscope. Nuclei were pelleted by centrifugation at 2000 rpm for 10 min and resuspended in 1 ml buffer B (20 mM HEPES pH 7.9, 25 % glycerol, 1.5 mM

MgCI2, 0.5 mM DTT, 0.5 mM EDTA, 0.5 mM PMSF, 10 pg/ml TLCK, 10 pg/ml

TPCK, 10 mM 3-glycerol phosphate, 10 mM levamisole, 1 pg/ml antipain, 1 pg/ml leupetin, 1 pg/ml aprotinin and 1 pg/ml pepstatin A). The concentration of NaCI was made up to 300 mM (final) by addition of 4 M NaCI. After incubating at 4°C for 30 min, followed by centrifugation at 20000 rpm for 20 ( ^7cx>ox£j min, the supernatant nuclear extract was divided into 10 pi aliquots and stored at -70°C.

2.2.15.2 Preparation of Gel:

Gels were 4 % acrylamide containing 0.25 x TBE pH 8.3 (4°C). Polymerization was achieved by addition of 60 pi TEMED and 400 pi freshly prepared ammonium persulphate (10 %, w/v) to 100 ml gel mix and poured into 12 x 14 cm siliconized plates with 3 mm spacers.

2.2.15.3 End Labelling of Oligonucleotides:

The end labelling of oligonucleotides w as, performed as follows: The reaction contained 0.5 pg oligonucleotide, 100 pCi [a3-p] ATP, 50 mM Tris/HCI pH 7.6, 10 mM MgCI2, 5 mM DTT, 0.1 mM EDTA and 2 units of Klenow fragment in a final volume of 25 pi. The reaction was allowed to proceed for 30 min at 30°C. Labelled oligonucleotide was separated from free [a3-p] ATP by chromatography on Sephadex G-25.

2.2.15.4 Formation of the DNA-Protein Complex:

Each 20 pi reaction mix contained 20 mM HEPES pH 7.9, 4Xglycerol, 1 mM MgCI2, 0.5 mM DTT, 50 mM KCI, 1 pg dldC, 100-200 cps of labelled

93 Materials and Methods

target DNA and 1 pi of cell nuclear extract prepared as above. The reaction mix was incubated at 4°C for 30 min and was loaded directly on the gel prepared as described above.

2.2.15.5 Electrophoresis:

Electrophoresis was performed in a cold room with a recirculating buffer

system. The gel was prerun at 35mA for 1 hr. A mixture of xylene cyanol and bromophenol blue was used as tracking dye. After the samples were loaded, 200 volts was applied for 5 min in order to obtain a rapid penetration of the samples in the gel and 160 volts were applied afterwards.

Electrophoresis was continued until the bromophenol blue reached the bottom of the gel.

2.2.15.6 Autoradiography:

Following electrophoresis, the gel was fixed in 10 % acetic acid, 10 % methanol and dried at 80°C using a vacuum gel-drier. Autoradiography was carried out at room temperature for 2 to 3 days using Kodak X-Omat S film.

2.2.16 Quantification of GST n in Cultured Cellsf o$HurJ-etai., /9$v

2.2.16.1 Preparation of Soluble Supernatant Fraction from Cultured Cells:

About 1 x 108 cultured cells were harvested by trypsinization and washed

twice with PBS. After centrifugation in a bench top centrifuge, the cell pellet was resuspended in 1 ml homogenization buffer containing 10 mM sodium phosphate pH 7.0, 0.16 M KCI, 1 mM DTT and 25 pM PMSF. Cell extracts were prepared by freeze-thawing the cell suspension three times by alternating between liquid nitrogen and a 37°C water bath followed by 45 min

94 Materials and Methods centrifugation at 45000xg. The supernatant was applied to a 0.5 ml hexylglutathione-agarose affinity column. After washing with 10 ml of homogenization buffer, GST was eluted with 2.4 ml Tris/HCI pH 9.1, 5 mM hexyl-glutathione, 5 mM GSH and applied to HPLC.

2.2.16.2 Analysis of GSTn Subunit by Reverse-Phase HPLC

HPLC analysis of GST n was carried out on a 10 cm x 0.8 cm Waters pBondapak C 18 reverse-phase column. The solvents were water containing 0.06% TFA (solvent A) and acetonitrile containing 0.06% TFA (solvent B). The column was washed with solvent A and 2 ml sample was injected. A linear gradient was run from 40% to 60% solvent B over 60 min with a flow rate of 1.5 ml/min. Polypeptides were monitored at 214 nm and peaks were integrated using a Hewlett Packard Intergrator 3390A. The protein content of peak was then obtained by reference to those obtained with purified GST standards.

95 CHAPTER III

THE G-SITE OF GLUTATHIONE TRANSFERASE n

96 The G-Site of GST %

Foreword

As discussed in Chapter I, the pi class GSTs possess a broad substrate

specificity, glutathione peroxidase activity toward lipid hydroperoxides but not cumene hydroperoxide and sensitivity to active oxygen species. They are associated with carcinogenesis and cancer chemotherapeutic drug resistance.

Therefore, the knowledge of the active site, especially the G-site of human GST 7u is important for both understanding the role of GSTrc in malignancy and

drug resistance, and the development of GST inhibitors that target the G-site. In this chapter, the effects of chemical modifications of GST n by group specific reagents are presented and the results are discussed.

3.1 Cysteine Residues

3.1.1 Modification of GST n by DTNB:

The thiol-specific reagent, DTNB, was used to study the involvement of cysteine residues of GST n in its active site. DTNB reacts with the thiol group of cysteine resulting in disulfide formation between the cysteinyl thiol and 2- nitro-5-thiol benzoic acid (Fig. 3.1).

COOH COOH COOH COOH >-NO, + N 0 2/ + E-SH

DTNB

Fig. 3.1 The Reaction of Cysteine residue with DTNB

As shown in Fig. 3.2, two cysteinyl thiol groups per subunit of GSTk react

with DTNB, but at different rates. At 12°C, one thiol group reacts with DTNB rapidly, whereas the other thiol group reacts very slowly. However, the latter also reacts rapidly at 37°C. The modification of the more reactive

97 The G-Site of GST %

100 - ~ 2.0 >* c □ -Q m D o JO ra o CD !c E H > 50 N C 1.0 LU

8 16 24

Time (min)

Fig. 3.2 DTNB Modification of GST n

GST n (6 |iM) was incubated in buffer A at 12°C with DTNB (0.5 mM) for 10 min and then the incubation temperature was raised to 37°C until no change in A412 was observed. Urea was added to a concentration of 6 M. The amount of thioi group modified was determined from A412. Aliquots were removed at time indicated to assay the enzyme activity towards CDNB. ( a ) remaining enzyme activity and (o) the number of thiol per subunit modified by DTNB.

98 The G-Site of GST k

thiol group alone caused complete inactivation of the enzyme (Fig. 3.2). No further reactive thiol groups were detected even in the presence of 6 M urea, although there are four cysteine residues per subunit of GST n (Ahmad etal., 1990). This may be due to the resistance of the relevant tertiary structure

to these denaturing conditions.

In order to determine the rate constant of modification of these thiol groups by DTNB, GST n was incubated with 0.14 mM DTNB at 12°C in buffer A. The results (Fig. 3.3) show that the reaction can be divided into a fast and a slow phase. Lines f and s are the tangents of the fast and slow reaction portions of the curve respectively. Line a, which was parallel to the abscissa, was drawn to pass through the intersection of the two tangents, giving an angle with line s as a. Line b was drawn from the origin to form an angle with line f which is equal to a. The A412 of the point of intersection of lines f and s represents the number of the more reactive thiol group modified by DTNB, which was found to be 1.05 per subunit. From line b, the T1/2 (half time of modification) and therefore k, (pseudo-first order rate constant, ^ = 0 .6 9 3 /7 ^ ) of the more reactive thiol group were determined to be 0.11 min and 6.3 min'1 respectively. Using the formula k ^ k /p T N B ], the second order rate constant k 2 of the more reactive thiol group was calculated to be 44000 M'1min *1. From line b, T1/2 and k, of the less reactive thiol group were determined as 30 min and 0.023 min'1 and k2, 162 M'1min'1.

3.1.2 Effect of GS-OCT on DTNB Modification of GST n:

Underthe condition described above (12°C, DTNB:GST n = 20:1), the addition of GS-OCT, over the concentration range of 14 pM to 1.4 mM, inhibited the modification of the more reactive thiol group by DTNB. The higher GS-OCT concentration, the lower k*. However, GS-OCT did not alter the reactivity of the less reactive thiol group with DTNB (Table 3.1). Since GS-OCT is a competitive inhibitor of GST n with respect to GSH, but a non-competitive

99 The G-Site of GST rc

c -Q ZJ C/5 o fE 0.5

0 2 4 6 8 10

Time (min)

Fig. 3.3 Determination of the Rate Constants of GST k Cysteine Residue Modification by DTNB

G ST k (7 jiM) was incubated in buffer A at 12°C with DTNB (0.14 mM). The number of modified thiol groups were determined by A412. The dashed lines f & s are the tangents of the fast and slow reaction portions of the curve respectively. The Tvz (half time of modification reaction) and (pseudo-first order rate constant) of the more reactive thiol group were determined from line b. The Ti/2 and k1 of the less reactive thiol group were determined from line s. The rate constant of each thiol group was calculated from the formula ^

= M D T N B ].

100 The G-Site of GST tc

inhibitor with respect to CDNB (Xia & Chen, 1989b), it is concluded that the more reactive thiol group participates in the G-site of GST n.

Table 3.1 The Effect of GS-OCT on the DTNB Modification of GST n

GST 7i (7 p.M) was incubated in buffer A at 12°C with 0.14 mM DTNB in the presence of various concentrations of GS-OCT. The number of modified thiol group per subunit was determined from A412. Calculation of the modification rate constant is given in Fig. 3.3.

[GST-OCT] k2 of more ^ of less (M) reactive thiol reactive thiol (M'1min‘1) (M‘1min'1)

0.0 44000 162 1.4x10*6 13400 158 1.4x1 O'5 7670 179 1.4x1 O'4 3640 144

3.1.3 Kinetics of NEMI Inactivation of GST n:

NEMI, which also reacts with the thiol group (Fig. 3.4), was used to confirm the results obtained with DTNB.

N-CH,CH3 + E-SH E-S n -c h 2c h 3

NEMI

Fig. 3.4 The Reaction of Cysteine Residue with NEMI

101 The G-Site of GST %

GST n gradually lost its activity in the presence of 1.4 x 10-6 to 1 x 10"4 M of NEMI (NEMI:GST tc = 55 to 440:1). A plot of log enzyme activity against time gave a set of curves shown in (Fig. 3.5a). In order to obtain the half time of the inactivation (T1/2) caused by NEMI at different concentrations, a set of tangents, passing through the origin of the inactivation curves were plotted (dotted lines) representing the initial rates of inactivation of GST n by NEMI at different respective concentrations. The pseudo-first order rate constants (k^ were determined by ^ = 0 .6 9 3 /^ . According to the fomula k1=k2[l]n or logk^logkg+nlogll] (Hollenberg, 1971), where kg represents the rate constant of the inactivation by covalent modification, [I] represents the concentration of reagent (in this case, NEMI) and n represents the number of molecules of reagent covalently binding to one subunit of enzyme. Plotting logk1 against log[NEMI] (Fig. 3.5b), gave a straight line with a slope (n) of 0.98. This indicated that one molecule of NEMI bound to one subunit of GST k causing the inactivation of the subunit. The rate constant of the modification inactivation of GST n by NEMI, calculated by plotting kt against the concentration of NEMI (Fig. 3.5c), was 1.35x103 M'1min'1.

3.1.4 Effects of GS-HEX, GS-OCT, Bilirubin and CDNB on NEMI Modification of GST n :

Results in section 3.1.2 have shown that the GS-OCT can block the reaction of the more reactive thiol group reacting with DTNB. In order to demonstrate whether GS-HEX and GS-OCT can protect GST n from inactivation by NEMI, GST n was incubated with NEMI under the condition mentioned above in the presence or absence of GS-HEX or GS-OCT. The results suggest that both GS-HEX and GS-OCT can completely protect the enzyme from modification by NEMI, thus providing further evidence that GS-HEX and GS-OCT have a protective effect on the more reactive thiol group (Fig. 3.6). A similar experiment with CDNB showed that it had no effect on the inactivation of GSTrc by NEMI (Fig. 3.6), suggesting that the essential thiol group is not

102 Time (min) CM LT3 LO O O) 0 0 3 0 O The G-Site of GST n GST of G-Site The CO o 3 0 00 Fig. 3.5 Kinetics of NEMI Modification of GST GST of Modification NEMI of Kinetics 3.5 Fig. 03 LC3 in LU + C3 ° a ! / a o 103 CM <0 k The G-Site of GST %

Fig. 3.5 Kinetics of NEMI Modification of GST n

a) Kinetics of inactivation of GST n by NEMI: GST n was incubated in buffer A at 18°C with NEMI at different concentrations. Aliquots were removed at 5 min intervals and assayed for the enzyme activity. The initial concentrations of NEMI were: (•) 12.5, (o) 25, (■) 50 and (□) 100 |iM. The broken lines are the tangents of the curves passing through the origin, b) Determination of the order of GST n inactivation by NEMI: The data are plotted as log^ (pseudo-first order rate constant) against log [NEMI]. c) Determination of rate constant of the inactivation: The data are plotted as k, against the initial concentration of NEMI. The rate constant of inactivation kg is calculated from the formula k^k^ N E M I], ie, the slope of the line.

involved in CDNB binding site. Interestingly, bilirubin can also inhibit the modification of GST n by NEMI, although it is a competitive inhibitor with respect to CDNB (Xia & Chen, 1989c). This may indicate that bilirubin binding occurs near to the thiol group of the active site.

3.1.5 The Involvement of Cysteine Residue in the G-Site

Studies by Desideri et al. (1991) also showed that only one thiol group per subunit of GST n reacted with a spin label derivative of maleimide and that activity was lost in parallel. Comparison of the amino acid sequences reveals that both human GST n and rat subunit 7 contain 4 cysteine residues at the same positions, ie. 14, 47, 101 and 189. While subunit 1 contains two cysteines at position 17 and 111, subunit 2 contains only one cysteine residue at position 211 and subunits 3 and 4 contain three at positions 86, 114 and 174 (Mannervik & Danielson, 1988). Analysis of a cDNA coding the mouse pi class GST suggests that it also possesses a cysteine residue at position 47

104 The G-Site of GST n

Time (min)

0 5 10 15 20 25 1.0

0.8

0.6 o > >

0 .4

0.2

Fig. 3.6 Effects of GS-OCT, GS-HEX, Bilirubin and CDNB on NEMI Modification of GST n

0.24 jiM GST k was pre-incubated alone or with 1 p.M GS-OCT, 5 pM GS-HEX, 500 |iM CDNB or 100 pM bilirubin for 5 min in Buffer A at 18°C. 50 pM NEMI was added to initiate the reactions. Aliquots were removed at 5 min intervals and assayed for the enzyme activity. (•JGSTrc + NEMI; ( a ) GST n + GS-HEX + NEMI; (■) GST k + GS-OCT + NEMI; ( a ) GST n + CDNB +

NEMI; (□) GST k + bilirubin + NEMI.

105 The G-Site of GST n

(Tamai et al., 1990). Thus, cysteine 47 appears to be a characteristic of the GSTs of pi family. Tamai etal. (1990, 1991) have shown that modification of Cys47 not only caused a loss of the enzyme activity but also abolished . its

binding ability to hexylglutathione affinity matrix, suggesting that this cysteine

residue plays an important role in the G-site. However, they have also

demonstrated that Cys47 itself was not essential for catalytic activity, since a mutant enzyme, in which cysteine 47 was replaced by alanine, retained its

activity (Tamai et al., 1991). This suggests that modification of a cysteine residue may alter conformation in such a manner as to abolish enzyme

activity.

3.2 Lysine Residues:

3.2.1 Modification by TNBS:

TNBS is a relatively specific reagent for primary amino groups, eg. the e- amino group of lysine (Fig. 3.7).

NO,

n o 2 n o 2

TNBS

Fig. 3.7 The Reaction of Lysine Residue with TNBS

Excess TNBS detects 12 lysyl amino groups per subunit in 6 M urea (the N- terminal amino residue of GST k is proiine). Studies of the relations between

TNBS modification and GST k inactivation showed that the enzyme was entirely inactivated after 10 of them were modified (Fig. 3.8a). As these 10

106 The G-Site of GST k

amino groups show similar reactivity with TNBS, the number of the amino groups which was essential for the enzyme activity, can be analyzed by the formula, 'Va=x, derived by Prof. Chengru Zhou, Academica Sinica Shanghai (personal communication), where x represents the percentage of the remaining amino group after TNBS modification; a represents the percentage of the remaining enzyme activity after TNBS modification; / represents the number of the amino groups which are essential for the enzyme activity. A plot of S/a against x at fixed / values (/=1,2,3...n), yields a straight line when the / value corresponds to the number of the essential amino residues. In this case (Fig. 3.8) /= 1, indicating that only one primary amino group per subunit is involved in the enzyme active site.

3.2.2 Effect of GS-OCT, bilirubin and CDNB on TNBS Modification of

GST k :

0.5 pM enzyme was incubated with 1 pM GS-OCT, 100 pM bilirubin or 500 pM CDNB in the presence of 50 pM TNBS. As shown in Fig. 3.9, GS-OCT can protect GST n from being inactivated by TNBS, whereas CDNB has no effect. GS-OCT is a competitive inhibitor of GST n with respect to GSH, but a non-competitive inhibitor with respect to CDNB (Xia & Chen, 1989b). This indicates that the amino group, which is essential for GST n activity, is involved in the G-site. Bilirubin slightly enhances the modification of the essential lysine residue by TNBS (Fig. 3.9). T/j&e data dm consistent with allosteric effect of bilirubin on GSH binding to GST n, since in the presence of bilirubin, the binding of GSH showed homotropic positive cooperation between two subunits of GST n (Xia & Chen, 1989c). Thus after the binding of bilirubin, GST n undergoes conformational change so that the G-site is more accessible to both GSH and G-site modifier. TNBS also has the ability to react with thiol groups. However, spectral analysis of the TNB-GST n complex excluded this possibility (data not shown).

107 i S

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108 The G-Site of GST %

Time (min)

0 5 10 15 20 25

1.0

0.8

0.6

0 . 4

0.2

Fig. 3.9 Effects of GS-OCT, Bilirubin and CDNB on TNBS Modification of GST n

0.5 fiM GST k was pre-incubated alone or with 1 jiM GS-OCT, 500 jiM CDNB or 100 pM bilirubin for 5 min in buffer B at 22°C. 50 p.M TNBS was added to initiate the reactions. Aliquots were removed at 5 min intervals and assayed for the enzyme activity. (•) GST n + TNBS; (■) GST n + GS-OCT

+ TNBS; (a ) GST k + CDNB + TNBS; (□) GST k + bilirubin + TNBS.

109 The G-Site of GSTk

3.3 Arginine Residues

3.3.1 Modification of GST n by DiAC:

The involvement of arginine residues in enzyme active site was studied by

using DiAC, which reacts specifically with the guanidino group of arginine

residue (Fig. 3.10).

CH3COCOCH3 + E-C = NH

Fig. 3.10 The Reaction of Arginine Residue with DiAC

In Fig. 3.11a, a concentration-dependent decrease of the activity of GSTrc upon incubation with DiAC was observed. However, there was no linear relation between logarithm of remaining activity and time. In order to obtain the k1 (pseudo-first order constant) and k2 (rate constant of GST n inactivation by DiAC), a set of tangents of the inactivation curves passing through the origin were plotted (dotted lines). The means of calculation of k, and Ic, are given in section 3.1.3. Results shown in Fig. 3.11b and c indicated that pseudo-first order rate constant (k j was proportional to [DiAC]1, suggesting that one guanidino group was associated with enzyme activity. k> = 1.8 M' min'1.

3.3.2 Effects of GSH, GS-HEX, GS-OCT, Bilirubin and CDNB on DiAC

Modification of GST n:

Results in Fig. 3.12 showed that 100 pM GSH or 10 pM GS-HEX or 1 jiM GS-

OCT inhibited the inactivation of GST n by DiAC, whereas CDNB had little

110 Time (min) O CM CM n i O n i o —I o O) The G-Site of GST % GST of G-Site The D 0 0 CD O n i Fig. 3.11 Kinetics of DiAC Modification of GST GST of ModificationDiAC Kineticsof Fig. 3.11 o CO CM os 5 a ! / a o 111 CO o o n i l n cu b O < n The G-Site of GST n

Fig. 3.11 Kinetics of DiAC Modification of GST n

a) Rates of inactivation of GST n by DiAC: GST n was incubated in Buffer C at 25°C with DiAC at different concentrations. Aliquots were removed at 5 min intervals and assayed for the enzyme activity. The initial concentrationsof DiAC were: (•) 0, (o) 20, (■) 50 and (a) 100 mM. The broken lines are the tangents of the curves passing through the origin, b) Determination of the order of GST k inactivation by DiAC: The data are plotted as log of k1 (pseudo-first order rate constant) against log [DiAC]. c) Determination of rate constant of inactivation: The data are plotted as k1 against the initial concentration of DiAC. The rate constant of inactivation Ic, is calculated from formula k^k/JDiAC], ie, the slope of the line.

effect and bilirubin enhanced the inactivation. This indicates that the guanidino group of arginine, which is essential for the enzyme activity, is associated with the G-site of GST n.

3.3.3 The Involvement of Arginine Residues in G-Site of GST n

Several models give a basis for the binding of GSH to GSH-dependent enzymes such as glyoxalase, GSSG reductase and GSH peroxidase. A common feature of these models is the involvement of positively charged arginines at the enzyme surface which interact with the carboxylate group at the end of GSH (Rosevear et al., 1984; Schasteen et al., 1983). As discussed above, one arginine residue is associated with the G-site of GSTtc. Arginine residues have also been demonstrated to be involved in the active sites of GST pi from bovine placenta (Schaffer et al., 1988) and rat liver microsomal GST (Andersson & Morgenstern, 1990). In the primary sequence of GST n, arginine residues which occur at positions 13, 18 and

112 The G-Site of GST k

Time (min)

0 5 10 15 20 25

0.8

0.6 o > >

0.4

0.2

Fig. 3.12 Effects of GSH, GS-OCT, GS-HEX, CDNB and Bilirubin on DiAC Modification of GST n

0.14 |j.M GST ie was pre-incubated alone or with 100 pM GSH, 10 pM GS-HEX, 1 pM GS-OCT, 50 pM CDNB or 100 pM bilirubin for 5 min in buffer C at 25°C. 50 mM DiAC was added to initiate the reactions. Aliquots were removed at 5 min intervals and assayed for the enzyme activity. ( • ) GST k

+ DiAC; (o) GST n + GSH + DiAC; (a ) GST k + GS-HEX + DiAC; (■) GSTti

+ GS-OCT +DiAC; ( a ) GST tt + CDNB + DiAC; (□) GST k + bilirubin +

DiAC.

113 The G-Site of GST k

186, are strictly conserved throughout the alpha, mu and pi families. However, replacement of these conserved arginine residues of GST B1B1 by site-directed mutagenesis has suggested that all of them were associated

with, but not essential, for the enzyme activity (Stenberg et al., 1991).

3.4 Carboxyl Groups:

3.4.1 Modification of GST n by EDAC:

EDAC can react specifically with carboxyl groups of glutamate or aspartate residues and the C-terminal carboxyl group (Fig. 3.13). Kinetics studies of the enzyme inactivation by EDAC indicated that the pseudo-first order rate constant (k j was proportional to the [EDAC]1 (Table 3.2), suggesting that only one carboxyl group was involved in the active site of the enzyme.

n h c h 2c h 3 o n h c h 2c h 3 0 = C/ +E-COOH------► E-COC^ +H20 XNHCH2CH2CH2N(CH3)2 NHCH2CH2CH2N(CH3)2

EDAC

Fig. S.13 The Reaction of Carboxyl Group with EDAC

The inactivation reaction rate constant was 4.0 M'1min‘1 according to the formula logk^logkg+nlogll] (details of calculation are explained in section 3.1.3).

3.4.2 The effects of GSH, GS-HEX, GS-OCT, bilirubin and CDNB on EDAC Modification of GST n:

Studies showed that 1 pM GS-HEX, 1 pM GS-OCT and 500 pM GSH

protected GST k from inactivation by 50 mM EDAC, whereas CDNB had no

114 The G-Site of GST %

Table 3.2 Modification of GST n by EDAC

The pseudo-first order rate constant (k j of

inactivation of GST n by EDAC

EDAC conc. (mM) k, (min)'1

10 0.038 20 0.091 50 0.192 100 0.385 200 0.910

effect and bilirubin again enhanced the modification (Fig. 3.14). This suggests that this carboxyl group may be also associated with the G-site.

3.5 Histidine and Tyrosine Residues:

3.5.1 Modification of GST n by DEPC:

DEPC reacts with the imidazole group of histidine residue to yield an N- carbethoxyhistidyl derivative (Fig. 3.15). This reaction can be conveniently followed spectrophotometrically by the increase in absorbance, which has a maximum between 230 and 250 nm.

(Ch 3c h 2o )2c = o + + c h 3c h 2o h *—N N-COCH2CH3 II DEPC 0

Fig. 3.15 The Reaction of Histidine Residue with DEPC

115 The G-Site of GST k

Time (min)

0 5 10 15 20 25 1.0

0.8

0.6

0.4

0.2

Fig. 3.14 Effects of GSH, GS-OCT, GS-HEX, CDNB and Bilirubin on EDAC Modification of GST n

0.27 pM GST 7i was pre-incubated alone or with 500 jiM GSH, 1 pM

GS-HEX, 1 jiM GS-OCT, 500 |iM CDNB or 100Abilirubin for 5 min in buffer D at 25°C. 50 mM EDAC was added to initiate the reactions. Aliquots were removed at 5 min intervals and assayed for the enzyme activity. ( • ) GST n

+ EDAC; (o) GST n + GSH + EDAC; ( a ) GST n + GS-HEX + EDAC; (■)

GST 7i + GS-OCT + EDAC; (a ) GST k + CDNB + EDAC; (□) GST k + bilirubin + EDAC.

116 The G-Site of GST n

However, DEPC can also react with tyrosine residue (Fig. 3.16).

0 II (CH3CH20)2C = 0 + E- OH * E‘\ \ /> o c o c h 2ch3 + CH3CH2OH

DEPC

Fig. 3.16 The Reaction of Tyrosine with DEPC

O-carbethoxylation of tyrosine is readily detected by a decrease in the absorbance at 278 nm. Comparison of the spectrum of the DEPC-modified GST n with the control (Fig. 3.17) between 230 and 300 nm, showed an increase in absorbance between 230 and 270 nm and a decrease between 270 and 300 nm indicating that both histidine and tyrosine residues of GSTtc were derivatized. When 12 pM GST n was treated with EDAC, the increase in absorbance at 246 nm was 0.0305, which is equivalent to 9.53 p.M of N- carbethoxy-histidyl derivative. This suggests that 0.8 histidine residue per subunit was derivatized. The decrease in absorbance at 278 nm was 0.0126, implying that 9.64 jiM tyrosine residues were modified by DEPC, equivalent to 0.86 tyrosine per subunit of GST n.

3.5.2 Modification of GST n by DEPC and Enzyme Activity:

The relationship between the number of histidine residues modified by DEPC per subunit of GST n and the enzyme activity indicated that the enzyme was entirely inactivated after the modification of one histidine residue (or tyrosine residue) (Fig. 3.18). This suggests that one histidine residue and/or tyrosine residue(s) are involved in the enzyme active site.

3.5.3 The Effects of GSH and CDNB on DEPC Modification:

GST n was preincubated with either GSH or CDNB before DEPC was added to the mixture. The results suggest that GSH completely protected the

117 The G-Site of GST %

X (nm)

250 275 300

0 .0 8

0 .0 6

0 .0 4

0.02

0.0

Fig. 3.17 Modification of GST n by DEPC

12 pM of GST 7T in 400 pi buffer E was mixed with 8 pi 250 mM DEPC freshly

made in ethanol and a reference blank was made by mixing 12 pM GST k in 400 pi buffer E with 8 pi ethanol. The control was prepared by mixing 400 pi buffer E with 8 pi 250 mM DEPC and a reference blank was made by mixing 400 pi buffer D with 8 pi ethanol. After 1 hour at 4 °C, both the sample and the control were scanned from 310 to 210 nm.

(— ) DEPC-modified enzyme. ( ------) DEPC control. 8246=3200 M‘1cm '\ e278=1300M‘1cm'1.

118 The G-Site of GST n

Modified-His/subunit

0.4 0.6 0.80.2 1.0 100

0 0.012 0.024 0.036

Fig. 3.18 DEPC Modification of GST n and Enzyme Activity

GST n (10 fiM) was incubated in buffer E at 4°C with various concentrations of DEPC. 0.1 M-l aliquot was removed after 30 min for enzyme activity assay. The extent of carbethoxylation of enzyme was quantitated by A246 (e=3200 M'1cm'1).

119 The G-Site of GST n

enzyme from inactivation by DEPC, whereas CDNB again had no effect. This indicates that either histidine or tyrosine or both modified by DEPC, are associated with the G-site.

3.5.4 Localization of DEPC-Modified Histidine of GST n

In order to obtain short peptides containing DEPC-modified histidine, the

DEPC modified GST n (DEPC-GST 71) was subjected to chymotrypsin digestion, which tends to cleave at aromatic or bulky aliphatic residues. The resulting peptides were separated by reverse phase HPLC. 5 peptides, which had an increased A240: A214i relative to that of GST n were sequenced (Fig. 3.19). The following two peptides were found to contain DEPC-His: peptide a: TILRHGR.. and peptide b: LRHGR.., both of them arise from a region of GST n sequence which contains His71. Together, they accounted for all of the expected yield of the His71. Peptide which contains other histidine residues were not found in peaks with increased A240 : A ^ . This indicates that only the His71 of GST n is modified by DEPC.

Sequence comparison reveals that histidine 71 is conserved between GSTtt and subunit 7. However, there is no histidine at this position in GST from other classes. It seems that this histidine is unique to pi class GST. Interestingly, a comparison of the complete sequence of human GSTs from all classes shows the occurrence of a region with significant hydrophobic character from about position 155 to 180, which harbours a histidine residue around position 162 (Ahmad et al., 1990). A histidine in this region is also conserved in the rat enzymes from the three classes. Replacement of histidine 159 of rat GST 1-1 by tyrosine or lysine, caused a decrease in the enzyme activity, whereas replacement by asparagine resulted in full retention of enzyme activity (Wang et al., 1991).

120 The G-Site of GST tu -a X3

eptide Analysis of DEPC-GST k The G-Site of GST k

Fig. 3.19 Peptide Analysis of DEPC-GST n

DEPC-treated GST n was digested with chymotrypsin and the resulting peptides were separated by reverse phase HPLC and analyzed by A214 (— ) and A242 (— ). Peptides yielding higher A ^ than the control (peaks a, b, c, d and e) were subjected to primary sequence analysis.

3.5.5 Localization of DEPC-Modified Tyrosine of GST n:

As stated in Chapter I, Tyr7 is one of the few residues that is conserved throughout all four classes GSTs, and it is possible that this residue plays a critical part in GST function. The N-terminal sequence of GST n is PPYTVVYFP, which contains Tyr3 and Tyr7. In order to see whether the Tyr7 is obtained at a reduced yield compared with that of Tyr3 due to DEPC modification, the N-terminal sequence of DEPC-modified GST t: was analyzed and the yields of both Tyr3 and Tyr7 were calculated based on the yield of the succeeding amino residue in the sequence, ie. for Tyr3, the yield was based on Thr4 and for Tyr7, it was based on Phe8. The data shown in Table 3.3 suggest that Tyr7 may be the target of DEPC. DEPC-Tyr7 has been observed during the sequencing of Tyr7, as an extra peak, corresponding to DEPC-Tyr, was detected at this stage of sequencing but not with that of Tyr3. As Tyr7 is one of the few residues which are conserved in all four classes GSTs (Fig. 1.7), it is very likely that Tyr7 of GST n is associated with enzyme activity. A very recent study by Stenberg et al. (1992) showed that replacement of Tyr8 of human GST B1B1 by Phe caused loss of 95-98% of the enzyme activity. Their studies have also shown that, compared with the wild type enzyme, the mutation of Tyr8 has a lower affinity towards GSH but a similar affinity towards hexylglutathione. This suggests that the mutation affects primarily chemical steps in the catalytic mechanism.

122 The G-Site of GST %

Table 3.3 Modification of Tyr7 by DEPC (% of expected yield)

Tyr3a Tyr7b

GST k 104.8 92.7

DEPC-GST k 89.7 55.2°

a based on the yield of T h r4;b based on the yield of Phe8. c This does not represent the true level of Tyr modification since DEPC-Tyr decomposes to yield Tyr during sequencing.

3.6 Tryptophan Residues

NBS can react with the indole group of tryptophan residues. Incubation of GST 7i with NBS showed that the enzyme lost 95% of its activity after treatment with NBS, suggesting that tryptophan residues may be associated with enzyme active site.

There are two tryptophan residues (Trp28 and 38) in GST n. Arduini et al. (1989) have investigated the environments of the tryptophan residues in GSTtc by fluorescence techniques. Their studies revealed that one tryptophan residue seems to be deeply embedded within the polypeptide in a rigid, weakly polar environment, characteristic of a (3-type secondary structure. The other one is located in a more polar site, probably near the surface, in a rather flexible region of the enzyme. A tryptophan residue has also been reported in the vicinity of the active site of rat GST 4-4 (van Ommen et al., 1989).

123 The G-Site of GST n

3.7 Summary

Amino acid residues which are essential for the enzyme activity of GST n have been identified by chemical modification of the enzyme with various group-specific reagents and the results are summarized in Table 3.4. The evidence indicates that these residues are involved in the G-site of the enzyme. However, such involvement could mean for each residue either a structural role in maintaining conformation of the G-site, a participatory role in binding substrate or involvement in the catalytic mechanism.

At this stage, a continuation of the work was considered, perhaps by the use of site-directed mutagenesis. However, it was felt that the interpretation of these results or results of site directed mutagenesis without the knowledge of the three dimensional structure of GST n remained supposition. Therefore, it was concluded that a change of direction to investigate the regulation of

GST n gene expression would be more profitable, particularly since the CRC Molecular Toxicology Research Group leads research in this area.

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125 CHAPTER IV

THREE-DIMENSIONAL STRUCTURE OF G-SITE

126 Foreword

Just before this thesis was finished, the three-dimensional structure of porcine GST pi was resolved by Professor Robert Huber and his colleagues at the Max Planck-lnstitut fur Biochemie, Martinstried bei Munchen. In collaboration with Professor Huber, the results of chemical modification studies discussed in Chapter III were analyzed in terms of the three-dimensional structure of

porcine GST pi. Co-ordinates of various modification reagents were prepared and computerized modelling was used to assess the following effects of each modification: 1). availability or accessibility for chemical modification; 2). loss of essential interaction with GSH either by charge alteration or conformational

change; 3). steric hindrance to GSH binding. Modelling was carried out

using the three-dimensional structure of porcine GST pi at 2.1 A resolution and the results are discussed in this chapter.

4.1 The Three-Dimensional Structure of Porcine GST pi in Complex with Glutathione Sulfonate (Reinemer et al., 1991)

Using X-ray crystallography, Reinemer et al. (1991) solved the three- in dimensional structure of a pig lung GST pi^complex with glutathione sulfonate (GSA) by multiple isomorphous replacement at 3 A resolution which has been refined to 2.3 A resolution. As shown in Fig. 4.1a, each subunit is folded into two domains. Domain I, which contains residues 1-74, consists of a central four-stranded 3-sheet flanked on one side by two a-helices and on the other side, facing the solvent, by a bent irregular helix structure. Domain II, which contains residues 81-207, consists of five a-helices, four 8-turns and a 310- helix (Table 4.1). The dimeric molecule is globular and between the subunits, and along the local diad, is a large cavity (Fig. 4.1b).

127 Three-dimensional structure of G-site

Fig. 4.1 Three-Dimensional Structure of Porcine GST pi (Reinemer et al., 1991, EMBO J. 10, 1997-2005)

a) Stereo-ribbon diagram of a subunit of porcine GST pi with the model of the inhibitor GSA included (thick line), b) Stereo drawing of the Ca positions of the dimeric GST pi molecule (thin line) with GSA included (thick line), c) Model of inhibitor GSA (thick line) and its next neighbours at the binding site.

128 Three-dimensional structure of G-site

Table 4.1 Secondary Structural Components in Porcine GST pi

Domain I Domain II

Structural Amino acid Structural Amino acid element residues element residues

8 strand 1 3-7 a helix D 81-107 8 turn 11-14 a helix E 109-132 a helix A 15-25 310 helix 135-137 8 strand 2 29-32 8 turn 140-143 a helix B 38-44 a helix F 148-163 8 turn 45-48 8 turn 164-167 8 strand 3 52-55 8 turn 168-171 8 strand 4 58-61 a helix G 172-182 a helix C 63-74 a helix H 185-192 8 turn 196-199

The dimeric GST pi binds two molecules of GSA, a competitive inhibitor with conformation similar to the X-ray determined structure of glutathione. GSA occupies a site (G-site) on domain I, which is situated in a cleft formed between the two intrasubunit domains. The cleft extends from a segment (residues 8-10, connecting 8-strand 1 and a-helix A) to about Ser63 at the N- terminal end of a-helix C. One end of the cleft opens out to the solvent, while the other, near Ser63, is adjacent to the cavity at the centre of the dimer. Side chains lining the G-site include Tyr7, Gly12, Arg13, Trp38, Lys42, Gln49,

Pro51, Gln62, Ser63 and Glu95 in one subunit and BAsp96 in the other subunit (Fig. 4.1c).

129 Three-dimensional structure of G-site

The enzyme bound GSA is orientated at G-site with itsT-glutamyl arm pointing downwards in the direction of the dimer’s large cavity, while the sulfonate moiety is pointing towards domain II, and glycine moiety pointing away from domain II (Fig. 4.1c). TheT-glutamyl moiety interacts with the side chains of Arg13, Gln49, Gln62 and BAsp96 (from the other subunit), whereas the glycine part makes contact with the side chains of Trp38 and Lys42. The sulfonate group of GSA interacts with the side chain of Tyr7 and the carbonyl oxygen and amide nitrogen of C yS 03' form interactions with the amide nitrogen and carbonyl oxygen of Leu50 respectively (Fig. 4.1c)

The sequence of human GST n has two extra residues, Glu40 and Gly41 with the result that the numbering of human GST n after 41 is different, ie. Lys44 and Cys47 in human GST n are equivalent to Lys42 and Cys45 in porcine GST pi. In this chapter, the numbering hereafter corresponds to the human enzyme.

4.2 His71 and Tyr7

Chemical modification study has indicated that one histidine residue was associated with the G-site of GST n. The peptide sequence analysis showed that His71 of GST n was derivatized by DEPC. As shown in Fig. 4.1c, His71 is located in the middle of a-helix C and is distal from the G-site. By computerized modelling, it can be shown that His71 is available for DEPC

modification since it does not form hydrogen bonds with other residues and there is space for the modification of His71 by DEPC without significantly altering the conformation of the enzyme, even in the vicinity of His71 itself (Fig. 4.2). Therefore, it is unlikely that this modification will affect activity.

Peptide analysis of DEPC-modified GST n also revealed that Tyr7 was a target of DEPC. Since Tyr7 is one of the few residues which are conserved in all four classes GSTs (Fig. 1.5), it is very likely that Tyr7 is associated with

130 Three-dimensional structure of G-site CO CNI O a *5 x: CL ’<75 .£ 2 o _ d) o “■ “ c c d> o LU ’> O 2 O LU CL ■o O (/) r^ H- c k. c £ o o 0) w k. o c o tz

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4.3 Lys44

As shown in Chapter III, one lysine residue is involved in the G-site of GSTrc. The results obtained from computerized modelling of the reaction of lysine residues with TNBS are consistent with chemical modification studies. Most of the lysine residues are located on the surface of the enzyme, thus are accessible to TNBS, but only the modification of Lys44 affects the enzyme activity. Lys44, located in the middle of a-helix B, is situated at the "funnel” which accommodates GSA, so there is plenty of room for the modification. As it has been shown that the glycine carboxylate of GSA interacts with Lys44 and Trp38 (Reinemer etal., 1991), the modification of Lys44 by TNBS would abolish the ionic interaction of the GSA with Lys44, therefore causing steric hindrance to GSA binding. The binding of GS-OCT to the enzyme would be expected to protect the modification of Lys44 by TNBS.

4.4 Arg13

Chemical modification studies have shown that one arginine residue was involved in the G-site. As shown in Fig. 4.1c, Arg13, located in a 8-turn between 8-strand 1 and a-helix A, is situated near the G-site and forms an

132 Three-dimensional structure of G-site il CO LU C/3 CL 5 d) a o 0. 1 1 d> £ = 5 Q O £ o o / H= 0 o is; .C LU .a o c E 0) c o

133 Three-dimensional structure of G-site E CO O 3 I ' 0. ’ ’ © CO o CO o c c co X < -Q £ 2 O 0. CO H T3 ° < Q) X o 5 c o o CO C

134 Three-dimensional structure of G-site interaction with Glu97. Using the computerized modelling system, it can be shown that the modification of Arg13 by DiAC would abolish its interaction with Glu97, therefore altering the position of a-helix A which is essential for the activation of GSH. This also causes inhibition by altering the position of the adjacent BAsp98 which interacts with the amino group of 7-glutamyl residue of GSA.

4.5 BAsp98 and Glu97

Modification of GST n by EDAC showsthat one carboxyl group is associated with G-site of GST n. Both Glu97 and BAsp98 are located in the middle of a- helix D and are accessible by EDAC. As shown in Fig. 4.1c, the carboxyl group of BAsp98 interacts with the 7-glutamyl residues of GSA. Modification of BAsp98 will not only destroy the ionic interaction but also cause steric hindrance to GSA binding. Glu97 is not available for modification as it forms an ionic interaction with Arg13. The results of protection studies are consistent with these propositions.

4.6 Trp38

Modification by NBS shows that tryptophan residues are involved in the active site of GST n. There are two tryptophan residues: Trp28 and Trp38. The three-dimensional structure of porcine GST pi shows that Trp38 participates in the G-site (Fig. 4.1c). It is located at the beginning of a-helix B and interacts with the glycine residue of GSA. Therefore, modification of Trp38 disrupts its interaction with GSA and cause steric hindrance to GSA binding.

4.7 Cys47

Chemical modification studies have demonstrated that one cysteine residue is associated with the G-site of GST n. There are four cysteine residues per

135 Three-dimensional structure of G-site

subunit of GST n, Cys 14, 47, 101 and 189 (Kano et al., 1987; Ahmad eta!., 1990). As shown in Fig. 4.1a and b, Cys 47 is located in the beginning of 8- turn (47-50) between a-helix B and 8-strand 3. Therefore, unlike the other

cysteine residues, it is situated at the surface and accessible to both to NEMI and DTNB. By computerised modelling (Fig. 4.5), it is shown that the DTNB modification of Cys47 has a large steric effect on the conformation of the loop between a-helix B and 8-strand 3, resulting in the movement of Gln51, the side chain of which is bound by the 7-glutamyl moiety of GSA through the 7- carbonyl group. This would weaken the ionic interactions which bind GSA to the protein. The reaction of NEMI with Cys47 has a similar steric effect, although not as large (Fig. 4.6). Although the human GST n has two extra residues at 40 and 41, comparison of the conformation of both porcine and human GSTrc around Cys47 (Cys45 for porcine) and Gln51 (Gln49 for porcine) revealed that both Cys47 and Gln51 located at identical positions in the three-dimensional structure at 2.1 A resolution (Reinemer et al., data not shown). This implies that the positions of Cys47 and Gln51 are crucial for the enzyme function. However, the role of Cys47 in GST n is not yet known. It is not clear why GS-OCT is able to prevent the inactivation by both NEMI and DTNB. It a possible that, in the absence of GSA, GSH or GS-OCT, the loop containing Cys47 becomes more mobile and that the thiol group of Cys47 is more accessible to reagents.

4.8 Summary

The results of chemical modification are consistent with the three-dimensional structure of porcine GST pi. The conclusions are summarized in Table 4.2. It is shown that Lys44, BAsp98 and Trp38 are involved in the binding of GSH and that Tyr7 is associated with the catalytic mechanism which facilitates the reduction of the pKa of the glutathionyl thiol. Arg13 is important in the catalytic mechanism as it helps stabilize a-helix A which produces an electrostatic field essential for the deprotonization of Tyr7.

136 Three-dimensional structure of G-site iZ 0) rr in 3 .C O □_ ‘to !E d) C CD k_ a> o 5 ° O) O (0 CD o o c c J= ’> i- - H z Q m 3 ■o O c >% 1

137 Three-dimensional structure of G-site CO cn o o •*- o 7 w ro o o c c .C z LU '■5 .2 T3 O a> c ° G) c E o >*

138 X 0 0 c O O 0 ' u > 0 0 0 0 0 0 Q. x : C LO 3 0 0 0 3 O ■O O) 0 0 ' c 3 3 S o O) 0 . t i 'C 0 0 O O ■♦—0 O 1 ° < ? * o "O O c o O 0 c c c g - § 0 0 0 ^ ■ f o K § 1 x : 05 < < Q_ a 0 _ 3 CO CO • 8 . c CO o c ■ S g o a a — 0 %—» *♦— r— o x : O ^ CO .2S *o2 a "co ’ > O ) 05 C 05 o •- > c c E O l 0 - C 2 .E ’•6 c c ^ c ■O V i o '■E CO o 0 0 o 0 o o 2 c c c g o c I®T > < T 3 S o XJ E r a o 0 o - co o : e o o 0 2 X E < < a> o ’c > » a > c o 0 CO 0 g B 'u o ~o o * E CL.2 .2 c O c O O C CD c ■- a 0 0 05 0 £5 0 O 0 o 2 . c o ^0 c 0 JC t> sz — x : 0 •*— 0 0 I g o o Q. * = JD ~ o H- 3 0 E 0 {- <2 g co o 0 CO o i_ SH z o 0 0 . ^ 0 CLO S.

> » M 0 >s c E c 0 5 0 a _ 3 a 1 . > » _3 _ > » 3 ® a ) a . 05 0 05 05 cog 3 < o O x : CNI CD O) 1 5 > 0 ■*-> C < c C C < a> o 0 . 2 CO o .2 CO . o < c •S < T > 0 o o t 5 C 5 .co CD h 0 0 M- 0 - i_ E o O 0 0 0 0 0 a . C c o c ° - _t t .s= 3 .= 3 c • | C O 2 2 o o § - o 2 CD c O ) ' c c o C 05 o CD E 0 03 > s — 05 . 2 > , 0 o < X 0 '<75 0 0 0 o 0 >< > 0 CO o 0 0 c CD -Q E E o c ® o o s— c o > O CO E p 0 c L L O 0 LL O ID s> S o . 2 CJ3 c/>

c o CO 0 co c 0 5 3 CL * o ■*t CO CO e 0 CO ■Q w 0 CD < CL 0 I d> > » GC CD 2 £ CHAPTER V

REGULATION OF GST n GENE EXPRESSION

140 Regulation of GST n Gene Expression

Foreword:

The gene encoding GST n has been sequenced. Its promoter region contains a consensus AP1 binding site together with two G/C boxes and a TATA box (Fig. 1.13). Studies have shown that the consensus AP1 binding site fails to respond to TPA and any regulatory elements extra to the promoter region have yet to be identified. In this chapter, a function of the consensus AP1 binding site is characterized and a downstream positive regulatory element is identified which appears to be part of the promoter. It is also demonstrated that retinoic acid suppresses GST k gene expression, whereas . insulin induces its expression.

5.1 A Function of the Consensus AP1 Binding Sequence

The promoter region of GST n contains a TATA box, two G/C boxes and a consensus AP1 binding site (Fig. 1.13). This consensus AP1 binding site is identical to the 7 bp motif (TGACTCA) present in the 5’ flanking region of the human metallothionein IIA (hMTIIA) gene (Angel et al., 19815). This latter sequence confers TPA inducibility when positioned immediately upstream of a heterologous promoter and, in synthetic multiple copies, it behaves as a TPA inducible enhancer. However, the GST n gene is not transcriptionally activated by TPA (Dixon et al., 1989). To investigate any function of the consensus AP1 binding site as well as the two G/C boxes, the effect of their deletion on basal promoter activity of the GST n gene was determined.

5.1.1 Construction of pB S**C A T plasmids:

The source plasmids used were: pSS0.2, constructed by inserting the Hind III / Sst I fragment of the GST n promoter region (nucleotides -99 to +72) into the multicloning site of pUC13. pICCAT, derived from the pSV2CAT (Appendix b & c) by replacing the SV40 early promoter-containing fragment

141 Regulation of GST n Gene Expression with double-stranded multicloning site oligonucleotide from pUC13, so designed that fragments, which will be inserted into the multicloning site, are placed immediately upstream of the chloramphenicol acetyl transferase (CAT) gene. The pBS**CATswere constructed as follow: pSS0.2 was digested with Hind III followed by Bal 31 exonuclease digestion and then blunt-ended with Klenow DNA polymerase before finally being treated with Sst I. The products namely small Sst I / blunt-end fragments were gel-purified and ligated into the Sst I and Sma I (which generates a blunt end) digested pICCAT. After transformation into E.coli JM83, about 200 colonies containing appropriate recombinants were detected by colony hybridization using a 32P-labelled GST n promoter fragment (nucleotides -99 to +72) as the probe. 60 recombinants were purified and sequenced by double-strand sequencing in both directions across the whole insert. Appropriate constructs (pBS**CAT, where "**" refers to the position of the nucleotide from which the upstream sequence was deleted; B stands for Bal 31 and S stands for Sst I) (Fig. 5.1 b) were selected and purified by two successive CsCI density gradient centrifugations.

5.1.2 Transfection of pBS**CATs into Cultured Human Cells: pBS**CATs were transiently transfected into EJ, MCF7 and HepG2 cells, which differ in their endogenous GST n expression. EJ cells express GSTtc at a moderate level whereas HepG2 cells (Dixon etal., 1989) and MCF7 cells (data not shown) express GST n at a very low level. pSVzCAT, which contains the SV40 promoter (Gorman etal., 1982), was used as the positive control and pICCAT, which has no promoter activity (Dixon etal., 1989), was used as the negative control. pSS0.2CAT, which contains the GST n gene promoter in a 171 bp Hind III and Sst I fragment (-99 to +72) cloned immediately upstream of CAT gene in pICCAT was used as the reference since it has been demonstrated that the promoter contained in pSS0.2C A Tis active not only in cell lines which express GST tt, such as HeZa, but is also active in cell lines which barely express GST n, such as HepG2 (Dixon etal.,

142 Regulation of GST n Gene Expression

j W

Fig. 5.1 Transfection of pBS**CATs into EJ, MCF7 and HepG2 cells.

143 Regulation of GST it, Gene Expression

Fig. 5.1 Transfection of pBS**CATs into EJ, MCF7 and HepG2 cells. A) The map of GST n promoter region. i tAv Consensus AP1 binding site^, G/C boxes and TATA box are indicated. B) The schematic representation of 5’ deletion constructs which were generated by Bal 31 digestion and cloned into the multicloning site of pICCAT directionally. C) The relative CAT activity expression of pBS**CAT\n EJ cells (depicted by open bar), MCF7 cells (depicted by striped bar) and HepG2 cells (depicted by filled bar). For every construct, each bar represents the mean of nine to twelve independent determinations.

1989). Experiments were performed in triplicate and were repeated with constructs from different preparations.

As illustrated in Fig. 5.1c and Fig. 5.2, the patterns of CAT marker gene expression from pB S**C A Tconstructs were very similar in all three cell lines. When pBS60CAT (containing only part of the consensus AP1 binding site), pBS46CAT (lacking one G/C box in addition to the consensus AP1 binding site) and pB S 43C A T(lacking both G/C boxes as well as the consensus AP1 binding site) were transfected into cells, the CAT expression in each case was very similar to the background obtained by transfection with pICCAT\ the negative control. This suggests that the consensus AP1 binding site is essential for the basal level promoter activity of GST n in all three cell lines

tested. Deletion in the sequence -99 to -71 resulted in some modest changes of the downstream CAT expression (Fig. 5.1; Fig. 5.2). There is therefore the possibility that weak enhancing and silencing elements are present adjacent to the consensus AP1 binding site. However, the fact that deletion of the surrounding sequence may affect the activity of the consensus AP1 binding site artificially can not be excluded.

144 Each CAT assay contained 10 to 50 jil cell extract, 0.2 uCi [*C] chloramphenicol, 0.4 mM acetyl CoA and 220 mM Tris/HCl (pH 7.8) in a final reaction volume of 150 jjlI. All components of the reaction except acetyl CoA were mixed and preincubated at 37°C for 5 min. The reactions were started by addition of acetyl CoA, incubated for 30 min and terminated by the addition of 2 ml of cold ethyl acetate. The acetate fraction was removed to a fresh tube and evaporated to dryness under vacuum. The residue was taken up in 5 vil ethyl acetate and was spotted onto a silica TLC plate. TLC separation was achieved by ascending chromatography in chloroformrmethanol (95:5). After sufficient development the silica plate was dried and autoradiography was performed. A standard result was shown as illustrated below.

solvent front ►

diacetyl-Cam

3-acetyl-Cam ► f ' 1-acetyl-Cam ►

Cam ►

origin ► Regulation of GSTtz Gene Expression

• •

••••••••

• • • • • • • •

1 2 3 4 5 6 7 8

Fig. 5.2 pB S**CAT Transfection of HepG2, EJ and MCF7 Cells

a) HepG2 cells; b) EJ cells; c) MCF7 cells.

Lane 1, pSS0.2CAT\ 2, pBS77CAT\ 3, pBS74CAT\ 4, pBS71CAT\ 5, pBS60CAT\ 6, pBS46CAT\ 7, pBS43CAT\ 8, pICCAT.

DefoCo of CAT assay a fie described in dkx note o* the oppos,r* pays.

145 Regulation of GST % Gene Expression

Since the deletion of the consensus AP1 binding sequence abolished the promoter activity in all of the cell lines tested, it is not possible to study the effect of the deletion of the two G/C boxes on the activity of the promoter at

present.

5.2 Analysis of Upstream and Downstream Regions of GST n Gene for Functionally Active cis-Acting Sequence:

Efforts have been made to localize any regulatory elements upstream of the

G ST n gene. It was demonstrated that a fragment of GST 71 corresponding to nucleotides -99 to -8 promoted the transcription of the downstream CAT gene. However, there was no evidence for any enhancer or silencer activity upstream of the promoter region between -99 to -6000 bp of GSTtc (Dixon et al., 1989). To expand the analysis, two fragments from the upstream region extending to -13500 bp and two fragments downstream from tsp extending to + 3000 have been tested for their c/'s-acting regulatory activities.

5.2.1 Restriction Mapping of the Fragment -3.0 to -13.5 kb:

DNA from cos3.5.1, which contains GSTn and 15 kb of its 5’ flanking region, was subjected to digestion with Apa I, Bgl II, EcoR I, Hinc II, Hind III , Pst I and Sst I. Fragments generated with a single enzyme digestion were purified and subjected to further digestion with a second enzyme. Analysis of the fragments resulted from single and double digestion with above enzymes allowed the construction of a map with respect to the site for the enzymes (Fig. 5.3a).

5.2.2 Repetitive Sequences Present in the 5’ Flanking Region of the

GST 71 Gene:

The 5’ flanking sequence of the GST n gene has been sequenced up to

146 Regulation of GST k Gene Expression .O CO co co o CO co in o co T3 •a X X CO CO L L X ■a -o X X •a Q_ LL X •a l i X in CO 12 LO U. ‘5) cc a co ) d O 0 (/> o C 0 . Q o 0 c c O) 0 o x 2 o 0 c 0 X ‘co < CD CD LL LL 0 CO o o c X 0 CO 0 i— 0 CO ■O 0 X ■o o CL . a 0 CO O) O O U CO c o o c co 0 c CO

147 Regulation of GST n Gene Expression

-2.8 kb from the transcriptional start site (I.G. Cowell unpublished results; Morrow etal., 1990). A search of the GenBank revealed homologies between these sequences and two repetitive insertion elements, Alu and LINE 1 (L1)

family of short and long interspersed repeats respectively. The Alu repeat occupies positions -520 to -820, whereas the L1 repeat stretches from the 5’ end of Alu sequence and extends as far as the sequence is available.

In order to study whether the region up&tmaM of -2.8 kb contains any

repetitive sequence, cos 3.5.1, which contains GST n and 15 kb of its 5’ flanking region, was digested with Sst I and a 6 kb Sst l/Sst I fragment that corresponding to 5’ flanking region of the GST n gene (-6 kb to +72 bp) was subjected to single or double digestion of Hind III, Hinc II, Pst I and Bgl II. The resulting fragments were separated by 0.8% agarose gel and blotted onto Hybond-N membrane. The blot was probed with Alu fragment as well as total human genomic DNA and a 400 bp Hind Ill/Sstl fragment containing a single copy DNA from the 5’ flanking region of the GST k gene (-350 to +72) was used as the negative control. The results showed that no additional Alu sequences can be detected up to 6 kb from the tsp (Fig. 5.4). However, the repetitive DNA extended to about -4.8 to -5.5 kb (Fig. 5.5). From the length of the repetitive sequence, it can be concluded that either the L1 element does not show the commonly observed 5’ truncation (Hattori et al., 1986) or that the upstream sequence contained clusters of repetitive DNA. From these results, it can be concluded that any cis -acting regulatory elements, if associated with the GST n gene, would be further than 6 kb upstream of the gene, within the gene itself or downstream of it.

5.2.3 Construction of PlasmidspUH3.5CATt pUP5.3CAT, pDSH1.8CAT and pDB2.3CAT:

To determine whether the 5’ flanking region (-13.5 to -6), the gene itself as well as its 3' sequence can modify the activity of the promoter of GST 71 gene,

148 Regulation of GST % Gene Expression

S B B Hd A P Hd He P He B

X - L - r : ____ ^ i f _2> I i 1 <

B. 1. Hind

2. Pst I

3. Hinc II

4. Bgl II I I L

Fig. 5.4 Detection of Aiu Sequence in the 5’ Flanking Region of GST 7r Gene.

A) Restriction map of the 5’ flanking region (-6.0 kb to +72 bp). The

sites for Apa I (A), Bgl II (B), Hinc II (He), Hind III (Hd), Pst I (P) and Sst I (S) are shown. B) Fragments generated by single or double digestion with above enzymes. The fragments which hybridized with 32P labelled Alu sequence are

indicated by arrows.

149 Regulation of GST % Gene Expression

S B B Hd A P Hd He P He B

'i—I c ^ i r J I L

1. Hind III »■

2. Pst I

3. Hinc II

4. Bgl II I I L

5. Bgl II / Pst I I I L

6. Hind III / Pst I J L J ______L

Fig. 5.5 Detection of Repetitive Sequence in 5’ Flanking Region of GST n Gene A) Restriction map of the 5’ flanking region of GST n gene (-6.0 kb to +72 bp). Sites for Apa I (A), Bgl II (B), Hinc II (He), Hind III (Hd), Pst I (P)

and Sst I (S) are shown. B) Fragments generated by single or double digestion of the above enzymes. Fragments which hybridized with human genomic DNA are

indicated with arrows.

150 Regulation of GST n Gene Expression a Hind III fragment (-8.3 to -4.8) and a Pst I fragment (-13.5 to -8.2, blunt- ended and ligated with Hind III linker) were inserted into the Hind III site of pSS0.2CAT\o generate p(JH3.5CATand pU P5.3C ATrespectively (Fig. 5.6b).

Fig. 5.7a shows the restriction map of the downstream region of GST n gene (Cowell, 1989, PhD dissertation). Two fragments from the downstream region, fragment Sst I / Hind III (+72 to +1.8 kb, the Sst I site was blunt-ended and ligated with Hind III linker) and fragment Bam HI /Bam HI (+1.8 to +3 kb, ligated with Hind III linker), were inserted into the Hind III site of pSS0.2CAT to produce pDSH1.8CAT and pDB2.3CAT respectively (Fig. 5.7b). All recombinants were purified by two successive gradient centrifugations.

5.2.4 The CAT Activities:

Plasmids pUP5.2CAT, pUH3.5CAT, pDSH1.8CAT and pDB2.3CAT were transfected into cultured Hela, HepG2 and EJ cells alone with pSV2CAT, p lC C A T and pSS0.2CAT. Cell lysates were tested for the CAT activities and the results showed that, in all three cell lines tested, the CAT expression from these four test constructs were not significantly different from that of pSS0.2CAT. Fig. 5.8 illustrates the results obtained from transfections of EJ cells. These indicate that no regulatory activity was detectable within these regions of the GST n gene in the three cell lines used. The region remaining untested is the fragment -8 to +72.

5.3 Identification of the Downstream cis-Acting Element of the GST n Gene:

The fragments used previously for basal promoter activity by Dixon et al. (1989) and Morrow etal. (1990) were nucleotides -99 to +72 and -80 to +314 respectively. Both fragments therefore included the first exon and part of the first intron which occupies region +35 to +318.

151 Regulation of GST n Gene Expression

A) 1Kb I 1

-13.5 -8.3 -6.0 -4.8 . 3.0 I I M I 3TI— 7TMTK -- T P Hd S E Hd Hd Hd P E S S B B Hd A P Hd

B) PUH3.5CAT = 1 _ 1 1 pUP5.3CAT

Fig. 5.6 Scheme for Generation of GST tc-CAT Constructs Containing Fragments from 5’ Flanking region of GST n gene

A) Restriction map of the 5’ flanking region of GST k gene. Restriction sites are as follow: Apa I (A), Bgl II (B), Eco Rl (E), Hinc II (He), Hind III (Hd), Pst I (P) and Sst I (S). B) Fragments used to generate constructs pUP5.3CAT and pUH3.5CAT.

152 Regulation of GST n Gene Expression

A. 1Kb I------1

1 2 3 4 5 6 7

—K I J t I hl1 A1 F X Bg T Sll SSI P P SI H B X P Bg b

B. pDSH 1.8CAT

pDB2.3CAT tzz - 1

Fig. 5.7 Scheme for Generation of GST 71-CAT Constructs

Containing Fragments from the Downstream Region of GST 71 Gene.

A) Restriction map of the GST n gene and the 3’ downstream region. Restriction sites are shown as follow: Bam HI (B), Bgl II (Bg), Eco Rl (E), Hind III (H), Pst I (P), Sst I (SI). Sst II (Sll), Stu I (S), Tthl III (T) and Xba I (X). Exons are indicated as numbered. B) Fragments used to generate the GST71-CAT constructs pDSH1.8CATand pDB2.3CAT.

153 Regulation of GST n Gene Expression

Fig. 5.8 Transfection of pUH3.5CAT, pUP5.3CAT, pDSH1.8CAT and pDB2.3CAT into EJ Cells

Lane 1, pSV2CAT\ 2, pSS0.2CAT,; 3, pUH3.5CAT\ 4, pUP5.3CAT\ 5, pDB2.3CAT\ 6, pDSH1.8CAT\ 7, pICCAT.

5.3.1 A c/s-Acting Regulatory Element Located in the Sequence +8 to +72, Downstream of the tsp :

The involvement of the downstream region (including nucleotides +8 to +72) in modulating the transcription, was tested by using three GST jt-CAT fusion genes, pSS0.2CAT, pSSO.ICAT and pCR0.15CAT, in transient transfection assays. The construction of pSS0.2CATand pSSO. 1CATwere described by

Dixon et al. (1989). pSS0.2CATcontains nucleotides -99 to +72 of the GST71 gene, whereas pSSO.ICAT contains nucleotides +8 to +72 of the GST tt gene (Fig. 5.9). pCRO.1 SCAT was constructed by the polymerase chain

154 Regulation of GST % Gene Expression

-99 - 8 + 1 + 8 +72

u - o o - n ------—» AP1 G/C G/C TATA lntron 1

PCR0.15CAT

pSSO.ICAT I -----

PSS0.2CAT

Fig. 5.9 GST n Promoter-CAT Chimeric Constructs

Consensus AP1 binding site, G/C boxes and TATA box are shown. The fragments shown were either isolated from restriction enzyme digestions or generated by PCR and were cloned directionally into the multicloning sites of plCC A T-

reaction (PCR) using pSS0.2CAT as the template. The PCR primers used were as follow: Primer 1, 5’-GCGGAGCTCCAGCGAAGGCCTCGC-3\ was made complementary to nucleotides -11 to +13 of the GST n gene fragment in pSS0.2C A Twith two nucleotides altered to generate an Sst I site at +5 to +10; Primer 2, 5’-CCGCATATGGTGCACTCTCA-3\ was made

155 Regulation of GST % Gene Expression complementary to nucleotides 542 to 562, at the 5’ end of the polylinker of pICCAT. The fragment generated by PCR was cleaved at its Hind III and Sst I sites, ligated into the Hind III and Sst I digested pICCAT, thereby yielding pCRO. 1 SCAT (Fig. 5.9). pCR0.15CAT, pSS0.2CAT, pSSO.ICAT * pSV2C A T {the positive control; and plCCAT(\he negative control) were transfected into four cell lines which differ in their endogenous GST k expression including Hela, EJ (both express G ST n at a modest level) (Dixon eta!., 1989) and HepG2, MCF7 (the GST n expression in both cell lines was hardly detectable) (Xia et a l, data not shown). Experiments were carried out in triplicate and were repeated with constructs from different preparations.

The result from transfection of pCR0.15CAT (nucleotides -99 to +8, the "minimal promoter") suggests that the 107 bp promoter-containing fragment of GST n gene actively promote the transcription of its downstream CAT gene. The resulting CAT activities were significantly above those obtained with pICCAT in all four cell lines tested. Compared with pSV2CAT, pCRO. 15CAT expressed CAT activities were 4%, 14%, 16% and 25% of those obtained from pS V fiA T , the positive control, in Hela, EJ, HepG2and MCF7 cells. CAT expression from pSSO .ICAT (nucleotides -8 to +72) were the same levels as the background generated from pICCAT. Interestingly, CAT transcription from pSS0.2C A T(nucleotides -99 to +72) gave results which were 2.9, 3.9, 4.0 and 5.4 fold higher than those from pC R 0.15C A Tin MCF7, EJ, Hela and HepG2 cell respectively (Fig. 5.10, Table 5.1). This indicates that there is a cis- acting element located in the sequence +8 to +72 downstream of the tsp which h> contributes^the promoter activity of the GST n gene and is functional in all four cell lines examined.

156 Regulation of GST n Gene Expression

Table 5.1 Relative CAT Activities Generated by GST k Promoter-CAT constructs

Values were calculated as percentage of chloramphenicol acetylation, corrected for protein concentration and are expressed relative to the activity obtained with pCR0.15CAT containing the GST n promoter. Transfections were carried out in triplicate and the results are the mean of three independent experiments.

Heta HepG2 MCF7 EJ pICCAT 0.0410.01 0.0810.06 0.0810.03 0.1010.02 pSSO.ICAT 0.0510.06 0.0810.05 0.0810.01 0.1010.04 pCR0.15CAT 1.0010.20 1.0010.13 1.0010.10 1.0010.10 pSS0.2CAT 4.0011.00 5.4012.70 2.9010.31 3.9010.08 pSV2CAT 26.014.80 6.9012.40 4.0810.70 7.210.480

5.3.2 The Downstream cis -Acting Element Binds Proteins in vivo and in vitro:

To study whether the downstream cis- acting element located at sequence +8 to +72 binds trans-acting factors, pS S 0.2C A T (nucleotides -99 to +72) was co-transfected with pSSO.ICAT (nucleotides -8 to +72) into EJ, HepG2 and MCF7 cells and the CAT activities observed were 2.8 to 4.8 fold lower than those co-transfected with pICCAT (Fig. 5.11a). This suggests that the fragment contained in pSSO. 1CATcan compete with that in pSS0.2CAT\or factors which can up regulate the promoter activity. Interestingly, pCR0.15CAT co-transfected with pSSO.ICAT, which has little overlapping sequence, gave CAT activities 4.6 to 7.0 fold lower than those obtained by

157 Regulation of GSTk Gene Expression

m • • # # • • i

12345 12345

HepG2 MCF7

• •

EJ Hela

Fig. 5.10 CAT Assay with HepG2, Hale, EJ and MCF7 Cells Transfected with GST n

Promoter-CAT Chimeric constructs

Transient assayswere performed in all four cell lines using p S V fiA T (lane 5), pCRO. 15CAT (lane 4), pSS0.2CAT (lane 3), pSSO.ICAT (lane 2) and pICCAT (lane 1).

158 Regulation of GST % Gene Expression

co-transfection of pCRO. 15CATand pICCAT (Fig. 5.11 b). This suggests that the trans-acting factor, which binds to the downstream cis -acting element, is related to a transcriptional factor active in the region -99 to -8, the minimal promoter.

In order to further investigate proteins which bind to the downstream cis -acting element and the DNA binding site, electrophoresis mobility shift assay was performed, using 32P end labelled fragment containing -8 to +72 of GST n gene as the probe. The cell nuclear extract was prepared from SVK14 cells (a human keratinocyte cell line). As shown in Fig. 5.12a, a shifted band was observed. The binding of the proteins to the fragment was specific, since it can not be competed out by the addition of high concentration of dldC (Fig. 5.12b). Further studies to identify the specific sites that are involved in protein binding are now under investigation.

5.3.3 The Function of the Downstream cis -Acting Element Is Position Dependent:

In order to determine whether the downstream cis -acting element is functional as a classically defined enhancer, ie position and orientation independent, a construct was made by PCR. pSSO.ICAT was used as the PCR template. The two primers used were primer 1 ,5’-TGGAAGCTTTGAGCCCCA-3\ which is complementary to nucleotides +63 to +72 of the GST n gene and +73 to +80 (the 3’ end of the polylinker of the vector) with two nucleotides altered to /generate a Hind III site at +72 to +77, and primer 2, 5’- CCGCATATGGTGCACTCTCA-3', which is complementary to nucleotides 542 to 562, at the 5’ end of the polylinker of pICCAT The fragment generated by PCR was digested with Hind III and ligated into the Hind III digested pCRO. 15CAT, thus generating pCR0.25CAT(Fig. 5.13a). Sequencing across the whole insert showed that the fragment -8 to +72 is present in inverse orientation in front of the promoter (Fig. 5.13). Transfection of pCR0.25CAT

159 Regulation of GSTk Gene Expression

•« • “ V . - t • m u H #• * • S S Co K' r : 3 \ 4 o m ■ W H in • * • C a U LO » • O a u E—1 a Ca a

r o LO • * o CN| O a #• aCD

£ 4 om m a

o in s w a a c\ •• a 4 Uu H w a • • #

m •■j W • • » m o

Fig. 5.11 The Effect of Co-transfection with pS S O .IC A T on p S S 0 .2 C A T and pC R 0.15C A T

160 Regulation of GST n Gene Expression

Fig. 5.11 The Effect of Co-transfection with pSSO .IC A T on pSS0.2C A T and pC R 0.15C A T A) pSS0.2CATan6 pCRO. 15CATwere co-transfected with pIC C A T{lanes 5,3) and pSSO. 1C A T(lanes 4,2) into EJ, HepG2 and MCF7 cells. pSSO.1 CAT was co-transfected with pICCAT as the negative control.^ B) The CAT gene expression of pSS0.2CAT and pCRO.1 SCAT are shown as activity relative to the pICCAT co-transfection control. The results were obtained in triplicates from two independent experiments.

as well as pSS0.2CAT and pCRO.15CAT into HepG2 cells showed that the level of the CAT transcription from pCR0.25CAT was about 6 fold lower than that from pSS0.2CAT, and was about the same level as that obtained from pCRO.15CAT (Fig. 5.13b). This suggests that the activity of cis- acting element located between +8 to +72 of GST n gene is position-dependent and thus is a downstream element integral to the promoter.

5.3.4 Effect of the Downstream c/s-Acting Element on mRNA Stability

To support the hypothesis that the downstream cis- acting element functions by regulating the upstream promoter activity rather than affectsthe stability of mRNA, the half-life of CAT mRNA transcribed from both pSS0.2CAT and pCRO.15CAT in MCF7 cells was evaluated after inhibition of RNA synthesis by addition of actinomycin D, a known inhibitor of RNA polymerase II. MCF7 cells were transfected with both pSS0.2CATand pCRO. 15CAT. 48 hours after transfection, fresh medium containing 10 pg / 10 ml of actinomycin D was added. Total RNA was extracted 1, 2 and 4 hours after actinomycin D addition and 50 pg of total RNA was used in slot blot analyses. The filter was

161 Regulation of GST n Gene Expression

Fig. 5.12 Gel Shift Assay Using Nucleotide -8 to +72 of GST n Gene as Probe

Experiment was carried out as described in section 2.2.15. A). All lanes contain 1 pg dldC. Lane 1, no protein; lane 2, 1 pg nuclear extract; lane 3, 5 pg nuclear extract. B). All lanes contain 1 pg nuclear extract. Lane 1, 10 pg dldC; lane 2, 1 pg dldC; lane 3, no dldC.

162 Regulation of GST n Gene Expression CD < • a) • • • • • < CN CJ co co o CL ^ O C N C u u cc o < i r o < IT) 0 o cc CL < CJ 0 CO 0 >~ Q. - ; ■ o o |- O CM o — n °- H O H n i K < 2 S ■D H X ■' CO ' *■ G & 0 D) c co 0 O o c C - 2 >* 1 1 E w 0 o CD 0 o o Hi E 0

0 CO ’= O CO < u_ c c L -C -4—' 0 ~ 0 O 0 o 0 E CO O 0 Q- ~ £ 0 0 0 0 C 0 o c ? o — o - E o o o c 00 o CO CD

X CO n *0 CO o ~ O CM _0 1- > < CO 00 O C\J CM 0 0 0 0 0 0 > L C 0 0 — i £ 0 E 0 c 0 L L L C in a C D C L C 0 3 0 0 c L C 0 CNJ

O X O O O < H CD in 1 CO O < I— O O O - CM ■M" 1- < 0 _ in L C 0 c 0 L C 0 C 0 0 L C C c 0 T ■'

163 Regulation of GSTk Gene Expression

hybridized with 32P-labelled CAT cDNA and the result is shown in Fig. 5.14. These data demonstrated that the level of mRNA of CAT gene transcribed from pSS0.2CAT in MCF7 cells is about 2.5 fold of that from pCRO.1 SCAT

and both CAT mRNAs display a halMife of about 2 hours. This indicates that

the downstream element, up-regulates the expression of the gene by enhancing transcription, rather than affecting the stability of the mRNA or the efficiency of translation.

5.4 Negative Regulation of The GST k Promoter Activity by Retinoic Acid (RA):

Chambon and his colleagues have showed that the transcription from rat

stromelysin gene (a member of the metailoproteinase family) is repressed by the vitamin A derivative retinoic acid (RA) (Nicholson eta/., 1990). Further studies indicated that the DNA sequence required for RA dependent repression co-localized with a consensus AP1 binding (TRE) site which is located at -72 to -65 of the gene and was essential for basal promoter activity. Comparison of the consensus AP1 binding site of GSTn promoter (Fig. 1.13) with that of the rat stromelysin reveals that the location and the sequence of the consensus AP1 binding site of the two genes are very similar (with TGAGTCA at -65 to -71 for the stromelysin gene and TGACTCA at -58 to -65 for the GST k gene) and both are essential for their own promoter activities. be To test whether the promoter activity of the GST n gene can also^ repressed by RA, construct pSS0.2CAT which contains the GST n promoter and the downstream c/s-acting element was co-transfected with hRARft (an expression vector encoding the human RA receptor 6 cDNA) into EJ cells. The preliminary experiment showed that the CAT expression was 2-3 fold lower in cells cultured in medium (with 2 % FCS) containing 1 (iM RA than those without RA (Fig. 5.15). This indicates that the promoter of the GST n gene may respond to RA-mediated repression. To further the investigation, the levels of GST n protein in cultured SVK14 cells, with or without treatment of

164 mRNA Expression Gene n GST of Regulation pSS0.2CAT pCRO.15CAT. rbd ih A cN. o a, row cDNA. CAT with probed used in slot blot assays. A) Autoradiograph of a slot blot blot slot a of was RNA Autoradiograph total of A) fig hours assays. 50 4 blot and and slot 2 in 1, used addition extracted was actinomycin RNA total after The jig/10ml). a add o h mdu (ia cnetain a 10 was concentration (final medium the to added was pCRO.15CAT. MCF7 cells were transfected with either either with transfected were cells MCF7 i. .4 lt lt nlss f RA Stability mRNA of Analysis Blot Slot 5.14 Fig. 12 4 2 1 4 2 1 b, ; ) eaie ees f RA a, mRNA. of levels Relative B) 8 or atrtaseto, cioyi D actinomycin transfection, after hours 48 pCRO.15CAT. (hours) 165 pSS0.2CAT\ 2 4 2 1 pSS0.2CAT o b, row or or Regulation of GSTk Gene Expression

• •• • ^ • •

+ h RARR -hRARR

+ RA -RA + RA -RA

Fig. 5.15 RA/?£-Mediated RA Repression of GST k Promoter Activity in EJ Cells.

pSS0.2CAT was co-transfected into EJ cells with (+) or without (-) hRARR. 4 hours after transfection, cells were cultured in medium (containing 2 % FCS) with or without 1 |iM RA (the final concentration of ethanol is 0.02%). 48 hours after RA stimulation, cellswere harvested and the CAT activities were determined.

166 Regulation of GST n Gene Expression

RA, were determined by reverse phase-HPLC analysis. The GST n level in cells transfected with hRARB and challenged with 1 pM RA, was compared with that of control cells transfected with anti-hRAR7 (contains cDNA of RAR7 in reverse order) and without RA treatment (Fig.5.16).

The result shows that the level of GST n in cells transfected with hRARB was 9.5 pg/1x108 cells, whereas in cells transfected with anti-hRARy, that level was 31 pg/1 x108 cells. This result indicates that RA represses the expression of the endogenous GST n gene. Further studies on the mechanism of the regulation of the GST n gene by RA are beu raj carried out.

5.5 Insulin Induction of the Promoter Activity of GST n Gene:

Carnovale et al. (1990) have shown that there was a direct relationship between the blood insulin levels and the GST activity towards CDNB in liver, kidney and intestine in male Wistar rats. To test whether insulin can influence the gene expression of GST n, MCF7 and SVK14 cells transfected •fhoe with pSS0.2CAT were cultured in serum^media with or without 1 pM insulin. The results showed that the CAT activities from MCF7 and SVK14 cells treated with insulin were about 3 to 5 fold higher than those of the controls (Fig. 5.17; data for SVK14 is not shown). In addition, using our GST rccDNA as the probe, Dr. Bulawela (Charing Cross Hospital Medical School) has demonstrated that the GST n mRNA level in SVK14 cells challenged with 1 pM insulin is much higher than the control cells (Fig. 5.18). These data suggest that insulin can up regulate the activity of the GST n gene. However, further controls to characterize the mechanism through which insulin yields this result have yet to be done.

167 Regulation of GSTk Gene Expression

20 30 40 5 0

Retention Time (min)

Fig. 5.16 Analysis of RA Repression of GST n expression by Reverse Phase HPLC

1x107 cultured SVK14 cells cultured in medium with 2 % FCS were transfected with RAR vectors. 4 hours after transfection, 2 pJ 1.5 mg/ml RA (in ethanol) or 2 jil ethanol were added. GST n levels were quantified by reverse phase HPLC as described in section 2.2.16. A) hRARB + RA; B) artti-hRARJ.

168 Regulation of GSTtz Gene Expression

• •

-I 4-1

Fig. 5.17 Induction of GST k Promoter Activity by Insulin

pSS0.2CATwas transfected into MCF7 cells. 4 hours after transfection, cells were cultured in serum free medium with or without 1 (iM insulin. 48 hours after insulin stimulation, cells were harvested and the CAT activities were determined.

169 Regulation of GSTk Gene Expression

1 2 3

Fig. 5.18 Northern Blot Analysis of the Induction of GST n mRNA by Insulin

Total RNA from cultured cells was prepared as described in section 2.2.14.1. The blot was probed with GST k cDNA. Lane 1, RNA from SVK14 cells; lane 2, RNA from SVK14 cells challenged with insulin; lane 3, RNA from 3T3 cells (negative control).

170 CHAPTER VI

DISCUSSION TO CHAPTER V

171 Discussion: Regulation of GST k Gene Expression

6.1 The Consensus AP1 Binding Site of the GST n Promoter

The regulation of many eukaryotic genes has been analyzed in recent years and the mechanisms which are involved appear to be substantially similar to those operating in prokaryotes, ie. they act through the interaction of proteins or trans-acting factors with short DNA sequences or cis -acting elements which are usually found flanking the 5’ end of the gene (Mitchell & Tjian, 1989).

Eukaryotes are unique, however, in the complex arrangements of the cis- acting elements and the large number of trans-acting factors that bind to them.

The TPA (phorbol 12-0-tetradecanoyl-13-acetate) responsive element (TRE), otherwise known as the AP1 binding site is a well-characterized cis -acting sequence found in the regulatory regions of many genes, such as the 72 base pair enhancer element of SV40, the promoters of human collagenase, rat stromelysin and human metallothionein IIA genes, all of which are responsive to transcriptional activation by TPA (Angel et al., 1987a,b; Lee et ai., 1987a,b), and it has been demonstrated to be the binding site for a number of related trans-acting factors such as the nuclear oncogene c-jun and c-fos complex or other similar protein complex (Angel e ta., l 1988; Bohmann etal., 1987; Chiu et al., 1988; Halazonetis et al., 1988; Rauscher et al., 1988; Sassone-Corsi et al., 1988). As has been mentioned in Chapter I, the promoter region of both human GST n and rat subunit 7 contain a consensus AP1 binding site. It is well documented that endogenous expression of rat subunit 7 gene is responsive to TPA (Sakai etal., 1988a). However, it is the upstream GPE I rather than the TRE sequence in the promoter region, which

appears to be responsible for TPA activation (Okuda et al., 1987). GPE I is an imperfect palindrome composed of two imperfect TRE-like sequences (Okuda et al., 1989). It is evident that both of them are essential to achieve the maximum level of TPA responsiveness. The distance between these two

172 Discussion: Regulation of GST k Gene Expression

TRE-like sequences and their relative orientation also affect the overall activity (Okuda et al., 1990). Thus it is suspected that the synergistic action of these two TREs is crucially important for their function (Okuda et al., 1990).

Studies by both Dixon et al. (1989) and Morrow et al. (1990), however, have

failed to demonstrate the responsiveness of the GST n gene to the transcriptional activating effect of TPA or jun and fos. A role, if any, for the consensus AP1 binding site was therefore not yet been elucidated by their work. In this thesis, by means of transient expression of 5’ deletion mutants

of the GST k promoter from -71 to -43, it is shown that the integrity of the consensus AP1 binding site is essential for the basal level activity of the GST n promoter. It is, therefore presumably available to c-jun or c-fos, but c-jun and c-fos are not essential for the GST n promoter activity since the GST n promoter can actively promote the downstream CAT transcription even in the F9 cell line, which expresses c-jun and c-fos at a very low level. In addition, co-transfection of c-jun and c-fos expression vectors into F9 cells has no effect on GST n promoter activity (Morrow et al., 1990; Okuda et al., 1990).

These paradoxical results may be due to the fact that some consensus AP1 binding sites can also be bound by trans-acting factors from the CREB/AFT families (Distel et al., 1990). Deutsch et al. (1988) have reported that the ability of AP1 binding site TGACTCA and the cAMP response element (CRE) TGACGTCA to confer inducibility to TPA or cAMP responsiveness can be

influenced by the sequence around the short motifs. Fink et al. (1991) have discovered that cAMP and TPA induced transcriptional activation of the human vasoactive intestinal peptide (VIP) gene are mediated by the same enhancer element which contains a TRE-like sequence. However, there is evidence that the GST k gene fails to respond to cAMP challenge in cultured cell (Dixon et al., unpublished result).

The AP1 binding site consensus in the promoter region of GST n gene is

173 Discussion: Regulation of GST n Gene Expression

presumably inappropriate to impart TPA inducibility on the gene. Nevertheless, it contributes to the basal level activity of GST n promoter. Angel etal. (1987) and Lee etal. (1987) have shown that one or more copies of AP1 binding site positioned upstream of a heterologous promoter increases

its basal level of expression. In addition, it is interesting to note that deletion

of the AP1 binding site from the rat GST subunit 7 gene promoter, which does not confer the inducibility of TPA, reduced the promoter’s activity significantly (Sakai etal., 1988a).

Recently, three papers have reported data which apparently agree with the results obtained in the thesis. Firstly, Timmers et al. (1990) identified a putative AP1 binding site (TGACTCC) in the promoter region (-52 to -46) of the rat JE gene (one of the cellular immediate early genes), which is essential for the basal activity of its promoter, but does not respond to TPA induction. Their results also indicate that c-jun and c-fos proteins are probably not involved in the activation of JE AP1 binding site since the sequence does not bind in vitro synthesized c-jun and c-fos. Secondly, as demonstrated by van Dan et al. (1990), the AP1 binding site of the c-jun gene is important for the basal level expression but does not effect the TPA-responsiveness of the gene. Thirdly, Buttice et al. (1991) have studied the role of the AP1 binding site (TGAGTCA) at -70 to -64 of the human stromelysin gene. Point mutations in any positions of the AP1 binding site reduced the basal expression of the gene, indicating that the AP1 site is required for the basal level activity of the promoter. However, their data also suggested that c-jun and c-fos or proteins of similar activity are required for the basal level expression since the promoter of stromelysin gene faiWto promote the down stream CAT gene expression in F9 cells.

The evidence provided here suggests that it is difficult to predict the function of a given element by merely relying on its sequence. The consensus AP1 binding site of GST n is such an example. Presumably, the organization of

174 Discussion: Regulation of GST k Gene Expression

the regulatory elements plays an important role in gene expression comparable to that of the element itself. This is to say that the transcriptional activity of elements, such as an AP1 binding site, may change depending on

the precise sequence and its DNA environment, ie., the context and

architecture of the gene. As stated by Muramatsu and his colleagues (Okuda et al., 1990), factors such as deviation from the consensus sequence, number of copies, combination of the relative orientations and distance

between them, its location on genes, other nearby trans-acting factor binding site and even chromatin structure may modulate the activity of an AP1 binding

site.

It will be interesting to confirm that trans-acting factors bind to the consensus AP1 binding site of GST n and to identify the proteins which transcriptionally activate the expression of GST n gene wathe recognition of the AP1 binding site.

The fact that the GST n promoter is active not only in cell lines such as EJ and SVK14, which express endogenous GST n, but also in cell lines such as HepG2 and MCF7, which do not express GST n at detectable levels, suggests that control of GSTn gene transcription may involve a repression mechanism. Silencers have been found in many genes, such as the rat insulin I gene (Laimins etal., 1986), the rat a-fetoprotein gene (Muglia etal., 1986) and the human retinol binding protein gene (Colantuoni et al., 1987) and especially the rat GST subunit 7 gene (Imagawa et al., 1991 a,b). Rat GST 7-7 is virtually silent in normal liver, but is specifically and strongly expressed during hepatocarcinogenesis (Sato et al., 1984; Suguoka et al., 1985; Pemble et al., 1986b). Therefore, the silencer of the rat GST subunit 7 gene may play a key role of the regulation of the gene in hepatocytes. The combination of a non-tissue-specific promoter and non-tissue-specific enhancer, with a tissue-specific silencer, which only functions in cells where the gene is not expressed, would presumably have the same effect as a

175 Discussion: Regulation of GST % Gene Expression

tissue-specific enhancer activating a gene in appropriate cells. In the case of GST n gene, which has a broad pattern of expression, but is silent in some

cells such as hepatocytes, the former model may suit better.

6.2 Regulation of the GST n Promoter by Retinoic Acid

The study of the repression effect of retinoic acid (RA) on GST n gene expression was triggered by the finding by Chambon and colleagues that the

DNA sequence, required for RA dependent repression, was co-localized with a consensus AP1 binding site, which is essential for the promoter activity of rat stromelysin gene (Nicholson etal., 1990). In this thesis, there is evidence that RA represses both the transient expression of CAT gene driven by GST 7t promoter and the endogenous GST n gene expression. Since the GST % promoter fragment used contains an AP1 binding site in addition to two G/C boxes and a TATA box, it is reasonable to suspect that the AP1 binding site is associated with the RA mediated repression.

As stated above, the AP1 binding site of GST k is essential for the basal level activity of the promoter. Thus it appears that RAR-RA complex may repress the transcription of GST tc gene by blocking activation by a positive regulatory factor which binds to the consensus AP1 binding site. Evidence provided by Schule et al. (1990) indicated that jun/fos and the receptor of retinoic acid (RAR) recognize a common response element in the human osteocalcin gene which contains arjAPI (TGACTCA) as its core sequence. Transfection of RAR expression vectors in cultured rat osteosarcoma cells activate heterologous promoters containing this sequence. On the other hand, co-transfection of jun and fos expression vectors suppresses the basal level transcription of osteocalcin gene and the induction by RA.

been Co-localization of regulatory elements has also^observed in other genes. For example, the co-localization of DNA elements required for glucocorticoid

176 Discussion: Regulation of GST % Gene Expression

dependent repression has been reported for TPA induction of proliferin gene expression (Mordocq & Linzer 1989), for cAMP induction of the glycoprotein hormone a-subunit gene (Akerblom et al., 1988) and for induction of the bovine prolactin gene by undefined activating factors (Sakai etal., 1988b).

RA exerts its biological effect by acting through at least two distinct classes of intracellular proteins including the RA receptors (RAR a, 8 and Y) and the retinoid X receptors ( RXRs ), both of which are members of the steroid receptor superfamily. RARs and RXRs, however, differ substantially in primary structures and in their response to synthetic retinoids indicating the existence of distinct additional regulatory networks through which RA may exert its biological effects. It is interesting to note that, unlike hRARB, cRXR, the retinoid X receptor from chicken, failed to mediate the repression of GST n gene expression by RA (Xia et al., data not shown). This indicates a significant difference between the two regulatory systems through which RA

effect gene transcriptions. Interestingly,Schule et al. (1991) has reported a similar phenomenon. All three members of the RAR subfamily (a, 8 andY) can down-regulate the transcriptional activation by c-jun of human collagenase gene or heterologous promoter containingthe collagenase promoter AP1 binding site, whereas the RXR fails to repress c-jun activation.

The repression mechanism is not clear. Gel mobility-shift has failed to demonstrate the binding of RAR-RA complex to the AP1 binding site of both rat stromelysin gene (Nicholson et al., 1990) and human collagenase gene (Schule et al., 1991). On the other hand, the DNA binding domain of RAR has been demonstrated to be essential to confer RA mediated repression

(Schule et al., 1991). It appears that RAR inhibits jun/fos or similar proteins binding to DNA by direct protein/protein interaction between RAR-RA complex and jun/fos since in gel shift assay, addition of RAR-RA complex can inhibit the binding of jun/fos to the AP1 site of human stromelysin gene.

177 Discussion: Regulation of GST n Gene Expression

Whatever the mechanisms are, the possibility that the co-localization of the sequence required for RAR-RA dependent repression with a consensus AP1 binding site, which is essential for the basal level expression of GST n gene

is very interesting indeed. GST k has been demonstrated to be elevated in many tumours and RA is known to inhibit cell growth and induce

differentiation. In some case, RA has been shown to be an effective therapeutic agent in treating certain human cancer. Therefore, further

exploiting the mechanism by which RA represses the GST n via the consensus AP1 binding site will presumably open up an approach to understand the mechanisms which lead^the over-expression of GST n in tumours and the potential linkage between the ability of RA to trigger cell differentiation and to block malignant progression.

6.3 Intron 1 and GST n Promoter Activity

•f Studies have shown that in each cell type, regardless^its endogenous GSTrc expression, the transient expression obtained with pCR0.15CAT (-99 to +8 nucleotides) was much lower than that obtained with pSS0.2CAT (-99 to +72 nucleotides) implying that a non-tissue specific transcriptionally active sequence is present at nucleotides +8 to +72. The finding that co-transfection of pSS0.2CAT with pSSO .ICAT (-8 to +72 nucleotides) resulted in CAT expression lower than the co-transfection with pICCAT supports this supposition. Transfection ofpCR0.25CAT, which contains nucleotides -99 to +8 with -8 to +72, placed upstream of -99, yields CAT activities of the same level as that of pCR0.15CAT, suggesting that the downstream c/s-acting sequence present at nucleotides -8 to +72 does not function as a classically defined enhancer, since its function is position and orientation dependent.

The possibility that the downstream c/s-acting sequence may affect the stability of the mRNA of the gene is ruled out by the fact that the level of mRNA from pSS0.2C A Tis about 2.5 fold higher than that from pCRO. 15CAT, and they display a similar half-life. Thus, it can be concluded that this

178 Discussion: Regulation of GST k Gene Expression downstream cis -acting element is integral to the upstream promoter.

Sequence comparison reveals that the rat GST subunit 7 and human GSTrc gene are 73 % conserved in the first 26 basepairs of intron 1, but there is little homology in exon 1 and in the remainder of intron t and other introns (Fig.

6.1). Interestingly, there is a G/C box motif (CCGCCC, the consensus SP1 binding site) positioned in the rat GST subunit 7 intron 1 between +83 to +88, while in human GST n intron 1, there is a G/C box-like motif (CGGCCC) located at the similar intron position from +46 to +51. However, the implication of this is yet to be elucidated. It will be interesting to see whether the corresponding part of intron 1 of rat GST subunit 7 gene has similar effect on its promoter. Preliminary gel mobility assays and co-transfection experiments have demonstrated that the fragment, +8 to +72, does bind to proteins or trans-acting factors both in vivo and in vitro. To further study the regulation of GST n gene, it is essential to identify these trans-acting factors.

A similar phenomenon has been observed in the human platelet derived growth factor-B (PDGF-B) or c-sis gene, where a cell specific positive regulatory activity within the first intron is position and orientation-dependent (Franklin eta!., 1991). Another example is that of the human smooth muscle (aortic type) a-actin-encoding gene, where the promoter region and a part of the first intron shows remarkable sequence conservation with the equivalent region of the chicken gene. In this case, the conserved region of the first intron also has a positive regulatory effect on its promoter (Nakano et al., 1991). A third example is that of a negative cis-acting element found in exon 1 of both the rat and the human myc genes (Bently & Groudion, 1986; 1988).

179 Discussion: Regulation of GST k Gene Expression

-10 0 -80 AP1 -60 G/C

n TCCGCGGGACCCTCCAGAAGAGCGGCCGGCGCCGTGACTCAGCA-CTGGGG 7 C -ATT ------CT TGT T C

G/C . TATA -2.0 . + 1 Jt CGGAGCGGGGCGGGACCACCCTTATAAGGCTCGGAGGCCGCGAGGCCTTCGC 7 G ------CATGC - C TCT G TAT

+ 20 + 3 0

71 TGGAGTTTCGCCGCCGCAG— TCTTC---- GCCACC------7 C — C - GT T - ATA ATCGT GCAGCTTTGAGTCCACA

+ 4 0 + 6 0 I------8------*------8 K ------AGTGAGTACGCGCGGCCCGCG-TCCCCGGGGA 7 CCTCTGTCTACGCAGCAGCT | A T_____ C____ AGCT______ACT

+ 80 Intron 1 + 1 0 0 • • • « ■ --- K TGGGGCTCAGAGCTCCCAGCATGGGGCCAACCCGCAGCATCAGGCCCGGGCT 7 ACCA TGC T TG GGCCT G TCT T C GGGCA TTGCAC A

Fig. 6.1 Comparison of the Promoter Region, Exon 1 and Intron 1 of the Human GST n and the Rat Subunit 7 Genes

Top sequence, human GST n gene; bottom sequence, rat subunit 7 gene. Only the bases which differ from those of human gene are shown for the rat gene. Gaps are introduced for maximum alignment. Numbering refers to the nucleotide position relative to the transcription initiation site (No.

1) of the GST k gene. The TATA box, G/C box core motifs, consensus AP1 binding site and intron 1 are boxed.

180 Discussion: Regulation of GST n Gene Expression

6.4 Regulation of the GST n Promoter by Insulin

In this thesis, it is demonstrated that insulin activatesthe GST n promoter by 3 to 5 fold. This finding is further supported by the observation that the mRNA of GST n in SVK14 cells is elevated after treatment by insulin. Carnovale etal. (1990) have established a direct relationship between plasma insulin level and GST activity towards CDNB in the male Wistar rat. Using Western and Northern blots, Hatayama etal. (1991) have demonstrated that levels of both the protein and mRNA of GST subunit 7, which are apparently undetectable in fleshly isolated hepatocytes, are remarkably induced when the primary cultured rat hepatocytes are treated with insulin.

Insulin has been shown to regulate genes which encode proteins involved in a variety of biological phenomena. Some of these genes code for not only proteins that have a well established metabolic connection with insulin, such as pyruvate kinase (Vaulont et al., 1986), glyceraldehyde-3-phosphate dehydrogenase (Nasrin et al., 1990) and growth hormone (Yamashita & Melmed, 1986), but also others such as c-fos (Stumpo & Blackshear, 1986) and c-jun (Mohn et al., 1990). Insulin initiates its action by binding to a specific cell surface receptor that possesses an intrinsic tyrosine kinase activity which is stimulated by insulin binding. It is evident that this enzyme plays a crucial role in mediating insulin action, however, its physiologically important substrates have not been identified (O’Brien & Granner, 1991). Therefore, the mechanism by which insulin regulates gene expression is under intensive investigation. Traditionally, it has been divided into three classes of effect: namely immediate metabolic effects, such as glucose metabolism; developmental effects, such as differentiation of keratinocytes; and growth effects, such as its requirement in the growth of cells in culture. Based on the fact that insulin transcriptionally inducesc-fos, it has been speculated that the stimulation by insulin ofc-fos gene expression may mediate insulin’s effect on

181 Discussion: Regulation of GST % Gene Expression

the expression of other genes (Stumpo & Blackshear, 1986). Recently, Burgering et al. (1991) have proposed that p21ras is an intermediate of the insulin signal transduction pathway involved in the regulation of the expression

of some genes, such as c-jun and c-fos , but not others such as phosphatidylinositol-3-kinase (PI-3-K). These two hypotheses suggest that insulin induction of genes which respond to jun/fos may be mediated by p21ras. The activation of p21ras could explain the growth factor effect of insulin, and this could also explain the regulation of rat GST subunit 7 by insulin in primary cultured rat hepatocytes, as the expression of both c-jun and c-fos mRNA were co-induced with GST subunit 7 mRNA by insulin treatment (Hatayama et al., 1991). In addition, GST subunit 7 gene expression has been demonstrated to responde to TPA or fos/jun (Sakai et al., 1988a). However, this hypothesis apfDrantly does not apply to that of GST n, which responds to neither TPA nor fos/jun (Dixon etal., 1989; Morrow etal., 1990).

Other approaches have been adopted to interpret the mechanism involved in insulin regulation of gene expression. Comparison of some of the sequences, which have been demonstrated to mediate the response of the gene expression to insulin, has revealed a common core motif TGG(T)TG(T)TTT(C)TG (O.Brien & Granner, 1991). However, whether this is a coincidence or a key to insulin action remains to be established. On the other hand, Nasrin et al. (1990) compared the minimal sequence contacted by the IRE-A binding protein of glyceraldehyde-3-phosphate dehydrogenase gene "CCCGCCTC" to the sequence from other genes, which were reported to be regulated by insulin, and showed that these eight bases are perfectly conserved in the 5’ flanking region of several genes including c-fos , c-myc and genes encoding phosphoribosyl transferase, glycerophosphate dehydrogenase

and p33. Proteins which bind to these two sequences, however, have not yet been identified.

There is little homology between the motif proposed by O ’Brien & Granner

182 Discussion: Regulation of GSTk Gene Expression

(1991) and the nucleotides of the 171 bp fragment, which confer the inducibility of insulin on GST n. However, a sequence CCCGCGTC, which differs only in one base compared with CCCGCCTC proposed by Nasrin et al. (1990), is located in the intron 1 between +49 to +55 of the GST n gene. This may imply that the intronic sequence of GST n is involved in mediating insulin induction of the gene. This finding is also in line with the observation that the CAT activities resulted from pSS0.2CAT were 3.9 fold over thexof pCRO. 15CAT in MCF7 cells cultured in full medium, and the CAT activity of pSS0.2CAT in MCF7 cells cultured serum containing insulin were 3 to 5~folM heh4'r -than that cultured in serum free medium.

The mechanism involved in the regulation of GST n by insulin is far from being understood. Nevertheless, the fact that the 171 base pair fragment of GST n confers the responsiveness to insulin induction of the gene opensup an approach to reveal at least one of the insulin signal transduction pathways leading to the regulation of gene expression.

183 APPENDIX

184 Appendix

1 10 MET-Pro-Pro-Tyr-Thr-Val-Val-Tyr-Phe-Pro-Val-Arg-Gly-Arg- 20- Cys-Ala-Ala-Leu-Arg-Met-Lue-Lue-Ala-Asp-Gln-Gly-Gln-Ser- 30 40 Trp-Lys-Glu-Glu-Val-Val-Thr-Val-Glu-Thr-Trp-Gln-Glu-Gly- 50 Ser-Leu-Lys-Ala-Ser-Cys-Leu-Tyr-Gly-Gln-Leu-Pro-Lys-Phe- 60 Gln-Asp-Gly-Asp-Leu-Thr-Leu-Tyr-Gln-Ser-Asn-Thr-Ile-Leu- 70 80 Arg-His-Leu-Gly-Arg-Thr-Leu-Gly-Leu-Tyr-Gly-Lys-Asp-Gln- 90 Gln-Glu-Ala-Ala-Leu-Val-Asp-Met-Val-Asn-Asp-Gly-Val-Glu- 100 110 Asp-Leu-Arg-Cys-Lys-Tyr-Ile-Ser-Leu-Ile-Tyr-Thr-Asn-Tyr- 120 Glu-Ala-Gly-Lys-Asp-Asp-Tyr-Val-Lys-Ala-Leu-Pro-Gly-Gln- 130 Leu-Lys-Pro-Phe-Glu-Tyr-Leu-Leu-Ser-Gln-Asn-Gln-Gly-Gly- 140 150 Lys-Thr-Phe-Ile-Val-Gly-Asp-Gln-Ile-Ser-Phe-Ala-Asp-Tyr- 160 Asn-Leu-Leu-Asp-Leu-Leu-Leu-Ile-His-Glu-Val-Leu-Ala-Pro- 170 180 Gly-Cys-Leu-Asp-Ala-Phe-Pro-Leu-Leu-Ser-Ala-Tyr-Val-Gly- 190 Arg-Leu-Ser-Ala-Arg-Pro-Lys-Leu-Lys-Ala-Phe-Leu-Ala-Ser- 200 Pro-Glu-Tyr-Val-Asn-Leu-Pro-Ile-Asn-Gly-Asn-Gly-Lys-Gln

Appendix A. The Primary Sequence of GST n subunit

185 Appendix

Pst I (2824) Pvu I (2000) A(n)

Pst I (1866)

Amp

Ori

Nde I Transcript (554) Sst I (5005/0) Sph I (198) AUG

CAT 72 21 A/T

Appendix B. The Structure of pSV2CAT pSV2CAT contains the bacterial chloramphenicol acetyl transferase (CAT) coding region, which is under the control of the SV40 viral early promoter/enhancer, in a pBR322 background including the ampiciilin resistance gene (ampr) and origin of replication. The filled section represents the SV40 early promoter region, shown enlarged bel ow (72, 21 and A/T refer to the 72 bp repeats of the enhancer, the 21 bp repeats and the A/T rich region of the SV40 early promoter respectively). The heavily stippled section (c), corresponds to the CAT coding region; the lightly stippled section (i), represents the SV40 small t intron; and open section (p) represents the polyadenylation site containing regions. The ampiciilin resistance gene (ampr) and the plasmid origin of replication (ori) are indicated.

186 Appendix

Pvu I Pst I

Pst I Amp

Orx

AUG

CAT Hind III Xba I Sma I Sal I BamH I Sst

Appendix C. The Structure of pIC C A T pIC C A Twas derived from pSV2CAT. The SV40 early promoter containing Acc I/Hind III fragment was excised and replaced with a multicloning polylinker from pUC13 (shown as a shaded box and is enlarged below).

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212 LIST OF PUBLICATIONSAND PRESENTATIONS

1. Xia, C .L & Chen, H .L (1989) Purification and Identification of Human Placental Glutathione Transferase (GST n) Acta. Biochem. Biophys. Sinica. 21, 57-64.

2. Xia, C .L & Chen, H .L (1989) Kinetics and Inhibition Studies of Human Placental Glutathione Transferase (GST n) Chinese Biochem. J. 5, 294-300.

3. Xia, C .L & Chen, H .L (1989) Allosteric Effect of Bilirubin on Human Placental Glutathione Transferase (GST n) Chinese Biochem. J. 5, 301-307.

4. Dixon, K.H., Cowell, I.G., Xia, C .L , Pemble, S.E., Ketterer, B. & Taylor, J.B. (1989) Control of Expression of the Glutathione S- Transferase % Gene Differs from the Rat Orthologue Biochem. Biophys. Res. Commun. 163, 815-822.

5. Xia, C .L , Cowell, I.G., Dixon, K.H., Pemble, S.E., Ketterer, B. & Taylor, J.B. (1991) Glutathione Transferase n: Its Minimal Promoter and Downstream c/'s-Acting Element. Biochem. Biophys. Res. Commun. 176, 233-240.

6. Xia, C .L , Meyer, D.J., Ketterer, B., Chen H .L , Rienemer, P. & Huber, R. Chemcal Modification of Human Glutathione Transferase P1-1. Eur. J. Biochem. Submitted.

7. Xia, C .L & Chen, H .L(1989) The Allosteric Effect of Bilirubin on Glutathione Transferase n. The Third International Conference on Glutathione Transferase and Drug Resistance.

8. Xia, C .L , Meyer, D.J., Ketterer, B. & Chen, H .L (1989) The Involvement of 1 Cysteine, 1 Lysine, 1 Histidine, 1 Arginine and 1 Carboxyl Residue in the Active-site of GST n. The Third International Conference on Glutathione Transferase and Drug Resistance.

9. Xia, C .L, Cowell, I.G., Dixon, K.H., Pemble, S.E., Ketterer, B. & Taylor, J.B. (1991) Glutathione Transferase n: Its Minimal Promoter and Downstream c/'s-Acting Element. Proc. Am. Asso. Cancer Res. 32, 11.

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