LIBRARY Mich‘QEIn State University PLACE IN RETURN BOX to remove this checkout from your record. TO AVOID FINES return on or before date due. MTE DUE DATE DUE DATE DUE 1m mumms—nu INCREASED MULTIDRUG RESISTANCE IN ADRIAMYCIN SENSITIVE MCF-7 BREAST TUMOR CELLS OVEREXPRESSING HUMAN PLACENTAL THIOLTRANSFERASE By Elizabeta Borer Meyer A DISSERTATION Submitted to Michigan State University in partial fiJlfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Biochemistry Department of Environmental Toxicology 1998 ABSTRACT INCREASED MULTIDRUG RESISTANCE IN ADRIAMYCIN SENSITIVE MCF-7 BREAST TUMOR CELLS OVEREXPRESSING HUMAN PLACENTAL THIOLTRANSFERASE By Elizabeta Borer Meyer Human placental thioltmnsferase (hpTT) cDNA was isolated including its 5'- and 3'- untranslated regions. The nucleotide sequence of the cDNA and the deduced amino acid sequence agreed with those of cDNAs isolated simultaneously from other human tissues. The deduced amino acid sequence in general is highly conserved throughout mammalian species. Human 'I'I‘ differs fi'om other mammalian Us by lacking an internal methionine residue and the presence of a cysteine rather than serine at residue 7. The recombinant hpTT cDNA was overexpressed in E. coli and purified; both the dithiol-disulfide oxidoreductase and DHA reductase activities were comparable to that of purified native erythrocyte TT. The purified recombinant hpTT also cross-reacted with antibodies generated against the purified human erythrocyte TT or the recombinant pig liver TT protein. To determine whether elevated levels of “IT would increase resistance to Adriamycin, MCF-7 WT breast tumor cells were transfected with the TT cDNA in an expression plasmid. Several stably transfected MCF-7 WT cell lines were established that constitutively overexpressed TT 9-66 fold and had 2 to lO-fold elevated resistance to Adriamycin, indicating a correlation between TT levels and drug resistance. In addition, Adn'amycin resistance in both MCF-7 WT and transfected MCF-7 WT cells was independent of L- ascorbate 2-phosphate, indicating that increased Adriamycin resistance is not related to DHA reduction. An alternate TT activity such as protein mixed disulfide:GSH exchange may contribute to the elevation in drug resistance. Dedicated to my mother, Dr. Katarina T. Borer, who was my inspiration and role model. iv ACKNOWLEDGMENT S At Michigan State University, I found faculty, stafi, and Students generous with time, reagents, and advice. First, I would like to thank the members of my doctoral committee, past and present, including Dr. Zachary Burton, Dr. David Dewitt, Dr. William Helferich, Dr. Lee Kroos, and Dr. William Smith. Without your assistance, this project would never have been completed. Secondly, I would like to thank Brian Smith-White for cloning assistance and the LE3 92 strain; Dr. Stacey Kraemer for RT-PCR assistance; Pappan for patient instruction assembling this document; and Dr.Burton for the pET strain. Special thanks go to fiiends that maintained my sanity and spirit, including Stacey, Marty Regier, Claire Vielle, Carol McCutcheon, Doug Weisner, Anandita, Carol Mindock, Bao-J en Shyong, Michelle Anderson, Laura Pence, Barb Harnd, John Boyse, Rev. Bill Dobbs, the Wells lab postdocs and students; Aaron, Anita, Melissa, Lori, Vicky, and Vivek, as well as Rajashree Krishnaswamy, DianPeng Xu, Leslie Dybas, Mike Washburn, and Chungle Dou. A large thank you to all the members of my family, my grandmother Borka Tomljenovic; my mother, Dr. Katarina Borer and her husband, Dr. Paul Wenger; my “little” brothers, Robert and Richard, and their spouses, Sherri and Laurie; my father, Dr. Robert C. Borer, Jr., and his wife, Kathryn. I love you all, and cannot possibly describe what your support has meant to me. To my mentor, Dr. William Wells; I will never forget your patience with headstrong students, and enthusiasm for research And the final thank you to my husband, Chris, and children, Nicholas and Christina, who made everything worthwhile. TABLE OF CONTENTS LIST OF TABLES .................................................. ix LIST OF FIGURES ................................................... x ABBREVIATIONS ................................................. xiii CHAPTER 1: LITERATURE REVIEW 1. Cellular Oxidative Stress ....................................... 1 II. Ascorbate ................................................. 13 A. DHA Reductases ...................................... 19 B. SDHA Reductases ..................................... 20 III. Glutathione ............................................... 21 IV. Thiol-disulfide Oxidoreductases ................................. 26 Protein Disulfide Isomerase ............................... 28 Thioredoxin ............................................ 35 Thioltransferase .......................................... 47 Postulated physiological roles for TT .......................... 54 V. Adriamycin ................................................ 61 VI. Drug Resistance ............................................ 65 VII. MCF-7 Breast Tumor Cells .................................... 72 CHAPTER 2: HUMAN TT SEQUENCE AND DISTRIBUTION Introduction .................................................. 79 Materials .................................................... 80 Methods ..................................................... 80 Pig liver TT probe generation ............................... 80 Southern analysis of human genomic DNA ..................... 8] Screening the human placental cDNA library ................... 81 Subcloning and sequencing the TT cDNA ...................... 83 Human erythrocyte TT purification ........................... 91 Protein assay ........................................... 92 TT activity assay ........................................ 92 DHA Reductase activity assay .............................. 93 Rabbit anti-TT antibody generation ........................... 93 SDS-PAGE and Western analysis of purified erythrocyte TT vi Western blots of pooled human tissues ........................ 94 HpTT probe generation ................................... 95 Northern blots of pooled human tissues ....................... 95 Results ...................................................... 97 Cloning the hpTT cDNA .................................. 97 Human genomic Southern analysis ........................... 97 Human erythrocyte TT purification ........................... 98 Anti-human erythrocyte TT antibody generation ................. 98 Tissue distribution of TT ................................. 110 Discussion .................................................. 11 1 CHAPTER 3: EXPRESSION AND PURIFICATION OF RECOMBINANT HPTT Introduction ................................................. 113 Materials ................................................... 113 Methods .................................................... 114 Construction of the recombinant hpTT expression vector ......... 114 Overexpression and purification of hpTT in E. coli .............. 1 15 Recombinant Purification l .......................... 1 15 Recombinant Purification 2 .......................... 118 SD S-PAGE and irnmunoblotting analysis ..................... 119 TT Activity Assays ...................................... 120 DHA Reductase Assays .................................. 120 Protein Assays ......................................... 121 Results ..................................................... 121 Discussion .................................................. 126 CHAPTER 4: INCREASED RESISTANCE TO ADRIAMYCIN IN MCF-7 BREAST TUMOR CELLS CONSTITUTIVELY OVEREXPRESSING hpTT Introduction ................................................. 128 Methods .................................................... 129 Culture of MCF-7 breast tumor cells ......................... 129 Partial fiactionation of cell extracts .......................... 129 Protein Assays ......................................... 130 SDS-PAGE and Western Blot Analysis ....................... 130 PCR amplification of the hpTT cDNA ....................... 131 Construction of the constitutive hpTT expression vector .......... 131 G-418 Dose-Response in MCF-7 cells ....................... 134 Transient transfection of MCF-7 cells with a luciferase expression plasmid ................................ 13 5 Stable transfection of MCF-7 cells with a TT overexpression plasmid ......................................... 13 7 Characterization of cell lines that constitutively overexpress hpTT .......................................... 13 8 vii RNA Isolation .................................... 138 RT-PCR ........................................ 138 TT Activity Assays ................................ 139 Adriarnycin Cytotoxicity Assays ............................ 140 Results ..................................................... I41 6-418 Dose-Response ................................... 141 Western Blot Analysis ................................... 141 Transient transfection .................................... 142 Constitutive stable cell lines ............................... 142 RT-PCR .............................................. 142 TT activity ............................................ 142 Adriarnycin Cytotoxicity .................................. 146 Discussion .................................................. 146 CONCLUSION .................................................... 1 53 BIBLIOGRAPHY .................................................. 157 Table 1 Table 2 Table 3 Table 4. LIST OF TABLES Relative DHA reductase activity ............................. 22 Redox Potentials of TDOR enzymes .......................... 34 MCF-7 WT and ADRR difl‘erences ........................... 78 Human erythrocyte TT purification .......................... 104 ix Figure 1. Figure 2. Figure 3. Figure 4. Figure 5. Figure 6. Figure 7. Figure 8. Figure 9. Figure 10. Figure 11. Figure 12. Figure 13. Figure 14. Figure 15. Figure 16. Figure 17. LIST OF FIGURES Structures of antioxidants .................................. 8 Oxidation-reduction relationships of AA, DHA and SDHA in a model cell ................................................... 10 Phase I and Phase II detoxification reactions .................... 14 AA synthesis ........................................... 17 Proposed mechanism of DHA reduction ....................... 23 GSH synthesis, functions, and the y-glutamyl cycle ............... 27 Predicted human TT structure .............................. 31 TDOR enzyme folding similarities ............................ 33 PDI primary structure ...................................... 36 PDI oxidoreduction ....................................... 38 TRX oxidoreduction ....................................... 40 TI amino acid sequence comparisons ......................... 51 TT dithiol—disulfide transfer mechanism ....................... 53 TT and TGF-B amino acid sequence similarities ................. 6O Adriarnycin structure ..................................... 63 GSSG reductase, GSH, and G6PDH interrelationships ............ 75 Generation of 423 bp pig liver TT cDNA probe ................ 82 Figure 18. Figure 19. Figure 20. Figure 21. Figure 22. Figure 23. Figure 24. Figure 25. Figure 26. Figure 27. Figure 28. Figure 29. Figure 30. Figure 31. Figure 32. Figure 33. Figure 34. Figure 35. Figure 36. Figure 37. Figure 38. Agtll structure and PCR amplification of cDNA inserts .......... Construction of pCR4 .................................... hpTT cDNA nucleotide and deduced amino acid sequence ......... Generation of the 425 bp hpTT probe ......................... EcoRI digestion of Agtll hpTT cDNA clones ................... Southern analysis of human genomic DNA .................... Thiopropyl sepharose 6B affinity purification of human erythrocyte TT oooooooooooooooooooooooooooooooooooooooooooooooooo SDS-PAGE and Western blot analysis of purified human erythrocyte TT .................................................. Human tissue Northern analysis for TT content ................ Human tissue Western analysis for TT content ................. Construction of pEThTT ................................. Time course of hpTT overexpression in E. coli ................. G-75 purification of recombinant hpTT ....................... SDS-PAGE and Western analysis of purified recombinant hpTT . . . . Construction of pTTeu ................................... Construction of pTTcmv ................................. G-418 Dose-response .................................... Western analysis of TT content in MCF-7 extracts .............. Transient transfection of MCF-7 WT and ADRR cells ............ TT activity in MCF-7 WT, ADR“, and transfected WT cells ....... Adriamycin cytotoxicity in MCF-7 WT, ADRR, and transfected WT cells ................................................. 86 88 9O 96 99 101 107 109 117 122 124 125 133 136 143 144 145 147 149 Figure 39. Effect of AAP on Adriarnycin cytotoxicity in MCF-7 WT, ADRR, and transfected WT cells ..................................... 150 xii ABC ADF AP-l ATP BCIP BSO CAMP cDNA CMV DEPC DHA DMSO DNA DTT EDTA EtBr FCS FSH G1 G41 8 G6P G6PDH GAPDH GSH GSH-PX GSSG GST HEPES hpTT IPTG kDa ABBREVIATIONS ascorbic acid L-ascorbate-Z-phosphate ATP-binding cassette protein adult T-cell derived leukemic factor activator protein-l adenosine triphosphate bis-4-chloro-indolyl phosphate L-buthionine-(S,R)—sulfoximine cyclic adenosine 5'-3'-monophosphate complementary DNA cytomegalovirus diethylpyrocarbonate dehydroascorbate dirnethylsulfoxide deoxyribonucleic acid dithiothreitol ethylenediarninetetraacetic acid endoplasmic reticulum ethidium bromide fetal calf serum follicle stimulating hormone growth arrested phase of cell cycle growth phase 1 of cell cycle Geneticin, arninoglycoside antibiotic glucose 6-phosphate glucose 6-phosphate dehydrogenase glyceraldehyde-3 -phosphate dehydrogenase glutathione glutathione peroxidase glutathione disulfide glutathione S-transferase N-(2-hydroxyethyl) piperazine-N’-(2-ethanesulfonic acid) human placental thioltransferase iso-l-thio-B-D—galactoside kilodalton Michaelis-Menten constant lambda bacteriophage xiii LH LI),o LTR MCF-7 WT MCF-7 ADRR MDR ITIWCO Tris leutinizing hormone lethal dose 50 long terminal repeat Michigan Cancer Foundation wild-type breast tumor cell line Michigan Cancer Foundation Adriamycin-resistant cell line multi-drug resistant mouse mammary tumor virus molecular weight cutofl' nicotinamide dinucleotide, reduced nicotinarnide dinucleotide phosphate, reduced nitro blue tetrazolium nuclear factor kappa B nuclear magnetic resonance polyacrylamide gel electrophoresis phosphate bufi‘ered saline polymerase chain reaction Protein disulfide isomerase plaque-forming units P-170 glycoprotein protein kinase C phenylmethylsulfonylfluoride reduction-oxidation ribonucleic acid reactive oxygen species Rosewell Park Memorial Institute reverse-transcriptase polymerase chain reaction DNA synthesis phase of cell cycle semi-dehydroascorbate sodium.dodecyl sulfate superoxide dismutase tris bufi‘ered saline thiol-disulfide oxidoreductase transforming growth factor-8 tumor necrosis factor-a 12-O-tetradecanoyl 13-acetate Tris-hydroxymethylaminomethane thioredoxin thioltransferase maximal enzyme velocity 5-bromo—4~chloro-3 -indolyl-B-D-galactoside xiv CHAPTER ONE LITERATURE REVIEW To avoid redundancy, this review is presented in seven major sections: 1. Cellular oxidative stress; 11. Ascorbate and related reductases; III. Glutathione and related enzymes; IV. Thiol-disulfide oxidoreductases and their postulated cellular fiinctions, including thioltransferase (TI, glutaredoxin), the enzyme of particular research interest; V. Adriarnycin, the antitumor drug of interest; VI. Drug resistance; and VII. MCF-7 cells, that provide the model system used. Extensive reviews already are available for each area; the discussion here is of details germane to the research question. The details presented here are designed to give the reader an understanding of reactive oxygen species (ROS) involved in Adriarnycin damage; the roles of various enzymes that detoxify these ROS in drug resistance, especially enzymes that are structurally or mechanistically related to TT; changes in these enzyme activities in the MCF-7 WT and ADRR cells that constitute the model system; and the roles of ascorbic acid (AA) and glutathione (GSH) in detoxification, particularly with respect to thioltransferase (TT). I. Cellular Oxidative Stress Cellular oxidative stress is modulated by the presence of many biological antioxidants and enzymes, many of which are altered in drug resistance. Responses to oxidative stress afi‘ect resistance to antitumor drugs that generate toxic oxygen radical species. Detailed here 2 for the reader are many of the mechanisms whereby R08 are generated in the cell, and an overview of cellular detoxification systems, particularly those changed in drug resistance, is presented. In recent years, there has been an increasing interest in oxygen fiee radicals and their reactions, especially with respect to biological systems. Mammalian cells are continuously subjected to ROS such as hydrogen peroxide (HZOZ), superoxide (02"), and hydroxyl radicals ('OH). When cellular mechanisms which detoxify ROS are exceeded, cytotoxic lipid peroxidation, nitroperoxidation, protein oxidation, DNA damage and cell lysis result. ROS play a role in inflammation, rheumatoid arthritis, atherosclerosis, hepatic diseases, aging, mutagenesis, chemotherapy, and xenobiotic metabolism (reviewed in 1). Cellular reduction- oxidation balance regulating the effects of ROS is a "housekeeping" function of the cell, as changes in oxidant or redth levels in the cell affect biosynthetic reactions, including activities of proteins regulating metabolism (Section IV); expression of cytokines (2), adhesion molecules (3), and protooncogenes (4); and the activities of several transcription factors (Section IV). Free radicals are extremely reactive molecules containing an unpaired electron in molecular orbitals. These radicals are produced in cells through a wide variety of reactions and external influences. Homolytic fission of covalent bonds results in two radical products. In biological systems, ROS result most commonly from reduction-oxidation (redox) reactions catalyzed by transition metals or enzymes and aerobic metabolism. F120;2 is generated in cells through several mechanisms. Dismutation of 02' generates H202 [1] (5)- [1] 20,- + 2H“ —+ H202 + 02 Hydrogen peroxide is also generated through mixed-function oxidase enzymic reactions such as that of D-arnino acid oxidase and urate oxidase (6). Activated neutrophils undergo a respiratory burst releasing Oz" and other noxious substances to destroy invading organisms (7). Electrons passing down electron transport chains can react with O2 rather than cytochrome c oxidase, producing 02" (8). Enzymatic reactions can generate Oz". Superoxide is generated during the metabolism of arachidonate to eicosanoids by lipoxygenases and cyclooxygenases (9-12). Xanthine oxidase (EC. 1.1.3.22), produces 02" during the oxidation of xanthine to uric acid (13). 02" can also be generated through redox cycling of quinones which are structural components of many chemotherapeutic agents (see Section V). At low pH, the superoxide radical is protonated (HOZ'). Superoxide and hydrogen peroxide generate the hydroxyl radical ('OH) through a metal-catalyzed Haber-Weiss reaction [2] (14). Fe2+ [2] o," + H202 _. 02 + 'OH + orr Free radical Fenton reactions (15) can also be initiated if Fe2+ comes into contact with hydrogen peroxide [3]. [3] Fe” + H20, —’ Fe” + 'OH + OH' Species such as H202 and 02" are not highly reactive, and may diffuse from generation 4 sites. H202 can cross membranes, thereby affecting more than one cellular compartment, where 0," cannot. H102 is a weak physiological oxidant and inactivates some enzymes such as glyceraldehyde—3-phosphate dehydrogenase (GAPDH) and fructose 1,6-bisphosphatase by oxidation of essential thiol groups (16). Oz" exerts toxic efi‘ects when converted to more reactive species such as 'OH and HOZ'. HOz' is a stronger oxidant than 02" and will directly attack polyunsaturated fatty acids, oxidizing membranes (17). Hydroxyl radicals are highly reactive, immediately interacting with biomolecules in the vicinity (18), often resulting in damage to cellular components. Hydroxyl radical attack on deoxyribose and nucleotide ring structures damages DNA, resulting in DNA strand breakage (19). If excessive, this strand breakage can lead to NAD“ depletion (20), since the adenosine diphosphate moiety in NAD+ is removed and placed on the damaged DNA by poly(ADP-ribose) as a repair signal. Membrane damage results fi'om ‘OH abstraction of a H atom from bilayer lipids leaving lipid radicals. These radical lipids react with molecular oxygen to form peroxyradicals, and continue chain oxidation reactions (21), resulting in significant membrane damage (22). Amino acid oxidation, decarboxylation, deamination, and inappropriate cleavage or cross-linking are results of radical-induced damage in proteins. Cysteine thiols can be oxidized to stable disulfides or sulfenic (SOH), sulfinic (S02), and sulfonic (803.) groups. Free radical damage to proteins is ofien irreversible, and can lead to inactivation and unfolding (reviewed in 23). Evidence strongly suggests that proteins damaged under oxidative conditions are insuficiently catabolized, and accumulate in the cell (24-27), leading to aging-related disease, such as cataracts in eye tissue (28). Biological antioxidants such as vitamins C (ascorbic acid, AA, Section II) and E (or- Tocopherol), glutathione (GSH, Section III), carotenoids, a-lipoic acid, and uric acid protect 5 cells fi'om ROS toxicity as do cellular enzyme systems including superoxide dismutase (SOD) (29), catalase (30), glutathione peroxidase (GSH-PX) (31,32), peroxyredoxins (previously known as thiol-specific antioxidant) (33), GSH-S-transferases (GSTs) (34), and ascorbate peroxidases (35), as well as thiol-disulfide oxidoreductases (Section IV). a-Tocopherol is membrane-bound, and protects against lipid peroxidation. The one- electron oxidation of a-Tocopherol results in the a—chromanoxyl radical. Cytoplasmic AA can reduce the a-chromanoxyl radical, recycling a-Tocopherol at the surface of biological membranes (3 6-3 8). AA is probably the most efl‘ective and least toxic antioxidant identified in mammalian cells (39,40). AA reduces semi-quinones, H202, Q", HQ ,' OH,1 9 , thiyl radicals and hypochlorous acid produced at sites of inflammation or during other cellular processes. Comparisons of cellular antioxidant 'OH radical scavenging abilities revealed that AA is superior to GSH and uric acid in scavenging ability (41). AA also proved to be the significant scavenger of membrane fatty acid nitroxide radicals when compared with other cellular detoxification systems such as catalase, GSH, GSH-PX, SOD, and a-tocopherol (42). GSH at high concentrations was able to enhance the AA scavenging ability, and AA was an insignificant radical scavenger when cellular levels fell below 0.1 mM. This suggests that nitroxide radical reduction may be impaired in cells that have low levels of AA, or insuflicient ability to recycle oxidized AA AA radical scavenging activity results in the oxidation products dehydroascorbate (DHA) or semi-dehydroascorbate (SDHA) (Fig.1), which are regenerated by cytoplasmic DHA reductases (Section II A) and membrane-bound NADH- dependent SDHA reductase (Section H B), respectively (Fig.2). GSH (Fig. 1) functions as an antioxidant directly, and as a cofactor or substrate in 6 many reactions. Reactive species reduced by glutathione include free radicals, H202, organic hydroperoxides, epoxides, alkenes, quinones, and aldehydes. One-electron reduction of radicals with GSH yields thiyl radicals; two-electron reductions using two moles of GSH results in glutathione disulfide (GSSG). GSH is a cofactor in several detoxification reactions, either through conjugation with reactive compounds, or as a substrate to reduce oxidized thiols. Many proteins form mixed protein disulfides with GSH under oxidizing conditions. GSH is involved in maintaining cysteine and coenzyme A as well as cellular proteins in their reduced state. Intracellular GSH levels are high (1-10 mM), but oxidative stress can shifi the thiolzdisulfide equilibrium elevating GSSG (43,44). A series of enzymes participate in the removal of ROS from cells. These enzymes are found in diverse cellular locations, as well as in varying levels, depending on the oxidative demands of the particular tissue. Superoxide dismutase (E.C. 1.15.1.6) catalyzes the disrnutation of Oz“ [1], which decreases cellular 'OH. Three types of superoxide dismutases (SOD) are lmown (reviewed in 45). Two are found in humans; a Cu/Zn-containing enzyme, originally termed hepatocuprein or erythrocuprein, and a Mrr-containing SOD. The Cu/Zn SOD is found in cell cytosol, lysosomes, between inner and outer mitochondrial membranes, and in the nucleus, whereas the Mn—containing SOD enzyme is found in the mitochondrial matrix. A third Fe- containing SOD is found in bacteria and plants, but not mammals. Glutathione reductase (46) catalyzes the regeneration of GSH from glutathione disulfide (GSSG) [4], Fig. 1. Structures of antioxidants. A. Ascorbic acid (AA) B. Dehydroascorbic acid (DHA), the two-electron oxidation product of ascorbic acid C. Semi-dehydroascorbic acid (SDHA), the one—electron oxidation product of ascorbic acid D. Glutathione (GSH) O O C2H502 \ HO OH B. \ o ’/ \\o C. Fig. 1. Structures of antioxidants. O O" §C|J/ H—CII—‘H III—H N—H 0:1: in. in. +H3N—CIII—H o¢c\0’ Fig. 2. Oxidation-reduction relationships of AA, DHA, and SDHA in a model cell. Intracellular AA (AAHZ) reacts with free radicals (R'), superoxide fiee radical (H02), hydrogen peroxide (HzOz) and other oxidants. Single-electron oxidation results in SDHA ('AAH), which may disproportionate into DHA and SDHA. ('AAH) also may be reduced back to AA using NADH-dependent SDHA reductase. Intracellular DHA may be recycled by DHA reductases such as IT, utilizing GSH generated by GS SG reductase. AA is transported into the cell through unknown mechanisms, and may difluse out of the cell. Extracellular AA in plasma or extracellular space is oxidized to DHA and transported into the cell through two types of transport mechanisms. Taken from Wells, WW. and Xu, D.-P. (1995) Dehydroascorbate reduction. J. Bioenerget. Biomemb. 26, 369-377, with permission. 10 o~1m+02~ Nonzu OuIN «ONI NON: 8mg 5_:__eo%~xm e ~I<< «5:829... 223:5 La ommuoaumm I ., ofinuoommocchEEow 382.com <10 +I+Io ‘—— n __/W L-gulanolactone H \/CCéO oxidase C I OH 0 OH OH 2-Keto L-gulonolactone L-Ascorbic Add Fig. 4. AA synthesis. AA is synthesized from glucose. L-gulonolactone oxidase catalyzes the formation of 1- gulonolactone fi'om 2-keto-L-gulonolactone, which then is spontaneously converted to AA. Humans, other primates, guinea pigs, fi'uit bats, insects, invertebrates and fishes lack L- gulonolactone oxidase and cannot synthesize AA. 18 Several enzymes have an AA requirement for effective catalysis. AA is required as a cofactor for proline hydroxylase (83,84) and lysine hydroxylase (84), enzymes that participate in collagen biosynthesis. AA is presumably required by the hydroxylases to keep the metal centers in the reduced state, and to protect the enzyme. If AA is deficient then individual collagen fibers are insufliciently hydroxylated, resulting in improperly formed fibers, which ultimately leads to poor wound-healing and fragile vessels. Other enzymes requiring AA as a cofactor are y-butyryl betaine hydroxylase (71), which converts y-butyryl betaine into carnitine, and dopamine-B-hydroxylase (84), which converts dopamine into norepinephrine. Interrelationships between AA and GSH synthetic processes have been demonstrated. Administration of L-buthionine-(SR)-sulfoximine (BSO), a specific 'y-glutamylcysteine synthetase inhibitor (85), results in glutathione deficiency in animals or cells in culture. When BSO-treated animals are supplemented with 2 mM AA/kg/day, mortality decreases and levels of GSH rise. When 2 mM DHA/kg/day is added instead of AA, GSH levels do not change, and mortality increases to 100% (reviewed in 86), indicating that AA efl'ects GSH metabolism. Studies also indicate that one antioxidant level (GSH or AA) increases when levels of the other antioxidant decrease under conditions of oxidative stress (87). When adult mice are GSH-depleted by BSO administration, AA synthesis is elevated, and sufficient to protect the mice fi'om toxic ROS. The AA level first doubles in 4 hours; then AA levels decrease and DHA levels rise. This finding is consistent with the inability to use GSH to recycle DHA [12]. In a related experiment, Vitamin C-depleted (scorbutic) guinea pigs were fed a GSH analog, glutathione monoethyl ester. The untreated scorbutic guinea pigs live 21-24 days, whereas the GSH-analog supplemented guinea pigs survive longer, suggesting that GSH may alter AA l9 metabolism (88). GSH depletion will afl‘ect AA recycling ability [12] and DHA levels will rise, however, AA and DHA levels were not measured under these conditions. GSH is required, at least in part, for AA recycling, in viva. Newborn rats are less capable of AA synthesis than adult rats. GSH-depleted newborn rats had tissue damage and mitochondrial swelling in lung (89), liver (90), brain (89), and lens (91), presumably due to the buildup of H202 produced as a metabolic byproduct of aerobic respiration In addition, intissues ofBSO-treated animals, AAwasdepleted andtheDHApercentage oftotal AAwas higher tlmn in untreated controls. This demonstrated both the in viva need for DHA recycling to AA, and indicated that non—GSH mechanisms of AA recycling are insuficient to regenerate cellular AA A. DHA Reductases Cellular sources of DHA include cytoplasmic radical scavenging by AA (92); SDHA free radical disproportionation (92) [11], and extracellular DHA transported into the cell (76,77, Fig.2). DHA is rapidly reduced to AA in the cell. DHA may be reduced by GSH chemically (61), or by glutathionezdehydroascorbate oxidoreductases (EC. 1.8.5.1)[12]. The extent of catalyzed and uncatalyzed DHA reduction in mammalian systems is unknown. Observations support a strong role for the enzymatic recycling of DHA In viva AA recycling was demonstrated in y-gulono lactone oxidase-deficient species where DHA supplementation prevented scurvy (90). In human lymphocytes and neutrophils, the rate of DHA uptake is proportional to that of cytosolic DHA reductase activities (93). Neutrophils have increases in cytoplasmic AA (up to 14 mM) after activation (75), although external AA stays at physiological levels (50-150 11M). This indicates that extracellular DHA is a source ofAA 20 Several mammalian DHA reductases have been partially or completely characterized since 1990. Two GSH-dependent DHA reductases, PDI and TT (80), also have dithiol- disulfide exchange activity (Section IV). PDI has TRX-like domains responsible for the dithiol-disulfide transfer activity, although TRX does not have DHA reductase activity. Three otha GSH-dependent DHA reductases without dithiol-disulfide oxidoreductase activity have been isolated; an incompletely characterized 31 kDa rat liver protein (94), a 32-kDa human DHA (95) and a 23 kDa spinach chloroplast protein (96). Lipoarnide dehydrogenase catalyzes the NADH-lipoic acid dependent reduction of DHA to AA (97), and 3a-hydroxy steroid dehydrogenase catalyzes the NADPH-dependent reduction of DHA (98). TI is the most eficient DHA reductase characterized to date (Kcat/Km) (Table 1). The mechanism of DHA reduction and whether a flea radical intermediate is required still need to be resolved. Site-directed mutagenesis studies (99,100) of the pig liver TT active site residues characterized the dithiol-disulfide exchange mechanism, and led to a postulated mechanism of DHA reduction (Fig.5). The proposed scheme for DHA reduction by TI is a nucleophilic attack followed by successive exchange with 2 molecules of GSH. First, the nucleophilic Cys22 attacks the DHA C2 carbonyl carbon, resulting in a thioherniketal intermediate. Then GSH displaces reduced AA, leaving an enzyme mixed disulfide, which is reduced with a second molecule of GSH. B. SDHA Reductases The primary reaction known to generate SDHA is the one-electron reduction of the a-chromanoxyl radical to a-Tocopherol by AA in membranes. SDHA has not typically been detected in the cytosol. Several groups have reported the presence of a NAD(P)H-dependent ascorbate fiee radical reductase (monodehydroascorbate reductase, EC. 1.6.5.4) (71)[13] 21 activity in cellular membranes such as chloroplasts (96,101-103), and mammalian mitochondria (104-106), microsomes (107,108), and Golgi (109). Several SDHA reductases have been purified to homogeneity; 47 kDa protein from cucumber fruit (110), 39 kDa protein fiom soybean root nodules (111), and a 66 kDa protein fiom Neuraspara (112). A 47 kDa SDHA reductase cloned fi'om pea (113) has a deduced amino acid sequence highly homologous to soybean and cucumber fi'uit SDHARS as well as microbial flavin oxidoreductases, containing the GXGXXG/A NAD(P)H- and FAD-binding domains (114) of oxidoreductases. A SKL motif in the carboxyl terminus presumably targets this pea SDHA reductase to the peroxisomes. Attempts at purification of mammalian SDHA fi'ee radical reductases have resulted in complete loss of activity, indicating that there is probably more than one protein involved in the electron flow from NADH to the ascorbyl radical (1 15). P-chloromercuribenzoate and 5,5'-dithiobisnitrobenzoic acid inhibit purified plant SDHARS, indicating that a thiol group is involved in the active site. 111. Glutathione Explained here for the reader are the various firnctions for glutathione (GSH), as an antioxidant, and substrate for many cellular detoxification reactions; as well as the synthesis and transport of GSH. This is germane to the research question, as oxidative stress changes cellular GSH levels, and changes in GSH-containing enzymes are noted in drug resistance. In addition, an enzyme structurally and mechanistically related to TT may modulate the rate of L-cysteine transport into cells, efl‘ecting levels of GSH synthesis. Finally, GSH is a substrate for the dithiol-disulfide reductase activity of TT. 22 Table 1 Relative DHA Reductase activity Parameter Pig TT“ Bovine Rat DHAR” Rat Rat Human PDI‘ 3aHSDH‘ LA/LDHd DHAR‘ M,(kDa) 11.7 57 31 37.5 32 Km", 0.26 2.8 0.245 4.6 1.4 0.21 (mM) kw/Km 2.4x 1x102 3.9x103 2.1x102 1.2x10‘ 1.5x (M'l/sec": 10‘ 10’ - f 'Taken from (454). l’I‘aken from (91). cTaken from (85). dTaken fi'om (88). eTaken from (86). TT = thioltransferase, PDI = protein disulfide isomerase, DHAR = dehydroascorbate reductasae, 30LHSDH = 3a hydroxy-steroid dehydrogenase, LA/LDH = lipoic acid/lipoamide dehydrogenase. 23 attach crizoii crizoti HO-C-H HO-C-H HO-C-H \C’O‘C=o ‘cro‘cso or -o H/ \ I H, \0\H/ I \ I n- (2.29 —* 9'? o 6) at s ‘— OH on (0HA>+ H I (AA) ’3' +cs-H a, +,s-sc ‘sli (our GSH ‘311 (OH) (OH) .8' cssc Rsrl (OH) Fig. 5. Proposed mechanism of DHA reduction. DHA reduction by TI‘ is proposed to involve a nucleophilic attack by C22 on the C2 carbonyl carbon of DHA. GSH then displaces AA from the thiohemiketal intermediate, leaving an enzyme mixed disulfide, which is reduced with a second molecule of GSH through a dithiol- disulfide transfer mechanism. From (100), with permission. 24 Glutathione (GSH), or L-y-glutamy1-L-cysteinyl-glycine (116), is a tripeptide at physiological pH with two negatively charged carboxyl groups and one positively charged amino group (Fig.1). The most reactive group of glutathione is the sulfllydryl of the cysteinyl residue, which can serve as an electron donor, therefore participating in reactions as a nucleophile, reductant and free radical scavenger. Evolutionary comparisons indicate that GSH emerged when the atmosphere changed from anaerobic to oxygen-containing (116), suggesting that part of the firnction of GSH is to detoxify the reactive products of oxygen metabolism (117) (section I). Glutathione is present in high intracellular concentrations (0.1-10 mM) in higher eukaryotes (43,44). GSH or analogous thiols appear to be ubiquitous in all species examined, indicating an important universal function. In addition to one- and two-electron reductions of ROS, GSH stores cysteine, and is a cofactor or substrate for many enzyme reactions. Tissue concentration of the cysteine is low, maintained in the range of 10-100 M (118), as high tissue levels of cysteine may result in toxicity. Spontaneous oxidation of cysteine leads to H202; cysteine may interfere with the transport or filnction of some metal ions; and cysteine can also form mixed disulfides with essential protein sulfllydryls. GSH is a cofactor for a large number of enzymes that perform a wide variety of metabolic firnctions, including glyoxalase, formaldehyde dehydrogenase, maleylacetoacetate isomerase, prostaglandin endoperoxide isomerase, and DDT dechlorinase (reviewed in 86). GSH is also a substrate for many enzymes that maintain cellular homeostasis or metabolize xenobiotics in plants and animals, including TT (Section IV C), GSTs and GSH-sz (Section I), and DHA reductases (Section II A). 25 The y glutamyl cycle first described by Alton Meister (119) is involved in the synthesis, degradation, and recycling of glutathione (Fig.6). Glutathione is synthesized by two enzymatic reactions [14,15]. 7 glutarnylcysteine synthetase [14] L-glu +‘L-cys + ATP -* L-y-glutamyl-L-cysteine + ADP + Pi glutathione synthetase [15] L-y-glutamyl-L-cysteine + gly + ATP -' GSH + ADP + P, GSH can be eficiently transported out of cells, and is postulated to firnction to protect cell membranes, and to provide a reducing environment in the immediate environment of the cell membrane in the extracellular space (reviewed in 86). GSH is not transported directly into cells, and only cells containing membrane-bound y-glutamyl transpeptidase (120) such as the liver and kidney import y-glutamyl amino acids. Most normal cells have excess GSH Studies selectively inhibiting y-glutamyl-cysteine synthetase [14] using BSO found that cellular GSH export continues even when there is insuficient GSH synthesis, resulting in decreasing GSH levels in BSO-treated cells (119). Cells that are slower to export GSH have slower decreases in GSH when administered BSO. GSH depletion to about 5% of normal results in oxidative damage and cell sensitivity to radiation or chemical agents (122). When erythrocytes are subjected to high levels of oxidative stress, intracellular GS SG levels initially increase, then excess GSSG is transported out of the cell into the plasma, 26 possibly to prevent protein mixed disulfide formation (123,124). GSSG can be transported out of the cell in an ATP-dependent manner through two incompletely characterized transport systems; one a low Kg high V“, transporter, the other a high K, low V“, transporter (125). There is some debate in the literature as to whether GS SG transport occurs through the same mechanisms used to transport GSH adducts out of the cell. The high aflinity, low v,“ transporter for GSH adducts is competitively inhibited by GS SG, whereas the low afinity, high V" transporter is not (126). IV. Thiol-disulfide Oxidoreductases Thiol-disulfide oxidoreductases (TDOR) are heat-stable enzymes postulated to be involved in cellular sulfllydryl homeostasis. TDOR enzymes include protein disulfide isomerase (PDI), thioredoxin (TRX), and TT. There has been a virtual explosion of information available about TDOR enzymes over the past five years; however, TT is least well characterized physiologically. Details about the related enzymes PDI and TRX are presented here as many activities and fimctions attributed to these other TDORS are postulated to apply to TT. A common structural feature of TDOR enzymes is an active-site pair of cysteine residues in a 14 atom 100p (CXXC) that can exist in the reduced (dithiol) and oxidized (Intramolecular disulfide) forms. These TDOR enzymes, together with their electron donors, reductases, and NADPH have been shown to catalyze the reduction of disulfide bonds in a wide variety of substrates. Three-dimensional models predict similar structures between TRX, TT, and PDI (127- 129). The 2.2 A crystal structure of pig liver TT (129) shows that the protein folds into an almost spherical tit/B structure with a four-stranded B-sheet in the core, flanked on either side 27 mama ® Transhydrogenases fadiials admins: 0,3” + A =3 A' + GSH @Peroxidasc KC? Catalytic functions Protective ........... functions \Glutathionc (GSH) "" \ © Feedback TGIWCYS -Gly AMCYSI] y-Glutamyl inhibition cycle y-Glu-CysH / Gly x ’ © Q - o c H-Gl -—< @ ys y - CysH Cys—Gly Glu © [y—Glu-CySzl @ Cys-X Metabolic and Y’alu‘AA (””90“ 5-Oxoproline @ 6) functions N-Ac-Cys-X y-Glu-Cys-X AA Fig. 6. Glutathione synthesis, functions, and the 'y-glutamyl cycle. Overview of the metabolism and fimction of GSH GSH is synthesized by y-glutamyl cysteine synthetase (4) and glutathione synthetase (5). GSH is exported by y-glutamyl transpeptidase (1 ). Precursors for GSH synthesis are recycled GSH degradation producs as well as L- cysteien, which is transported into the cell by y-glutamyl transpeptidase (l). GSH participates in many cellular defense mechanisms; as a substrate for GSTs (8), and GHS-sz (9), and fimctions directly as an antioxidant (10). GSH also is required as a cofactor in other reactions (13) such as those catalyzed by formaldehyde dehydrogenase, glyoxylase, maleylacetoacetate isomerase, DDT-dechlorinase, and prostaglandin endoperoxide isomerase. (AA= amino acids, X= compounds forming GSH-conjugates) (Taken, with permission, fi'om Holmgren, A., Branden, C .-I., Jomvall, H., and Sjoberg, B.- M., eds, Thioredoxin and Glutaredaxin Systems: Structure and Function, Raven Press, New York, 1986, p.340) 28 by helices. The active site disulfide bridge protrudes from the protein surface flanked by a hydrophobic area on one side, and by a cluster of charged amino acids on the other (Fig.7). The folding pattern is similar in TRX (130) and the TRX-like domains in PDI (128) (Fig.8), although the only sequence similarity is within the active sites. Interestingly, glutathione peroxidase (131) also has a similar folding pattern and a similar active site disulfide placement (132), even though it does not have dithiol-disulfide reductase activity. Even with similar tertiary structures, antibodies to TT, TRX and PDI do not cross-react (133). PDI is predominantly located in the lumen of the rough and smooth endoplasmic reticulum (ER) and is loosely associated with the ER surface (134,135). TT and TRX are found primarily as soluble enzymes in the cytosol (136-145), although localization of TRX to the cytoplasmic membrane has been reported (145). The endoplasmic reticulum has a redox environment more oxidative than cytoplasm (44). Differences in cellular location, substrate specificity and redox potential of these TDOR enzymes, in addition to the redox states of the cellular compartments in which they are located, suggest separate physiological firnctions (147-149) (Table 2). little is known about the regulation of these TDOR enzymes. A. Protein Disull'lde Isomerase Protein disulfide isomerase (EC 5.3.4.1, PDI), also known as glutathione-insulin transhydrogenase (EC 1.8.4.2, GIT) (150), is a multi-firnctional protein first independently identified by Anfinsen (151), Straub (152) and Tomizawa (153). PDI has been purified to homogeneity fiom a variety of tissues and species (154-158). The enzyme is a homodirner with subunits of approximately 57 kDa, and is extremely acidic with a pI between 4.2 and 5.0 (155). Amino acid sequences from rat, bovine, chicken and human PDIS, and are identical in 406 of the 493 residues that comprise the mature subunits (reviewed in 159), indicating a 29 protein highly conserved throughout evolution. The PDI cDNA has been cloned from yeast (160), sea urchin eggs (161), rat pancreas (162) and human liver (163-166). Each monomer is comprised of several domains (reviewed in 159, Fig.9). At the N-terrrrinal of the protein is a classical signal peptide sequence, followed by an internal domain highly homologous to TRX. A second internal domain following has high homology to mammalian estrogen receptors, which is followed by 200 residues of internal repeat sequences. Towards the C- terminal end of the protein another domain with high TRX homology is observed, adjacent to a very acidic region. Like other ER proteins, PDI possesses a carboxy-tenninal KDEL sequence (167) which targets PDI to the lumen. PDI is widely distributed throughout animal and plant tissues. Highest levels of PDI activity and protein are in tissues where synthesis of disulfide-containing proteins is predominant, such as wheat endosperm, chick embryo, as well as mammalian liver, pancreas, and lymphoid tissues (168). PDI has been reported to be induced during lymphocyte maturation when a strong immunogenic stimulus, bacterial lipopolysaccharide is administered (169), indicating that PDI may be induced under conditions of oxidative stress. PDI catalyzes thiol-disulfide exchange reactions (153) in vitra in a broad range of protein substrates, leading to the isomerization of intramolecular disulfide bonds and the associated folding of nascent secretory proteins (168,170,171). PDI also functions in vitra as a chaperone-antichaperone (172,173), a glycosylation site binding protein (174), a thyroid hormone-binding protein (175), a Ca2*-binding protein (176), and a DHA reductase (80, Section HA). The mechanism of PDI oxidoreduction (Fig. 10) uses TRX as the electron donor, and a second molecule of TRX to regenerate PDI (177). Oxidized TRX is regenerated by TRX 30 Fig. 7. Predicted human TT structure. A Three-dimensional structure of pig liver TT. The active site cysteine residues (C22,C25) are shown in yellow (with a disulfide bridge), as are the two other conserved mammalian cysteine residues (C78,C82). P24 and F26 side chains are not shown; R26 and K27 are shown in red and orange, respectively. B. Three-dimensional structure of human TT using the X-ray coordinates for pig liver TT and replacing non-conserved amino acids. The overall structure is the same as that of other thiol- disulfide oxidoreductases in the general folding pattern; an almost spherical a/D structure, with a four-stranded [3 sheet in the core, flanked by helices on either side. The active site cysteine residues, shown in yellow, form an intramolecular disulfide bridge. The three other cysteine residues are shown in yellow; the conserved C78, C82, and the N—terminal C7 near the active site. The other active site residues are shown in red (PZ3 ), green (Y24), and orange (R26,R27). Fig. 7. Predicted human TT structure. 31 32 Fig. 8. TDOR enzyme folding similarities. Schematic representations comparing polypeptide chain folds of (A) Thioltransferase, (3.) T4 bacteIiOphage thioredoxin, and (C.) E. caIi thioredoxin. The folding patterns and placement of active site residues are similar, despite difi‘erent primary structures. B-strands are depicted as thick arrows, and cl-helices are rectangular boxes. Striped helices are forward of the B-sheet, and clear helices are behind. The active site is represented by a circle. ( From Katti, SK, Robbins, AH, Yang, Y.Y., and Wells, W.W. (1995) Protein Science, 4, 1998- 2005, with permission.) 34 Table 2 Redox Potentials of TDOR Enzymes Reduction potential Pig liver thioltransferase -0. 159 :1; 0.004 V E cali glutaredoxin -0.260 V Proteln disulfide lsomerase _ -0.11 to .91 V The redox potentials of the three TDOR enzymes indicate that for dithiol-disulfide oxidoreduction, TRX and TT are more efi‘ective in the cytoplasm, which is a more reducing environment than the ER; whereas PDI is more efi‘ective in the ER, which has a more oxidizing environment. (From Jung, C.-H., and Thomas, IA. (1996) Arch. Biochern. Biophys. 335, with permission.) ‘ 35 reductase, an NADPH-dependent enzyme. Four WCGHCK sequences (17 8) homologous to the WCGPCK found in TRX (179) exist in native PDI. Mutagenesis studies have demonstrated that the cysteine residues in these motifs are the redox-active groups, that only one redox-active cysteine residue is required for efficient protein folding (180), and that the N- and C-terrninal TRX-like domains are firnctionally non-equivalent (181), although each domain filnctions independently in catalysis (182,183). Whether or not there is cooperativity between these redox-active domains is not yet known. B. Thioredoxin TRX (TRX), an enzyme of approximately 12 kDa, has been purified, cloned and sequenced from a variety of species and tissues (reviewed in 137). Amino acid sequences have been determined for purified TRX from Anabena Sp 7119 (184), Rhadabacter sphaeraides Y (185), Rat liver (186), calf liver (187), human leukemic cells (ADF, 188), T4 phage (189,190), and a joint TRX-TRX-reductase in Mycabacterium leprae (191). TRX has been cloned fi'om bacteria, yeast, plants, and mammals, including E. cali (192), Bacillus brevis (193), cyanobacteriurn Anacysris' ridulans (194), yeast (195,196), spinach (197), and several human tissues (198,199). TRX amino acid sequences from archaebacteria to yeast and humans are between 27 - 69°/o identical to E. cali TRX (127). The active site, highly conserved between prokaryotes and eukaryotes, is comprised of the amino acid sequence VDFXAXWCGPC(KIM/I)XP and conserved residues distant in primary structure. Mammalian TRXs also contain two additional C-terminal cysteine residues which easily undergo oxidation to disulfides with subsequent aggregation and inactivation of the protein (200). TRX is an acidic protein, with a redox active Cys containing an acidic pK, (around 6.7 in human TRX). TRX uses NADPH 36 A B C D E F N- TRX-1 '52Fl repeats TRX-2 KDEL -c Fig. 9. PDI primary structure. PDI is a homodirner with subunits of approximately 57 kDa. Each monomer is composed of several domains. A. N-tenninal signal sequence. B. TRX-like domain 1. C. Estrogen- receptor-like domain. D. Internal repeat sequences. E. TRX-like domain 2. F. Acidic region with KDEL that targets PDI to the ER 37 as an electron donor, and oxidized TRX is directly regenerated in a NADPH-dependent manner by TRX reductase (Fig. 1 1). TRX is found in multiple systems involved in the redox regulation of cellular functions. Physiological roles have been postulated for TRX including dithiol reducing activity, protein posttranslational modification for secretion, growth stimulation, facilitation of interactions between transcription factors and their target DNA sequences, signal transduction, cytokine activity, and a role in cellular protection from cytotoxic agents. TRX firnctions as an endogenous reducing agent (201). Sulfate reduction in E. cali relies upon either TT or TRX (202). Deletion of both TRX genes in yeast results in methionine auxotrophy indicating that in yeast, TT cannot assimilate sulfate (195). Several mammalian species such as mouse contain one filnctional TRX gene, and a processed pseudogene (203), but the biological significance is not yet understood. Plants contain multiple TRXs essential for photosynthetic growth (204). TRX regulates photosynthetic chloroplast enzymes such as fructose 1,6 bisphosphatase, phosphofi'uctokinase, glucose 6-phosphate dehydrogenase, glyceraldehyde 3-phosphate dehydrogenase, and NADP+-malate dehydrogenase by dithiol-disulfide exchange (205). E. cali TRX reduces exposed S-S bridges in a variety of proteins in vitra (137), including oxidized nbomrclease (152), choriogonadotropins (137), proteolytic enzymes (206), factor VIII and other coagulation factors (207,208), glucocorticoid receptor (209), Vitamin K0 and Vitamin K reductase (210), and insuhn (211,212). Mammalian TRX has broader substrate specificity than the E. cali enzyme and will react with non-thiol compounds such as alloxan, menadione (213), and selenite (214). TRX also firnctions as an electron donor to plasma GSH-Px (215), involved in detoxification of peroxides (Section I). TRX reductase 38 NADP+ TRX (red) >< <——TFtX reductase NADPH + H+ ' TRX (red) TRX (ox) V /5H protein > Protein A SH PDI (red) pol (ox) > <'I'FtX (red) PDI (red) TRX (ox) Fig. 10. PDI oxidoreduction. TRX donates electrons to PDI, which catalyzes the reduction of protein disulfides. Oxidized PDI is regenerated by a second molecule of TRX. TRX is reduced by NADPH—dependent TRX reductase. 39 will directly reduce lipid hydroperoxides by NADPH, a new pathway for detoxification which is strongly stimulated by selenols (216). These selenols are then reduced by TRX. TRX preferentially reduces oxidized protein monothiols that have been converted to sulfenic or sulfinic residues over thiols oxidized to disulfides (217). TRX is found in high levels in nerve cells and axons corresponding to areas of neurotransrnittor synthesis and secretion (138), indicating that TRX may be involved in the posttranslational modification of proteins prior to secretion. Irnmunolocalization of TRX in secretory cells revealed that TRX is associated with intracellular membranes (145). TRX has been found localized to plasma membranes of nucleated cells (218). TRX-like domains are found in PDI (Section IV A), a multi-functional ER protein involved in disulfide formation, protein secretion, and protein folding. There is growing evidence that TRX is involved in eukaryotic grth control. Exogenous TRX functions as a growth factor in many cell types. 12’I TRX added to media is taken up by cultured cells (219). Recombinant TRX stimulates proliferation in murine 3T3 fibroblasts and human cancer cell lines when applied at 100 nM levels (reviewed in 220). Exogenous TRX added to serum-free lymphoblastoid and Burkitt's cell lines stimulates DNA synthesis (221,222) and redox-dependent proliferation (223,224). In contrast, elevated TRX secretion results in growth arrest and morphological changes in human HepG2 hepatoma cells (225). TRX levels are increased in tumors (226-229) when compared with those in normal tissues, suggesting that TRX may filnction as a growth factor for human cancers. Redox activity of TRX is essential in lymphocyte irnmortalization by human T-lymphocyte virus-1 and Epstein-Barr virus (230). TRX is expressed at high levels in rapidly dividing cells, such 40 TRX reductase NADPH + H+ NADP+ TRX (red) TRX (ox) TRX (red) V Protein-802' ——* Protein-SCH Fig. 11. TRX oxidoreduction. TRX reduces oxidized protein sulflrydryl groups, especially those in sulfenic and sulfonic oxidation states. The electron donor for TRX is NADPH. Oxidized TRX is reduced by NADPH-dependent TRX reductase. 41 as human lymphoid and hematopoietic cells, and at low levels in resting cell types, such as lymphocytes or monocytes (231). TRX can be excreted by normal and tumor cells in culture (232) using a leaderless secretory pathway (233). For example, CD4+ T-cells secrete TRX, promoting growth in normal and leukemic B cells (234). ADF, an autocrine growth factor identical to TRX, is produced in human T-lymphocyte virus I-infected cells. ADF synergizes with interleukin-1 and interleukin-2, cytokines involved in proliferation (223,23 5-23 6), induces interleukin 2- receptor a chain production and stimulates cell growth (23 7-23 8). Interestingly, interleukin 2-receptor a chain itself is under strong control of NFxB, a redox-sensitive transcription factor. Several mechanisms are prOposed for growth control by TRX First, reduced TRX may activate protein kinase C (PKC), which subsequently activates phophoinositol-specific phospholipase C activity (221). PKC has several thiol-rich regions in each of the catalytic and regulatory subunits containing critical cysteine residues that are extremely sensitive to oxidative modification (239-241). PKC activity has been shown to be activated and inactivated through oxidative changes (242,243). The zinc-thiolate structure of PKC is required to bind phorbol ester and DNA (244). TRX increases B-cell proliferation through a PKC-dependent mechanism, as the PKC inlubitors staurosporin and calphostin C both block TRX-induced proliferation. A second potential mechanism is based on the observation that TRX promotes L- cysteine transport into cells, increasing intracellular GSH content. Thiol compounds such as L-cysteine and GSH are involved in the activation and cell cycle progression of stimulated lymphocytes (224,245). There is a close association between the TRX and GSH systems, and 42 redox regulation by these systems plays an important role in regulating cell proliferation and activation. Oxidation of cellular thiols including TRX with diamide induces apoptosis in Jurkat T cells and human lymphoblasts (246). Observations in yeast support TRX involvement in cell cycle regulation. There are two TRX genes in yeast, which are 74% identical. Loss of either of these genes afi‘ects neither cell growth nor morphology, however, deletions in both alter cell cycle and morphology. G1 phase is absent, and the S phase is 3-fold longer, suggesting slow DNA replication. The overall generation time increases by 33%, and there is a significant increase in cell size, and a greater proportion of budded cells (195). These observations in yeast also support the theory that TRX is involved in DNA synthesis and hence regulates the rate of proliferation. TRX catalyzes the reduction of ribonucleotide reductase in bacteria and mammals (247), and is therefore involved in providing deoxynucleotides critical for the DNA synthesis that occurs in the S phase of the cell cycle. TRX has other efl‘ects related to development and growth in varied systems. A Drasaphila TRX homolog, "deadhead", also indicates a role for TRX in cell cycle and deve10pment. "Deadhead" is not essential for viability, but is essential for female meiosis and early embryonic development in viva (248). Drasaphila "deadhead" eggs are fertilized, but cannot complete mitosis. TRX is involved in the regulation of eosinophil migration (249), and is expressed in the ovary throughout the menstrual cycle (250). TRX also serves a role in bacteriophage replication and assembly. T7 DNA polymerase contains p5 protein and TRX in a 1:1 relationship, where TRX confers processivity to the oligomeric complex (251). The redox activity of TRX is separate from the role in T7 replication. TRX may help keep the p5 bil NT exp. 301} ram Ihe'll j 43 protein in the DNA binding clefi, similar to the function for the "thumb-like" domain in E. cali DNA polymerase I Klenow fragment (reviewed in 251). Filamentous phage assembly also requires host TRX. TRX-like domains are found in a phosphoinositide-specific phospholipase C (252) and in two gonadotropin hormones (253), introducing the possibility that TRX is involved in signal transduction. Insulin-like growth factors and their binding proteins contain vicinal cysteine residues similar to those found in PDI and TRX, and have dithiol-disulfide exchange activity (254). Thiol redox reactions play a role in regulating conformational changes in both the insulin receptor and possibly in the insulin-like growth factor receptor, indicating that intrinsic dithiol-disulfide exchange may be important in activating these receptor classes (254). Redox changes in thiols are involved in lymphocyte activation (236). T-cell Fc receptor signal transduction occurs via tyrosine kinase and TRX redox regulation (255). Redox regulation of p56”, a member of the src family of tyrosine kinases also occurs in T cells (256). TRX may have a role in transcription In vitm, TRX can modulate transcription factor 111C DNA binding (257), and activate the human immunodeficiency virus-1 enhancer binding protein via thiol-redox mechanisms (258) as well as modulate glucocorticoid receptor steroid binding (209). Glucocortocoid receptor DNA binding requires reduced -SH groups (259). TRX is postulated to have effects on both the activation and the DNA-binding of NFxB and activator protein-1 (AP-1), transcription factors involved in the inducible expression of genes responsive to oxidative stress as well as celiUlar defense mechanisms. Both AP-l and NFxB are easily inhibited by the oxidation of sulflrydryl groups, and reactivated by thiols (260). Redox effects regulate the amount of these transcription factors, their DNA-binding afinities, and their nuclear or cytoplasmic distributions. 44 The NFxB/Rel/dorsal oncoprotein/transcription factor family binds target DNA sequences through a conserved basic region with a single cysteine flanked by basic residues (RXXRXRXXC) (261). NFxB is composed of two subunits, p65 and p50. Transcriptional activation of NFxB-dependent genes is usually induced by the dissociation of the inhibitory IxB protein from the cytoplasmic heterodimer, and the subsequent translocation of active NFxB into the nucleus. DNA damaging agents such as oxidative stress and UV irradiation induce cellular immediate early genes including c-fas and c-jun. Fos and Jun are individual DNA-binding subunits that interact with each other through a leucine-zipper to form AP-l. DNA binding by F as and Jun (AP-1) is regulated posttranslationally by phosphorylation of the C-terminal of Fos as well as redox changes (260). A lysine-cysteinearginine sequence in the C-terminal DNA-binding domain is conserved in all the members of the family except the constitutively active viral protooncogene, v-jun. The cationic environment surrounding the critical cysteine residue in both transcription factors renders the thiol highly reactive and particularly susceptible to oxidation. This cysteine binds DNA when reduced; oxidized cysteine is not permissive for DNA binding. Oxidative conditions potentiate the activation of NFxB and AP-l in intact cells, and have mixed efi‘ects on DNA binding activity in vitra. In tissue culture, NFKB and AP-l activities (both DNA binding and transcriptional transactivation) are modulated by exogenous application or transient expression of TRX in a dose-dependent manner (262). GS SG also modulates activation and binding in intact cells and in virra (263). Experiments suggest that the two transcription factors difier in the redox potentials of their critical cysteine residues, their sensitivity to oxidative inactivation, and responses to antioxidants and exogenous TRX 45 (23 1,263 -269). Activation by TRX involves PKC-independent de nova transcription of c-jun and c- fas (262). TRX afi‘ects in vitra AP-l DNA binding (264) through the ubiquitous nuclear redox factor Ref-1. Ref-1 is subject to redox control, and stimulates Fos-Jun and NFch interactions as well as apurinic/apyrimidinic endonuclease DNA repair activity. Ref-1 activity is augmented ‘by TRX. Another series of TRX firnctions are cytoprotective. TRX protects human macrophages fiom human irmnunodeficiency vinrs expression (270), prevents TNFa-induced cytotoxicity (271), and inactivates some toxic venoms (272). All venom neurotoxins inactivated by TRX (snake, scorpion, and bee) are disulfide-containing proteins. In vitra reduction of these toxins increases their susceptibility to tryptic proteolysis, and decreases toxin activity, whether via phospholipase-A, (B-bungarotoxins in bee and snake venoms) or acetylcholine receptor (a-bungarotoxins) mechanisms. TRX attenuates ischemia-reperfilsion injury in culture (273). TRX expression is elevated in gerbil astroglial cells afier transient global ischemia (274). Neuroprotection fiom ischemia and reperfusion injury in culture by central nervous system glial cells has been correlated to secreted TRX, increased cellular GSH, and requires reducing conditions (275). GSH and TT are incapable of protecting neurons subjected to injury without TRX. Exogenously added B-mercaptoethanol, a reducing agent, increased cellular survival, whereas added BSO, which reduces GSH levels, decreased survival. TRX also increases resistance to cis-diamine dichloroplatinum (ID-induced cytotoxicity (276). Antisense stable transfection of TRX into drug-resistant kidney cells found decreased TRX expression correlates with increased sensitivity to drugs that generate ROS, 46 such as cisplatin, mitomycin C, doxorubicin, and etoposide (277). One mechanism for this resistance is that TRX functions as an endogenous radical scavenger (278), and protects endothelial cells from injury by H202 and other ROS released by activated neutrophils (279). TRX is induced by a number of stimuli in viva and in vitra. Oxidative stresses such as H202 and ischemia result in TRX induction in rat retina (280), keratinocytes, lymphoid cells (281), and retinal pigment epithelial cells (282). TRX induction was localized to the mitochondria in the retinal pigment epithelial cells (284). Retinal stimulated TRX expression 8-10 fold after 4 hours in monkey conducting airway epithelial cells (283) without concurrent protein synthesis. In yeast, the YAPl transcription factor responds to oxidative stress by elevating expression levels of TRX2, resulting in resistance to hydroperoxides and thioloxidants (216). Yeast cells deficient in the YAPl were hypersensitive to thioloxidants and hydroperoxides. Site-directed mutagenesis and NMR studies have determined protein conformations and filnctions for several active Site residues (284-290). Replacement of the TRX active site proline with histidine, mimicking the PDI active site, increased the disulfide isomerase activity 10-fold (291). The redox-active cysteine 31 in human TRX has been determined to be responsible for both the ROS-reducing (HzOz) and the protein refolding activities, indicating a redox requirement for radical and ROS reduction (284). Replacement of either active site cysteines in human TRX competitively inhibited TRX reductase and the mitogenic activity (290). Replacement of lysine 36 in human TRX affected growth rates and reduction rates but did not change the redox activity (289). As most of these experiments have been carried out in vitra, or in tissue culture conditions, the biological firnctions of TRX are continuing to be investigated by many 47 research groups. It remains to be seen how significant these TRX activities are in viva. C. Thioltransferase Transhydrogenases, thiol-disulfide oxidoreductases or. thiol-disulfide exchange enzymes were first documented by Racker in 1955. Racker found an enzyme of approximately 12 kDa molecular weight in beef liver cytosol that catalyzed the conversion of homocystine to homocysteine in the presence of GSH, GSSG reductase, and NADPH which he termed glutathione cystine transhydrogenase (EC 1.8.4.1) (292). Since then it has been determined that the GSH-dependent reduction of low-molecular weight thiol substrates was actually not a transhydrogenase reaction [16], [16] RSSR + 2 GSH -* 2 RSH + GSSG but actually two consecutive ionic SN2 displacement reactions [17,18], [17] RSSR + GSH -' RSSG + RSH [18] RSSG + GSH -* GSSG + RSH and the resulting GSSG is reduced by GSSG reductase using NADPH as the electron donor (293) [19], regenerating the reduced GSH. [19] GSSG + NADPH + H” _. zosrr + NADP+ 48 Elucidation of this mechanism resulted in a enzyme title change from "transhydrogenase" to ”thioltransferase" (EC. 1.8.4.1). Glutaredoxin, another cytosolic protein of approximately 12 kDa that also catalyzes the GSH-dependent reduction of mixed protein-disulfides and uses GSSG reductase and NADPH to regenerate GSH, was discovered in E. cali TRX deletion mutants that still could reduce ribonucleotide reductase (294). Comparison of amino acid sequences (295), catalytic activities, size, irnmunoreactivities, pIs, and substrate preferences determined that TT and glutaredoxin were two names for the same enzyme. Currently, TT has been cloned from many varied Species, including three different genes in E. cali (296,297), as well as from Haemaphilus influenzae (298), Pyracaccus firfiasus (299), T4 bacteriophage (300,301), vaccinia virus (302), T rypanasama cruzii (303 ), yeast (304), rice (305), castor bean (306), pig liver (307), and several human tissues (308- 312) with 48.6 to 90.7% similarity and 25.3 to 82.2% identity to human placental TT. TT is found in all GSH-containing organisms, as well as in one GSH-negative Species, methanabacterium thermautraphum (313). TT amino acid sequences are 80-91% conserved among mammalian species (Fig. 12). The active site amino acid consensus sequence (314) CP(Y/F)C is conserved in bacterial, manunalian and plant TTs (Fig. 12). Mutagenesis studies on recombinant pig liver '1'1‘ revealed that these cysteine residues (C22 and C”) are the redox active residues, and that C22 is essential for catalytic activity (100). Pig liver TT C22 has an unusually acidic pK, of 3.8 (314), facilitated by the proximal R26. Two other C-terrninal half-cystine residues exist in all mammalian TT, but site-directed mutagenesis of pig TT, replacing these residues with alanine determined that they were not redox active. Human TT contains a third half-cystine residue 49 at residue 7, possrbly increasing the protein susceptibility to oxidation (315), and decreasing the heat stability of the protein (316). In the presence of GSH, TT catalyzes the formation. of GSSG from disulfide substrates such as cystine, S-SO,-cysteine, hydroxyethyldisulfide, cysteinyl-bovine serum albumin mixed disulfide, oxidized ribonucleotide reductase and various GSH-containing mixed disulfides (317-319). Cystearnine is an alternate reductant for TT (320). TT displays selectivity for glutathionyl substrates and catalyzes their reduction more efi'lciently than TRX (321). Site-directed mutagenesis and isotope studies revealed a potential mechanism for dithiol-disulfide transfer (99,100) (Fig. 13). Reduced TT first reacts with a dithiol substrate, such as a protein mixed disulfide, then with a thiol substrate such as GSH. Oxidized TT is regenerated by a second molecule of GSH, producing GS SG, which is then reduced by GS SG reductase. Structural studies have supported this model. Two-dimensional NMR results Show few changes between oxidized and reduced TRX, as major differences are associated only with active site and neighboring residues (323). TT-mixed disulfides formed with GSH using recombinant E. cali TT are visible by NMR (324,325). Solution structures show the GSH- binding site is conserved between human and E. cali TT (310). Studies investigating TT- catalyzed GSH and protein-GSH mixed disulfides determined that the y-L-glutamyl-L— cysteinyl moiety of GSSG and GSH-mixed disulfides is an essential determinant for recognition by TT (326). TT inhibitors include diamide and other sulflrydryl-oxidizing agents. Anti- inflammatory and anti-histaminic drugs also inhibit purified TT non-competitively (327), as 50 Fig. 12. TI‘ amino acid sequence comparisons. TT amino acids sequences are highly conserved, ranging from 48.6 to 90.7% similarity to human TT in species as diverse as GSH-negative bacteria, viruses, yeast, plants, and mammals. The active site CPY/F C is conserved in all species, and all mammalian TTs sequenced to date contain an additional conserved C-terrninal CIGGC motif. Amino acid sequences and deduced amino acid sequences are aligned for human (deduced, 308), pig (deduced, 307), bovine (295), rabbit (322), yeast (deduced, 304), vaccinia virus (deduced, 302), castor bean (deduced, 306), rice (deduced, 305), H. influenzae (deduced, 298), E. cali (deduced, 297,298), Pyracaccus furiousus (deduced, 299), and Methanabacterium thermautraphum (deduced, 313) using the Genetics Computer Group (GCG, Wisconsin) programs. yeast vaccinia castor rtce haemoph ecolr pyrof methano yeast vaccrnra castor rrce haemOph ecoli pyrof methane human P19 bovine rabbit yeast vaccinia caster race haemoph ecolt pyrof methane human 919 bovrne rabbrt yeast vaccinia caster race haemoph ecolt pyrof methane human Pig bovine rabbit yeast vaccrnia caster rrce haemoph ecoli pyrof methane oooooooooo .......... ssssssssss eeeeeeeeee uuuuuuuuuu oooooooooo oooooooooo oooooooooo uuuuuuuuuu 51 .......... .......... oooooooooo ssssssssss oooooooooo .......... .......... cccccccccc .......... cccccccccc cccccccccc ssssssssss ssssssssss .......... .......... oooooooooo cccccccccc cccccccccc oooooooooo aaaaaaaaaa MGLISDADKK VIKEEFFSKM VNPVKLIVFV RKDHCQYCDQ LKQLVQELSE LTDKLSYEIV oooooooooo .......... eeeeeeeeee .......... eeeeeeeeee .......... .......... .......... oooooooooo cccccccccc ...MAQEFVN ...MAQAFVN ....AQAFVN ....AQEFVN VSQETVAHVK ...MAEEFVQ ...MAMTKTK ...MALAKAK 181 NEIODYLQQL NEIQDYLQQL SEIQDYLQQL SEIQDYLQQL SEIQDALEEI NELRDYFEOI SEIQTALAEW SELQSALAEW KE...DLSKS KE...DLQQK VEAIEYPEWA IDIMVDREKA 241 ITA .......... .......... oooooooooo .......... oooooooooo eeeeeeeeee oooooooooo .......... oooooooooo eeeeeeeeee CKIQPGKVVV SKIQPGKVVV SKIQPGKVVV SKIQPGKVVV DLIGQKEVFV QRLANNKVTI ELVSSNAVVV ETVASAPVVV ..... MFVVI ..... MQTVI NIDQDVRILV ....VVKIEV TGAR. TGAR. TGAR. TGAR. SGQK. TGGR. TGQR..TVPN TGQR..TVPN VGKPVETVPQ AGKPVETVPQ DQYNVMAVPK IDYGLMAVPA .TVPR .TVPR .TVPR .TVPR .TVPN .TVPR .......... .......... oooooooooo .......... .......... .......... .......... .......... .......... oooooooooo FIKPTCPYCR FIKPTCPFCR FIKPTCPYCR FIKPTCPYCR AAKTYCPYCK FVKYTCPFCR FSKTYCPYC. YSKSYCPFC. FGRPGCPYC. FGRSGCPYC. FVTPTCPYCP FTSPTCPYCP VFIGKDCIGG VFIGKECIGG VFIGQECIGG VFLGKDCIGG VYINGKHIGG IFFGKTSIGG VFIGGKHIGG VFINGKHIGG IFIDEKPIGG IFVDQQHIGG IVIQVNGEDR IAI....DGV oooooooooo eeeeeeeeee .......... .......... nnnnnnnnnn .......... .......... eeeeeeeeee .......... ssssssssss .RAQEILSQL .KTQELLSQL .KTQELLSQL .KTQEILSEL ATLSTLFQEL .NALDILNKF TSVKKLLDQL VRVKKLFGQL VRAKNLAEKL VRAKDLAEKL ..... LAVRM ..... MAIEV CSDLVSLQQS CTDLESMHKR CTDLVNMHER CSDLIAMQEK NSDLETLKKN YSDLLEIDNM CDSTTAKHSQ CDDTLALNNE CTDFEALMKE YTDFAAWVKE VEFEGAYPEK VRFVGAPGRE Fig. 12. TI amino acid sequence comparisons. .......... oooooooooo .......... eeeeeeeeee cccccccccc .......... .......... .......... eeeeeeeeee uuuuuuuuuu PIKQGLLE.. PFKEGLLE.. PFKQGLLE.. PFKQGLLE.. NVPKSKAL.. SFKRGAYE.. G...AKYK.. G...ATFK.. KGEVADFDYR SNERDDFQYQ AHKFAIENTK VD....EAKK GELLTRLKQI GELLTRLQQI GELLTRLKOM GELLARLKBM GKLAEILKPV DALGDILSSI GQLVPLLTEA GKLVPLLTEA QFGIVA.... NLDA ...... MFLEKLLSAL ELFEAISDEI .......... eeeeeeeeee eeeeeeeeee .......... cccccccccc eeeeeeeeee .......... oooooooooo eeeeeeeeee oooooooooo 180 FVDITATNHT FVDITATSDT FVDITAAGNI FVDITATSDM VLELDEHSNG IVDIKEFXPE VVELDTESDG AIELDGESDG YVDIHAEGIT YVDIRAEGIT AGKGKILGDM EFGDKIDVEK 240 GALQ ...... GALK ...... GALQ ...... GALRQ ..... FQ ........ GVLRTC.... GAV ....... GAIASSAKTT cccccccccc uuuuuuuuuu S ......... 52 Fig. 13. IT dithiol-disulfide transfer mechansim. E = TT, S'= thiolate anion of Cn (11‘), SH = sulfhydryl of C25 and OH = the hydroxyl group of a C25S TT mutant. RSSR = disulfide substrate, RSH = reduced product, GSH = glutathione, GSSG = glutathione disulfide, 1AM = iodoacetamide, and HI = hydriodic acid. Studies in pig liver TT resulted in this proposed mechanism of action. (1,5): Reduced TT preferentially reacts with glutathionyl-containing dithiol substrates, such as protein mixed disulfides. (4): The enzyme mixed disulfide then reacts with a molecule of GSH to produce reduced protein/product, and a "IT mixed disulfide with GSH. (5) A second molecule of GSH reduces TT, producing GSSG, which can be regenerated by GSSG reductase (not shown). From (100) with permission. 53 N:zoua :8 39:” I Gala 29. 2mm 50:5 / ”film 5 £95 m M A .e.\ _ :2 «me... n <_ / 422868 \ Fig. 13. TT dithiol-disulfide transfer mechanism. 54 well as chlorarnphenicol, an antibiotic (328), and cisplatin, an antitumor drug (329). No specific TT inhibitor has been discovered to date. D. Postulated physiological roles for 'IT: As this doctoral research commenced, no in viva function for TT was established. Postulated physiological functions include a role in protein sulflrydryl maintenance, protection of cytoplasmic proteins from oxidative stress, regulation through posttranslational modification of critical cysteine residues, signal transduction, and cancer through growth regulation and resistance to anti-cancer drugs. A structurally and mechanistically related protein, TRX, has been examined in more detail to date; many of these functions postulated for TT are thus extensions of functions attributed to TRX. 1. Protein processing: TT may be involved in degradation and posttranslational modification of proteins. Protein degradation may involve reduction of disulfides which maintain the native structure of proteins by TT (330). TT may complete posttranslational processing of cytosolic protein domains produced in the ER lumen. Since the ER is a more oxidizing environment (44) than the cytosol, nascent protein sulflrydryl groups could have GSH adducts (protein-SSG). As the protein enters the cytoplasm, 'I'I‘ may catalyze the GSH-dependent reduction of protein-SSG derivatives (146). 2. Protection against cellular oxidative stress: Proteins form mixed disulfides as an early response to oxidative stress (reviewed in 331). This has been termed S-thiolation, and occurs primarily with GSH through a mechanism other than dithiol-disulfide transfer, possibly as an oxidation-reduction reaction with protein thiyl radicals. 'I'I-catalyzed reduction or deglutathionylation of protein-SSG disulfides formed 55 under oxidative stress could return proteins to their native state. TT is an eficient deglutathionylase under physiological conditions (146,331-335), removing GSH adducts from carbonic anhydrase II, actin, creatine kinase, GAPDH, phosphorylase b and glutathione S- transferase. For example, TT can efi‘ectively catalyze the reduction of hemoglobin-glutathione disulfide adduct, a byproduct— of oxidative stress in red blood cells (336). TI has a deglutathionylation rate more than 10-fold that of TRX and PDI. The specificity of deglutathionylation depends on the type of thiol modification; TT is more efi‘ective with protein-GSH mixed disulfides, whereas TRX and PDI are more efi‘ective with sulflrydryls in varied oxidation states, such as RSOH, RSOZ', and RSO; (146,321). A second role in protection fi'om oxidative stress is related to the DHA reductase activity (Section II) of TI (80). TT regenerates AA, a cellular antioxidant, from DHA TT is responsible for much of the DHA reduction in normal human neutrophils (312). IT, GSSG reductase and GST are inducible in liver by a variety of agents including phenobarbital and the dietary antioxidant BHA (2,3-t-butyl-hydroxyanisole) (337). TT is highly inducible by ultraviolet B radiation in rat keratinocytes (338), characteristic of immediate early genes, and genes induced by DNA damage. Finally, immune complexes have been shown to stimulate TT release from rabbit polymorphonuclear leukocytes (3 28), under conditions of oxidative stress. TT release was not dependent on cytolysis, indicating an extracellular function for TT in response to oxidative stress. 3. Redox regulation of enzyme function: Thiol-disulfide exchange reactions may regulate enzymatic activities in a manner analogous to phosphorylation-dephosphorylation mechanisms of control. Studies investigating 56 enzyme regulation by thiol oxidation/reduction support this theory. Bulk transfer of GSH- equivalents to and from total cellular protein was demonstrated (339). Disulfide exchange catalyzed by TT has reactivated many oxidized (inactive) proteins in vitra, such as ribonucleotide reductase (340), pyruvate kinase (316), phosphofructokinase (217,316), GAPDH (217,341), ornithine carboxylase (342), and glutathione S-transferase (341,343). Phosphofiuctoldnase is inhibited by thiol oxidation, whereas the opposing regulatory enzyme, fructose 1,6-bisphosphatase is activated (344,345). TT and TRX both reactivate phosphofiuctokinase and ribonucleotide reductase in vitra, however TT is more emcient than TRX with respect to both substrates (149). TI may play a role in the oxidative activation/deactivation of metabolic and regulatory enzymes. 4. Role in signal transduction: TI' could affect signal transduction pathways by reducing cytosolic enzyme, receptor or transcription factor disulfides to modulate function in response to intracellular redox changes. Several observations support dithiol-disulfide exchange and oxidation-reduction reactions in horrnone-induced receptor activation. First, TT enhances L-triiodothyronine binding to its receptor in vitra, supporting a role in growth and difl‘erentiation pathways (346). Secondly, two gonadotropic hormones, leutropin B subunit (LH) and follicotropin (FSH), have tetrapeptides homologous to that of the TDOR CXXC motif; (CGPC) and (CGKC), respectively (347). Structural and functional similarities also exist; the LH and FSH tetrapeptide CGXC motif is predicted to be located in a B-tum similar to that of the TDOR enzyme active sites; and LH and FSH were 60 and 300-fold more active than TRX, 57 respectively, when dithiol-disulfide exchange activities are measured with the standard ribonuclease reactivation assay (347). Receptors for these gonadotropins are composed of disulfide-containing subunits (348), suggesting that LH and F SH may utilize thiol-disulfide exchange to modify and subsequently activate their receptor. The gonadotropin CGXC motif is also immediately adjacent to the "determinant loop” (253) proposed as responsible for biological specificity. Thirdly, TT has a marked similarity to conserved regions between the TGF-B factors (349), a family of proteins involved in the control of cell growth and differentiation, and implicated in inflammatory processes (Fig. 14). Specific regions of similarity are around the TT redox active CXXC residues, and the second C-terminal dithiol site, CXXXC. TGF-B signals through contacting two distantly related transmembrane receptors that are serine/threonine kinases (350). TGF-B binds receptor 11, a constitutively active kinase, which then starts the signal transduction cascade. The predicted receptor II structure includes a single hydrophobic transmanbrane domain, a cytoplasmic serine/threonine kinase domain, and a cysteine-rich extracellular domain (351). Activation of the TGF-B receptor may involve dithiol-disulfide interchange. Finally, several firnctions of TRX (Section IV B), another TDOR enzyme with similar dithiol-disulfide exchange activity may also be functions of TT. TRX has been shown to restore the ligand-binding capability of oxidized glucocorticoid receptor, in vitra (209), and modulate F, receptor signal transduction (255). TRX modulates in virra DNA binding abilities of transcription factors Fos/Jun (AP-1), and NFItB (263,352). The GS SG/GSH ratio also efl‘ects transcription factor DNA binding (263 ); DNA binding was activated by low levels of GS SG, and inactivated by high levels of GSSG, 58 indicating these transcription factors may form mixed disulfides under oxidizing conditions. Since TT has greater glutathionyl substrate specificity than TRX (321), future experiments may Show TI to be the more significant enzyme with respect to NFKB and AP-l activation. 5. Role in cancer: Based on the involvement of TT in maintaining cellular redox homeostasis, TT may firnction both positively and negatively in cancer by regulating growth and differentiation as well as modulating drug resistance. TT has not directly been shown to function as a growth factor, however several related proteins have a demonstrated role in growth and differentiation. TRX (ADF) is detected in many tumor tissues, and is a growth-promoting factor (227). TGF-B has structural similarities to TT, and regulates the action of cyclin-dependent kinase inhibitors, affecting cell cycle progression (353). Finally, thiols other than GSH are indicated in redox stimulation of apoptosis in T-lyrnphocytes (246). Chemotherapy involves treatment of cells with alkylating agents (such as BCNU, bis- cloroethylnitrosourea) or quinones such as Adriarnycin (doxorubicin) that generate ROS including electrophiles, radicals, oxidants, and peroxides (3 54), which result in DNA, lipid and protein damage (Section I and V). Systems to detoxify ROS have already been mentioned (Section I); typically GSH and various enzymes associated with sulfllydryl biochemistry that serve to conjugate, degrade or excrete the toxic species. Anti-neoplastic-dcpendent inhibition and induction of GSH-utilizing enzymes has also been studied, although seldom including the thiol-disulfide oxidoreductase enzymes. Sulfllydryl enzymes important in proliferating cells are inactivated via oxidation or alkylation. Many cancer cells have depressed levels of repair and detoxification enzymes (356), resulting 59 Fig. 14. TI‘ and TGF-B amino acid sequence similarities. Several TTS (II) are aligned with the C-terrninal domain of the TGF-B family members (I). Residues around the TT active site consensus CPY/F C and around the C-terminal CIGGC motif are conserved between TTS and TGFBs. Residues at the bottom of the figure are the residues in the TGF-B consensus pattern for the first similar region in that domain that are also conserved in TTS. With the exception of a large gap, almost all the residues in the TGF-B amino acid consensus sequence are conserved in TTS. Residues marked with ** are residues absolutely conserved between both TTS and TGF-Bs. Residues marked * are equally or more conserved between TGF-Bs and TTS, than within TGF-Bs. Taken from (349) with permission. 60 — idolat— u o u o a a a gut. Ear-gen H§F¢F~ gas..- a gage—O nngS—hanflflughu asserts... Easiness; U E h 01% all“ $0: r519 1607\— It g g I! gigrrg‘ginggaugg flung ark; 8?! gm“) :6 l8 ggggtrgha—gggufigzaa Egg fishy-Pug E 40080) is g Egg rrgg‘ggtgé 2. rings and... 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It; rage—gm ”:54: macaques» Bxhrrrrrrrrrrrrsrrrrrrrrru=8r§§~hdr§fih mug" "taut-r 000)! fig;— unfit—3°: rt00fl( Arc—4:023:38; rdgxhuhmlm gghfluldrg ~0¢§ S; guru's-4%).“ r:§§s=8 Ire—089. ggggg §u8¢8uarhnuSIg§ g5: §;~§r rgt tact 0hr Bunsen :§ ugguflhe—"rr uggggzuu r flagerhsdrguuz gauze-88 mgr Eggsuflu ggr rghn?g§gu8~h (:8 3:80... r:§0r 0:: mlmrrrrrrrrrrrrrrrrrrrrrtmunaguerbgsrhgglfiu g8 :84)?" :85 :gu as? sugar “Vision :gs. lughlr rug (ash £>~§arbseur§§ grgghgu Sgt 003: Ornlrggdggg—ghtrhugghgrr Ir r r I II rrtr trr rrrrrtgugfinrgdtgxrr guargfigugugrrUgalrflt tgggugmscgfirrhgggaxrghrrrrrrrrr rrtrrrrrrrrrr<>=5§Hr§JI§0mnrt Fig. 14. TI and TGF-B amino acid sequence similarities. 61 in decreased protection fi'om ROS. Many studies have shown that changes associated with elevation in the levels of these enzymes, substrates and cofactors in cancer cell populations contribute to drug resistance (3 57-361, Section VI). By protecting cells from alkylating or oxidative processes, GSH and TDOR enzymes may decrease the cytotoxic efl‘ects of chemotherapeutic agents. Generation of ROS such as O," and' OH may be involved in tumor promotion (reviewed in 362). Antioxidants may inhibit tumor promotion; ROS generation is an immediate early event involved in the stimulation of cell growth; and H202 and 02" induce c-fos mRNA expression and DNA synthesis. As previously descnbed, TT reactivates oxidized enzymes, including GST (341), an important electrophile scavenger (355), and functions as an electron donor to GSH-PX (215), as well as reduces oxidized AA (80). V. Adriamyciu Adriarnycin (doxorubicin) is an anthracycline antitumor antibiotic most commonly used for the treatment of malignancies in acute leukemias, non-Hodgkin's lymphomas, Hodgkin's disease, and sarcomas (3 63), and is considered to be the most effective single agent in the treatment of advanced breast cancer (3 64). Adriarnycin was originally isolated from Streptomycespeucetuis caesins (365) and contains an aminosugar, daunosamine, linked through a glycosidic bond to a napthacenequinone, adriamycinone (Fig. 15). The mechanism of action of anthracyclines is unknown, although effects on cells include: 1) intercalation between DNA bases; 2) DNA strand scission; 3) altered plasma membrane structure; 4) disruption of electron transport; and 5) redox cycling between quinone and semiquinone, generating toxic ROS such as 02" in the process. Adriarnycin forms complexes with nucleotides, proteins, and a broad range of 62 biologically active compounds such as NAD+ and cafl‘eine (3 66) in aqueous solution at 37 molecular O2 to 02" in the presence of physiological thiol levels (3 86-3 88). Thiols, such as GSH, are'believed to reduce the complexed Fe3+ to the Fe” state, which reacts with H202 produced by 0; disrmitation forming OH via Fenton and Haber-Weiss reactions (Section I). Adriamycin—copper complexes analogously cause ROS-dependent damage (3 89). In addition, the Adriarnycin quinone moiety is enzymatically reduced to a semiquinone fiee radical form by NADH dehydrogenase (3 90,391), xanthine oxidase and cytochrome P450 reductase (3 92). The semiquinone free radical rapidly reacts with 02 to produce 02" (393-3 97). Both the C. Adriarnycin intercalates into DNA readily; the tetracyclic rings insert between and parallel to adjacent DNA base pairs, and the amino sugar ionically interacts with the minor groove agar-phosphate backbone (367). Interestingly, intercalation requires a reducing agent (3 68). Adriamycin-DNA interactions such as intercalation and adducts disrupt transcription and DNA replication (3 69,370). Adriarnycin has been shown to cause toxicity by action at the cell surface at or below the concentration causing other biochemical efi‘ects in the cell in vitro (371,372). Adriamycin inserts into the membrane bilayer by intercalation between anionic phospholipids (3 73), and modulates many membrane characteristics including lectin interaction (3 74), glycoprotein synthesis (375), phospholipid structure and organization (3 76), fluidity (3 77), fusion properties (3 78), transport of small molecules and ions (3 79), expression of hormones and receptors (377), spectrin and cardiolipin binding (380), and Na+ permeability (381). ROS are involved in most proposed mechanisms of Adriarnycin action. Adriarnycin is an efi'ective chelator of iron (3 82), abstracting Fe3+ from fenitin (383,384) and myoglobin (3 85). Adriarnycin-iron complexes are redoxrcatalysts capable of non- enzymatically reducing 63 Fig. 15. Adriarnycin structure. Adriamycin contains an amino sugar, daunosamine, linked through a glycosidic bond to a tetracyclic napthacequinone moiety, adn'amycinone. 64 enzymatic and non-enzymatic reactions can redox cycle, producing large amounts of ROS from one molecule of Adriarnycin (383,398,399). Hydroxyl radicals damage and cleave DNA, and cause lipid peroxidation and protein oxidation (Section 1). Further DNA damage results from Adriarnycin-stabilized topoisomerase II-DNA complexes (400), which cleave DNA Quinones react with sulfhydryl groups on GSH and proteins; therefore Adriamycin may interfere with cellular defense and repair systems by inactivation of GSH-dependent enzymes and various TDOR enzyme system components. Other cellular effects of Adriarnycin include calcium release from the sarcoplasmic reticulum and impaired mitochondrial calcium sequestration. Of-damaged cell membranes result in increased membrane permeability to Ca” and interference with mitochondrial electron transport. Adriarnycin-mediated 02" production occurs in the initial steps of electron transport (397), leading to the possible formation of a large number of free radicals. Increased cytoplasmic and nuclear calcium has been linked with Adriamycin-induced apoptosis (401- 403) High cardiotoxicity is a side effect of Adriarnycin chemotherapy, the etiology of which has not been clearly determined. A single dose of Adriarnycin rarely causes clinical heart failure, instead cardiac dysfunction generally develops slowly over a period of several weeks (reviewed in 404). Murine models indicate cardiac ROS defense mechanisms differ from other tissues (405,406), depending primarily on SOD and GSH peroxidase, as cardiac catalase activity is low. Adriarnycin treatment results in a rapid drop in cardiac GSH peroxidase. Cardiac toxicity is suggested to be due to a combination of decreased ROS detoxification and abundant cardiac mitochondria, which contain unusually active electron transport chains, generating large amounts of 02". 65 Cell culture studies adding antioxidants, or free radical scavengers (Section I) support a mechanism of action involving ROS. Addition of SOD and catalase to cell culture medium reduced Adriarnycin toxicity, supporting a mechanism for oxygen radical toxicity outside cells (407). AA addition significantly decreases Adriamycin-elevated lipid peroxide levels (408). Interestingly, AA administration to mice and guinea pigs prevented Adriamycin-induced cardiomyopathy (409). VI. Drug resistance Tumor cells are highly susceptible to anticancer drugs and radiation when compared with normal cells (356). The mechanism for this differential drug sensitivity is undetermined, although two popular theories exist: rapidly dividing cells are more susceptible to antitumor drugs which target DNA replication and cell cytoskeleton; and these drugs are more effective against tumors as they accumulate to higher concentrations inside tumor cells than in normal cells. Chemotherapy improves long-term survival in metastatic breast cancer where there is a possibility of tumor reoccurrence (reviewed in 410). However, most metastatic cancers are either intrinsically resistant to chemotherapy or respond to chemotherapy with a cell subpopulation resistant to the chemotherapeutic agent. Multidrug resistance (MDR) is a broad term for tumor cell mechanisms evading cytotoxic drug efi‘ects. MDR tumor cells have decreased sensitivity to a broad spectrum of drugs with no similar intracellular targets nor obvious structural homology. Drugs commonly involved in the MDR phenotype are generally natural products or their derivatives and include anthracyclines, vinca alkaloids, epidophyllotoxins, and actinomycin D (411). MDR cells are typically more resistant to the treatment drug than others (412,413). While the MDR 66 mechanism is unknown, many cellular changes correlate with the MDR phenotype. Common changes observed in cells that develop MDR are a decreased accumulation of cytotoxic drugs; changes in activity and expression of certain proteins; and changes in cellular physiology (reviewed in 414). In any MDR cell any or all of these changes may be noticed, suggesting that there may be more than one mechanism giving the MDR phenotype. The most common change in cells acquiring MDR is a decreased cellular accumulation of cytotoxic drugs; via either increased drug efllux (415-420), decreased drug influx (415,416,421-426), or decreased cytosolic (427-429) or subcellular (429,430) drug retention. Proposed mechanisms of MDR fall into seva'al categories. Drug emux models include ATP-dependent drug transport or increased exocytosis rate which removes drugs fi'om the cell; drug distribution models including reduced drug accumulation through an alkaline shift of cytoplasmic pH or changes in nuclear or plasma membrane permeability, and compartrnentation in cellular organelles. Theories include altered cellular fimctions changing drug sensitivity, such as changes in drug-DNA interactions; altered DNA repair; and changes in expression and function of detoxification enzymes (reviewed in 410,414). None of the proposed mechanisms can account for all of the MDR phenotypes observed. The predominant hypothesis is that chemotherapeutic agents difl‘use into the cell down a concentration gradient and that the drugs are removed from membranes or cytoplasm by ATP-driven "pumps" or "flippases" (reviewed in 410). Several potential drug transport proteins with strong homology to ATP-binding cassette (ABC) protein membrane transporters found in yeast, E. coli (431-433), the human major histocompatibility antigen peptide transporter (434), and the human cystic fibrosis membrane regulator (43 5-43 9) are overexpressed in MDR. P-glycoprotein (ng), the product of the human [MDR] gene, is a 67 170-Kda membrane glycoprotein associated with both the plasma membrane and internal organelles (440). MDR-associated protein, a 180 Kda protein, is found on intracellular organelles (441-443), and a 110 Kda protein is found primarily in lysosomes (444). A vacuolar I-F-A'I'Pase subunit is also overexpressed in MDR (445). ABC proteins are involved in translocating ligands, especially proteins, across membranes. Overexpression of ABC-type proteins also is associated with increased ion channels (435-43 9). The cystic fibrosis membrane regulator is a cAMP-controlled Cl' channel which regulates secretory activity in epithelia. Most of the research on MDR phenotypes has focused on these drug "transport" proteins, especially the ng, as ng is most commonly associated with MDR Studies demonstrated that ng can transport drugs (446,447); antibodies and ATP-pump inhibitors stop drug efilux (448-450); site-directed mutagenesis of ng residues results in drug specificity changes and alters efllux (451-453); and both drugs and MDR reversal agents (chemosensitizers) can bind the ng (448-450,4S4,455). ng is highly overexpressed in normal tissues such as adrenal gland, kidney, and pancreas, suggesting a fimction of normal secretion of metabolites into the bile, urine, and lumen of the gastrointestinal tract (456,457). ng overexpression is not associated with all MDR phenotypes. The broad ng substrate specificity is unusual for a membrane pump, as most membrane pumps show high substrate specificity. MRP expression also confers MDR to NIH-3T3 cells (458). Antitumor drugs are weak bases with pK,s between 7.4 and 8.2 (459-461) that readily traverse membranes when neutral in charge. Once protonated in the cell, antitumor drugs are retained and biologically active. Cellular pH afl‘ects both retention and exocytosis of drugs: binding of cytosolic targets such as DNA (462-467), RNA (466,468) and tubulin (469,470), 68 has an acidic optimum pH. The nucleus is the primary drug target although major drug accumulation sites are the trans-Golgi and the lysosomes (471,472), which are acidic cellular compartments involved in cellular exocytosis and endocytosis (429,43 0,473,474). Microscopic observations comparing cellular drug fluorescence in drug-sensitive and MDR lines find a shift in drug distribution in MDR cells fi'om nuclear to peripheral locations (429,475-480), with the drugs distributed primarily in the Golgi and lysosomes (429,471,472,474,475,481-487). Drug translocation across the nuclear membrane may facilitate these changes. Drug-sensitive tumor cells have a pH more acidic (6.85) than that of normal cells (7.6) (487) whereas the pH in MDR cells (7 .30-7.6) (481,488-490) resembles normal pH. Increased cellular pH correlates with elevated drug resistance in tumor cells (481), and raising the cytosolic pH of drug-sensitive cells using ammonium chloride or CO2 without MDR protein overexpression resulted in increased drug resistance (429). In addition, the MDR-like pH quantitatively accounted for the level of drug efflux seen in MDR cells. However, there are MDR cells with acidic cellular pH similar to the drug-sensitive lines (482). It is possible that in MDR, subcellular compartment pH changes occur undetectable when measuring total cellular pH, or that altered pH only can explain a subpopulation of MDR phenotypes. An alternate hypothesis is that these ABC-like proteins alter drug accumulation by influencing transrnernbrane ionic equilibria or secretory mechanisms. Transfection of cells with MRI results in both the MDR phenotype (550,551) and increased Cl‘ channel activity (432). ng overexpression may facilitate elevated cellular pH by functioning as a Cl‘ channel (414), decreasing proton entry into cells. Supporting this theory, drug-sensitive cells transfected with PgP have alkaline pH shifis (488) and increased Cl‘ conductance (435) in addition to the 69 display of MDR phenotypes. Changes in secretion have also been noted: ng overexpression in vinblastine-resistant human lymphoblastic leukemia cells correlates with enhanced exocytosis¥mediated secretion of lysosomal enzymes (491). Decreased drug influx is often observed in MDR cell lines (415-419,421-426). Since most antiturnor drugs are lipophilic and cross lipid membranes freely, hypotheses are that the plasma membrane structure has changed, limiting drug influx, or altering drug binding, afl‘ecting the transrnembrane signaling leading to cytotoxicity. Studies on membrane changes in resistant cell lines found that plasma membrane composition and binding characteristics change in the MDR phenotype (493,494). Anthracyclines such as Adriarnycin and Daunomycin bind to the plasma membrane of cells and exert a cytotoxic efl‘ect without entering the cell (3 72,404,492-497). Treatment of cells with difl‘erent detergents stimulates ng ATPase activity (498) and modifies the accumulation, binding (497), and cytotoxicity of antitumor drugs (426,499,500). MDR cells with enhanced membrane recycling (501,502) remove plasma membrane components to endosomes, vacuolar compartments and intracellular organelles (485,503), resulting in more drug ”sinks” in the cytoplasm that separate drug from target. The membrane recycling is inhibitable by Ca++ channel blockers and MDR reversal agents. MDR reversal agents or chemosensitizers cause physiological changes which alter drug accumulation in MDR cells. Chloroquine causes an alkaline shift of organelle pH, decreasing drug trapping in organelles (reviewed in 414). Amiloride (481) and veraparnil (483) acidify the cytoplasm, decreasing drug efllux, and accumulate in the lysosomes (484,485), altering lysosomal structure and function (430,486). This is accompanied by increased cytoplasmic and nuclear fluorescence intensity (429,47 5-479), indicating changes 70 in drug distribution and accumulation. In addition to previous mechanisms described, elevations in metabolic enzymes, detoxification enzymes, PKC activity (357-361), topoisomerase II activity, and altered GSH metabolism relate to the MDR phenotype without changes in transport processes that extrude drugs or drug-adducts. As anticancer drugs tend to be either strong alkylating agents, or quinones that participate in redox cycling (Sections I and V), MDR cells exhibit altered repair and protective processes. Decreases in drug metabolic activation resulting from downregulation of monooxygenase and related enzyme activities (504) also increase resistance in murine leukemia cells. Anticancer drugs such as acridines, actinomycins and anthracyclines inactivate replication and transcription by inducing topoisomerase II-mediated single-strand breaks in the DNA (505,507) (Section VI). Decreased topoisomerase II activity (SOS-509) reduces drug efl‘ects in some MDR phenotypes. Correlations have been observed between enhanced ng expression, thymidylate synthase, and metallothionein (Section I) (reviewed in 414). Enzymes commonly found overexpressed in MDR phenotypes include GSH-PX, GSH S-transferases, glucose 6- phosphate dehydrogenase (G6PDH, Section I), TRX (Section IV B), and TT (Section IV C). GSH-Px detoxifies radicals generated by redox active anticancer agents and GSH S- transferase protects cells by forming GSH adducts with alkylating agents. TT and TRX would presumably reverse the damage from oxidizing agents and fiee radicals. Glutathione disulfide reductase (Section I) provides for adequate GSH by reducing GSSG using NADPH generated by the oxidative portion of the pentose phosphate pathway [5,6], which includes G6PDH (Fig.16). 71 GSH levels have a significant role in cellular responses to antitumor agents. Cells exposed to oxidative anticancer agents or xenobiotics decrease the GSSG/GSH ratio during oxygen stress, probably due to the induction of the two synthetases involved in GSH production (Section III) [14,15]. Many cancer cells in both tumors and tissue culture express high levels of GSH-dependent enzymes, especially the class Pi GSTs and GSH-PX (510). Tumors that have acquired elevated resistance against cytotoxic drugs often display firrther increases in GSH and GSH-dependent enzymes (511-516). The demand for reduced GSH and NADPH is the ultimate support for the GSH- dependent enzyme systems. Numerous studies support MDR mechanisms involving GSH- and NADPH-based detoxification. GSH is elevated in many MDR cell lines (517). Mouse leukemia cells resistant to Adriarnycin had G-6-P levels and 6-PG levels that were nearly doubled (518,504) in addition to 4-fold elevations in glucuronyltransferase activity (504); and a human MDR line exposed to oxidizing agents had activated levels of pentose phosphate pathway enzymes (519), increased GSH, and large increases in GSH-PX activity. Many studies correlate changes in various GSH-dependent enzymes with MDR (407,520-523). An MDR human lung cancer line with no elevation in PgP and a 6-fold lower ' GSH concentration than the drug-sensitive line had elevations in GST and GS 86 reductase activities (521). An unusual GST with elevated organic peroxidase activity was increased along with Adriarnycin resistance (522) in human breast cancer cells. A study that mechanically disrupted human MCF-7 cell membranes transiently to introduce GST and GSH- Px without phenotype or genotype changes found increased resistance to Adriarnycin and quinones (407). In a vincristine-resistant human MCF-7 cell line, GST Pi was overexpressed, and increased activity of GST, GSH-PX, and PKC was noted (523). 72 GSH depletion increases tumor sensitivity to the effects of sulfliydryl-reactive chemotherapeutic drugs (524). MDR breast tumor cells preincubated with BSO showed increased drug sensitivity, where MDR cells given BSO and etoposide together had no changes in drug sensitivity (525). Neither drug emux nor drug retention changed significantly, and the efl‘ects were identical, although quantitatively smaller with drug sensitive breast tumor cells. Chemosensitization was therefore believed to be due to increased intracellular protein binding by sulfliydryl-reactive drugs. Overexpression of enzymes such as TRX and TT, capable of regenerating oxidized proteins via dithiol-disulfide exchange activity, also correlates with MDR phenotypes. A human MDR kidney cell line was transfected with TRX antisense DNA. MDR kidney cells that underexpressed TRX had decreased drug resistance (277). MCF-7 ADRR breast tumor cells have 4-fold elevations in TT activity over the MCF-7 drug-sensitive cells. Supplementation with an AA derivative, L-ascorbate 2-phosphate, resulted in firrther increases in resistance in the MDR line (526), indicating that perhaps both dithiol-disulfide transfer and DHA reduction play a role in resistance (Section VII). One common denominator in these studies is that individual enzyme activity levels and the changes in drug resistance are not quantitatively correlated, indicating that there are additional or synergistic mechanisms responsible for the MDR phenotype. VII. MCF-7 breast tumor cells: MCF-7 breast adenocarcinoma cells are a well-characterized line of cells preserving many of the biochemical and endocrinological characteristics of breast tissue. A sub- population of MCF-7 breast adenocarcinoma cells (MCF-7 ADR“) have developed a pleiotropic resistance to antitumor drugs after being cultured in the presence of increasing 73 doses of Adriarnycin (527), and demonstrate up to a lOOO-fold increased viability in the presence of Adriarnycin as compared to the parental sensitive MCF-7 strain. Grth of these MCF-7 ADRR cells in culture is hormone-independent; when transplanted into nude mice, MCF-7 ADRR cells promote estrogen-independent tumor growth. Membrane characteristics changed in MDR include elevated epidermal grth factor receptor levels, increased ng levels, and decreased estrogen receptor levels. Previous studies on the mechanism of Adriarnycin cytotoxicity in MCF-7 WT cells have implicated the generation of toxic ROS (407,517,528-542). As greater doses of Adriarnycin are administered to MCF-7 cells, increasing levels of ROS are apparent by electron spin resonance (3 93). Pleiotropic mechanisms producing increased Adriarnycin resistance in MCF-7 ADRR cells include decreased cellular drug accumulation, especially in the nucleus; increased drug detoxification, and increased DNA repair activity. In addition, MCF-7 ADRR cells generate several-fold less OH from Adriarnycin redox cycling (528), are less sensitive to extracellular and intracellular ROS (529), and are sensitized to Adriarnycin cytotoxicity after GSH depletion with BSO (436) (Section III). Postulated mechanisms for these changes in the MCF-7 ADRR cells include the increased expression of both seleno-cysteine containing GSH- Px (543), GST with intrinsic peroxidase activity (522), increased expression of PgP drug transporter protein (544), decreased estrogen dependence, diminished drug-activating enzyme cytochrome P-4501al activity (545), moderately increased SOD activity (543), and elevated cytosolic pH (429) (Table 3). Studies transfecting the GST Pi (546), p. and a (547) demonstrated that overexpression of these isozymes alone is not suflicient to confer resistance to Adriarnycin or other anticancer agents that generate ROS. 74 Fig. 16. GSSG reductase, GSH, and G6PDH interrelationships . In aerobic cells, NADPH is generated primarily in the pentose phosphate pathway via (1) G6PDH and (2) 6-phosphogluconate dehydrogenase. The NADPH generated is required for biosynthesis; reduction of GSSG to GSH via (3) GSSG reductase; metabolism of Adriarnycin to Adriamycinol via (4) daunorubicin reductase, and activation of Adriarnycin to a semi- quinone radical by (5) various NADPH-dependent flavoprotein reductases such as cytochrome P450 reductase. Reduced GSH is used to (*) either reduce glutathionylated protein -SH groups or disulfide bonds by (6) TDOR enzymes such as 'IT, and PDI, or to detoxify H202 by (7) GSH peroxidases. (8) SOD dismutation of O2 (generated fi'om Adriamycin redox cycling between quinone and semiquinone in the presence of 01) produces H202. NADPH and GSH production are essential for these processes removing ROS. Abbreviations: G6P=glucose 6 phosphate, G6PDH= glucose 6-phosphate dehydrogenase, 6PG= 6-phosphogluconate, R5P= rrbulose S-phosphate, GSH= glutathione, GSSG= glutathione disulfide, SOD= superoxide dismutase, NADPH= nicotinarnide dinucleotide phosphate, reduced, TDOR= thiol-disulfide oxidoreductase. 75 +QDEm_au< No m £26523. two W .mo +I N cozmmacoo :_o>Em_.6< Ew=onEoE / .oc_o>Em:u< Iw/ £995 wamv :m\ V .o wWESEd . » oE>~co mock V iv ONI N Gmww eh NONI Imam mmm +1 + Ian—<2 . ; +QD80°/o) across mammalian species (rabbit, cow, pig) was noted for 'IT (307). Predominant characteristics for Us are the highly conserved active site residues CPF/Y C, which have been shown to be essential for function by site-directed mutagenesis studies (100); two additional half-cystines (CXXXC) found in the C terminal which are not essential for function (100); and high amino acid conservation throughout with a less conserved C-terminal. The recently determined crystal structure of pig liver TI‘ determind at 2.2 A (129) showed the N-terminal to be near the active site, both of which are located on the protein surface. To explore human 11‘ structure and distribution, the cDNA was cloned fi'om a human placental cDNA library, and sequenced. The tissue distribution of TT mRNA expression was examined Human erythrocyte TT was purified, and used to generate polyclonal antibodies. These polyclonal antibodies were then used to screen pooled protein samples from various human tissues. 79 £— 80 Materials Restriction endonucleases were purchased from New England Biolabs (NEB), Boehrringer- Mannheim Biochemical (BMB), and Gibco-BRL. DNA fragments were purified through Spin-X 22 um cellulose acetate membranes (Costar). The 1 kb DNA ladder was purchased from Gibco-BRL. Competent DH5a E. Coli were purchased fi'om Clontech. Standard reagents were purchased from Sigma, Difco Laboratories, BMB, Gibco-BRL and NEB. Glutathione disulfide (GSSG) reductase, glutathione (GSH), and reduced nicotinarnide dinucleotide phosphate (NADPH) were purchased fiom BMB, and L-ascorbate (AA) was purchased fiom Sigma Chemical Co. Dehydroascorbate (DHA) was generated by the bromine oxidation of 20 mM Ascorbic acid (AA), following the procedure of Bode et al (552). LE3 92 E. coli host strain was a gift from Brian Smith-White. Methods Pig liver TT probe generation: The expression vector pKK233-2 containing the pig liver TT cDNA insert (553) was amplified in JMIOS E. coli and isolated using standard alkaline lysis followed by ion-exchange chromatography (554, Qiagen, Promega). Purified plasmid DNA was then digested with Neal and PW” restriction endonucleases to release a 423 bp cDNA fragment containing 318 bp of coding sequence, and 105 bp of 3' untranslated sequence (Fig. 17). This 423 bp cDNA fragment was isolated following electrophoresis and purification through Spin-X membranes (Costar), then radiolabelled (by random priming) with a-32P dCTP (3000 Ci/mmol, DuPont New England Nuclear, NEN) as in Feinberg and Vogelstein (554). 81 Southern analysis of human genomic DNA: Human genomic DNA (Novagen, cat# 69237-1, lot #1) was individually digested with restriction endonucleases EcoRI, HindIII, Neal and )0201 overnight, denatured in 10 mM Tris-HCl, pH 8.0, 1 mM EDTA at 65 °C for 10 min. Digested DNA (20 pg of each digest), together with Ficoll loading bufl‘er (0.25% bromophenol blue, Sigma, 0.25% xylene cyanol FF , Sigma, 15% Ficoll, type IV, Pharmacia) was electrophoresed on a 0.8% agarose gel in 0.5X TBE (45 mM Tris-borate, 1 mM EDTA) with 0.125 mg/ml ethidium bromide for 16 h at 20 mA. The DNA in the gel was then depurinated 15 min. using 0.25 N HCl, and transferred to Hybond N+ (Amersham) positively charged nylon membranes for 90 min using 0.5 N NaOH as the vehicle using a Hoefl‘er TransBlot vacuum transfer apparatus. Transferred blots were rinsed with 0.1X SSC (150 mM NaCl, 15 mM sodium citrate, pH 8.0) to remove excess salt, and baked at 80°C for 20 min under vacuum. Once dry, the membranes were prehybridized for 2 h with 5x Denhardt’s (0.01% Ficoll, Type 400, Pharmacia, 0.01% polyvirrylpyrrolidone, 0.01% BSA, Fraction V, Sigma), 0.5% SDS, 6X SSC (0.9 M NaCl, 90 mM sodium citrate, pH 8.0) at 65°C. Hybridization was performed in the same bufl‘er ' containing 5 x 10‘ dpm/ml”P radiolabeled probe at 60°C for 20 h. Successive washes at 65°C in 2X SSC, 0.1% SDS, 1X SSC, 0.5% SDS, and 0.5X SSC, 0.5% SDS were followed by aroom temperature rinse in 0.1 X SSC. Membranes were exposed to fihn at -70°C for 24 h. Screening the human placental cDNA library: A human placental Agtll cDNA library with a high proportion of low molecular weight cDNAs (Clorrtech) was screened with the 423 bp pig liver 'IT cDNA fiagment probe. LE 392 E. coli host cells were grown overnight in LB media in the presence of 0. 1% maltose. 82 EcoRl 5251 Ncol 4977 HinDlll Sphl .. Pstl pLTT Hlncll Accl val 1053 ’0’" pLTT/pKK233-2 Xmal 5255 9P 50°F“ AmpR Pvull 1694 Pvul 3363 Fig. 17. Generation of 423 bp pig liver 11‘ probe. Plasmid pKK233-2 DNA containing the pig liver TT cDNA insert and M13 polylinker sequences (552) was subjected to restriction digestion with N001 and M11. A 423 bp cDNA fragment containing the entire 318 bp coding sequence (fiom +1 to +318 relative to the translational start site) and 105 bp of untranslated sequence 3' of the coding sequence was isolated as described in Methods. 83 Approximately 10‘ pfu of A phage we‘e then added to 200 pl bacteria, and incubated 15 min at 37°C. The infected host cells were then plated in 0.7% LB agarose on 1.5% LB agar plates, and'ineubated 6-8 h at 37 °C until plaques were evident. Colony-Plaque membranes (NEN) were used in duplicate for immobilization of phage DNA. These membranes were thentneated with 0.1NNaOl-1, 1.5 MNaCl for 2 min to lyse the bacteria, neutralized twice with 1.0 M Tris-HCl, pH 7.5 for 3 min, then rinsed in 2X SSC (0.3 M NaCl, 30 mM sodium citrate, pH 8.0) and air dried to fix the DNA to the membrane. Once dry, the membranes were prehybridized 2 h with 5x Denhardt’s (0.1% Ficoll, Type 400, Pharmacia, 0.1% polyvinylpyrrolidone, 0.1% BSA, Fraction V, Sigma), 0.5% SDS, 6X SSC at 65°C, then hybridized in the same bufi‘er containing 5 x 10‘ dpm/ml 32P radiolabeled 423 bp probe at 65°C for 16-24 h Positive signals on duplicate filters were used to locate the corresponding positiveplaques. Plaqueswereextractedwithasterilepipetbase into SM (100 mMNaCl, 8.1 mM MgSO,, 50 mM Tris-Cl, pH 7.5, 0.01% gelatin), and left at 4°C overnight. Four positive plaques were found in 10,000 independent plaques. These were purified to homogeneity by four rounds of re-screening and plaque isolation, as described above. Subcloning and sequencing the TT cDNA: Liquid culture lysate method (556) was used to generate large amounts of phage DNA LE 392 E. coli were grown to stationary phase in LB with 0.1% maltose, then 200 ul bacterial growth were infected with 10“ plaque-forming units (pfu) of phage for 15 min at 37 °C to allow phage to adsorb. Four ml NZCY media was added, and the samples were agitated at 37°C for 6-12 h to completely lyse the bacteria. Lysed bacteria were treated with 2 drops of chloroform for 15 min at 37 °C, and bacterial debris was removed afier centrifugation at 4000 x g at 4°C for 10 min The supernatant solution containing the phage was treated with 1 drop 84 of chloroform, and placed at 4°C until phage DNA was harvested. lambda DNA was purified using the MagicTM DNA purification system (Promega) according to maufacturer's instructions. Human 'I'T inserts were PCR amplified from A clone DNA using a Perkin-Elmer 9600 Thermocycler with 25 rounds of therrnocycling at 95°C for 30 secs, followed by 55 °C for 30 sees, and then 72°C for 1 min using Taq DNA polymerase (BMB) and Agtll B- galactosidase sequencing primers (5'-dGGTGGCGACGACTCCTGGAGCCCG-3' and 5'- dTTGACACCAGACCAACTGGTAATG-3', NEB) which flank the TT cDNA insert on either side (Fig. 18). PCR-generated fi'agrnents of approximately 1 kb were isolated and subcloned into the PCRII plasmid (Invitrogen) as follows. Linear PCRII DNA and 38-40 ng of the PCR fiagments were ligated in a 1:3 molar ratio at 16°C overnight. Eight or nine ng of the resultant constructs designated pCRl, pCR2, pCR3, and pCR4 (Fig. 19) were then used to transform INVaF' supercompetent E coli (Invitrogen) and plated on LB plates containing 100 rig/ml arnpicillin and 75 rig/ml X-gal. Four separate recombinant plasmids were isolated, amplified in E. coli, and purified using the Wizard1M (Promega) purification system according to manufacturer's recommendations. Complete sequencing in both orientations of the TT inserts from the double-stranded templates was performed using a combination of 35S dATP, Sequenase 2.0 enzyme and reagents and automated fluorescent sequencing by the MSU- DOE-PRL Plant Biochemistry Facility using the ABI Catalyst 800 for Taq cycle sequencing and the ABI 373 A Sequencer for the analysis of products. Primers used for this sequencing were M13 primers and internal human TT specific nt sequence primers corresponding to A”‘QEILSQ35 and T”RLKQIGAL‘°‘ (Macromolecular Structure Facility, MSU, Fig.20). All four approximately 1 kb inserts were sequenced completely. 85 Fig. 18. PCR amplification of cDNA inserts. A. gtll contains two phage arms of approximately 24,100 and 19,600 bp, and insert cDNA, cloned in at EcoRI sites. The E coli B-galactosidase gene is interrupted by the cDNA insert. hpTT cDNA inserts were PCR amplified as described in Methods using the B-galactosidase sequencing primers for kgtll (NEB). PCR-generated fi'agments of approximately 1 kb were isolated and subcloned into pCRII to garerate pCRl, pCR2, pCR3, and pCR4, as described in Methods. Lane 1: 1 kb DNA ladder, lane 2-5 Purified DNA from pCRl, pCR2, pCR3, and pCR4, respectively, was digested with EcoRI to release the cDNA insert. pCRII is a cloning vector for PCR products that contains anEcoRIsiteon either side ofthe A-overhangs. DigestionwithEcoRIwill result in the release of any insert. All the hpTT cDNA clones are the same size, of approximately 1 kb. 86 3054— 2036— 1636 — 1018 — 506,517; —TT Fig. 18. PCR amplification of cDNA inserts. 87 Fig. 19. Construction of pCR4. PCR-generated fragments of approximately 1 kb from four separate Agtll clones were subcloned into pCRII as described in Methods. PCRII contains 5’ T-overhangs to facilitate ligation to Taq polymerase-generated arnplicons with 3'A-overhangs. The resultant constructs were designated pCRl, pCR2, pCR3, and pCR4. " C a,a 6' v .‘1' 3 ~. ColElori '7 Seal 2439 __ lNsilHinalllenLSacLBaml-WEEHI] ColElori LacZ * insert A R pCR4 mp 4850 bp . K F "a. . ll orl Nco|1881 Seal 2439 Fig. 19 Construction of pCR4. EcoRl BstXl Notl Aval PaeR71 Xhol Nsil Xbal Apal 89 Fig. 20. Hp'I'I‘ cDNA sequence and deduced amino acid sequence. The entire 318 bp coding region of the hpTT cDNA is shown (uppercase) and the corresponding deduced arrrino acid sequence (below). In addition, 106 bp of 5'-UTK and 438 bp of 3' UTR were also sequenced (lowercase). The translational start and stop codons are highlighted in hatched boxes. All numbers shown are relative to the putative ATG translational start site. The 3'-UTR is 438 nucleotides long with a putative polyadenylation tract (uppercase) 19 nucleotides after. The conserved active site CnPYC” is shown in bold type. Major difl‘erences between human TT and that of other mammalian TTs include the lack of an internal methionine residue and an additional cysteine at position 7. Primers used for sequencing are denoted by lines. 90 5'-gacaccaga ccaactggta atggtagcga ccggcgctca gctggttaaa atacctgcaa ctgaggattc ttcccgggga gaccgcagcc GCT CAR GAG TTT GTG AAC TGC AAA.ATC CAG CCT A Q E GTT GTT TTC V V F ATC GTC TAT TTT TCT ATT K Q I aatgttcaac atgaaaagca agaggctgtg taatcctgaa aagcatgaaa agtgtatctg tcatgaagtt tctagtgaca F V N C K I Q P ATC AAG CCC ACC TGC CCC TAC TGC I K P T C P I C CTC AGT CAA TTG CCC ATC AAA CAA L s Q L P I K Q GAT ATC ACA GCC ACC AAC CAC ACT D I T A T N H T TTG CAA CAG CTC ACG GGA GCA AGA LQQLTGAR ATT GGT AAA GAT TGT ATA.GGC GGG I G K D C I G G TTG CAA CAG AGT GGG GAA CTT CTT L Q Q S G E L L GGA GCT CTT CAG TAA ccaccacaga G A L Q * aattctgtga aaggtcacag gacccaattg tagttggtct tggtgtcata tggatcagag gtcatgcgga acactctgtt atttaagatg cactgtgtat ttattttatt tagactacca tgtaaaacat ctgataaaac ttacagcccc tgaaagagct cctacacttt gaaaacttaa tgcctgttct agaattgtaa gttgttaatt acacttaatt tctttctAAT AAAAAAAAcc tcagtgAAAA AAAAAAAA Fig. 20. HpTT cDNA sequence and deduced amino acid sequence. tcccctagca catcggc ATG It GGG RAG GTG G K V AGG AGG GCC R R A GGG CTT CTG G L L AAC GAG ATT N E I ACG GTG CCT T V P TGC AGT GAT C S D ACG CGG CTA T R L tctcatagga gagaaatcat gcacaagtgc gctatccaga gcaaagatta ctacaccaag gaatccctta tccttcaatc tatagatgat -101 -51 45 [14] 87 [28] 129 [42] 171 [56] 213 [70] 255 [84] 297 [98] 347 [105] 397 447 497 547 597 647 697 747 765 91 Human erythrocyte '1‘1‘ Purification: The human ‘I'l‘ purification procedure followed a modified procedure of Terada et a]. (316). Six units of outdated packed red cells were received from the Red Cross, placed on ice, and washed twice with 0.9% NaCl. Each wash was followed by centrifugation at 4080 x g in a Sorvall RC2B centrifuge with a GSA rotor for 15 nrin at 4°C, and the supernatants fi'om each wash were discarded. Erythrocytes were hernolyzed with 2 volumes of 10 mM NaHzPO“ pH 6.0 (Buffer A). The hemolysate pH was subsequently adjusted to 6.0 with 7% acetic acid, then heat treated at 65°C for 5 min with subsequent cooling at 4°C overnight. Two volumes of Buffer A were added to the heat-treated hemolysate prior to centrifugation at 10,800 x g for 15 min at 4°C. The 840 ml supematant solution was treated with 105 g pre-swelled CM- cellulose or CM-Sepharose to remove hemoglobin. 4 l of hemolysate supernatant were precipitated with 85% ammonium sulfate and pelleted at 16,000 x g for 15 min at 4°C. The precipitated pellets were resuspended in Bufi‘er A and dialyzed using Spectrapor 1 (mwco 6000-8000) tubing against four 4-l Buffer A changes. After centrifugation at 10,000 x g for 15 nrin at 4°C to remove particulate matter, the dialyzed sample was applied in 20 consecutive 100-160 ml aliquots to a Sephadex G-75 column (85 cm x 6 cm). TT was fractionated fi'om larger proteins using a running buffer of 50 mM Nal‘IZPO4 (pH 6.0). Fractions were assayed for T1“ activity as described below and active fractions were collected, pooled and concentrated under 50 psi N2 using a Diaflo PM 10,000 mwco membrane and an Arrricon concentrator. The partially purified fi'actions were then reduced with 5 mM DTT at 30°C for 30 min, dialyzed against two 2 1 buffer changes of Nz-saturated 100 mM Tris-Cl, pH 7.5, 5 mM EDTA, 5 M NaCl (Buffer B) and applied at 10 ml/h to a thiopropyl sepharose 63 column (2 ml bed volume, Pharmacia) equilibrated with 50 ml of Bufl‘er B. After a 100 92 ml wash with Nz-saturated Buffer B, 100 ml 100 mM NaOAc, pH 4.6, with 5 mM 0- mercaptoethanol was applied to the column. No TT activity was noted in either of these washes. 'IT was eluted with a 160-ml 20 mM-to-50 mM DTT gradient in a 100 mM Tris-Cl, pH 8.0 bufi‘er. Fractions were assayed for TT activity as described below. Protein Assay: Protein samples and bovine serum albumin (BSA) standards were precipitated in ice-cold acetone at -20 C for 30 nrin, and then pelleted to remove the D'IT. After acetone evaporation, the samples and BSA controls were resuspended in 0.01 N NaOH, and protein concentrations were determined by the bicinchinoninic acid (BCA) protein assay protocol according to the maunfacturer’s directions (Pierce Chemical Co.). TT Activity Assay: The enzyme activity was assayed as described by Gan and Wells (314). IT activity was measured as the GSH-dependent reduction of the prototype substrate S-sulfocysteine (Cys- SO3‘). The standard TI‘ assay mixture contained enzyme, 0.5 mM GSH, 1.4 U of glutathione reductase, 2.5 mM S-sulfocysteine (Cys-$0,“), 0.35 mM NADPH, 0.137 M sodium phosphate bufl‘er, pH 7.5. The reaction was initiated by the addition of Cys-SO31 Formation of GS SG was coupled to NADPH oxidation by glutathione reductase and measured spectrophotometrically by a decrease in Am at 30°C relative to a blank reaction without enzyme simultaneously monitored. One unit of TT activity is defined as that amount of enzyme catalyzing the formation of l umole of GS SG per nrin under standard conditions (314). Samples were measured in triplicate. 93 DHA Reductase activity assay: DHA reductase (DHAR) assays followed the direct spectrophotometric assay of Stahl er al. (557) following the relative change in absorbance at 265.5 nm as DHA is reduced to AA. Standard assay conditions Were enzyme, 0.137 M sodium phosphate bufi‘er, pH 6.8, 1 mM EDTA, 2 mM GSH, and 1 mM DHA incubated at 30°C. Blanks were run without the enzyme. The reaction was initiated by the addition of DHA, and was linear up to 2 min at 30°C. One unit of DHA reductase activity is defined as that amount of enzyme catalyzing the formation of one pmole of AA per rrrin under standard conditions (557). Rabbit anti-'I'l‘ antibody generation: Polyclonal antibodies were raised by subcutaneous immunization of two female New Zealand rabbits with 240-360 pg of human erythrocyte TT together with 0.5 ml Freund's complete adjuvant, followed four weeks later with 225 pg TT in 0. 5 ml Freunds' incomplete adjuvant. Blood was collected approximately every two to three weeks for seven months and antisera retained. Antisera were titered using dot blots against 0.01-1.00 pg recombinant pig liver TT, purified human erythrocyte TT and purified recombinant human placental TT. Rabbits were boosted with approximately 250 pg TT for increased immune response each time the titer decreased substantially. SDS-PAGE and Western analysis of purified erythrocyte 'IT: Laemmli SDS-PAGE analysis (558), using a 6% stacking gel and 15% separating gel was used. Samples were loaded in 2X SDS loading buffer, and electrophoresed for 1 h at 200V using a Tris-glycine-SDS buffer. One gel was stained with Coomassie Brilliant Blue R-250, while proteins from a duplicate gel were electrophoretically transferred to a nitrocellulose filter for 1 h at 100V using a 25 mM Tris-HCl (pH), 92 mM glycine, 20% methanol bufi‘er. 94 Non-specific binding was blocked by incubating the membrane overnight at 4 C in 0.1% Tween-20-TBS (Buffer A) with 5% non-fat dry milk. After blocking, a 4 rrrin wash in bufl‘er A was performed followed by incubation with the primary rabbit anti-human TT antibody (1:20,000) in Buffer A for 60 nrin at room temperature. The blot was then rinsed twice in Bufi‘er A for 5 nrin at room temperature, and incubated with the secondary alkaline phosphatase-conjugated goat anti-rabbit antibody (1:3000) in Buffer A for 60 min at room temperature. After the blot was rinsed two times for 5 min, the blot was incubated with BCIP and NBT (BioRad) to visualize immobilized TT bands. Western blots of pooled human tissues: A commercially prepared Western blot (Clontech) containing 75 pg total protein fi'om each tissue (skeletal muscle, liver, heart, lung, brain, kidney) per lane and stained with Ponceau S was photographed (Fig.26), rehydrated in 100% MeOI-I, then equilibrated with water. The membrane was then rinsed with 1X phosphate-bufi‘ered saline (PB S) for 10 nrin and blocked for 2 h in PBS containing 5% nonfat dry milk at room temperature. After blocking, a 5 min wash in 0.2% Tween-20, TBS (Buffer A) was performed followed by incubation with the primary rabbit anti-human TT antibody (1 :20,000) in Bufi‘er A containing 1% milk (Bufl‘er B) for 60 nrin at room temperature. The blot was then rinsed twice in Bufl‘er A at room temperature for 15 min, and incubated with secondary HRP-conjugated goat anti-rabbit antibody (1 12000, Arnersharn) in Bufi‘er B for 60 rrrin at room temperature. Alter briefly rinsing in Buffer A, the blot was washed once for 15 min, then 3 times for 5 min each in Bufl‘er A The ECL chemiluminescent detection system (Amersham) was used to visualize the signals for one min, and the blot was exposed to ECL-Hyperfilm (Amersham) for 90 sec. 95 HpTT probe generation: The plasmid PCR4 containing the human TT cDNA insert was digested with EcoRI and Sac! to release a 304 bp cDNA fi'agment containing the entire coding region except for the nucleotides corresponding to the five terminal amino acids. This fragment was subcloned into pT7T318U (Pharmacia) at the EcoRI and Sac] sites (F ig.21), and was purified and random-primed to generate a probe as previously described for the pig cDNA fiagment. Northern blots of pooled human tissues: Connnercially prepared Northern blots (Clontech) containing poly A+ mRNA fiom pooled human tissues (pancreas, testis, ovary, brain, colon, small intestine, skeletal muscle, prostate, liver, thymus, lung, peripheral blood leukocyte, spleen, placenta, kidney, and heart) were probed using a radiolabeled 304 nt human placental TT cDNA probe. Prehybridization of the membranes at 42°C for 6 h in 10X Denhardt's (0.02% Ficoll, Type 400, Pharmacia, 0.02% polyvinylpyrrolidone, 0.02% BSA, Fraction V, Sigma), 5X SSPE (0.75 M NaCl, 0.05 M NaH,PO,, 5 mM EDTA, pH 7 .4), 50% formarnide, 2% SDS and 100 pg/ml freshly denatured salmon sperm DNA was followed by hybridization for 18 h with 5 x 10’ dpm/ml 32P radiolabeled 304 nt human placental TT cDNA fi'agrnent, then washed twice with 2X SSC (300 mM NaCl, 30 mM sodium citrate) containing 0.05% SDS at room temperature. After exposure to X-OMAT film (Kodak) for 48 h at -70°C, the membrane was stripped in 0.01% SDS at 60°C for 30 min, and reprobed with 5 x 10’ dpm/ml 32P radiolabeled S-actin cDNA probe (Clontech) in hybridization buffer, washed as previously described, and exposed to film for 24 h at -70°C. Densitometry using the Biolrnage Visage 110 system (Milligen, Ann Arbor, MI) was used to quantitate the signal intensity in order to determine relative amounts of TT expression. TT signal intensities were normalized to mRN A concentration, as poly 96 Hmdm Psfl SaH HMcH it“: a W' BamHl vull Aval F10” Bar“ Kpnl " " 13le Sad Sad Trace L392 nl pTT500-2 3176 99 Pqu Pvul AmpR pBR3220fl Bam Fig. 21. Generation of the 425 bp hp'I'I‘ probe. Purified pCR4 DNA was subjected to digestion by EcoRI and Sac]. Digestion of pCR4 DNA withEcoRI and Sac] generates four fiagmerrts; the EcoRI-EcoRI plasmid fiagment (3 977 nt); a central SacI-Sacl fiagment (299 nt); an EcoRI-Sacl fragment (153 nt) corresponding to 3'- UTR sequences and 5 nucleotides from the pCR4 vector that flank the cDNA insert to the 3' EcoRI site; and the EcoRI-Sacl probe fiagment (425 nt) corresponding to the entire 5'- UTR (106 nucleotides) and coding sequence (+1 nt to + 311 nt relative to the translational start site) of hpTT, as well as 3 nt 5' from the pCR4 vector that flank the cDNA insert to the EcoRI site. This 425 bp fiagment was then subcloned into purified EcoRI and Sac] -digested pT7T318U, as described in methods, and designated pTT500-2. The probe was isolated by EcoRI-Sac] digestion from pT7T3 18U and purified as described in Methods. 97 A+ mRNA arnormts were adjusted by the manufacturer to yield a detectable B-actin signal in every lane. Results Cloning the hpTT cDNA: Four Agtll clones containing human placental TT were purified to homogeneity through repeated rounds of screening. Upon phage DNA isolation and purification, EcoRl digestion of the cloned phage DNA yielded unexpected results. Because EcoRI restriction sites flank the cloned cDNA insert in the A phage arms, it is expected that EcoRI digestion would result in the release of the cDNA insert fi'om the two arms of the 1 phage, generating two phage arms of approximately 24,100 and 19,600 bp in length, and the insert cDNA (Fig. 18). However, EcoRI digests of the four human TT clones apparantly partially digested the phage DNA, producing fragments appearing to be greater than 23000 bp with no smaller bands observable (F ig.22). Because each of the four isolated human TT 1 clones displayed similar refiactory properties to EcoRI digestion, a possible problem with the library construction, and not an isolated problem with an individual A clone is hypothesized. In order to circumvent this digestion problem, primers flanking the cDNA insert were used to PCR amplify the inserts in these four human 'IT cDNA clones. PCR amplified fragments were then subcloned into pCRII and sequenced. The sequences of all four clones were identical, indicating that there were no PCR-introduced errors in this amplification and isolation of the human placental 11‘ cDNA inserts (Fig.20). Human genomic Southern analysis: Southern analysis showed that genomic fiagments encoding hpTT. Hindi]! and Ned digestion of human genomic DNA resulted in approximately 4.5 kb fragments that putatively 98 encoded the entire human 11‘ gene. )flrol digestion resulted in a approximately 6 kb fragment hybridizing to the full-length cDNA, where EcoRI digestion resulted in several bands (10, 8, 7 .5, 6, and 3.1 kb, approximately). The Southern did not indicate the presence of any pseudogenes, but did indicate that there may be EcoRI sites in the promoter region or in introns (Fig. 23). There are no EcoRI sites in the hpTT cDNA. Human erythrocyte 'I‘I‘ purification: TT was purified from 6 units of packed human erythrocytes donated by the American Red Cross (Table 4, Fig.24). The purified enzyme produced a single band upon both Coomassie -stained SDS-PAGE (Fig.25) and western analysis using rabbit anti-recombinant pig liver TT antibodies (Fig.26). The apparent molecular weight of 11,300, as determined by gel electrophoresis agrees with that previously determined for human TT (317). The purified protein had an associated thiol-disulfide exchange activity of 118.7 U/mg comparable to that previously reported (321) and DHA reductase activity of 22.4 comparable to that reported by (310). T'l‘s are in general quite stable proteins; resistant to heat treatment, and stable upon storage. Purification of TTs are therefore generally straightforward. Human TT is an exception, being very susceptible to oxidation during the purification, resulting in denaturation and aggregation. Reducing human TT with DTT during the purification decreased the oxidation problem, but resulted in difliculty interpreting protein concentrations and DHA reductase activity by interfering with assay measurements. 1. Anti-human erythrocyte 'I'T antibody generation: Polyclonal antibodies were produced against purified human erythrocyte TT. All antisera exhibited cross-reactivity towards pig liver 'IT and human TT. Antisera collected at different 99 123456789 5090 _ 4072 - 3054 '- 2036 — 1636 — 1018— ‘ PLTT 506,517— Fig. 22. [£le digestion of Agtll hpTT cDNA clones. Shown here are EcoRI digests of purified lambda DNA from three hpTT thllclones, compared to EcoRI digestion of purified lambda DNA fiom the pig liver TT hgtll clone. Lane l-blank, lane 2- 1 kb molecular weight marker, lane 3- blank, lanes 4—7: EcoRI-digested Agtll DNA lane 4- hpTT clone 1, lane 5- hpTT clone 2, lane 6- pig liver TT, lane 7- hpTT clone 4. Not shown' kgtll hpTT clone 3, result is the same as the other three human clones. None of the lgtll human cDNA clones had inserts that were released by EcoRI digestion, whereas the pig kgtll cDNA was released from lambda DNA. The hpTT 1g t11 clones are partially digested by EcoRI, as two lambda arms of high molecular weight are evident. A problem with the library construction is hypothesized, as the same problem existed for all the hpTT lambda clones. 100 Fig. 23. Southern analysis of human genomic DNA. A. Ethiduim bromide stained 0.8% agarose gel with human genomic DNA digests. Lane 1- EcoRI, lane 2-HindIII, lane 3- Non], lane 4- XhoI, lane 5- blank, lane 6-1 kb DNA ladder. Genomic smears were readily produced by digestion with EcoRI, HindIII, and N001. Xhol digestion did not produce a strong visible smear. B. Southern blot. The gel in A was transferred to a Hybond N+ membrane (Amersham), and probed with a 423 pig liver TT probe as described in Methods. Neal and Hincflll digests indicate that the entire gene for human TT is encoded in approximately 4.5 kb. Xhol digestion resulted in a single, larger genomic fragment of about 6 kb. EcoRI digestion gave multiple bands, ranging fi'om 10, 8, 7,5, 6, and 3.1 kb, approximately. There may be an EcoRI site in the promoter region or in the intron structure of the TT gene. There is no indication that multiple genes or pseudogenes for TT exist. B 1 2 3 4 T —’— o — T 15 — .. 1o — .4 6 — -- W 5090 4'5 — ~ —4072 3-1 — —3054 —2036 . O o —1636 i —1018 Fig. 23. Southern analysis of human genomic DNA. 102 Fig. 24. Thiopropyl sepharose 6B purification of human erythrocyte 'I'l‘. Reduced human erythrocyte TT was eluted fi'om a thiol-disulfide affinity exchange column using a 20-50 mM DTT gradient as described in Methods. TT activity and elevated protein levels correspond to the same eluted fractions. Protein concentration is on the left (blue) and TT activity is on the right (red). ‘ 103 TTase Purification Profile thiopropyl sepharose GB column 610 0.25 A v 81 oz 2 .5 ._ i E E 5. 015 g r: .. . 5 8 4 . 0.1 > c a: 8 " . ' 3: g 2 i W‘ J\ (50.05 8 § 0 -fl—l—i—F+—H—++H+++++++WHO : 0- 1 4 7101316192225283134 Tube number Fig. 24 Thiopropyl sepharose 6B purification of human erythrocyte TT. 104 Table 4. Human erythrocyte 'I'I‘ purification. CM-cellulose Sephadex G75 and Diaflo (10,000 mwco) Thiopropyl . . harose 6B TT activity and protein levels were assayed as described in Methods. ' Specific Activity is measured in units of thioltransferase per mg of protein where a unit of thioltransferase is defined as catalyzing the formation of one nricromole of GSSG per nrin at 30°C. ° Total protein is relative to milligrams of BSA. 105 1 2 3 4 5 6 1 2 3 4 5 . ~—' 1oo—r ' 1%)- .— _-; 50 ' _ an. Cl- "C 50 _ 35_‘:' 35- "' ; 25- "’ ' 4 15— v" "ca>ca>aA5cx5cx>cx>cx>cx>cx>ca>cx>is>cx ligation to pET23dt vector EcoRI BstXl Notl Aval PaeFl71 Nail Xbal Apal (@Ncol/BamHl sites) Nco T7 promoter TTase T7 terminate !, ll F1 ori h I | °°' pEThTT 5.70 Kb Amp ”.33 00151 ori Fig. 28. Construction of pEThTT. l’ 118 supernatant was retained after centrifugation at 5,500 x g for 10 min at 4°C. After heat treatment for 5 min at 65 °C, a high-speed supernatant was collected (20,000 x g for 30 min at 4°C), and fiactionated by 40-85% ammonium sulfate. The pellet was resuspended in 60 ml 10 mM NaHzPOb pH 6.0, 2 rnM EDTA, 2 rnM DTT, applied to a Sephadex G-75 column (Pharmacia, 85 cm x 6 cm), and eluted with the same buffer. Active fractions were pooled into two groups, than concentrated with Cerrtricon concentrators (Amicon, mwco 3000), and stored at -70°C with 25% glycerol added until used. Recombinant purification 2: A 3 1 culture of 3121(DE3) bacteria containing the pEThTT plasmid were induced and harvestedasdescribedabove. Thebacterialpelletwasresuspendedin 300 mlsof50 mM Tris- Cl (pH 7.5), l rnM EDTA, 2 mM DTT and 1 mM PMSF, sonicated 3 times for 30 sec at 70%power onice, andsupernatarrtwasretairredthroughtwo successive centrifugation steps at 5,500 x g for 10 min at 4°C, and 18,000 x g for 30 min at 4°C, respectively. The high- . speed supernatant was fi'actionated with ammonium sulfate, and the 40-85% ammonium sulfate pellet was resuspended in 30 ml of50 rnM Tris-Cl (pH 7.5), 1 mM EDTA, 0.1mM DTT(Bufl‘erA)anddialyzedagainst4lofBufi‘erA Thedialyzed crude enzyme fi'actionwas then loaded onto aDEAE-Seplmrose column (13.5 cm x 2.5 cm) equilibrated with Bufi‘er A The 28 5-m1 flow-through fractions containing TT activity were collected, concentrated to 15 nrls using Cerrtriprep 3 fiactionators (Amicon, mwco 3000), and then reduced with 5 rnM DTT for 30 min at 37°C, and finally dialyzed against Nz-saturated 100 mM Tris-Cl (pH 7.5), 5 rnM EDTA, 5 M NaCl (Bufl‘er B). The dialysate was loaded onto a thiopropyl sepharose 6B (Pharmacia) column (10 m1 bed volume), and washed with 200 ml Bufl‘er B, followed by 300 mls of 0.1 M NaOAc (pH 4.6) plus 5 rnM B-mercaptoethanol. No TT activity came 119 through the column on either wash. Bound hpTT was eluted with a 20-50 rnM DTT gradient in 100 rnM Tris-Cl, pH 8.0. Fractions containing TI‘ activity were concentrated in a Centricon 3 microconcentrator (Amicon, mwco 3000) to less than 1 ml and flown with 15% (v/v) glycerol at -80°C until use. SDS-PAGE and Immunoblotting analysis: SDS-PAGE (558) using a 6% stacking gel and 15% separating gel was performed. Two pooled samples from the G-75 fractionation of Purification 1 were electrophoresed (20 ug each), and stained with 0.25% Coomassie Brilliant Blue R-250 in 50% methanol and 10% acetic acid for 20 mimrtes at 50% power in a conventional microwave oven. The gel was then destained 3 times for 20 nrinutes at 50% power in a conventional microwave. Proteins from a duplicate gel were electrophoretically transferred to a nitrocellulose filter for 1 hour at 100V using a 25 mM Tris-Cl (pH 8.3), 92 rnM glycine, 20% methanol bufi‘er. Non-specific binding was blocked by incubating the membrane overnight at 4°C in 0.1% Tween-TBS (Bufibr A) with 5% non-fat dry milk. After blocking, a 5 nrin wash in Buffer A was performed followed by incubation with the primary rabbit anti-human TT antibody (1:20,000) in Buffer A for 60 min at room temperature. The blot was then rinsed twice in Buffer A at room temperature for 15 min, and incubated with the secondary alkaline phosphatase-conjugated goat anti-rabiit antibody (1:3000) in Buffer A for 60 min at room temperature. After the blot was rinsed 2 times for 5 min, the blot was incubated with BCIP and NBT (BioRad) to visualize immobilized TT bands. Purified protein eluted fiom the thiopropyl sepharose 63 column in Purification 2 was treated in the same manner. 120 TT Activity Assays: TT enzyme activity was assayed as described for pig liver TT (314). Briefly, the reaction mixture contained 0.5 rnM GSH, 1.4 units of glutathione reductase, 2.5 mM S-sulfocysteine (Cys-S03) prepared by the method of Segle and Johnson (560), 0.35 mM NADPH, 0.137 mM sodium phosphate buffer, pH 7 .5.TT activity was measured as the GSH-dependent reduction of the prototype substrate Cys-SO; in a coupled spectrophotometric assay. The reaction was initiated by the addition of Cys-803'. Formation of glutathione disulfide (GS SG) was coupled to NADPH oxidation by GSSG reductase and measured spectrophotometrically by a decrease in A340 at 30°C. A decrease in A3,0 due to the conversion of NADPH to NADP+ corresponds to the amount of Cys-SO; reduced and GSH oxidized by TT. A blank reaction without enzyme was monitored simultaneously and subtracted from enzyme- catalyzed reaction rates. One unit of T1" was defined as that amount of enzyme catalyzing the formation of 1 pmol of GSSG/min under standard conditions (314). DHA Reductase Assays: DHA reductase assays followed the direct spectrophotometric assay of Stahl et a]. (557) based on the change in absorbance at 265.5 nm and 30°C as DHA was reduced to AA. The standard assay was 0.137 M sodium phosphate buffer, pH 6.8, 1 rnM EDTA, 2 mM GSH, 1 rnM DHA, and various amounts of enzyme in a total volume of 500 pl. Blanks run simultaneously without the addition of the enzyme were subtracted from enzyme-catalyzed reaction rates. The reaction was initiated by the addition of DHA, and was linear up to 2 rrrin at 30°C. DHA was generated by the bromine oxidation of 20 mM ascorbic acid, following the procedure of Bode et al (552). One unit of TT was defined as that amount of enzyme catalyzing the reduction of one pmol of AA per min under standard conditions. 121 Protein Assays: Protein samples and bovine serum albumin (BSA) standards were precipitated in ice-cold acetone at -20°C for 30 min, and then pelleted to remove the DTT. Afier acetone evaporation; the samples and BSA controls were resuspended in 0.01 N NaOH, and protein concentrations were determined by the bicinchinoninic acid (BCA) protein assay protocol according to the manufacturer's direction (Pierce Chemical Co) . Results Human placental 'IT in pEThTT was expressed at about 3-10% of soluble protein 3 h after IPTG induction (Fig. 29). The soluble TT was purified using two protocols, and activity was assayed to determine if the cDNA encoded a functional protein with native TT activity. The first purification strategy, (Purification l), i.e., sonication followed by heat treatment for 5 nrin at 65°C, resulted in almost 50% losses in enzyme activity. Ammonium sulfate fi'actionation resulted in a small purification, and gel filtration using a G-7 5 resin resulted in 3-fold increased purity. TT co-eluted from the G-75 column together with two major protein peaks, and was not resolved from either peak (F ig.30). The recombinant enzyme extract possessed both dithiol-disulfide oxidoreductase and DHA reductase activities, indicating a functional protein. The second purification strategy, (Purification 2) was applied to determine whether using DEAE Sephacel under conditions where TT did not bind would result in a greater purification from E. coli proteins than the standard protocol of CM Sepharose cation exchange resin. The DEAE Sephacel treatment resulted in only a 4.2-fold purification, and 122 Fig. 29. Timecourse of hp'I'I‘ expression in E. coli. Bacteria were sampled at various times after induction, adjusted to the same OD“, and electrophoresed as described in Methods. Lanes 1 and 10 are molecular weight markers; corresponding molecular weights are on the lefi. Lane 2: non-induced, lanes 3-9 induction of (h) 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, and 3.5, respectively. The induced band that corresponds to recombinant hpTT is indicated on the right. 123 Fig. 30. G-75 purification of recombinant hp'I'I‘. G-75 gel filtration was performed as described in Methods. Recombinant TT elutes between two peaks. Protein concentration (as measured by 0D,“) is indicated on the left Y axis, and as a line. TT activity is indicated on the right Y axis, and is indicated in bars. Fraction numbers are indicated on the X axis. Active fractions were separated into two pools, corresponding to the peaks they co-eluted with, and assayed for purity by SDS-PAGE and irnmunoblotting. 124 100 K 580- A 8 <3, 60 «r 8 l .s 40! """ (I) § 201—1 --------- ° 1 0 . 2040 hpTT purification G-75 column fraction number TT activity (U/mg) Fig. 30 G-75 purification of recombinant hp'I'I‘. 125 .‘U—e 50-1 a! 25- o - a m, . .4....-, 1 10 100 Adriarnycin dose (pM) Fig. 39. Effect of AAP on Adriarnycin cytotoxicity in MCF-7 WT transfected cell lines. Relative Adriarnycin cytotoxicity is shown on the left. A representative WT transfected cell line is shown. Fluorescent cytotoxicty assays were performed as described in Methods. I represents WT transfected with no AAP added, A represents WT transfected with 2 mM AAP added, V represents WT transfected cells with 5 rnM AAP added. The values represent the averages : standard error of the mean of three separate experiments, run in triplicate. Cytotoxicity comparisons were performed at the LDso levels using the statistical package PRISMTM by Graph Pad. 151 associated with hpTT overexpression in MCF-7 ADRR cells. When matched numbers of MCF-7 WT and MCF-7 ADRR cells were transfected, only the MCF-7 WT cells integated the constitutive overexpression pTTcmv plasmid into genomic DNA. The TT activity levels in 10 MCF-7 WT cell lines transfected with pTTcmv ranged from 9- to 66-fold above that of the untransfected MCF-7 WT cell line. Several of these cell lines, in fact, had TTactivity significantly exceeding that of the MCF-7 ADRR lines. Since TT activity in MCF-7 ADRR cell lines is already 6.5- to 23-fold higher than in the MCF-7 WT line, one explanation for the inability to isolate any pTTcmv stably transfected MCF-7 ADRR colonies is that the strong constitutive cytomegalovirus promoter may elevate TT dithiol-disulfide activity levels to where they are toxic to the MCF-7ADRR cell. MCF-7 ADRR cells have multiple measureable differences fiom the parental MCF-7 WT cell lines, including hormone independent gowth (545), altered membrane receptor levels (544), decreased drug accumulation, increased drug detoxification (522, 543 ), increased DNA repair activity, elevated cytosolic pH (429), and alterations in various GSH-dependent enzymes that detoxify ROS (526, 543, 545). TT and a related dithiol-disulfide oxidoreductase, TRX, have been demonstrated to modulate several receptor and enzyme activities, in vitro, including enzymes that are altered in the MCF-7 ADRR cell lines. Therefore, elevations in TT activity may adversely efi‘ect the MCF-7 ADRR cell lines, and not the MCF-7 WT cell lines. An alternative explanation of decreased transfection eficiency of the MCF-7 ADRR cells is not sufficient, since the luciferase control plasmid (pGL) was easily and similarly transfected into both MCF-7 WT and MCF-7 ADRR cell types. Future studies beyond the scope of this dissertation may determine if high levels of TT induction are lethal to MCF-7 ADRR cells using inducible promoters rather than constitutive promoters. 152 In order to test the hypothesis that increases in TT activity increase resistance to Adriarnycin, Adriamyciu dose-response was compared between the extremely Adriamycin- sensitive MCF-7 WT cell line and 10 MCF-7 WT pTTcmv-transfected cell lines which constitutively overexpress hpTT. All pTTcmv-transfected MCF-7 WT cell lines overexpressing TT activity were more resistant to Adriarnycin than untransfected MCF-7 WT cells. Our experimental results demonstrated a possible association of TT activity with Adriarnycin resistance independent of the levels of L-ascorbate 2-phosphate administered. Unlike MCF-7 ADRR cells, neither MCF-7 WT nor pTTcmv-transfected MCF-7 WT cell lines showed increased drug resistance when AAP was administered, and in fact, several lines even had slightly decreased resistance when 2 or 5 mM AAP was administered. This indicates the DHA reductase activity of TT is not correlated with increased drug resistance, as previously suggested (526). Adriarnycin resistance apparantly involves another intrinsic activity of TT, such as thiolzdisulfide oxidoreduction. Work published after these studies commenced supports a possible thiolzdisulfide oxidoreduction function in dnlg resistance (277). TRX, another protein of the same approximate molecular weight also catalyzing dithiol-disulfide transfer was shown to be related to drug resistance in cisplatin-resistant T4 bladder cancer cells (277). TRX levels are also elevated 2-fold in the MCF-7 ADRR cells (526). Perhaps both TT and TRX together modulate cellular oxidation-reduction status to increase cellular viability in the presence of drugs known to generate reactive oxygen species. CONCLUSION The cloning, sequencing and overexpression of human TT (308-312) has provided novel information about the human ‘I‘T structure. Human TT is the only characterized and cloned mammalian TT to have an extra half-cystine residue, although the adaptive value of such a cysteine is as yet undetermined. This cysteine is predicted to be on the protein surface near the conserved active site C”PY(F)C2’, by analogy with the pig liver TT crystal structure (129), and may be involved in the decreased stability of the human enzyme relative to other mammalian TTs. Mutagenesis studies of the recombinant protein have determined that if this cysteine residue at position 7 is replaced with a serine, the catalytic activity of TT for both DHA reduction and dithiol-disulfide oxidoreduction stays the same, however, the stability of the protein increases to that of other mammalian Us (315). TT protein and activity has been known to be distributed differently in varied tissues in other mammals (136-145). The examination of TT distribution in human tissues has not yet been reported. Both mRNA and protein levels indicate that TT is distributed differentially in human tissues; the tissues with the lowest expression and protein are endocrine glands; whereas tissues that are habitually exposed to highest levels of ROS are those with the highest levels of TT, such as heart, liver, lung, kidney, spleen, placenta, and peripheral blood leuk- ocyte. Recently, it was determined that stresses such as ultraviolet light (UV) B can induce the expression of TT in rat keratinocytes (338). UVB'induction mechanisms are not well 153 1 54 characterized; related UVC light can induce transcription of genes through NFch, serum response elements, and src tyrosine kinases, in addition to DNA repair mechnisms (reviewed in 338). The induction of TT as an immediate-early response indicates that TT is important for repair or regeneration of damaged cellular components at an early stage. T'T induction brings to 1 light certain questions about TI‘ regulation. How is IT regulated, both transcriptionally, and possibly post-transcriptionally? Is TT regulation tissue-specific? What role does "IT regulation play in normal development and gowth, as well as oxidative stress, and do aberrations result in pathophysiological conditions? What is the promoter structure of TT, and how does this relate to 'IT regulation? Can the UVB induction of TT in rat keratinocytes be seen in other tissues and from difi‘erent species? A recent explosion of research on the thiol-disulfide oxidoreductase enzymes has illuminated the fact that redox mechanisms are important in both maintaining cellular homoestasisaswellasrespondingto acutestresses, such as oxidative stress. The study ofTT follows the more advanced study of PDI and TRX, other members of the same class. At the same time, the recent interest in cellular antioxidants has prompted increased research on enzymes that recycle oxidized AA, such as the DHA and SDHA reductases. Here, also, much of the mechanism and function of TT remains to be elucidated. Clearly, recent research has demonstrated that TT is important in maintaining both reduced AA (79,80), as well as deglutatfiolation ofproteins (331-333). Other biological or physiological roles for TT remain to be determined, and may lead to many research avenues on TT firnction in cells. The overexpression of human TT in cells is novel. MCF-7 breast tumor cells that typically are highly sensitive to the anti-cancer drug, Adriarnycin, were found to have increased resistance when overexpressing TT. The resistance is apparently not related to the 155 DHA reductase ability of TT, as elevated levels of AAP were added, with no net effect on cell- viability. IF DHA reductase activity were part of the resistance mechanism, expected results would be that cells administered the AA precursor, AAP, would have geatly enhanced resistance due to the large amounts of AA available for scavenging Adriarnycin-generated oxyradials. The postulate, therefore, is that the dithiol-disulfide oxidoreductase activity of TT may be a sigrificant part of the drug resistance mechanism. In support of a role for thiol-disulfide oxidoreduction in drug resistance, and cell gowth, TRX, a related thiol-disulfide oxidoreductase of very similar molecular weight has very recently been reported to have an efi‘ect on drug resistance (277, 570). Reducing the TRX expression levels in MDR kidney cells resulted in a loss of drug resistance (277). Eliminating TRX dithiol-disulfide oxidoreduction with an active site mutant in MCF-7 WT breast tumor cells eliminated the transformed phenotype (5 70); and TRX overexpression in MCF-7 WT cells increased cell proliferation rate. This leaves interesting questions as to the potential mechanism for TT-increased drug resistance in MCF-7 WT cells. Does TT also filnction to stimulate cell proliferation? 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