ECTO-NOX Proteins

D. James Morré ● Dorothy M. Morré

ECTO-NOX Proteins

Growth, Cancer, and Aging D. James Morré Dorothy M. Morré Mor-NuCo, LLC Mor-NuCo, LLC Purdue Research Park Purdue Research Park West Lafayette, IN, USA West Lafayette, IN, USA

ISBN 978-1-4614-3957-8 ISBN 978-1-4614-3958-5 (eBook) DOI 10.1007/978-1-4614-3958-5 Springer New York Heidelberg Dordrecht London

Library of Congress Control Number: 2012941093

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Springer is part of Springer Science+Business Media (www.springer.com) Preface

One does not discover new lands without consenting to lose sight of the shore for a very long time.

André Gide

The discovery of the ENOX proteins was inexorably interwoven with the con- cept of a plasma membrane electron transport fi rst indicated from the fi ndings of Cathy Frantz, a masters student at Purdue University. Cathy found microsomal elec- tron transport activities associated with highly purifi ed plasma membranes from rat livers. The fi rst report of these fi ndings in 1973 at the American Society of Cell Biology meetings was greeted with indifferent disbelief. Clear exceptions were Prof. Fred Crane of Purdue University, a member of Cathy’s examining committee and Fred’s close friend and colleague, Prof. Hans Löw of the Karolinska Institute in Sweden. The Morrés continued to pursue these observations in their efforts to solid- ify the fl ow-differentiation model of Golgi apparatus functioning whereas Fred and Hans set upon a quest to discover the elusive plasma membrane electron transport system. That quest culminated with two co-edited volumes, one on animals in 1990

v vi Preface and one on plants in 1991 entitled Oxidoreduction at the Plasma Membrane: Relation to Growth and Transport published by CRC Press (Crane et al. 1990b, 1991). The existence of growth-related NADH oxidases of the plant and animal plasma membrane were fi rst reported in 1986 from plants (Morré et al. 1986a) but it was not until 1990 (Morré and Crane 1990; Fig. 4.8; cover) that their role as the terminal oxidases of the plasma membrane electron transport chain was correctly formulated. We thank our good friends and colleagues Frederick L. Crane and Hans Löw for championing the concept that the plasma membranes might contain redox active proteins with important functions in growth and disease even in the face of general non-acceptance of that notion by the overall scientifi c community and a universal lack of enthusiasm by extramural funding agencies for studies of plasma membrane electron transport no matter how well conceived. It is a singular but sometimes lonely privilege to write a book on a potentially important new family of proteins virtually single handedly identi fi ed, cloned, char- acterized and clinically implemented with minimal independent outside confi rmation at the time. While masquerading as intractable proteins, they have and continue to offer remarkable opportunities for research, commercial development and outside con fi rmation (Chap. 1 1 ; Table 11.1). The latter underscores the complexity of these proteins and the many unusual diffi culties especially in their assay. We recall a visit from Dr. Warren McKellar of Eli Lilly to our laboratory in the very early days of anti-cancer sulfonylurea research to carry out a spectrophotometric assay on his own to validate the activity. He was pleased to see a rate only to remark a few moments later that it had stopped momentarily only to start up again a few minutes later. Several more years were required before the true meaning of that observation and its subsequent reproduction with other systems fi nally led to the still controver- sial conclusion that ENOX activities were oscillatory. The ENOX proteins were discovered as a result of a search for a growth-related protein at the cell surface that was the target for immobilized forms of the anticancer drug doxorubicin (Adriamycin® ). In the late 1970s several groups demonstrated that doxorubicin exhibited an enhanced anti-cancer activity if it was fi rst conjugated to an impermeant support and not permitted to enter the cell (Chap. 1 1). As doxorubicin is a redox-active quinone site inhibitor, a redox protein was sought that was at the cell surface and cancer-speci fi c. The search led eventually to discovery of the ECTO- NOX (ENOX) family of external hydroquinone oxidases, also capable of oxidizing external NADH. One subset of the ENOX proteins, the tumor-specifi c tNOX or ENOX2 proteins, was inhibited by doxorubicin and other quinone site-targeted anti- cancer drugs, was cancer-speci fi c, was absent from the surface of non-cancer cells and tissues and was the fi rst ENOX protein to be cloned (Chueh 1997; Chueh et al. 2002b). Three additional family members including the constitutive human ENOX1 in 2008 (Jiang et al. 2008) followed with the most recent, an age-related ENOX protein, cloned in 2010 and a constitutive ENOX1 from plants in 2011. Being proteins of the external cell surface and lacking trans -membrane helices to anchor the protein in the mem- brane, ENOX proteins were found to be shed and to appear in soluble form in patient sera and urine where they serve as early diagnostic markers for cancer presence and Preface vii organ site to permit very early intervention strategies prior to advanced disease and metastatic spread (Chap. 12 ). Findings of Chap. 8 suggest that the cancer- or tumor-related ENOX2 (tNOX) proteins are all splice variants from a single gene. More importantly, each major type of human cancer is characterized by characteristic transcript variants of unique molecular weight and isoelectric point (Chap. 12). Using a proteomics approach and a recombinant antibody specifi c for a common exon, it is possible not only to detect cancer but to diagnose it as well. The purpose of this book is to document this unique family of cell surface pro- teins (the ECTO-NOX or ENOX protein family) involved in growth, biological time keeping, cancer, aging and viral infections and having properties of prions. The ENOX proteins are the exclusive discovery (subsequently con fi rmed by others) of the authors, Drs. D. James and Dorothy Morré, and their students and research asso- ciates at Purdue University. Roles in plasma membrane electron transport (Chap. 4 ), growth (Chap. 5), biological time keeping (Chap. 6), cancer (Chaps. 8 , 1 1 and 12 ), prevention of viral infections (Chap. 7), crop production through control of plant growth (Chap. 10 ), and coronary artery disease and skin aging (Chap. 9 ), are among the many developing opportunities for new discovery and commercialization sur- rounding the ENOX proteins. The book provides an ENOX-based mechanism for how cells become larger (increase in size) that is both unique and well documented with applications not only to cancer and cancer therapy but for production agriculture as well (Chap. 10 ) with increase of biomass for biofuel production as one exciting future prospect. Finally, the concept of and the evidence for oscillations in the ratios of electron spin pairs de fi ning ortho and para water as the basis for highly coordinated popula- tions of coherent water that appear vital to water’s biological and physical proper- ties is completely new, of interest to the biological and physical sciences and now becoming widely accepted by the physical scientists involved with the study of the properties of water (Chap. 6 ). Special mention is accorded to Michael Berridge, Frederick Crane, Iris Sun, Rita Barr and Hans Löw who have unwaveringly promoted plasma membrane redox and a functional role of ENOX proteins in the overall process, to the late Albert Overhauser for encouragement to seek an explanation of the oscillatory patterns of ENOX proteins at the atomic level (Chap. 6 ), to Michael Böttger for assistance with pivotal growth measurements (Chap. 5), to Ron Brightmore for his inspiring sur- veys of the literature and to Profs. Jacob Levitt and Hale Fletchall of the University of Missouri for planting the initial seeds of inquiry. Special thanks to Don Lee, Tom Shelton, Graham Kelly and Richard Greaves for recognizing the commercial poten- tial of the ENOX protein family. We express our appreciation to the many colleagues, postdoctorals, graduate stu- dents, undergraduate assistants, and technicians whose invaluable assistance made possible the experimental studies especially as graduate students, Andrew Brightman, P.-J. Chueh, Chinpal (James) Kim, Xiaoyu Tang and Ziying Jiang for ground break- ing protein puri fi cation and molecular cloning efforts. Appreciation is extended as well to the even greater numbers who challenged and criticized the work to force us viii Preface to work even more diligently to distinguish among possible interpretations of the fi ndings. We thank Peggy Runck for manuscript preparation and Aya Ryuzoji for preparation of the fi gures. We are especially indebted to the unwavering support of the Morré children, Connie, Jeffrey and Suzanne, and grandchildren, Christopher, Eric and Katherine Chalko, Matthew, Timothy and Nicholas Miner, Suzanna Morré and Aren and Mariah Rudder. May our ENOX proteins always oscillate in synchrony.

West Lafayette, IN, USA D. James Morré Dorothy M. Morré Contents

1 The ENOX Protein Family ...... 1 1.1 The ENOX Protein Family Members ...... 1 1.2 ENOX Proteins Are Associated with the External Cell Surface as Ecto Proteins and Are Shed into the Environment ...... 4 1.3 Two Activities of ENOX Proteins Alternate ...... 6 1.4 ENOX Proteins Participate Directly in the Enlargement Phase of Cell Growth ...... 7 1.5 ENOX Proteins Are Resistant to Degradation and Tend to Form Insoluble Aggregates ...... 7 1.6 ENOX Proteins Are Dicopper Proteins Lacking Both Iron and Flavin ...... 8 1.7 The Oscillatory Behavior Complicates Assays of ENOX Activities ...... 10 1.8 The Distinctive 2 + 3 Pattern of ENOX Oscillations Is a Unifying Characteristic of All Family Members ...... 11 1.9 ENOX Proteins Differ Markedly from the NOX Proteins of Host Defense ...... 13 1.10 ENOX Proteins Are of Low Specific Activity ...... 14 1.10.1 Natural Electron Donors and Acceptors for Cell Surface-Associated ENOX Proteins ...... 14 1.10.2 Hydroquinones as Natural Electron Donors ...... 15 1.10.3 Reduced Pyridine Nucleotides as Artificial Electron Donors ...... 15 1.10.4 Protein Thiols and Tyrosines as Electron Donors for arNOX Proteins and Generation of Superoxide ...... 15 1.10.5 Aggregation and Formation of Amyloid ...... 16 1.11 Why an External NADH Oxidase? ...... 17 1.12 Summary ...... 17

ix x Contents

2 Measurements of ECTO-NOX (ENOX) Activities ...... 19 2.1 Spectrophotometric Assay of NADH Oxidase ...... 21 2.2 Statistical Analysis ...... 22 2.3 Data Reduction Methods ...... 23 2.3.1 Diode Array Instruments ...... 30 2.4 Measurement of Hydroquinone Oxidase Activity

with Reduced Coenzyme Q10 or Phylloquinone as Substrate ...... 32

2.4.1 Enzyme Assay for Reduced Coenzyme Q10 Oxidase ...... 32 2.4.2 Enzyme Assay for Reduced Phylloquinone Oxidase ...... 33 2.5 Dissolved Oxygen Measurement ...... 35 2.6 Estimation of Protein Disulfide-Thiol Interchange Activity ...... 38 2.7 Preparation of Scrambled RNase Substrate ...... 38 2.8 Estimates of Protein Disulfide-Thiol Interchange from Enzymatic Assay of Dipyridyl-Dithio Substrate Cleavage ...... 41 2.9 Measurement of Trans-Plasma Membrane Redox by Reduction of Cell-Impermeable Dyes ...... 42

2.9.1 CoQ1 Can Function as an Intermediate Electron Carrier in WST-1 Reduction...... 42 2.9.2 Measurement of Plasma Membrane Electron Transport Based on WST-1 Reduction ...... 42 2.10 Summary ...... 44 3 The Constitutive ENOX1 (CNOX) ...... 47 3.1 ENOX1 Function ...... 47 3.2 ENOX1 Cloning ...... 50 3.3 ENOX1 Characterization ...... 53 3.4 ENOX1 Activity Requires the Presence of Copper ...... 57 3.5 Copper Binding and Site-Directed Mutagenesis of Potential Copper-Binding Sites ...... 58 3.6 Response to Nucleotides ...... 60 3.7 Aggregation and Electron Microscopy ...... 61 3.8 ENOX1 Fulfills Essential Roles in Cell Enlargement and Cellular Time-Keeping ...... 62 3.9 ENOX1 of Human Platelets ...... 62 3.10 Summary ...... 62 4 Role in Plasma Membrane Electron Transport ...... 65 4.1 Composition of the PMET ...... 65 4.1.1 NADH Coenzyme Q Reductases ...... 66 4.1.2 Hydroquinones ...... 68 4.1.3 Terminal Oxidases ...... 69 4.2 Electron Donors and Acceptors ...... 73 4.3 Rates of PMET ...... 73 4.4 Energetics of PMET ...... 76 4.5 PMET Driven Outward Proton Pumping and Alkalinization of the Cytoplasm ...... 78 Contents xi

4.6 PMET Function in Electron Import ...... 80 4.7 PMET and Growth ...... 81 4.7.1 Cell Cycle Check Point Control of Cell Enlargement ...... 81 4.7.2 PMET Activity and Growth Are Correlated ...... 82 4.8 Regulation of PMET ...... 82 4.8.1 Feedback Regulation of PMET ...... 85 4.8.2 ENOX Cell Surface Receptor Proteins ...... 86 4.9 PMET and Glycolysis ...... 86 4.9.1 PMET and Glycolysis of Cancer Cells ...... 86 4.10 PMET Links to Major Signaling Pathways ...... 88 4.10.1 Sirtuins ...... 89 4.10.2 Sphingolipid Rheostat ...... 91 4.10.3 NADH Modulation of PTEN Provides Link of PMET to Ras-Raf-Mek-Erk, PI3-AKT-mTOR and NF-k B ...... 91 4.10.4 AMP-Activated Protein Kinase ...... 93 4.10.5 Hypoxia ...... 93 4.11 NAD+ Homeostasis ...... 95 4.12 Summary ...... 95 5 Role in the Enlargement Phase of Cell Growth ...... 97 5.1 Cell Enlargement Linked to ENOX Activities ...... 97 5.2 ECTO NADH Oxidase of Liver Plasma Membranes Stimulated by Hormones and Growth Factors ...... 98 5.3 ECTO NADH Oxidase of Rat Hepatoma Plasma Membrane Constitutively Activated and No Longer Growth Factor or Hormone-Responsive ...... 98 5.3.1 Thiol Reagents ...... 100 5.4 Relationship to Growth ...... 101 5.4.1 Plants ...... 101 5.4.2 Vertebrate Cells ...... 113 5.5 Pathological Implications ...... 123 5.5.1 Apoptosis ...... 123 5.6 Physical Membrane Displacements ...... 124 5.7 ATP- and p97 AAA-ATPase-Dependent and Drug-Inhibited Vesicle Enlargement Reconstituted Using Synthetic Lipids and Recombinant Proteins ...... 124 5.8 Summary ...... 138 6 Roles as Ultradian Oscillators of the Cells Biological Clock ...... 141 6.1 Time Keeping Properties ...... 141 6.2 Molecular Studies ...... 142 6.3 Studies with Deuterium Oxide ...... 148 6.4 The Role of Copper ...... 149 6.5 The Copper Clock ...... 150 xii Contents

6.6 EXAFS Investigations ...... 152 6.7 Oscillations Inherent in the Structure of Water ...... 154 6.8 Period Length Determined by Ionic Radius of Liganded Cation ...... 158 6.9 Spectral Evidence for Disequilibrium of ortho:para Spin States in Liquid Water That Oscillate ...... 161 6.10 Other Mechanisms Proposed for ortho/para Conversions and Departures from Their Equilibrium Ratio of 3:1 ...... 164 6.11 The 24-min Period Has Properties of a Carrier Wave Generated from the Basic Underlying ortho–para Water Oscillations? The Heart Rate Model ...... 166 6.11.1 Growth Oscillations of Elongating Pollen Tubes ...... 168 6.12 Phasing of the Rhythm ...... 168 6.12.1 EMF Sets the Copper Clock ...... 169 6.13 A Mechanism to Explain How Oscillations of Redox Potential of Aqueous Solutions Become Synchronous and Remain So ...... 172 6.14 ENOX Clock and Cancer ...... 177 6.15 Are ENOX Oscillators Linked to the Drivers of the Circadian Clock and How Are They Linked? ...... 178 6.16 Why Oscillate? A Consequence of Active Sites in Metalloproteins? ...... 184 6.17 Summary ...... 185 7 Other Potential Functional Roles of ENOX Proteins ...... 187 7.1 Cell Cycle Control ...... 187 7.2 Gene Regulation ...... 187 7.3 Endomembrane Function, Membrane Displacements, Vesicle Budding ...... 188 7.3.1 Membrane Budding ...... 188 7.3.2 Energy Requirements for Physical Membrane Displacement ...... 190 7.4 Endocytosis and Autophagy ...... 192 7.5 Host Defense ...... 193 7.6 pH Control ...... 193 7.7 Lipid Oxidation ...... 195 7.7.1 arNOX Inhibitors and Prevention of Coronary Artery Disease ...... 197 7.8 Life Extension and Calorie Restriction ...... 197 7.9 Control of Apoptosis and Cell Survival ...... 198 7.10 Neurodegenerative Disorders ...... 200 7.11 Memory ...... 201 7.12 Gametogenesis ...... 202 Contents xiii

7.13 Role in Viral Pathogenesis ...... 203 7.13.1 ENOX2 Inhibitor (−)-Epigallocatechin-3-Gallate Blocks Virus Infections Alone and in Combination with Capsicum Vanilloids and Other Green Tea Catechins ...... 205 7.13.2 Brefeldin A and Antitumor Quassinoids ...... 207 7.14 Summary ...... 209 8 ENOX2 (tNOX) and Cancer ...... 211 8.1 ENOX2 Discovery ...... 211 8.2 ENOX2 Activity ...... 213 8.2.1 Biochemistry ...... 217 8.3 Sequence ...... 218 8.4 Structural Properties ...... 222 8.4.1 ENOX2 Protein Phosphorylation ...... 228 8.5 ENOX2 Presence and Cancer ...... 228 8.5.1 ENOX2 Autoantibodies Generated in Cancer Patients ...... 230 8.5.2 ENOX2 Gene Present in Genome as a Single Copy ...... 230 8.5.3 ENOX2 Lacks Intrinsic Membrane-Binding Motifs ...... 230 8.5.4 ENOX2 Has Properties of a Prion and Is Protease Resistant ...... 231 8.6 ENOX2 Has Characteristics of an Oncofetal Protein ...... 233 8.7 Transgenic Mouse Strain Overexpressing ENOX2 ...... 235 8.8 Alternative Splicing as Basis for Specific ENOX2 Localization to the Cell Surface ...... 242 8.8.1 Full-Length ENOX2 MRNA Identical to That of Cancer Cells Exists in Human Non-cancer Cells and Tissues ...... 247 8.8.2 Full-Length 71 kDa ENOX2 Protein Not Translated ...... 248 8.8.3 Cancer-Specific Expression of ENOX2 ...... 250 8.8.4 Splice Variants of ENOX2 Were Found in Cancer Cells ...... 250 8.8.5 Expression of Exon 4 Minus and Exon 5 Minus Forms of ENOX2 in COS Cells ...... 250 8.8.6 Delivery of 34 kDa ENOX2 Protein to the Plasma Membrane ...... 252 8.8.7 Mutation of Met 231 Blocked Expression of the Exon 4 Minus Splice Variant ...... 252 8.8.8 Subcellular Localization of E4m ENOX2-EGFP and Full-Length ENOX2-EGFP Fusion Proteins ...... 253 8.8.9 Regulation of ENOX2 Expression ...... 256 8.9 hnRNP F Splicing Factor Directs Formation of the Exon 4 Minus Variant of ENOX2 ...... 257 8.10 Summary ...... 259 xiv Contents

9 Age-Related ENOX Proteins (arNOX) ...... 261 9.1 arNOX Discovery ...... 264 9.2 Measurement of Superoxide Formation by arNOX ...... 266 9.3 Characteristics ...... 268 9.4 arNOX Cloning ...... 272 9.5 Characterization of Recombinant arNOX Proteins ...... 279 9.6 arNOX as a Biomarker of Aging ...... 284 9.7 Role in Skin Aging ...... 286 9.8 Role in Oxidation of Serum Lipoproteins ...... 291 9.9 arNOX Activity Correlates with Life Span in Sea Urchins ...... 297 9.10 arNOX in Plants ...... 298 9.11 arNOX Inhibitors ...... 299 9.11.1 Coenzyme Q ...... 299 9.11.2 Botanical Sources of arNOX Inhibitors ...... 305 9.12 Beneficial Biological Function Associated with Superoxide Production: Physiological Roles of Superoxide ...... 307 9.13 NQO1 (Cytoplasmic NAD(P)H: Quinone Oxidoreductases, DT-Diaphorase EC 1.6.99.2) and Plasma Membrane Electron Transport ...... 309 9.14 Summary ...... 310 10 The Auxin-Stimulated ENOX and Auxin Stimulation of Plant Growth ...... 313 10.1 Early Evidence for Auxin-Modulated Enzymes Involved in Plant Cell Enlargement ...... 313 10.2 The Plasma Membrane as the Subcellular Location of the Auxin-Responsive Mechanism ...... 317 10.3 Direct Effects of Auxin on Signaling Molecules Fail to Parallel Those of Mammalian Growth Factors ...... 318 10.4 Evidence for a Redox-Related Plasma Membrane-Located Auxin Target ...... 321 10.4.1 Separation of Auxin-Activated and Constitutive NADH Oxidase Activities ...... 327 10.5 Auxin-Stimulated NADH Activity and Growth Oscillates with a Period Length of 24 min ...... 327 10.6 Golgi Apparatus Transport Important to Sustained Cell Enlargement but not Specifically Required for Auxin-Induced Cell Enlargement ...... 331 10.7 Response of ENOX of Isolated Plasma Membrane Vesicles to Osmotica ...... 331 10.8 Inhibitors of the Auxin-Stimulated NADH Oxidase of Plants ...... 332 10.9 Cell Elongation Oscillates with a Period of 24 min and Exhibits a Second set of Oscillations in Response to 2,4-D ...... 335 Contents xv

10.10 The Auxin-Stimulated ENOX Has Properties of a Prion: How 2,4-D Kills Plants ...... 337 10.11 Summary ...... 347 11 Cancer Therapeutic Applications of ENOX2 Proteins ...... 345 11.1 PMET as a Target for Anticancer Drug Development ...... 348 11.1.1 Arsenicals as Unspecific Anticancer PMET Inhibitors ...... 349 11.2 Inhibition of PMET and Induction of Apoptosis ...... 350 11.2.1 Mechanism of Induction of Apoptosis When Plasma Membrane Electron Transport Is Inhibited ...... 351 11.3 Mechanism of Growth Arrest When Plasma Membrane Electron Transport Is Inhibited ...... 355 11.3.1 Elevation of Ceramide ...... 355 11.3.2 Links for Elevated Ceramide and Cell Cycle Arrest ...... 355 11.3.3 ENOX2 Inhibitors Slow the Growth of HeLa Cells and Induce Apoptosis in Cancer But Not in Noncancer Cells ...... 356 11.3.4 ENOX Inhibitors Increase Cytosolic NADH Levels ...... 358 11.3.5 Increased NADH Resulting from ENOX2 Cell Surface Inhibition Inhibits Plasma Membrane- Associated Sphingosine Kinase (SK) and Lowers Levels of Prosurvival Sphingosine-1-Phosphate ...... 359 11.3.6 Sphingomyelinase ...... 359 11.4 ENOX2 Inhibitors ...... 362 11.4.1 Vanilloids (Capsaicinoids) as PMET Inhibitors ...... 363 11.4.2 Anthracycline Antibiotics ...... 368 11.4.3 Cisplatin Targets ENOX2 of the PMET ...... 377 11.4.4 Antitumor Sulfonylureas ...... 378 11.4.5 Antitumor Quassinoids Target ENOX2 ...... 387 11.4.6 Acetogenins ...... 390 11.4.7 EGCg ...... 391 11.4.8 Phenoxodiol Targets the PMET Through Inhibition of ENOX2 ...... 400 11.4.9 Sulforaphane ...... 408 11.4.10 Suramin ...... 409 11.4.11 Callipeltin ...... 409 11.5 Nonsteroidal Anti-inflammatory Drugs ...... 410 11.6 Retinoids and Calcitriol Agents of Differentiation ...... 411 11.6.1 Retinoic Acid Inhibition of PMET ...... 411 11.6.2 Retinoid Inhibition of ENOX2 ...... 411 11.7 ENOX2-Directed Therapeutic Antibodies ...... 412 xvi Contents

11.8 Antisense ...... 414 11.9 ENOX Inhibitors Enhance the Response of Tumors to Radiation ...... 415 11.10 ENOX2 as a Target for Cancer Prevention Through Early Intervention ...... 415 11.11 Summary ...... 416 12 Cancer Diagnostic Applications of ENOX2 Proteins ...... 419 12.1 Cancer Cell Surface ENOX2 Shed into Sera as Biomarkers of Cancer Presence ...... 420 12.1.1 Biomarker Discovery Based on ENOX2 Activity ...... 421 12.1.2 Characteristics of ENOX2 as Cancer Biomarker Based on Activity ...... 421 12.1.3 Transcript Variants Detected by Two- Dimensional Gel-Western Blot Analysis Are Cancer Site-Specific Biomarkers ...... 424 12.1.4 Transcript Variants of ENOX2 ...... 425 12.2 Two-Dimensional Gel-Western Blot Cancer Detection System ...... 425 12.2.1 Two-Dimensional Gel-Western Blot Analysis of ENOX2 Transcript Variants Provide for Very Early Detection ...... 428 12.3 Early Intervention ...... 430 12.4 ENOX2 Autoantibodies May Preclude Conventional ELISA Tests for Early Appearance of ENOX2 in Serum or Plasma ...... 430 12.5 Lipid-Associated Sialic Acid (LASA) Fractions from Sera of Cancer Patients Contain ENOX2 Fragments as Major Nonlipid Constituents ...... 432 12.6 RTPCR Detection of Cancer Cells in Blood Based on Presence of ENOX2 Splice Variant mRNA ...... 433 12.7 Summary ...... 433

Epilogue—Remaining Challenges ...... 435

Appendix Detailed Description of Two Dimensional Gel Electrophoresis-Western Blot Early Cancer Detection Protocol ...... 441

References ...... 443

Index ...... 493