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Author(s): George F. Vande Woude; George Klein (Eds.)
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Advances in
CANCER
RESEARCH
Volume 104

Clusterin, Part A
Advances in
CANCER
RESEARCH
Volume 104
Edited by
Saverio Bettuzzi
Dipartimento di Medicina Sperimentale,
Sezione di Biochimica Biochimica
Clinica e Biochimica dell’Esercizio Fisico
Via Volturno 39-43100
Parma and Istituto Nazionale
Biostrutture e Biosistemi
(I.N.B.B.) Rome, Italy
Sabina Pucci
Department of Biopathology,
University of Rome
‘‘Tor Vergata,’’ via Montpellier
1 00-133 Rome, Italy
AMSTERDAM • BOSTON • HEIDELBERG • LONDON
NEW YORK • OXFORD • PARIS • SAN DIEGO
SAN FRANCISCO • SINGAPORE • SYDNEY • TOKYO
Academic Press is an imprint of Elsevier
Series Editors
George F. Vande Woude
Van Andel Research Institute
Grand Rapids
Michigan
George Klein
Microbiology and Tumor Biology Center
Karolinska Institute
Stockholm
Sweden

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herein. Because of rapid advances in the medical sciences, in particular, independent
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ISBN: 978-0-12-374772-3
ISSN: 0065-230X
For information on all Academic Press publications
visit our website at www.elsevierdirect.com
Printed and bound in USA
09 10 11 12 10 9 8 7 6 5 4 3 2 1

Contents
Contributors to Volume 104 ix
Introduction
Saverio Bettuzzi
I. Introduction 1
References 7
Clusterin (CLU): From One Gene and Two
Transcripts to Many Proteins
Federica Rizzi, Mariangela Coletta, and Saverio Bettuzzi
I. Introduction 10
II. CLU Gene Organization, Promoter Region, and
Transcription Products 11
III. CLU Protein Forms 18
IV. Conclusions 19
References 21
The Shifting Balance Between CLU Forms
During Tumor Progression
Sabina Pucci and Saverio Bettuzzi
I. Introduction 25
II. Shifting of Cell Metabolism in Tumorigenesis 26
III. Shifting of CLU Forms During Tumor Progression 27
IV. Concluding Remarks 31
References 31
v

Regulation of Clusterin Activity by Calcium
Beata Pajak and Arkadiusz Orzechowski
I. Introduction 34
II. Ca2þ
Signal 34
III. Ca2þ
Contribution to CLU-Mediated Effects 35
IV. Ca2þ
Influx Through TRPM-2 Channels and CLU Location 37
V. Ca2þ
and Cell Proliferation 38
VI. Is CLU a Ca2þ
-Dependent Pro- or Antiapoptotic Gene? 39
VII. Ca2þ
Homeostasis and CLU Transcription 43
VIII. CLU Translocations in Response to Ca2þ
Signal 47
IX. ER Stress, Unfolded Protein Response, Ca2þ
, and CLU 48
X. Ca2þ
, Clusterin, and DNA Repair 50
XI. Conclusions 51
References 52
Nuclear CLU (nCLU) and the Fate of the Cell
Saverio Bettuzzi and Federica Rizzi
I. Introduction: Where is CLU? 60
II. Preliminary Remarks Before Dealing with nCLU: The Possible
Action of sCLU as Differentiating Agent 62
III. A Brief History of nCLU 64
IV. CLU Expression and Localization in the Regressing Rat Ventral Prostate 65
V. CLU was Found Positively Linked to Apoptosis and Negatively Linked to
Proliferation: The Early Years (1993–1995) 67
VI. CLU and Follicular Atresia, Another Model of Tissue Involution Linked to
Apoptosis 69
VII. CLU Expression was Found Associated to Nonapoptotic Cell Death 69
VIII. CLU Expression was Found Associated to Lesions in Central
and Peripheral Nervous System 70
IX. Increased CLU Expression is Linked to Inhibition of Proliferation and
Induction of Cell Differentiation 71
X. Early Studies on Translational Upregulation of CLU During Apoptosis:
The Role of NF1 72
XI. CLU Gene Expression and Cell Death Induced by Vitamin D (Calcitriol)
and Tamoxifen (TAM): Implication in Chemioresistance 73
XII. Early Observations: CLU Expression is Induced by TGF-Beta 1;
NF-Kappa B Inhibitors Blocked the Secretion of CLU 74
XIII. 1996: nCLU Finally Appears in the Literature 75
XIV. A Role For nCLU in the Nucleus: Binding to Ku70 77
XV. nCLU Today: The Dark Side of the Moon Revealed 78
XVI. The Molecular Mechanisms of Production of nCLU are Still Sketchy 80
XVII. In the End: Is Targeting of CLU to the Nucleus an Experimental Artifact? 83
XVIII. Conclusions 84
References 85
vi Contents

The Chaperone Action of Clusterin and Its Putative Role
in Quality Control of Extracellular Protein Folding
Amy Wyatt, Justin Yerbury, Stephen Poon,
Rebecca Dabbs, and Mark Wilson
I. Introduction 90
II. Clusterin as a Chaperone 92
III. In Vivo Insights into the Chaperone Action of Clusterin 96
IV. Other Extracellular Chaperones (ECs) and a Model For Quality Control (QC)
of Extracellular Protein Folding 98
V. Future Research Directions 103
VI. Conclusion 107
References 107
Cell Protective Functions of Secretory
Clusterin (sCLU)
Gerd Klock, Markus Baiersdörfer, and Claudia Koch-Brandt
I. Introduction 115
II. sCLU—A Component of High-Density Lipoproteins 116
III. sCLU in Apoptosis—Signaling Toward Cell Survival? 117
IV. sCLU in the Removal of Dead Cells and Cellular Debris 120
V. sCLU in Immune Modulation 123
VI. Role of sCLU in Amyloid Beta (A ) Clearance in
M. Alzheimer 126
VII. Conclusions and Perspectives 128
References 129
Clusterin: A Multifacet Protein at the Crossroad of
Inflammation and Autoimmunity
Géraldine Falgarone and Gilles Chiocchia
I. Introduction 140
II. Inflammation, the Danger Signal, and Autoimmunity 141
III. Immune System Homeostasis and Regulation: The Common Purpose
of Autoimmunity and Cancer 142
IV. Inflammation 142
V. The Place and Role of CLU In Autoimmune Diseases 150
VI. Concluding Remarks 161
References 163
Contents vii

Oxidative Stress in Malignant Progression: The Role of Clusterin,
A Sensitive Cellular Biosensor of Free Radicals
Ioannis P. Trougakos and Efstathios S. Gonos
I. Introduction 172
II. Molecular Effects of Altered Oxidative Load in Human Cells 173
III. Oxidative Injury in Ageing and Age-Related Diseases 177
IV. Oxidative Stress in Malignant Progression 179
V. Clusterin as a Sensitive Cellular Biosensor of Oxidative Stress 181
VI. Concluding Remarks—Perspectives 191
References 195
Index 211
Color Plate Section at the end of the book
viii Contents

Contributors
Numbers in parentheses indicate the pages on which the authors’ contributions begin.
Markus Baiersdörfer, Institute of Biochemistry, Joh.-Gutenberg University
of Mainz, Becherweg 30, D-55099 Mainz, Germany (115)
Saverio Bettuzzi, Dipartimento di Medicina Sperimentale, Sezione di
Biochimica, Biochimica Clinica e Biochimica dell’Esercizio Fisico, Via
Volturno 39-43100 Parma and Istituto Nazionale Biostrutture e Biosistemi
(I.N.B.B.), Rome, Italy (1, 9, 25, 59)
Gilles Chiocchia, AP-HP, Hôpital Ambroise Paré, Service de Rhumatologie,
Boulogne-Billancourt F-92000, France; Département d’Immunologie,
Inserm, U567, Paris F-75014, France; and Institut Cochin, Université
Paris Descartes, CNRS (UMR 8104), Paris F-75014, France (139)
Mariangela Coletta, Dipartimento di Medicina Sperimentale, Sezione di
Volturno 39-43100 Parma and Istituto Nazionale Biostrutture e Biosistemi
(I.N.B.B.), Rome, Italy (9)
Rebecca Dabbs, School of Biological Sciences, University of Wollongong,
Wollongong, New South Wales 2522, Australia (89)
Géraldine Falgarone, Rheumatology Department, AP-HP, Hôpital Avicenne,
Bobigny F-93009, France; and EA 4222, University of Paris 13, Bobigny,
France (139)
Efstathios S. Gonos, Institute of Biological Research and Biotechnology,
National Hellenic Research Foundation, Athens 11635, Greece (171)
Gerd Klock, Institute of Biochemistry, Joh.-Gutenberg University of Mainz,
Becherweg 30, D-55099 Mainz, Germany (115)
Claudia Koch-Brandt, Institute of Biochemistry, Joh.-Gutenberg University
of Mainz, Becherweg 30, D-55099 Mainz, Germany (115)
Arkadiusz Orzechowski, Department of Physiological Sciences, Faculty of
Veterinary Medicine, Warsaw University of Life Sciences (SGGW),
Nowoursynowska 159, 02-776 Warsaw, Poland; and Department of Cell
Ultrastructure, Mossakowski Medical Research Center, Polish Academy
of Sciences, Pawinskiego 5, 02-106 Warsaw, Poland (33)
ix

Beata Pajak, Department of Cell Ultrastructure, Mossakowski Medical
Research Center, Polish Academy of Sciences, Pawinskiego 5, 02-106
Warsaw, Poland (33)
Stephen Poon, School of Biological Sciences, University of Wollongong,
Sabina Pucci, Department of Biopathology, University of Rome ‘‘Tor
Vergata,’’ Rome, Italy (25)
Federica Rizzi, Dipartimento di Medicina Sperimentale, Sezione di
Volturno 39-43100 Parma and Istituto Nazionale Biostrutture
e Biosistemi (I.N.B.B.), Rome, Italy (9, 59)
Ioannis P. Trougakos, Department of Cell Biology and Biophysics, Faculty of
Biology, University of Athens, Panepistimiopolis Zografou, Athens 15784,
Greece (171)
Mark Wilson, School of Biological Sciences, University of Wollongong,
Amy Wyatt, School of Biological Sciences, University of Wollongong,
Justin Yerbury, School of Biological Sciences, University of Wollongong,
x Contributors

Introduction
Saverio Bettuzzi
Dipartimento di Medicina Sperimentale, Sezione di
Biochimica, Biochimica Clinica e Biochimica dell’Esercizio Fisico,
Via Volturno 39-43100 Parma and Istituto Nazionale
Biostrutture e Biosistemi (I.N.B.B.), Rome, Italy
I. Introduction
References
Since the beginning, Clusterin (CLU) was revealed not as simple to study, and
certainly not a single protein. The growing research interest on CLU soon produced
many contributions by independent laboratories working in different systems. Thus,
many different names or acronyms have been given to CLU in the early years after
its discovery. Now, a general consensus recommend the name Clusterin and the
abbreviation CLU. CLU was first described as a glycoprotein found nearly ubiquitous
in tissues and body fluids. This early knowledge is mostly related to the secretory form of
CLU (sCLU), which is exported from the cell and released in secretions acting as an
extracellular chaperone. But CLU can also enter the nucleus. The detection of nCLU
(nuclear CLU), which is usually associated to cell death, is now emerging as a very
important event making this issue even more complex. This may explain why CLU is still
often described as an “enigmatic” protein. The use of the term “enigmatic” is a clear
indication that too many aspects related to the biological function(s) of CLU and its
possible role in pathogenesis have been obscure, or very difficult to interpret, for long
time. Contradictory findings on CLU are also present in the literature, sometimes due to
technical biases or alternative interpretation of the same result. The aim of the book is
ambitious: through a careful review of old data, in the light of novel information and up
to date methods and hypotheses, we will try to simplify the picture for the reader and
bring more light in a field still perceived to be too obscure to fully appreciate its
importance and potential implementation in the clinical setting. This introduction
will provide a brief general history and a critical view of the discovery of CLU with the
aim to underline what is new in the field and what is now obsolete. In the rest of the
book, conclusions and “take home messages” will also be provided to the reader
particularly focusing on possible clinical implementations and how all this knowledge
will very likely bring novelty in the fight against cancer. # 2009 Elsevier Inc.
I. INTRODUCTION
Clusterin (CLU) was first described as a glycoprotein found nearly ubiqui-
tous in tissues and body fluids. This knowledge is mostly related to the
secretory form of CLU (sCLU), which is exported from the cell and released
in secretions. In the literature there is a general consensus about the apparent
Advances in CANCER RESEARCH 0065-230X/09 $35.00
Copyright 2009, Elsevier Inc. All rights reserved. DOI: 10.1016/S0065-230X(09)04001-9
1

involvement of CLU in most important biological processes including sperm
maturation, tissue differentiation, tissue remodeling, membrane recycling,
lipid transportation, cell–cell or cell–substratum interaction, cell prolifera-
tion, cell survival, and cell death. For these reasons, CLU is generally
believed to be involved in many and diverse pathological states, including
neurodegeneration, ageing, and cancer (Rizzi and Bettuzzi, 2008; Rosenberg
and Silkensen, 1995; Shannan et al., 2006; Trougakos and Gonos, 2002;
Wilson and Easterbrook-Smith, 2000).
CLU was firstly isolated from ram rete testis fluid (Fritz et al., 1983). Ram
rete testis fluid is known to elicit clustering of Sertoli cells (in suspension)
and erythrocytes from several species. In their pioneer work, the authors
showed that a heat-stable, trypsin-sensitive protein was responsible to
aggregate cells. Thus, they named this extracellular protein “Clusterin,”
suggesting that it may play important roles in cell–cell interactions. Then,
CLU was purified from the same system (Blaschuk et al., 1983; Fritz et al.,
1983) and found to be a glycoprotein with a molecular mass of about 80
kDa and an isoelectric point of 3.6. Using reducing conditions they discov-
ered that CLU dissociates into subunits of about 40 kDa, also showing that
CLU exists both in dimeric and tetrameric forms at neutral pH and low salt
concentrations. In addition, the amino acid composition of CLU was
reported. Further, extracellular CLU was found to contain 4.5% glucos-
amine. In the same work it was suggested that Sertoli cells are the potential
source of CLU, since primary cultures of rat Sertoli cells secreted CLU in the
medium. One year later, different isoelectric forms of CLU were isolated
(Blaschuk and Fritz, 1984). Thus, since the beginning, CLU was revealed not
as simple to study, and certainly not a single protein. This basic knowledge is
still true, but when addressing this issue we need to keep in mind that the
most commonly reported descriptions of CLU only apply to the secreted
form of CLU exported in the extracellular compartment (sCLU).
Thefirstimmunolocalization ofCLU dates 1985, whenTung and Fritzraised
the first monoclonal antibodies against CLU to investigate its distribution in
the adult ram testis, rete testis, and excurrent ducts (Tung and Fritz, 1985).
Since this first reports, the CLU gene was found expressed in a wide range of
tissues (Choi et al., 1989; de Silva et al., 1990; Fischer-Colbrie et al., 1984;
James et al., 1991), although with very different levels of expression. The
research interest on CLU soon produced many contributions by independent
laboratories working in different systems. Therefore, in the early years after its
discovery many different names or acronyms have been given to CLU (Table I).
Now, the name Clusterin and the abbreviation CLU is recommended, thanks
to a general consensus among the main researchers on the field.
In humans, CLU was first described by Jenne and Tshopp in 1989 (Jenne
and Tschopp, 1989) as complement cytolysis inhibitor (CLI), a component of
soluble terminal complement complexes in human serum, bearing complete
2 Saverio Bettuzzi

identity to sulfated glycoprotein 2 (SGP2), one of the most important
glycoprotein produced by Sertoli cells (Collard and Griswold, 1987). SGP2
was found, in turn, to be identical to Clusterin and suggested to play a role in
sperm maturation. In the ventral prostate of the rat, CLU was first identified
as testosterone repressed prostate message 2 (TRPM2) (Montpetit et al.,
1986). At this time, TRPM2 was described as a truncated cDNA with no
correspondent protein. In an independent work published on 1989,
after complete cDNA cloning, sequencing, and comparison, Bettuzzi and
coworkers found that TRPM2 was instead fully homologous to full-length
SGP-2 cDNA (Bettuzzi et al., 1989). Only one year before, Cheng et al. found
that SGP-2 was identical to Clusterin, the serum protein involved in aggrega-
tion of heterologous erythrocytes already described by Fritz et al. in 1983
(Cheng et al., 1988). Finally, in 1990, an apolipoprotein named ApoJ
associated with discrete subclasses of high-density lipoproteins, to which it
was bound through amphiphilic helices present in its structure, was found to
be the human analogue of SGP2 (de Silva et al., 1990).
Therefore,thesamecDNA/protein,nownamedCLU,wasfinallyfoundtobe
implicated in important biological phenomena as different as erythrocyte
aggregation, complement activity, lipid transport in the blood, sperm
maturation, and prostate gland involution driven by androgen depletion.
Historically, the first review on CLU is dated 1992 (Jenne and Tschopp, 1992).
Table I List of Proteins That Have Been Found Homologue of Clusterin (CLU)
Source Species Name Function Reference
Rete testes fluid Ram Clusterin Reproduction Blaschuk et al. (1983)
Adrenal
medulla
Bovine GPIII Chromaffin
granules
Fischer-Colbrie et al. (1984)
Prostate Rat TRPM-2 Apoptosis Leger et al. (1987)
Prostate Rat SGP-2 Reproduction Bettuzzi et al. (1989)
Neuroretinal
cells
Quail T64 Cell
transformation
Michel et al. (1989)
Serum (liver) Human SP-40,40 Complement
modulation
Kirszbaum et al. (1989)
Serum (liver) Human CLI Complement
modulation
Jenne and Tschopp (1989)
Blood Human ApoJ Lipid transport de Silva et al. (1990)
Blood Human NA1/NA2 Lipid transport James et al. (1991)
Retina Human K611 Retinitis
pigmentosa
Jones et al. (1992)
Homologues have been isolated and/or cloned by different groups working in widely divergent areas.
For this reason, different names and acronyms have been originally given to the same gene/protein, today
named CLU. The CLU gene was located in all mammalians species studied and its sequence was found
highly conserved among species, while the CLU gene products are several proteins of different molecular size
and structure also depending on the experimental system used.
Introduction 3

The gene coding for CLU was mapped on chromosome 8 in the human
genome (Purrello et al., 1991; Slawin et al., 1990; Tobe et al., 1991) in
position 8p12! p21 (Dietzsch et al., 1992) and found to be present in single
copy. The CLU gene was then found to be present in all mammalian genomes
studied, with very high homology among species.
Since the beginning it appeared clear that CLU, although being a single
copy gene, was actually coding for different protein products. AWestern blot
analysis of CLU expression in the rat ventral prostate system revealed that
CLU has to be considered more as a family of protein products rather than a
single protein (Fig. 1). Several antirat CLU-positive bands resolved by
electrophoresis, with different molecular weights, are detectable under
basal level of expression (Fig. 1, lane N) in the prostate gland. The proteomic
profile of CLU is even more complex when CLU gene is potently induced in
the regressing prostate following androgen depletion caused by surgical
castration (Fig. 1, lane 1).
In spite of the growing number of papers on this issue (for instance, using
Clusterin or SGP-2 or TRPM2 or ApoJ as key words, 1552 research papers
and 139 reviews can be found on PubMed to date. . .). CLU is still often
described as an “enigmatic” protein. The use of the term “enigmatic” is a
clear indication that too many aspects related to the biological function(s) of
RVP
N 1
RVP=rat ventral prostate
N=normal gland
1=4-day castrated
-75kDa
-50kDa
-35kDa
-30kDa
Fig. 1 Western blot analysis of CLU expression in the rat ventral prostate (RVP). N, normal
prostate; 1, regressing prostate gland 4 days after androgen ablation caused by surgical castra-
tion. Several immunopositive CLU protein bands of different molecular weight are present both
under basal conditions (N) and when potently induced by castration (1). During the regression
of the rat prostate mainly due to massive apoptosis, extra protein bands are produced from the
same gene.
4 Saverio Bettuzzi

CLU and its possible role in pathogenesis have been obscure, or very difficult
to interpret, for long time. Contradictory findings on CLU are also present in
the literature. All this do not really help our understanding of the topic.
Following a tradition which started time ago, the 5th Clusterin/Apolipo-
protein J (CLU) Workshop has been held in Spetses (Greece) in June
2008. The next (6th) Workshop will be held in Parma (Italy) in 2011. The
Spetses meeting brought together the most active researchers in this field.
They presented their more recent data and discussed about the mechanisms
through which this unique protein would be executing the fate of the
cell. The debate mostly focused about whether CLU has a key role in
tumor prevention or tumor promotion. The fruitful discussion started to
render some consensus in the field. A scientific report on this event has
been recently published (Trougakos et al., 2009). The idea to write this
book has emerged during the meeting in Spestes as a great opportunity to
bring together experimental data, novel ideas, scientific hypotheses, and
opinions. Our hope is that the contribution of different senior researchers,
focusing on the possible role of CLU in tumorigenesis, presented in this
book would help the reader to understand more in this complex field.
The aim of the book is ambitious: through a careful review of old data, in
the light of novel information and up to date methods and hypotheses,
we will try to simplify the picture for the reader and bring more light in a
field still perceived to be too obscure to fully appreciate its importance and
potential implementation also in the clinical setting. After this introduction,
in which a brief general history and critical view of the discovery of CLU has
been provided, every chapter will be preceded by a specific introduction to
better introduce the reader to the specific topics that will be further dis-
cussed. The book starts with a description of the novel knowledge and the
complexity of the regulation of CLU gene expression, showing how from a
single copy gene multiple transcripts and protein products can be originated
in mammalian cells (Chapters “Clusterin: From one gene and two tran-
scripts to many proteins”, “The shifiting balance between CLU forms during
tumor progression” and “Regulation of Clusterin activity by calcium”)
including the nuclear form nCLU.
Because of the identification of many protein products with different
molecular weights in association to intracellular processing and maturation
of CLU, we will use the term “secreted CLU” (sCLU) when the most of CLU
is fully maturated in the Golgi apparatus and exported from the cell as
glycosylated protein in the extracellular compartment. But its secretion
can be completely abolished. Under these conditions, CLU is not detectable
any longer in the culture medium and the cells will get fully loaded with
CLU. Now CLU can be mostly found in the cytoplasm of the cell, but also in
the nucleus. The condition in which CLU is exclusively present in the
cytoplasm, as well as that in which CLU is solely present in the nucleus,
Introduction 5

are very rare. The most common situation is the detection of an “intracellu-
lar” staining showing CLU both in the cytoplasm and in the nucleus in
intact, fixed cells. Therefore, cytoplasm and nuclear localization may often
coexist. In any case, the detection of even a small amount of CLU in the
nucleus of intact cells by appropriate methods is here defined as “nuclear
CLU” (nCLU). While we can track CLU with appropriate antibodies, we do
not have definitive information about the structural differences between
different protein products besides changes in the molecular weight as
revealed by electrophoresis. Therefore, the detection of the protein forms
sCLU or nCLU is here intended more as a cell functional condition rather
than the identification of a structurally defined molecular form. This is why
we will avoid the use of the term “isoform” in this book for CLU proteins,
waiting for more definitive experimental data on the issue. The term
isoform will be used instead for different transcripts coding for CLU, on
which more is known because they have been identified and sequenced.
Then the discussion will continue with a critical review of what is known
about sCLU and nCLU (Chapters “Nuclear CLU and the fate of the cell”,
“The chaperone action of Clusterin and its putative role in quality control of
extracellular protein folding” and “Cell protective functions of secretory
Clusterin”) with a focus on their potential biological action. Then, we will
address what is known today about the involvement of CLU in important
physiological and pathological conditions such as inflammation and immu-
nity (Chapter “Clusterin: A multifacet protein at the crossroad of inflamma-
tion and autoimmunity”), oxidative stress (Chapter “Oxidative stress in
malignant progression: The role of Clusterin, a sensitive cellular biosensor
of free radicals”), and some of the most diffuse and important kind of
cancers (Chapters “CLU and prostate Cancer”, “CLU and breast Cancer”,
“CLU and colon Cancer” and “CLU and lung Cancer” of Volume 105).
The possibility that sCLU plays an important role in chemioresistance to
anticancer drug is also discussed (Chapter “CLU and chemoresistance” in
Volume 105), as well as the possible diverse role of CLU protein(s) and CLU
gene expression in dependence of the local tissue context and microenviron-
ment (Chapter “CLU and tumor microenvironment” of Volume 105).
Chapter “Regulation of CLU gene expression by oncogenes and epigenetic
factors: implication for tumorigenesis” of Volume 105 is the discussion
arena in which several senior authors in the field will try to merge their
expertise and ideas from different fields in a general consensus to provide an
integrated view on CLU gene expression from a novel point of view, that is,
epigenetic regulation, also discussing how CLU gene expression is affected
by oncogenes during cell transformation.
Finally, Chapter “Conclusion and Perspectives: CLU for novel therapies
and advanced diagnostic tools?” of Volume 105 will try to draw some
conclusions and “take home messages”, dealing with what is now obsolete
6 Saverio Bettuzzi

in the field, which clinical implementations have been already attempted,
how successful they have been in consideration of the most up to date
information on this issue and how all this knowledge will very likely bring
novelty in the fight against cancer.
ACKNOWLEDGMENTS
Grant sponsor: FIL 2008 and FIL 2009, University of Parma, Italy; AICR (UK) Grant No.
06–711; Istituto Nazionale Biostrutture e Biosistemi (INBB), Roma, Italy.
REFERENCES
Bettuzzi, S., et al. (1989). Identification of an androgen-repressed mRNA in rat ventral prostate
as coding for sulphated glycoprotein 2 by cDNA cloning and sequence analysis. Biochem. J.
257, 293–296.
Blaschuk, O. W., and Fritz, I. B. (1984). Isoelectric forms of clusterin isolated from ram rete
testis fluid and from secretions of primary cultures of ram and rat Sertoli-cell-enriched
preparations. Can. J. Biochem. Cell Biol. 62, 456–461.
Blaschuk, O., et al. (1983). Purification and characterization of a cell-aggregating factor
(clusterin), the major glycoprotein in ram rete testis fluid. J. Biol. Chem. 258, 7714–7720.
Cheng, C. Y., et al. (1988). Rat clusterin isolated from primary Sertoli cell-enriched culture
medium is sulfated glycoprotein-2 (SGP-2). Biochem. Biophys. Res. Commun. 155,
398–404.
Choi, N. H., et al. (1989). A serum protein SP40,40 modulates the formation of membrane
attack complex of complement on erythrocytes. Mol. Immunol. 26, 835–840.
Collard, M. W., and Griswold, M. D. (1987). Biosynthesis and molecular cloning of sulfated
glycoprotein 2 secreted by rat Sertoli cells. Biochemistry 26, 3297–3303.
de Silva, H. V., et al. (1990). Purification and characterization of apolipoprotein J. J. Biol.
Chem. 265, 14292–14297.
Dietzsch, E., et al. (1992). Regional localization of the gene for clusterin (SP-40,40; gene symbol
CLI) to human chromosome 8p12–> p21. Cytogenet. Cell Genet. 61, 178–179.
Fischer-Colbrie, R., et al. (1984). Isolation and immunological characterization of a glycopro-
tein from adrenal chromaffin granules. J. Neurochem. 42, 1008–1016.
Fritz, I. B., et al. (1983). Ram rete testis fluid contains a protein (clusterin) which influences cell-
cell interactions in vitro. Biol. Reprod. 28, 1173–1188.
James, R. W., et al. (1991). Characterization of a human high density lipoprotein-associated
protein, NA1/NA2. Identity with SP-40,40, an inhibitor of complement-mediated cytolysis.
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Jenne, D. E., and Tschopp, J. (1989). Molecular structure and functional characterization of a
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Introduction 7

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Kirszbaum, L., et al. (1989). Molecular cloning and characterization of the novel, human
complement-associated protein, SP-40, 40: a lin between the complement and reproductive
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Michel, D., et al. (1989). Expression of a novel gene coding a 51.5 kD precursor protein is
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8 Saverio Bettuzzi

Clusterin (CLU): From One
Gene and Two Transcripts to
Many Proteins
Federica Rizzi, Mariangela Coletta, and Saverio Bettuzzi
Dipartimento di Medicina Sperimentale, Sezione di Biochimica, Biochimica
Clinica e Biochimica dell’Esercizio Fisico, Via Volturno 39-43100 Parma and
Istituto Nazionale Biostrutture e Biosistemi (I.N.B.B.), Rome, Italy
I. Introduction
II. CLU Gene Organization, Promoter Region, and Transcription Products
III. CLU Protein Forms
IV. Conclusions
References
Clusterin (CLU) has kept many researchers engaged for a long time since its first
discovery and characterization in the attempt to unravel its biological role in mammals.
Although there is a general consensus on the fact that CLU is supposed to play
important roles in nearly all fundamental biological phenomena and in many human
diseases including cancer, after about 10 years of work CLU has been defined as an
“enigmatic” protein. This sense of frustration among the researchers is originated by
the fact that, despite considerable scientific production concerning CLU, there is still a
lack of basic information about the complex regulation of its expression. The CLU
gene is a single 9-exon gene expressed at very different levels in almost all major tissues
in mammals. The gene produces at least three protein forms with different subcellular
localization and diverse biological functions. The molecular mechanism of production
of these protein forms remains unclear. The best known is the glycosylated mature form
of CLU (sCLU), secreted with very big quantitative differences at different body sites.
Hormones and growth factors are the most important regulators of CLU gene expres-
sion. Before 2006, it was believed that a unique transcript of about 1.9 kb was
originated by transcription of the CLU gene. Now we know that alternative transcrip-
tional initiation, possibly driven by two distinct promoters, may produce at least two
distinct CLU mRNA isoforms differing in their unique first exon, named Isoform 1 and
Isoform 2. A third transcript, named Isoform 11036, has been recently found as one of
the most probable mRNA variants. Approaches like cloning, expression, and function-
al characterization of the different CLU protein products have generated a critical mass
of information teaching us an important lesson about CLU gene expression regulation.
Nevertheless, further studies are necessary to better understand the tissue-specific
regulation of CLU expression and to identify the specific signals triggering the expres-
sion of different/alternative transcript isoforms and protein forms in different cell types
at appropriate time. # 2009 Elsevier Inc.
Advances in CANCER RESEARCH 0065-230X/09 $35.00
Copyright 2009, Elsevier Inc. All rights reserved. DOI: 10.1016/S0065-230X(09)04002-0
9

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“The canine teeth of the males are long, strong and
pointed, but this is not the case with the females. The
structure, therefore, of the canine teeth is to be regarded
in the light of a sexual peculiarity, and not as having any
connection with the nature of the food.”
The teeth of man are inferior in strength to those of the
anthropoid apes, but the cause of this is to be sought not so much
in their original character as in the fact that they have been
weakened and degenerated by the use of cooked food for thousands
of years.
It may perhaps be objected that anthropoid apes, which have
been cited as typical frugivorous animals, are not so much so as I
have contended; that, while their chief food is doubtless fruits and
nuts, they do occasionally feed upon all kinds of substances—roots,
insects, small animals when they can catch them, etc. Thus
Professor Robert Hartmann in his “Anthropoid Apes” p. 255 says:
“Although they are for the most part content with
vegetable diet, gibbons sometimes eat animal food, such
as lizards; and Bennet saw a siamang seize and devour
one of these animals whole.... They do not, however,
display the keenness of scent and quickness of sight
which distinguish some animals of a lower order; such as
canine beasts of prey and ruminants manifest in many
different ways.” (p. 256).
Now, it will be noticed in the above connection that (1) these apes
are, by reason of their peculiar anatomical and physiological
construction, incapable of competing with the carnivora for food of
that character—and hence naturally disqualified to live upon it; and
(2) these animals do not naturally live upon this food by choice,
when other and, to them, more natural food is forthcoming. Only in
the last stages of hunger do they resort to food of this nature, which
they are obviously driven to by extremity, and are disqualified to eat
by reason of their peculiar construction. An animal can be driven to
eat anything if he is hungry enough. That does not prove that what

he eats is his natural food, nevertheless! Instinct, and other
considerations, must determine that.
The Frugivora.—The orang and the gorilla are perhaps the best
examples of this class of animals. Some bats and kangaroos may be
included in it also. Animals belonging to this class have thirty-two
teeth—sixteen in each jaw; four incisors or cutting teeth; two
pointed teeth, known as cuspids, four small molars, known as
bicuspids, and six molars. The eye-teeth project somewhat beyond
the others and fit into a blank space in the lower row, the other
teeth articulating uniformly. I have referred to the uses of this large
eye-tooth elsewhere (p. 29).
The Teeth of Man.—Now when we come to consider the teeth of
man, we are at once struck by the fact that they correspond, in
almost every particular, with the teeth of the gorilla and other
frugivorous animals; and the fact they do not at all resemble or
correspond to the teeth of any other animal! To the teeth of the
herbivora, the carnivora, the omnivora, etc., they bear but the
slightest resemblance, while they agree in almost every respect with
the teeth of frugivorous animals. If we compare the teeth of man
with those of the orang, the gorilla, or other frugivorous animal, we
find that the number, the arrangement, the structure, the nature,
and the size of the teeth are almost identical; while they bear but
the smallest resemblances to the teeth of any other animal or
genera. The complete absence of intervening spaces between the
human teeth characterises man as the highest and purest example
of the frugivorous animal. Man possesses no long, canine tooth,
capable of catching and holding a captured prey; he possesses no
tusks, like the omnivorous animals, and in every other way bears no
resemblance whatever to any other animal—while his teeth do bear
the very greatest and most detailed resemblance to the teeth of the
apes and frugivorous animals generally. Bearing all these facts in
mind, then, we surely can have no hesitation in classifying man as a
frugivorous animal—so far as his teeth are concerned. Considered
from that point of view, man must be classed with the pure
frugivora.

Not only in the number and structure of the teeth, but also in the
manner of masticating the food—in the movements of the teeth and
jaws themselves—there is a distinct resemblance between man and
the apes and other frugivora, and a radical distinction between him
and all other animals. In herbivorous animals the jaws have three
distinct motions—a vertical, or up-and-down motion; lateral or
sidewise; and forward and backward. These movements are
frequent and free, the result being that food eaten by these animals
undergoes a thorough grinding process well suited to the nature of
their food. In the carnivorous animals, on the other hand, the
movements of the jaws are in one direction only—they open and
shut “like a pair of scissors,” as one author said, and are well
adapted for tearing and biting off food that is to be swallowed more
or less en masse, to be acted upon by the powerful gastric juices of
the stomach. No such limited action is the case with man. With him
also the jaws can move in three directions—as in the case of the
herbivora—but the extent of such motion is much more limited. In
other words, the jaws of man are adapted to a diet necessitating
more or less grinding, and he may be classed with the herbivora on
that account. Whatever might be said, however, by way of
associating man with the herbivora, he is certainly as distinct as
possible from the carnivora, and resembles other animals far more
than he resembles them. He is certainly not carnivorous, whatever
else he may be!
Having thus passed in review the evidence presented by the teeth
for the naturally frugivorous nature of man, we must now turn and
examine the evidence afforded by the other organs of the body; and
see how far comparative anatomy affords proof of the nature of
man’s diet—as derived from a study of the other portions of his
bodily frame. I shall review these in turn. First let us consider the
extremities.

The Extremities.—According to Huxley, there are three great
divisions in the animal kingdom, as regards the extremities—viz.
those which possess hoofs, those possessing claws, and those
possessing hands. To the first division belong the herbivora and the
omnivora. Almost all animals possessing claws are carnivorous, while
animals possessing hands are almost invariably frugivorous. To this
rule there are very few exceptions. Since man certainly belongs to
the class possessed of hands, he is certainly frugivorous by nature.
The reason for this becomes apparent when we stop to consider the
habits of the various animals. The herbivora have no need for hands;
they have merely to walk about the grassy plains, and partake of
what nature has offered to them in abundance. The carnivorous
animal, on the other hand, takes his food by violence—suddenly
springing upon some defenceless and unresisting animal, and
tearing it to pieces with its sharp teeth and claws. For this reason
they are developed to the size and extent we see—capable of
inflicting such terrible injuries. And here I would again call attention
to what I said before—as to the carnivorous traits and characteristics
of the cat as compared with those of the dog. The teeth and claws
are far more developed in the former than in the latter. In man, of
course, his teeth and claws are entirely unfitted for any such office.
The soft, yielding nails are absolutely unlike the long, sharp claws of
the carnivora: nothing could be more dissimilar. But if we compare
the hands and extremities of man with those of other frugivorous
animals, there is a very close similarity between them. The reason
for this is that man (like the apes) can and should go out into the
open fields and forests and pick his food off the trees. The human
hand is eminently adapted to this end and for this purpose; but is
quite unadapted for any such purposes as the claws of the carnivora
are adapted for. I may remark here, incidentally, that all carnivorous
animals drink by lapping up the water or other liquid with their
tongues; while man, and all vegetarian animals, drink by suction—by
drawing up the fluid directly into the mouth. This is a very
distinguishing characteristic, to which there are few if any
exceptions. Needless to say, since man drinks by suction, he is

eminently a vegetarian animal, and is quite distinct from the
carnivora in this respect, as in all others.
The Alimentary Canal.—“One of the most interesting
comparisons,” says Dr Kellogg,[5] “which has been made
by comparative anatomists is the length of the alimentary
canal. This is very short in the carnivora, and long in the
herbivora. When compared to the length of the body of
the different classes of animals, the proportion is found to
be as follows:—In the carnivora, the alimentary canal is
three times the length of the body; in the herbivora, as
the sheep, thirty times the length of the body; in the
monkey, twelve times; in the omnivora ten times; in man,
as in the frugivora, twelve times. Here, as before, we see
that anatomy places man strictly in the frugivorous class.
Some writers have made the amusing blunder of making
the proportionate length of the alimentary canal in man 1
to 6, instead of 1 to 12, by doubling the height through
measuring him while standing erect. This measurement is
evidently wrong, for it includes the length of the lower
extremities, or hind legs, whereas in other animals the
measurement is made from the tip of the nose to the end
of the backbone. In omnivorous animals, the alimentary
canal is shorter than in the apes and in man, thus
affiliating this class more nearly with the carnivora than
with the herbivora.
“A curious fact had recently been observed by Kuttner,
as related by him in an article published in Virchow’s
Archives. This author has made extensive anatomical
researches respecting the lengths of the small intestine in
different classes of persons. He finds that in the
vegetarian peasants of Russia, the small intestine
measures from twenty to twenty-seven feet in length,
while among Germans, who use meat in various forms
quite freely, the length of the small intestine varies

between seventeen and nineteen feet. The author
attributes the difference in these two classes of persons to
the difference in diet. Of course differences of this sort
must be the influence of the diet exerted through many
generations. This observation would seem to suggest that
the special anatomical characteristic of the carnivorous
class of animals is due to the modifying influence of their
diet, acting through thousands of years. If the length of
the intestine in man may be shortened by the use of flesh,
with other foods, for a few hundred years, more extensive
modifications may easily result from the longer experience
of animals that subsist upon an exclusively carnivorous
diet.”[6]
The Stomach.—The position and form of the stomach are also of
significance. In the carnivora, it is only a small roundish sack,
exceedingly simple in structure; while in the vegetable feeders it is
oblong, lies transversely across the abdomen, and is more or less
complicated with ringlike convolutions—according to the nature of
the food. This appears conspicuously in the primates, which include
man, in the Rodentia, Edentata, Marsupials, and, above all, in the
Ruminants. In the latter, it presents a series of from four to seven
wide, adjoining and communicating sacks.
At a first superficial glance at the exteriors of the stomachs of the
carnivora and that of man, we apparently perceive a far closer
resemblance than between man’s stomach and that of a herbivorous
animal. In one sense, there can be no question that there is a closer
similarity; in another sense, it is not so. In man this organ is simple,
but is divided into a cardiac and pyloric portion—thus occupying, as
in many other anatomical respects, a middle line between the
carnivorous and herbivorous mammalia. The inner surface of the
stomach is covered with rugæ, or wrinkles, formed by the mucous
membrane, which lines the whole intestinal canal, and which forms
valvular folds; while in the carnivora the stomach is a simple globular
sac, without these corrugations. As Dr Trall observed[7]:

“Some may imagine, at a first glance, a closer
resemblance between the human stomach and the lion’s
than between the human and that of the sheep. But when
they are viewed in relation to their proper food, their
closer resemblance will vanish at once. It should be
particularly observed that, so far as mere bulk is
concerned, there is a greater similarity between the food
of frugivorous animals and carnivorous animals than
between frugivorous and herbivorous. The digestion and
assimilation of coarse herbage, as grass, leaves, etc.,
requires a more complicated digestive apparatus than
grains, roots, etc., and these more so than flesh and
blood. The structure of the stomach, therefore, in such
cases, seems precisely adapted to the food we assume
Nature intended for it.”[8]
The Liver.—Dr John Smith, in calling attention to the many
distinctions between the bodily structure of man and that of the
carnivora, pointed out the following differences among others:—
“In the carnivora and rodentia, which present the most
complex form of liver among the mammalia, there are five
distinct parts; a central or principal lobe, corresponding
with the principal part of the liver in man; a right lateral
lobe, with a lobular appendage, corresponding to the
‘lobulus Spigelii’ and the ‘lobus caudatus,’ and a small lobe
or lobule on the left side. Through the whole animal
series, however, the magnitude of the liver varies in
inverse ratio to the lungs.
“In man, the liver is much less developed than the same
organ in many other mammalia; and presents, as
rudimentary indications, certain organs which are in other
animals fully developed. Europeans, and the inhabitants of
Northern climes, who partake more of animal food, have
the liver much larger, and its secretions more copious,
than the inhabitants of warm climates. Perhaps this, in

some measure, depends upon the amount of non-azotised
articles taken along with the flesh of animals, by which
means the system is supplied with more carbon than is
needed. So that the enlarged liver is attributable to gross
living on mixed diet, rather than to an exclusively animal
diet.”
This author also says elsewhere (p. 79):
“The temporal and masseter muscles, by which the
motion of the lower jaw is effected, are of immense size in
carnivorous animals. The temporal muscle occupies the
whole side of the scull, and fills the space beneath the
zygomatic arch, the span and spring of which are
generally an index of the volume of this muscle; while the
extent and strength of the arch indicate the development
of the masseter muscle. On the contrary, the pterygoid
muscles, which aid the lateral movement of the jaw, are
extremely small. The zygoma is of great size and strength
in the carnivora; consisting of a long process of the
masseter bone, overlaid by the usual process of the
temporal bone, which is equally strong. The arch extends
not only backward but upward, by the bending down of
the extremity; the line of anterior declination falling
precisely on the centre of the carnassière tooth—the point
in which the force of the jaws is concentrated, and where
it is most required for cutting, tearing, and crushing their
food. In ruminants, the zygomatic arch is short, and the
temporal muscles are small; but the masseter muscle on
each side extends beyond the arch, and is attached to the
greater part of the side of the maxillary bone. The
pterygoid fossa is ample, and its muscles are largely
developed. The arch is small in man, the temporal
muscles moderate, and the force of the jaws
comparatively weak.”

The Placenta.—Let us now turn to another important distinction
between the carnivorous and non-carnivorous animals. Of these,
perhaps the most important is the character of the placenta—one of
the most distinguishing marks or characteristics of any species of
animal. This subject has been so well and ably summed up by
Professor Schlickeysen, in his “Fruit and Bread” (pp. 48-57), that I
cannot do better than quote the main portion of the argument, as
stated by this learned and able author. He says:
“We now come to consider the peculiar structure, form
and size of the placenta, as well as the exact method by
which, through it, in different species of animals, the
nourishment is effected. One of the most striking
differences presented in placental animals relates to the
method of union between the mother and the fœtus.
There are two very distinct types of the placenta, and,
according to Professor Huxley, no transitional forms
between them are known to exist. These types are
designated as follows:—
1. The non-deciduate placenta of the Herbivora.
2. The deciduate placenta, of which there are two
kinds:
(a) The zonary deciduate placenta of the
Carnivora.
(b) The discoidal deciduate placenta of the
Frugivora.
“The deciduate placenta is a distinct structure,
developed from the wall of the uterus, but separated from
it at parturition, and constituting what is known as the
‘after birth’; of this the human placenta is regarded by
Huxley as the most perfect example; while, of the non-
deciduate placenta, that of the pig and horse are the
typical representatives. The word ‘decidua’ signifies ‘that
which is thrown off.’

“The Non-Deciduate Placenta.—This form is thus
described by Professor Huxley: ‘No decidua is developed.
The elevations and depressions of the unimpregnated
uterus simply acquire a greater size and vascularity during
pregnancy, and cohere closely to the chorionic villi, which
do not become restricted to one spot, but are developed
from all parts of the chorion, except at its poles, and
remain persistent in the broad zone thus formed
throughout fœtal life. The cohesion of the fœtal and
maternal placentæ, however, is overcome by slight
maceration; and at parturition the fœtal villi are simply
drawn out, like fingers from a glove, no vascular
substance of the mother being thrown off.’ To this class
belong all the ruminants and Ungulata (hoofed
quadrupeds); the camel, sheep, goat and deer; the ant-
eater, armadillo, sloth, swine, tapir, rhinoceros, river-
horse, sea-cow, whale, and others.
“The Zonary Deciduate Placenta.—A zonary placenta
surrounds the chorion, in the form of a broad zone,
leaving the poles free. This form characterises all the land
and sea carnivora, and thus includes the cat, hyena,
puma, leopard, tiger, lion, fox and wolf; the dog and bear,
the seal, sea-otter and walrus. It includes, also, certain
extinct species, as the mastadon and dinotherium, which,
although not wholly carnivorous, were, to judge from their
teeth, partially so. The elephant, the only living species of
these ancient animals, is also of this class.
“The Discoidal Deciduate Placenta.—The discoidal
placenta is a highly developed vascular structure, lying on
one side of the fœtus, in the form of a round disc, leaving
the greater part of the chorion free. It is thus united only
on one side, at one circular point, with the mucous
membrane of the uterus, from which, as already
mentioned, it is separated at parturition. The orders of the
animals characterised by this form of placenta are the

rodentia, ant-eaters, bats, and various species of apes,
and man. All these are very closely united by homologous
anatomical forms. The human placenta does not differ, in
its general character, from that of the others, and there is
no good reason for separating man from his placental
classification.”
Relations between placental forms and Individual Characteristics.
—From our entire knowledge of the development of races and of
individuals, we may conclude, upon the basis of Huxley’s
classification, that an intimate relation exists between the form and
character of the placenta and the entire nature of the individual. We
find among the non-deciduata, besides the toothless sloths, only the
Ungulata, or hoofed quadrupeds, and others developed from them.
The arrangement of their teeth, as of their entire digestive
apparatus, marks them as belonging to a single family—namely, the
herbivora.
The zonary placenta characterises a very large family of animals
whose peculiarities are distinctly marked, especially with regard to
their teeth and digestive apparatus. These belong to the widely
diffused and numerous orders of the carnivora. But the most
interesting and important group, with reference to our present study,
is that characterised by the discoidal placenta; for, since it includes
man and the fruit-eating apes, it gives occasion for the comparison
between these and other placental animals from the standpoint of
dietetics.
We observe here at once that the majority of animals having a
discoidal placenta subsist chiefly upon fruits and grains, and that the
typical representatives of this class, namely, those whose plactental
formation is most distinctly discoidal, are also the most exclusively
frugivorous.

Here, as elsewhere in nature, an exact line cannot be drawn.
Transitional forms exist everywhere, and to this the placenta is no
exception. The most striking accordance, however, exists between
the placenta of man and that of the tailless apes—namely, the
gorilla, orang, chimpanzee and gibbon. Between other discoidal
species, the differentiation, though minute, is clearly marked; but
between man and these apes the resemblance is so exact as to
stamp them plainly as members of the same family.
The completely developed placenta is in the form of a circular
disc, about eight inches broad, one inch thick and weighing about
two pounds. Its manner of development is identical in the human
subject and that of the above-named anthropoid apes. Its exact
formation is thus described by Huxley:
“From the commencement of gestation, the superficial
substance of the mucous membrane of the human uterus
undergoes a rapid growth and textural modification,
becoming converted into the so-called decidua. While the
ovum is yet small, this decidua is departable into three
portions: The decidua vera, which lines the general cavity
of the uterus; the decidua reflexa, which immediately
invests the ovum; and the decidua serotina, a layer of
especial thickness, developed in contiguity with those
chorionic villi which persist and become converted into the
fœtal placenta. The decidua reflexa may be regarded as
an outgrowth of the decidua vera the decidua serotina as
a special development of a part of the decidua vera. At
first, the villi of the chorion are loosely implanted into
corresponding impressions of the decidua; but, eventually,
the chorionic part of the placenta becomes closely united
with and bound to the uterine decidua, so that the fœtal
and maternal structures form one inseparable mass.”
The fœtus thus united to the mother is nourished by means of
numerous arterial and venous trunks, which traverse the deeper
substance of the uterine mucous membrane, in the region of the

placenta. These are connected with the placenta by means of the
umbilical cord, which consists of two arteries and two veins. The
length of this cord is greater in the case of man and the anthropoid
apes than in any other animals, reaching in them a length of about
two feet. The strict accordance which thus appears between the
placental structure of man and the ape indicates, upon the basis of
Huxley’s principles of classification, the same physiological functions
and the same dietetic character. There exists a complete similarity
between the corresponding organs in each: Their extremities end in
hands and feet. Their teeth and digestive apparatus indicates a
frugivorous diet. Their breasts and manner of nursing suggest the
same tender care of the new-born creature; while the brain and
mental capacity are also of a like character—differing only in degree;
indeed, the difference between the ape and animals of the next
lower grade is much greater than between the ape and man; there
being in the latter case really no essential anatomical or
physiological differences.
The fact that man has four cuspid teeth affords no evidence
whatever that he is either partially or wholly carnivorous as regards
his dietary. If in diet he is naturally omnivorous, his teeth should
have the structure and arrangement of those of omnivorous animals
—as exhibited in the hog, for example.
That the cuspid teeth do not indicate a flesh dietary, either in
whole or in part, is shown by the presence of the so-called cuspids
in purely herbivorous animals—as in the stag, the camel and the so-
called “bridle-teeth” of the horse.
I am convinced that no animals were created to eat flesh, but that
so-called carnivorous animals were originally nut-eating animals (see
p. 55). The squirrel eats birds as well as nuts, which closely
resemble meat in composition. This view readily explains the close
resemblance in many particulars existing between the human
digestive apparatus and that of the so-called carnivorous animals. It
is reasonable to suppose that these nut-eating animals were at some
remote time forced by starvation to slay, and eat, by the failure of

their ordinary food supply—just as the horses of the Norwegian
coast have been known to plunge into the sea and catch fish, when
driven to this extremity by starvation. Suppose the carnivorous
animal’s natural diet to be nuts, in the absence of his normal food he
would find nothing else so closely resembling his ordinary food as
the flesh of animals, since the two have about the same proteid
percentages.
Dr Kellogg, in his excellent little book, entitled “Shall We Slay to
Eat?” (pp. 30-32), sums up a number of remarkable facts in favour
of a fruitarian diet, or at least in favour of a non-flesh diet, as
follows:—
“In carnivorous, herbivorous and omnivorous animals,
the mammary glands are located upon the abdomen,
while in the higher apes and man they are located on the
chest. This is an interesting anatomical fact to which there
is no exception.
“In carnivorous animals the colon is smooth and non-
sacculated. In the higher apes and man the colon is
sacculated. In herbivorous animals the colon is sacculated,
as in man.” (The great importance and significance of this
fact will be apparent presently, when we come to consider
the physiological arguments against flesh-eating.)
“In carnivorous animals the tongue is very rough,
producing a rasping sensation when coming in contact
with the flesh. In the higher apes and man the tongue is
smooth.
“In carnivorous animals the skin is not provided with
perspiratory ducts—hence the skin does not perspire in
the dog, the cat, and allied animals. In the ape, the skin is
provided with millions of these glands, and in man they
are so numerous that if spread out, their walls would
cover a surface of eleven thousand square feet. In the pig,
an omnivorous animal, only the snout sweats. In horses,

cows and other vegetable-eating animals, the whole skin
sweats, as it does in man.” (The great importance of this
fact will be apparent when we come to consider the
physiological arguments against a flesh-diet: see p. 55.)
“Carnivorous, herbivorous and omnivorous animals are
all supplied with an extension of the backbone—a tail. In
the higher apes, as well as in man, the tail is wanting.
“Carnivorous, herbivorous and omnivorous animals go
on all fours, and their eyes look on either side, while many
of the higher apes walk nearly or entirely upright, as does
man, and their eyes look forward.[9]
“Carnivorous animals have claws, herbivorous and
omnivorous have hoofs, while apes and men have flat
nails, not found in any other animal. Carnivorous,
herbivorous and omnivorous animals are all quadrupeds,
or four-footed, while the higher apes and man are
provided with two hands and two feet. The hinder or
lower extremities of the ape are sometimes erroneously
called hands; according to Dr Huxley, they are, from both
bony and muscular structure, properly classified as feet,
and not as hands.
“In carnivorous animals, the salivary glands are small,
and the saliva which they secrete has little effect upon
starch, while in the apes and man the glands are well
developed and the saliva is active” (see pp. 47-48).
In addition to all the facts that have been pointed out, there are
others of lesser interest, but all of which, nevertheless, go to confirm
the fact that man is closely related to the apes, and consequently
intended for a fruitarian diet, and that he is in no wise related to the
carnivora or their diet. Metchnikoff has summarised many of these
facts, extending the work of Darwin, Huxley, Haekel, etc. These
other, minor, facts might perhaps be summarised as follows:—

There is an exact agreement between the skeleton of man and the
higher apes—all the bones corresponding, each to each, while there
is a great dissimilarity between man and any other animal whatever.
The nerves, the viscera, the spleen, the liver, the lungs, the brain,
the skin, nails and hair—all present the closest possible analogy and
similarities. The eyes are strikingly similar, while the chemical and
microscopical character of the blood is also very similar in man and
the higher apes. This fact is of especial importance and significance,
when we bear in mind that only apes and men are subject to certain
blood diseases—to which all other animals are impervious. In
structure, as in habits, man and the apes are in many respects
remarkably alike, and proportionately dissimilar to all other animals.

CHAPTER III
THE ARGUMENT FROM PHYSIOLOGY
“After structure—function!” Having seen in the last chapter that
man is constructed throughout for a diet composed entirely of fruits,
nuts, grains, and other non-flesh foods, we now turn to a
consideration of the functions of the various organs of the body—the
chemical composition of the organic tissues, secretions, etc.—in
order to see if these will further bear us out in our argument. There
can be no question that the most important argument of all, on this
subject of diet, is the argument based upon comparative anatomy—
since that argument places man in his right class immediately, and in
a manner that cannot be evaded by any amount of argument. But
other aspects of the question are also of importance, and afford
strong proof of the natural character of man’s diet. The next
argument we should consider, therefore, is the physiological, and we
shall first of all consider the secretions.
The Saliva.—The differences between the saliva of man and that
of any of the carnivora is striking. In man, this secretion is alkaline—
though only slightly so, in a healthy man. Nevertheless, that is its
normal reaction, and to this there is no exception. In the carnivora,
on the other hand, the reaction is acid, and because of this fact is
capable of dissolving the food more or less whole, and without the
long process of mastication necessary for the herbivora and
frugivora. The saliva in the human being effects many chemical
changes in the food—notable among these being the conversion of
starch. Were man intended to live on flesh, the saliva would be acid
also—instead of alkaline as it is.

The Gastric Juice.—Dr Schlickeysen says of this:[10]
“A leading element of the gastric juice is lactic acid. This
excites a slight fermentation of the chyme, and thus
exerts an influence upon the digestion of vegetable, but
not upon that of animal, food. It is far too weak to act
upon the fibres of animal flesh. All fats are insoluble in
water, spirits of wine, and acids. Flesh, when eaten by
man, tends to undergo a process of decay in the stomach,
causing a scrofulous poisoning of the blood. In this
unnatural action lies the cause of many complaints and
disturbances of the system: as bad breath, heartburn,
eructions and vomiting. In the case of the carnivora, the
gastric juice exerts a decomposing influence upon flesh,
and causes its assimilation and excretion. Since the
pancreatic juice of the duodenum, into which the chyme
passes from the stomach, bears a close resemblance to
the saliva, it follows that the chyme here, also, can have
only a slightly acid property, which it indeed can only have
when it is of a vegetable character. Bile, which is here
poured into the intestines, has only a slight alkaline
reaction, and its use seems to be limited to the prevention
of decay; which, however, can only occur in the case of
flesh-food; so that the effort of nature to maintain flesh-
food in its proper condition by the secretion of bile must
be excessive, and must eventually cause an excitement
and weakening of the whole organism.”
And Dr Kellogg has pointed out[11]:
“Another property possessed in a high degree by the
gastric juice of carnivorous animals is its antiseptic or
germicidal quality. When exposed to the conditions of
warmth and moisture, flesh, whether that of mammals
birds or fish, readily decomposes or decays, giving rise to

poisonous substances of the most offensive character. The
gastric juice of the dog is capable of preventing this
putrefactive change while the food is undergoing the
process of stomach digestion. That such changes occur
later, however, while the food residue is lying in the colon
previous to expulsion from the body, is evidenced by the
extraordinarily offensive character of the fæcal matters of
this class of animals.”
In man, this secretion is very weak, comparatively speaking, and
hence of small value in preventing such putrefactive changes as
those mentioned above. Take any piece of meat, and expose it for
some considerable period to an environment of heat and moisture,
and see the result! Putrefaction soon occurs—except where the meat
is “embalmed” or preserved by powerful chemicals—thus rendering
it unfit for human food. But it will be seen that just such conditions
prevail in the human alimentary tract as are most suitable for the
speedy and deadly decomposition of the food eaten; and, in the
case of flesh-foods, the resulting products are poisonous in the last
degree. The gastric juice of the human stomach being so far weaker
than that of the carnivorous animal, the flesh is far less completely
acted upon and digested in the stomach—much more work being
passed on to the intestines, in consequence. Now comes in a most
important factor. The bowel of the carnivorous animal is, as we have
seen, short, (three times the length of the body) when compared to
the frugivora, whose alimentary tract is about twelve times the
length of the body. That is, the digestive tract in man is, roughly,
about four times as long as in the carnivorous animal. The result of
this is that any food eaten would take, ceteris paribus, four times as
long to pass through the tube in the one case as in the other. This
fact alone is sufficient to condemn the use of flesh-foods in any form
for frugivorous animals, since the less active antiseptic and
germicidal properties of the gastric juice in these animals render
unsafe the long retention of such easily decomposable substances as
flesh.

But more than that, and worse still; the character of the internal
structure of the tract is not alike in the two cases! In the carnivora,
this is smooth, and offers but few impediments to the free passage
of the food through it. In man, on the contrary, as with the higher
apes and the herbivora, the intestine is corrugated or sacculated—
this being for the express purpose of retaining the food as long as
possible in the intestine, and until all possible nutriment has been
abstracted from it. This is admirably suited to such foods as the
herbivora and frugivora enjoy, but is quite unsuited for flesh-foods of
all kinds—being, in fact, the worst possible receptacle for such
foods. The intestine, in the carnivora, is suited for its particular food
—it is short and smooth, and well adapted to dissolve the food
quickly and pass it out of the system as rapidly as possible; while in
frugivora, on the other hand, the intestine is adapted to retain the
food a much longer time—the sacculated surface retaining the food
as long as possible. The result of this is that, when flesh-foods are
eaten, disastrous results are sure to follow.
As previously shown, the liver is much larger, proportionately, in
the carnivora; and not only is this the case, but the amount of bile
secreted is far greater in the carnivora than in man. It has been
found, by careful experiments upon dogs, that the quantity of bile
might increase fifty per cent., and even more, under a purely meat
diet; but rapidly decreased when the quantity and proportion of the
meat was reduced. Thus it appears that the use of a meat diet
requires a far greater degree of activity on the part of the liver than
any other diet. This is amply provided for in the carnivore by the
increased size and power of that organ, but in man and the frugivora
such is not the case, and the result is that if meat be eaten by man,
the liver is called upon to do an extra amount of work, and this may
ultimately result in its premature breakdown.
The kidneys also are greatly affected by the diet. It is now well
known that uric acid is created in large quantities by a flesh diet—
the measured excretions showing that from three to ten times as
much uric acid is secreted when flesh is eaten as when no meat is
ingested; and when we bear in mind the exceedingly disastrous

effects of uric acid upon the system, and what a powerful disease-
producing agency it is, I think that we must conclude that this
symptom is strongly suggestive, and strongly indicative of the fact
that man cannot eat meat without running grave chances of
diseasing and ruining his organism.
The Excretions.—There is also a marked difference in the
excretory products of the various animals. While, in the carnivora,
the action of the urine is acid, it is alkaline in the herbivora (or
should be). In man it is frequently acid—though this varies with the
nature of the food. Thus, if the diet be largely one of flesh, the urine
will become far more acid, and will also become very offensive; the
perspiration will also be tainted, and very noticeable to those with a
keen sense of smell, and who do not eat meat themselves! This has
frequently been observed, and may account for the fact that flesh-
eating animals will always eat a horse or a sheep in preference to
man, if it be possible. Doubtless, their keen sense of smell detects
the fact that man is (usually) largely carnivorous in his habits, and
their instinct teaches them that the flesh of the purely herbivorous
animal is for this reason superior to that of man. Has anyone
thought why it is that a cat will kill a mouse, and eat it, while a dog
will kill a cat, but will not eat it? It is because the mouse is a
vegetarian animal, and the cat is a carnivorous animal. Instinct
teaches the cat that the tissues of the mouse’s body are more or less
pure and inoffensive—owing to the nature of the diet; while the
same instinct teaches the dog that the cat’s body is impure and
more or less poisonous, for the reason that its flesh is tainted and
full of poisons, because of its diet. If any animal lives upon flesh,
that animal’s body is bound to be tainted more or less in
consequence; and those animals which prey upon others know that
fact, by reason of their sense of smell and instinct. This is a
remarkable and most instructive fact; a rule which will rarely be
found to fail. Its significance and interpretation is obvious. Professor

Schlickeysen also informs us that “the overloading of the blood with
flesh-food causes, in order to effect their decomposition, an
excessive consumption of oxygen, and hence the difficulty of
breathing, and asthmatical affections of many flesh-eaters, and their
excessive excretion of carbonic acid.” I have referred to some of
these poisons, formed within the system, and the harm they must
doubtless exert upon the organism, elsewhere.
In addition to all these arguments, there are other forcible reasons
for considering man as one of the non-flesh-eating animals—which
reasons may be included in this chapter. The habits of any animal
are distinctive; and they, collectively, indicate man’s position—though
this argument must always be confirmatory, and not proof in itself.
For instance, all naturally carnivorous animals sleep in the daytime,
and prowl about in search of their prey at night; while with the
vegetarian animals (man included) this is not the case. The manner
of eating and especially of drinking, is also highly characteristic—all
carnivorous animals lapping their liquids—while the herbivora and
frugivora drink—as I have previously pointed out. The peculiar mode
of functioning of various organs might also be pointed out and
insisted upon. But one of the most striking arguments is that based
upon the anatomical structure of the skin. As before stated, this
perspires, in the case of all vegetarian animals, while the glands are
atrophied and inactive in all carnivora. Let us now consider the
significance of this fact.
“Recent researches show us that uric acid arises from
the decay of cell nuclei. That portion of uric acid which
has its origin in the digestive organs is, like other alloxanic
bases, changed into urea—or rather should be. But a
diseased liver (or a healthy one which is overworked,
owing to an excessive ingestion of food containing cell
nuclei, and therefore an excessive amount of uric acid) is
unable to transform all the uric acid formed into urea. The
quantity of uric acid arising from the normal decay of the
tissue is small; in fever, when there is a more rapid decay
of cells, the quantity of uric acid and other related

alloxanic bodies is considerably increased. The greater the
quantity of useless body-material, and the worse (more
dysæmic) it is in quality, the greater is the danger of a
more rapid decay of cells, and a precipitation of uric acid
and related products taking place.... The uric acid, passing
through the liver, may perhaps be transformed into urea
by a special action of the cells; but the uric acid drawn
directly from the digestive canal, and that formed directly
from the assimilated food or from the body-material, has
to be oxidised, in order to be excreted in the innocuous
form of urea. An organism possessed of the faculty of
oxidation is protected against a precipitation of uric acid,
but in a dysæmic organism, the faculty of transforming
uric acid into urea is lessened.... It is a fact well worth
considering that the urine of carnivorous animals—e.g.
dog and cat—is often quite free from uric acid, while
human urine varies in this respect according to the food
taken: if vegetable food alone is consumed, the urine will
contain, like the urine of herbivorous animals, only traces
of uric acid (from ·2 to ·7 grammes in 24 hours); but if a
large proportion of flesh-food be taken, the urine will
contain 2 grammes or more. Man is the only creature
which suffers from the uric acid diathesis; is it not likely
that this arises from a wrong choice of food?
“Now, if the excretion of the uric acid always took place
easily, we should not have much trouble about its
formation, but it is this excretion which constitutes the
difficulty. Uric acid and the acid salts of the uric acid
dissolve with difficulty in cold water; but more easily in
warm; still, one gramme of uric acid requires from 7 to 8
litres of water at the temperature of the body for its
solution. The acid urate of soda dissolves in 1100 parts of
cold and 124 parts of boiling water. The ammonia salts
and the salts of the alkaline earths do not dissolve nearly
so easily.

“The ‘warm water’ which keeps the uric acid and the
uric acid salts dissolved in the body is the blood and tissue
fluids. Serious disturbances must take place if this fluid
becomes cooler or diminished in quantity; for a deposit of
crystalline uric acid would occur in the body.
“A person who has to daily excrete 2 grammes of uric
acid, is constantly liable to this precipitation, as he may at
any time lose large quantities of water through
perspiration. It is, therefore, undoubtedly safer to have
the uric acid combined with soda, as an acid urate; but
where is soda to be obtained if it is absent from the blood,
owing to dysæmia?
“The more acid the urine is, the more easily will a
precipitation of the uric acid occur in the organism—for
instance, in the kidneys or bladder. The urine of a person
eating flesh contains a large amount of uric acid, as we
have seen before; it is also strongly acid in reaction
whereas the urine of herbivorous animals is generally
alkaline in reaction....
“A very acid urine rich in uric acid is also produced by
salt meat and salt fish, because in the process of salting,
the basic salts (basic alkaline phosphates and carbonates)
pass into the pickle water and neutral common salt takes
their place. Russian physicians have told me that in certain
parts of Russia, where the people eat a great deal of salt
fish, urine stones are frequent.... Now, if we wish to
prevent by the use of alkalies the formation of uric acid
sediments, or gradually to dissolve such concretions as
have already formed in the bladder, it is certainly more
rational to prescribe a diet of fruits and potatoes than to
order alkaline mineral waters—which, when taken
constantly, may produce all sorts of disturbances.
“If, then, it is true that our ordinary diet consists chiefly
of foods rich in albumen and phosphoric acid but poor in

soda, and that in consequence of this a tendency towards
the accumulation of uric acid in the body is pretty
generally found, the very slightest extra strain on the
system will be sufficient to cause a precipitation of uric
acid and uric acid salts in the body. This result is very
often brought about by a chronic acid catarrh of the
stomach, which in its turn depends upon dysæmia, and is
in 95 out of 100 cases the predecessor of gout. The
fermentation acids, especially oxybutyric acid (which is
found in the urine both in acid catarrh of the stomach and
in diabetes mellitus), combine with some of the alkalies of
the blood, and thus lessen its alkalescence (basic
character); and as catarrh of the bowels and periodic
diarrhœas are frequently associated with acid catarrh of
the stomach, these bases may be even directly excreted in
the stools, and thus the quantity of alkalies in the blood
be further diminished.
“Now we find that men consuming vegetable food form
only small quantities of uric acid, herbivorous animals as
well as carnivorous hardly any, but men living on flesh-
food very large quantities, we must come to the
conclusion that men cannot properly manage flesh-food.
The organism of the flesh-eating animal has the faculty of
completely digesting flesh-food, whereas the organism of
man is unable to accomplish this. Consequently man
cannot be classed as carnivorous and cannot eat flesh
unpunished....
“To illustrate this further, we may mention another
important point here. Carnivorous animals have atrophied,
inactive sweat glands, whilst man and herbivorous animals
possess well-developed sweat glands. There is no doubt,
therefore, that the herbivora must have preceded the
carnivora in point of time—the carrion feeders being the
connecting link between them.[12] The carnivora have

retained the sweat glands as atrophied (rudimentary)
organs, and as a sign of their origin, but have given up
the habit of sweating, or, in other words, have adapted
their skin to the changed conditions of feeding. An animal
whose food contains large quantities of urea as well as of
creatin, creatinin, xanthin, hypoxanthin, guanin, etc. (the
early stages of uric acid), and thus increases the quantity
of urea and uric acid already present in the body, must
take care always to keep these substances in solution. But
the urea and uric acid can only be dissolved in
comparatively large quantities of warm water (blood).
Such an animal must, therefore, be exempt from the
possibility of suddenly losing a large part of its blood and
tissue fluid by sweating—or else a precipitation of the
above substance will take place. Nor should an organism
allow of any sudden cooling down of portions of the skin—
such as might be caused by evaporation of the sweat, or
else a precipitation would again take place. In a word,
such an animal must not be subject to sweating, or else it
would be troubled with acute and chronic rheumatism,
gout, etc....
“Now as man is subject to sweating, it is evident that he
was not intended to live on flesh, but on vegetables, or
rather on fruits, for he was never meant to live on
cereals.... Man may eat a limited amount of meat and
cereals without doing himself much harm; but he must
always remember that they ought never to form his
principal food.
“As soon as it is really understood that we were never
intended to live on flesh and cereals, the uric acid
diathesis as a trouble of mankind will disappear. We must,
of course, not forget to restrict the consumption of
common salt and to use such vegetable foods as are rich
in food salts, and not those which are rich in albumen; for
a diet consisting of bread, pulses, and cereals, and

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