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 Advances in
CANCER
RESEARCH

Volume 112
 Intratumor Diversity and
Clonal Evolution in Cancer

Advances in
CANCER
RESEARCH
Volume 112

Series Editors Edited by

Kenneth D. Tew David Gisselsson

Cell and Molecular Pharmacology, Department of Clinical Genetics
John C. West Chair of Cancer Research, Lund University, University Hospital
Department of Cell and Molecular Lund, Sweden
Pharmacology & Experimental Therapeutics
at MUSCCharleston, South Carolina, USA

Paul B. Fisher
Department of Human & Molecular
Genetics, VCU Institute of Molecular Medicine
Richmond, Virginia, USA
Academic Press is an imprint of Elsevier
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ISBN: 978-0-12-387688-1
ISSN: 0065-230X

For information on all Academic Press publications visit

our website at www.elsevierdirect.com

Printed and bound in USA

11 12 13 14 10 9 8 7 6 5 4 3 2 1
 Contributors

Numbers in parentheses indicate the pages on which the authors’ contributions begin.

Batoul Y. Abdallah, Center for Molecular Medicine and Genetics, Wayne

State University School of Medicine, MI, USA (217)
Jennifer Alami, Department of Pathology and Molecular Medicine,
Queen’s University, Kingston, ON, Canada (183)
Steven W. Bremer, Center for Molecular Medicine and Genetics, Wayne
State University School of Medicine, MI, USA (217)
Daniela Cimini, Department of Biological Sciences, Virginia Tech,
Blacksburg, VA, USA (43)
Daniel Dom
ınguez, Cell Biology Unit, Department of Cell Biology,
Physiology and Immunology, Bioscience School, Universitat
Autònoma de Barcelona, Bellaterra, Spain (11)
Andrew Evans, Department of Pathology, University Health Network,
Toronto, Canada (183)
Cristina Fr
ıas, Cell Biology Unit, Department of Cell Biology, Physiology
and Immunology, Bioscience School, Universitat Autònoma de
Barcelona, Bellaterra, Spain (11)
Macoura Gadji, The University of Manitoba, Manitoba Institute of Cell
Biology, Cancer Care Manitoba, The Genomic Centre for Cancer
Research and Diagnosis, Winnipeg, Manitoba, Canada; The Cheikh
Anta Diop University of Dakar, Laboratory of Haematology
and Immunology, National Centre of Blood Transfusion of Dakar,
Senegal (77)
Anna Genesc
a, Cell Biology Unit, Department of Cell Biology, Physiology
and Immunology, Bioscience School, Universitat Autònoma de
Barcelona, Bellaterra, Spain (11)
David Gisselsson, Departments of Clinical Genetics and Pathology, Lund,
University, Lund, Sweden (1)

ix
x Contributors

Sverre Heim, Centre for Cancer Biomedicine, University of Oslo, Oslo,

Norway; Section for Cancer Cytogenetics, Institute for Medical
Informatics, The Norwegian Radium Hospital, Oslo University
Hospital, Oslo, Norway; Medical Faculty, University of Oslo, Oslo,
Norway (127)
Henry H.Q. Heng, Center for Molecular Medicine and Genetics, Wayne
State University School of Medicine, MI, USA; Karmanos Cancer
Institute, Wayne State University School of Medicine, MI, USA;
Department of Pathology, Wayne State University School of
Medicine, MI, USA (217)
Mattias H€ oglund, Department of Oncology, Clinical Sciences, Lund
University, Lund, Sweden (151)
Anthony M. Joshua, Department of Medical Biophysics, University of
Toronto, Toronto, Canada; Division of Applied Molecular Oncology,
Ontario Cancer Institute, Princess Margaret Hospital, Toronto,
Canada; Department of Medical Oncology, Princess Margaret
Hospital, Toronto, Canada (183)
Ludger Klewes, The University of Manitoba, Manitoba Institute of Cell
Biology, Cancer Care Manitoba, The Genomic Centre for Cancer
Research and Diagnosis, Winnipeg, Manitoba, Canada (77)
Hans Knecht, Division of Haematology/Oncology, CHUS University
Hospital, Sherbrooke (Qu
ebec), Canada (77)
Narisorn Kongruttanachok, The University of Manitoba, Manitoba
Institute of Cell Biology, Cancer Care Manitoba, The Genomic
Centre for Cancer Research and Diagnosis, Winnipeg, Manitoba,
Canada (77)
David Lindgren, Center for Molecular Pathology, Department of
Laboratory Medicine, Lund University, SUS Malm€ o, Malm€ o, Sweden
(151)
Guo Liu, Center for Molecular Medicine and Genetics, Wayne State
University School of Medicine, MI, USA (217)
Sabine Mai, The University of Manitoba, Manitoba Institute of Cell
Biology, Cancer Care Manitoba, The Genomic Centre for Cancer
Research and Diagnosis, Winnipeg, Manitoba, Canada (77)
Joshua M. Nicholson, Department of Biological Sciences, Virginia Tech,
Blacksburg, VA, USA (43)
Judit Pampalona, Cell Biology Unit, Department of Cell Biology,
Physiology and Immunology, Bioscience School, Universitat
Autònoma de Barcelona, Bellaterra, Spain (11)
Paul C. Park, Department of Pathology and Molecular Medicine, Queen’s
University, Kingston, ON, Canada (183)
Contributors xi

Alexander Pietras, Center for Molecular Pathology, CREATE Health,


Department of Laboratory Medicine, Lund University, Skane
University Hospital, Malm€ o, Sweden (255)
Jeremy A. Squire, Department of Pathology and Molecular Medicine,
Queen’s University, Kingston, ON, Canada (183)
Christiaan Righolt, The University of Manitoba, Manitoba Institute of
Cell Biology, Cancer Care Manitoba, The Genomic Centre for Cancer
Research and Diagnosis, Winnipeg, Manitoba, Canada; Department of
Imaging Science and Technology, Delft University of Technology, Delft,
The Netherlands (77)
Joshua B. Stevens, Center for Molecular Medicine and Genetics, Wayne
State University School of Medicine, MI, USA (217)
Manuel R. Teixeira, Department of Genetics, Portuguese Oncology
Institute, Porto, Portugal; Biomedical Sciences Institute (ICBAS),
University of Porto, Porto, Portugal; Centre for Cancer Biomedicine,
University of Oslo, Oslo, Norway (127)
Laura Tusell, Cell Biology Unit, Department of Cell Biology, Physiology
and Immunology, Bioscience School, Universitat Autònoma de
Barcelona, Bellaterra, Spain (11)
Rhea Vallente, The University of Manitoba, Manitoba Institute of Cell
Biology, Cancer Care Manitoba, The Genomic Centre for Cancer
Research and Diagnosis, Winnipeg, Manitoba, Canada (77)
Johan Vallon-Christersson, Department of Oncology, Clinical Sciences,
Lund University, Lund, Sweden; CREATE Health Strategic Center
for Translational Cancer Research, Lund University, Lund, Sweden
(151)
Mark D. Vincent, Department of Medical Oncology, London Regional
Cancer Centre, London Health Sciences Centre, Ontario, Canada
(283)
Landon Wark, The University of Manitoba, Manitoba Institute of Cell
Biology, Cancer Care Manitoba, The Genomic Centre for Cancer
Research and Diagnosis, Winnipeg, Manitoba, Canada (77)
Julia L. Williams, Department of Pathology and Molecular Medicine,
Queen’s University, Kingston, ON, Canada (183)
Christine J. Ye, Department of Internal Medicine, Wayne State University
School of Medicine, MI, USA (217)
Maisa Yoshimoto, Department of Pathology and Molecular Medicine,
Queen’s University, Kingston, ON, Canada (183)
 Intratumor Diversity and Clonal
Evolution in Cancer—A Skeptical
Standpoint
David Gisselsson
Departments of Clinical Genetics and Pathology, Lund
University, Lund, Sweden

I. Introduction
A. Is Intratumor Diversity a Feature of All Neoplasms?
B. To What Extent Does Genomic Instability Lead to Genomic Intercellular Diversity?
C. To What Extent Does Genetic Variation Correspond to Phenotypic Variation?
D. Is genomic Instability a Self-Sustaining Process?
E. Do Diverse Tumor Cell Populations Always Stem from a Single Cell?
Acknowledgment
References

Clonal evolution in cancer is intimately linked to the concept of intratumor

cellular diversity, as the latter is a prerequisite for Darwinian selection at the
micro-level. It has been frequently suggested in the literature that clonal evolution
can be promoted by an elevated rate of mutation in tumor cells, so-called genomic
instability, the mechanisms of which are now becoming increasingly well charac-
terized. However, several issues need clarification before the presumably complex
relationship between mutation rate, intratumor diversity, and clonal evolution can
be understood sufficiently well to translate into models that predict the course of
tumor disease. In particular, it has to be clarified which of the proposed mechanisms
for genomic instability that are able to generate daughter cells with sufficient
viability to form novel clones, how clones with different genomic changes differ
phenotypically from each other, and what the selective forces are that guide com-
petition among diverse clones in different microenvironments. Furthermore, stan-
dardized measurements of mutation rates at the chromosome level, as well as
genotypic and phenotypic diversity, are essential to compare data from different
studies. Finally, the relationship between clonal variation brought about by geno-
mic instability, on the one hand, and cellular differentiation hierarchies, on the
other hand, should be explored to put genomic instability in the context of the
tumor stem cell hypothesis. # 2011 Elsevier Inc.

Advances in CANCER RESEARCH, Volume 112 0065-230X/10 $35.00

Copyright 2011, Elsevier Inc. All right reserved. DOI: 10.1016/B978-0-12-387688-1.00001-6
1
2 David Gisselsson

I. INTRODUCTION

Intratumor diversity is currently discussed primarily in the contexts of

genomic instability, clonal selection leading to chemotherapy resistance,
and tumor cell differentiation hierarchies, including so-called tumor-
initiating cells or cancer stem cells. The issue of intercellular heterogeneity
in tumors is undoubtedly complex, not least because many of the terms
used in this field lack consensus definitions. As a consequence, experimen-
tal work is challenged by the fact that few standardized approaches are
available to measure core parameters such as the rate of chromosomal
instability or the degree of intercellular heterogeneity. Yet another prob-
lem is that much of the published work has been performed on established
cancer cell lines, representing systems optimized for in vitro growth in a
stable microenvironment, in stark contrast to the challenging and shifting
milieu in which human tumors develop. Few methods are currently avail-
able to thoroughly dissect mutation rates and intercellular variation
in vivo. A third point that needs clarification is the presumed connection
between genomic instability and clonal evolution in cancer. This is based
on the premise that Darwinian evolution requires heritable and variable
traits, and that cell populations with sufficient heritable variation for an
evolutionary process to take place are more likely to arise with an
increased mutation rate. Even though natural selection is a well-consolidated
mechanism behind species evolution, a number of items still require clarifi-
cation before it can be linked to genomic instability and widely applied to the
development of neoplastic cells (Fig. 1). Some of the more evident ones are as
follows.

A. Is Intratumor Diversity a Feature of All Neoplasms?

It is generally assumed that clonal selection is a key process in carcino-
genesis, but the experimental basis for this remains to be extended. There
are of course already several levels at which experimental studies have
shown tumor cells to exhibit a higher degree of diversity than their non-
neoplastic counterparts: (1) morphology, (2) gene expression including
protein markers, and (3) genomic alterations. However, the extent to
which cells exhibiting different morphologies and gene expression signa-
tures in fact constitute functionally different cells with stable, distinct
phenotypes over time is less clear. It is therefore possible that some of
these ostensibly distinct cell populations may continually change charac-
teristics and cross into each other’s phenotypic spectra. That such a change
of basic characteristics is not only limited to more stem cell-like tumor cells
differentiating into more mature cells over time is indicated by recent
Intratumor Diversity and Clonal Evolution in Cancer—A Skeptical Standpoint 3

[(Fig._1)TD$IG]

Fig. 1 A simplified model connecting genomic instability and clonal evolution in cancer.
There are several crucial points in need of clarification in this process, including: What is the
primary generator of genomic diversity—an increased mutation rate or an increased cellular
tolerance for mutations? Is genomic instability self-sustaining, for example through aneuploidy
giving rise to dysregulation of mitotic checkpoints, resulting in further aneuploidy? Which of the
described mechanisms for genomic instability are compatible with clonogenic survival? Which
of the genetic alterations brought about by genomic instability do in fact result in an altered
cellular phenotype through an altered gene expression? What microenvironmental selective
forces guide the selection and clonal expansion of phenotypically different cells? And, finally, is
the process a result of clonal expansion from one or several founder cells? See text for details.
(For color version of this figure, the reader is referred to the web version of this book.)
4 David Gisselsson

studies showing that nonstem cell-like tumor cells can spontaneously

change to a more stem-like state (Chaffer et al., 2011). Such bidirectional
interconversions would make it difficult to confidently delineate stable
and distinct tumor cell populations within a tumor and stress the impor-
tance of including a temporal aspect in studies of phenotypic diversity. In
contrast, genetic variability constitutes a robust parameter to pinpoint the
relationship between different cell populations in a tumor. However, not
all tumors have been shown to contain cell populations that are genetically
distinct. While many of the common cancers such as adenocarcinomas of
the breast, colon, and lung frequently show cell populations with different
genomic changes within the same tumor, this is far from always the case in
leukemias, lymphomas, and embryonic childhood tumors (Bielas et al.,
2006; Mitelman et al., 2011). Whether all of these latter tumor groups will
in fact be shown to contain genetically diverse cell populations when
subjected to novel high-resolution genetic analyses, such as next-genera-
tion sequencing, remains to be seen.

B. To What Extent Does Genomic Instability Lead to

Genomic Intercellular Diversity?
A first prerequisite to be met for genomic instability to have a role in
promoting variation and ultimately clonal evolution is the survival of cells
having undergone mutational events, such as missegregation of one or
several chromosomes. The few studies addressing the survival of cells with
aneuploidy as a result of chromosomal missegregation have so far shown
that the capacity for clonogenic survival varies greatly among cells having
undergone different types of chromosome missegregations. For example,
there is substantial evidence for multipolar mitosis giving rise to three or
more aneuploid daughters not resulting in viable clones (Ganem,
Godinho, and Pellman, 2009; Stew
enius et al., 2005). On the other hand,
multipolar metaphase cells that reorient to a bipolar configuration or at
least undergo bipolar cytokinesis have the capacity to generate aneuploid
clonogenic daughters at least in vitro (Ganem et al., 2009; Gisselsson et al.,
2010). This amply illustrates that not all types of so-called chromosomal
instability can give rise to genetic diversity, thus disqualifying some of
them as key mechanisms behind clonal evolution. This must be kept in
mind when surveying the vast number of reports of molecular mechanisms
giving rise to chromosomal segregation errors, few of which experimen-
tally address the actual fate of cells having attained the respective types of
genomic instability. It cannot be excluded that the tendency to missegre-
gate chromosomes at a high rate, in some cancers, is simply a side effect of
deregulation of cell cycle checkpoint systems (either epigenetically or by
sporadic mutation), and that this checkpoint deregulation confers a
Intratumor Diversity and Clonal Evolution in Cancer—A Skeptical Standpoint 5

growth advantage distinct from that of chromosomal variation. In that

case, it is possible that cells having undergone gross genomic alterations
rarely reproduce in comparison to cells managing to segregate their chro-
mosomes correctly despite the checkpoint deficiency. If so, the latter cells
will be the ones that predominantly contribute to tumor growth, while the
former would continuously reach evolutionary dead ends. Hence, there
could be little genomic variation in a tumor cell population despite an
elevated rate of chromosome missegregation. Finally, one needs to con-
sider whether the increased number of mutations observed in tumor cells
could in some cases merely reflect an increased cellular tolerance to certain
mutations, rather than a consequence of an increased mutation rate.

C. To What Extent Does Genetic Variation Correspond

to Phenotypic Variation?
For Darwinian selection to function, heritable genetic variation must
also result in different phenotypic traits, associated with variable degrees
of fitness. The extent to which different genomic changes affect cellular
fitness has been very little explored. Recent studies on murine cells have
indicated that alterations in fitness resulting from whole chromosome
aberrations are unpredictable and vary from chromosome to chromosome
(Williams et al., 2008). A similar complex picture has started to appear
regarding the relationship between differentiation, on the one hand, and
genomic alterations, on the other. At least in some tumors, for example,
childhood tumors where differentiation hierarchies can be unequivocally
assessed, a complex scenario is emerging, where some large-scale genomic
alterations (such as trisomies) appear to exist without correlation to dif-
ferentiation hierarchies, while smaller genomic imbalances (such as dele-
tions of key embryonic genes) may exist only in cell populations at certain
maturation levels (Gisselsson et al., 2010; Holmquist Mengelbier et al.,
2010). Furthermore, in breast neoplasms it has been shown that stem-cell-
like and more differentiated tumor populations can be highly genetically
distinct (Park et al., 2010), questioning a straightforward differentiation
hierarchy in which maturing cells are invariably the offspring of stem cell-
like cells. Evidently, a thorough examination of the relationship between
genetic diversity and differentiation hierarchies, spanning multiple tumor
types, would be of value to further assess the mechanisms responsible for
phenotypic diversity among cells in a certain tumor.

D. Is genomic Instability a Self-Sustaining Process?

Many authors equal aneuploidy (the state of a chromosome number that
is not a multiple of the haploid set, in humans n = 23) to genomic, or at
6 David Gisselsson

least chromosomal instability (an increased rate of change of the chromo-

some complement). Some studies indicate that once an aneuploid state has
been established, this confers a high probability of developing chromo-
somal instability because of the alterations in gene dosage brought about
by abnormal chromosome copy numbers (Duesberg et al., 1998, 2000).
However, few studies have sought to reproduce these first experiments,
and at least one study argues strongly against the idea of such autocatalytic
aneuploidy, based on the finding that extra copies generated by chromo-
some transfer do not induce chromosomal instability (Lengauer et al.,
1997). The issue remains contested. A major objection that can be raised
against a positive feedback loop for aneuploidy is that patients with
constitutional aneuploidy, such as Down syndrome, do not typically show
overt evidence of chromosomal instability such as mosaicism, when sub-
jected to routine cytogenetic analysis. However, fairly recent findings
indicate that some low-grade elevation of the chromosome mutation fre-
quency may in fact exist in trisomic individuals (Lightfoot and Hoog,
2004; Thomas et al., 2008) although the parameters studied mainly cor-
relate to structural and not numerical chromosomal instability. Another
objection is that several types of tumors, in particular those harboring gene
fusions driving pathogenesis, are largely monoclonal cytogenetically also
when they carry numerical changes in the stem line (Mitelman et al.,
2011). However, one may argue that routine cytogenetics is not suffi-
ciently sensitive to detect small subclones harboring the additional numer-
ical changes that would be expected if aneuploidy indeed led to additional
chromosomal alterations. In fact, increasing the number of analyzed
tumor cells have in some neoplasms been shown to result in detection of
an unexpectedly high number of clones carrying different configurations
of chromosome changes (Gorunova et al., 1995, 1998). Furthermore,
detailed in situ analyses of tumor tissue have revealed that chromosome
aberrations assigned to the tumor stem line of ostensibly monoclonal
tumors by cytogenetic and or genomic array analyses are in fact restricted
to certain tumor subcompartments, indicating that there is a degree of
genomic intercellular variation that failed detection by standard techni-
ques (Gisselsson et al., 2010; Holmquist Mengelbier et al., 2010).
Notwithstanding the fact that the degree of chromosomal variation may
be underestimated both in tumor tissue and in patients with aneuploidy
syndromes, it remains to be clarified to what extent aneuploidy in itself
leads to chromosomal instability. Does this differ between different pat-
terns of aneuploidy? Is there a threshold of aneuploidy that has to be
overcome for the triggering of genomic instability? Presently, one cannot
exclude that some aneuploid cancers obtain their abnormal chromosome
number through a single event or a transient phase of instability, after
which the mutation rate returns to that of nonneoplastic cells.
Intratumor Diversity and Clonal Evolution in Cancer—A Skeptical Standpoint 7

E. Do Diverse Tumor Cell Populations Always Stem

from a Single Cell?
The notion that tumors originate from a single cell that acquires sequen-
tial somatic mutations is now well established in the cancer research com-
munity, and remains largely unchallenged. However, there is accumulating
cytogenetic as well as molecular genetic (based on X chromsome inactiva-
tion) evidence that this scenario is not always applicable (Davidsson et al.,
2005; Heim et al., 1997; Johansson et al., 1999; Parsons, 2008; Teixeira
et al., 2001), as unrelated neoplastic clones have been found in several tumor
types. This casts a new light on the presumed connection between genomic
instability, intercellular diversity, and clonal selection. Genetic diversity in
tumor tissue that occurs primarily because of parallel evolution of multiple
clones is to some extent different from the scenario where the diversity stems
from genomic instability in a single proliferating clone. On the other hand,
some degree of instability may still be acquired for the development of
somatic mutations within each unrelated clone. Evidently, the issue of
mono- versus polyclonality needs to be thoroughly addressed before
detailed models can be made of the role played by genomic instability in
the generation of intercellular diversity and clonal evolution in tumors.
The current special volume of Advances in Cancer Research, for which this
chapter serves as an introduction, is an attempt to deliver updated informa-
tion on some of the key questions mentioned above. The chapters are
arranged to cover the field from genotype to phenotype, including mechan-
isms behind chromosomal missegregation, genomic intratumor diversity,
diversity in relation to differentiation hierarchies, and evolutionary models
of carcinogenesis. It will be apparent to the reader that the field of genomic
instability and clonal evolution is still a juvenile one, with mind-challenging
hypotheses flourishing amidst the patches of raw experimental data.

ACKNOWLEDGMENT

Grant sponsors: Swedish Childhood Cancer Foundation, Swedish Cancer Society, Swedish
Research Council, Crafoord Foundation, Gunnar Nilsson Cancer Foundation, the Royal
Physiographic Society, and the Medical Faculty at Lund University.

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 Role of Telomere Dysfunction in
Genetic Intratumor Diversity
Anna Genesc

a, Judit Pampalona, Cristina Frı́as,
Daniel Domı́nguez, Laura Tusell
Cell Biology Unit, Department of Cell Biology, Physiology
and Immunology, Bioscience School, Universitat Autònoma
de Barcelona, Bellaterra, Spain

I. Introduction
II. Chromosome End Protection: The Shelterin Complex
III. Telomere Maintenance, Cell Proliferation, and Telomere Uncapping
A. Telomere length declines with cell divisions in most cells
B. Telomere dysfunction triggers the dna damage response
IV. Telomeres, checkpoints, and cancer
A. When telomeres are too short, lessons from mice
B. Cancer arises when cell-cycle checkpoint defects accompany short telomeres
V. Telomere Uncapping as a Source of Genomic Instability
A. Telomere lengths are highly heterogeneous within somatic cells
B. Initiation of breakage-fusion-bridge cycles by dysfunctional telomeres
C. Missegregation of anaphase-bridged chromatin generates aneuploid cell
populations
D. Telomere uncapping also fuels changes in cell ploidy
VI. Shortening Telomeres in Aged Human Cells
A. Telomere attrition occurs in epithelial compartments
B. Does tumor suppressor gene expression also decline with age?
C. Bfb-intermediates and short telomeres in early preneoplastic lesions
D. Toward a malignant phenotype
VII. Summary
Acknowledgments
References

Most solid tumors are unable to maintain the stability of their genomes at the
chromosome level. Indeed, cancer cells display highly rearranged karyotypes con-
taining translocations, amplifications, deletions, and gains and losses of whole
chromosomes, which reshuffle steadily. This chromosomal instability most likely
occurs early in the development of cancer, and may represent an important step in
promoting the multiple genetic changes required for the initiation and/or progres-
sion of the disease. Different mechanisms may underlie chromosome instability in
cancer cells, but a prominent role for telomeres, the tip of linear chromosomes, has

Advances in CANCER RESEARCH, Volume 112 0065-230X/10 $35.00

Copyright 2011, Elsevier Inc. All right reserved. DOI: 10.1016/B978-0-12-387688-1.00002-8
11
12 Anna Genesc

a et al.

been determined. Telomeres are ribonucleoprotein structures that prevent natural

chromosome ends being recognized as DNA double-strand breaks, by adopting a
loop structure. Loss of telomere function appears from either alteration on telo-
mere-binding proteins or from the progressive telomere shortening that normally
occurs under physiological conditions in the majority of cells in tissues.
Importantly, unmasked telomeres may either trigger the senescent phenotype that
has been linked to the aging process or may initiate the chromosome instability needed
for cancer development, depending on the integrity of the DNA damage checkpoint
responses. Telomere dysfunction contributes to chromosome instability through end-
to-end chromosome fusions entering breakage-fusion-bridge (BFB) cycles. Resolution
of chromatin bridge intermediates is likely to contribute greatly to the generation of
segmental chromosome amplification events, unbalanced chromosome rearrange-
ments, and whole chromosome aneuploidy. Noteworthy is the fact that telomere
length heterogeneity among individuals may directly influence the scrambling of the
genome at tumor initiation. However, reiterated BFB cycles would randomly reorga-
nize the cell karyotype, thus increasing the genetic diversity that characterizes tumor
cells. Even though a direct link is still lacking, multiple evidence lead one to believe
that telomere dysfunction directly contributes to cancer development in humans. The
expansion of highly unstable cells due to telomere dysfunction enhances the genetic
diversity needed to fuel specific mutations that may promote cell immortalization and
the acquisition of a tumor phenotype. # 2011 Elsevier Inc.

I. INTRODUCTION

Several lines of evidence indicate that tumorigenesis in humans is a

multistep process in which a succession of genetic changes leads to the
progressive conversion of normal human cells into cancer cells. Within a
developing tumor, mutations accumulate over time giving rise to variant
cell populations that present the six biological endpoints that collectively
dictate malignant growth: sustaining proliferative signaling, evading
growth suppressors, resisting cell death, limitless replicative potential,
inducing angiogenesis, and tissue invasion and metastasis (Hanahan and
Weinberg, 2000, 2011).
Another feature that characterizes a high proportion of cancers is the
presence of a highly complex karyotype. Studies over a century have
shown that cancer cells experience gains and losses of whole chromosomes
as well as changes in chromosome structure, including reciprocal and
nonreciprocal translocations, inversions, deletions, duplications, and
amplifications (Bayani et al., 2007; Camps et al., 2005; Roschke et al.,
2002). Hematologic malignancies and pediatric neoplasms are mainly
characterized by relatively simple primary aberrations, which are usually
distinctive, recurrent, and associated with a particular cancer type. In
contrast, most human cancers in the adult population have an epithelial
origin and display a myriad of complex chromosomal aberrations that are
Role of Telomere Dysfunction in Genetic Intratumor Diversity 13

not always shared by cells of the same tumor or linked to a particular

tumor type, which suggest constant genetic reshuffling. Thus, cancer cell
heterogeneity in a tumor is the rule rather than an exception. This chro-
mosomal diversity among the tumor cell population is thought to be
acquired through chromosomal instability (CIN), which can be defined
as the continuous formation of new structural and numerical chromosome
aberrations, and is one of the most frequent forms of instability in human
cancers (Lengauer et al., 1998).
Genomic instability is a prominent characteristic of most cancer types
but at what stage of cancer development it arises and what its molecular
basis is are questions to which we are only beginning to get answers
(Negrini et al., 2010). By using mouse modeling approaches it is becoming
clear that CIN is not simply a passenger phenotype but probably plays a
causative role in the onset of a substantial proportion of malignancies
(Schvartzman et al., 2010). In humans, a large amount of data collected
from tumor biopsies also suggest that CIN has a founder effect in tumor-
igenesis since it is present in all stages of cancer; from precancerous lesions,
even before TP53 mutations are acquired (Bartkova et al., 2005;
Gorgoulis et al., 2005), to advanced cancers (Lengauer et al., 1997;
Nowell, 1976). In addition, the level of CIN and chromosomal aberrations
correlates with tumor grade and prognosis (Carter et al., 2006), that is,
chromosomal aberrations tend to be more numerous in malignant tumors
than in benign ones, and both aneuploidy and CIN are associated with
poorer prognoses as well as aggressive histopathologic features (Blegen
et al., 2003; Habermann et al., 2007; Heilig et al., 2010; Nishizaki
et al., 2002; Reshmi and Gollin, 2005; Ried et al., 1999; Risques et al.,
2003; Selvarajah et al., 2008;). Collectively these data suggest that
genomic instability enables evolving populations of premalignant cells
to become malignant by accelerating the accumulation of genetic
changes that are responsible for cancer cell evolution (Hanahan and
Weinberg, 2000).
The presence of genomic instability reflects defects in cellular processes
that maintain the integrity of the genome. To date, the specific molecular
mechanisms that underlie CIN in cancer cells remain obscure. A network
of genome surveillance mechanisms and DNA repair pathways ensures the
integrity of the genome and cell viability. Moreover, equal chromosome
segregation during cell division is controlled by mitotic checkpoints,
which monitor the complement of chromosomes being passed to daughter
cells. Accordingly, disruption of any of the multiple pathways that regu-
late mitotic fidelity would lead to an increased instability of the genome
(Roschke and Kirsch, 2010). However, data analysis of different high-
throughput sequencing studies to determine cancer-relevant genes sug-
gested that mutations in DNA repair and mitotic checkpoint genes
14 Anna Genesc

a et al.

probably do not account for the presence of genomic instability in many

sporadic cancers (Negrini et al., 2010). In contrast, they propose onco-
gene-induced DNA replication stress or telomere erosion as responsible
mechanisms for the presence of genomic instability in these tumors
(Negrini et al., 2010).
In this respect, growing evidence correlates the telomere shortening that
occurs normally during the aging process with the onset and progression
of neoplasia. The ongoing chromosome instability that occurs when telo-
mere function is lost could increase the mutation rate and thus alter the
copy numbers of oncogenes and tumor-suppressor genes, which in turn
could promote the tumorigenic process (Maser and DePinho, 2002).
Underscoring the importance of telomeres in cancer comes from a recent
study where leukocyte telomere length was measured over a follow-up
period of 10 years in 787 individuals (Willeit et al., 2010). Remarkably,
individuals who had shorter telomere length were more likely to develop
cancer, even after adjusting for more conventional cancer risk factors,
including age. Those with the shortest telomere length had more than a
triplicated risk of developing cancer, and those in the middle group had
twice the risk compared to those with the longest telomeres. And not only
that, those in the short telomere group also had a higher risk of dying from
their malignancy than those with longer telomeres, probably because
reduced telomere length was also associated with more aggressive cancers,
such as tumors of stomach, lung, and ovary (Willeit et al., 2010). All of this
indicates that telomere dysfunction is one of the sparks that lit the flame
before the fire.

II. CHROMOSOME END PROTECTION:

THE SHELTERIN COMPLEX

Telomeres cap the ends of linear chromosomes in a variety of different

species to distinguish a chromosome’s natural end from a DNA break. In
eukaryotes, telomeric DNA consists of a double-stranded region rich in
GC, which terminates at the 30 end in a single-stranded overhang
(Blackburn, 1984; Henderson and Blackburn, 1989) (Fig. 1). The telo-
meric sequence is highly conserved in evolution, suggesting that it is an
efficient system of genome protection. All vertebrates share the repetitive
sequence TTAGGG, but its length varies between different species; human
telomeres range between 5 and 15 kb while laboratory mouse telomeres
are remarkably longer at 40–60 kb (Lejnine et al., 1995; Meyne et al.,
1989; Zijlmans et al., 1997).
 Role of Telomere Dysfunction in Genetic Intratumor Diversity 15

[(Fig._1)TD$IG]

Fig. 1 The structure of mammalian telomeres. Telomeres consist of many kb of TTAGGG/

AATCCC repeats ending in a G-rich overhang (top right). The telomeric DNA is decorated by
shelterin proteins that modulate telomere functions and homeostasis. The protective function
of the telomere is achieved through the formation of a lariat structure (bottom right). The 30
overhang invades the double telomeric track with the aid of the shelterin proteins, thus
avoiding being recognized as a double-strand break (DSB). (For color version of this
figure, the reader is referred to the web version of this book.)

Telomere DNA sequences, including the overhang, serve as a platform

for the assembly of specific protein complexes, which modulate the chro-
mosome extremity properties (Liu et al., 2004). Some of these proteins are
maintained at the telomeres during all cell cycle phases and do not func-
tion anywhere else in the nucleus. This is the case for the shelterin com-
plex, consisting of the following proteins: TRF1 (TTAGGG Repeat Factor
1), TRF2 (TTAGGG Repeat Factor 2), POT1 (Protection of Telomeres 1),
TIN2 (TRF1-Interacting Nuclear protein 2), TPP1 (POT1 and TIN2 orga-
nizing protein), and RAP1 (Repressor-Activator Protein 1). This complex
is crucial to form and maintain sealed chromosome ends, suppress DNA
repair processes, and regulate telomeric DNA length (reviewed in Palm
and de Lange, 2008). In particular, the protective function of telomeres is
accomplished through remodeling the terminal linear DNA into a lariat
conformation. Incubation of a linear telomeric model, consisting of dou-
ble-stranded TTAGGG repeats ending in a 30 overhang, with the shelterin
protein TRF2 presumably produced the invasion of the single-stranded
overhang inside the double helix of the telomeric track located further
back (Griffith et al., 1999). Indeed, at least in vitro, TRF2 induces positive
supercoils that can promote unwinding and invasion (Amiard et al.,
2007). But most importantly, this closed chromatin loop was also
observed in vivo after psoralen cross-linking purified telomeric DNA
(Griffith et al., 1999) and in telomere-enriched chromatin (Nikitina and
Woodcock, 2004). As a whole, these studies elucidated for the first time
16 Anna Genesc

a et al.

the end structure of the chromosome terminus and provided a new vision
of how the protective function of the telomere is achieved. Modeling of the
linear telomeric track into a loop physically hides the chromosome ends
from inappropriate DNA damage responses (DDRs).
In addition to structural functions, shelterin proteins can also regulate
the telomere length by controlling the accessibility of telomeres for telo-
merase—the enzyme that synthesizes telomeres de novo. Telomerase does
not act on every telomere in each cell cycle but instead exhibits an increas-
ing preference for telomeres as their length declines (Teixeira et al., 2004).
Thus, it was proposed that availability for telomerase elongation would
probably depend on the amount of shelterin on the telomere track (Lei
et al., 2004; Loayza et al., 2004). In this regard, a model where the
telomere alternates between an open and closed state has been proposed.
In this model, the switch for the conformational change depends on the
likelihood that POT1 components can position themselves on the 30 ter-
minus, through TPP1 interactions (de Lange, 2005).
Although telomeres accommodate a core of six proteins that coat and
shape chromosome ends ensuring their integrity, telomeric proteome stud-
ies have listed a plethora of other proteins associated with mammalian
telomeres (Dejardin and Kingston, 2009) that might also influence chro-
mosome end protection and dynamics. Importantly, some of them func-
tion in DNA damage signaling and repair such as the MRN complex—
composed of MRE11, RAD50 and NBS1-, DNA-PKcs, ATM, or ATR. In
telomeres, these proteins are required for efficient replication and proper
trimming-processing of extremities after replication, and help in double-
strand invasion to form the D-loop structure (Verdun and Karlseder,
2007; Gilson and Geli, 2007).

III. TELOMERE MAINTENANCE, CELL PROLIFERATION,

AND TELOMERE UNCAPPING

A. Telomere Length Declines with Cell Divisions

in Most Cells
Telomerase is a specialized ribonucleoprotein complex that includes
two essential components: an RNA template (TR or TERC) and a
reverse transcriptase catalytic subunit (TERT) (Greider and
Blackburn, 1985, 1989; Lingner et al., 1997). In addition to these essen-
tial components, a number of other telomerase-associated proteins have
been identified, that is, dyskerin, NOP10, NHP2, GAR1, and TCAB1
(Meier, 2005; Venteicher and Artandi, 2009). These proteins are not
indispensable for the catalytic activity of the enzyme in vitro; however,
Role of Telomere Dysfunction in Genetic Intratumor Diversity 17

some likely regulate telomerase activity, assembly, and function in vivo

(Artandi and DePinho, 2010). Elongation of telomeres by telomerase
occurs during the S-phase of the cell cycle coincident with DNA repli-
cation. But both fork progression and accessibility of telomerase to the
chromosome end are prevented by the presence of the t-loop structure.
This topological barrier is remodeled upon the action of telomere bind-
ing proteins through co-factors that allow fork progression, control
nuclease processing at the 50 end needed for proper G-tail formation,
facilitate telomere elongation when possible, and eventually recap the
telomere (Gilson and Geli, 2007).
Considering that the expression of the telomerase enzyme is highly
regulated and most somatic cells possess low levels or do not possess
any detectable telomerase activity at all (Wright et al., 1996), a reduc-
tion of the telomere track occurs at each cell division owing to the
failure of conventional DNA polymerases to fully replicate the lagging
strand terminus. Already predicted by Watson (1972) and Olovnikov
(1973), this ‘‘end replication problem’’ causes a shortening in the newly
synthesized chain of few base pairs/end/cell division. Besides this phe-
nomenon, in humans and in mice, the active degradation of the 50
chromosome ends necessary for proper folding to occur after replica-
tion supposes, in the absence of a compensatory mechanism, a loss of
50–200 base pairs/end/cell division (Huffman et al., 2000). Of note,
telomere length may also decline through replication-independent path-
ways such as the presence of reactive oxygen species (Kawanishi and
Oikawa, 2004).

B. Telomere Dysfunction Triggers the DNA

Damage Response
Importantly, the physiological telomere shortening that occurs in many
somatic cells with cell division can eventually halt cell proliferation. It
appears that for proper telomere function a minimum length of telomere
repeats is required. Sustained telomere shortening in proliferating cells
produces critically short telomeres that activate the DDR rendering a
permanent cellular arrest—senescence- or apoptosis, which prevents loss
of genome integrity (Fig. 2). Evidence for a relationship between exces-
sively short telomeres and replicative arrest comes from the observance
that the ‘‘end-replication problem’’ can be circumvented by telomerase.
Ectopic expression of hTERT arrests DDR signaling in senescent human
cells and permits, and in some cases apparently promotes, the continued
replication and expansion of cell populations (Bodnar et al., 1998; Shay
and Wright, 2007). Thus it appears that telomeres track the number of cell
divisions through which a cell passes and control cell proliferation by
 18 Anna Genesc

a et al.

[(Fig._2)TD$IG]

Fig. 2 Cellular response to dysfunctional telomeres. Telomere dysfunction, through either

replicative shortening or disruption of telomeric proteins, elicits a DDR similar to that
triggered after a DSB is produced. Activation of ATM/ATR at dysfunctional telomeres
results in a p53-mediated cell-cycle arrest, replicative senescence, or apoptosis. (For color
version of this figure, the reader is referred to the web version of this book.)

halting cell division once telomeres shorten at a certain predetermined

length (Wright and Shay, 2001).
Another source of telomere damage that may lead to telomere uncap-
ping is the sudden loss of telomeric proteins. Removal of TRF2 from
telomeres, either by a dominant negative allele or by targeted mutation
in mice, results in the loss of the 30 overhang leading to acute telomere
uncapping and, within a few divisions, all chromosome ends are detected
as DNA breaks and then fuse with each other (Smogorzewska and de
Lange, 2002; van Steensel et al., 1998). The collapse of t-loops eliminates
the discriminatory capacity that normal cells have to distinguish natural
chromosome ends from double strand breaks (DSBs) even in the presence
of long stretches of TTAGGG repeats. Accordingly, dysfunctional telo-
meres, either in cells where TRF2 function is disrupted or by excessive
shortening, accumulate proteins involved in DNA damage recognition
and/or repair distinctive of conventional DSBs, such as 53BP1, gH2AX,
MRE11, and the phosphorylated forms of ATM and ATR (d’Adda di
Fagagna et al., 2003; Takai et al., 2003) (Fig. 2). All these observations
indicate that upon a DNA insult—either DSBs or uncapped telomeres—
activation of the classic DDR occurs resulting in cell death via an apoptotic
response or senescence.
Role of Telomere Dysfunction in Genetic Intratumor Diversity 19

In this context, it has been proposed that the slow accumulation of

senescent cells with aging could eventually affect tissue fitness, thus con-
tributing to age-related diseases. In humans, a decline in the telomere
length correlates with a rise in the mortality rate from age-dependent
diseases (Cawthon et al., 2003). But also of relevance, limiting the prolif-
erative potential of damaged cells would prevent the outgrowth of cell
populations at risk of evolving into neoplasia. Consequently, the senescent
phenotype has been suggested to be a potent tumor-suppressor
mechanism.

IV. TELOMERES, CHECKPOINTS, AND CANCER

A. When Telomeres are Too Short, Lessons from Mice

The generation of mice deficient in the telomerase RNA gene, mTerc/
(Blasco et al., 1997) allowed the impact of specific reductions in telomere
length to be studied at the organismal and cellular levels. These mice
exhibit a progressive reduction in the telomere length throughout the cell
divisions, as occurs in human somatic cells with age, and in subsequent
generations. Early generations of these animals show no obvious patho-
logical phenotype, probably because their cells still possess sufficiently
long telomeres. In contrast, advanced generation mTerc/ mice often
show defects in highly proliferative tissues such as the hematopoietic
and the reproductive system or the intestinal epithelium, in addition to
presenting signs of premature aging (Lee et al., 1998; Rudolph et al.,
1999). Overall, the decrease in tissue fitness was due to the loss of cell
viability and an increase of apoptosis associated with telomere dysfunc-
tion, which in turn was manifested by the presence of signal free chromo-
some ends, end-to-end fusions and anaphase bridges. Both in mice and
humans, cell protection from a variety of genotoxic insults is achieved by
activating the p53 pathway, which prevents inappropriate proliferation of
damaged cells. Accordingly, removal of the p53 function restored many of
the cellular defects associated with the accumulation of short dysfunc-
tional telomeres by attenuating their detrimental effect (Chin et al., 1999).
All these observations indicated that a minimum telomeric length is nec-
essary to maintain the functions of tissues and fuelled speculation that
short telomeres would dramatically limit tumor growth in the absence of
telomerase when a fully functional p53 pathway is present. According to
this view, a reduced incidence of spontaneous tumors was found in the
mTerc/ mouse model (Blasco et al., 1997; Feldser and Greider, 2007).
To further examine the contribution of short dysfunctional telomeres in
neoplasia, telomerase-knockout mice were mated with different mouse
20 Anna Genesc

a et al.

models bearing defective proteins related to cancer such as ApcMin+/—a

model for multiple intestinal neoplasia (Rudolph et al., 2001); Ink4a/

/Arf/—mice prone to mesenchymal and lymphoid tumors
(Greenberg et al., 1999); Atm/—mice that develop thymic lymphoma
(Qi et al., 2003; Wong et al., 2003); or Alb-uPA/—a hepatocellular
carcinoma model (Farazi et al., 2003) among others. In all these mouse
models, the impact of telomere shortening also produced a reduction of
tumor incidence. Nevertheless, these investigations importantly contrib-
uted to the knowledge of the operating pathways in cancer development.
In particular, studies in the intestinal neoplasia ApcMin+/ model allowed
telomere dysfunction to be associated with early stages of malignancy.
While mTerc//ApcMin+- mice developed more early-stage microadeno-
mas than ApcMin+/ mice with intact telomere function, growth and pro-
gression of adenomas was significantly suppressed in the double mutant
because of the inability to activate telomerase (Rudolph et al., 2001).
These studies made it possible to establish that telomere dysfunction is
necessary at first, to promote the CIN capable of generating preneoplastic
lesions. But later, to allow for tumor progression, it is necessary to reac-
tivate telomerase or alternative recombination mechanisms in order to
reach the adequate telomere length compatible with cell survival. If this
does not happen, excessive CIN becomes unsustainable and most cells die
by apoptosis. Together these results show that dysfunctional telomeres
inhibit tumor formation in vivo in the setting of an intact DNA damage
p53 signaling pathway.

B. Cancer Arises When Cell-Cycle Checkpoint Defects

Accompany Short Telomeres
Seminal studies proposing a model where telomere dysfunction together
with impaired cell-cycle control promote cellular transformation come
from crossing mTerc/ mice with mice that were haploinsufficient in
p53 (Artandi et al., 2000). In this setting, loss of the p53 function accel-
erated tumorigenesis and produced a shift of the tumor spectrum. In
contrast to initial generations of mTerc//p53+/ mice with intact telo-
mere function, which normally succumb to lymphomas and sarcomas, the
mTerc//p53+/ mice with dysfunctional telomeres showed a shift in
their tumor spectra toward epithelial cancers, the tumor type prevalent
in adult humans. In addition, the genomic profile of such murine carcino-
mas changed, showing complex structural chromosomal abnormalities
similar to those found in human carcinomas, which are generally rare in
mouse tumors. Similar results were obtained when telomere dysfunction
was induced directly without telomere shortening but through a mutation
of the shelterin protein TPP1. In a context of impaired p53 function,
Role of Telomere Dysfunction in Genetic Intratumor Diversity 21

Tpp1acd/acd mice became highly prone to epithelial carcinogenesis

(Else et al., 2009). In all, these findings demonstrate that telomere uncap-
ping, either through loss of the G-rich overhang itself or by critically
shortening the (TTAGGG)n tract of DNA, can trigger, in the setting of
p53 deficiency, the genome instability that promotes the development of
epithelial cancers.

V. TELOMERE UNCAPPING AS A SOURCE OF

GENOMIC INSTABILITY

Given that loss of telomere repeats has been implicated in both tumor-
igenesis and the aging process, great efforts have been made to measure in
human cells and model organisms the minimal length required for a
functional telomere, as this could be a biomarker forecasting an indivi-
dual’s longevity and risk of disease (Cawthon et al., 2003).

A. Telomere Lengths are Highly Heterogeneous

within Somatic Cells
It has been thought that the uncapping signal is generated when the
average telomere length reaches a threshold of about 2 kb (Levy et al.,
1992). However, examination of the telomere lengths of primary human
fibroblasts at senescence by single telomere length analysis (STELA) has
demonstrated the presence of very few telomeres that are virtually devoid
of telomere repeats (Baird et al., 2003). These results support the notion
that it is not the average length of short telomeres but a few sentinel
telomeres, those with the shortest lengths, that preferentially trigger cell
cycle arrest (Zou et al., 2004).
Various studies have paid attention to unraveling whether a specific
telomere length at individual chromosome ends exists in cells and tissues.
In humans, the distribution of telomere lengths among the different chro-
mosome arms is heterogeneous, with some chromosome arms having
longer telomeres than others. Importantly, this telomere signature is a
heritable trait that is genetically determined at conception and maintained
throughout life (Graakjaer et al., 2004), and consequently it is also shared
within families (Graakjaer et al., 2006a, 2006b). So, when a telomere
length distribution in a cell is fixed, the time point at which these particular
chromosome arms become uncapped will depend on the specific telomere
shortening rate occurring in each cell type or tissue. As a whole, given that
each human being has a specific profile of telomere lengths, the risk of a
particular chromosome being unstable also differs amongst individuals
22 Anna Genesc

a et al.

and will probably be reproduced in a particular organ for a given

individual.

B. Initiation of Breakage-Fusion-Bridge Cycles

by Dysfunctional Telomeres
Importantly, studies in mouse and human telomerase-deficient cells
have demonstrated that the particular chromosomes with a critical telo-
mere length in each animal or individual are those most frequently
involved in end-to-end fusion events, namely ring or dicentric chromo-
somes (Deng et al., 2003, 2004; der-Sarkissian et al., 2004; Plug-
DeMaggio et al., 2004; Soler et al., 2005). These unstable chromosome
configurations can set in motion BFB cycles, as already noted in maize by
McClintock (McClintock, 1939), a mechanism capable of producing
rapid and widespread changes in gene dosage as well as complex chromo-
some rearrangements. When fused chromosomes—interchromosome
dicentrics—or sister chromatids—isodicentric chromatids—are pulled to
opposite poles during cell division, anaphase bridges are likely to occur
(Fig. 3A and 3B). However, the probabilities of bridging and the chromo-
some aberrations eventually formed after bridge resolution are different
depending on the initial substrate for joining. While isodicentric chroma-
tids always bridge at anaphase as they proceed from a continuous DNA
molecule, dicentric chromosomes may only contribute to genomic insta-
bility if a twist between the two centromeres occurs during division. Thus,
dicentric chromosomes with a larger intercentromeric distance present
higher probabilities of twisting and bridging.
Studies using real-time imaging have shown that anaphase bridges usu-
ally break during cell division (Hoffelder et al., 2004; Pampalona et al.,
2010a; Shimizu et al., 2005; Titen and Golic, 2008). Upon breakage of an
isodicentric chromatid a partial duplication on a chromosome end in one
daughter cell, and a terminal deletion on the same chromosome in the
other daughter cell, are generated (reviewed in Murnane and Sabatier,
2004) (Fig. 3A1). Such new uncapped broken chromosomes may again
fuse with their replica after DNA synthesis if no other substrate for joining
exists, resulting in further amplification of the genes that are close to the
break point or fusion point (Lo et al., 2002; Tusell et al., 2008). In
contrast, resolution of interchromosome dicentrics results in partial
losses—terminal deletions—and partial gains of the involved chromo-
somes that are also susceptible to be repaired by fusing with other unpro-
tected extremities afterwards (Soler et al., 2005) (Fig. 3B1). As a result,
broken extremities can be healed in the form of stable chromosome aber-
rations such as nonreciprocal translocations or other nonbalanced rear-
rangements, diminishing CIN. But remarkably, chromosomes can also
Role of Telomere Dysfunction in Genetic Intratumor Diversity 23

undergo prolonged periods of chromosome instability, involving repeated

BFB cycles, if new dicentric chromosomes are formed. Thus, in the case of
a telomere-dysfunction background, CIN is initiated by the formation of
end-to-end fusions that can bridge and break during cell division. But
relevantly, the continuous proliferation of cells showing increasing telo-
mere dysfunction enhances the formation of new unstable chromosome
aberrations, which can perpetuate BFB cycles eventually leading to mas-
sive structural CIN.

C. Missegregation of Anaphase-Bridged Chromatin

Generates Aneuploid Cell Populations
A wide variety of cancer cells, besides anaphase bridges, also exhibit
micronuclei (MN) and nuclear buds (Gisselsson et al., 2001a). The pres-
ence of nuclear buds has been related to chromatin regression after ana-
phase bridge breakage (Pampalona et al., 2010a) although this abnormal
nuclear morphology has also been related to the budding, outside of the
nucleus, of highly amplified DNA sequences (Shimizu et al., 1998).
Regarding micronuclei, these have always been considered to originate
from improper segregation of chromosomes during cell division. At telo-
phase, lagging whole chromosomes or chromosome fragments are even-
tually enclosed by a nuclear membrane separated from that of the daugh-
ter nuclei. Specifically, MN containing parts of chromosomes have been
shown to originate from fragmented chromosome material when ana-
phase bridges are formed, stretched, and broken during telophase in car-
cinoma cell lines (Hoffelder et al., 2004) or in human epithelial cells
showing telomere dysfunction (Pampalona et al., 2010a). But relevantly,
a significant increase in centromere-labeled MN has also been observed
along with telomere dysfunction, thus raising the possibility that bridged
dicentric chromatids may also lead to migration defects at anaphase
(Pampalona et al., 2010b).
Pioneering work by Barbara McClintock in 1938 gave an early sugges-
tion that anaphase bridges resulting from interlocked ring chromosomes
can be excluded from the telophase nuclei, thus leading to whole chromo-
some losses (McClintock, 1938). In order to directly evaluate the contri-
bution of telomere dysfunction in missegregation events we took advan-
tage of the cytokinesis-blocked micronucleus assay (Pampalona et al.,
2010b). Segregation pattern analysis of specific chromosomes in binucle-
ated human mammary epithelial cells demonstrated a preferential errone-
ous distribution of chromosomes with critically short telomeres.
Abnormal segregation of bridged end-to-end fusions resulted in both
nondisjuntion and anaphase loss, thus supporting the notion that the
mechanical tension of bridged chromatin could result from defective
24 Anna Genesc

a et al.

[(Fig._3)TD$IG]

Fig. 3 Chromosome instability mediated by dysfunctional telomeres

(A) Sister chromatid fusion generates a continuous DNA molecule that always bridges at
anaphase. (1) Resolution of the bridge through breakage would lead to inverted repeats
(yellow triangles) on the end of a chromosome in a daughter cell and a deletion (Del) in
the other. These chromosome aberrations are again susceptible to reorganize, leading to
nonreciprocal translocations (NRT) or new dicentric chromosomes. While the forma-
tion of a NRT avoids further instability, the new dicentric may bridge again at anaphase,
thus perpetuating chromosome instability. (2) Anaphase bridges may also erroneously
segregate chromosomes between daughter cells. This results in the formation of aneu-
ploid cells. Whereas the loss of a chromosome results in a real hypodiploid progeny, the
gain of an unstable chromosome will produce a broad range of abnormalities since it can
reorganize further.
(B) Fusion of chromosomes before the S-phase results in conventional interchromosome
dicentrics, which will bridge at anaphase only if a twist between the two centromeres is
produced. The two bridged dicentric chromatids can either break or missegregate. (1)
Rather than amplification events, breakage of both chromatids generates partial gains
and losses in the daughter cells. Again, the broken extremities can further reorganize in
the form of stable or unstable aberrations, and consequently new BFB cycles can be
initiated. (2) In the case that one dicentric chromatid breaks and the other missegregates,
partial and whole chromosome losses and gains will be produced. Eventually, a hypo-
diploid clone of cells will emerge after proliferation of the monosomic cell, whereas the
gained dicentric could re-enter into the BFB cycle.
(C) Persistent telomere damage can induce bypass of mitosis and re-entry into S-phase,
resulting in a tetraploid cell.
(D) Tetraploidization mediated by telomere dysfunction might also be produced by persis-
tent chromatin bridges during mitosis. The presence of chromatin at the cleavage plane
could induce furrow regression and cytokinesis failure, eventually leading to tetraploid
cells.
 26 Anna Genesc

a et al.

[(Fig._3)TD$IG]
Fig. 3 (Cont.) (See Page 4 in Color Section at the back of the book.)

microtubule–kinetochore attachments that eventually end in an abnormal

chromosome segregation (Pampalona et al., 2010b). Consistent with these
results, a strong correlation between telomere shortening and losses of
both chromosome arms and centromeres was observed in biopsies of
colon mucosa from ulcerative colitis progressors (O’Sullivan et al.,
2002). Also of note is the fact that the frequency of anaphase bridges in
the epithelium of these individuals was highly significant. Finally, studies
in colorectal cancer cell lines also indicated that detachment of anaphase
bridges could contribute to aneuploidy by loss of chromosomes during cell
division (Stewenius et al., 2005).
All of this points to the notion that shortening of telomeric DNA repeats
results in anaphase bridges that, through BFB cycles, are able to generate
unbalanced structural chromosome rearrangements. But in addition,
bridged chromatin may also unfaithfully segregate between the daughter
cells resulting in aneuploid progeny (Fig. 3A2 and 3B2). According to an
anaphase bridge-mediated increase in genome complexity, tumors with
anaphase bridges typically had complex karyotypes with both numerical
and structural changes. In contrast, tumors without anaphase bridges had
either few (<3) cytogenetic changes or numerical changes only (Galli et al.,
2004).

D. Telomere Uncapping also Fuels Changes in Cell Ploidy

It is clear that anaphase bridges underlie CIN in the form of structural
and numerical aberrations when telomeres become critically short and
prone to end fusions. In addition, telomere uncapping has been recently
Role of Telomere Dysfunction in Genetic Intratumor Diversity 27

associated with the emergence of whole genome duplication events

(Davoli et al., 2010), through anaphase-bridge-independent mechanisms.
Depletion of the shelterin proteins POT1a and POT1b, in p53 defective
mice, produced a generalized telomere dysfunction leading to the progres-
sive accumulation of polyploid cells containing duplo- and quadruplo-
chromosomes. Polyploidization in the form of endomitosis was also
described in TRF2 depleted mouse fibroblasts deficient in Lig4 and p53
(Davoli et al., 2010; Smogorzewska et al., 2002). In these cases, polyploi-
dization was induced by persistent DNA damage signaling of uncapped
telomeres, as repair is repressed by TRF2 presence at telomeres or by
blocking the nonhomologous end-joining repair pathway. These cells
experienced a prolonged G2 arrest due to ATM- or ATR-pathways acti-
vation but eventually skip mitosis and re-entered cell cycle, thus duplicat-
ing the whole genome (Fig. 3C). In this setting, polyploidization was
dependent on the DDR as inactivation of the DNA damage signaling
cascade of ATM/ATR diminished the tetraploid phenotype.
It is clear that massive telomere damage can fuel polyploidization
through endoreduplication; however, this does not exclude other possible
causes of mitotic failure mediated by telomere dysfunction. In this sense, it
can be envisaged than anaphase bridges resulting from end-to-end chro-
mosome fusions may remain unbroken during mitosis, thus mechanically
preventing cell abscission (McClintock, 1938; Mullins and Biesele, 1977)
(Fig. 3D). The presence of chromatin occluding the cleavage plane could
induce furrow regression giving rise to tetraploid cells. In yeast, the NoCut
pathway monitors the clearance of chromatin from the midzone to ensure
that cytokinesis completes only after all chromosomes have migrated to
the poles (Mendoza et al., 2009; Norden et al., 2006). A similar mecha-
nism has recently been observed in human cells, in which the protein
Aurora B remains phosphorylated when chromatin occludes the cleavage
plane, t allowing extra time for the chromatin to be released
(Steigemann et al., 2009). Although more studies are needed to support
this possibility, it is tempting to speculate that uncleared bridged chroma-
tin from the midzone could prevent cytokinesis and result in tetraploid
cells.
In the context of neoplasia, different studies support the view that
tetraploid cells act as a source of cancer cell precursors. Polyploid cells
are often found in tumors of all stages (Storchova and Kuffer, 2008) and
tetraploidy renders an increase in CIN (Fujiwara et al., 2005; Mayer and
Aguilera, 1990; Storchov a et al., 2006) that could constitute an important
step in early cell transformation. It has been hypothesized that while
diploid cells might die after losing multiple chromosomes, the presence
of extra chromosome sets in polyploid cells would buffer the effects of
deleterious mutations, allowing cells with DNA damage to survive longer
28 Anna Genesc

a et al.

until a crucial growth-enhancing or transforming mutation occurs

(Ganem et al., 2007).
The overall conclusion from these studies is that telomere-dependent
instability could drive the formation of rearranged karyotypes showing
both structural and numerical chromosome aberrations and also changes
in ploidy levels, all of which are a hallmark of cancer cells. But more
importantly, the inherent instability of these polyploid aberrant cells,
through BFB-cycles and errors in merotelic kinetochore-microtubule
attachments (Ganem et al., 2009; Silkworth et al., 2009), can result in
additional mutations essential for progression toward malignant
transformation.

VI. SHORTENING TELOMERES IN AGED HUMAN CELLS

There are a large number of studies demonstrating the harmful impact of

relatively short telomeres in cells. Excessive telomere shortening is a rel-
evant feature of both premature aging inherited diseases, often related to
genetic defects in telomerase function (Monaghan, 2010), and cancer, a
common age-related disease where initial stages have also been linked to
eroded telomeres (Aubert and Lansdorp, 2008).

A. Telomere Attrition Occurs in Epithelial Compartments

Multicellular organisms with renewing epithelia need to maintain tissue
fitness throughout the life of individuals through a cycle of stem cell
proliferation and replacement. Nevertheless, telomerase expression in
most adult stem cell compartments is not sufficient to maintain telomere
homeostasis, and therefore telomere length of almost all epithelial tissues
decreases with human aging (Takubo et al., 2010). Coincident with these
observations, most prevalent cancers in adult humans arise from epithelial
compartments and their frequency increases exponentially with age.
Studies in mouse models have provided clues about the influence of
excessive telomere shortening with organismal age and with cell divisions
in culture (Fig. 4). When one or a few telomeres reach a critically short
length, the DDR signals to cells to either senesce or die through apoptosis.
Senescent cells remain viable but arrest growth and cannot initiate DNA
replication in response to physiological mitogens (Cristofalo and Pignolo,
1993; Stanulis-Praeger, 1987). Because senescence stops incipient cancer
cells from proliferating, it was proposed to be a powerful tumor-suppressive
mechanism (Krtolica and Campisi, 2002). In this context, the senescence
response is considered beneficial because it protects organisms from cancer,
 Role of Telomere Dysfunction in Genetic Intratumor Diversity 29

[(Fig._4)TD$IG]

Fig. 4 Model for telomere-driven carcinogenesis. Telomere length diminishes during

successive rounds of cell division in an environment where telomerase cannot compensate
the end-replication problem. When a few sentinel chromosomes become uncapped, the p53-
and Rb-dependent pathways induce a permanent cell cycle arrest known as senescence.
Abrogation of these surveillance mechanisms would allow proliferation of cells carrying
dysfunctional telomeres. The fusion of uncapped ends will fire the BFB cycles, which
would later perpetuate, leading to structural, numerical, and ploidy chromosome
abnormalities. Massive chromosome instability will frequently end in crisis, a phase of
high rates of cell death. However, the high rate of mutation in these cells could also result
in the activation of the mechanisms required to stabilize the chromosome ends. The reduction
of chromosome instability to a permissible rate would fuel the genetic diversity needed for
eventually acquiring a tumor phenotype. (For color version of this figure, the reader is
referred to the web version of this book.)

a major life-threatening disease. However, it must be considered that aging

itself could deteriorate the p53 and retinoblastoma (Rb) protein-p16 path-
ways that govern the senescent growth arrest. Abrogation of these pathways
would allow cells, which are destined to senesce to propagate, thus further
reducing the length of their telomeres and promoting CIN (Fig. 4). In
preneoplastic lesions and cancer cells, components of the signaling cascade
governed by p53 and Rb, such as the cyclin-dependent kinase inhibitors
30 Anna Genesc

a et al.

p21CIP1 and p16INK4a, are frequently disrupted (Gemma, 2002). This high-
lights the possibility that many more mutations could contribute to faulty
cell-cycle checkpoints with age. Or, in other words, that advancing age
could contribute to an environment more permissive for carcinogenesis.

B. Does Tumor Suppressor Gene Expression

also Decline with Age?
Aging is a natural process with gradual decline of many biological
functions, which may enhance mutation frequency. Compelling evidence
in mice indicates that the functions of critical proteins involved in DNA
repair, cell-cycle control, and apoptosis decline in an age-dependent man-
ner (Feng et al., 2007). In virtually all tissues examined, p53 activity was
significantly lower in old mice compared to young. Moreover, less effi-
cient apoptosis was exhibited in the old tissues, thus indicating a reduced
functionality of p53 with age. Importantly, the activity levels of the
upstream DNA damage sensor kinase ATM, which phosphorylates p53
in the presence of DNA damage, was also found to be reduced after
irradiation in older mice. Similarly, in humans, different reports have
shown a progressive loss of the ability to repair DNA damage with age
in vitro (Lambert et al., 1979; Nette et al., 1984). This reduced repair
capacity with age correlated well with the increase of mutation rate in vivo
with human aging (King et al., 1994; Robinson et al., 1994).
Yet, another condition leading to disabled cell-cycle checkpoints
involves epigenetic changes in chromatin, such as DNA methylation
or histone modification, which may modify gene expression patterns.
There is now substantial evidence that aging also affects DNA methyl-
ation of specific loci, including cancer-related genes (reviewed in Fraga
and Esteller, 2007). In this sense, silencing of tumor suppressor and
tumor-related genes, such as APC, CDH1, and CDKN2A, by hyper-
methylation increases with age in nonneoplastic tissues of individuals
(Waki et al., 2003). But most importantly, hypermethylation of different
tumor suppressor genes was more frequent in nonneoplastic epithelial
tissue of cancer patients than in those individuals without cancer. In
agreement with these observations, an age-related increased methyla-
tion level has been associated with cancer susceptibility in the elderly
(So et al., 2006).
All these studies indicate that the aging process is linked not only to
telomere shortening and chromosome uncapping but also to harmful
inactivation or defects in surveillance mechanisms that normally monitor
genomic integrity, thus potentially increasing the possibilities for preneo-
plastic cells to proliferate indiscriminately and ultimately acquire a malig-
nant phenotype.
Role of Telomere Dysfunction in Genetic Intratumor Diversity 31

C. BFB-Intermediates and Short Telomeres in Early

Preneoplastic Lesions
In humans, excessive telomere shortening is observed in some chronic
human diseases associated with high cell turnover and an increased risk of
cancer, such as liver cirrhosis (Kitada et al., 1995) or pancreatitis
(van Heek et al., 2002). In an analogous fashion, very short telomeres
have been reported to be an early alteration in many human cancers, such
as those from the urinary bladder, esophagus, large intestine, oral cavity,
uterine cervix, and prostate (Meeker et al., 2002, 2004). But more impor-
tantly, in Barrett’s esophagus, the preneoplastic state that precedes esoph-
ageal adenocarcinoma, the samples with the shorter telomeres were also
those with the highest degree of chromosomal abnormalities (Finley et al.,
2006). Similarly, in ulcerative colitis, a chronic inflammatory disease of
the colon, a mechanistic link between telomere shortening and CIN was
supported by a higher frequency of anaphase bridges in cancer precursors
(O’Sullivan et al., 2002). Collectively, such observations suggest that
precancerous cells go through a period of excessive telomere erosion that
may initiate CIN, thus increasing the mutability of the genome.
Therefore, as a general rule, it is expected that telomere dysfunction fires
the initial BFB cycles that will go on to self-perpetuate through the forma-
tion of new unstable chromosome aberrations. Of course, the characteristic
telomere length distribution of each individual chromosome could have a
direct impact on the way chromosome instability is initiated. At first, only
aberrations involving specific chromosomes with the shortest telomeres are
evident, but later the random scrambling of the karyotype, through
repeated BFB cycles, allows for the genetic divergence of the genome that
fosters the characteristic karyotype heterogeneity within tumors. Strong
evidence supporting the existence of a period of telomere-crisis before
cancer onset comes from the evolution of the chromosome breakpoints
during tumor progression in pancreatic carcinoma and osteosarcoma, two
tumor types characterized by multiple complex aberrations. While cases
with few chromosomal aberrations showed preferential clustering of
breakpoints to the terminal bands, tumors harboring complex, reorganized
genomes showed a gradual shift of breakpoints to interstitial and centro-
meric regions (Gisselsson et al., 2001b). The high rate of terminal break-
points in low rearranged tumors suggested an early disruption of the
telomere integrity, and was consistent with persistent BFB cycles gradually
eroding the chromosome arms (McClintock, 1941) during tumor evolu-
tion. Accordingly, both chromosomes lacking telomeric FISH signals and
anaphase bridges were observed in cells from low-grade tumor biopsies.
Together, this study further strengthened the possible relationship of telo-
mere dysfunction with cancer initiation in human cells.
32 Anna Genesc

a et al.

D. Toward a Malignant Phenotype

While genomic instability generated through telomere dysfunction
might be necessary for tumor development, too much instability can be
detrimental to tumor growth. In vitro models have shown that rampant
telomere-driven instability threatens cell viability as cells accumulate very
short telomeres, telomeric fusions, and anaphase bridges, and display a
high degree of apoptosis (Wright and Shay, 1992) (Fig. 4). Most cells that
enter this second proliferative block, termed crisis, die. That is why this
state, along with replicative senescence, is also considered a barrier for
dangerous cell proliferation. However, the constant reshuffling of the
karyotype during telomere-dependent instability could provide the genetic
diversity needed for some cells to acquire certain genetic alterations, which
subvert the proliferation barriers to continue growth and eventually trans-
form (Shay and Wright, 2005).
Overcoming the second proliferative barrier is mostly achieved by
upregulating telomerase expression or, less frequently, via an alternative
recombination-based telomere maintenance mechanism (Hanahan and
Weinberg, 2011), as stabilization of chromosome ends, at least to a
permissible level, avoids widespread cell death. Telomerase is expressed
in the majority of advanced human carcinomas (Kim et al., 1994; Shay
and Bacchetti, 1997), suggesting that telomerase reactivation is a criti-
cally important step for initiated lesions to progress to malignancy, since
it removes telomere shortening’s inhibitory barrier to tumor progres-
sion. Of note, studies in human cell lines have established that karyotype
evolution correlates with the lifetime of the cells prior to telomerase
reactivation. Cultures immortalized after a short period of telomere-
dependent CIN had very few abnormalities whereas cell lines immor-
talized after a long instability period were characterized by having
multiple gross chromosomal abnormalities (der-Sarkissian et al.,
2004). All of this indicates that a delayed acquisition of telomerase
function could be a mandatory step to boost malignancy (Hanahan
and Weinberg, 2011), since acquiring much more CIN would enhance
the probabilities of generating tumor-promoting mutations. Telomerase
reactivation per se does not cause cells to transform into a tumorigenic
state and, at least in vitro, conversion to a fully tumorigenic state occurs
when normal cells inactivate the p53 and Rb pathways, abrogate PP2A
(protein phosphatase 2A) function and express oncogenic H-Ras and
hTERT (Hahn, 2002; Hahn et al., 1999). Therefore, in order to become
tumorigenic, cells with telomerase must acquire additional mutations to
evolve into malignancy.
Importantly, after subsequent upregulation of telomerase expression,
telomere-instability does not disappear but decreases to a point where it
Role of Telomere Dysfunction in Genetic Intratumor Diversity 33

affects cellular fitness to a sufficiently small extent to allow tumor devel-

opment. In many tumor types, this chromosome instability is translated
into a stepwise accumulation of cytogenetic changes during tumor pro-
gression (Al-Mulla et al., 1999; Mitelman et al., 1997; Nishizaki et al.,
1997), through a continuous cycle of DNA DSB formation and repair.
This is evidenced from the presence of DSB markers, such as phosphory-
lated histone H2AX and 53BP1 nuclear foci (Bartkova et al., 2005), as
well as anaphase bridges and/or mitotically unstable chromosomes
(Gisselsson et al., 2004; Rampazzo et al., 2010) in cancer cells. At this
stage, both residual traces of the initial telomere-dependent instability and
newly attained CIN mechanisms could be responsible for the accumula-
tion of cytogenetic changes. In this context, even though telomerase is
expressed, recurrent instability allows for evolution and adaptation of the
cancerous cells to the environment as well as the development of the
cancer hallmarks that eventually lead to metastatic spread.

VII. SUMMARY

Accumulated data support the notion that loss of telomere repeats con-
tributes to human carcinogenesis; however, the nature of this link remains
elusive. It is not an easy issue to demonstrate, since telomere-driven insta-
bility should occur early in the neoplastic process and the subsequent
derivation of the genome would mask telomere-dysfunctional traces.
However, evidence from model organisms, human in vitro cell systems,
and tissue samples has revealed the existence of scars that denote the flow
through a state of telomere instability.
The physiological shortening of telomeres with age in human tissues
may detonate CIN through anaphase bridge intermediates when
uncapped chromosomes fuse with each other. Massive remodeling and
scrambling of the genome, in proliferating cells, is then achieved through
repeated BFB cycles of unstable chromosomes resulting from the joining of
both initially broken chromosomes and those that later became unpro-
tected. As a result, telomere dysfunction serves as a generator of genomic
instability where an arbitrary pattern of structural, numerical and
ploidy abnormalities can occur. Adding more complexity to the ongoing
telomere-driven instability, the presence of merotelic attachments in
polyploid cells, may further increase chromosome missegregation in
an already reorganized genome. Thus, telomere-driven genome instabil-
ity could well be one of the sources leading to a hypermutable hetero-
geneous genome prone to accumulating the genetic changes that drive
cells into malignity.
34 Anna Genesc

a et al.

ACKNOWLEDGMENTS

Research in the Tusell lab is supported by grants from the Consejo de Seguridad Nuclear;
Instituto de Salud Carlos III (RD06/0020/1020) and the Generalitat de Catalunya
(2009SGR-282).

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 How Mitotic Errors Contribute to
Karyotypic Diversity in Cancer
Joshua M. Nicholson and Daniela Cimini
Department of Biological Sciences, Virginia Tech,
Blacksburg, VA, USA

I. Introduction
II. Intratumor Diversity at the Karyotypic Level
A. Preneoplastic aneuploidy
B. The Effects of aneuploidy
C. Chromosome instability and the cancer karyotype: between order and disorder
III. How Mitotic Errors Contribute to the Karyotypic Diversity of Cancer Cells
A. Kinetochore–microtubule attachments
B. Spindle assembly checkpoint (SAC)
C. Multipolarity and spindle geometry
D. Microtubule dynamics
E. Polyploidization
IV. Conclusion
Acknowledgments
References

Aneuploidy is a common feature of cancer cells, and is believed to play a critical

role in tumorigenesis and cancer progression. Most cancer cells also exhibit high
rates of mitotic chromosome mis-segregation, a phenomenon known as chromo-
somal instability, which leads to high variability of the karyotype. Here, we describe
the nature, nuances, and implications of cancer karyotypic diversity. Moreover,
we summarize recent studies aimed at identifying the mitotic defects that may be
responsible for inducing chromosome mis-segregation in cancer cells. These include
kinetochore attachment errors, spindle assembly checkpoint dysfunction, mitotic
spindle defects, and other cell division inaccuracies. Finally, we discuss how such
mitotic errors generate karyotypic diversity in cancer cells. # 2011 Elsevier Inc.

I. INTRODUCTION

At the crux of carcinogenesis lies complexity. Amongst a single tumor,

cell-to-cell variability is manifest in many different properties: karyotype,
morphology, enzyme receptors, ability to metastasize, mutations, and

Advances in CANCER RESEARCH, Volume 112 0065-230X/10 $35.00

Copyright 2011, Elsevier Inc. All right reserved. DOI: 10.1016/B978-0-12-387688-1.00003-X
43
44 Joshua M Nicholson and Daniela Cimini

drug resistance. While seemingly chaotic per cell, the tumor as a whole is
much like a mosaic displaying interdependency and organization. For this
reason it has been called a species (Duesberg and Rasnick, 2000; Duesberg
et al., 2011; Huxley, 1956; Klein et al., 2010; Van Valen and Maiorana,
1991; Vincent, 2010), a society of cells (Heppner, 1984), and a complex
ecosystem (Heppner, 1984; Merlo et al., 2006; Sachs and Hlatky, 2010).
Whereas it is likely that a myriad of mechanisms contribute to cancer
complexity, in this chapter we specifically focus on how mitotic defects
contribute to the complexity of the cancer cell karyotype. It has long been
known that cancer cells are characterized by aneuploid karyotypes
(Bayreuther and Klein, 1958; Chu and Giles, 1958; Hauschka and
Levan, 1958), and an increasing body of work has unveiled a causal
relationship between aneuploidy and tumorigenesis [reviewed in
(Cimini, 2008; Pavelka et al., 2010a; Sen, 2000)]. A key role of aneuploidy
in tumorigenesis and tumor progression is also indicated by the fact that
measuring DNA indices or ploidy is clinically informative as a prognostic
indicator in various cancers (Atkin and Kay, 1979). For instance, ploidy
measurements in different types of cancer (Frankfurt et al., 1985; Grote
et al., 2001; Jakobsen, 1984; Jakobsen et al., 1988; Kallioniemi et al.,
1987; Susini et al., 2011) are as, if not more, accurate when predicting
survival than other widely used measures such as the prostate-specific
antigen (PSA) test (Stamey, 2004; Vickers et al., 2011).
Karyotypic analysis also indicates that there is wide variability in the
chromosome number within the same cancer cell population, suggesting
that errors in mitotic chromosome segregation are recurrent in cancer
cells. In the remaining part of this chapter, we will start by reviewing what
is known about intratumor karyotypic diversity. Next, we will describe
the mitotic mechanisms that promote chromosome mis-segregation in
cancer cells, and thus can produce karyotypic diversity.

II. INTRATUMOR DIVERSITY AT THE

KARYOTYPIC LEVEL

A. Preneoplastic Aneuploidy
Aneuploidy is ubiquitous in cancer (Gebhart and Liehr, 2000;
Mertens et al., 1997; Mitelman et al., 2011; Weaver and Cleveland,
2006), and was proposed to have a causal role in cancer already over a
century ago (Boveri, 1902, 1914). Over the years, the idea that aneu-
ploidy plays a causal role in the origin of cancer has been supported by
substantial experimental evidence. For example, random aneuploidy
How Mitotic Errors Contribute to Karyotypic Diversity in Cancer 45

appears after carcinogen application but before transformation in

Chinese hamster (Duesberg et al., 2000a), mouse (Conti et al., 1986),
rat (Sudilovsky and Hei, 1991), and human cells (Hittelman, 2001;
Hittelman et al., 1996). Furthermore, although the results were occa-
sionally ambiguous (see below for further discussion), a number of
studies in mouse models carrying mutations/deletions that cause aneu-
ploidy showed a correlation between aneuploidy and tumorigenesis
(Babu et al., 2003; Diaz-Rodriguez et al., 2008; Iwanaga et al., 2007;
Michel et al., 2001; Sotillo et al., 2007; Weaver et al., 2007). For
aneuploidy to play a causal role in tumorigenesis, it would be expected
to occur in undifferentiated dividing cells within the organism’s tissues.
Unfortunately, the rates of chromosome mis-segregation and the fre-
quencies of aneuploidy in situ are very difficult to assess due to the
inaccessibility of primary tissues. One exception in humans is repre-
sented by peripheral blood lymphocytes, which can be easily isolated.
Isolated lymphocytes can be either analyzed directly by chromosome- or
centromere-specific fluorescence in situ hybridization (FISH) or used to
establish short-term cultures. Cultured lymphocytes can be used for
metaphase spread analysis (Minissi et al., 1999), micronucleus assay
(Countryman and Heddle, 1976), or cytokinesis-block assay (Fenech,
1993) to determine spontaneous rates of chromosome mis-segregation
and aneuploidy. Studies using these approaches have yielded a large
amount of information, and indicate that the basal frequencies of aneu-
ploidy for individual chromosomes in human lymphocytes range
between 0.03% and 10% (Carere et al., 1998; Catalan et al., 2000;
Hayes et al., 2000; Minissi et al., 1999; Ramirez et al., 2007; Rehen
et al., 2005; Zhang et al., 1998;). Furthermore, studies in brain cells
have found aneuploidy for individual chromosomes to be 4%
(Kingsbury et al., 2006; Rehen et al., 2005). Such frequencies are sur-
prisingly high. Indeed, if the same aneuploidy rates were occurring in all
dividing cells, hundreds of thousands aneuploid cells would be produced
every day in each individual, given that 2.5  108 cells are dividing at
any given time (Alberts et al., 1994). If such frequencies were realistic,
cancer would be expected to occur at much higher rates in the popula-
tion. So, how can these high frequencies of aneuploidy be reconciled
with the relatively low incidence of cancer if indeed aneuploidy is a
cause of cancer? One possibility is that the rates of aneuploidy observed
in peripheral blood lymphocytes and neurons are not representative of
all cell types. However, it is noteworthy that the frequency of aneu-
ploidy in peripheral blood lymphocytes of healthy individuals correlates
with cancer risk (Hagmar et al., 1994; Olaharski et al., 2006; Rossner
et al., 2005). In addition, aneuploidy in human peripheral blood
 Another Random Document on
Scribd Without Any Related Topics
footman in his brown livery assisted her into the carriage.
Then she smiled merrily, and bowed as I raised my hat, and
she was borne away westward in the stream of fine
equipages, hers the smartest of them all.

A week later, having seen nothing further of her, I wrote

and received a prompt response. Then in the happy autumn
days that followed we contrived to meet often, and on each
occasion I grew deeper and deeper in love with her. Since
that evening when we had stood together beneath the
street lamp in Kensington, she had made no mention of the
pencil-case or of its owner. Indeed, it seemed that her
sudden identification of it had betrayed her into
acknowledging that its owner had been her lover, and that
now she was trying to do all she could to remove any
suspicion from my mind.

Nevertheless, the remembrance of that crime and of all the

events of that midnight adventure was ever within my
mind, and I had long ago determined to make its
elucidation the chief object of my life. I had placed myself
beneath the thrall of some person unknown, and meant to
extricate myself and become again a free agent at all costs.

On several occasions I had seen the cabman West on the

rank at Hyde Park Corner, but although he had constantly
kept his eyes open in search of Edna, his efforts had all
been in vain. I had seen also the old cab-driver who bore
the nickname “Doughy,” but it turned out that it had not
been his cab which my mysterious protectress had taken
after parting from me. One point, however, I settled
satisfactorily. On one of our walks together I contrived that,
the man West should see Mabel, but he afterwards declared
that the woman of whom he was in search did not in the
least resemble her. Therefore, it was certain that Mabel and
Edna were not, as I had once vaguely suspected, one and
the same person.

Sometimes I would meet my idol after her studies at the

Royal Academy of Music, and accompany her across the
park; at others we would stroll together in the unfrequented
part of Kensington Gardens, or I would walk with her
shopping and carry her parcels, all our meetings being, of
course, clandestine ones.

One morning in the middle of November I was overfed at

receiving an invitation from Mrs Anson to dine at The
Boltons, and a couple of days later the sum of my happiness
was rendered complete by finding myself seated beside
Mabel in her own home.

The house possessed an air of magnificence and luxury

which I scarcely expected. It was furnished with great
elegance and taste, while the servants were of an even
more superior character than the house itself. Among the
homes of my many friends in the West End this was
certainly the most luxurious, for money seemed to have
been literally squandered upon its appointments, and yet
withal there was nothing whatever garish nor any trace of a
plebeian taste. There was a combined richness and
quietness about the whole place which impressed one with
an air of severity, while the footman who ushered me in was
tall, almost a giant in stature, and solemn as a funeral
mute.

Mrs Anson rose and greeted me pleasantly, while Mabel, in

a pretty gown of coral-pink, also shook my hand and raised
her fine dark eyes to mine with a glance of pleasure and
triumph. It was, no doubt, due to her that I had been
bidden there as guest. A red-headed, ugly-faced man
named Hickman, and a thin, angular, irritating woman,
introduced to me as Miss Wells, were my only fellow-guests.
The man regarded me with some suspicion as I entered,
and from the first I took a violent dislike to him. It may
have been his forbidding personal appearance which caused
my distrust. Now that I reflect, I think it was. His face was
bloated and deeply furrowed, his eyes large, his lips thick
and flabby, his reddish beard was ill-trimmed and scanty.
He was thick-necked; his face was further disfigured by a
curious dark-blue scar upon the left jaw, and I could not
help remarking within myself, that if some faces resembled
those of animals, his was closely allied to that of a savage
bulldog. Indeed, I had never before seen such an eminently
ugly face as his.

Yet he spoke with the air and perfect manner of a

gentleman. He bowed with refined dignity as I was
introduced, although I thought his smile seemed
supercilious, while I was almost certain that he exchanged a
curious, contemptuous look with Mabel, who stood behind
me.

Was he aware of our little exchanges of confidences? Had

he secretly watched us in our walks along the leafy byways
of Kensington Gardens, and detected that I loved her? It
seemed very much as though he had, and that he had
endeavoured to disparage me in her eyes.

At Mrs Anson’s invitation, I took Mabel in to dinner, and sat

next her, while opposite us sat the dog-faced man with the
irritating spinster. The latter was a fitting companion for
him, bony of countenance, her back straight as a board, her
age uncertain, and her voice loud, high-pitched, and
rasping. She wore a number of bangles on her left wrist;
one of them had pigs and elephants hanging on it, with
hearts, crosses, bells, and framed and glazed shamrock
leaves mixed in. That would not have mattered much had
she not been eating, but as dinner progressed the room
grew a trifle warm, and she unfortunately had a fan as well
as those distressing bangles, which fan she rhythmically
waved to and fro, playing the orchestra softly when fanning
herself, or loudly as she plied her knife and fork “click-clack,
jingle-jingle, tinkle-tinkle, click-clack!” until the eternal
music of those pigs, elephants, crosses, hearts and bells
prevented anything beyond a jerky conversation. She
turned and twisted and toyed with her menu, tinkling and
jingling the whole time like a coral consoler or an infant’s
rattle. Little wonder, I thought, that she remained a
spinster. With such an irritating person to head his
household, the unfortunate husband would be a candidate
for Colney Hatch within a month. Yet she was evidently a
very welcome guest at Mrs Anson’s table, for my hostess
addressed her as “dear,” and seemed to consider whatever
positive opinion she expressed as entirely beyond dispute.

I liked Mrs Anson. Although of that extremely frigid type of

mother, very formal and unbending, observing all the rules
of society to the letter, and practically making her life a
burden by the conventionalities, she possessed,
nevertheless, a warm-hearted affection for her child, and
seemed constantly solicitous of her welfare. She spoke with
the very faintest accent with her “r’s,” and I had, on the
first evening we had met at the colonel’s, wondered
whether she were of Scotch, or perhaps foreign, extraction.
The general conversation in the interval of the Irritating
Woman’s orchestra turned upon foreign travel, and
incidentally, in answer to an ingenious question I put to her,
she told me that her father had been German, but that she
had nearly all her life lived in England.

The Irritating Woman spoke of going to the Riviera in

December, whereupon Mabel remarked—
“I hope mother will go too. I’m trying to persuade her.
London is so dull and miserable in winter compared with
Cannes or Nice.”

“You know the Riviera well, I suppose?” I inquired of her.

“Oh, very well,” she responded. “Mother and I have spent

four winters in the south. There’s no place in Europe in
winter like the Côte d’Azur—as the French call it.”

“I much prefer the Italian Riviera,” chimed Miss Wells’s

high-pitched voice. She made it a point of honour to differ
with everybody. “At Bordighera, Ospedaletti, San Remo, and
Alassio you have much better air, the same warmth, and at
about half the price. The hotels in Nice and Cannes are
simply ruinous.” Then, turning to Mrs Anson, she added,
“You know, dear, what you said last year.”

“We go to the Grand, at Nice, always,” answered Mrs Anson.

“It is dear, certainly, but not exaggeratedly so in comparison
with the other large hotels.”

“There seems of late to have been a gradual rise in prices

all along the Riviera,” remarked Hickman. “I’ve experienced
it personally. Ten or twelve years ago lived in Nice for the
season for about half what it costs me now.”

“That exactly bears out my argument,” exclaimed the

Irritating Woman, in triumph. “The fact is that the French
Riviera has become far too dear, and English people are,
fortunately for themselves, beginning to see that by
continuing their journey an extra twenty miles beyond Nice
they can obtain just as good accommodation, live better,
breathe purer air, and not be eternally worried by those
gaudy tinsel-shows called Carnivals, or insane attempts at
hilarity miscalled Battles of Flowers.”
“Oh, come, Miss Wells,” protested Mabel, “surely you won’t
condemn the Battles of Flowers at Nice! Why, they’re
acknowledged to be amongst the most picturesque
spectacles in the world!”

“I consider, my dear, that they are mere rubbishy ruses on

the part of the Niçois to cause people to buy their flowers
and throw them into the roadway. It’s only a trick to
improve their trade.”

We all laughed.

“And the Carnival?” inquired Hickman, much amused.

“Carnival!” she snorted. “A disgraceful exhibition of a town’s

lawlessness. A miserable pageant got up merely to attract
the unsuspecting foreigner into the web spread for him by
extortionate hotel-keepers. All the so-called fun is
performed by paid mountebanks; the cars are not only
inartistic, but there is always something extremely offensive
in their character, while the orgies which take place at the
masked balls at the Casino are absolutely disgraceful. The
whole thing is artificial, and deserves no support at all from
winter visitors.”

Mrs Anson, for once, did not agree with this sweeping
condemnation, while Mabel declared that she always
enjoyed the fun of the battles of flowers and paper confetti,
although she admitted that she had never had the courage
to go out on those days when the pellets of lime, or “harp
confetti,” are permitted. Both Hickman and myself
supported Mabel in defence of the annual fêtes at Nice as
being unique in all the world.

But the Irritating Woman was not to be convinced that her

opinions were either ill-formed or in the least distorted. She
had never been present at a Carnival ball, she admitted, but
it had been described to her by two estimable ladies who
had, and that was, for her, sufficient. They were a pair of
pious souls, and would, of course, never exaggerate to the
length of a lie.

Dinner over, the ladies retired, and Hickman and myself

were left to smoke and gossip. He was certainly a very ugly
man, and at times asserted an overbearing superiority in
conversation; but having watched him very closely, I at
length arrived at the conclusion that this was his natural
manner, and was not intended to be offensive. Indeed, ever
since that first moment when I had entered and been
introduced, he had shown himself to be very pleasant and
affable towards me.

“Poor Miss Wells!” he laughed, after the door had closed.

“She’s so infernally positive about everything. It would be
as good as an entertainment to induce her to expound her
views upon religious matters.”

“Any argument seems utterly useless,” I remarked.

“Do you know Nice well?” he inquired, after reflecting a

moment.

“I’ve spent three winters there,” I answered.

“And at Monte Carlo, I suppose?”

“Yes, of course,” I responded, laughing. “I suppose scarcely

any man goes to Nice without going over to Monty and
risking a few louis.”

“Were you lucky?”

“So, so. One season I won five thousand francs. In fact, I’ve
never lost on the whole season. I’ve always left the Riviera
with some of the bank’s money.”

“Then you can heartily congratulate yourself,” he said, “I’m

the reverse. I generally lose. Do you believe in any system
at roulette?”

“No; they are all frauds,” I answered promptly. “Except

one,” he interposed. “There’s one based on the law of
averages, which must turn up in your favour if you’re only
patient enough. The reason why it is so difficult is because
it’s such a long and tedious affair.”

“Explain it,” I urged, for a new system that was infallible

was, to me, of greatest interest. I had, in the days before
my blindness, made a study of the chances at roulette, and
had played carefully upon principles which had, to me,
appeared most natural. The result had been that with care I
had won—not much, it was true—but it was better than
leaving one’s money to swell the company’s dividends.

“The system,” he said, tossing off his glass of curaçoa at

one gulp, “is not at all a complicated one. If you study the
permanences of any table—you can get them from the
Gazette Rose—you’ll find that each day the largest number
of times either colour comes up in succession is nine. Now,
all you have to do is to go to a table at the opening of the
play, and taking one colour, red or black it makes no
difference, stake upon it, and allow your money to
accumulate until it is swept away. If the colour you stake
upon comes up eight times in succession, and you have
originally staked twenty francs, your gains lying on the table
will amount to two thousand five hundred and sixty francs.
Even then, don’t touch it. The colour must, in the law of
averages, come up nine times in succession each day,
taking the week through. If it comes up, you’ll win five
thousand and twenty francs for the louis you staked, and
then at once leave the table, for it will not come up nine
times again that day. Of course, this may occur almost at
the opening of the play, or not until the table is near
closing, therefore it requires great patience and constant
attendance. To-day it may not come up nine times, but it
will probably come up nine times on two occasions to-
morrow, and so the average always rights itself.”

His theory was certainly a novel one, and impressed me.

There might, I thought, be something in it. He had never
had patience to try it, he admitted, but he had gone
through a whole year’s “permanences,” and found that only
on three or four occasions had it failed.

For half an hour or so he sat lucidly explaining the results of

his studies of the game with the air of a practised gambler.
In these I became at once interested—as every man is who
believes he has found the secret of how to get the right side
of the bank; but we were at length compelled to put down
our cigars, and he led the way into the drawing-room,
where the ladies awaited us.

The room was a large, handsome one, elegantly furnished,

and lit by two great lamps, which shed a soft, subdued light
from beneath their huge shades of silk and lace. Mabel was
sitting at the open grand piano, the shaded candlelight
causing the beautiful diamond star in the coils of her dark-
brown hair to flash with a dazzling iridescence, and as I
entered she turned and gave me a sweet smile of welcome.

A second time I glanced around that spacious apartment,

then next instant stood breathless—transfixed.

I could not believe my own eyes. It seemed absolutely

incredible. Yet the truth was beyond all doubt.
In the disposition of the furniture, and in the general
appointments of that handsome salon, the home of the
woman I so dearly loved, I recognised the very room which
I had once explored with my keen sense of touch—the room
in which had been committed that ghastly, mysterious,
midnight crime!
 Chapter Fifteen.
What I Saw.

“How you men gossip!” Mabel exclaimed, tingling upon the

piano-stool, and laughing merrily.

“I wasn’t aware that we had been very long,” I answered,

sinking into a low armchair near her. “If so, I’m sure I
apologise. The fact is, that Mr Hickman was explaining a
new system of how to break the bank at Monte Carlo.”

“Oh, Mr Hickman!” she cried, turning at once to him. “Do

explain it, and I’ll try it when we go to the Riviera.”

“Mabel, my dear,” exclaimed her mother, scandalised, “you’ll

do nothing of the kind. You know I don’t approve of
gambling.”

“Oh, I think it’s awfully good fun,” her daughter declared.

“If you win,” I added.

“Of course,” she added; then, turning again to Hickman, she

induced him to explain his new and infallible system just as
he had explained it to me.

The trend of the conversation was, however, lost to me. My

ears were closed to all sound, and now that I reflect I am
surprised that I succeeded in retaining my self-possession. I
know I sat there rigid, as one held motionless in terror; I
only replied in monosyllables to any remark addressed to
me, and I knew instinctively that the colour had left my
countenance. The discovery was as bewildering as it was
unexpected.
Every detail of that handsome room was exactly as I had
pictured it. The blind, with their keen sense of touch, are
quick to form mental impressions of places and things, and
the general character of this apartment I had riveted upon
my mind with the fidelity of a photograph.

The furniture was of gilt, just as I had detected from its

smoothness, and covered with a rich brocade in wide stripes
of art green and dull red-brown—an extremely handsome
pattern; the carpet was dark, with a pile so thick that one’s
feet fell noiselessly; the three long windows, covered by
heavy curtains of brocade to match the furniture, reached
from the high-painted ceiling to the ground, exactly as I
had found them in my blind gropings. About the room were
two or three tables with glass tops, in trays beneath which
were collections of choice bric-à-brac, including some
wonderful Chinese carvings in ivory, while before the
fireplace was spread the great tiger-skin, with paws and
head preserved, which I so well remembered.

I sat there speechless, breathless. Not a single detail was

there wanting. Never before, in all my life, had amazement
held me so absolutely dumbfounded.

Close to where I saw was a spacious couch, over the centre

of which was thrown an antimacassar of silken crochet-
work. It was covered with the same brocade as the rest of
the furniture, and I stretched forth my hand, with feigned
carelessness and touched it. Its contact was the same, its
shape exact; its position in the room identical.

Upon that very couch I had reclined while the foul tragedy
had been enacted in that room. My head swam; I closed my
eyes. The great gilt clock, with its pendulum representing
the figure of a girl swinging beneath the trees, standing on
the mantelshelf, ticked out low and musically, just as it had
done on that fateful night. In an instant, as I sat with head
turned from my companions and my eyes shut, the whole of
that tragic scene was re-enacted. I heard the crash, the
woman’s scream, the awe-stricken exclamation that
followed in the inner room. I heard, too, the low swish of a
woman’s skirts, the heavy blow struck by an assassin’s
hand, and in horror felt the warm life-blood of the unknown
victim as it trickled upon my hand.

Mabel suddenly ran her white fingers over the keys, and the
music brought me back to a realisation of my true position.
I had at length discovered the actual house in which the
mysterious tragedy had been enacted, and it became
impressed upon me that by the exercise of greatest care I
might further be enabled to prosecute secret investigations
to a successful issue, and at length solve the enigma.

My eyes fixed themselves upon the couch. It was the very

spot where I had rested, sightless, helpless, while those
strange events had taken place about me. Was it any
wonder that I became filled with apprehensions, or that I
sat there petrified as one turned to stone?

The square, dark-green antimacassar had been placed in

the exact centre of the couch, and sewed down in order to
keep it in its place. Where I was sitting was fortunately in
the shadow, and when Mabel commenced playing I rose—
unsteadily I think—and reseated myself upon the couch, as
being more comfortable. Then, while the woman who held
me entranced played a selection from the “Trovatore,” I,
unnoticed by the others, succeeded in breaking the stitches
which tacked the antimacassar to the brocade. The feat was
a difficult one, for one does not care to be detected tearing
the furniture of one’s hostess. Nevertheless, after ten
minutes or so I succeeded in loosening it, and then, as if by
the natural movement of my body, commenced to work
aside.

The music ceased, and even though all my attention was

now centred upon my investigations, I congratulated Mabel
upon her accurate execution. Hickman was standing beside
her, and together they began to search for some piece he
had requested her to play, while Miss Wells, with her hearts
and elephants jingling, turned to me and commenced to
talk. By this I was, of course, interrupted; nevertheless,
some ten minutes later, I rose, and naturally turned back to
straighten the rumpled antimacassar. In doing so I
managed to lift it and glance beneath.

In an instant the truth was plain. Concealed beneath that

square of green crochet-work was a large dark-brown stain
upon the brocade. It was the mark of the life-blood of that
thin, well-dressed, unknown victim, who had, in an instant,
been struck to the heart!

The shock at its discovery caused me to start, but next

instant I smoothed out the antimacassar into its former
place without attracting any attention, and passed across
the room with the motive of inspecting an object which I
well remembered discovering when I had made my blind
search. Upon a pedestal of black marble stood an exquisite
little statuette of a Neapolitan dancing-woman, undoubtedly
the work of some Italian master. Without pausing to
examine it, I took in its every detail as I passed. It was
exactly as I had felt it, and in the self-same spot as on that
fatal night.

Beside the couch, as I turned again to look, I saw that a

large skin rug had been thrown down. Without doubt it had
been placed there to conceal the ugly stain of blood upon
the carpet.
And yet there, on the scene of one of the foulest and most
cowardly assassinations, we were actually spending the
evening quietly, as became a respectable household. The
thing seemed absolutely incredible. A dozen times I
endeavoured to persuade myself that the whole discovery
was but a chimera, arising from my disordered imagination.
Nevertheless, it was impossible to disguise from myself the
fact that in every detail the truth was borne out. In that
very room the unknown man had been struck dead. The
marks of his blood still remained as evidence of the truth.

I saw that beside the high lamps at that moment in use,

there was a magnificent candelabra suspended from the
ceiling, and in this were electric lamps. Then, at the door, I
noticed the switch, and knew that it was the same which I
had heard turned off by the assassin before leaving the
house.

At the end of the room, too, were the folding doors, now
concealed by curtains. It was through those very doors that
Edna, my mysterious protectress, had passed and repassed
to that inner room whence had come the sound of
champagne being uncorked and the woman’s piercing
scream.

Mabel leaned over and spoke to me, whereupon I sank

again into the chair I had previously occupied. She began to
chat, but although her beautiful eyes held me fixed, and her
face seemed more handsome than any I had ever seen, the
diamonds in her hair dazzled my eyes, and I fear that my
responses were scarcely intelligible.

“You are not quite yourself to-night, I think,” she remarked

at last, rising from the piano, and taking the low chair that I
drew up for her. “Are you unwell?”
“Why?” I asked, laughing.

“Because you look rather pale. What’s the matter?”

“Nothing,” I answered, as carelessly as I could. “A slight

headache. But it has passed now.”

My eyes wandered to those curtains of green plush. How I

longed to enter that room beyond!

At that moment she took out her handkerchief. Even that

action added to the completion of the mental picture I had
formed. Her tiny square of lawn and lace exhaled a sweet
odour. It was that of peau d’Espagne, the same subtle
perfume used by the mysterious Edna! It filled my nostrils
until I seemed intoxicated by its fragrance combined with
her beauty.

Her dress was discreetly decolleté, and as she sat chatting

to me with that bright vivaciousness which was so
charming, her white neck slowly heaved and fell. She had, it
seemed, been striving all the evening to get a tête-à-tête
chat with me, but the chatter of that dreadful Irritating
Woman and the requests made by Hickman had prevented
her.

As she gossiped with me, now and then waving her big
feather fan, she conveyed to my mind an impression of
extreme simplicity in the midst of the most wonderful
complexity. She seemed to take the peculiar traits from
many characters, and so mingle them that, like the
combination of hues in a sunbeam, the effect was as one to
the eye. I had studied her carefully each time we had met,
and had found that she had something of the romantic
enthusiasm of a Juliet, of the truth and constancy of a
Helen, of the dignified purity of an Isabel, of the tender
sweetness of a Viola, of the self-possession and intellect of
a Portia—combined together so equally and so
harmoniously that I could scarcely say that one quality
predominated over the other. Her dignity was imposing, and
stood rather upon the defensive; her submission, though
unbounded, was not passive, and thus she stood wholly
distinct in her sweetness from any woman I had ever met.

The following day was one on which she was due to take
her music-lesson, and I inquired whether I might, as usual,
meet her and escort her across the Park.

“You are really very kind,” she responded; “but I fear I take
up far too much of your time.”

“Not at all,” I hastened to assure her. “I always enjoy our

walks together.”

She smiled, but a moment later said—

“I fear that I shall be prevented from going to Hanover

Square to-morrow, as I shall be making calls with mother.
We’ve been neglecting to call of late, and have such a host
to make.”

“Then I shan’t see you at all to-morrow?” I said in deep

disappointment.

“No, I fear not,” she answered. “As a matter of fact, my

movements for the next few days are rather uncertain.”

“But you’ll write and tell me when you are free?” I urged
earnestly.

“If you wish,” she responded, smiling sweetly. Apparently

she was in no wise averse to my companionship, a fact
which had become to me more apparent now that she had
induced her mother to invite me to their table.
I endeavoured to extract from her some appointment, but
she only whispered—

“Remember that our meetings are clandestine. Don’t let

them overhear us. Let’s change the subject.” And then she
began to discuss several of the latest novels.

She had apparently a wide knowledge of French fiction, for

she explained how a friend of hers, an old schoolfellow, who
had married a French baron and lived in Paris, sent her
regularly all the notable novels. Of English fiction, too, she
was evidently a constant reader, for she told me much
about recent novels that I was unaware of, and criticised
style in a manner which betrayed a deep knowledge of her
subject.

“One would almost think you were a lady novelist, or a

book-reviewer,” I remarked, in response to a sweeping
condemnation which she made regarding the style of a
much-belauded writer.

“Well, personally, I like books with some grit in them,” she

declared. “I can’t stand either the so-called problem novel,
or a story interlarded with dialect. If any one wants nasty
problems, let them spend a few shillings in the works of
certain French writers, who turn out books on the most
unwholesome themes they can imagine, and fondly believe
themselves realists. We don’t want these queue-de-siècle
works in England. Let us stick to the old-fashioned story of
love, adventure, or romance. English writers are now
beginning to see the mistake they once made in trying to
follow the French style, and are turning to the real
legitimate novel of action—the one that interests and grips
from the first page to the last.”
She spoke sensibly, and I expressed my entire accord with
her opinion. But this discussion was only in order to hide
our exchange of confidences uttered in an undertone while
Hickman and the two ladies were chatting at the further end
of the room.

All the time I was longing to get a sight of the interior of the
adjoining apartment, the room whence had burst forth that
woman’s agonised cry in the stillness of the night. I racked
my brain to find some means of entering there, but could
devise none. A guest can hardly wander over his hostess’s
house on the first occasion he receives an invitation.
Besides, to betray any interest in the house might, I
reflected, arouse some suspicion. To be successful in these
inquiries would necessitate the most extreme caution.

The fragrant odour of peau d’Espagne exhaled by her

chiffons seemed to hold me powerless.

The gilt clock with its swinging girl had already struck
eleven on its silver bell, and been re-echoed by another
clock in the hall playing the Westminster chimes, when
suddenly Mrs Anson, with a book in her hand, looked across
to her daughter, saying—

“Mabel, dear, I’ve left my glasses on the table in the library.

Will you kindly fetch them for me?”

In an instant I saw my chance, and, jumping to my feet,

offered to obtain them. At first she objected, but finding me
determined, said—

“The library is the next room, there. You’ll find them on the
writing-table. Mother always leaves them there. It’s really
too bad to thus make a servant of you. I’ll ring for Arnold.”
“No, no,” I protested, and at once went eagerly in search of
them.
 Chapter Sixteen.
The Inner Room.

The adjoining room was, I found, in the front part of the

house—a rather small one, lined on one side with books,
but furnished more as a boudoir than a library, for there
were several easy-chairs, a work-table, and a piano in a
corner. At this instrument the mysterious player had on that
night sat executing Chopin’s “Andante-Spinato” the moment
before it became interrupted by some tragic and
unexpected spectacle. I glanced around and noted that the
furniture and carpet were worn and faded, that the books
were dusty and evidently unused, and that the whole place
presented an air of neglect, and had nothing whatever in
keeping with the gorgeousness of the other handsome
apartments.

The glasses were, as Mrs Anson had said, lying beside the
blotting-pad upon a small rosewood writing-table. I took
them up, and, having made a tour of inspection, was about
to leave the place, when suddenly, on the top of some
books upon a shelf close to the door, I espied a small
volume.

The curious incident of the birthday book occurred to me;

therefore I took down the little volume and found that it
really was a birthday book. No name was inscribed on the
title-page as owner, but there were many names scribbled
therein. In swift eagerness I turned to the page of my own
birthday—the 2nd of July. It was blank.

I stood pondering with the book still in my hand. The

absence of my name there proved one or two things, either
I had not signed a birthday book at all, or, if I had, it was
not the one I had discovered. Now, there are frequently two
birthday-books in one house, therefore I resolved, ere I
gave the matter reflection, to prosecute my investigations
further and ascertain whether there was not a second book.

With this object I made a second tour around the room,

noting the position of every article of furniture. Some music
lay scattered beside the piano, and, on turning it over, I
found the actual copy of Chopin’s “Andante” which had been
played on the night of the tragedy. The cover had been half
torn away, but, on examining it closely beneath the light, I
detected plainly a small smear of blood upon it.

Truly the house was one of mystery. In that room several

persons had drunk champagne on that memorable night
when blind Fate led me thither; in that room a woman had,
according to the man’s shout of alarm, been foully done to
death, although of this latter fact I was not altogether sure.
At any rate, however, it was plain that some tragic event
had previously taken place there, as well as in that room
beyond where I had reclined blind and helpless. It was
strange also that the apartment should remain neglected
and undusted, as though the occupants entertained some
dislike to it. But I had been absent long enough, and,
returning to the drawing-room with the missing glasses,
handed them to Mrs Anson.

Hickman had, in my absence, crossed to Mabel, and was

sitting beside her in earnest conversation, therefore I was
compelled to seat myself with my hostess and the Irritating
Woman and chat with them. But ere long I contrived again
to reach the side of the woman whom I adored, and to
again press her for an appointment.
“It is far better forme to write to you,” she answered,
beneath her breath. “As I’ve told you, we have so many
calls to make and cards to leave.”

“Your mother tells me that you have a box for the Prince of
Wales’s on Saturday night, and has asked me to join you,” I
said. Her eyes brightened, and I saw that she was delighted
at the prospect. But she expressed a hope that I wouldn’t
be bored.

“Bored!” I echoed. “Why, I’m never bored when in your

company. I fear that it’s the other way about—that I bore
you.”

“Certainly not,” she responded decisively. “I very soon

contrive to give persons who are bores their congé. Mother
accuses me of rudeness to them sometimes, but I assure
you I really can’t help being positively insulting. Has mother
asked you to dine on Saturday?”

“Yes,” I answered. “But shan’t I see you before then?”

“No; I think it is very unlikely. We’ll have a jolly evening on

Saturday.”

“But I enjoy immensely those walks across the Park,” I

blurted forth in desperation.

“And I also,” she admitted with a sweet frankness. “But this

week it is utterly impossible to make any arrangements.”

Mention of the theatre afforded me an opportunity of

putting to her a question upon which, during the past
couple of hours, I had reflected deeply.

“You’ve, of course, been to the Exhibition at Earl’s Court,

living here in the immediate vicinity,” I said.
“I’ve only been once,” she answered. “Although we’ve had
this house nearly two years, exhibitions don’t appeal to me
very much. I was there at night, and the gardens were
prettily illuminated, I thought.”

“Yes,” I said. “With the exception of the gardens, there is far

too much pasteboard scenic effect. I suppose you noticed
that serrated line of mountains over which the eternal
switchback runs? Those self-same mountains, repainted
blue, grey, or purple, with tips of snow, have, within my
personal knowledge, done duty as the Alps, the Pyrenees,
the Rockies, and the Atlas, not counting half a dozen other
notable ranges.”

She laughed, slowly fanning herself the while.

By her reply I had obtained from her own lips a most

important fact in the inquiry I intended now to prosecute,
namely, that this house had been her home for nearly two
years. Therefore it had been in Mrs Anson’s possession at
the time of the tragedy.

Since the moment when I had first recognised that; room

as the one in which I had been present on the night of the
mysterious assassination, the possibility had more than
once occurred to me that Mrs Anson might have;
unwittingly taken it ready furnished after the committal of
the crime. Such, however, was not the fact. Mabel had
asserted that for nearly two years she had lived, there.

Again, even as I sat there at her side, deep in admiration of

her magnificent figure in that striking toilette of coral-pink,
with its soft garniture of lace and chiffons, I could not help
reflecting upon the curious fact that she should have
recognised the dead man’s pencil-case. And she had, by her
silence, assented to my suggestion that he had been her
lover. That little gold pencil-case that I had found in his
pocket when he lay dead at that very spot where we were
now sitting had been one of her love-gifts to him.

The mystery hourly grew more puzzling and bewildering.

Yet so also each hour that I was at her side I fell deeper and
deeper in love with her, longing always for opportunity to
declare to her the secret of my heart, yet ever fearing to do
so lest she should turn from me.

Our unexpected meeting at Grosvenor Gate, after I had

received that letter from my anonymous correspondent,
combined with the startling discovery that it was actually in
her house that the mysterious tragedy had been enacted;
that in that very room the smart, refined young man who
had been her lover had fought so fiercely for life, and had
yet been struck down so unerringly, formed an enigma
inscrutable and perplexing.

The mystery, however, did not for one moment cause me to

waver in my affection for her. I had grown to love her fondly
and devotedly; to adore her as my idol, as the one who held
my whole future in her hands, therefore whatever suspicion
arose within my mind—and I admit that grave suspicion did
arise on many occasions—I cast it aside and fell down to
worship at the shrine of her incomparable beauty.

Miss Wells’s carriage was announced at last, and the

Irritating Woman, tinkling and jingling, rose with a wearied
sigh and took her leave, expressing her thanks for “a most
delightful evening, my dear.”

Mabel, mischievous as a school-girl, pulled a grimace when

the music of the bangles had faded in the hall outside, at
which we laughed in merry chorus.
With Hickman I remained ten minutes or so longer, then
rose, also declaring that it was time we left. The grave man-
servant Arnold served us with whiskies and sodas in the
dining-room, and, Mabel having helped me on with my
covert-coat, we shook hands with our hostess and her
daughter, and left in company.

The night was bright and starlit, and the air refreshing.
Turning to the left after leaving the house, we came
immediately to a road which gave entrance to that secluded
oval called The Boltons. I looked at the name-plate, and
saw it was named Gilston Road. It must have been at this
corner that I had been knocked down by a passing cab
when, on my first adventurous journey alone, I had
wandered so far westward.

I turned to look back, and noticed that from the dining-

room window of the house we had just left any occurrence
at the corner in question could be distinctly seen. Edna had
explained that she had witnessed my accident from that
window, and in this particular had apparently told me the
truth.

The remarkable and unexpected discoveries of that evening

had produced a veritable tumult of thoughts within my
brain, and as I walked with Hickman I took no note of his
merry, irresponsible gossip, until he remarked—

“You’re a bit preoccupied, I think. You’re pondering over

Mabel’s good looks, I suppose?”

“No,” I answered, starting at this remark. Then, to excuse

myself, I added, “I was thinking of other things. I really beg
your pardon.”

“I was asking your opinion of Mabel. Don’t you consider her

extremely handsome?”
“Of course,” I answered, trying to suppress my enthusiasm.
“She’s charming.”

“A splendid pianist, too.”

“Excellent.”

“It has always been a wonder to me that she has never

become engaged,” he remarked. “A girl with her personal
charms ought to make an excellent match.”

“Has she never been engaged?” I inquired quickly, eager to

learn the truth about her from this man, who was evidently
an old friend of the family.

“Never actually engaged. There have been one or two little

love-affairs, I’ve heard, but none of them was really
serious.”

“He’d be a lucky fellow who married her,” I remarked, still

striving to conceal the intense interest I felt.

“Lucky!” he echoed. “I should rather think so, in many

ways. It is impossible for a girl of her beauty and nobility of
character to go about without lots of fellows falling in love
with her. Yet I happen to know that she holds them all aloof,
without even a flirtation.”

I smiled at this assertion of his, and congratulated myself

that I was the only exception; for had she not expressed
pleasure at my companionship on her walks? But
recollecting her admission that the victim of the assassin’s
knife had been her lover, I returned to the subject, in order
to learn further facts.

“Who were the men with whom she had the minor love-
affairs—any one I know?” I inquired.
“I think not, because it all occurred before they returned to
live in England,” he answered.

“Then you knew them abroad?”

“Slightly. We met in a casual sort of way at Pau, on the

Riviera, and elsewhere.”

“Both mother and daughter are alike extremely pleasant,” I

said. “In high spirits Mrs Anson is sometimes almost as
juvenile as Mabel.”

“Quite so,” he laughed. “One would never believe that she’s

nearly sixty. She’s as vivacious and merry as a woman half
her age. I’ve myself been surprised at her sprightliness
often and often.”

Again and again I endeavoured to turn the conversation

back to the identity of Mabel’s former lover, but he either
did not know or purposely refused to tell me. He spoke now
and then with an intentional vagueness, as though his
loyalty to the Ansons prevented him from betraying any
confidences reposed in him as a friend of the family.
Indeed, this cautiousness showed him to be a trustworthy
man, and his character became thereby strengthened in my
estimation. On first acquaintance I had instantly
experienced a violent aversion to him, but now, on this walk
together along the Fulham Road, I felt that we should
probably end by becoming friends.

He walked with long strides and a swinging, easy gait that

seemed almost military, while his air of careless merriment
as he laughed and joked, smoking the choice cigar which
the man had handed to him in the hall just before our
departure, gave him the aspect of an easy-going man-
about-town.
“I fully expect, my dear fellow,” he laughed—“I fully expect
that you’ll be falling in love with the pretty Mabel if you’re in
her company very much.”

“You’re chaffing,” I protested, echoing his laugh.

“Not at all,” he asserted. “Only take care. Love-making with

her is a dangerous pastime—devilish dangerous, I assure
you.”

“Dangerous to the man’s heart—eh?”

“Yes,” he responded in a vague tone, glancing at me

curiously; “if you like to put it in that way.”

We had passed from the Fulham Road into the King’s Road,
Chelsea, and at that moment he halted suddenly at the
corner of a street of high, regularly built houses, most of
which were in darkness, saying—“I live down here. Come in
and have a final whisky and soda with me; then you can
take a cab back to the Strand. There are cabs all night on
the rank in Sloane Square.”

“I fear it’s too late,” I protested, glancing at my watch, and

finding it past one o’clock.

“No, no, my dear fellow, come along,” he urged. “You’ll want

a drink before you get home;” and, thus persuaded, I
accompanied him up the street to one of the high houses,
each exactly similar to its neighbour, with a flight of
hearthstoned steps leading up to its front door, and a deep,
grimy basement protected by a few yards of iron railings.

In the hall, although the gas had been extinguished, there

remained a small hand-lamp alight, evidently placed there
for his use. This he took, and conducted me to a front
room, upon what the landlady of such a residence would
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