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PROGRESS IN
Molecular Biology
and Translational Science
Volume 95
This page intentionally left blank
PROGRESS IN
Molecular Biology and

Translational Science
Molecular Biology
of Cancer:Translation
to the Clinic
edited by
Raymond W. Ruddon, M.D., Ph.D.

Department of Pharmacology,
University of Michigan Medical School,
Ann Arbor, Michigan, USA
Volume 95
AMSTERDAM • BOSTON • HEIDELBERG • LONDON

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10 11 12 13 10 9 8 7 6 5 4 3 2 1
Contents
Contributors..................................................................................... ix
Preface............................................................................................. xi
Introduction to the Molecular Biology of Cancer:

Translation to the Clinic . . . . . . . . . . . . . . . . . . . . . . . . 1
Raymond W. Ruddon
Molecular Biology and Anticancer Drug Discovery. . . . . 9

John S. Lazo
I. Introduction ................................................................................ 9
II. Phenotypic Targets........................................................................ 12
III. Molecular Targets ......................................................................... 16
IV. Other Contemporary Issues in Anticancer Drug Discovery ................... 26
V. Conclusions ................................................................................. 27
References .................................................................................. 27
Targeting Chemokine (C-C motif) Ligand 2 (CCL2) as an

Example of Translation of Cancer Molecular Biology
to the Clinic . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
Jian Zhang, Lalit Patel, and Kenneth J. Pienta
I. Biology of CCL2........................................................................... 32
II. CCL2 in Prostate Cancer ............................................................... 37
III. CCL2 Development as a Therapeutic Target...................................... 41
IV. Conflicting Reports on the Roles of CCL2 in Cancer........................... 43
V. Conclusions ................................................................................. 43
References .................................................................................. 45
v
vi contents
Chromosomal Aberrations in Solid Tumors . . . . . . . . . . 55
Arul M. Chinnaiyan and Nallasivam Palanisamy
I. Introduction .............................................................................. 56
II. Historical Background: Discovery of Chromosome Aberrations
in Cancer .................................................................................. 57
III. Discovery of Gene Fusions in Cancer............................................. 58
IV. New Approaches for Gene Fusion Identification .............................. 61
V. Methods for the Characterization of Chromosome Aberrations
in Solid Tumors.......................................................................... 70
VI. Next-Generation Sequencing Technology........................................ 79
VII. Structural Classification of Gene Fusions ........................................ 82
VIII. Functional Classification of Gene Fusions ....................................... 83
IX. Mechanism of the Formation of Gene Fusions in Cancer................... 84
References ................................................................................ 86
Circulating Tumor Cells . . . . . . . . . . . . . . . . . . . . . . . 95

Daniel F. Hayes and Jeffrey B. Smerage
I. Introduction ................................................................................ 96
II. What Are the Technological Issues? ................................................. 96
III. What Are the Clinical Utilities of CTCs Detection and Enumeration? .... 100
IV. CTC Characterization ................................................................... 106
V. Summary .................................................................................... 107
References .................................................................................. 108
Stem Cells in Normal Development and Cancer . . . . . . 113

Rosemarie Chirco D’Angelo and Max S. Wicha
I. Introduction of Cancer Stem Cells and the Cancer Stem
Cell Hypothesis ........................................................................... 114
II. Comparison of Normal Stem Cells with Cancer Stem Cells ................. 115
III. Definition of Cancer Stem Cells and Identification of Cancer Stem
Cell Markers............................................................................... 117
IV. Identification of Cancer Stem Cells ................................................. 123
V. Activation of Signaling Pathways and Targeted Therapies for Cancer
Stem Cells.................................................................................. 129
VI. Therapeutic Implications for Targeting Cancer Stem Cells .................. 143
VII. Conclusions ................................................................................ 144
References ................................................................................. 145
contents vii
Bioinformatics and Systems Biology of Cancers . . . . . . . 159
Gilbert S. Omenn
I. Introduction .............................................................................. 160
II. The Cancer Biomedical Informatics Grid (caBIG) ............................ 162
III. TCGA: The Cancer Genome Anatomy Project ................................. 165
IV. Alternative Splicing: Discovery of a New Class of Protein Cancer
Biomarker Candidates ................................................................. 172
V. Concepts Tools for Systems Biology Analysis .................................... 181
VI. Determining the Activity of All 21,000 Protein-Coding Genes in the
Human Genome......................................................................... 183
VII. Bioinformatics and Systems Biology of Metastasis: The Case of
Lung Cancers ............................................................................ 184
VIII. Special Resources for Pharmacogenomics of Cancer Therapies ........... 185
IX. Conclusion ................................................................................ 187
References ................................................................................ 188
Progress in Cancer Nanotechnology. . . . . . . . . . . . . . . 193

Istvan J. Majoros, Brent B. Ward, Kyung-Hoon Lee,
Seok Ki Choi, Baohua Huang, Andrzej Myc, and
James R. Baker
I. Introduction and Historical Perspective .......................................... 194
II. Targeted Therapy........................................................................ 195
III. Computer Simulations as an Approach to Develop Nanotechnology
in Cancer .................................................................................. 196
IV. Nanomolecular Carriers for Drugs and Imaging Agents ..................... 200
V. Nanotechnology in Cancer-Targeted Delivery of Therapeutic Agents .... 207
VI. Targeted Imaging........................................................................ 216
VII. Apoptosis Sensors ....................................................................... 219
VIII. Future Direction in Research and Technology.................................. 227
References ................................................................................ 228
Applications of Molecular Imaging . . . . . . . . . . . . . . . . 237

Craig J. Galbán, Stefanie Galbán, Marcian E. Van Dort,
Gary D. Luker, Mahaveer S. Bhojani, Alnawaz Rehemtulla,
and Brian D. Ross
I. Optical Imaging............................................................................ 238
II. Magnetic Resonance Imaging.......................................................... 257
III. Nuclear Imaging........................................................................... 271
References .................................................................................. 286
viii contents
Cancer Epigenetics . . . . . . . . . . . . . . . . . . . . . . . . . . 299
Wendell Weber
I. Introduction ................................................................................ 299
II. First, a Little History..................................................................... 300
III. Epigenetic Patterns in Normal Cells ................................................ 302
IV. Epigenetic Patterns in Cancer......................................................... 326
V. Epigenetic Therapies for Cancer ..................................................... 337
VI. Prospects for the Future of Cancer Epigenetics ................................. 342
References .................................................................................. 344
Molecular Targets and Clinical Cancer Risk Reductive

Interventions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 351
Madhuri Kakarala and Dean E. Brenner
I. Defining Cancer Risk Reductive Intervention (Chemoprevention) ....... 351
II. Cellular Transformational Molecular Events as Targets for CRRIs ....... 352
III. Inherited Genetic Mutations (Cancer Susceptibility Syndromes) ......... 352
IV. Special Features of CRRI Development ......................................... 355
V. Molecular Intermediates as Biomarkers for Cancer Risk
Reductive Efficacy ..................................................................... 356
VI. Future Approaches to Molecular Biomarker Applications to CRRI ...... 358
VII. Standards for Biomarkers as Endpoints for Cancer Risk
Reductive Efficacy ..................................................................... 358
VIII. Examples of CRRIs and Their Molecular Targets ............................. 358
IX. Nutritional Products ................................................................... 363
X. Multiagent CRRIs ...................................................................... 364
XI. Molecular Viral Targets for Cancer Risk Reduction ........................... 364
References ................................................................................ 366
Index ....................................................................................... 377
Contributors
Numbers in parentheses indicate the pages on which the authors’ contributions begin.
James R. Baker, Michigan Nanotechnology Institute for Medicine and

Biological Sciences, University of Michigan, Ann Arbor, Michigan, USA (193)
Mahaveer S. Bhojani, Department of Radiology, University of Michigan,
Center for Molecular Imaging, Ann Arbor, Michigan, USA (237)
Dean E. Brenner, University of Michigan Medical Center and VA Medical
Center, Ann Arbor, Michigan, USA (351)
Arul M. Chinnaiyan, Michigan Center for Translational Pathology, and
Department of Pathology, and Howard Hughes Medical Institute, Maryland,
and Department of Urology; and Comprehensive Cancer Center, University
of Michigan, Ann Arbor, Michigan, USA (55)
Seok Ki Choi, Michigan Nanotechnology Institute for Medicine and
Biological Sciences, University of Michigan, Ann Arbor, Michigan, USA
(193)
Rosemarie Chirco D’Angelo, Department of Internal Medicine, Division of
Hematology and Oncology, University of Michigan Comprehensive Cancer
Center, University of Michigan, Ann Arbor, Michigan, USA (113)
Craig J. Galbán, Department of Radiology, University of Michigan, Center
for Molecular Imaging, Ann Arbor, Michigan, USA (237)
Stefanie Galbán, Department of Radiation Oncology, University of Michigan,
Daniel F. Hayes, Breast Oncology Program, University of Michigan
Comprehensive Cancer Center, Ann Arbor, Michigan, USA (95)
Baohua Huang, Michigan Nanotechnology Institute for Medicine and
(193)
Madhuri Kakarala, University of Michigan Medical Center and VA Medical
Center, Ann Arbor, Michigan, USA (351)
John S. Lazo, Department of Pharmacology and Chemical Biology, University
of Pittsburgh Drug Discovery Institute and Cancer Institute, University of
Pittsburgh, Pittsburgh, Pennsylvania, USA (9)
Kyung-Hoon Lee, Michigan Nanotechnology Institute for Medicine and
Biological Sciences and Department of Chemistry, University of Michigan,
Ann Arbor, Michigan, USA (193)
Gary D. Luker, Department of Radiology, University of Michigan, Center for
Molecular Imaging, Ann Arbor, Michigan, USA (237)
ix
x contributors
Istvan J. Majoros, Michigan Nanotechnology Institute for Medicine and

Biological Sciences, University of Michigan, Ann Arbor, Michigan, USA (193)
Andrzej Myc, Michigan Nanotechnology Institute for Medicine and
(193)
Gilbert S. Omenn, Department of Internal Medicine, Department of Human
Genetics, School of Public Health, Center for Computational Medicine and
Bioinformatics, University of Michigan, Ann Arbor, Michigan, USA (159)
Nallasivam Palanisamy, Michigan Center for Translational Pathology, and
Department of Pathology; and Comprehensive Cancer Center, University of
Michigan, Ann Arbor, Michigan, USA (55)
Lalit Patel, Department of Medicine; and Department of Urology, Michigan
Center for Translational Pathology and the University of Michigan Comprehen-
sive Cancer Center, University of Michigan, Ann Arbor, Michigan, USA (31)
Kenneth J. Pienta, Department of Medicine; and Department of Urology,
Michigan Center for Translational Pathology and the University of Michigan
Comprehensive Cancer Center, University of Michigan, Ann Arbor,
Michigan, USA (31)
Alnawaz Rehemtulla, Department of Radiation Oncology, University of
Michigan, Center for Molecular Imaging, Ann Arbor, Michigan, USA (237)
Brian D. Ross, Department of Radiology, University of Michigan, Center for
Molecular Imaging, Ann Arbor, Michigan, USA (237)
Raymond W. Ruddon, Department of Pharmacology, University of Michigan
Medical School, Ann Arbor, Michigan, USA (1)
Jeffrey B. Smerage, Breast Oncology Program, University of Michigan
Comprehensive Cancer Center, Ann Arbor, Michigan, USA (95)
Marcian E. Van Dort, Department of Radiology, University of Michigan,
Brent B. Ward, Oral and Maxillofacial Surgery, University of Michigan
Hospitals, Ann Arbor, Michigan, USA (193)
Wendell Weber, Department of Pharmacology, University of Michigan
Medical School, Ann Arbor, Michigan, USA (299)
Max S. Wicha, Department of Internal Medicine, Division of Hematology and
Oncology, University of Michigan Comprehensive Cancer Center, University
of Michigan, Ann Arbor, Michigan, USA (113)
Jian Zhang, Department of Medicine; and Department of Urology, Michigan
Center for Translational Pathology and the University of Michigan Comprehen-
sive Cancer Center, University of Michigan, Ann Arbor, Michigan, USA (31)
Preface
The purpose of this volume in the Progress in Molecular Biology and Transla-
tional Science series is to explore some of the most exciting recent advances in
basic research on the molecular biology of cancer and how this knowledge leads
to advances in the diagnosis, treatment, and prevention of cancer.
The chapter topics include introduction to the molecular biology of cancer
(Ruddon), molecularly targeted approaches to the development of anticancer
drugs (Lazo), targeting chemokine ligands and their role in cancer metastasis
(Pienta), discovery of cancer cell fusion genes in solid cancers (Chinnaiyan),
role of circulating tumor cells in cancer diagnosis, disease progression and
response to therapy (Hayes), cancer stem cells (WIcha), bioinformatics and
systems biology of cancers(Omenn), progress in cancer nanotechnology
(Baker), molecular imaging (Ross), cancer epigenetics (Weber), and cancer
prevention (Brenner).
The senior investigators represented here are all University of Michigan
faculty, with the exception of John Lazo, from the University of Pittsburgh, but
even he has a U of M genealogy, having received his Ph.D. in Pharmacology
from the Ruddon lab at U of M. However, all of these scientists are leaders in
their fields of research. Thus, this is not just a parochial concatenation of
narrowly focused local research, but it is, in fact, representative of the most
advanced research in the fields discussed here.
R. W. RUDDON, ANN ARBOR
xi
Introduction to the Molecular
Biology of Cancer: Translation
to the Clinic
Raymond W. Ruddon, M.D., Ph.D.
Department of Pharmacology, University of
Michigan Medical School, Ann Arbor,
Michigan, USA
Advances in molecular biology over the last several decades are being steadily
applied to our understanding of the molecular biology of cancer, and these
advances in knowledge are being translated into the clinical practice of oncology.
Many examples can be cited to demonstrate this. Some of them are listed
below. Everyone has their favorite list, of course; however, everyone would
probably agree that the items on this list should be included in any such list of
advances.1
1. Techniques of modern molecular biology: These include PCR, DNA

microarrays, proteomics, molecular imaging, identification of cancer
stem cells, and the analysis of DNA methylation in cancer epigenetics.
Other advances are in methods for metabolomic measurements, nano-
technology, systems biology, cancer immunology and monoclonal anti-
body production, and bioinformatics. Many of these advances will be
discussed in this volume.
2. Cancer susceptibility genes: In the early 1900s, it was known that familial
clustering of some cancers occurred, for example, with colon cancer and
breast cancer, but the genes involved in this were not known. The APC,
BRCA-1, BRCA-2, and p53-inherited mutations, for example, were not
known until more recently. Research in this area has identified a number
of genes involved in cancer susceptibility, and with modern cloning
techniques, more are identified every few months.
3. Genes involved in cancer initiation and promotion: It has been known
for a long time that chemicals and irradiation could damage DNA and
initiate cancer in animals and humans, but what genes were altered was
almost completely unknown until the advances in molecular biology
were applied. We now know a lot about what genes are involved at
various stages of a number of cancers. For example, the work of Bert
Vogelstein and his colleagues have defined a pathway, sometimes called
the ‘‘Vogelgram,’’ for the progression of colon cancer.2 We knew that
Progress in Molecular Biology Copyright 2010, Elsevier Inc.

and Translational Science, Vol. 95 1 All rights reserved.
DOI: 10.1016/S1877-1173(10)95001-1 1877-1173/10 $35.00
2 RAYMOND W. RUDDON
DNA repair was important and that heritable conditions of defective

DNA repair (e.g., xeroderma pigmentosum) could lead to cancer, but
the ideas about the mechanisms of DNA repair were primitive until
fairly recently.3
4. The identification of oncogenes: This did not really take off until the early
1980s. The src gene was identified in 1976 by Stehelin et al. and erb,
myc, and myb oncogenes in the late 1970s, but this was about the limit of
our knowledge (reviewed in Ref. 4).
5. Tumor suppressor genes: The term ‘‘tumor suppressor gene’’ was not
even coined until the early 1980s, although their existence had been
implied from the cell fusion experiments of Henry Harris, who showed
that if you fused a normal cell with a malignant cell, the phenotype was
usually nonmalignant (reviewed in Ref. 5). The RB gene was the first
one cloned, in 1986 by Friend et al.6 P53 was originally thought of as an
oncogene. It was not realized until 1989 that wild-type p53 could
actually suppress malignant transformation. A number of tumor
suppressor genes have, of course, been identified since.
6. Cell cycle checkpoints: These were identified in yeast starting in the
1970s by Lee Hartwell and colleagues, but the identification of many of
the human homologs of these genes did not occur until the late 1980s.
7. Tumor immunology: The mechanism of the immune response and the
ability to manipulate it with cytokines, activated dendritic cells, vaccines,
and drugs was not in the treatment armamentarium until recently.
Advances in identification of tumor antigens and in the techniques to
produce monoclonal antibodies are now leading to newer treatment
modalities.
8. The viral etiology of cancer: This was still being widely debated in the
1980s. The involvement of Epstein–Barr virus in Burkitt’s lymphoma
and of hepatitis B virus in liver cancer was becoming accepted, but the
role of viruses in these diseases and in cervical cancer, Kaposis’ sarcoma,
and in certain T-cell lymphomas became clearer much later.
9. Growth factors that affect cancer: Even though growth factors that
stimulate cell replication, such as IGF-1 and 2, FGF, NGF, PDGF,
and EGF, have been known for a long time, knowledge about their
receptors and signal transduction mechanisms have been greatly
expanded. Importantly, it is now known that the signal transduction
mechanisms that cancer cells use are overlapping and redundant.
Thus, cancer cells can become resistant to anticancer drugs by
upregulating alternate pathways.
INTRODUCTION TO THE MOLECULAR BIOLOGY OF CANCER 3
The explosion of knowledge about signal transduction mechanisms and

how these pathways interact have also been a tremendous boon to our
understanding of how cells respond to signals in their microenvironment and
communicate with one another.
10. Regulation of gene expression: Current information on the packaging of

chromatin, transcription factors, coinducers and corepressors, inhibi-
tory RNA (siRNA), and micro RNA is expanding our knowledge of how
gene expression is regulated in cancer.
Several decades of advances in cancer cell molecular biology have led to a

rich pipeline of anticancer agents aimed at membrane-bound receptor protein
kinases, intracellular signaling kinases, epigenetic abnormalities, as well as to
agents that affect protein folding and degradation, tumor vasculature, and the
tumor cell microenvironment (reviewed in Ref. 7).
The history of chemotherapy has seen its ups and downs since the intro-
duction of nitrogen mustard after World War II.8 The focus until recently has
been almost exclusively on toxic chemicals that somewhat nonselectively kill
dividing cells. Now, however, there is a large focus on what has been called
‘‘molecularly targeted agents.’’ Two successful examples are trastuzumab
(Herceptin) and imatinib (Gleevec). The number of successes for this approach
to date is small, however, and the success of imatinib in treatment of chronic
myelogenous leukemia (CML) may be the exception rather than the rule,
arguing for the need to continue to search for agents that are toxic to dividing
cells (albeit, hopefully, more selectively toxic ones).9
The other school of thought argues that targeted therapies can be devel-
oped against specific targets that are selectively overexpressed or mutated in
cancer cells and thus be less toxic to normal tissues.10 To this end, a detailed
understanding of the target’s structure and function will be necessary to
identify a targeted drug’s mechanism of action and the mechanisms of
resistance development to the drug. In addition, the use of cytotoxic drugs in
combination with targeted drugs will most likely continue to be the most
efficacious approach to treating cancer.
In Chapter 2 of this volume, John Lazo expands on these concepts.
Of course, all successful approaches to new cancer treatment depend on
the adaptation of molecular findings into the clinic, so-called translational
research. In Chapter 3, Ken Pienta et al. describe a beautiful example of how
this is done. They have shown how the monocyte chemoattractant protein
MCP-1 (CCL2) is involved in the lethal phenotype of prostate cancer cells,
that it is increased in prostate cancer bone metastases, and that it is a
4 RAYMOND W. RUDDON
pro-survival and pro-angiogenic factor that leads to metastasis.11 They have

also shown that monoclonal antibody targeted to CCL-2 inhibits bone
metastasis and increases survival.
In Chapter 4, Nallasivam Palanisamy and Arul Chinnaiyan discuss the
recent discovery of gene fusions in ‘‘solid cancers’’ such as prostate, breast,
and lung cancers. Up until this research, it was generally believed that gene
fusion events occurred typically in hematologic malignancies and rare bone and
soft-tissue tumors, but not in tumors such as prostate, lung, breast, and colon.
Novel gene fusions were discovered utilizing integrative analysis of high
throughput long- and short-read transcriptome sequencing of cancer cells.
The finding in human prostate cancers that a fusion (TMPRSS2-ERG) occurs
between an oncogene and an androgen regulated gene was a seminal observa-
tion. Subsequently, gene fusion events have been found in a variety of other
human solid cancers, including breast, lung, and pancreatic cancers.12
In the Chapter 5, Dan Hayes and Jeffrey Smerage discuss the technique to
isolate cancer cells from the blood of cancer patients and how the monitoring of
serial changes in circulatory breast cancer cells can be used as an index of
cancer progression in both murine xenograft models of human breast cancer13
and in patients with breast cancer.14
In this method, an iron-tagged (ferrofluid) monoclonal antibody to epithe-
lial cell surface markers is used to isolate epithelial cells from red and white
blood cells. Since epithelial cells do not normally circulate, these cells are often
of tumor origin. Hayes et al.14 have shown that the number of circulatory tumor
cells (CTCs) obtained before treatment is an independent predictor of pro-
gression-free survival and overall survival in patients with metastatic breast
cancer.
The concept that cancer stem cells (discussed in chapter 6 by D’Angelo and
Wicha) are the most aggressive cell type in a tumor cell population is an area of
evolving research. The concept is based on studies that indicate that only a very
small subset of cells (often less than 0.1%) of cells in a tumor have the ability to
generate a new tumor from an implanted human cancer cell population in
immunodeficient (SCID) mice.15 The stem cells in a human breast cancer cell
population have a specific stem cell population phenotype: CD44þ/CD24/
aldehyde dehydrogenase 1þ. These findings have important diagnostic and
therapeutic implications. For example, as noted earlier, currently available
chemotherapeutic drugs were developed largely on the basis of their ability
to shrink a tumor mass in experimental models and clinical trials. This essen-
tially predicts the ability of a drug to kill the bulk of cells in a cancer population,
potentially leaving the more aggressive, drug-resistant cells behind. Thus,
drugs more specifically targeted to the cancer stem cell population would
most likely result in more effective and durable responses.
As more is learned about the phenotype and genotype of cancer cells, it is

becoming apparent that cancer cells are extremely complex. They not only
utilize many of the same genes and proteins that normal cells express but can
up- or downregulate functions in order to survive extraordinary environmental
threats such as toxic drugs or radiation therapy. They have redundant and
overlapping signal transduction pathways that enable them to circumvent
many challenges to their viability. The relatively new science of systems biology
is beginning to unravel much of this complexity.
Systems biology is a conceptual framework to quantify and integrate the
types of biological information contained in cells, tissues, organisms, and popu-
lations of individuals. It is these interacting networks that modulate and regulate
life. This includes the study of genomics, transcriptomics, proteomics, metabo-
lomics, and most other ‘‘-omics’’ that have yet to be so named. Systems biology
attempts to delineate and integrate the dynamic relationships between DNA as
it is packaged in chromalin, RNA (including mRNA, rRNA, siRNA, and micro-
RNA), gene regulatory networks, protein–protein interactions, and cellular
communication systems as well as interactions at the tissue and organ levels.
This is a tremendously complicated business and requires the analysis and
integration of enormous data sets. Thus, the science of bioinformatics is playing
a crucial role in this new way to look at cancer cell biology. For example, even in
lower organisms, such as yeast, Caenorhabditis elegans, and Drosophila, the
interactions of gene and protein networks are high (reviewed in Ref. 16).
The global mapping of a yeast genetic interaction network containing 1000
genes revealed over 4000 interactions. A single large network of 1548 proteins
showed 2538 interactions. In C. elegans, more than 5500 protein–protein
interactions were identified. In Drosophila, a total of 10,623 gene transcripts
produced a map of 7048 proteins with 20,405 predicted interactions.
Some of the issues of determining the complicated interactions in cells and
the use of bioinformatics in solving these puzzles are described by Gil Omenn
in Chapter 7.
Studies in the relatively new field of metabolomics are beginning to reveal a
lot about the cancer cell phenotype. Metabolomics is at the same time less
complicated and more complicated than its ‘‘-omics’’ cousins genomics, tran-
scriptomics, and proteomics. It is less complicated in the sense that whereas
there are 25,000–30,000 genes in the human genome, 100,000 transcripts, and
1,000,000 proteins, there are only about 1800 compounds that constitute the
metabolome. It is more complicated in that these 1800 compounds are in a
constant, rapid state of flux depending on absorption of dietary substances,
hormone levels, drug intake, exercise, body temperature, and the presence of
disease states such as diabetes, infections, or cancer. NMR and mass spectrom-
etry are the typical tools used in the study of metabolomics.17
6 RAYMOND W. RUDDON
Multiple complex metabolic events characterize cancer development and

progression. Recently, sarcosine, an N-methyl derivative of the amino acid
glycine, was identified in urine as a metabolite that differentiates prostate
cancer from benign prostate tissue and as a biological marker that was greatly
increased during prostate cancer progression.18
The molecular profile of cancer cells can also be used to target delivery of
drugs to cancer cells. One way to take advantage of this is the use of nanopar-
ticles bound to anticancer drugs to improve a chemotherapeutic response
(see Baker et al., Chapter 8 in this volume).
Nano- is a prefix for something that is a one billionth part (10 9 of a
specified unit, e.g., nanometer, nanosecond, etc.). Baker and colleagues have
designed dendrimer nanoparticles that target the intracellular folate receptor
in cancer cells (that use an uptake mechanism for folate different from normal
cells) to selectively deliver the anticancer drug methotrexate. This kills 100-fold
more cultured cancer cells than free, unbound methotrexate.19
Brian Ross et al. (Chapter 9) discuss the use of molecular imaging as an
early biomarker of cancer treatment response. There is a critical need for such
biomarkers because it is very difficult for a clinical oncologist to know with any
high degree of accuracy if and when a patient is responding to a therapeutic
regimen. In this context, a biomarker is defined as a biochemical entity that can
be measured in plasma, urine, ascites fluid, or other body fluids or in tissue by
molecular imaging. These biomarkers can be used as an indicator of pathologi-
cal processes or as an indicator of therapeutic response. Ross and colleagues
have used an innovative technique of measuring diffusion coefficients in tissue,
which is a measure of the degree of water Brownian motion (and hence, tissue
‘‘flexibility’’) as a measure of drug response. The diffusion coefficient varies
between an untreated solid cancer (e.g., a glioblastoma) and one undergoing
necrosis in response to treatment.
Not all characteristics of cells, including cancer cells, are totally regulated
by the DNA sequence of genes; far from it. A number of regulatory events are
modulated by DNA modifications such as DNA methylation and by modifica-
tion of histones bound to chromatin such as methylation and acetylation. This
phenomenon is called epigenetics. DNA methylation status and level of histone
acetylation, for example, determine which genes may be expressed or
repressed. Increased DNA methylation of tumor suppressor genes is frequently
observed in cancer cells that have lost or diminished expression of these genes.
In Chapter 10, Wendell Weber discusses how epigenetics affects the
normal function of cells and what epigenetic changes are seen during carcino-
genesis and that occur in already transformed cancer cells. The chapter also
describes the influence of epigenetic profiling in diagnosis, therapy response,
and prognosis.
In spite of the fact that remarkable progress has been made over the past
few decades in the understanding of the molecular, cellular, and tissue process-
es involved in precancer and during cancer progression, the development of
effective and safe modalities for prevention of cancer remains slow, inefficient,
and expensive. One of the problems is that treatment with cancer preventative
agents usually means that individuals need to take a chemopreventive agent for
many years, if not for a lifetime. This means that such treatments must be
extremely nontoxic and safe. Secondly, pharmaceutical companies are loath to
fund such clinical trials because the trials must go on for years if not decades
and involve very large numbers of individuals. All this costs a lot of money. Of
course, initially such clinical trials can be focused on high-risk individuals,
which would cut down on the scale of the trial. Also, if accurate surrogate
markers for effectiveness could be developed, the amount of time and number
of patients could be reduced significantly.20
Similarly, the application of epidemiology to cancer prevention can provide
significant information on cancer risk and the usefulness of preventive strate-
gies.21 For example, the elucidation of clues to cancer causation flows from
observed associations of population exposures to tobacco, diet, environmental
chemicals, and other exogenous factors with the development of cancer in
patients. Indeed, the one real success story for cancer prevention has come
about through smoking cessation.
Nevertheless, everyone wants the ‘‘magic pill’’ that will allow them to keep
their life styles and at the same time enable them to avoid getting cancer. As
numerous studies have shown, alterations in diet or ingestion of mega doses of
antioxidants and vitamins have so far proved to be of little avail. As better
molecular models of cancer initiation and promotion become available, the
hope is that effective chemopreventive agents may be developed. The history
of cancer prevention for the past 100 years has been recently reviewed.22
Current clinical data for ‘‘cancer risk reductive interventions’’ (also previously
known as ‘‘chemoprevention’’) are discussed by Kakarala and Brenner in
Chapter 11.
References
1. Ruddon RW. Cancer Biology. 4th ed. London: Oxford University Press; 2007. p. 1–16.
2. Fearon ER, Vogelstein BA. A genetic model for colorectal tumorigenesis. Cell 1990;61:759–67.
3. Hoeı̈jmakers JHJ. Genome maintenance mechanisms for preventing cancer. Nature
2001;411:366–74.
8 RAYMOND W. RUDDON
6. Friend SH, Bernards R, Rogelj S, Weinberg RA, Rapaport JM, Albert DM, et al. A human DNA
segment with properties of the gene that predisposes to retinoblastoma and osteosarcoma.
Nature 1986;323:643–6.
7. Ma WW, Adjei AA. Novel agents on the horizon for cancer therapy. CA Cancer J Clin
2009;59:111–37.
8. DeVita Jr. VT, Chu E. A history of cancer chemotherapy. Cancer Res 2008;68:8643–53.
9. Hambley TW. Is anticancer drug development heading in the right direction? Cancer Res
2009;69:1259–62.
10. Hait WN. Targeted cancer therapeutics. Cancer Res 2009;69:1263–7.
11. Li X, Loberg R, Liao J, Ying C, Snyder LA, Pienta KJ, et al. A destructive cascade mediated by
CCL2 facilitates prostate cancer growth in bone. Cancer Res 2009;69:1685–92.
12. Maher CA, Kumar-Sinha C, Cao X, Kalyana-Sundaram S, Han B, Jing X, et al. Transcriptome
sequencing to detect gene fusions in cancer. Nature 2009;458:97–101.
13. Eliane J-P, Repollet M, Luker KE, Brown M, Rae JM, Dontu G, et al. Monitoring serial
changes in circulating human breast cancer cells in murine xenograft models. Cancer Res
2008;68:5529–32.
14. Hayes DF, Cristofanilli M, Budd GT, Ellis MJ, Stopeck A, Miller MC, et al. Circulating tumor
cells at each follow-up time point during therapy of metastatic breast cancer patients predict
progression-free and overall survival. Clin Cancer Res 2006;12:4218–24.
15. Al-Hajj M, Wicha MS, Benito-Hernandez A, Morrison SJ, Clarke MF. Prospective identifica-
tion of tumorigenic breast cancer cells. Proc Natl Acad Sci USA 2003;100:3983–8.
17. Nicholson JK, Lindon JC. Metabonomics. Nature 2008;455:1054–6.
18. Sreekumar A, Poisson LM, Rajendiran TM, Khan AP, Cao Q, Yu J, et al. Metabolomic profiles
delineate potential role for sarcosine in prostate cancer progression. Nature 2009;457:910–4.
19. Quintana A, Raczka E, Piehler L, Lee I, Mhyc A, Majoros I, et al. Design and function of a
dendrimer-based therapeutic nano-device targeted to tumor cells through the folate receptor.
Pharmacol Res 2002;19:1310–6.
20. Kelloff GJ, Bast Jr. RC, Coffey DS, D’Amico AV, Kerbel RS, Park JW, et al. Biomarkers,
surrogate end points, and the acceleration of drug development for cancer prevention and
treatment: an update. Clin Cancer Res 2004;10:3881–4.
21. Greenwald P, Dunn BK. Landmarks in the history of cancer epidemiology. Cancer Res
2009;69:2151–62.
22. Lippman SM, Hawk ET. Cancer prevention: from 1727 to milestones of the past 100 years.
Cancer Res 2009;69:5269–84.
Molecular Biology and
Anticancer Drug Discovery
John S. Lazo
Department of Pharmacology and Chemical
Biology, University of Pittsburgh Drug
Discovery Institute and Cancer Institute,
University of Pittsburgh, Pittsburgh,
Pennsylvania, USA
I. Introduction .................................................................................. 9
II. Phenotypic Targets .......................................................................... 12
III. Molecular Targets ........................................................................... 16
A. General Issues ........................................................................... 16
B. Drug Resistance......................................................................... 17
C. Oncogene and Nononcogene Addiction ........................................... 18
D. Synthetic Lethal ......................................................................... 20
E. Combination Chemotherapy ......................................................... 23
F. Nontraditional Targets ................................................................. 23
IV. Other Contemporary Issues in Anticancer Drug Discovery ....................... 26
V. Conclusions ................................................................................... 27
References .................................................................................... 27
The profound impact of molecular biology on the philosophy of how one

should seek new cancer therapeutics cannot be overstated. It has enabled the
discovery of unique drugs as well as the identification of new drug targets and
biomarkers and the creation of powerful animal models. Nevertheless, the
process of cancer drug discovery remains inherently complex and inefficient.
This is partially a consequence of the requirement of any successful therapy to
show differential effects toward tumor cells relative to nonmalignant cells. The
goal of this chapter is to outline the impact of molecular biology on modern
approaches to anticancer drug discovery and to highlight the continuing
challenges.
I. Introduction
Contemporary cancer drug discovery largely focuses on identifying new
therapies by leveraging advances in our understanding of the molecular biology
of cancer. This has been facilitated by the enormous increase in our knowledge
concerning the molecular pathogenesis of cancer in the past two decades, which

DOI: 10.1016/S1877-1173(10)95002-3 1877-1173/10 $35.00
10 JOHN S. LAZO
was stimulated by the profound investment in research funding from both private
and public sources. In addition to great improvements in diagnostics and imaging,
there are now more than 200 approved drugs for cancer; hundreds are in clinical
development. Nonetheless, our ability to control, much less cure, cancer has
been disappointing. Oncology has among the worst success rates (5%) for clinical
drug development compared to all other major disease sectors.1 Drug discovery
and development in cancer remains a challenging, high-risk, inefficient, and
complex process. Why is that so?
There are several reasons for the poor success in anticancer drug discovery
and development. First, as mentioned in Chapter 1, tumors evolve through a
multistep process in which cancer cells frequently acquire a large number of low-
frequency genetic or epigenetic changes that can contribute to oncogenesis.
These mutations affect the expression or functionality of oncogenes or tumor
suppressors. The substantial number of alterations makes it challenging to distin-
guish the critical changes that are worthy of being targeted with drugs from the
nonessential ones or ‘‘passengers’’ that are a legacy of the disease process. Some
changes may have been important for the initial stages of oncogenesis but now are
completely dispensable for the oncogenic phenotype and, therefore, unworthy of
being drug targets. A second problem is that many of the oncogenes represent
overexpressed versions of normal proteins or they contain mutations that are only
subtly different from the normal gene product. Thus, selective targeting of cancer
cells may be considered problematic. Generally, drugs affect gain-of-function
abnormalities, making it at least conceptually challenging to design drugs that
will replace the loss of a tumor suppressor. Finally, the fundamental genetic
instability of cancer produces inherent plasticity in the disease that promotes
rapid drug resistance.
There is no doubt that the cancer drug discovery landscape has benefited
from developments in a host of disciplines and technologies. Advances in
organic chemistry have facilitated the complete synthesis of complex natural
products with powerful pharmacological activities.2 Combinatorial chemistry
and diversity-oriented synthesis have expanded greatly the numbers of drug-
like compounds that now can be regularly probed for anticancer activities.
Publically accessible databases of compounds and biological actions, such as
PubChem, PharmGKB, ChemSpider, and ChemExper, provide information on
tens of millions of compounds.3 Advances in automated liquid handling plat-
forms have enabled high throughput screening of hundreds of thousands of
compounds for bioactivity. Similarly, high content platforms have empowered
investigators to directly test compounds for cellular actions. Computational and
structural biology have generated tools for molecular docking and visualization
of drug or ligand interactions with putative targets.4 Innovation in drug delivery
and nanotechnology offer hope for better pharmacokinetics and drug targeting
to tumors.2 The emergence of systems biology as a discipline has created new
ANTI-CANCER DRUG DISCOVERY 11
tools to visualize signaling pathways controlling attributes that are vital for the
neoplastic phenotype. How are these developments in various disciplines
integrated into the process of discovering a new anticancer drug?
Fundamentally, there are two broad approaches used to identify new drugs:
Forward Pharmacology and Reverse Pharmacology.5 These approaches are not
unique to anticancer drug discovery, but there is some merit to the argument that
the field of oncology drug discovery has uniquely benefited from the advances of
molecular biology with the cataloging of vast numbers of oncogenes and tumor
suppressors, which has accelerated the transition from Forward Pharmacology to
Reverse Pharmacology. The oldest strategy, Forward Pharmacology, relies on
observing phenotypic changes of cells, organs, or organisms by chemical sub-
stances. Even before the acquisition of any fundamental understanding of the
regulation of physiological systems or the nature of pathological processes, one
could readily measure the inhibition of cancer cell proliferation in a culture dish.
This molecular target-unbiased approach merely focused on the desired biology
without any knowledge of how the process was regulated. There are at least six
phenotypic attributes associated with tumor cells that can be examined in culture
(Table I). Indeed, a majority of our existing clinically used anticancer drugs were
discovered using a phenotypic assay and with little knowledge of how they might
be selectively toxic to cancer cells. In contrast, Reverse Pharmacology is
practiced with the goal of finding a modulator, usually an inhibitor, of a molecular
target that is believed to be critical for the cancer phenotype.5 Clearly, molecular
biology has profoundly influenced this branch of pharmacology and anticancer
TABLE I
EXAMPLES OF PHENOTYPIC ATTRIBUTES OF CANCER CELLS AND COMPOUNDS THAT ALTER THE PROCESS
Phenotypic Proposed mechanism Drug or

target of action compound References
Proliferation DNA damage, Cisplatin, 6,7

Cyclin-dependent kinase, AT7519, P276-00,
Dihydrofolate reductase Methotrexate
Angiogenesis Vascular endothelial growth Bevacizumab, 8
factor A (VEGF-A), VEGF Sunitinib
receptor
Metastasis/ Increased activating transcrip- Sulindac 9
invasion tion factor 3 (ATF3)
Senescence DNA damage Doxorubicin 10
Stem cell Potassium ionophore (?) Salinomycin 11
Differentiation Induction of CCAAT/enhanc- All-trans retinoic acid 12
er binding protein (Tretinoin)
12 JOHN S. LAZO
drug discovery. Molecular biology and molecular genetics have been critical in
identifying potential causal factors in neoplasia. Once identified, the molecular
target (generally an enzyme) can be produced in a recombinant form permitting
in vitro interrogation for inhibitors. This class of assays has the distinct advantage
of generally being precise, rapid, and easier to conduct than assays requiring
animal, organs, or cells. One can sometimes even determine at an atomic level
the interactions between the compound and the target, making the generation of
a chemical structure–activity relationship practical and the synthesis of more
potent analogs rational. Target specificity and modes of inhibition assays are also
conceptually easier to perform with a known target than with phenotypic assays.
For molecular targeted anticancer drugs, it is desirable that most, if not all, vital
cell types in the human body not depend on the target for survival. Otherwise,
the drug is likely to have a narrow therapeutic window owing to the requirement
for the target in normal cells. Although currently there is enormous enthusiasm
for target-driven anticancer drug discovery, phenotypic screening approaches
are regaining popularity in part because they can be designed to ensure com-
pound entry into cells and stability, the failure of which can lead to the demise of
compounds identified by more reductionist assays.
II. Phenotypic Targets

As mentioned above, efforts to identify compounds that kill cancer cells in
culture have existed for more than half a century and have produced what are
now commonly called the ‘‘cytotoxic drugs.’’ Murine cell lines, such as P388
leukemia, L1210 leukemia, and B16 melanoma, dominated the early years of
cancer cell testing both in culture and in mice, but with the successful culturing
of human cancer cells, such as HeLa, and the development of immunosup-
pressed mice, such as nude mice, there was a gradual movement to the
use of human-derived cancer cells and xenografts. The establishment by the
National Cancer Institute in 1990 of a panel of 60 human cell lines (NCI60)
for compound interrogation marked a major innovation in anticancer high
throughput drug screening and profiling. The NCI60 comprises cell lines
from nine cancer types: six leukemias, nine melanomas, nine non-small-cell
lung, seven colorectal, six CNS, seven ovarian, six breast, two prostate, and
eight renal. Despite this diversity, the NCI60 has not been valuable in predict-
ing which specific human tumor types will be responsive to an experimental
drug. Some have argued that a much larger cell panel, perhaps with thousands
of cultured cell types, would be required to achieve that goal.13 Others14 have
suggested that human tumor cell lines, which have been adapted to grow under
the extremely artificial conditions of serum, culture medium, and high oxygen,
acquire new mutations resulting in a population that is inherently different
from primary tumors. After all, most tumors isolated from patients do not yield
cells that can grow productively in culture. Moreover, cell lines adapt distinc-
tive phenotypes after multiple passages in different laboratories. Thus, a HeLa
cell that has been extensively passaged in one laboratory is not necessarily the
same as that studied in another laboratory. Nonetheless, the US National
Cancer Institute has established a large publicly available annotated database,
which can be mined with programs, such as the COMPARE algorithm
(see http://dtp.nci.nih.gov/docs/compare/compare.html), to examine the profile
of drug sensitivity to the panel. Studies using this algorithm have clustered
compounds with common mechanisms of action, identified compounds with
new mechanisms of action, and exposed agents that are substrates for multiple
drug resistance.13 The landmark work of the National Cancer Institute has also
spawned other tumor cell line panels in an effort to develop an ideal tumor cell
screening panel.13
All cell proliferation or cell death assays depend on the assay protocol but
most importantly on the endpoint that is being measured. Some assays simply
measure the total number of cells on the plate after a fixed time point. Others
determine the number of viable cells using, for example, Trypan blue exclusion
or Alamar blue reduction. In most cases, however, there is little distinction
between cell growth inhibition and cell death. There is a growing interest
in developing assays that will differentiate between apoptosis, necrosis, necrop-
tosis, anoikis, autophagy, and other processes associated with cell death.
Obviously, it is desirable to establish preferential toxicity against cancer cells
versus normal cells, but the question of selecting the appropriate normal cell
type for the comparison often is difficult to answer.
Phenotypic cytotoxicity assays clearly are only biological models: the com-
position of the culture medium, the presence of fetal bovine serum, and the
absence of other stromal cells make the environment extremely artificial.
Additionally, the nonphysiological oxygen concentrations and the lack of a
normal extracellular matrix and stromal cells prevent a precise recapitulation
of the environment in which tumor cells find themselves in vivo. The impor-
tance of the tumor microenvironment in response to therapy is well estab-
lished.13 Studies with three-dimensional cultures, tumor spheroid systems, and
mixed stromal cell substrates may provide improved platforms for future drug
testing systems.
Although drugs identified with phenotypic assays or Forward Pharma-
cology are generally regarded in the current parlance as ‘‘cytotoxics’’ and
not targeted, all probably function by influencing a molecular target. Potent
inhibitors of dihydrofolate reductase, topoisomerase I, topoisomerase II, ribo-
nucleotide reductase, microtubule stabilizers, and tubulin disruptors were first
identified as inhibitors of cancer cell proliferation and only later were their
mechanisms of action, namely molecular targets, determined (often with the
14 JOHN S. LAZO
assistance of molecular biology tools). For example, methotrexate binds to its

target dihydrofolate reductase at picomolar concentrations and has orders of
magnitude preference for this target over its secondary target, thymidylate
synthase. Conversely, many drugs identified with molecular targets in mind,
such as proteosome inhibitors (bortezomib) or histone deacetylase inhibitors
(vorinostatin), are not easily distinguished from the traditional cytotoxics and
might reasonably be referred to as ‘‘neocytotoxics.’’ Nonetheless, the term
‘‘targeted therapy’’ has become closely associated with drugs that are identified
with Reverse Pharmacology processes aimed at attacking enzymes thought to
be essential for neoplasia.
In addition to cell proliferation as a phenotypic endpoint, there are a
number of other cancer-associated properties that have been employed to
identify potentially new and useful anticancer agents. These include assays to
measure inhibition of tumor cell invasion and metastasis,15 angiogenesis,8
differentiation,16,17 and senescence10,18 (Table I). Successful metastasis only
occurs if positive and negative regulatory mechanisms, governing angiogenesis,
proteolysis, motility, host defense systems, and cellular adhesion events are
concomitantly deregulated. Because metastasis is a complex multifactorial
event, it is extremely difficult to evaluate in vitro. Therefore, there are few
available robust assays and most of the research effort has focused on identify-
ing agents that alter cell migration and invasion. One common cell migration
and invasion assay uses a Boyden chamber or a Transwell device, which either
lack or contain a coating of an extracellular matrix emulator.15 Cells are placed
in an upper chamber and the lower chamber is loaded with medium containing
serum. The migration of cells into the lower chamber is then quantified at
various times. There are a growing number of molecular targets associated with
tumor cell invasion and metastases, which have been studied. These include
enzymes involved in the cleavage of collagens, namely the metalloproteinases.
The cell culture invasion assays have been quite useful to validate these
compounds.
The recognition of the critical role of angiogenesis in cancer and the
advanced knowledge concerning biological factors regulating tumor-stimulated
angiogenesis and the emergence of clinically approved drugs have stimulated
the development of a number of innovative assays aimed at discovering new
antiangiogenic agents.8 One of the most sophisticated automated in vivo assays
exploits transgenic zebrafish to quantify the microscopic effects of drugs on
blood vessel formation.19
Differentiation therapy is generally defined in oncology as the use of small
or large molecular entities that induce the reversion of malignancy with the
restoration of mature cells of the same histological lineage. Clinically, the
poster child for differentiation therapy has been all-trans retinoic acid
(ATRA) for the treatment of acute promyelocytic leukemia (APL), which has
dramatically improved the prognosis for patients with this disease. Exposure of
APL cells in culture to ATRA leads to differentiation into mature neutrophils,
most likely due to the induction of CCAAT/enhancer binding proteins.12 There
is preclinical evidence that methyltransferase inhibitors, such as azacytidine,
and the histone deactylase inhibitor sodium phenylbutrate can also induce
differentiation in APL.17 In addition, arsenic trioxide has been documented
to induce differentiation in APL cells, leading to clinical remissions, although
an incontrovertible mechanism of action is lacking.
In contrast to differentiation, tumor cell senescence is the loss of replicative
ability that is accompanied by cellular enlargement, flattened morphology with
an increased cytoplasmic area, increased granularity, extensive cytoplasmic
vacuoles, and multinucleation. In addition, senescent cells have changes in
gene expression that is largely independent of cell type, including an increase in
the expression senescence-associated b-galactosidase, cyclin D, and cyclin-
dependent kinase inhibitors, p21 and p16. Senescent cells also have a reduction
in the tumor suppressor protein Rb. A surprisingly large number of clinically
used, mechanistically distinct anticancer agents, including DNA alkylators,
topoisomerase poisons, antimetabolites, and microtubule stabilizers, as well
as irradiation, have been reported to induce senescence.10 Typically, senes-
cence is seen with cultured human tumor cells at low drug concentrations,
while apoptosis is observed with higher drug concentrations, presumably
because of the greater cell damage. For example, treatment of human hepato-
ma cells with a low concentration of the indirect DNA-damaging agent doxo-
rubicin (< 100 nM) causes senescence, whereas higher concentrations (20 mM)
produces frank apoptosis. It should be emphasized, however, that the impor-
tance of the phenomenon of senescence for the clinical activity of any drug
remains to be determined.
Interest also exists in identifying drug-like compounds that can disrupt the
essential dialogue between tumor cells and stromal cells, but currently, there
are no clinically used agents that faithfully act in this manner. As will be
mentioned in Chapter 6, there is considerable enthusiasm for identifying
drugs that selectively kill cancer stem cells. Unfortunately, useful in vitro assays
for identifying agents that specifically kill epithelial cancer stem cells have not
been available because of the paucity of these cancer stem cells within tumor
cell populations and their instability in culture. The recent development of an
innovative assay by Gupta et al.11 may have overcome this barrier, however.
Using this assay, Gupta et al.11 discovered several compounds, including sali-
nomycin, with selective toxicity for breast cancer stem cells. Treatment of mice
with salinomycin inhibited mammary tumor growth in vivo and induced
increased epithelial differentiation of tumor cells. Thus, it may be possible to
target what is thought to be a critical cancer cell population normally resistant
to existing therapies.
16 JOHN S. LAZO
III. Molecular Targets

A. General Issues
Remarkable advances in our fundamental knowledge of the oncogenic
process catalyzed by the tools of molecular biology have enabled the current
so-called ‘‘targeted’’ therapy approach. There is no doubt that a majority of all
current anticancer drug discovery programs are now driven by molecular
targets. Not only does this reductionist approach provide a pleasant feeling of
rationality that is frequently absent in the phenotypic approach mentioned
above but it also facilitates ligand–target interaction studies. The approach
has also been propelled by the availability of enormous databases annotating
gene expression profiles, protein expression, metabolomic patterns, cancer
mutations, and other changes in human tumors. Finally, the clinical success
of the tyrosine kinase inhibitor, imatinib, has been a primal stimulus for
molecular targeted drug discovery.
The tools of molecular biology have armed investigators with many
approaches to identify potential molecular targets. Perhaps the most powerful
has been small interference RNA (siRNA) screens. MacKeigen et al.,20 using
siRNA libraries aimed at suppressing all of the known cellular kinases and
phosphatases, demonstrated that 73 of the 650 known or putative kinases (11%)
were essential for the survival of HeLa cells and 72 of the 222 known or putative
protein phosphatases or their regulatory protein (32%) were essential for cell
survival. Each of these enzymes represent at potential molecular targets for novel
anticancer therapies. Clearly, there are many hurdles that must be overcome for
the individual kinase or phosphatase to be fully validated as a cancer drug target,
perhaps most importantly, the need to document selective toxicity against malig-
nant cells versus nonmalignant cells. In some cases, it appears that the actions of
even highly selective kinase inhibitors, such as the RAF inhibitors, are exquisitely
dependent on the cellular context in which the kinase target is expressed.21
Before considering potential anticancer drug discovery targets, it might be
instructive for the oncology community to review the lessons learned from
another genomic-derived, target-based drug discovery program, namely the
infectious disease field. The sequencing of the first complete bacterial genome
in 1995 heralded a new era of hope for antibacterial drug discoverers, who now
had powerful tools to search entire genomes for new antibacterial targets.
Armed with knowledge about pathogen genomes, several companies launched
genomics-derived, target-based approaches to screen for new classes of drugs
with unique modes of action. One company identified 160 genes that were vital
for pathogenic bacterial survival and, thus, were considered ideal drug targets.
They cleverly narrowed the list further by excluding those genes that encoded
proteins that had close human homologs. Over a 7-year period, they
systematically screened many of these seemingly ideal targets with large

260,000–530,000 compound libraries for selective inhibitors.22 Despite com-
pleting 67 high throughput screening campaigns on these highly attractive and
novel antibacterial targets, no credible development candidate emerged. It is
sobering to recognize that the disappointing results from that well-conceived
genome-derived, target-based approach in the infectious disease sector by one
company have been shared by other pharmaceutical and biotechnology com-
panies. This reflects the significant challenges of target-based drug discovery
even with seemingly ‘‘simple’’ diseases. How can failures be avoided or at least
be reduced in our target-based strategies in oncology? The answer is not
necessarily immediately obvious. Certainly, the composition of the chemical
library is critical.22 It is also clear from this exercise that selection of tractable
pharmacologically validated targets is critical. Extracellular targets, such as
receptors, transporters, exoenzymes, cell surface antigens, proteoglycans, and
extracellular matrix components, are much more likely to be attractive
than intracellular molecular targets. Being fully validated is more important
than being new. Finally, whole cell assays probably provide better discrimina-
tors of drug-like compounds than simple in vitro assays for intracellular molec-
ular targets because the compound must easily enter cells in order to be
efficacious.
It is axiomatic that any successful anticancer therapy, whether designed to
cure or to just control cancer, must demonstrate preferential actions on tumor
cells relative to normal cells in the patient. This forms the basis of the thera-
peutic index. Almost all of the cytotoxic and most of the current molecularly
targeted drugs have remarkably narrow therapeutic indices limiting the
amount of drug that can be administered.
B. Drug Resistance
Soon after the first clinical trials with cytotoxic anticancer drugs in the
middle of the last century, it became apparent that human tumors rapidly
acquired resistance to chemotherapy. Indeed, modern combination chemo-
therapy, which uses multiple agents with distinct mechanisms of actions, is
structured on the concept of deterring drug resistance. Despite considerable
preclinical and clinical efforts, however, we have been remarkably unsuccessful
in identifying drugs that reverse drug resistance. Drugs, such as verapamil,
ZNRD1, biricodar, and INF271, which inhibit the multidrug resistance ABC
cassette protein ABCB1 (also known as MDR1 or p-glycoprotein), have not
emerged as viable clinical supplements for the existing chemotherapeutic
substrates of this membrane transporter. This could be due to the lack of
efficacy of the agents tested, unacceptable toxicity associated with inhibition
of ABCB1 in critical normal tissues, or a relatively minor role for ABCB1 in
human drug resistance. In contrast, an understanding of the mechanism of
18 JOHN S. LAZO
drug resistance has enabled the successful design and development of second
generation anticancer drugs that retain efficacy in tumor cells with acquired or
innate resistance to the first generation agents. For example, soratinib is
effective against tumor cells resistant to imatinib. Premetrexate inhibits multi-
ple folate-related targets, some of which are involved in resistance to metho-
trexate. Many of the second generation compounds now have embedded in
them multiple targets, some of which are considered resistance mechanisms or
participants in the oncogenic phenotype.
C. Oncogene and Nononcogene Addiction

If cancer-causing genes products are not unique to tumor cells but are
exploited normal proteins required for development or normal cell signaling,
then how can one target them? This is the conundrum facing the medical
oncologist. Surprisingly, basic cell biological studies have revealed a remark-
able dependency of cancer cells on the expression of certain oncogenes or the
lack of expression of some tumor suppressors. Weinstein23 first coined the term
‘‘oncogene addiction’’ as the enhanced state of dependency on the presence of
an oncogene; he later extended the term to include tumor suppressor gene
hypersensitivity.24 The Weinstein hypothesis is that the malignant phenotype of
a cell with one or more oncogenes or with the absence of a tumor suppressor
protein mandates an extensive alteration in cellular signaling processes. Once
the adaptation occurs, tumor maintenance depends on the continued activity of
an oncogene or the absence of the tumor suppressor. Tumor addiction to
oncogenes could help explain the sensitivity of cancer cells to some targeted
therapies (Table II). This anthropomorphic notion of ‘‘tumor addiction’’ has
been expanded to a dependency on existing intracellular pathways that include
participating proteins that may themselves not be oncogenes or tumor sup-
pressors.6 For example, elevation in a proximal tyrosine kinase activity in an
oncogenic signaling pathway, such as the mitogen-activating protein kinase
pathway, could lead to making other members of the pathway, which are not
themselves oncogenes, required because they now are rate-limiting in the
pathway. Alternatively, cells with an oncogene-mediated hyperactive mitogen-
activating protein kinase pathway may become more dependent on normal
protein phosphatases to regulate the hyperactivity. These alterations, which are
required for the maintenance of the oncogenic state, become very logical and
attractive molecular targets.
Many oncogenes paradoxically induce both pro-mitogenic signals and anti-
mitogenic or pro-apoptotic signals. Oncogene activation presumably stimulates
growth because the former is dominant. As long as the oncogene signal is
sustained, the proliferative signal, which promotes mitogenesis, survival, or
both, dominates. According to the theory, acute inactivation of the oncogene
TABLE II
EXAMPLES OF ONCOGENE AND NONONCOGENE ADDICTION TARGETS AND AGENTS
Target Agent
Oncogene targets
BCR-Abl, c-Kit Imatinib, Dasatinib
Retinoic acid receptor, Retinoic X receptor ATRA (Tretinoin)
ERBB2 Trastuzumab
Epidermal growth factor receptor Erlotinib, Gefitinib
MDM2 Nutlin-3
BCL-2 Oblimersen, ABT-737
PI3K GDC-0941, BEZ235
Nononcogene targets
mTOR Temsirolimus, Everolimus
Topoisomerase I Topotecan, Irinotecan
Dihydrofolate reductase Methotrexate, Pemetrexed
Vascular endothelial growth factor Bevacizumab
PARP1 AZD2281, ABT-888, AG014699
Heat shock protein-90 Geldanamycin, Alvespimycin
Mitotic Spindles Vinblastine, Vincristine, Paclitaxel, Epothilone B
Chk1 PF-00477736
causes growth cessation or death when the antimitogenic or pro-apoptotic

signals decay more slowly than the mitogenic signals, perhaps due to differ-
ences in mRNA and/or protein half-life.
There are now multiple examples supporting the notion that cancer cells
in vitro are dependent on or addicted to certain activated oncogenes; similar
evidence is mounting for tumor cells becoming addicted to the loss of the
functionality of tumor suppressor genes. Oncogene addiction is thought to be
the basis of the success of the tyrosine kinase inhibitor imatinib for CML in
which BCR–ABL is the oncogene and gastrointestinal stromal tumors in which
KIT is the oncogene.
Considering the large number of identified oncogenes and tumor suppres-
sors, how does one determine what to target? This is particularly important when
one remembers that tumor onset and progression frequently mandate the
accumulation of multiple genetic and epigenetic lesions. Computational and
experimental methods are emerging that permit the identification of the so-
called ‘‘dominant’’ and ‘‘recessive’’ changes.25 Using the Met receptor as a model
system, Bertotti et al.25 combined multiplex phosphoproteomics, genome-wide
20 JOHN S. LAZO
expression profiling, and functional assays to deconvolute signatures that are

required for sustained oncogene addiction, and these would seem to represent
attractive drug targets. This is one of what will no doubt be many attempts to
deconvolute the profile associated with oncogene-addicted human tumors.
The concept of oncogene addiction occurring in vivo has been established
with several oncogenes. For example, lymphomas, osteosarcomas, and papillo-
mas formed with a chemically induced MYC oncogene were shown to regress
when MYC expression is terminated by removal of the inducing chemical.26 A
comparable phenomenon has been documented with HRAS, BCL-ABL, and
KRAS oncogenes. Similarly, reintroduction of a tumor suppressor, such as p53,
to tumors in which there is loss of p53 through gene deletion, mutation, or
epigenetic silencing, causes tumor regression.27 It is critical for the oncology
community to identify the complete constellation of oncogene and tumor
suppressors to which human tumors are addicted. Some might consider the
term ‘‘addiction’’ to be too vague and not sufficiently quantitative to ensure
therapeutic success. Indeed, the term is not necessarily favored in the field of
drug abuse, because it describes a human behavior rather than a physiologically
measurable phenomenon. In the context of cancer, however, the long-term
regression of tumors after the withdrawal of an oncogene or the reexpression of
a tumor suppressor appears to provide a sound quantitative method to validate
an addiction participant.
How can we pharmacologically exploit the concept of tumor suppressor
addiction? Loss of the tumor suppressors Rb, p16, p21, or p27—all cause an
increase in cyclin-dependent kinase activity, which permits cell cycle progres-
sion. Therefore, one could theorize that such tumors should be hypersensitive
to inhibitors of cyclin-dependent kinases. Tumors that have lost the tumor
suppressor and lipid phosphatase PTEN, which normally acts to restrain
PI3K signaling, should theoretically be hypersensitive to PI3K inhibitors.
D. Synthetic Lethal
If oncogene expression and tumor suppressor loss alter the networks upon
which tumor cells depend, is there a strategy to uncover the proteins and
pathways? An unbiased experimental approach termed synthetic lethal for
identifying molecular targets or chemosensitivity nodes provides a powerful
conceptual framework. The synthetic lethal strategy evolved from yeast genetic
studies in which investigators were interested in identifying genes that colla-
borated with yeast functionality. Two genes were labeled to be ‘‘synthetic
lethal’’ if deletion or mutation of either gene alone did not result in death but
loss of both genes caused death (Table III). Yeast synthetic lethal interactions
have most commonly been described for loss-of-function alleles but they also
can involve gain-of-function genes. In Saccharomyces cerevisiae, approximate-
ly 20% of genes are individually essential, but synthetic lethal screens reveal
TABLE III
GENETIC SYNTHETIC LETHALITY
Gene A Gene B Phenotype
Deletion/Mutation Viable
Deletion/Mutation þ Viable
Deletion/Mutation þ Viable
Deletion/Mutation þ þ Death
TABLE IV
MIXED DRUG AND GENETIC SYNTHETIC LETHALITY
Drug A Gene A Phenotype
Treatment/Deletion/Mutation Viable
Treatment/Deletion/Mutation þ Viable
Treatment/Deletion/Mutation þ Viable
Treatment/Deletion/Mutation þ þ Death
that a large fraction of the remaining genes can collaborate in death process-
es.28,29 Many of the targets for existing cytotoxic agents are essential gene
products such as tubulin or topoisomerase. First principles would teach us
that they are not attractive because they are essential for both normal and
malignant cells; this is why existing compounds have such a narrow therapeutic
index. If the synthetic lethal concept from yeast genetic screens could be
extended to human tumor cells, which have preexisting mutations, deletions,
and epigenetic abnormalities, it might be possible to identify innovative drug
targets. It might even be possible that some of our current cytotoxic drugs are
efficacious because they inadvertently exploit synthetic lethality. In this model,
one of the gene deletions or mutations is replaced with a drug (Table IV).
One attractive example is the sensitivity of cancers carrying BRCA1 mutations,
which is responsible for homologous DNA recombination repair. These tumors
are exceptionally sensitive to DNA-damaging and cross-linking agents such as
temozolomide and cisplatin.6 These tumors are heavily reliant on orthogonal
forms of DNA repair, such base-excision repair that is mediated by poly-ADP-
ribose polumerase (PARP1). PARP1 facilitates repair of single-strand breaks.
In nonmalignant cells, endogenous DNA damage generated by PARP1 inhibi-
tion by agents, such as ABT-888, is well tolerated because of functional
compensation from homologous recombination-mediated repairs. This is an
22 JOHN S. LAZO
TABLE V
DRUG–DRUG SYNTHETIC LETHALITY
Drug A Drug B Phenotype
Treatment Viable
Treatment þ Viable
Treatment þ Viable
Treatment þ þ Death
excellent example of nononcogene addiction of tumor cells with BRCA1 muta-

tions on PARP1. The synthetic lethality model can also be extended by repla-
cing a gene deletion with a second drug (Table V). This could obviously be a
powerful strategy for identifying novel drug combinations in an unbiased
manner (see below).
Molecular biology has provided a long list of human oncogenes and tumor
suppressors. A number of groups have established isogenic pairs of normal and
malignant cell lines suitable for screening of compound libraries or plant, soil,
and marine extracts, which are complex mixtures of Natural Products, for
agents that selectively inhibit cells with cancer-relevant genetic alterations in
a high throughput manner. For example, compounds have been identified in
marine sponge extracts that preferentially inhibited Trp53/ mouse embryonic
fibroblast proliferation relative to wild-type mouse embryonic fibroblasts.30
Colon cancer cells with the KRAS mutant alleles have been created and
engineered to produce a blue fluorescent protein reporter. An isogenic coun-
terpart was also created in which the mutant KRAS allele was eliminated by
homologous recombination; these latter cells were engineered to produce
yellow fluorescent protein. This allowed the investigators to monitor the drug
treated pair of cells for differential killing using the ratio of blue/yellow fluores-
cence.31 The availability of the isogenic pair enabled the investigators to
identify a novel cytosine nucleoside, which was selectively toxic to cells contain-
ing mutant KRAS. The general synthetic lethal approach has been expanded
further based on the recent recognition of the minimal genes required for
transformation. Thus, primary human cells have been transformed with human
telomerase reverse transcriptase, RAS, and oncoproteins that affect pRB, p53,
and/or protein phosphatase 2A (PP2A).32 A number of compounds that were
identified included several clinically useful inhibitors of topoisomerase I and II,
which help validate the notion that existing cytotoxic agents found by Forward
Pharmacology were likely discovered unknowingly with a synthetic lethal
approach. Obviously, it is also possible to exploit the synthetic lethal strategy
for new drug combinations.
E. Combination Chemotherapy
Almost all regimens for the treatment of cancer rely on drug combinations.
They have been created almost exclusively by empirically amalgamating drugs
with different mechanisms of action, untoward effects, and mechanisms of
resistance. Molecular biology has provided new information about the inter-
connections of the anticancer targets that could provide some theoretical
guidance for the formulation of drug combinations. Nonetheless, even with
the introduction of targeted therapies where there is a quantifiable pharmaco-
logical effect, it is most common to develop drug combinations in the clinic by
escalating the individual agents to the maximum tolerated dose until the
aggregate effect of toxicity is considered to be excessive. This approach pre-
sumes, perhaps incorrectly, that the maximum tolerated dose is the maximum
effective dose. There is no incontrovertible clinical evidence to support this
notion. It has been extremely challenging to provide any in vitro guidance for
this important question, especially as we see the use of combinations containing
multiple agents. We know each of the individual drugs is likely to exert multiple
pharmacological effects at different concentrations and each has a unique
pharmacokinetic profile that alters the plasma and tumor concentration over
time. Thus, it is important to address this issue, because there is compelling
evidence that the anticancer effect of drug combinations can be profoundly
dependent on the ratio of the individual drugs.33
F. Nontraditional Targets
As mentioned above, the primary targets for almost all current drugs are the
catalytic site of enzymes or ligand binding sites. Many enzymes, however, depend
on allosteric regulation to be fully active, and there is a growing interest in
disrupting this process. We know that the intracellular spatial, kinetic, and
substrate specificity regulation of enzymes can be controlled by multiple
protein–protein interactions (PPIs). These are widely believed to control all
major cellular functions, including the maintenance of DNA topology, DNA
replication, mRNA transcription, protein translation and its proper folding, the
assembly and maintenance of morphological structures, and the regulation of
cellular metabolism and signaling pathways. The protein interactions comprise
identical or homotypic binding and nonidentical or heterotypic binding between
two or more polypeptides; they can be characterized thermodynamically and
kinetically as anything from high affinity stable contacts to low affinity transient
interactions. Some are critical for functionality and others are gratuitous.
It is easy to imagine how the critical PPIs might provide valuable potential
molecular targets that would be mechanistically distinct from the common active
sites or ligand binding drug discovery targets. While PPIs might superficially
seem attractive, until recently it was widely believed that PPI surfaces were not
24 JOHN S. LAZO
druggable because they were large and amorphous.34 Refined structural eluci-
dation of many protein–protein complexes indicate that the protein-binding
interfaces can be dissected into discrete patches with critical residues, termed
‘‘hot spots,’’ that are vital for binding.34 Biochemical PPI assays exploiting
methods, such as co-precipitation, co-purification, affinity chromatography, ul-
tracentrifugation, nuclear magnetic resonance, surface plasmon resonance, mass
spectrometry, and isothermal titration calorimetry, have been developed.34,35
Additionally, higher throughput biochemical PPI screening assay formats have
been developed, including capture ELISAs, cell surface binding, fluorescence
polarization, time-resolved fluorescence, ligand-induced changes in thermal
stability, bead-based technologies, such as AlphaScreen and Luminex, and reso-
nance energy transfer assays.34,35 Cell-based PPIs assays exploiting two-hybrid
transcriptional reporter systems have also been developed in mammalian cells.
Several imaging-based high content screening assay formats have also been
employed to study PPIs, such as the co-localization of fluorescently labeled
protein partners, fluorescent resonance energy transfer measurements between
PPI partners bearing donor and acceptor fluorescently labeled protein, protein
fragment complementation assays, and positional biosensors that measure the
PPI-induced redistribution of fluorescently labeled protein partners.
Perhaps one of the most successful discovery examples of PPI inhibitors
has been with p53 and MDM2. The p53 tumor suppressor is a transcriptional
activator that regulates the expression of target genes involved in processes that
serve to restrict the initiation, progression, or survival of cancer cells. p53
controls cell cycle arrest, DNA damage repair, apoptosis, senescence, metasta-
sis, and angiogenesis. In more than half of all human cancers, p53 is inactivated
by single-point missense mutation in the DNA-binding domain, resulting in
deficient regulation of p53 target genes. In a significant proportion of the
remaining cancers where wild-type p53 is functional, MDM2 is overexpressed
and blocks the tumor suppressor activity of p53. In this case, MDM2 binds to
the N-terminal transactivation domain of p53, thereby inhibiting activation of
p53 target genes. Because MDM2 contains a E3-ubiquitin-ligase activity, it also
tags p53 for degradation by the proteasome.36 The structure of the protein–
protein binding interfaces between p53 and MDM2 has been comprehensively
mapped and characterized; there are three amino acid residues in the p53 N-
terminus (Phe19, Trp23, and Leu26) that bind to a small hydrophobic pocket
on the surface of MDM2. This protein–protein binding interface was targeted
using a combination of structure-based drug design and an SPR assay that
measured the disruption of the p53-MDM2 binding.37 A cis-imidazoline com-
pound, called nutlin, was identified as a small molecule that occupied the
hydrophobic pocket on the surface of MDM2 disrupting the p53-MDM2
PPI. Nutlin caused stabilization of p53 in cells and suppressed tumor growth
in xenograft models.37 The initial success of nutlin has spawned several other
groups to investigate and identify small molecules that focus on disrupting the
p53–MDM2 interaction. Benzodiazepene analogs such as TDP665759 have
been shown to disrupt p53–MDM2 interactions in vitro with low micromolar
concentrations.36 They have also been found to suppress the growth of p53
wild-type tumor cells in culture. Treatment of normal mice with TDP665759
produces an increase in the p53-dependent transcript, cyclin-dependent kinase
inhibitor p21 in liver. It is therapeutically interesting that TDP665759 had a
synergistic growth-inhibitory effect with doxorubicin both in culture and in
xenografts. Potent spirooxindoles such as MI-215 have been synthesized that
inhibit the p53–MDM2 interaction in vitro in multiple tumor cell lines. MI-219
displays good pharmacokinetic and bioavailability properties in mice and
induces tumor regression without obvious reported untoward effects.
There is a vast array of potential PPI targets. It seems likely that the initial
success with p53-MDM2 PPI inhibitors will stimulate the field. There are
already efforts to generate PPI inhibitors for MYC-MAX38 and BCL2-BAX39
heterodimers. One would expect that some of these will enter advance preclin-
ical development in the near future. In addition, others are attempting to
produce inhibitors of protein–DNA interactions using analogs of natural anti-
biotics like netropsin and distamycin.40 These compounds might be able to
regulate transcription in tumor cells but the preclinical development of these
has been quite slow probably due to poor uptake of the compounds and the
lack of the requisite cancer selectivity.
Additional strategies are being pursued to reactivate p53 tumor suppressor
activity as potential cancer therapeutics. These include searching for com-
pounds that inhibit MDM2 ubiquitin E3 ligase activity or restore the thermal
stability and DNA-binding activity of p53 DNA-binding mutants. The core
domain of wild-type p53 is somewhat unstable, with a melting temperature of
44 C and a short half-life of nine minutes. Some of the known p53 mutations
add to the thermal instability of the protein, leading to the loss of DNA binding
and the p53 response. Small molecules that could stabilize p53 in its active
biological conformation and, thus, restore its DNA-binding functionality, could
potentially rescue wild-type p53 function. Proof-of-principle studies were
provided with antibodies that bind the C-terminus of p53 and with synthetic
peptides derived from the C-terminal domain.41 The antibodies and peptides
showed a stimulatory effect on the DNA-binding ability of p53. Because these
antibodies and peptides lack ideal pharmacological attributes, drug discovery
campaigns have been conducted to identify small molecules that would emu-
late the antibodies and peptides.
One compound, CP31398, was identified using an in vitro assay for confor-
mational refolding of mutant p53 to a wild-type shape.41 CP31398 induced the
expression of both reporter and endogenous p53 target genes. More recent
results suggest that CP31398 did not act as a PPI inhibitor but rather
26 JOHN S. LAZO
intercalated with DNA, altering and destabilizing the p53-DNA core domain
complex. CP31398 also decreased sequence-specific DNA binding of wild-type
p53 and the His273 mutant p53. It also produced cellular toxicity that was
independent of mutant p53. Therefore, there continues to be a challenge with
respect to reactivating mutant p53 with small molecules. If such compounds
were discovered, they will probably need to target a large fraction of the known
mutation observed in p53 to be clinically interesting.
IV. Other Contemporary Issues in Anticancer Drug Discovery

In addition to the complexities of anticancer drug discovery mentioned
above, it is useful to consider some of the other aspects that will affect how
future drugs will be identified and developed. There is enormous interest in the
potential role of fusing diagnostics with therapeutics leading to personalized
medicines. There is considerable debate about the statistical metrics that
should be used to distinguish a driver from a noncontributing passenger
mutation. Clearly, there is enormous complexity in the patterns of mutations
in tumors among individuals and even within an individual.
Every tumor harbors a complex combination of low-frequency mutations
thought to drive the cancer phenotype. Does this mean one will require
thousands of drugs to successfully treated tumors or are there chemosensitivity
nodes that can be exploited? Should we be seeking highly potent and selective
agents or multitargeted and promiscuous inhibitors? Highly potent and selective
enzyme inhibitors have been the mantra for cancer drug discoverers for the last
two decades. With the recognition that the complex signaling pathways often
have collateral participants that can function as redundant elements when the
targeted protein is inactivated, multitargeted drugs become more attractive.
What are the appropriate animal models for studying anticancer drugs? The
lack of reliable animal models for predicting the clinical efficacy of new anticancer
drugs has long plagued oncology.13,42,43 The most promising solution remains
highly controversial. While xenografts of human cancer cell lines have been useful
for determining the pharmacological properties of new agents, including their
pharmacokinetics, they have not been very successful in predicting drug effica-
cy.44 It is possible that orthotopic models will improve the predictability of human
xenografts. An extracellular matrix-embedded hollow fiber assay system has been
promoted as one convenient approach to emulate the three-dimensional tumor
microenvironment in vivo.45 Recent reports have argued in favor of genetically
engineered mouse models as being superior to xenograft approaches.46–48 Genet-
ically engineered mouse models of cancer appear to recapitulate many aspects of
sporadic human tumors. Nevertheless, the research community has been slow to
adopt genetically engineered mouse models of cancer for drug discovery. There
are several practical and philosophical reasons for this reluctance. One concern
with the genetically engineered mouse models is that the resulting tumors are
thought to be genetically too homogenous. While this uniformity may concep-
tually be beneficial for preclinical evaluation of drugs, some believe that the
tumors lack the complex genetic heterogeneity seen in human tumors, which
may be an important factor determining the responsiveness to therapy. Using
genetically engineered mouse models of cancer required the establishment of a
substantial infrastructure and commitment of resources. In general, there is the
need to use a larger number of mice than with xenograft models to generate the
appropriate preclinical dataset. Recent studies with conditional mutant KRAS-
driven non-small-cell lung cancer and pancreatic adenocarcinoma models illus-
trate the power and perhaps the superiority of genetically engineered mouse
models.48 The investigators examined standard-of-care agents both alone and in
combination with clinically used agents that block the Epidermal Growth Factor
receptor and the Vascular Endothelial Growth Factor using noninvasive imaging
methods and conventional survival endpoints. They found a concordance be-
tween the preclinical results and existing clinical data leading them to conclude
that the genetically engineered mouse platform model human disease well and
that they had predictive value for anti-cancer drug development.
V. Conclusions
Molecular biology has irreversibly altered the manner in which new anti-
cancer drugs are discovered. It has provided numerous validated molecular
targets that have been the subject of high throughput drug screens. It has also
provided reagents to conduct imaging-based cellular screens and to generate
new animal models of cancer. Although the inherent complexities of human
cancer remain, the new knowledge obtained about the processes permitting
the development of cancer should enable the creation of more efficacious drugs
for the treatment of hematological and solid tumors.
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Targeting Chemokine
(C-C motif) Ligand 2 (CCL2)
as an Example of Translation
of Cancer Molecular Biology
to the Clinic
Jian Zhang,*,{ Lalit Patel,*,{
and Kenneth J. Pienta*,{
*Department of Medicine, Michigan Center
for Translational Pathology and the
University of Michigan Comprehensive
Cancer Center, University of Michigan,
{
Department of Urology, Michigan Center
for Translational Pathology and the
University of Michigan Comprehensive
Cancer Center, University of Michigan,
I. Biology of CCL2 ............................................................................. 32

A. CCL2 Basics ............................................................................. 32
B. CCL2 Expression ....................................................................... 33
C. CCL2 Functions ........................................................................ 33
D. CCR2, The Functional Receptor for CCL2....................................... 34
E. Regulation of CCL2 and CCR2 ..................................................... 35
II. CCL2 in Prostate Cancer .................................................................. 37
A. Proliferation and Survival ............................................................. 37
B. Angiogenesis ............................................................................. 37
C. Migration, Invasion, and Metastasis ................................................ 38
D. Macrophage Infiltration ............................................................... 39
E. Osteoclast Recruitment and Activation ............................................ 39
III. CCL2 Development as a Therapeutic Target ......................................... 41
A. Preclinical Animal Models ............................................................ 41
B. Clinical Studies .......................................................................... 42
IV. Conflicting Reports on the Roles of CCL2 in Cancer ............................... 43
V. Conclusions ................................................................................... 43
References .................................................................................... 45
Chemokines are a family of small and secreted proteins that play pleiotropic
roles in inflammation-related pathological diseases, including cancer. Among
the identified 50 human chemokines, chemokine (C-C motif) ligand 2 (CCL2)
is of particular importance in cancer development since it serves as one of the
key mediators of interactions between tumor and host cells. CCL2 is produced

DOI: 10.1016/S1877-1173(10)95003-5 1877-1173/10 $35.00
32 ZHANG ET AL.
by cancer cells and multiple different host cells within the tumor microenvi-
ronment. CCL2 mediates tumorigenesis in many different cancer types. For
example, CCL2 has been reported to promote prostate cancer cell prolifera-
tion, migration, invasion, and survival, via binding to its functional receptor
CCR2. Furthermore, CCL2 induces the recruitment of macrophages and
induces angiogenesis and matrix remodeling. Targeting CCL2 has been
demonstrated as an effective therapeutic approach in preclinical prostate
cancer models, and currently, neutralizing monoclonal antibody against
CCL2 has entered into clinical trials in prostate cancer. In this chapter,
targeting CCL2 in prostate cancer will be used as an example to show
translation of laboratory findings from cancer molecular biology to the clinic.
I. Biology of CCL2
A. CCL2 Basics
Chemokines, a family of chemoattractant cytokines, are classified into four
sub-families as CXC, CC, CX3C, and C based upon the number and location of
the cysteine residues at the N-terminus of the protein. Chemokine (C-C motif)
ligand 2 (CCL2), also known as monocyte chemotactic protein-1 (MCP-1), is a
small, secreted protein that belongs to the CC chemokine family. CCL2 was
purified and cloned in 1989 from human gliomas and myelomonocytic cells by
two independent research groups based on its ability to chemoattract mono-
cytes.1,2 Subsequent to its cloning, it was confirmed that this protein was also
identical to the product of the human JE gene. The JE gene, originally identi-
fied in mouse fibroblasts, is a platelet-derived growth factor (PDGF)-inducible
gene. Since then, CCL2 has been shown to display chemoattractic activity for
not only monocytes, but also memory T cells, natural killer (NK) cells, and
perhaps dendritic cells, resulting in recruiting of these cells to sites of tissue
injury and inflammatory responses.3,4 The human CCL2 cDNA encodes a 99
amino acid residue precursor protein with a hydrophobic signal peptide of 23
amino acids and a mature peptide of 76 amino acids.5,6 The CCL2 gene is
located on the chromosome 17 where many of the genes of the CC chemokine
family are located. The mouse or rat CCL2 gene has about 75% homology to
humans. CCL2 functions through binding to a functional chemokine receptor
CCR2, although it also binds to CCR4.7 The roles of CCL2 have been impli-
cated in the pathogenesis of various diseases that associate with monocyte
infiltration, for example, rheumatoid arthritis, atherosclerosis, and multiple
types of cancer [see review in Ref. 8].
TARGETING CHEMOKINE (C-C MOTIF) LIGAND 2 (CCL2) 33
B. CCL2 Expression
CCL2 is expressed in a wide array of tissues. It can be produced by multiple
cell types, including fibroblasts, macrophages, lymphocytes, astrocytes, mast
cells, endothelial cells, and osteoblasts.1,9–14 In addition, CCL2 can also be
produced by a variety of human and murine malignant cells [see review in Refs.
15–17]. In prostate cancer, determined by immunohistochemical staining on a
human tissue microarray, CCL2 positive staining was located mostly in epithe-
lial and fibromuscular stromal cells18; however, CCL2 positive staining was also
observed in the extracellular areas surrounding neoplastic glands and epithelial
cells,18 suggesting both autocrine and paracrine production of CCL2 in the
tumor microenvironment. In at least one report, CCL2 expression levels
positively correlated with Gleason score (a measure of tumor aggressiveness)
and pathologic stages.18–20 CCL2 production has been determined by enzyme-
linked immunosorbent assay (ELISA) in conditioned media collected from
prostate cancer cell lines and compared to primary prostate epithelial cells.18
Prostate cancer cells produce higher amounts of CCL2 as compared to non-
malignant prostate epithelial cells.18 Higher production of CCL2 at the sites of
bone metastases was demonstrated by a clinically related report in which
Loberg et al. collected tumor bone metastatic and normal bone specimens
from vertebral lesions in three patients with prostate cancer.21 Total protein
lysates were isolated and analyzed by cytokine array. Elevated CCL2 produc-
tion was identified in the tumor-bone microenvironment compared to normal
bone microenvironment, suggesting that CCL2 plays a critical role in prostate
tumorigenesis in bone metastases.21 In vitro, it has been further reported that
human osteoblasts and bone marrow endothelial cells produced higher amount
of CCL2 compared to prostate epithelial cells.13 CCL2 can also be produced by
osteoclasts.22–25 Its production can be induced by Receptor Activator of NF-kB
ligand (RANKL) and tumor necrosis factor alpha (TNFa).22–25 To date, it
remains unclear which cell type(s) play a major role in the production of
CCL2 in the tumor microenvironment.
C. CCL2 Functions
CCL2 functions as a chemoattractant through binding to its receptor on
monocytes, macrophages, and lymphocytes [see review in Ref. 26]. The exis-
tence of CCL2/CCR2 axis has been validated using CCR2 knockout animals.27
In acute inflammatory response, CCL2 has been shown to actively recruit
monocytes to the site of inflammation.28,29 CCL2 also plays important roles
in T-cell immunity, and CCL2 expression is associated with Th2 response.30–32
For example, CCL2 is overproduced in an animal model of Th2 immune-
mediated asthma.33 CCL2 is a potent factor in the polarization of Th0 cells
toward a Th2 phenotype.34 It has been demonstrated that CCL2 induced
34 ZHANG ET AL.
interleukin-4 (IL-4) production through direct activation of IL-4 promoter in T

cells.35 CCL2 knockout mice demonstrated impaired Th2 immunity, but intact
Th1 immune response.36 There is a large body of evidence showing the crucial
roles of CCL2/CCR2 axis in various chronic inflammatory conditions that are
associated with macrophage infiltration such as atherosclerosis,37,38 multiple
sclerosis,39 rheumatoid arthritis,40 glomerulonephritis,41 pulmonary hyperten-
sion,42 and pulmonary fibrosis.43 It has been reported that CCL2 is expressed
at the site of tooth eruption and bone resorption.44 It has been shown that
CCL2 can induce osteoclast maturation and formation as represented by the
formation of Tartrate Resistant Acid Phosphatase (TRAP)-positive, multinucle-
ar cells in the absence of RANKL, but the produced osteoclasts lack the ability
to cause bone resorption.25,45 In addition, CCL2 has been reported to cause
the degranulation of basophiles and migration of mast cells.46,47 This effect can
be enhanced by pretreatment with IL-3 and other cytokines.48 In tumor devel-
opment, stimulation of infiltrating macrophages has been shown to augment
antitumor activity or promote tumor development, depending on the type of
cancer [see review in Ref. 49].
D. CCR2, The Functional Receptor for CCL2

CCR2, a G protein-coupled receptor, is the key functional receptor for
CCL2. The activation of the ligand-receptor binding leads to the activation of
intracellular signaling cascades that mediate chemotactic response. CCR2 has
both pro-inflammatory (mediated by APC and T cells) and anti-inflammatory
(mediated by regulatory T cells) effects [see review in Ref. 8]. CCR2-deficient
mice have been shown to have altered inflammatory responses in an allergic
asthma model.50
CCR2 can be expressed by both hematopoietic cells such as macrophages
and nonhematopoietic cells such as endothelial cells,51 fibroblasts,52 and mesen-
chymal stem cells.53 In prostate cancer, it has been shown that CCR2 expression
correlates with prostate cancer progression and metastasis as determined by in
situ immunohistochemical staining.54,55 Specifically, CCR2 expression correlated
with Gleason score and pathological stages; however, these published reports
were not able to distinguish which cell type(s) may produce CCR2 transcripts in
the metastatic sites. In prostate cancer cell lines, differential expression of CCR2
has been reported.21,54 In particular, more aggressive cancer cells express greater
levels of CCR2 compared with the less aggressive cancer cells or nonneoplastic
cells.54 These findings suggest that the CCL2/CCR2 axis may be a target for
prostate cancer treatment. It has been recognized that CCR2 antagonists are
potential therapeutic agents in preventing, treating, or ameliorating a CCR2-
mediated inflammatory syndrome or disease such as psoriasis, uveitis,
rheumatoid arthritis, multiple sclerosis, asthma, obesity, and chronic obstructive
pulmonary disease [see review in Ref. 56]. In a prostate cancer study, a CCR2
antagonist has been shown to diminish the prostate cancer cell proliferation and
invasion in vitro.18
Recently, CCR2 was reported as a key factor in balancing the bone
remodeling process.57 It was shown that CCR2 knockout mice had high bone
mass and stability (biomechanical properties by compression) due to a decrease
in number, size, and function of osteoclasts.57 RANK expression is diminished
in CCR2 knockout mice, and CCL2 enhances RANK expression via NFkB and
ERK1/2 pathways57; therefore, CCR2 could become a therapeutic target in
postmenopausal bone loss.
E. Regulation of CCL2 and CCR2

1. CCL2 REGULATION
CCL2 production is elevated in various diseases that are associated
with chronic inflammation and macrophage infiltration. Similar to other
inflammation-associated soluble factors, CCL2 production can be induced by
oxidative stress, cytokines, and growth factors. In prostate cancer, it has been
reported that serum CCL2 levels are elevated in patients with skeletal metas-
tases compared to localized prostate cancer.58 One of the key regulators has been
suggested to be parathyroid hormone-related protein (PTHrP), a 141-amino
acid protein that has limited homology to PTH, but binds the same receptor as
PTH with similar biological activity.59,60 It was also reported that PTHrP is
highly expressed in metastatic bone lesions, compared to a moderate expression
on localized prostate cancer tissues and cell lines.61–63 PTHrP has been shown to
enhance bone metastases in animal models of both prostate cancer64 and breast
cancer.65–68 It has been demonstrated that PTHrP treatment of osteoblastic
cells upregulates CCL213,69 which can be blocked by a PTHrP antagonist,70,71
suggesting that prostate cancer cell-derived PTHrP plays an important role in
elevation of osteoblast-derived CCL2. It was further demonstrated that PTHrP
induces CCL2 production in human bone marrow endothelial (HBME) cells.13
Investigation of the mechanisms through which CCL2 is upregulated in osteo-
blasts and HBME cells is needed to provide a better understanding of the roles
of tumor microenvironment in skeletal metastasis.
The CCL2 gene is regulated in a tissue-specific and stimulus-
specific manner.13 In its promoter region, there are a pair of C/EBP-binding
sites ( 2591 to 2579; 3118 to 3107) that are important for the response
to insulin activation of PI3K,72 a pair of NFkB-binding sites ( 2639 to 2630;
2612 to 2603) that are important for interleukin-1 (IL-1) and TNFa
stimulation, and a GC box ( 64 to 59) that binds Sp1 and is important for
CCL2 basal expression.73 Using CCL2 reporters that were transfected
into human fetal osteoblast (hFOB) cells, PTHrP induced CCL2 promoter
36 ZHANG ET AL.
activity in hFOB cells through NFkB and C/EBP activation13; however, it

remains unknown (a) whether PTHrP can also upregulate the CCL2 promot-
er-luciferase reporter set in the HBME cells, as well as in prostate cancer cell
lines, (b) whether PTHrP does so via NFkB and C/EBP activations as with
hFOB, (c) whether other stimuli such as IL-6 or TNFa or RANKL can also
upregulate this promoter in these cells, and, (d) if so, via which transcription
factor, NFkB and/or C/EBP. It is worthy to note that TNFa has been reported
to induce CCL2 expression in sensory neurons74–76 and RANKL induces
CCL2 expression in osteoclasts at transcriptional levels.25,57
Recently, a gene expression profile in individual human prostate cancer
specimens before and after exposure to chemotherapy (docetaxel treatment)
was determined.77 In that study, several genes, including CCL2, were upregu-
lated after chemotherapy treatment. In addition, docetaxel was shown to
induce CCL2 expression in prostate cancer cell lines in vitro. CCL2-specific
siRNA inhibited prostate cancer cell proliferation and enhanced the growth-
inhibitory effect of low-dose docetaxel. This protective effect of CCL2 was
associated with activation of the ERK/MAP kinase and PI3K/AKT.77 These
findings suggest a mechanism of chemotherapy resistance mediated by cellular
stress responses involving the induction of CCL2 expression and indicate that
inhibiting CCL2 activity could enhance therapeutic responses to taxane-based
therapy. To date, elevated serum CCL2 levels in cancer patients have not been
demonstrated to be specific enough to correlate with disease activity, tumor
burden, or response to therapy.
2. CCR2 REGULATION
Little is known about the regulation of the CCR2 gene in normal or
cancerous tissues. It is downregulated as monocytes move down the macro-
phage differentiation pathway while other related chemokine receptors are
not.78 IFNgþ M-CSF or PMA þ ionomycin downregulate CCR2 expression in
monocytes, and this can be replicated with a 1220/þ115 hCCR2 promoter-
pGL3 luciferase reporter.78 Peroxisome proliferator-activated receptor-gamma
(PPAR-g) ligands (i.e., Rosiglitazone) also downregulate CCR2 in circulating
monocytes, while cholesterol slightly upregulated CCR2.79 While proinflam-
matory cytokines rapidly reduce CCR2 expression in monocytes, they upregu-
late CCR2 expression in the brain.80 Constitutive tissue-specific expression of
CCR2 in THP-1 monocyte cells has been shown to be dependent upon a 31-bp
region ( 89 to 59) adjacent to the TATA box that contains an Oct-1 binding
site and a pair of tandem C/EBP binding sites located in the 50 UTR (þ 50 to
þ 77 bp).81 In addition to the Oct-1 and C/EBP binding sites that function in
monocyte CCR2 expression, the hCCR2 50 flank and UTR contains an array of
possible binding sites for PPAR/RXR, SREBP, GATA, STAT, NFAT, and AP-1.
It remains unknown whether these sequences are sufficient for positive
regulation in prostate cancer cells, but the monocyte expression of CCR2

suggests that they should function in osteoclast precursor cells, for example,
RAW264.7 cells.
II. CCL2 in Prostate Cancer

A. Proliferation and Survival
CCL2 has been shown to promote prostate cancer cell proliferation and
invasion in vitro via the phophatidylinositol 3-kinase (PI3K)/AKT signaling
pathway.18,21 CCL2 induces Akt phosphorylation in prostate cancer cells. In
addition, CCL2 stimulates p70-S6 kinase phosphorylation, a downstream tar-
get of Akt, resulting in actin rearrangement, a critical step in the formation of
the migratory phenotype of the tumor cells.21 Activation of p70-S6 kinase alters
the actin cytoskeleton microstructure82 and the binding of CCL2, and CCR2
has been linked to the actin skeleton through interactions with FOUNT, a novel
activator of C-C chemokines.83–85 Constitutive activation of this PI3K/AKT
pathway has been implicated in prostate cancer progression,86–89 and activation
of AKT pathway further induces survival benefits for the tumor cells.90 The
later protective roles of CCL2 have shown to upregulate survivin gene expres-
sion; therefore, CCL2 plays an important role for the survival of tumor cells,
possibly through reduction of autophagosome formation.90,91 Survivin has been
demonstrated to serve as a key molecule that protects the tumor cells from
autophagic death.90,92
B. Angiogenesis
Chemokines play an important role in the maintenance of hematopoietic
homeostasis, regulation of cell proliferation, tissue morphogenesis, and angio-
genesis.93 In human breast cancer, it was reported that CCL2 levels in the
excised breast cancer tissue were correlated significantly with the levels of
angiogenic factors, including vascular endothelial growth factor (VEGF), thy-
midine phosphorylase, TNFa, and IL-8.94 Transfection of colon cancer cells
with the CCL2 gene induces angiogenesis in a murine model.95 It has been
demonstrated that both CCL2 and VEGF expression positively correlates with
TAM infiltration and angiogenesis in breast cancer.96 In prostate cancer, it has
been reported that CCL2 induces tumor cells to produce the pro-angiogenic
factor VEGF-A, which indirectly induces sprout formation in human bone
marrow endothelial cells.71 In vivo, it has been shown that administration of
neutralizing antibody against CCL2 significantly reduces tumor blood vessel
density and decreases the prostate cancer tumor burden (Fig. 1)71,97; therefore,
CCL2 is a key mediator of tumor angiogenesis.
Other documents randomly have
different content
mandó que se volviesen á sus pueblos, y luego les mandó salir de
Méjico.
Dejemos á los mensajeros, que luego salieron, y los mejicanos
por tres partes con la mayor furia que hasta allí habiamos visto, y se
vienen á nosotros, y en todos tres reales nos dieron muy recia
guerra; y puesto que les heriamos y matábamos muchos dellos,
paréceme que deseaban morir peleando, y entónces cuando más
recios andaban con nosotros pié con pié peleando, nos decian:
—«Tenitoz Rey Castilla, Tenitoz Ajaca;» que quiere decir en su
lengua: «¿Qué dirá el Rey de Castilla? ¿Qué dirá ahora?»
Y con estas palabras tirar vara y piedra y flecha, que cubrian el
suelo y calzada.
Dejemos esto, que ya les íbamos ganando gran parte de la
ciudad, y en ellos sentiamos que, puesto que peleaban muy como
varones, no se remudaban ya tantos escuadrones como solian, ni
abrian zanjas ni calzadas; mas otra cosa tenian muy cierta, que al
tiempo que nos retraiamos nos venian siguiendo hasta nos echar
mano; y tambien se nos habia acabado ya la pólvora en todos tres
reales, y en aquel instante habia venido á la Villa-Rica un navío que
era de una armada de un licenciado Lúcas Vazquez de Aillon, que se
perdió y desbarató en las islas de la Florida, y el navío aportó á
aquel puerto, como dicho tengo, y venian en él ciertos soldados y
pólvora y ballestas y otras cosas; y el teniente que estaba en la Villa-
Rica, que se decia Rodrigo Rangel, que tenia en guarda á Narvaez,
envió luego á Cortés pólvora y ballestas y soldados.
Y volvamos á nuestra conquista, por abreviar: que mandó y
acordó Cortés con todos los demás capitanes y soldados que les
entrásemos todo cuanto pudiésemos hasta llegalles al Tatelulco, que
es la plaza mayor, adonde estaban sus altos cues y adoratorios; y
Cortés por su parte y Sandoval por la suya, y nosotros por la
nuestra, les íbamos ganando puentes y albarradas, y Cortés les
entró hasta una plazuela donde tenian otros adoratorios.
En aquellos cues estaban unas vigas, y en ellas muchas cabezas
de nuestros soldados que habian muerto y desbaratado en las
batallas pasadas, y tenian los cabellos y barbas muy crecidas, más
que cuando eran vivos, y no lo habia yo creido si no lo viera desde
tres dias, que como fuimos ganando por nuestra parte dos aberturas
y puentes, tuvimos lugar de las ver, é yo conocia tres soldados mis
compañeros; y cuando las vimos de aquella manera se nos saltaron
las lágrimas de los ojos; y en aquella sazon se quedaron allí donde
estaban, más desde á doce dias se quitaron, y las pusimos aquellas
y otras cabezas que tenian ofrecidas á otros ídolos, y las enterramos
en una iglesia que se dice ahora los Mártires, que nosotros hicimos.
Dejemos desto y digamos cómo fuimos batallando por la parte de
Pedro de Albarado y llegamos al Tatelulco, y habia tantos mejicanos
en guarda de sus ídolos y altos cues, y tenian tantas albarradas, que
estuvimos bien dos horas que no se lo pudimos tomar; y cómo
podian ya correr caballos, puesto que les hirieron á los más; mas
nos ayudaron muy bien y alancearon muchos mejicanos; y como
habia tantos contrarios en tres partes, fuimos las tres capitanías á
batallar con ellos; y á la una capitanía, que era de un Gutierre de
Badajoz, mandó Pedro de Albarado que subiese en el alto cu de
Huichilóbos, y peleó muy bien con los contrarios y muchos papas
que en las casas de los adoratorios estaban, y de tal manera le
daban guerra los contrarios, que le hacian venir las gradas abajo; y
luego Pedro de Albarado nos mandó que le fuésemos á socorrer y
dejásemos el combate en que estábamos; é yendo que íbamos, nos
siguieron los escuadrones con quien peleábamos, y todavía les
subiamos sus gradas arriba.
Aquí habia bien que decir en qué trabajo nos vimos los unos y los
otros en ganalles aquellas fortalezas, que ya he dicho otras veces
que eran muy altas; y en aquellas batallas nos tornaron á herir á
todos muy malamente, y todavía les pusimos fuego á los ídolos, y
levantamos nuestras banderas, y estuvimos batallando en lo llano,
despues de le haber puesto fuego, hasta la noche, que no nos
podiamos valer de tanto guerrero.
Dejemos de hablar en ello, y digamos que como Cortés y sus
capitanes vieron en aquella sazon desde sus barrios y calles en sus
partes léjos del alto cu, y las llamaradas en que el cu mayor ardia, y
nuestras banderas encima, se holgó mucho, y se quisieran hallar en
él; mas no podian, porque habian un cuarto de legua de la una parte
á la otra, y tenian muchas puentes y aberturas de agua por ganar, y
por donde andaba le daban recia guerra, y no podian entrar tan
presto como quisieran en el cuerpo de la ciudad; mas dende á
cuatro dias se juntó con nosotros, así Cortés como Sandoval, é
podiamos ir desde un real á otro por las calles y casas derrocadas y
puentes y albarradas deshechas y aberturas de agua todo ciego; y
en este instante se iban retrayendo Guatemuz con todos sus
guerreros en una parte de la ciudad dentro de la laguna, porque las
casas y palacios en que vivia ya estaban por el suelo; y con todo
esto, no dejaban cada dia de salir á nos dar guerra, y al tiempo de
retraer nos iban siguiendo muy mejor que de ántes; é viendo esto
Cortés, que se pasaban muchos dias, y no venian de paz ni tal
pensamiento tenian, acordó con todos nuestros capitanes que les
echásemos celadas.
Y fué desta manera: que de todos tres reales se juntaron hasta
treinta de á caballo y cien soldados los más sueltos y guerreros que
conocia Cortés, y envió á llamar de todos tres reales mil tlascaltecas,
y nos metimos en unas casas grandes que habian sido de un señor
de Méjico, y esto fué muy de mañana, y Cortés iba entrando con los
demás de á caballo que le quedaban, y sus soldados y ballesteros y
escopeteros por las calles y calzadas como solia; y ya llegaba Cortés
á una abertura y puente de agua, y entónces estaban peleando con
los escuadrones de mejicanos que para ello estaban aparejados, y
muchos más que Guatemuz enviaba para guardar la puente; y como
Cortés vió que habia gran número de contrarios, hizo que se retraia
y mandaba echar los amigos fuera de la calzada, porque creyesen
que de hecho se iban retrayendo; y le iban siguiendo al principio
poco á poco, y cuando vieron que de hecho hacia que iba huyendo,
van tras él todos los poderes que en aquella calzada le daban
guerra; y como Cortés vió que habia pasado algo adelante de las
casas á donde estaba la celada, tiraron dos tiros juntos, que era
señal de cuándo habiamos de salir de la celada, y salen los de á
caballo primero, y salimos todos los soldados y dimos en ellos á
placer; pues luego volvió Cortés con los suyos y nuestros amigos los
tlascaltecas, é hicieron gran matanza.
Por manera que se hirieron y mataron muchos, y desde allí
adelante no nos seguian al tiempo del retraer; y tambien en el real
de Pedro de Albarado les echó una celada, mas no tan buena como
esta; y en aquel dia no me hallé yo en nuestro real con Pedro de
Albarado por causa que Cortés me mandó que para la celada
quedase con él.
Dejemos desto, y digamos cómo estábamos ya en el Tatelulco, y
Cortés nos mandó que pasásemos todas las capitanías á estar con
él, é que allí velásemos, por causa que veniamos más de media
legua desde el real á batallar con los mejicanos; y estuvimos allí tres
dias sin hacer cosa que de contar sea, porque nos mandó que no les
entrásemos más en la ciudad ni les derrocásemos más casas, porque
les queria tornar á requerir con las paces; y en aquellos dias que allí
estuvimos en el Tatelulco envió Cortés á Guatemuz rogándole que se
diese y no hubiese miedo, y con grandes ofrecimientos que le
prometia que su persona seria muy acatada y honrada dél, y que
mandaria á Méjico y á todas sus tierras y ciudades como solia; y les
envió bastimentos y regalos, que eran tortillas y gallinas y cerezas y
tunas y caza, é que no tenian otra cosa; y el Guatemuz entró en
consejo con sus capitanes, y lo que le aconsejaron fué, que dijese
que queria paz, é que aguardarian tres dias, é que al cabo de los
tres dias se verian el Guatemuz y Cortés, y se darian los conciertos
de las paces; y en aquellos tres dias tenian tiempo de aderezar
puentes y abrir calzadas y adobar piedra y vara y flecha y hacer
albarradas; y envió Guatemuz cuatro mejicanos principales con
aquella respuesta; é creiamos que eran verdaderas las paces, y
Cortés les mandó dar muy bien de comer y beber, y les tornó á
enviar á Guatemuz, y con ellos les envió más refresco como de
ántes; y el Guatemuz tornó á enviar á Cortés otros mensajeros, y
con ellos dos mantas ricas, y dijeron que Guatemuz vernia para
cuando estaba acordado; y por no gastar más razones sobre el caso,
él nunca quiso venir, porque le aconsejaron que no creyese á Cortés,
y poniéndole por delante el fin de su tio el gran Montezuma y sus
parientes y la destruccion de todo el linaje noble de los mejicanos, é
que dijese que estaba malo, é que saliesen todos de guerra, é que
placeria á sus dioses, que les darian vitoria contra nosotros, pues
tantas veces se la habia prometido.
Pues como estábamos aguardando al Guatemuz y no venia, vimos
luego la burla que de nosotros hacia; y en aquel instante salian
tantos batallones de mejicanos con sus divisas, y dan á Cortés tanta
guerra, que no se podia valer; y otro tanto fué por nuestra parte de
nuestro real; pues en el de Sandoval lo mismo; y era de tal manera,
que parecia que entónces comenzaban de nuevo á batallar; y como
estábamos algo descuidados, creyendo que estaban ya de paz,
hirieron á muchos de nuestros soldados, y tres fueron heridos muy
malamente, y el uno dellos murió, y mataron dos caballos y hirieron
otros más; é ellos no se fueron mucho alabando, que muy bien lo
pagaron; y como esto vido Cortés, mandó que luego les tornásemos
á dar guerra y les entrásemos en su ciudad á la parte donde se
habian recogido; y cómo vieron que les íbamos ganando toda la
ciudad, envió Guatemuz á decir á Cortés que queria hablar con él
desde una gran abertura de agua, y habia de ser Cortés de la una
parte y el Guatemuz de la otra, y señalaron el tiempo para otro dia
de mañana; y fué Cortés para hablar con él, y no quiso Guatemuz
venir al puesto, sino envió á muchos principales, los cuales dijeron
que su señor Guatemuz no osaba venir por temor que cuando
estuviese hablando le tirarian escopetas y ballestas y le matarian; y
entónces Cortés les prometió con juramento que no les enojaria en
cosa ninguna, y no aprovechó, que no le creyeron.
En aquella sazon dos principales de los que hablaban con Cortés
sacaron de un fardalejo que traian tortillas é una pierna de gallina y
cerezas, y sentáronse muy de espacio á comer, porque Cortés los
viese y entendiese que no tenian hambre; y desde allí le envió á
decir á Guatemuz, que pues no queria venir, que no le daba nada y
que presto les entraria en todas sus casas, y veria si tenia maíz,
cuanto más gallinas; y desta manera se estuvieron otros cuatro ó
cinco dias que no les dábamos guerra; y en este instante se salian
de noche muchos pobres indios que no tenian qué comer, y se
venian al real de Cortés y al nuestro, como aburridos de hambre; y
cuando aquello vió Cortés, mandó que en bueno ni en malo no les
diésemos guerra, é que quizá se les mudaria la voluntad para venir
de paz, y no venian; y en el real de Cortés estaba un soldado que
decia el mismo que él habia estado en Italia en compañía del Gran
Capitan, y se halló en la chirinola de Garayana y en otras grandes
batallas, y decia muchas cosas de ingenios de la guerra, é que haria
un trabuco en el Tatelulco, con que en dos dias que con él tirase á la
parte y casas de la ciudad adonde el Guatemuz se habia retraido,
que las haria que luego se diesen de paz; y tantas cosas dijo á
Cortés sobre ello, que luego puso en obra hacer el trabuco, y
trajeron piedra, cal y madera de la manera que él la demandó, y
carpinteros y clavazon, y todo lo perteneciente para hacer el
trabuco, é hicieron dos hondas de recias sogas, y trujeron grandes
piedras, y mayores que botijas de arroba; é ya que estaba armado el
trabuco segun y de la manera que el soldado dió la órden, y dijo que
estaba bueno para tirar, y pusieron en la honda una piedra hechiza,
lo que con ella se hizo es, que no pasó adelante del trabuco, porque
fué por alto y luego cayó allí donde estaba armado; y desque
aquello vió Cortés hubo mucho enojo del soldado que le dió la órden
para que lo hiciese, y tenia pesar en sí mismo, porque él creido tenia
que no era para en la guerra ni para en cosa de afrenta, y no era
más de hablar, que se habia hallado de la manera que he dicho; y
segun el mismo soldado decia, que se decia Fulano de Sotelo,
natural de Sevilla, y luego Cortés mandó deshacer el trabuco.
Dejemos desto, y digamos que como vió que el trabuco era cosa
de burla, acordó que con todos doce bergantines fuese en ellos
Gonzalo de Sandoval por capitan general y entrase en el rincon de la
ciudad adonde se habia retraido Guatemuz, el cual estaba en parte
que no podian entrar en sus palacios y casas sino por el agua, y
luego Sandoval apercibió á todos los capitanes de los bergantines; y
lo que hizo diré adelante cómo y de qué manera pasó.
CAPÍTULO CLVI.
CÓMO SE PRENDIÓ GUATEMUZ.
Pues como Cortés vido que el trabuco no aprovechó cosa

ninguna, ántes hubo enojo con el soldado que le aconsejó que lo
hiciese, y viendo que no queria paces ningunas Guatemuz y sus
capitanes, mandó á Gonzalo de Sandoval que entrase con los
bergantines en el sitio y rincon de la ciudad adonde estaban
retraidos el Guatemuz con toda la flor de sus capitanes y personas
más nobles que en Méjico habia, y le mandó que no matase ni
hiriese á ningunos indios, salvo si no le diesen guerra, é que aunque
se la diesen, que solamente se defendiese, y no les hiciesen otro
mal, y que les derrocase las casas y muchas barbacanas que habian
hecho en la laguna; y Cortés se subió luego en el cu mayor del
Tatelulco para ver cómo entraba Sandoval con los bergantines, y les
fueron acompañando Pedro de Albarado y Luis Marin, y Francisco de
Lugo y otros soldados.
Y como el Sandoval entró con los bergantines en aquel paraje
donde estaban las casas de Guatemuz, cuando se vió cercado el
Guatemuz, tuvo temor no le prendiesen ó le matasen, y tenia
aparejadas cincuenta grandes piraguas para si se viese en aprieto
salvarse en ellas y meterse en unos carrizales, é ir desde allí á tierra,
y esconderse en unos pueblos de sus amigos; y asimismo tenia
mandado á los principales y gente de más cuenta que allí en aquel
rincon tenia, y á sus capitanes, que hiciesen lo mismo; y como
vieron que les entraban en las casas, se embarcan en las canoas, é
ya tenian metida su hacienda de oro y joyas y toda su familia, y se
mete en ellas, y tira la laguna adelante, acompañado de muchos
capitanes y principales; y como en aquel instante iba la laguna llena
de canoas, y Sandoval luego tuvo noticia que Guatemuz con toda la
gente principal se iba huyendo, mandó á los bergantines que
dejasen de derrocar casas y siguiesen el alcance de las canoas, é
que mirasen que tuviesen tino é ojo á qué parte iba el Guatemuz, y
que no le ofendiesen ni le hiciesen enojo ninguno, sino que
buenamente procurasen de le prender.
Y como un Garci-Holguin, que era capitan de un bergantin, amigo
de Sandoval, y era muy gran velero su bergantin, y llevaba buenos
remeros, le mandó que siguiese hácia la parte que le habian dicho
que iba el Guatemuz y sus principales y las grandes piraguas, y le
mandó que si le alcanzase, que no le hiciese mal ninguno más de
prendelle, y el Sandoval siguió por otra parte con otros bergantines
que le acompañaban; é quiso Dios Nuestro Señor que el Garci-
Holguin alcanzó á las canoas é grandes piraguas en que iba el
Guatemuz, y en el arte dél y de los toldos é piragua, y aderezo dél y
de la canoa, le conoció el Holguin y supo que era el grande señor de
Méjico, y dijo por señas que aguardasen, y no querian, y él hizo
como que les queria tirar con las escopetas y ballestas, y hubo el
Guatemuz miedo de ver aquello, y dijo:
—«No me tiren, que yo soy el Rey de Méjico y desta tierra, y lo
que te ruego es, que no me llegues á mi mujer ni á mis hijos, ni á
ninguna mujer, ni á ninguna cosa de lo que aquí traigo, sino que me
tomes á mí y me lleves á Malinche.»
Y como el Holguin le oyó, se gozó en gran manera y le abrazó, y
le metió en el bergantin con mucho acato, á él, á su mujer y á veinte
principales que con él iban, y les hizo asentar en la popa en unos
petates y mantas, y les dió de lo que traia para comer; y á las
canoas en que iba su hacienda no les tocó en cosa ninguna, sino
que juntamente las llevó con su bergantin; y en aquella sazon el
Gonzalo de Sandoval se puso á una parte para ver los bergantines, y
mandó que todos se recogiesen á él, y luego supo que Garci-Holguin
habia prendido al Guatemuz, y que le llevaba á Cortés; y como el
Sandoval lo supo, mandó á los remeros que llevaba en su bergantin
que remasen á la mayor priesa que pudiesen, y cuando alcanzó á
Holguin le dijo que le diese el prisionero, y el Holguin no se lo quiso
dar, porque dijo que él lo habia prendido, y no el Sandoval; y el
Sandoval dijo que así era verdad, y que él era general de los
bergantines, y que el Holguin venia debajo de su dominio é mando,
y que por ser su amigo se lo habia mandado, y tambien porque era
su bergantin muy ligero, más que los otros; é mandó que le
siguiesen y le prendiesen, y que al Sandoval, como á su general, le
habia de dar el prisionero; y el Holguin todavía porfiaba que no
queria; y en aquel instante fué otro bergantin á gran priesa á Cortés
á demandalle albricias, que, como dicho tengo, estaba muy cerca,
en el Tatelulco, mirando desde el cu mayor cómo entraba el
Sandoval; y entónces le contaron la diferencia que traia Sandoval
con el Holguin sobre tomalle el prisionero; y cuando Cortés lo supo,
luego despachó al capitan Luis Marin y á Francisco de Lugo para que
luego hiciesen venir al Gonzalo de Sandoval y al Holguin, sin más
debatir, é que trajese al Guatemuz, y á la mujer y familia con mucho
acato, porque él determinaria cúyo era el prisionero y á quien se
habia de dar la honra dello; y entre tanto que le fueron á llamar, hizo
aderezar Cortés un estrado lo mejor que pudo con petates y mantas
y otros asientos, y mucha comida de lo que Cortés tenia para sí, y
luego vino el Sandoval y Holguin con el Guatemuz, y le llevaron ante
Cortés; y cuando se vió delante dél le hizo mucho acato, y Cortés
con alegría le abrazó, y le mostró mucho amor á él y á sus
capitanes; y entónces el Guatemuz dijo á Cortés:
—«Señor Malinche, ya yo he hecho lo que estaba obligado en
defensa de mi ciudad y vasallos, y no puedo más; y pues vengo por
fuerza y preso ante tu persona y poder, toma luego ese puñal que
traes en la cinta y mátame luego con él.»
Y esto cuando se lo decia lloraba muchas lágrimas con sollozos, y
tambien lloraban otros grandes señores que consigo traia; y Cortés
le respondió con doña Marina y Aguilar, nuestras lenguas, y dijo muy
amorosamente que por haber sido tan valiente y haber vuelto y
defendido su ciudad se le tenia en mucho y tenia en más á su
persona, y que no es digno de culpa ninguna, é que ántes se lo ha
de tener á bien que á mal; é que lo que Cortés quisiera, fué que,
cuando iban de vencida, que porque no hubiera más destruicion ni
muerte en sus mejicanos, que vinieran de paz y de su voluntad; é
que pues ya es pasado lo uno y lo otro, y no hay remedio ni
enmienda en ello, que descanse su corazon y de sus capitanes; é
que mandará á Méjico y á sus provincias como de ántes lo solian
hacer; y Guatemuz y sus capitanes dijeron que se lo tenian en
merced; y Cortés preguntó por la mujer y por otras grandes señoras
mujeres de otros capitanes, que le habian dicho que venian con
Guatemuz; y el mismo Guatemuz respondió y dijo que habia rogado
á Gonzalo de Sandoval y á Garci-Holguin que les dejase estar en las
canoas en que estaban, hasta ver lo que el Malinche ordenaba; y
luego Cortés envió por ellas, y les mandó dar de comer de lo que
habia lo mejor que pudo en aquella sazon; y luego, porque era tarde
y queria llover, mandó Cortés á Gonzalo de Sandoval que se fuese á
Cuyoacoan, y llevase consigo á Guatemuz y á su mujer y familia y á
los principales que con él estaban; y luego mandó á Pedro de
Albarado y á Cristóbal de Olí que cada uno se fuese á sus estancias
y reales, y luego nosotros nos fuimos á Tacuba, y Sandoval dejó á
Guatemuz en poder de Cortés en Cuyoacoan, y se volvió á
Tepeaquilla, que era su puesto y real.
Prendióse Guatemuz y sus capitanes en 13 de Agosto, á hora de
vísperas, dia de señor San Hipólito, año de 1521, gracias á nuestro
Señor Jesucristo y á nuestra Señora la Vírgen Santa María, su
bendita Madre, amen.
Llovió, y tronó y relampagueó aquella noche, y hasta media
noche mucho más que otras veces.
Y como se hubo preso Guatemuz, quedamos tan sordos todos los
soldados, como si de ántes estuviera uno puesto encima de un
campanario y tañesen muchas campanas, y en aquel instante que
las tañian cesasen de las tañer; y esto digo al propósito, porque
todos los noventa y tres dias que sobre esta ciudad estuvimos, de
noche y de dia daban tantos gritos y voces é silbos, unos
escuadrones mejicanos apercibiendo los escuadrones y guerreros
que habian de batallar en la calzada, é otros llamando las canoas
que habian de guerrear con los bergantines y con nosotros en los
puentes, y otros apercibiendo á los que habian de hincar palizadas y
abrir y ahondar las calzadas y aberturas y puentes, y en hacer
albarradas, y otros en aderezar piedra y vara y flecha, y las mujeres
en hacer piedra rolliza para tirar con las hondas; pues desde los
adoratorios y casas malditas de aquellos malditos ídolos, los
atambores y cornetas, y el atambor grande y otras bocinas
dolorosas, que de continuo no dejaban de se tocar, y desta manera,
de noche y de dia no dejábamos de tener gran ruido, y tal, que no
nos oiamos los unos á los otros: y despues de preso el Guatemuz
cesaron las voces y el ruido, y por esta causa he dicho como si de
ántes estuviéramos en campanario.
Dejemos desto, y digamos cómo Guatemuz era de muy gentil
disposicion, así de cuerpo como de faiciones, y la cara algo larga y
alegre, y los ojos más parecian que cuando miraba que eran con
gravedad y halagüeños, y no habia falta en ellos, y era de edad de
veinte y tres ó veinte y cuatro años, y el color tiraba más á blanco
que al color y matiz de esotros indios morenos, y decian que su
mujer era sobrina de Montezuma, su tio, muy hermosa mujer y
moza.
Y ántes que más pasemos adelante, digamos en qué paró el
pleito del Sandoval y del Garci-Holguin sobre la prision de
Guatemuz; y es que, Cortés le dijo que los romanos tuvieron otra
contienda de la misma manera que esta, entre Mario y Lucio
Cornelio Sila, y esto fué cuando Sila trajo preso á Yugurta, que
estaba con su suegro el Rey Ibócos; y cuando entraba en Roma
triunfando de los hechos y hazañas heróicos, pareció ser que Sila
metió en su triunfo á Yugurta con una cadena de hierro al pescuezo,
y Mario dijo que no le habia de meter Sila, sino él; é ya que le metia,
que habia de declarar que el Mario le dió aquella facultad y le envió
por él para que en su nombre le llevase preso, y se le dió el Rey
Ibócos; pues que el Mario era capitan general y debajo de su mano
y bandera militaban, y el Sila, como era de los patricios de Roma,
tenia mucho favor; y como Mario era de una villa cerca de Roma,
que se decia Arpino, y advenedizo, puesto que habia sido siete veces
cónsul, no tuvo el favor que el Sila, y sobre ello hubo las guerras
civiles entre Mario y el Sila, y nunca se determinó á quién se habia
de dar la honra de la prision de Yugurta.
Volvamos á nuestro propósito, y es, que Cortés dijo que haria
relacion dello á su majestad, y á quien fuese servido de hacer
merced se le daria por armas, que de Castilla traerian sobre ello la
determinacion; y desde á dos años vino mandado por su majestad
que Cortés tuviese por armas en sus reposteros ciertos Reyes, que
fueron Montezuma, gran señor de Méjico; Cacamatzin, señor de
Tezcuco, y los señores de Iztapalapa y de Cuyoacoan y Tacuba, y
otro gran señor que decian que era pariente muy cercano del gran
Montezuma, á quien decian que de derecho le venia el reino y
señorio de Méjico, que era señor de Matalacingo y de otras
provincias; y á este Guatemuz, sobre que fué este pleito.
Dejemos desto, y digamos de los cuerpos muertos y cabezas que
estaban en aquellas casas adonde se habia retraido Guatemuz; y es
verdad, y juro amen, que toda la laguna y casas y barbacoas
estaban llenas de cuerpos y cabezas de hombres muertos, que yo no
sé de qué manera lo escribia.
Pues en las calles y en los mismos patios de Tatelulco no habia
otras cosas, y no podiamos andar sino entre cuerpos y cabezas de
indios muertos.
Yo he leido la destruicion de Jerusalen; mas si en ella hubo tanta
mortandad como esta yo no lo sé; porque faltaron en esta ciudad
gran multitud de indios guerreros, y de todas las provincias y
pueblos sujetos á Méjico que allí se habian acogido, todos los más
murieron; que, como he dicho, así el suelo y la laguna y barbacoas,
todo estaba lleno de cuerpos muertos, y hedia tanto, que no habia
hombre que sufrirlo pudiese; y á esta causa, así como se prendió
Guatemuz, cada uno de los capitanes se fueron á sus reales, como
dicho tengo, y aun Cortés estuvo malo del hedor que se le entró por
las narices en aquellos dias que estuvo allí en el Tatelulco.
Dejemos desto, y pasemos adelante, y digamos cómo los
soldados que andaban en los bergantines fueron los mejor librados é
hubieron buen despojo, á causa que podian ir á ciertas casas que
estaban en los barrios de la laguna, que sentian que habria oro, ropa
y otras riquezas, y tambien lo iban á buscar á los carrizales, donde lo
iban á esconder los indios mejicanos cuando les ganábamos algun
barrio y casa; y tambien porque, so color que iban á dar caza á las
canoas que metian bastimentos y agua, si topaban algunas en que
iban algunos principales huyendo á tierra firme para se ir entre ellos,
otomites, que estaban comarcanos, les despojaban de lo que
llevaban.
Quiero decir que nosotros los soldados que militábamos en las
calzadas y por tierra firme no podiamos haber provecho ninguno,
sino muchos flechazos y lanzadas y heridas de vara y piedra, á causa
que cuando íbamos ganando alguna casa ó casas, ya los moradores
dellas habian salido y sacado toda la hacienda que tenian, y no
podiamos ir por agua sin que primero cegásemos las aberturas y
puentes; y á esta causa he dicho en el capítulo que dello habla, que
cuando Cortés buscaba los marineros que habian de andar en los
bergantines, que fueron mejor librados que no los que batallábamos
por tierra; y así pareció claro, porque los capitanes mejicanos, y aun
el Guatemuz, dijeron á Cortés, cuando les demanda el tesoro del
gran Montezuma, que los que andaban en los bergantines habian
robado mucha parte dello.
Dejemos de hablar más en esto hasta más adelante, y digamos
que, como habia tanta hedentina en aquella ciudad, que Guatemuz
le rogó á Cortés que diese licencia para que se saliese todo el poder
de Méjico á aquellos pueblos comarcanos, y luego les mandó que así
lo hiciesen.
Digo que en tres dias con sus noches iban todas tres calzadas
llenas de indios é indias y muchachos, llenos de bote en bote, que
nunca dejaban de salir, y tan flacos y sucios é amarillos é hediondos,
que era lástima de los ver; y despues que la hubieron
desembarazado, envió Cortés á ver la ciudad, y estaban, como dicho
tengo, todas las casas llenas de indios muertos, y aun algunos
pobres mejicanos entre ellos, que no podian salir, y lo que purgaban
de sus cuerpos era una suciedad como echan los puercos muy flacos
que no comen sino yerba; y hallóse toda la ciudad arada, y sacadas
las raices de las yerbas que habian comido cocidas: hasta las
cortezas de los árboles tambien las habian comido.
De manera que agua dulce no les hallamos ninguna, sino salada.
Tambien quiero decir que no comian las carnes de sus mejicanos,
sino eran de los enemigos tlascaltecas y las nuestras que apañaban;
y no se ha hallado generacion en el mundo que tanto sufriese la
hambre y sed y contínuas guerras como esta.
Dejemos de hablar en esto, y pasemos adelante: que mandó
Cortés que todos los bergantines se juntasen en unas atarazanas
que despues se hicieron.
Volvamos á nuestras pláticas: que despues que se ganó esta
grande y populosa ciudad, y tan nombrada en el universo, despues
de haber dado muchas gracias á Nuestro Señor y á su bendita
Madre, ofreciendo ciertas promesas á Dios Nuestro Señor, Cortés
mandó hacer un banquete en Cuyoacan, en señal de alegrías de la
haber ganado, y para ello tenian ya mucho vino de un navío que
habia venido al puerto de la Villa-Rica, y tenia puercos que le
trujeron de Cuba; y para hacer la fiesta mandó convidar á todos los
capitanes y soldados que le pareció que era bien tener cuenta con
ellos en todos tres reales; y cuando fuimos al banquete no habia
mesas puestas, ni aun asientos para la tercia parte de los capitanes
y soldados que fuimos, y hubo mucho desconcierto, y valiera más
que no se hiciera, por muchas cosas no muy buenas que en él
acaecieron, y tambien porque esta planta de Noé hizo á algunos
hacer desatinos, y hombres hubo en él que, despues de haber
comido, anduvieron sobre las mesas, que no acertaban á salir al
patio; otros decian que habian de comprar caballos con sillas de oro,
y ballesteros hubo que decian que todas las saetas que tuviesen en
su aljaba que habian de ser de oro, de las partes que les habian de
dar, y otros iban por las gradas rodando abajo.
Pues ya que habian alzado las mesas, salieron á danzar las damas
que habia, con los galanes cargados con sus armas, que era para
reir, y fueron las damas pocas, que no habia otras en todos los
reales ni en la Nueva-España; é dejo de nombrarlas por sus nombres
é de referir cómo otro dia hubo sátira; porque quiero decir que,
como hubo cosas tan malas en el convite y en los bailes, el buen
fraile fray Bartolomé de Olmedo lo murmuraba, é le dijo á Sandoval
lo mal que le parecia, é que bien dábamos gracias á Dios para que
nos ayudase adelante; é el Sandoval tan presto le dijo á Cortés lo
que fray Bartolomé murmuraba é gruñia, y el Cortés, que era
discreto, le mandó llamar é le dijo:
—«Padre, no excusaba solazar y alegrar los soldados con lo que
vuestra reverencia ha visto é yo he hecho de mala gana; ahora resta
que vuestra reverencia ordene una procesion, y que diga Misa é nos
predique, y diga á los soldados que no roben las hijas de los indios,
y que no hurten ni riñan pendencias é que hagan como católicos
cristianos, para que Dios nos haga bien.»
É fray Bartolomé se lo agradeció á Cortés; que no sabia lo que
habia dicho Albarado, y pensaba que salia del buen Cortés, su
amigo; y el fraile hizo una procesion, en que íbamos con nuestras
banderas levantadas y algunas cruces á trechos, y cantando las
letanías, y á la postre una imágen de nuestra Señora; y otro dia
predicó fray Bartolomé, é comulgaron muchos en la Misa despues de
Cortés y Albarado, é dimos gracias á Dios por la vitoria.
Y dejemos de más hablar en esto, y quiero decir otras cosas que
pasaron que se me olvidaba, y aunque no vengan ahora dichas sino
algo atrás, sin propósito; y es, que nuestros amigos Chichimecatecle
y los dos mancebos Xicotengas, hijos de D. Lorenzo de Vargas, que
se solia llamar Xicotenga el viejo y ciego, guerrearon muy
valientemente contra el poder de Méjico, y nos ayudaron muy
esforzada y extremadamente de bien; y asimismo un hermano del
señor de Tezcuco D. Hernando, que se decia Suchel, que despues se
llamó don Cárlos; este hizo cosas de muy esforzado y valiente varon;
y otro capitan natural de una ciudad de la laguna, que no se me
acuerda su propio nombre, tambien hacia maravillas, y otros muchos
capitanes de pueblos que nos ayudaban, todos guerreaban muy
poderosamente; y Cortés les habló y les dió muchas gracias y loores
porque nos habian ayudado, con muchas buenas palabras y
promesas de que el tiempo andando les daria tierras y vasallos y les
haria grandes señores, y les despidió; y como estaban ricos de ropa
de algodon y oro, y otras muchas cosas ricas de despojos, se fueron
alegres á sus tierras, y aun llevaron hartas cargas de tasajos
cecinados de indios mejicanos, que repartieron entre sus parientes y
amigos, y como cosas de sus enemigos, la comieron por fiestas.
Agora, que estoy fuera de los recios combates y batallas de los
mejicanos, que con nosotros, y nosotros con ellos teniamos de
noche y de dia, por que doy muchas gracias á Dios, que dellas me
libró, quiero contar una cosa muy temeraria que me acaeció, y es,
que despues que vide abrir por los pechos y sacar los corazones y
sacrificar á aquellos sesenta y dos soldados que dicho tengo que
llevaron vivos de los de Cortés, ofrecelles los corazones á los ídolos,
y esto que agora diré, les parece á algunas personas que es por falta
de no tener muy grande ánimo; y si bien lo consideran, es por el
demasiado ánimo con que en aquellos dias habia de poner mi
persona en lo más recio de las batallas, porque en aquella sazon
presumia de buen soldado y era tenido en esta reputacion, y habia
de hacer lo que más osados y atrevidos soldados suelen hacer, y en
aquella sazon yo hacia delante de mis capitanes; y como de cada dia
via llevar á nuestros compañeros á sacrificar, y habia visto, como
dicho tengo, que les aserraban por los pechos y sacalles los
corazones bullendo, y cortalles piés y brazos, y se los comieron á los
sesenta y dos que dicho tengo, temia yo que un dia que otro habian
de hacer de mí lo mismo, porque ya me habian asido dos veces, y
quiso Dios que me escapé; y acordóseme de aquellas muertes, y por
esta causa desde entónces temí desta cruel muerte; y esto he dicho
porque ántes de entrar en las batallas se me ponia por delante una
como grima y tristeza grandísima en el corazon; y encomendándome
á Dios y á su bendita Madre nuestra Señora, y entrar en las batallas,
todo era uno, y luego se me quitaba aquel temor, y tambien quiero
decir qué cosa tan nueva era agora tener yo aquel temor no
acostumbrado, habiéndome hallado en muchos rencuentros muy
peligrosos, ya habia de estar curtido el corazon y esfuerzo y ánimo
en mi persona agora á la postre más arraigado que nunca; porque,
si bien lo sé contar y traer á la memoria, desde que vine á descubrir
con Francisco Fernandez de Córdoba y con Grijalva, y volví con
Cortés, y me hallé en lo de la Punta de Cotoche y en lo de Lázaro,
que por otro nombre se dice Campeche, y en Potonchan y en la
Florida, segun que más largamente lo tengo escrito cuando vine á
descubrir con Francisco Fernandez de Córdoba.
Dejemos desto, y volvamos á hablar en lo de Grijalva y en la
misma de Potonchan, y con Cortés en lo de Tabasco y la de
Cingapacinga, y en todas las guerras y rencuentros de Tlascala y en
lo de Cholula, y cuando desbaratamos á Narvaez me señalaron para
que les fuésemos á tomar la artillería, que eran diez y ocho tiros que
tenian cebados y cargados con sus pelotas de piedra, los cuales les
tomamos, y este trance fué de mucho peligro; y me hallé en el
primer desbarate cuando los mejicanos nos echaron de Méjico, ó por
mejor decir, salimos huyendo cuando nos mataron en obra de ocho
dias ochocientos y cincuenta soldados; y me hallé en las entradas de
Tepeaca y Cachula y sus rededores, y en otros rencuentros que
tuvimos con los mejicanos cuando estábamos en Tezcuco sobre
coger las mielpas de maíz, y en lo de Iztapalapa cuando nos
quisieron anegar, y me hallé cuando subimos en los peñoles, y ahora
los llaman las fuerzas ó fortaleza que ganó Cortés, y en lo de
Suchimileco, é otros muchos rencuentros; y entré con Pedro de
Albarado con los primeros á poner cerco á Méjico, y les quebramos
el agua de Chalputepeque, y en la primera entrada que entramos en
la calzada con el mismo Pedro de Albarado; y despues desto, cuando
desbarataron por la misma nuestra parte y llevaron seis soldados
vivos, y á mí me llevaban, é ya se hacia cuenta que eran siete
conmigo, segun me llevaban engarrafado á sacrificar; y me hallé en
todas las demás batallas ya por mí memoradas, que cada dia y de
noche teniamos, hasta que vi, como dicho tengo, las crueles
muertes que dieron delante de mis ojos á aquellos sesenta y dos
soldados nuestros compañeros; ya he dicho que agora que por mí
habian pasado todas estas batallas y peligros de muerte, que no lo
habia de temer como lo temia agora á la postre.
Digan agora todos aquellos caballeros que desto del militar
entienden, y se han hallado en trances peligrosos de muerte, á qué
fin echarán mi temor, si es á mucha flaqueza de ánimo ó á mucho
esfuerzo; porque, como he dicho, sentia yo en mi pensamiento que
habia de poner por mi persona, batallando en parte que por fuerza
habia de temer la muerte más que otras veces, y por esto me
temblaba el corazon y temia la muerte; y todas aquestas batallas
que aquí he dicho donde me he hallado, verán en mi relacion en qué
tiempo y cómo y cuándo y dónde y de qué manera otras muchas
entradas y rencuentros tuvo Cortés y muchos de nuestros capitanes,
sin estos que aquí tengo dichos que no me hallé yo en ellos, porque
eran de cada dia tantos, que aunque fuera de hierro mi cuerpo, no
lo pudiera sufrir, en especial que siempre andaba herido y pocas
veces estaba sano, y á esta causa no podia ir á todas las entradas;
pues aun no han sido nada los trabajos y peligros y rencuentros de
muerte que de mi persona he recontado, que despues que ganamos
esta fuerte y gran ciudad pasé otros muchos, como adelante verán
cuando venga á coyuntura.
Y dejemos ya, y diré y declararé por qué he dicho en todas estas
guerras mejicanas cuando nos mataron nuestros compañeros, digo
lleváronlos, y no digo matáronlos, y la causa es esta: porque los
guerreros que con nosotros peleaban, aunque pudieran matar luego
á los que llevaban vivos de nuestros soldados, no los mataban luego,
sino dábanles heridas peligrosas porque no se defendiesen, y vivos
los llevaban á sacrificar á sus ídolos, y aun primero les hacian bailar
delante de Huichilóbos, que era su ídolo de la guerra; y esta es la
causa porque he dicho los llevaron.
Y dejemos esta materia, y digamos lo que Cortés hizo despues de
ganado Méjico.
CAPÍTULO CLVII.
CÓMO MANDÓ CORTÉS ADOBAR LOS CAÑOS DE CHALPUTEPEQUE, É OTRAS MUCHAS
COSAS.
La primera cosa que mandó Cortés á Guatemuz fué que adobasen

los caños del agua de Chalputepeque, segun y de la manera que
solian estar ántes de la guerra, é que luego fuese el agua por sus
caños á entrar en aquella ciudad de Méjico; é que luego con mucha
diligencia limpiasen todas las calles de Méjico de todas aquellas
cabezas y cuerpos de muertos, que todas las enterrasen, para que
quedasen limpias y sin que hubiese hedor ninguno en toda aquella
ciudad; y que todas las calzadas y puentes que las tuviesen tan bien
aderezadas como de ántes estaban, y que los palacios y casas que
las hiciesen nuevamente, y que dentro de dos meses se volviesen á
vivir en ellas; y luego les señaló Cortés en qué parte habian de
poblar, y la parte que habian de dejar desembarazada para en que
poblásemos nosotros.
Dejémonos agora destos mandados y de otros que ya no me
acuerdo, y digamos cómo el Guatemuz y todos sus capitanes dijeron
á nuestro capitan Cortés que muchos capitanes y soldados que
andaban en los bergantines, y de los que andábamos en las calzadas
batallando, les habiamos tomado muchas hijas y mujeres de algunos
principales; que le pedian por merced que se las hiciese volver; y
Cortés les respondió que serian muy malas de las haber de poder de
los compañeros que las tenian, y puso alguna dificultad en ello; pero
que las buscasen y trajesen ante él, é que veria si eran cristianas ó
si querian volver á casa de sus padres y de sus maridos, y que luego
se las mandaria dar; y dióles licencia para que las buscasen en todos
tres reales, é un mandamiento para que el soldado que las tuviese
luego se las diese si las indias se querian volver de buena voluntad
con ellos; y andaban muchos principales en busca dellas de casa en
casa, y eran tan solícitos, que las hallaron, y las más dellas no
quisieron ir con sus padres ni madres ni maridos, sino estarse con
los soldados con quien estaban, y otras se escondian, y otras decian
que no querian volver á idolatrar, y aun algunas dellas estaban ya
preñadas; y desta manera, no llevaron sino tres, que Cortés mandó
expresamente que las diesen.
Dejemos desto, y digamos que luego mandó hacer unas
atarazanas y fortaleza en que estuviesen los bergantines, y nombró
alcaide que estuviese en ellas, y paréceme que fué á Pedro de
Albarado, hasta que vino de Castilla un Salazar que se decia de la
Pedrada.
Digamos de otra materia: cómo se recogió todo el oro y plata y
joyas que se hubieron en Méjico, é fué muy poco, segun pareció,
porque todo lo demás hubo fama que lo mandó echar Guatemuz en
la laguna cuatro dias ántes que se prendiese; é que demás desto,
que lo habian robado los tlascaltecas y los de Tezcuco y Guaxocingo
y Cholula, y todos los demás de nuestros amigos que estaban en la
guerra; y demás desto, que los que andaban en los bergantines
robaron su parte; por manera que los oficiales del Rey decian y
publicaban que Guatemuz lo tenia escondido, y Cortés holgaba dello
de que no lo diese, por habello él todo para sí; é por estas causas
acordaron de dar tormento á Guatemuz y al señor de Tacuba, que
era su primo y gran privado; y ciertamente le pesó mucho á Cortés,
porque á un señor como Guatemuz, Rey de tal tierra, que es tres
veces más que Castilla, le atormentasen por codicia del oro, que ya
habian hecho pesquisas sobre ello, y todos los mayordomos de
Guatemuz decian que no habia más de lo que los oficiales del Rey
tenian en su poder, y eran hasta trecientos y ochenta mil pesos de
oro, porque ya lo habian fundido y hecho barras; y de allí se sacó el
real quinto, é otro quinto para Cortés; y como los conquistadores
que no estaban bien con Cortés vieron tan poco oro, y al tesorero
Julian de Alderete le decian algunos dellos que tenian sospecha que
por quedarse Cortés con el oro no querian que prendiesen al
Guatemuz ni le diesen tormento; y porque no le achacasen algo á
Cortés, y no lo podia excusar, consintió que le diesen tormento á
Guatemuz, como al señor de Tacuba; y lo que confesaron fué, que
cuatro dias ántes que le prendiesen lo echaron en la laguna, ansí el
oro como los tiros y escopetas y ballestas, y otras muchas cosas de
guerra que de nosotros tenian de cuando nos echaron de Méjico y
cuando desbarataron agora á la postre á Cortés; y fueron adonde
Guatemuz habia señalado, y entraron buenos nadadores y no
hallaron cosa ninguna; y lo que yo vi, que fuimos con el Guatemuz á
las casas donde solia vivir, y estaba una como alberca grande de
agua honda, y de aquella alberca sacamos un sol de oro como el
que nos hubo dado el gran Montezuma, y muchas joyas y piezas de
poco valor, que eran del mismo Guatemuz; y el señor de Tacuba dijo
que él tenia en unas casas suyas grandes, que estaban de Tacuba
obra de cuatro leguas, ciertas cosas de oro, é que le llevasen allá é
que diria dónde estaba soterrado y lo daria; y fué Pedro de Albarado
y seis soldados con él, é yo fuí en su compañía; y cuando llegamos
dijo que por morirse en el camino habia dicho aquello, é que lo
matasen, que no tenia oro ni joyas ningunas; y ansí nos volvimos sin
ello, y ansí se quedó, que no hubimos más oro que fundir; verdad es
que la recámara del Montezuma, que despues poseyó el Guatemuz,
no se habia llegado á muchas joyas y piezas de oro, que todo ello
tomó para que con ello sirviésemos á su majestad; y porque habia
muchas joyas de diversas hechuras y primas labores, y si me parase
á escribir cada cosa y hechura dello por sí, seria y es gran prolijidad,
lo dejaré de decir en esta relacion; mas dijeron allí muchas
personas, é yo digo de verdad, que valía dos veces más que la que
habia sacado para repartir el real quinto de su majestad; todo lo cual
enviamos al Emperador nuestro señor con Alonso de Ávila, que en
aquel tiempo vino de la isla de Santo Domingo, y con Antonio de
Quiñones; lo cual diré adelante cómo y dónde, en qué manera y
cuándo fueron.
Y dejemos de hablar dello y volvamos á decir que en la laguna,
donde decia Guatemuz que habia echado el oro, entré yo y otros
soldados á zabullidas, y siempre sacábamos pecezuelos de poco
precio, lo cual luego nos lo demandó Cortés y el tesorero Julian de
Alderete; y ellos mismos fueron con nosotros adonde lo habiamos
sacado, y llevaron consigo buenos nadadores, y sacaron obra de
noventa ó cien pesos de sartalejos de cuentas y ánades y perrillos y
pinjantes y collarejos y otras cosas de nonada, que ansí se puede
decir, segun habia la fama en la laguna del oro que de ántes habia
echado.
Dejemos de hablar desto, y digamos cómo todos los capitanes y
soldados estábamos algo pensativos de ver el poco oro que parecia
y las partecillas que dello nos daban; y el padre fray Bartolomé de
Olmedo, de la órden de la Merced, y Alonso de Ávila, que entónces
habia vuelto de la isla de Santo Domingo de cuando le enviaron por
procurador, y Pedro de Albarado y otros caballeros y capitanes,
dijeron á Cortés que, pues que habia poco oro, que las partes que
habian de caber á todos que las diesen y repartiesen á los que
quedaron mancos y cojos y ciegos y tuertos y sordos, y á otros que
se habian quemado con la pólvora, y á otros que estaban dolientes
de dolor de costado, que á aquellos les diese todo el oro, y que para
aquellos seria bien dárselo, é que todos los demás que estábamos
sanos lo habriamos por bien; y si esto le dijeron á Cortés, fué sobre
cosa pensada, creyendo que nos daria más que las partes que nos
venian, porque habia mucha sospecha que lo tenian escondido todo;
y lo que respondió fué, que veria las partes que cabian, é que visto,
en todo pondria remedio; y como todos los capitanes y soldados
queriamos ver lo que nos cabia de parte, dábamos priesa para que
se echase la cuenta y se declarase á qué tantos pesos saliamos; y
despues que lo hubieran tanteado, dijeron que cabian los de á
caballo á cien pesos, y á los ballesteros y escopeteros y rodeleros
que no se me acuerda bien; y de que aquellas partes nos señalaron,
ningun soldado lo quiso tomar; y entónces murmuramos de Cortés y
del tesorero Alderete, y el tesorero por descargarse decia que no
podia haber más, porque Cortés sacaba otro quinto del monton,
como el de su majestad, para él, y se pagaba de muchas costas de
los caballos que se habian muerto, y tambien dejaban de meter en
el monton otras muchas piezas que habiamos de enviar á su
majestad; y que riñésemos con Cortés, y no con él: y como en todos
tres reales habia soldados que habian sido amigos y paniaguados del
Diego Velazquez, gobernador de Cuba, de los que habian pasado
con Narvaez, que no estaban bien con Cortés, como vieron que no
les daban las partes del oro que ellos quisieran, no lo quisieron
recibir lo que les daban; y como Cortés estaba en Cuyoacan y
posaba en unos grandes palacios que estaban blanqueados y
encaladas las paredes, donde buenamente se podia escribir con
carbon y con otras tintas, amanecian cada mañana escritos motes,
unos en prosa y otros en versos, algo maliciosos, á manera como
masepasquines é libelos; y unos decian que el sol y la luna y el cielo
y estrellas y la mar y la tierra tienen sus cursos, é que si algunas
veces salen más de la inclinacion para que fueron criados más de
sus medidas, que vuelven á su ser, y que ansí habia de ser la
ambicion de Cortés en el mandar; y otros decian que más
conquistados nos traian que la misma conquista que dimos á Méjico,
y que no nos nombrásemos conquistadores de Nueva-España, sino
conquistados de Hernando Cortés; y otros decian que no bastaba
tomar buena parte del oro como general, sino tomar parte de quinto
como Rey, sin otros aprovechamientos que tenia; y otros decian:
—«¡Oh, qué triste está el alma mia hasta que la parte vea!»
Otros decian que Diego Velazquez gastó su hacienda é descubrió
toda la costa hasta Pánuco, y la vino Cortés á gozar; y decian otras
cosas como estas y aun decian palabras que no son para decir en
esta relacion.
Y como Cortés salia cada mañana y lo leia, y como estaban unas
chanzonetas en prosa y otras en metro, y por muy gentil estilo y
consonancia cada mote y copla á lo que iba inclinada y á la fin que
tiraba su dicho, y no como yo aquí lo digo; y como Cortés era algo
poeta, y se preciaba de dar respuestas inclinadas á loas de sus
heróicos hechos, y deshaciendo los del Diego Velazquez y Grijalva y
Narvaez, respondia tambien por buenos consonantes y muy á
propósito en todo lo que escribia; y de cada dia iban más
desvergonzados los metros, hasta que Cortés escribió:
—«Pared blanca, papel de nécios.»
Y amanecia más adelante:
—«Y aun de sábios y verdades.»
Y aun bien supo Cortés quién lo escribia, y fué un Fulano Tirado,
amigo de Diego Velazquez, yerno que fué de Ramirez el viejo, que
vivia en la Puebla, y un Villalóbos, que fué á Castilla, y otro que se
decia Mansilla, y otros que ayudaban de buena para Cortés á los
puntos que le tiraban; y de tal manera andaba la cosa, que fray
Bartolomé de Olmedo le dijo á Cortés que no permitiese que aquello
pasase adelante, sino que con cordura vedase que no escribiesen en
la pared.
Fué buen consejo, y mandó Cortés que no se atreviese ninguno á
poner letreros ni perques de malicias; que castigaria á los
desvergonzados que escribiesen con graves penas, y á fe que
aprovechó.
Dejemos desto, y digamos que, como habia muchas deudas entre
nosotros, que debiamos de ballestas á cuarenta y á cincuenta pesos,
y de una escopeta ciento, y de un caballo ochocientos, y mil, y á
veces más, y una espada cincuenta, y desta manera eran tan caras
las cosas que habiamos comprado; pues un cirujano que se llamaba
maestre Juan, que curaba algunas malas heridas y se igualaba por la
cura á excesivos precios, y tambien un médico que se decia Murcia,
que era boticario y barbero, tambien curaba; y otras treinta trampas
y zarrabusterías que debiamos, demandaban que les pagásemos de
las partes que nos daban; y el remedio que Cortés dió fué, que puso
dos personas de buena conciencia, que sabian de mercaderías, que
apreciasen qué podian valer las mercaderías y cosas de las que
habiamos tomado fiado, y que lo apreciasen; llamábanse los
apreciadores el uno Santa Clara, persona muy honrada, y el otro se
decia fulano de Llerena; y se mandó que todo aquello que aquellos
apreciadores dijesen que valía cada cosa de las que nos habian
vendido, y las curas que nos habian hecho los cirujanos, que
pasasen por ello; é que si no teniamos dineros, que aguardasen por
ello tiempo de dos años.
Otra cosa tambien se hizo: que todo el oro que se fundió echaron
tres quilates más de lo que tenia de ley, porque ayudasen á las
pagas, y tambien porque en aquel tiempo habian venido mercaderes
y navíos á la Villa-Rica, y creyendo que en echarle los tres quilates
más, que ayudasen á la tierra y á los conquistadores; y no nos
ayudó en cosa ninguna, ántes fué en nuestro perjuicio; porque los
mercaderes, porque aquellos tres quilates saliesen á la cabal de sus
ganancias, cargaban en las mercaderías y cosas que vendian cinco
quilates, y ansí anduvo el oro de tres quilates tepuzque, que quiere
decir en la lengua de indios cobre; y ansí agora tenemos aquel modo
de hablar, que nombramos á algunas personas que son
preeminentes y de merecimiento el señor don fulano de tal nombre,
Juan ó Martin ó Alonso, y otras personas que no son de tanta
calidad les decimos no más de su nombre, y por haber diferencia de
los unos á los otros, decimos á fulano de tal nombre tepuzque.
Volvamos á nuestra plática: que viendo que no era justo que el
oro anduviese de aquella manera, se envió á hacer saber á su
majestad para que se quitase y no anduviese en la Nueva-España; y
su majestad fué servido de mandar que no anduviese más, é que
todo lo que se le hubiese de pagar en almojarifazgo y penas de
cámara que se le pagase de aquel oro malo hasta que se acabase y
no hubiese memoria dello, y desta manera se llevó todo á Castilla.
Y quiero decir que en aquella sazon que esto pasó ahorcaron dos
plateros que falseaban las marcas y las echaban cobre puro.
Mucho me he detenido en contar cosas viejas y salir fuera de mi
relacion.
Volvamos á ella, y diré que, como Cortés vió que muchos
soldados se le desvergonzaban y le pedian más partes, y le decian
que se lo tomaba todo para sí, y le pedian prestados dineros, acordó
de quitar de sobre sí aquel dominio y de enviar á poblar á todas las
provincias que le pareció que convenia que se poblasen.
Á Gonzalo de Sandoval mandó que fuese á poblar á Tutepeque, é
que castigase unas guarniciones mejicanas que mataron cuando
salimos de Méjico sesenta personas, y entre ellas seis mujeres de
Castilla que allí habian quedado de los de Narvaez; é que poblase á
Medellin, é que pasase á Guacacualco é que poblase aquel puerto, y
tambien mandó que fuese á conquistar la provincia de Pánuco; y á
Rodrigo Rangel que se estuviese en la Villa-Rica, y en su compañía
Pedro de Ircio; y á Juan Velazquez Chico mandó que fuese á Colima,
y á un Villa-Fuerte á Zacatula, y Cristóbal de Olí que fuese á
Mechoacan; ya en este tiempo se habia casado Cristóbal de Olí con
una señora portuguesa, que se decia doña Filipa de Araujo; y envió
á Francisco de Orozco á poblar á Guaxaca, porque en aquellos dias
que habiamos ganado á Méjico, como lo supieron en todas estas
provincias que he nombrado que Méjico estaba destruida, no lo
podian creer los caciques y señores dellas, como estaban léjos, y
enviaban principales á dar á Cortés el parabien de las vitorias, y á
darse y ofrecerse por vasallos de su majestad, y á ver cosa tan
temida como dellos fué Méjico si era verdad que estaba por el suelo;
y todos traian grandes presentes de oro, que daban á Cortés, y aun
traian consigo á sus hijos pequeños, y les mostraban á Méjico, y
como solemos decir:
—«Aquí fué Troya;» y se lo declaraban.
Dejemos desto, y digamos una plática que es bien que se declare;
porque me dicen muchos curiosos letores que ¿qué es la causa que
los verdaderos conquistadores que ganamos la Nueva-España y la
grande y fuerte ciudad de Méjico, por qué no nos quedamos en ella
á poblar y nos veniamos á otras provincias? Tienen razon de lo
preguntar; quiero decir la causa por qué, y es esto que diré.
En los libros de la renta de Montezuma mirábamos de qué partes
le traian el oro, y dónde habia minas y cacao y ropa de mantas; y de
aquellas partes que veiamos en los libros que traian los tributos del
oro para el gran Montezuma, queriamos ir allá, en especial viendo
que salia de Méjico un capitan principal y amigo de Cortés, como era
Sandoval; y tambien como viamos que en todos los pueblos de la
redonda de Méjico no tenian minas de oro ni algodon ni cacao, sino
mucho maíz y maqueyales, de donde sacaban el vino, y á esta causa
la teniamos por tierra pobre, y nos fuimos á otras provincias á
poblar, y en todas fuimos muy engañados.
Acuérdome que fuí á hablar á Cortés que me diese licencia para
que fuese con Sandoval, y me dijo:
—«En mi conciencia, hermano Bernal Diaz del Castillo, que vivís
engañado; que yo quisiera que quedárades aquí conmigo; mas si es
vuestra voluntad ir con vuestro amigo Gonzalo de Sandoval, id en
buena hora, é yo tendré siempre cuidado de lo que se os ofreciere,
más bien sé que os arrepentireis por me dejar.»
Volvamos á decir de las partes del oro, que todo se quedó en
poder de los oficiales del Rey, por las esclavas que habiamos sacado
en las almonedas.
No quiero poner aquí por memoria qué tantos de á caballo ni
ballesteros ni escopeteros ni soldados, ni en cuantos dias de tal mes
despachó Cortés á los capitanes para que fuesen á poblar las
provincias por mí arriba dichas, porque seria larga relacion; basta
que digo pocos dias despues de ganado Méjico é preso Guatemuz, é
de ahí á otros dos meses envió otro capitan á otras provincias.
Dejemos ahora de hablar de Cortés, y diré que en aquel instante
vino al puerto de la Villa-Rica, con dos navíos, un Cristóbal de Tapia,
veedor de las fundaciones que se hacian en Santo Domingo, y otros
decian que era alcaide de aquella fortaleza que está en la isla de
Santo Domingo, y traia provisiones y cartas misivas de don Juan
Rodriguez de Fonseca, Obispo de Búrgos é se nombraba arzobispo
de Rosano, para que le diésemos la gobernacion de la Nueva-España
al Tapia; é lo que sobre ello pasó diré adelante.
CAPÍTULO CLVIII.
CÓMO LLEGÓ AL PUERTO DE LA VILLA-RICA UN CRISTÓBAL DE TAPIA QUE VENIA
PARA SER GOBERNADOR.
Pues como Cortés hubo despachado los capitanes y soldados por

mí ya dichos á pacificar y poblar provincias, en aquella sazon vino un
Cristóbal de Tapia, veedor de la isla de Santo Domingo, con
provisiones de su majestad, guiadas y encaminadas por D. Juan
Rodriguez de Fonseca, Obispo de Búrgos y Arzobispo de Rosano,
porque ansí se llamaba, para que le admitiesen á la gobernacion de
la Nueva-España; y demás de las provisiones, traia muchas cartas
misivas del mismo Obispo para Cortés y para otros muchos
conquistadores y capitanes de los que habian venido con Narvaez,
para que favoreciesen al Cristóbal de Tapia; y demás de las cartas
que traia cerradas y selladas del Obispo, traia otras en blanco para
que el Tapia en la Nueva-España pusiese todo lo que quisiese y le
pareciese, y en todas ellas traia grandes prometimientos que nos
haria muchas mercedes si dábamos la gobernacion al Tapia, y por
otra parte muchas amenazas, y decia que su majestad nos enviaria
á castigar.
Dejemos desto, que Tapia presentó sus provisiones en la Villa-
Rica de la Veracruz delante de Gonzalo de Albarado, hermano de
Pedro de Albarado, que estaba en aquella sazon por teniente de
Cortés, porque un Rodrigo Rangel, que solia estar allí por alcalde
mayor, no sé qué desatinos habia hecho cuando allí estaba, y le
quitó Cortés el cargo; y presentadas las provisiones, el Gonzalo de
Albarado las obedeció y puso sobre su cabeza como provisiones y
mando de su rey y señor; é que en cuanto al cumplimiento, que se
juntarian los alcaldes y regidores de aquella villa é que platicarian y
verian cómo y de qué manera eran ganadas y habidas aquellas
provisiones, é que todos juntos las obedecian, porque él solo era
una persona, y tambien porque querian ver si su majestad era
sabidor que tales provisiones enviasen; y esta respuesta no le
cuadró bien al Tapia, y aconsejáronle que se fuese luego á Méjico,
adonde estaban Cortés con todos los capitanes y soldados, y que
allá las obedecerian; y demás de presentar las provisiones, como
dicho tengo, escribió á Cortés de la manera que venia por
gobernador; y como Cortés era muy avisado, si muy buenas cartas
le escribió el Tapia, y vió las ofertas y ofrecimientos del Obispo de
Búrgos, y por otra parte las amenazas; si muy buenas palabras y
muy llenas de cumplimientos él le escribió, otras muy mejores y más
halagüeñas y blandosamente y amorosas y llenas de cumplimientos
le escribió Cortés en respuesta; y luego Cortés rogó y mandó á
ciertos de nuestros capitanes que se fuesen á ver con el Tapia, los
cuales fueron Pedro de Albarado y Gonzalo de Sandoval y Diego de
Soto el de Toro y un Valdenebro y el capitan Andrés de Tapia, á los
cuales envió á llamar por la posta que dejasen de poblar por
entónces las provincias en que estaban, é que fuesen á la Villa-Rica,
donde estaba el Cristóbal de Tapia, y con ellos mandó que fuese un
fraile que se decia fray Pedro Melgarejo de Urraca.
Ya que el Tapia iba camino de Méjico á se ver con Cortés,
encontró con nuestros capitanes y con el fraile por mí nombrados, y
con palabras y ofrecimientos que le hicieron, volvió del camino para
un pueblo que se decia Cempoal, y allí le demandaron que mostrase
otra vez las provisiones, y que verian cómo y de qué manera lo
mandaba su majestad, y si venia en ellas su real firma ó era sabidor
dello, é que los pechos por tierra las obedecerian en nombre de
Hernando Cortés y de toda la Nueva-España, porque traian poder
para ello; y el Tapia les tornó á notificar y mostrar las provisiones; y
todos aquellos capitanes á una las obedecieron y pusieron sobre sus
cabezas como provisiones de nuestro rey y señor, é que en cuanto al
cumplimiento, que suplicaban dellas para ante el Emperador nuestro
señor; y dijeron que no era sabidor dellas ni de cosa ninguna, é que
el Cristóbal de Tapia no era suficiente para ser gobernador, é que el
Obispo de Búrgos era contra todos los conquistadores que serviamos
á su majestad, y andaba ordenando aquellas cosas sin dar verdadera
relacion á su majestad, y por favorecer al Diego Velazquez, y al
Tapia por casar con uno dellos á una doña Fulana de Fonseca,
sobrina del mismo Obispo; y luego que el Tapia vió que no
aprovechaban palabras ni provisiones ni cartas de ofertas ni otros
cumplimientos, adoleció de enojo; y aquellos nuestros capitanes le
escribian á Cortés todo lo que pasaba, y le avisaron que enviase
tejuelos de oro y barras, é que con ellos amansaria la furia de Tapia;
lo cual el oro vino por la posta, y le compraron unos negros y tres
caballos y el un navío, y se volvió á embarcar en el otro navío y se
fué á la isla de Santo Domingo, de donde habia salido; é cuando allá
llegó, la audiencia real que en ella residia y los frailes jerónimos que
estaban por gobernadores notaron muy bien su vuelta de aquella
manera, y se enojaron con él porque ántes que saliese de la isla
para ir á la Nueva-España le habian mandado expresamente que en
aquella sazon no curase de venir, porque seria causa de quebrar el
hilo y conquistas de Méjico, y no les quiso obedecer; ántes, con
favor del Obispo de Búrgos, D. Juan Rodriguez de Fonseca, se
resolvió; que no osaban hacer otra cosa los oidores sino lo que el
Obispo de Búrgos mandaba, porque era presidente de Indias,
porque su majestad estaba en aquella sazon en Flandes, que no
habia venido á Castilla.
Dejemos esto del Tapia, y digamos cómo luego envió Cortés á
Pedro de Albarado á poblar á Tustepeque, que era tierra rica de oro.
Y para que bien lo entiendan los que no saben los nombres
destos pueblos, uno es Tutepeque, adonde fué Gonzalo de Sandoval,
y otro es Tustepeque, adonde en esta sazon va Pedro de Albarado; y
esto declaro porque no me culpen que digo que dos capitanes
fueron á poblar una provincia de un nombre, y son dos provincias; y
tambien habia enviado á poblar el rio de Pánuco, porque Cortés tuvo
noticia que un Francisco de Garay hacia grande armada para venirla
á poblar; porque, segun pareció, se lo habia dado su majestad al
Garay por gobernacion y conquista, segun más largamente lo he
dicho y declarado en los capítulos pasados cuando hablaba de todos
los navíos que envió adelante Garay, que desbarataron los indios de
la misma provincia de Pánuco, é hízolo Cortés porque si viniese el
Garay la hallase por Cortés poblada.

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Cancer Biology 4th Edition Raymond W. Ruddon M.D.

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International Review of Cell and Molecular Biology 1st

The Biology of Cancer 1st Edition Robert A. Weinberg

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The Biology of Cancer 2nd Edition Janice Ann Gabriel

Molecular Biology and

Raymond W. Ruddon, M.D., Ph.D.

AMSTERDAM • BOSTON • HEIDELBERG • LONDON

This book is printed on acid-free paper. ⬁

Copyright ß 2010, Elsevier Inc. All Rights Reserved

For information on all Academic Press publications

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Introduction to the Molecular Biology of Cancer:

Molecular Biology and Anticancer Drug Discovery. . . . . 9

Targeting Chemokine (C-C motif) Ligand 2 (CCL2) as an

Circulating Tumor Cells . . . . . . . . . . . . . . . . . . . . . . . 95

Stem Cells in Normal Development and Cancer . . . . . . 113

Progress in Cancer Nanotechnology. . . . . . . . . . . . . . . 193

Applications of Molecular Imaging . . . . . . . . . . . . . . . . 237

Molecular Targets and Clinical Cancer Risk Reductive

James R. Baker, Michigan Nanotechnology Institute for Medicine and

Istvan J. Majoros, Michigan Nanotechnology Institute for Medicine and

R. W. RUDDON, ANN ARBOR

1. Techniques of modern molecular biology: These include PCR, DNA

Progress in Molecular Biology Copyright 2010, Elsevier Inc.

DNA repair was important and that heritable conditions of defective

The explosion of knowledge about signal transduction mechanisms and

10. Regulation of gene expression: Current information on the packaging of

Several decades of advances in cancer cell molecular biology have led to a

pro-survival and pro-angiogenic factor that leads to metastasis.11 They have

As more is learned about the phenotype and genotype of cancer cells, it is

Multiple complex metabolic events characterize cancer development and

The profound impact of molecular biology on the philosophy of how one

Progress in Molecular Biology Copyright 2010, Elsevier Inc.

Phenotypic Proposed mechanism Drug or

Proliferation DNA damage, Cisplatin, 6,7

II. Phenotypic Targets

assistance of molecular biology tools). For example, methotrexate binds to its

III. Molecular Targets

systematically screened many of these seemingly ideal targets with large

C. Oncogene and Nononcogene Addiction

causes growth cessation or death when the antimitogenic or pro-apoptotic

expression profiling, and functional assays to deconvolute signatures that are

Gene A Gene B Phenotype

Drug A Gene A Phenotype

Drug A Drug B Phenotype

excellent example of nononcogene addiction of tumor cells with BRCA1 muta-

IV. Other Contemporary Issues in Anticancer Drug Discovery

I. Biology of CCL2 ............................................................................. 32

Progress in Molecular Biology Copyright 2010, Elsevier Inc.

interleukin-4 (IL-4) production through direct activation of IL-4 promoter in T

D. CCR2, The Functional Receptor for CCL2

E. Regulation of CCL2 and CCR2

activity in hFOB cells through NFkB and C/EBP activation13; however, it