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 MOLECULAR BIOLOGY INTELLIGENCE UNIT 15

Mikhail V. Blagosklonny
BLAGOSKLONNY

Cell Cycle Checkpoints

and Cancer
MBIU
15
Cell Cycle Checkpoints and Cancer
 MOLECULAR
BIOLOGY
INTELLIGENCE
UNIT 15

Cell Cycle Checkpoints

and Cancer

Mikhail V. Blagosklonny
Bethesda, Maryland, U.S.A.

LANDES BIOSCIENCE EUREKAH.COM

GEORGETOWN, TEXAS AUSTIN, TEXAS
U.S.A. U.S.A.
 CELL CYCLE CHECKPOINTS AND CANCER
Molecular Biology Intelligence Unit

Eurekah.com
Landes Bioscience

Copyright ©2001 Eurekah.com

All rights reserved.
No part of this book may be reproduced or transmitted in any form or by any means, electronic or
mechanical, including photocopy, recording, or any information storage and retrieval system,
without permission in writing from the publisher.
Printed in the U.S.A.
Please address all inquiries to the Publishers:
Eurekah.com / Landes Bioscience, 810 South Church Street
Georgetown, Texas, U.S.A. 78626
Phone: 512/ 863 7762; FAX: 512/ 863 0081
www.Eurekah.com
www.landesbioscience.com

ISBN: 1-58706-067-1

Library of Congress Cataloging-in-Publication Data

Cell cycle checkpoints and cancer / [edited by] Mikhail V. Blagosklonny.

p.; cm. -- (Molecular biology intelligence unit; 15)
ISBN 1-58706-067-1 (alk. paper)
1. Cancer cells--Regulation. 2. Cell cycle. 3. Cellular signal transduction. 4. Cell transformation.
5. Carcinogenesis.
[DNLM: 1. Cell Transformation, Neoplastic. 2. Cell Cycle. QZ 202 C39253
2001] I. Blagosklonny, Mikhail V. II. Series.
RC269.7 .C455 2001
616.99´407--dc21 2001002072
 CONTENTS
Preface ................................................................................................. xii
1. Autocrine Transformation: Cytokine Model .......................................... 1
James A. McCubrey, Xiao-Yang Wang, Paul A. Algate,
William L. Blalock and Linda S. Steelman
Abstract ................................................................................................. 1
Cytokine Regulation of Growth ............................................................ 1
2. Signal Transduction Pathways:
Cytokine Model ................................................................................... 17
James A. McCubrey, William L. Blalock, Fumin Chang,
Linda S. Steelman, Steven C. Pohnert, Patrick M. Navolanic,
John G. Shelton, Paul E. Hoyle, Phillip W. Moye,
Stephanie M. Oberhaus, Martyn K. White, John T. Lee
and Richard A. Franklin
Abstract ............................................................................................... 17
Cytokine-Induced Signal Transduction Resulting in Growth
and the Prevention of Apoptosis ...................................................... 17
Adaptor Proteins that Couple Receptors
with Downstream Pathways ............................................................ 19
The Jak-STAT Pathway ...................................................................... 19
The PI3K/Akt Pathway ....................................................................... 22
The Ras/Raf/MEK/ERK Signal Transduction Pathway ....................... 22
The Ras/Raf/MEK/ERK Pathway: Downstream Kinase Activation ..... 26
Interactions Between the Raf/MEK/ERK
and the PI3K/Akt Pathways ............................................................ 28
The Ras/Raf/MEK/ERK Pathway: A Tether Enhancing Signal
Transduction ................................................................................... 28
The Ras/Raf/MEK/ERK Pathway: Regulation of Downstream
Transcription Factors ...................................................................... 28
Induction of Autocrine Gene Expression by Altered Raf/MEK
and PI3K/Akt Expression ................................................................ 29
Mutations of Ras/Raf/MEK/ERK Cascade
which Result in Neoplasia ............................................................... 29
Regulatory Phosphatases of the Ras/Raf/MEK/ERK Pathway .............. 29
Alternative MAPK Pathways Activated by Stress ................................. 31
Default Pathways which Dampen Signaling ........................................ 31
Jak/STAT Inhibitors ........................................................................... 33
PI3K/p70S6K Inhibitors ..................................................................... 33
Ras/Raf/MEK/ERK Pathway Inhibitors .............................................. 33
PKC Inhibitors ................................................................................... 33
Cytokine Regulation of Cell Cycle Progression ................................... 34
Links Between the Ras/Raf/MEK/ERK Pathway
and Cell Cycle Proteins ................................................................... 34
Cytokine Regulation of Apoptosis and Cell Death .............................. 34
Apoptotic Mediators: The Caspases ..................................................... 34
 Roles of Bcl-2 Family Members in Cytokine-Mediated Regulation
of Apoptosis .................................................................................... 35
Mitochondrial Regulated Apoptosis .................................................... 35
Interactions Between Cytokine Signaling Pathways and Apoptosis ...... 36
Phosphorylation of Bcl-2: Positive and Negative Effects ...................... 36
Future Remarks ................................................................................... 36
Acknowledgments ............................................................................... 37
3. The Restriction Point of the Cell Cycle ................................................ 52
Mikhail V. Blagosklonny and Arthur B. Pardee
Mitogen-Dependent and -Independent Phases of the Cell Cycle ......... 52
The Restriction Point .......................................................................... 52
In Search of Mediators of the Restriction Point ................................... 53
Cyclins: From Mitogen Signaling to the Restriction Point .................. 54
The Restriction Point: a Knot of Mitogen and Inhibitory Signaling .... 55
Growth Arrest versus Proliferation ....................................................... 57
From Restriction- to “Check”-Points .................................................. 58
The Restriction Point and G1 Checkpoint .......................................... 59
The Restriction Point and Therapy ..................................................... 60

4. DNA Damage, Cell Cycle Control and Cancer .................................... 65

Jens Oliver Funk, Temesgen Samuel and H. Oliver Weber
Abstract ............................................................................................... 65
Introduction ........................................................................................ 65
Origins of DNA Damage .................................................................... 66
DNA Damage of Intrinsic Origin ....................................................... 66
DNA Damage of External Origin ........................................................ 66
Upstream DNA Damage Signaling ...................................................... 66
ATM-Dependent Signaling Pathways ................................................. 67
CHK2—The Next Line of Defense ..................................................... 67
p53—The Core of the DNA Damage Pathways ................................. 68
Regulatory Effects Converging on p53 ................................................ 69
The G1/S Checkpoint ......................................................................... 70
p21CIP1—A Two-Tailed Cell Cycle Regulator .................................. 70
The G2/M Checkpoint ....................................................................... 71
Control of the Unperturbed G2/M Transition .................................... 71
Regulation of the CDC25C Phosphatase ............................................ 72
DNA Damage and the G2/M Transition ............................................ 72
Links to Cancer and Genetic Instability .............................................. 73

5. DNA-Damage-Independent Checkpoints from Yeast to Man .............. 79

Duncan J. Clarke, Adrian P.L. Smith and Juan F. Giménez-Abián
Abstract ............................................................................................... 79
Budding Yeast versus Higher Eukaryotes ............................................. 79
S-Phase Checkpoint ............................................................................ 81
Topoisomerase II-Dependent Checkpoint ........................................... 86
 Checkpoint Control in Prophase ......................................................... 87
Spindle Assembly Checkpoint ............................................................. 87
Checkpoint Control of Mitotic Exit .................................................... 93
Oncological Implications of Mitotic Checkpoint Homologs ............... 99
6. The Regulation of p53 Growth Suppression ...................................... 106
Ronit Vogt Sionov, Igal Louria Hayon and Ygal Haupt
Abstract ............................................................................................. 106
Introduction ...................................................................................... 106
Regulation of p53 .............................................................................. 107
Regulation of Intracellular Distribution of p53 ................................. 110
p53-Mediated Growth Regulatory Functions .................................... 112
The Choice Between Growth Arrest and Apoptosis ........................... 115
Cell Type-Dependence ...................................................................... 116
7. Functional Interactions Between BRCA1 and the Cell Cycle ............. 126
Timothy K. MacLachlan and Wafik El-Deiry
Introduction ...................................................................................... 126
BRCA1 Protein and mRNA during the Cell Cycle ............................ 126
Subcellular Localization ..................................................................... 127
Activity at Cell Cycle Checkpoints .................................................... 129
Interactions with Cell Cycle Proteins ................................................. 130
Transcription of Cell Cycle Genes ..................................................... 131
Conclusion ........................................................................................ 132

8. The Role of FHIT in Carcinogenesis ................................................. 135

Yuri Pekarsky, Kay Huebner and Carlo M. Croce
Abstract ............................................................................................. 135
Chromosomal Changes in Cancer ..................................................... 135
FHIT Loci is the Target of Chromosomal Abnormalities at 3p14.2 .. 136
Inactivation of FHIT mRNA and Protein Expression in Cancer. ..... 137
The Tumor Suppressor Activity of FHIT .......................................... 138
Toward Fhit Function ....................................................................... 139
Conclusions ....................................................................................... 140

9. Hypoxia and Cell Cycle ...................................................................... 143

Rachel A. Freiberg, Susannah L. Green and Amato J. Giaccia
Introduction ...................................................................................... 143
Cell Cycle and Check Points ............................................................. 144
Hypoxia-Induced Arrest .................................................................... 146
Mechanisms Underlying Cell Cycle Arrest By Hypoxia ..................... 147
Hypoxia-Induced Inhibition of CDK2 Activity and Resistance
to Chemotherapy .......................................................................... 151
Acknowledgments ............................................................................. 152
10. G2 Checkpoint and Anticancer Therapy ............................................ 155
Zoe A. Stewart and Jennifer A. Pietenpol
Abstract ............................................................................................. 155
Introduction ...................................................................................... 155
G2 Checkpoint Activation ................................................................ 157
G2 Checkpoint Maintenance ............................................................ 162
Modulation of the G2 Checkpoint—Therapeutic Implications ......... 165
Future Directions .............................................................................. 169
Acknowledgments ............................................................................. 169

11. p53, Apoptosis and Cancer Therapy .................................................. 179

Rosandra Kaplan and David E. Fisher
Abstract ............................................................................................. 179
Introduction ...................................................................................... 179
p53’s Emergence as a Key Death Regulator ....................................... 181
Clinical Aspects of p53 ...................................................................... 183
Cell Cycle Arrest ............................................................................... 184
Apoptosis .......................................................................................... 184
Regulating p53 Activation in the Stress Response .............................. 187
Cell Cycle Arrest vs Death ................................................................. 187
Therapy ............................................................................................. 188
12. Non-Apoptotic Responses to Anticancer Agents:
Mitotic Catastrophe, Senescence and the Role of p53 and p21 .......... 196
Igor B. Roninson, Bey-Dih Chang and Eugenia V. Broude
Abstract ............................................................................................. 196
Can Apoptosis Account for Tumor Cell Response
to Anticancer Agents? .................................................................... 196
p53 as a Negative Regulator of Mitotic Catastrophe .......................... 199
Induction of Senescence by DNA-Damaging Agents ......................... 200
Role of p53 and p21 in Damage-Induced Senescence and Abnormal
Mitosis .......................................................................................... 202
Paracrine Activities of Senescent Cells: Implications for Treatment
Outcome and Side Effects of Cancer Therapy ............................... 203
Mitotic Catastrophe and Senescence as Target Responses in Cancer
Treatment ..................................................................................... 203

13. Small Molecule Inhibitors of Cyclin-Dependent Kinases ................... 208

Geoffrey I. Shapiro
Introduction ...................................................................................... 208
Flavopiridol ....................................................................................... 208
The Paullones .................................................................................... 219
Purine Derivatives ............................................................................. 220
UCN-01 ............................................................................................ 221
Novel Selective Cdk Inhibitors .......................................................... 226
Conclusion ........................................................................................ 228
14. Cell Cycle Molecular Targets and Drug Discovery ............................. 235
John K. Buolamwini
Abstract ............................................................................................. 235
Introduction ..................................................................................... 235
Events in Cell Cycle Progression ....................................................... 236
Regulatory Pathways ......................................................................... 237
Oncogenic Cell Cycle Targets ........................................................... 239
Cell Cycle Molecular Target-Based Cancer Drug Discovery .............. 239
Cancer Drug Development of Small Molecule CDK Inhibitors ........ 241
Other Targets .................................................................................... 241
Index .................................................................................................. 247
 EDITOR

Mikhail V. Blagosklonny
Bethesda, Maryland, U.S.A.
Chapter 3

CONTRIBUTORS
Paul A. Algate Fumin Chang
Corixa Corporation Department of Microbiology
Seattle Washington, U.S.A. and Immunology
Chapter 1 East Carolina University School
of Medicine
William L. Blalock Greenville, North Carolina, U.S.A.
Department of Microbiology Chapter 2
and Immunology
East Carolina University School Duncan J. Clarke
of Medicine, The Scripps Research Institute
Greenville, North Carolina, U.S.A. La Jolla, California, U.S.A.
Chapters 1, 2 Chapter 5

Eugenia V. Broude Carlo M. Croce

Department of Molecular Genetics Kimmel Cancer Center
University of Illinois Thomas Jefferson University
Chicago, Illinois, U.S.A. Philadelphia, Pennsylvania, U.S.A.
Chapter 12 Chapter 8

John K. Buolamwini Wafik El-Deiry

Department of Pharmaceutical Departments of Medicine, Genetics
Sciences, and Pharmacology
College of Pharmacy Howard Hughes Medical Institute,
University of Tennessee University of Pennsylvania School
Health Sciences Center, of Medicine
Memphis, Tennessee, U.S.A. Philadelphia, Pennsylvania, U.S.A.
Chapter 14 Chapter 7

Bey-Dih Chang David E. Fisher

Department of Molecular Genetics Division of Pediatric
University of Illinois Hematology/Oncology
Chicago, Illinois, U.S.A. Boston Children’s Hospital
Chapter 12 & Dana Farber Cancer Institute
Boston, Massachusetts, U.S.A.
Chapter 11
Richard A. Franklin Ygal Haupt
Department of Microbiology Lautenberg Center for General
and Immunology and Tumor Immunology
East Carolina University School The Hebrew University
of Medicine, Hadassah Medical School
Greenville, North Carolina, U.S.A. Jerusalem, Israel
Chapter 2 Chapter 6

Rachel A. Freiberg Igal Louria Hayon

Stanford University School of Medicine Lautenberg Center for General
Division Radiation Biology/ Department and Tumor Immunology
of Radiation Oncology Stanford, The Hebrew University
California, U.S.A. Hadassah Medical School
Chapter 9 Jerusalem, Israel
Chapter 6
Jens Oliver Funk
Laboratory of Molecular Tumor Biology Paul E. Hoyle
Department of Dermatology Department of Microbiology
University of Erlangen-Nuremberg and Immunology
Erlangen, Germany East Carolina University School
Chapter 4 of Medicine,
Greenville, North Carolina, U.S.A.
Amato J. Giaccia Chapter 2
Stanford University School of Medicine,
Division Radiation Biology/Dept. Rosandra Kaplan
Radiation Oncology Department of Medicine
Stanford, California, U.S.A. Boston Children’s Hospital
Chapter 9 Boston, Massachusetts, U.S.A.
Chapter 11
Juan F. Giménez-Abián
Centro de Investigaciones Biológicas Ronit Vogt Sionov
Consejo Superior de Investigaciones Lautenberg Center for General
Científicas and Tumor Immunology
Velázquez, Madrid, Spain The Hebrew University
Chapter 5 Hadassah Medical School
Jerusalem, Israel
Susannah L. Green Chapter 6
Stanford University School of Medicine
Division Radiation, Biology/Dept. John T. Lee
Radiation Oncology Department of Microbiology
Stanford, California, U.S.A. and Immunology
Chapter 9 East Carolina University
School of Medicine,
Greenville, North Carolina U.S.A.
Chapter 2
Timothy K. MacLachlan Jennifer A. Pietenpol
Departments of Medicine Department of Biochemistry
Genetics and Pharmacology Center in Molecular Toxicology
Howard Hughes Medical Institute and the Vanderbilt-Ingram
University of Pennsylvania School Cancer Center
of Medicine, Philadelphia Vanderbilt University
Pennsylvania, U.S.A. School of Medicine,
Chapter 7 Nashville, Tennessee, U.S.A.
Chapter 10
James McCubrey
Department of Microbiology Steven C. Pohnert
and Immunology Department of Biochemistry
East Carolina University School East Carolina University School
of Medicine, of Medicine,
Greenville, North Carolina, U.S.A. Greenville, North Carolina, U.S.A.
Chapters 1, 2 Chapter 2

Phillip W. Moye Igor B. Roninson

Department of Microbiology Department of Molecular Genetics
and Immunology University of Illinois
East Carolina University School Chicago, Illinois, U.S.A.
of Medicine, Chapter 12
Greenville, North Carolina, U.S.A.
Chapter 2 Temesgen Samuel
Laboratory of Molecular Tumor Biology
Patrick M. Navolanic Department of Dermatology
Department of Microbiology University of Erlangen-Nuremberg
and Immunology Erlangen, Germany
East Carolina University School Chapter 4
of Medicine,
Greenville, North Carolina, U.S.A. Geoffrey I. Shapiro
Chapter 2 Department of Adult Oncology
and Lowe Center for Thoracic
Stephanie M. Oberhaus Oncology
Department of Microbiology Dana-Farber Cancer Institute
and Immunology and Department of Medicine
East Carolina University School Brigham and Women’s Hospital
of Medicine, Greenville Harvard Medical School
North Carolina, U.S.A. Boston, Massachusetts, U.S.A.
Chapter 2 Chapter 13

Arthur B. Pardee John G. Shelton

Dana Farber Cancer Institute Department of Microbiology
Harvard Medical School and Immunology
Boston, Massachusetts, U.S.A. East Carolina University School
Chapter 3 of Medicine,
Greenville, North Carolina, U.S.A.
Chapter 2
Adrian P.L. Smith Xiao-yang Wang
The Scripps Research Institute Department of Microbiology
La Jolla, California, U.S.A. and Immunology
Chapter 5 East Carolina University School
of Medicine,
Linda S. Steelman Greenville, North Carolina, U.S.A.
Department of Microbiology Chapter 1
and Immunology
East Carolina University School H. Oliver Weber
of Medicine, Laboratory of Molecular Tumor Biology
Greenville, North Carolina, U.S.A. Department of Dermatology
Chapters 1,2 University of Erlangen-Nuremberg
Erlangen, Germany
Zoe A. Stewart Chapter 4
Department of Biochemistry
Center in Molecular Toxicology, Martyn K. White
Vanderbilt-Ingram Cancer Center, Department of Pathology, Anatomy and
Vanderbilt University School Cell Biology
of Medicine, Jefferson Medical College,
Nashville, Tennesee, U.S.A. Philadelphia, Pennsylvania , U.S.A.
Chapter 10 Chapter 2
 PREFACE
Advances in Research and Challenges in Therapy
Mikhail V. Blagosklonny

T
he ultimate goal of cancer research is the development of effective anticancer
therapy. During the last several decades, the discovery of oncogenes, tumor
suppressors, growth factors, signal transduction pathways has dramatically
escalated our understanding of cancer cell biology and mechanisms of cell
transformation.1-3 Hundreds of cellular proteins and pathways have been logically
considered as molecular targets in a mechanism-based approaches of anticancer drug
development.4-6
Yet, the progress in cancer treatment has not paralleled these dramatic achieve-
ments in basic research. Certainly, a delay must exist between identification of mo-
lecular targets and their clinical applications. However in many other fields of medi-
cine, effective drugs had been found prior to identification of their molecular mecha-
nisms, such as aspirin, anti-malaria drugs and antibiotics. Vaccination against viruses
such as smallpox had been developed almost two centuries before the immune system
and viruses were described. The most relevant parallel to anticancer drug
development is the discovery of antibiotics. Penicillin had revolutionized the
treatment of bacterial diseases long before its molecular target was identified. The
bacterial wall, a structure that does not exist in human cells, allows penicillin to kill a
bacteria without affecting a human cell, thus exercising dramatic selectivity. In this
light, the absence of the magic bullet against cancer is consistent with the lack of a
cancer-selective target. We have learned that there are very few molecules in cancer
cells that are dispensable or absent in normal cells. One of these few, Bcr-Abl, is a
selective target in Bcr-Abl-positive leukemia, even though molecular therapeutics that
inactivate Bcr-Abl have additional targets 7 Mutant p53 is another example of a
potential cancer-selective target.8, 9 Although telomerase exists in normal cells, its
functional significance in cancer cells allows us to consider this enzyme as a
reasonably-selective target. 10 However, these and other examples do not alter the gen-
eral conclusion: proto-oncogenes and signal transduction molecules are required for
proliferation and survival of normal cells and therefore most mechanism-based thera-
peutics (e.g., inhibitors of kinases) will be also toxic for certain normal cells. The
absence of cancer-selective targets is the most important problem of the anticancer
drug-screen, because compounds toxic to cancer cells also kill normal cells, therefore
side-effects are inevitable. Besides, natural compounds are synthesized by microor-
ganisms, plants and animals in order to kill other organisms. They are not intended to
discriminate between normal and cancer cells and cannot selectively kill cancer cells.
In light of the low probability of finding a “magic bullet”, it is not surprising that
alternative approaches emerge. These range from the targeting of endothelial cells to
protection of normal cells, from a selective delivery of drugs using tissue-specific
markers to exploiting hypoxia and drug-resistance.
 Loss of cell cycle checkpoints is the most universal alteration in human can-
cer.11-13 Furthermore, as emphasized by Hanahan and Weinberg, the large and
diverse collection of cancer-associated genes can be tied to the operations of a small
group of regulatory circuits. 2 In other words, although numerous genetic alterations
may cause loss of normal checkpoints, common strategies might be developed against
a wide variety of cancers. As suggested by Paul Nurse, this would present a more
promising approach than unspecific attempts to block cell cycle progression, which
are less likely to distinguish between cancerous and normal cells.14 Aiming at defective
cell cycle checkpoints is different from targeting cancer-specific molecules. In the check-
point approach, it is not necessary to target cancer-promoting or key-functional mol-
ecules (e.g., CDK), nor the molecule which is altered in cancer (mutated, overexpressed,
etc). A target may lie upstream of the affected function or may belong to parallel
pathways. Although the same molecule will be targeted in both cancer and normal
cells, the functional outcome can be different in cells with defective checkpoints. For
example, loss of the G1 checkpoint is common in cancer cells with mutant p53. In
response to DNA damage, such cancer cells are arrested in G2. The arrest at G2/M is
dramatically sensitive to even one double strand break because failure to arrest would
lead to the irreversible loss of chromosome fragments.15 Since G2 arrest in cells lack-
ing p53 depends on the Chk1 kinase, inhibition of this kinase results in abrogation of
the G2 checkpoint exclusively in cancer cells lacking p53.16-18 Following treatment
with DNA damaging drugs, mitotic progression of cancer cells will result in selective
killing of cells with defective checkpoints.19, 20
Even currently used chemotherapy, such as DNA-damaging and microtubule ac-
tive drugs, is effective in the treatment of some malignancies, especially of
apoptosis-prone leukemia and lymphomas, and some solid tumors such as testicular
cancer. Of course, as expected, the toxicity to normal cells limits effectiveness of chemo-
therapy in many cases. More intriguingly however is the question of why these drugs
are useful and in some cases may cure the disease. Although most of these drugs target
nonselective and even nonmechanism-based targets, such as DNA, topoisomerases,
or tubulin, their ultimate effects converge on targeting checkpoints. These drugs indi-
rectly target checkpoints.
Modulation of cell cycle checkpoints may result in treatment regimens with im-
proved therapeutic indices by exploiting the disruption of checkpoints in tumor cells.21,
22
Loss of the G2/M delay might be more consequential to a cell carrying a defect in a
G1/S checkpoint than to an otherwise wild-type cell.15 Pharmacological abrogation of
the G2 checkpoint can increase sensitivity to chemotherapy in G1-checkpoint-deficient
cells, whereas cells with normal checkpoints may take refuge in G1. Furthermore, loss
of checkpoints could be used for selective protection of normal cells.23-27 Recently it has
been shown that inhibitors of CDK can prevent chemotherapy-induced hair loss in rats.28
Exploiting defective checkpoints is only in its infancy of development. However, as is
often in the history of medicine, unintentional exploitation of checkpoint loss in cancer
might be responsible for the effectiveness of standard therapies. The link between check-
point control and apoptosis also tempts novel therapeutic approaches.29-31 Rational de-
sign based on the understanding of cell cycle control coupled with utilizing novel
mechanism-based therapeutics for manipulating the cell cycle will bring anticancer che-
motherapy to a new level.32
The Book Overview
This book “Cell Cycle Checkpoints and Cancer” addresses mechanisms of normal
and cancer cell cycling, checkpoint control, the link of mitogenic signaling and cell
cycle machinery. Considerable attention is devoted to the analysis of checkpoint mecha-
nisms from yeast to man allowing us to understand the logic of the cell cycle. Applica-
tions to current and future anticancer therapies is discussed throughout the book and
especially in last Chapters.
Mitogenic signaling is normally initiated on the cellular membrane by mitogens,
growth factors and cytokines.33 Not surprisingly, autocrine production of mitogens is
common in malignant transformation. Autocrine and paracrine growth factor synthe-
sis contribute to angiogenic and metastatic properties of transformed cells. In the
following Chapter, James McCubrey et al review the mechanisms of autocrine pro-
duction of cytokines and growth factors. The cytokine model illustrates deregulation
of autocrine cytokine expression on several levels with potential therapeutic approaches.
In additional Chapter, McCubrey et al discuss signal transduction from cytokine
receptors to cell cycle machinery via Ras/Raf-1/MEK/ERK, PI3K/Akt, Jak-STAT and
other pathways. The Chapter spotlights links between mitogenic signaling and apoptotic
machinery and mechanisms that allow cancer cell to evade apoptosis.
In normal cells, growth factors are necessary to initiate and maintain the transition
through G1 phase leading to S phase. The point at G1 at which commitment occurs
and a cell no longer requires growth factors to complete the cell cycle has been termed
the restriction point by Arthur Pardee in 1974. This discovery shaped the main direc-
tion of the research in cell cycle regulation culminating in the discovery of cyclins and
cyclin-dependent kinases. It is important that following growth-regulating stimuli, both
inhibitors and stimulators of CDKs are simultaneously induced. The choice between
proliferation and growth arrest is determined by the state of the restriction point. The
Chapter discusses that the restriction point could be considered as a prototype of cell
cycle checkpoints.
By arresting the cell cycle, activation of checkpoints presumably allows cells to
repair DNA. In “DNA damage, cell cycle control, and cancer” Jens Oliver Funk et al
describes series of events that is triggered in cells upon DNA damage as well as a
framework for the understanding of the functions of the core components involved in the
cell cycle response to DNA damage.
Cell cycle checkpoints are not restricted to DNA damage.34 As discussed by Dunkan
Clarke et al, checkpoints are mechanisms that establish dependence relationships be-
tween biochemically unrelated cellular processes. For example, the S-phase check-
point ensures that genome duplication is completed before cell division. The
topoisomerase II-dependent checkpoint ensures that the topology of the newly repli-
cated DNA has been correctly organized before cells begin mitosis. Distinct checkpoints
monitor mitotic spindle assembly, preventing the onset of chromosome segregation
until all the chromosomes are correctly aligned, and prevent exit from mitosis until
anaphase chromosome segregation has been completed.
The p53 tumor suppressor play a key role in checkpoint control in mammalian
cells. Levels of p53 are regulated by the Mdm-2-dependent protein degradation.35
p53 can induce growth arrest and/or apoptosis. Intriguingly, p53-mediated
apoptosis involves both transcription-dependent and independent mechanisms.36
In this book, R. Vogt Sionov, I. L. Hayon and Ygal Haupt discuss mechanisms of
p53 induction and its effect on cell cycle checkpoints.
As emphasized, p21 is an important regulator of cell cycle checkpoints. The iden-
tification of p21 (also named WAF1 by Wafik S.El-Deiry) as a p53-inducible protein
had culminated the search for a mediator of the p53 tumor suppressor by Bert Vogelstein
and his colleagues.37 Later, Wafik S. El-Deiry and coauthors have demonstrated that
p21 is transactivated by another tumor suppressor, BRCA1.38 In famalies that
inherit breast and ovarian cancer, BRCA1 mutations account for close to 100% of
resultant cancers. As discussed in this book by Timothy MacLachlan and Wafik
El-Deiry, among other qualities of BRCA1, it is influenced by and affects directly the
position of the cell cycle and the transition from phase to phase in the cell cycle inti-
mately involves BRCA1. Yet, many functions of BRCA1 are not clear. The authors
summarize recent advances leading to new hypothesis.
Discovered in 1994, BCRA1 is not the last tumor suppressor identified to date.
The logic of the discovery of tumor suppressors is illustrated in the Chapter by Carlo
M. Croce and coauthors. The novel tumor suppressor FHIT, fragile histidine triad
protein, is normally expressed in epithelial tissues and is inactivated in most common can-
cers including lung and breast cancer. It is inactivated in more than 50% of these
tumors. FHIT is the most common genetic alteration in human cancer.
Amato J. Giaccia and his colleagues discuss hypoxia and the cell cycle. When tu-
mors are more than 150 µm or approximately ten cells in diameter, they exceed their
ability to obtain sufficient oxygen by diffusion alone; and hypoxia develops. As hy-
poxia plays important roles in both tumor response to therapy and malignant progres-
sion, it is essential to understand how hypoxia affects cell cycle and molecular mecha-
nisms involved in this process. The Chapter provides insights in the cell cycle control
by hypoxia.
Recent studies spotlight the importance of G2 checkpoint.39 Stewart and Pietenpol
discuss DNA-damage induced G2 checkpoint signaling pathways. The Chapter discuss
mechanism of G2 checkpoint activation and G2 checkpoint maintenance. The au-
thors analyzed how knowledge of these signaling pathways may lead to more efficient
use of current anticancer therapies and the development of novel agents.
As emphasized by Rosandra Kaplan and David E. Fisher in “p53, apoptosis, and
cancer therapy”, the challenge in cancer therapy focuses fundamentally on the paucity
of therapeutic exploitable differences between cancer cells and normal cells. The ac-
tions of p53 likely mediates the successful treatment responses in those few tumors in
which chemotherapy produces durable cures.
p53 and p21 act as positive regulators of accelerated senescence in tumor cells, but
they are not absolutely required for this response.40 By contrasting the functions of
p53 as a positive regulator of apoptosis and as a negative regulator of mitotic catastrophe
with secondary cell death, Igor B. Roninson et al explain conflicting and paradoxical
results in the literature. In their provocative Chapter, the authors raised a prospect
that induction of program of accelerated senescence in tumor cells may be a feasible and
biologically justified approach to cancer therapy and that the induction of permanent
cytostatic arrest could be the primary mode of treatment response in certain clinical cases.
 Geoffrey I. Shapiro reviews preclinical and clinical development of small molecule
inhibitors of cyclin-dependent kinases. As more potent and selective CDK inhibitors
are now eagerly anticipated, it is important to review the preclinical and clinical
results with the agents presently under development. According to Dr. Shapiro,
as novel CDK inhibitors are developed, with improved potency and selectivity, it
will be critical to determine whether they induce cytotoxicity, or whether they
are primarily cytostatic, and to continually evaluate the selectivity of CDK inhi-
bition for transformed cell types.
In the final Chapter “Cell Cycle Molecular Targets and Drug Discovery” John K.
Buolamwini focuses on potential molecular targets in cell cycle regulatory pathways
and their exploitation for small molecule drug design and discovery. The inhibition of
kinase catalytic activity has been successfully achieved with small molecules that have
advanced into clinical trials for cancer therapy. More potential anticancer molecular
targets are emerging including critical oncogenic kinases and regulatory proteins iden-
tified in the progression through mitosis. These include aurora kinases, polo-like kinases,
and the anti-apoptotic protein survivin.
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1. Hunter T. Oncoprotein networks. Cell 1997; 88:333-346.
2. Hanahan D, Weinberg RA. The hallmarks of cancer. Cell 2000; 100:57-70.
3. Sherr CJ. The Pezcoller lecture: Cancer cell cycle revisited. Cancer Res. 2000; 60:3689-3695.
4. Kaelin WGJ. Choosing anticancer drug targets in the postgenomic era. J Clin Invest 1999;
104:1503-1506.
5. Shapiro GI, Harper JW. Anticancer drug targets: cell cycle and checkpoint control. J Clin Invest
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6. Gibbs JB. Mechanism-based target identification and drug discovery in cancer. Science 2000;
287:1969-1973.
7. Druker BJ, Lydon NB. Lessons learned from the development of an Abl tyrosine inhibitor for
chronic myelogenous leukemia. J Clin Invest 2000; 105:3-7.
8. Blagosklonny MV. p53 from complexity to simplicity: mutant p53 stabilization,
gain-of-function, and dominant-negative effect. FASEB J 2000; 14:1901-1907.
9. Sigal A, Rotter V. Oncogenic mutations of the p53 tumor suppressor: the demons of the
guardian of the genome. Cancer Res. 2000; 60:6788-6793.
10. Hahn WC, Stewart SA, Brooks MW et al. Inhibition of telomerase limits the growth of human
cancer cells. Nat Med 1999; 5:1164-1170.
11. Pardee AB. A restriction point for control of normal animal cell proliferation. Proc Natl Acad Sci
USA 1974; 71:1286-1290.
12. Hartwell LH, Kastan MB. Cell cycle control and cancer. Science 1994; 266:1821-1828.
13. Zhou B-BS, Elledge SJ. The DNA damage response: putting checkpoints in perspective.
Nature 2000; 408:433-439.
14. Nurse P. A long twentieth century of the cell cucle and beyond. Cell 2000; 100:71-78.
15. Paulovich AG, Toczyski DP, Hartwell LH. When checkpoints fail. Cell 1997; 88:315-321.
16. Graves PR, Yu L, Schwarz JK et al. The Chk1 protein kinase and the Cdc25C regulatory pathways
are targets of the anticancer agent UCN-01. J Biol Chem 2000; 275:5600-5605.
17. Jackson JR, Gilmartin A, Imburgia C et al. An indolocarbazole inhibitors of human checpoint
kinase (Chk1) abrogates cell cycle arrest caused by DNA damage. Cancer Res 2000; 60:566-572.
18. Monks A, Harris ED, Vaigro-Wolff A et al. UCN-01 enhances the in vitro toxicity of clinical
agents in human tumor cell lines. Invest. New Drugs 2000; 18:95-107.
19. Wahl AF, Donaldson KL, Fairchild C et al. Loss of normal p53 function confers sensitization to
Taxol by increasing G2/M arrest and apoptosis. Nature Med 1996; 2:72-79.
20. Bunz F, Dutriaux A, Lengauer C et al. Requirement for p53 and p21 to sustain G2 arrest after
DNA damage. Science 1998; 282:1497-1501.
21. Fisher DE. Apoptosis in cancer therapy: crossing the threshold. Cell 1994; 78:539-542.
22. Kastan MB, Canman CE, Leonard CJ. P53, cell cycle control and apoptosis: Implications for
cancer. Cancer Metastasis Reviews 1995; 14:3-15.
23. Pardee AB, James LJ. Selective killing of transformed baby hamster kidney (BHK) cells. Proc Natl
Acad Sci USA 1975; 72:4994-4998.
24. Darzynkiewicz Z. Apoptosis in antitumor strategies: modulation of cell-cycle or differentiation. J
Cell Biochem 1995; 58:151-159.
25. Blagosklonny MV, Robey R, Bates S, Fojo T. Pretreatment with DNA-damaging agents permits
selective killing of checkpoint-deficient cells by microtubule-active drugs. J Clin Invest 2000;
105:533-539.
26. Blagosklonny MV, Bishop PC, Robey R et al. Loss of cell cycle control allows selective microtu-
bule-active drug-induced Bcl-2 phosphorylation and cytotoxicity in autonomous cancer cells. Cancer
Res 2000; 60:3425-2428.
27. Chen X, Lowe M, Herliczek T et al. Protection of Normal Proliferating Cells Against Chemo-
therapy by Staurosporine-Mediated, Selective, and Reversible G(1) Arrest J Natl Cancer Inst 2000;
92:1999-2008.
28. Davis ST, Benson BG, Bramson HN et al. Prevention of chemotherapy-induced alopecia in rats by
CDK inhibitors. Science 2001; 291:134-137.
29. Reed JC. Dysregulation of apoptosis in cancer. J. Clin. Oncol. 1999; 17:2941-2954.
30. Sellers WR, Fisher DE. Apoptosis and cancer drug targeting. J Clin Invest 1999; 104:1655-1661.
31. Lowe SW, Lin AW. Apoptosis in cancer. Carcinogenesis 2000; 21:485-495.
32. Lees JA, Weinberg RA. Tossing monkey wrenches into the clock: new ways of treating cancer. Proc
Natl Acad Sci USA 1999; 96:4221-4223.
33. McCubrey JA, May WS, Duronio V, Mufson A. Serine/Threonine phosphorylation in cytokine
signal transduction. Leukemia 2000; 14:1060-1079.
34. Clarke DJ, Gimenez-Abian JF. Checkpoints controlling mitosis. Bioessays 2000; 22:351-363.
35. Haupt Y, Maya R, Kazaz A, Oren M. Mdm2 promotes the rapid degradation of p53. Nature 1997;
387:296-299.
36. Haupt Y, Rowan S, Shaulian E et al. p53 mediated apoptosis in HeLa cells: transcription depen-
dent and independent mechanisms. Leukemia 1997; 11:337-339.
37. El-Deiry WS, Tokino T, Veculescu VE et al. WAF1, a potential mediator of p53 tumor suppres-
sion. Cell 1993; 75:817-825.
38. Somasundaram K, Zhang H, Zeng YX et al. Arrest of the cell cycle by the tumour-suppressor
BRCA1 requires the CDK-inhibitor p21WAF1/CIP1. Nature 1997; 389:187-190.
39. Chan TA, Hwang PM, Hermeking H et al. Cooperative effects of genes controlling the G2/M
checkpoint. Genes Dev 2000; 14:1584-1588.
40. Chang BD, Broude EV, Fang J et al. p21Waf1/Cip1/Sdi1-induced growth arrest is associated with
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recovering cells. Oncogene 2000; 19:2165-2170.
CHAPTER 1

Autocrine Transformation: Cytokine Model

James A. McCubrey, Xiao-Yang Wang, Paul A. Algate, William L. Blalock
and Linda S. Steelman

Abstract
Autocrine growth factor secretion by cells is a frequent event involved in malignant transfor-
mation. Constitutive growth factor gene expression can in turn result in the deregulation of
survival. Furthermore, autocrine and paracrine growth factor synthesis can also contribute to
the enhanced angiogenic and metatastatic properties of transformed cells converting them into
more malignant tumors. We will discuss three fundamental mechanisms which can result in
autocrine transformation; first, mutations of the cytokine or growth factor genes themselves,
second, the aberrant expression of upstream receptors, kinases, or downstream transcription
factors which can induce autocrine growth factor synthesis and third, retrovirally induced cy-
tokine gene expression. We will discuss possible therapeutic strategies designed to inhibit these
events. We will use as a model the interleukin-3 (IL-3) gene and discuss how the aberrant
regulation of this gene can result in the prevention of apoptosis and lead to autocrine trans-
formation.

Cytokine Regulation of Growth

Cytokine usually refers to growth factors which often affect the hematopoietic system.
Some cytokines were initially called lymphokines because they were produced by lymphocytes
and often, but not always, functioned on lymphocytes. Even though some cytokines such as
IL-3 and granulocyte/macrophage colony stimulating factor (GM-CSF) have quite different
sounding names, they share many properties, are closely genetically linked, and were most
likely derived from a common ancestral gene which underwent tandem duplication. In this
Chapter we will use the term cytokine more frequently than growth factor. However, it should
be kept in mind that IL-3 and GM-CSF are often referred to interchangeably as lymphokines,
cytokines, and growth factors.
Cytokines can stimulate cell cycle progression, proliferation, and differentiation, as well
as, inhibit apoptosis of hematopoietic and other types of cells.1-3 Peripheral blood cells are
generated from self-renewable, pluripotential hematopoietic stem cells in the bone marrow.
Cytokines such as IL-3, GM-CSF, stem cell factor (SCF, a.k.a. steel factor, c-Kit-L, macrophage
growth factor), FL (a.k.a. Flt-3L, the ligand for the flt2/3 receptor), erythropoietin (EPO), and
others affect the growth and differentiation of these early hematopoietic precursor cells into
cells of the myeloid, lymphoid, and erythroid lineages.1-4 This Chapter will concentrate on the
regulation of IL-3 since much of the knowledge of how cytokines affect cell growth, signal
transduction, cell cycle progression, and apoptosis has been elucidated from research with IL-3
and IL-3-dependent cell lines. IL-3 was initially defined over 20 years ago by its ability to
induce the enzyme 20-α-hydroxysteroid dehydrogenase in cultures of splenic lymphocytes from
nude mice.5 However, it soon became apparent that IL-3 was being studied by a number of

Cell Cycle Checkpoints and Cancer, edited by Mikhail V. Blagosklonny. ©2001 Eurekah.com.
2 Cell Cycle Checkpoints and Cancer

investigators under a variety of aliases. It was called persisting cell-stimulating factor (PSF),6
mast cell growth factor (MCGF), 7 hematopoietic cell growth factor (HCGF), 8
histamine-producing cell-stimulating factor,9 multi-colony stimulating factor (Multi-CSF),10
Thy-1-inducing factor,5 and burst promoting activity (BPA).11 All of these growth stimula-
tory activities were subsequently identified as the same protein and renamed IL-3. It is appar-
ent that the many names by which this cytokine was known reflected its diverse biological
properties. There are over 7000 citations in the Medline® database which use IL-3 as a key-
word. Interestingly, IL-3 has remained one of the most intensively studied growth factors for
over 20 years. This may be due, in part, to its strong anti-apoptotic activities.
IL-3 acts on both myeloid and lymphoid lineages. In vivo administration of pharmaco-
logical doses of recombinant IL-3 to mice resulted in the increased production of red blood
cells, leukocytes, and platelets.12 Moreover, over-expression of the IL-3 gene in hematopoietic
progenitors via retroviral transduction of bone marrow cells resulted in a noneoplastic,
myeloproliferative syndrome in vivo.13 Infection of primary hematopoietic cells with retroviruses
encoding IL-3 does not normally result in malignant transformation, as over-expression of a
second gene is often required which will synergize with IL-3 and lead to autonomous growth.
Experimentally, the hox2.4 gene product has been shown to synergize with IL-3 and result in
the transformation of certain hematopoeitic cells.14
In addition to stimulating proliferation and differentiation of hematopoietic cells, cytokines
such as IL-3 also promote cell survival. IL-3-dependent cells undergo apoptosis after with-
drawal of IL-3 for a prolonged period of time (12 to 48 hours, depending upon the cell type
and species of origin).15 However, addition of IL-3 to IL-3-deprived cells can prevent apoptosis
in a significant proportion of these cells.15 This anti-apoptotic function of IL-3 remains inten-
sively studied today. Investigation of the effects of IL-3 on apoptosis has contributed signifi-
cantly to the apoptosis/programmed cell death field. In fact, the initial clues to the function of
Bcl-2 came after the observation that over-expression of Bcl-2 prolonged the survival of
IL-3-dependent cells cultured in the absence of cytokines.16

Regulation of IL-3 Expression: TCR Ligation and Mitogen Induced IL-3

Expression
Most hematopoietic cells do not usually synthesize the 26-kDa IL-3 protein. In those cells
that do express the IL-3 gene, the gene is normally under stringent controls.17-43 In peripheral
blood, activated T cells, natural killer cells, mast cells and some megakaryocytic cells can syn-
thesize IL-3.17-19 For optimal IL-3 expression, T cells must be activated via the T-cell receptor
(TCR)/CD3 pathway or by agents that mimic this pathway, e.g., the combination of the phorbol
ester, phorbol 12-myristate 13-acetate (PMA) and calcium ionophores.17-19 When a T cell is
activated, aggregation of the TCR/CD3 complex occurs. Receptor aggregation is followed by a
complex series of biochemical events leading to the activation of protein kinase C (PKC) and a
rise in the intracellular concentration of Ca2+.1,20 The activated serine/threonine PKC kinase
can phosphorylate the repressor protein, inhibitor κB (I-κB), which is subsequently ubiquitinated
and degraded, thus permitting I-κB to disassociate from nuclear factor-κB (NF-κB).21 The
unmasked NF-κB nuclear localization signals present on NF-κB allow it to enters the nucleus
and transactivate cytokine gene expression.
In addition, there is a complex of proteins which also phosphorylates I-κB: the I-κB
kinases α and β (I-κKα and I-κKβ).22 I-κK phosphorylates I-κB on serine residues. The I-κB
kinases can be also activated through serine/threonine phosphorylation by the NF-κB induc-
ing kinase (NIK), the mitogen-activated protein kinase kinase kinase-1 (MEKK1), and Akt
(see below).23 In summary, there are multiple mechanisms to activate I-κK, which in turn
regulates I-κB and subsequently NF-κB. An illustration of the regulation of IL-3 gene expres-
sion is presented in Figure 1. Similar mechanisms mediate the expression of IL-2, GM-CSF,
and other T-cell derived cytokines (Fig. 1).
Autocrine Trasformation: Cytokine Model 3

Fig. 1 Activation of IL-3 and GM-CSF expression. The effects of diacylglycerol (DAG) and Ca2+ on PKC
activation and the subsequent activation of calmodulin, calcineurin, NF-κB and NF-AT. Moreover the
effects of activation of the Ras/PI3K anti-apoptotic cascade are indicated. NF-κB can also be activated by
NIK. I-κB can be targeted for degradation by serine/threonine phosphorylation mediated by PKC Akt,
I-κKα and I-κKβ. The activated transcription factors are indicated in clear ovals. Once activated, NF-κB
and NF-AT enter the nucleus and stimulate IL-3 and GM-CSF expression. The sites of inhibition by the
immunosuppressive drugs CsA, FK506 and DSG are also shown in black on this diagram.

There are other transcription factors which modulate the expression of cytokine genes.
Increased levels of intracellular Ca2+ following TCR aggregation allows calmodulin to activate
calcineurin, a serine/threonine phosphatase.20 Activated calcineurin dephosphorylates the
cytoplasmic (c) form of the transcription factor, NF-ATc (nuclear factor of activated T
cells) enabling NF-AT to translocate to the nucleus (n). This results in the transactivation
of cytokine gene expression, including IL-3 and GM-CSF whose promoters contain NF-AT
binding sites.24-42
There are also additional kinase cascades which regulate cytokine gene expression. PKC
can also activate the Ras pathway by inactivating the GTPase activating protein (GAP), a
negative regulator of Ras.26,35-37 Ras is a member of a large multi-gene family, which encodes
small GTP-binding proteins that serve as molecular switches. Inactivation of GAP stimulates
Ras activity, which results in the enhancement of activator-protein-1 (AP-1) binding activity as
discussed below.26, 35-37 A diagram of where some of these transcription factors bind the IL-3
promoter region is presented in Figure 2. AP-1 can then stimulate cytokine gene expression,
including IL-3. Interestingly, the neurofibromatosis-1 (NF1) gene, a tumor suppressor
frequently lost in juvenile chronic myelogenous leukemia (CML), is functionally related to
GAP.44,45 NF1 likely serves to block Ras activation, thus its loss leads to constitutive Ras acti-
vation and contributes to the generation of CML.

Regulation of IL-3 Expression: Transcription Factor Binding Sites

The cis-acting elements of the human IL-3 promoter include two activation regions sepa-
rated by an inhibitory region.24-43 These genetic elements lie within a region that extends to
4 Cell Cycle Checkpoints and Cancer

Fig. 2 (See Figure legend on opposite page)

~300 bp upstream of the transcription start site (Fig. 2, Panel A). An inhibitory element,
nuclear inhibitory protein (NIP), has been described that binds to the IL-3 promoter, which
suppresses IL-3 transcription. This binding site for this transcription factor is located between
bp -271 to -250.24 Unfortunately no further information has been provided about this factor
and the role that deletion of this transcription factor binding site plays in leukemia.
Autocrine Trasformation: Cytokine Model 5

Fig. 2. (opposite page) Transcription regulation of IL-3 and GM-CSF. Panel A, The effects of activated Ras,
PKC, KSR and Src family kinases on the Raf/MEK/ERK signal transduction pathway and IL-3 expression.
Ras activation can be induced by external stimuli but inhibited by NF1 or GAP (black ovals). Active Ras
and downstream kinases are indicated in gray ovals. Ras can further transmit the signal to Raf, MEK, ERK
and p90Rsk which can result in the activation of the AP-1 and CREB transcription factors (shown in clear
ovals) which bind the promoter region of the IL-3 and GM-CSF genes. Activation of Ras can also result in
the activation of PI3K and the subsequent activation of PDK1, PDK2 and Akt which can phosphorylate
and activate IκK. Raf can also be activated by KSR and Src family tyrosine kinases. Other transcription
factors (e.g., c-Jun, Elf-1) are activated by other kinases such as JNK and p38 that in turn are activated by
MKK4, MEKK1 and MKK3/6 and SEK. The NF-κB and NF-AT as well as the Oct-1, AML1 and CREB
transcription proteins bind to the ACT-1 region. Possible control mechanisms for NF-κB, I-κB and NF-AT
(NIK, IκK and calcineurin) were presented in Figure 1. Also shown in this picture are the negative effects
of the NIP protein which binds the NIP region and suppresses transcription. In addition, there are CK1
and CK2 transcription factors, which bind to the CK1 and CK2 regions, as well as the Tax, EGR1, EGR2,
DB1 and AML1 transcription factors, which bind to the CT/GC rich region. Panel B, This panel depicts
the binding of NF-AT molecules to the intergenic region between the IL-3 and GM-CSF genes. The binding
of these proteins to the intergenic region influences the chromatin configuration of this gene cluster.

The IL-3 promoter region contains sequence motifs common to many cytokine promot-
ers, including CK (cytokine)-1 and CK-2/GC elements. These sequences appear to be dispens-
able for the activity of the IL-3 promoter.26-30 A CT/GC-rich region located between bp -76 to
-47 confers a basal transcriptional activity to the IL-3 promoter and responds to trans-activation
by the human T-cell leukemia virus type I-encoded Tax protein.31 At least four transcription
factors, (AML1, EGR1, EGR2 and DB1) have been shown to bind to this region.28-31,40-42
The binding of EGR1 and EGR2 to these sites increases IL-3 promoter activity when an ap-
propriate cell is activated.34 In contrast to EGR1 and EGR2, DB1 binds constitutively and
enhances the transcriptional activity of the IL-3 promoter when trans-activated by Tax.31 The
AML1 transcription factor also binds this region, but it has a higher affinity binding site in
the -175 to -135 region.40-42
Two regions of the IL-3 promoter play important positive regulatory roles in the response
of T cells to mitogens. One region is called Act-1 and is located between -175 to -135 bp.27, 28
The 5´ part of this region binds a mitogen-inducible, T-cell specific, octamer-1-associated
protein (Oct-1).27 Nuclear factor-IL3A (NF-IL3A) binds the middle region, while the 3´ Act-1
sequence contains a consensus-binding site for a cAMP responsive element binding protein
(CREB).30,38 The role of the Act-1 region is to coordinate the functions of several cis-acting
transcription factors, which leads to a maximal effect upon IL-3 transcription.27,28 The AML1
transcription factor also binds to this region.40-42 The other important transcriptional regulatory
region is located between bp -315 to -274 and contains AP-1 and Elf-1 binding sites.19,25 The
c-Jun and c-Fos heterodimer (AP-1) binds to the AP-1 site,19 while the Elf-1 site is bound by an
Ets-related transcription factor, Elf-1.25,35 Tissue specific expression of the IL-3 gene may re-
sult from interactions between the Act-1 and this latter region.35-37
There are many kinases which regulate the activity of the transcription factors that bind
the IL-3 promoter region. Signal transduction cascades originating from extracellular signals
(including cell stress) often regulate the activities of these kinases (MEKK1, MKK4, JNK,
ERK, MKK3/6, p38MAPK, p90Rsk, and others) which in turn can regulate IL-3 expression.34, 45-47
In addition to the cis-acting elements 5´ to the IL-3 transcription start site, there is
another set of cis-acting elements found in the intergenic region between the IL-3 and GM-CSF
genes which are sensitive to the immunosuppressive drugs CsA and FK-506 (Fig. 2, Panel B).39
This region contains four NF-AT sites, which are bound by NF-AT transcription factors upon
mitogen activation.
Proto-oncogenes, which are sometimes mutated in human cancer, (e.g., c-Fos, c-Jun NF-κB,
EGR, and Ets-related transcription factors) can bind the IL-3 promoter region.45, 46 This suggests
that abnormal expression of these oncoproteins may result in autocrine transformation and
6 Cell Cycle Checkpoints and Cancer

lead to leukemia. In many transformed cells, the pathways controlling the activities of these
transcription factors are dysregulated.45,46 For example, constitutive activation of the Ras/Raf/
MEK/ERK (extracellular regulated kinase) cascade can alter the activity of
transcription factors, to induce autocrine growth factor synthesis.48,49

Genetic Influences on IL-3 Expression: DNA Methylation

Genomic DNA demethylation is also believed to influence the propagation of specific T-
cell cytokine profiles. The extent of methylation of certain cytokine genes, such as IL-3 and
interferon-γ (IFN-γ), may contribute to distinct patterns of cytokine gene expression in T-cell
clones. Demethylation of the IL-3 promoter was shown to be confined to specific CpG sites
within the same clones.50 This is a potential mechanism that could lead to the ability of certain
T-cell clones to express specific cytokines.

Therapeutic Approaches Based Upon Reducing Cytokine Gene Expression

We have discussed the mechanisms by which T-cell activation can result in the transcrip-
tion of the IL-3 gene. Now we will discuss therapies that exploit the inhibition of IL-3 tran-
scription. There may be therapeutic approaches to inhibit the activity of NF-κB, which will
decrease cytokine gene expression. 15-Deoxyspergualin (DSG), an immunosuppressive drug
which has been through Phase I/II clinical trials, inhibits the localization of heat shock protein
70 (Hsp 70) to the nucleus in response to heat stress, as well as the intranuclear activation of
NF-κB, through its interaction with Hsp70.51 Another approach to inhibiting NF−κB activation
involves introducing adenoviral vectors into leukemic cells which overexpress I-κB.52 This would
result in a decrease in NF-κB activity and cytokine gene synthesis. This gene therapeutic ap-
proach may prove beneficial in the suppression of tumor growth. Decreasing the levels of
NF-ATc would suppress cytokine gene expression. The immunosuppressive drugs cyclosporin
A (CsA) and FK506 mediate their activity by inhibiting calcineurin activation, thereby
preventing the dephosphorylation of NF-ATc.39,53 Another approach is to treat leukemic
patients with immunosuppressive drugs. This approach would have to be carefully monitored
as it could render the patient susceptible to life-threatening microbiological infections.
Other targets to inhibit cytokine gene synthesis include the upstream signal transduction
cascades. Ras is frequently targeted by anti-neoplastic drugs including farnesyl transferase (FT)
inhibitors (see below). Addition of a farnesyl group is necessary for Ras localization to the
cytoplasmic membrane. Drugs, which block Ras farnesylation, are currently being developed
by pharmaceutical companies for therapeutic use (e.g., Janssen, Merck).54 Inhibiting Ras could
decrease cytokine gene expression.
Pharmacological companies have developed inhibitors to some of the kinases involved in
regulation of cytokine gene expression (e.g., SB203580 is a p38MAPK inhibitor developed by
the Smith Klein Beecham Company).55 Blocking p38MAPK activity would suppress some of the
transcription factors involved in cytokine synthesis. The critical question remains: How do we
target these inhibitors exclusively to malignant cells rather than normal cells? It may be possible
to control, either directly or indirectly to control the activities of these important regulatory
molecules in transformed cells.26,34,45,51,56

Regulation of IL-3 Expression: Post-Transcription Regulation

We have described how TCR ligation and mitogen stimulation can activate kinase path-
ways resulting in the activation of transcription factors, which bind the IL-3 promoter region
and induce expression of the IL-3 gene. The next point of IL-3 regulation to be discussed is the
control of IL-3 synthesis due to post-transcriptional mechanisms. IL-3 mRNAs are very un-
stable and decay within one-half to one hour after their synthesis.57-75 This property appears to
be critical for their normal function, since the degradation of IL-3 mRNA, as well as other
cytokine mRNAs, is stringently controlled.57-75 An AU-rich element (ARE) found within the
3' untranslated region (UTR) of the IL-3 and other cytokine mRNAs is involved in the regu-
lation of IL-3 mRNA stability (Fig. 3).58-63 These evolutionarily conserved ARE sequences
Autocrine Trasformation: Cytokine Model 7

Fig. 3. Post-transcriptional regulation of normal and mutated IL-3 expression. Panel A, The wild-type IL-3
ARE is shown which binds the indicated proteins. This IL-3 mRNA would be induced in T cells after TCR
ligation. The binding of these proteins results in mRNA with a short mRNA half-life. p36 and p95 are the
only proteins that were demonstrated by northwestern analysis to bind directly to the IL-3 gene.3,53,55 p95
is depicted as a larger sphere due to artistic constraints. The exact sequences where p36 and p95 bind the
IL-3 UTR are not known, nor is the stoichiometry of binding. Panel B, Calcium ionophores disrupt the
binding of RNA- binding proteins to the IL-3 ARE resulting in conditional stabilization of IL-3 mRNA.
This stabilized IL-3 mRNA would be detected after treatment of T-cell lines with calcium ionophores. Panel
C, The RNA-binding proteins are prevented from binding the truncated IL-3 gene in tumorigenic autocrine
transformed cells which contain an IAP provirus inserted into the IL-3 ARE.3,58-62 The prevention of
binding of these proteins results in the continuous stabilization of IL-3 mRNA. The sizes of the coding and
noncoding IL-3 sequences are not drawn to scale.

serve to tightly regulate cytokine expression, a critical function due to the potent growth
stimulatory and anti-apoptotic effects of cytokines. A diagram of the post-transcriptional regu-
lation of IL-3 is presented in Figure 3.
Electrophoretic mobility shift assays (EMSAs) and UV-crosslinking experiments have iden-
tified the proteins that bind to cytokine AREs (62, 64-71). These included proteins with ap-
parent molecular weights of 36-, 40-, 43-, 46-, 55-, 57-, 68- and 95-kDa. The adenine/uridine
binding protein (AUF1, also known as heterogeneous nuclear ribonuclear protein D [hnRNP
D]) was shown to bind to the IL-3 ARE, by an EMSA followed by immunoprecipitation of
IL-3 ARE binding proteins with a specific α-AUF1 antibody.62
All three isoforms of hnRNP D, which exhibit apparent molecular weights of 40-, 43-,
and 46-kDa, bind to the IL-3 ARE.62 hnRNP C also binds to the IL-3 ARE region.62 Calcium
ionophore treatment prevents/reverses binding of these proteins to the IL-3 ARE and results in
stabilized IL-3 mRNA62 (Fig. 3, Panel B). The affinities of the hnRNP D proteins for their
RNA substrates were shown to be negatively correlated with mRNA stability.69
8 Cell Cycle Checkpoints and Cancer

Therapeutic Approaches Based Upon Decreasing Cytokine mRNA Stability

CsA and FK506 decrease IL-3 production by certain mast cells via mRNA destabilization
as well as affecting NF-ATc activation.74 These results suggest three possibilities: 1) CsA and
FK506 may have additional targets besides calcineurin which regulate mRNA stability, 2)
calcineurin may have other targets besides NF-AT which regulate mRNA stability or 3) NF-AT
may regulate the expression of additional genes besides cytokines, which regulate mRNA stability.
The immunosuppressive drug rapamycin, which has a different biochemical target
than CsA, also destabilizes IL-3 mRNA in certain autocrine transformed cells.75 Rapamycin
is primarily thought to affect p70 ribosomal S6 kinase (p70S6K) phosphorylation, which
subsequently modulates the efficiency of protein translation (see below). This is believed
to result from rapamycin inhibiting the mammalian target of rapamycin (mTor), which is
downstream of phosphatidylinositol-3 kinase (PI3K) but upstream of p70S6K. The mecha-
nisms by which the immunosuppressive drugs CsA, FK506, and rapamycin prevent the bind-
ing of proteins to the IL-3 ARE are unknown. The drugs may alter the phosphorylation states
of ARE binding proteins, preventing them from interacting with the IL-3 ARE.

Chromosomal Translocations which may Inhibit IL-3 Expression

We have discussed how IL-3 mRNA is synthesized and regulated in normal cells. Now we
will discuss how IL-3 can be abnormally expressed in certain leukemias and lymphomas.
Chromosomal translocations have been linked to aberrant IL-3 expression. In certain human
B-cell lymphomas, chromosomal translocations between the immunoglobulin heavy chain (IgH)
locus on chromosome 14 and the IL-3 gene on chromosome 5 [t (5; 14)(q31; q32)] were
detected.76,77 The IgH enhancer, a strong tissue specific enhancer involved in many chromosomal
translocations in hematopoietic cells (e.g., Burkitt’s lymphoma, follicular B-cell lymphomas
involving Bcl-2) induces the transcriptional activation of the IL-3 gene. The role IL-3 plays in
the growth of B-cells remains controversial. IL-3-dependent pro-B cell lines have been
available since 1985. These cell lines offer support to show that IL-3 can play a role in the
growth of some early hematopoietic cells.4 It is also possible that in human B-cell lymphomas
containing a translocated IL-3 gene, IL-3 serves as a paracrine growth factor to support the
growth of neighboring cells. This, in turn, provides the necessary growth factors for the B-cell
lymphoma. For example, the IL-3 produced by the B-cell lymphoma may stimulate the expres-
sion of: IL-4, IL-5, IL-6, IL-7, in either neighboring cells or the B-cell lymphoma, which in
turn supports the lymphoma growth. Such complicated cytokine circuitry is common, al-
though often we do not know which growth factor plays the critical role.

Abnormal IL-3 Expression: Inhibition Due to Chromosomal Translocations

The AML1 transcription factor is normally a transcriptional activator which binds the
promoter region of genes such as IL-3 and stimulates its expression.40-42 Some chromosomal
translocations may result in the creation of chimeric transcription factors which suppress IL-3
expression. The AML-ETO fusion protein that is generated by the t:(8; 21) chromosomal
translocation encodes a transcriptional repressor which has been shown to suppress IL-3 pro-
moter activity in in vitro promoter activity assays. Moreover, the t:(12: 21) translocation
encodes the chimeric TEL-AML protein which also represses transcription of the IL-3 and
other genes as measured by in vitro promoter activity assays. This chromosomal translocation
is the most commonly identified molecular abnormality in childhood acute lymphoblastic
leukemia (ALL). In freshly isolated human ALL cells which have the TEL-AML1 fusion pro-
tein, no IL-3 was detected.40-42 Thus some chromosomal translocations appear to suppress
IL-3. The roles that these chimeric transcription factors play in leukemogenesis are being inves-
tigated in “knock-in” mice.43 It may be that suppression of IL-3 synthesis is unrelated to the
leukemic properties of the cells and that the real targets of suppression by these chimeric
transcription factors are other genes involved in the induction of differentiation.
Autocrine Trasformation: Cytokine Model 9

Abnormal Cytokine Gene Expression Due to Retroviral Infection

Retroviruses, such as human T-cell leukemia virus-I (HTLV-I), encode sequences which
can regulate gene expression. The tax gene product of HTLV-I is a transactivator which can
induce the expression of many genes including: IL-2, IL-3, IL-15, GM-CSF, TNF, c-fos, c-jun,
and IL-2Rα chain.31 The tax protein can induce genes whose promoter regions contain CREB,
Ap-1, and serum responsive element (SRE) sequences. Although most studies on HTLV-I
infection of hematopoietic cells have focused primarily upon IL-2 and IL-15 expression, there
may be some cases where HTLV-I infection can result in abnormal autocrine IL-3 expression
in certain early hematopoietic cells which lead to autocrine transformation. Recent studies
have shown that both IL-2 and IL-15 expression are necessary for autocrine transformation; as
treatment of cells with antibodies to either cytokine by themselves did not fully inhibit the
growth of the HTLV-I-infected T cells.79 In contrast, when antibodies to both IL-2 and IL-15
were added, a greater level growth inhibition was observed.78,79

Autocrine Cytokine Gene Expression Due to Activated Raf and MEK1

Expression
We have observed that introduction of activated Raf and MEK1 genes into
cytokine-dependent cells resulted in autocrine transformation.80-89 Initially, we observed the
synthesis of GM-CSF, but not IL-3 transcripts, in cells which would grow in response to either
Raf or MEK1 expression. Moreover, the GM-CSF cytokine was detected in the supernatants,
which supported the proliferation of the parental cells. However, when we treated the
autocrine-transformed cells with neutralizing antibodies to GM-CSF, the highest level of growth
inhibition observed was approximately 50%.80,81 When we examined the expression of other
cytokines, we noticed that mRNAs encoding additional cytokines were detected in the Raf and
MEK1 transformed cells including IL-5, IL-6, IL-10, and IL-12. Some of these cytokine tran-
scripts (e.g., IL-5 & IL-6) were detected in uninfected cells and were observed to be regulated
in the cells by the addition of either GM-CSF or IL-3 to the growth medium. In contrast,
IL-10 and IL-12 were only detected in the cells which grew in response to activated Raf and
MEK1. The contribution of these additional cytokines to autocrine responsive growth is
being determined. Thus, the activation of downstream signal transduction cascades by Raf
and MEK1 resulted in the activation of cytokine genes that were not detected in the parental
cytokine-dependent cells.

Abnormal Regulation of IL-3 Expression: Biological Consequences of IL-3

ARE Disruption
We have characterized autocrine-transformed cells, which secrete IL-3 and have an
intracisternal type A particle (IAP) transposed into the IL-3 ARE. 58,59,62 In these
autocrine-transformed FL-IL-3R cells, only two AUUUA motifs adjacent to the IL-3 gene remain
intact due to the IAP transposition in the parental FL5.12 cell line (Fig. 4, Panel A). IL-3 mRNA
isolated from these cells has a much longer half-life (T1/2 = 16 to 24 hours) than wild-type IL-3
mRNA (T1/2 = 0.5 to 1 hour). Moreover, the IL-3-secreting hematopoietic cells expressing the
mutated IL-3 mRNAs were tumorigenic upon injection into immunocompromised nude mice
(Fig. 4).57, 59-61
To determine the regions of the rearranged IL-3 gene that were responsible for the abrogation
of cytokine-dependency, chimeric IL-3 gene constructs containing portions of the wild-type
and IAP-disrupted IL-3 genes were made, transfected into IL-3-dependent parental FL5.12
cells, and examined for their ability to abrogate cytokine-dependency. The resulting
factor-independent cells were then examined for their tumorigenicity upon injection into im-
munocompromised mice.59-61 Recombinant IL-3 constructs were also made which tested the
abilities of various IAP-LTRs and exogenous retroviral LTRs (e.g., Moloney-Murine Leukemia
Virus, Mo-MuLV) to affect IL-3 expression and factor-dependency. In Figure 4, Panel B, we
10 Cell Cycle Checkpoints and Cancer

Fig. 4. (see figure legend on opposite page)

have illustrated the recombinant IL-3 constructs and their abilities to abrogate the
cytokine-dependency of the parental FL5.12 cells.
Transfection of cells with a germline IL-3 gene did not result in the frequent isolation of
factor-independent cells. In those cells that were factor-independent, amplification of the
introduced GIL3 construct was detected.60,61 In contrast after transfection with the RIL3
construct factor-independent cells were detected. These transfected cells had not inherited a
Autocrine Trasformation: Cytokine Model 11

Fig. 4. (opposite page) Effects of LTRs and ARE deletions on IL-3 expression and tumorigenicity. Structures
of germline and rearranged IL-3 genes that are contained in the FL-IL3-R2 cell line and modified IL-3 genes.
Panel A. Maps of the germline (G) IL-3 locus present in FL5.12 cells and the rearranged (R)IL-3 locus
contained in FL-IL3-R cells. The black thick line is the germline IL-3 locus from start of transcription to
termination of transcription. The open thick line is the rearranged IL-3 gene from start to termination of
transcription. Boxes indicate the five IL-3 exons. Panel B. The germline and rearranged IL-3 genes as well
as various constructs containing deletions of the AUUUA regions as well as additions of different LTR and
other genetic sequences were inserted into the pSV2neo expression vector.59-61 The constructs were trans-
fected into IL-3 dependent FL5.12 cells, and their abilities to abrogate cytokine dependence were deter-
mined and compared. Relevant restriction sites are indicated (E = EcoRI, B = Bam HI, N = NcoI, H = Hae
III, K = KstI). LS-IAP-LTR = Lymphocyte specific IAP-LTR (identical to the IAP-LTR contained in the rIL3
gene), Bacterial DNA = insertion of a 450 bp piece of Bacteriodes fragilis DNA. Mo-MuLV -LTR = Moloney
Murine Leukemia Virus LTR, PCR-DNA at -0.2 is the parent construct for the other LTR insertion
constructs which contain the different LTRs at -0.25. The 5´G + 3´R and 5´R + 3´G are chimeric IL-3
constructs which contain respective portions of the germline and rearranged IL-3 genes. Key to induction
of factor independence following transfection of IL-3 constructs: (–) = no or very low level of
factor-independence, ++ moderate level of factor-independence, +++ = higher level of factor-independence,
++++ = highest level of induction of factor-independence. Key to tumorgenicity: (–) no tumors or very few
(sporadic) tumors, (+) tumors in all mice examined.

high copy number of the rIL3 construct indicating that inheritance of a single rIL3 construct
was sufficient to abrogate cytokine-dependency.
The effects of the 5´ and 3´ regions of the germline and rearranged regions of the IL-3
genes were examined by creating chimeras of these genes by cleaving them in the middle with
the Bam HI (B) restriction endonuclease. This resulted in two constructs, 5´R + 3´G and 5´G
+ 3´R. The 5´R + 3´G construct, which contained the wild-type ARE sequence, did not readily
abrogate the cytokine-dependency of FL5.12 cells, whereas the construct (5´G + 3´R) which
contained the IAP-truncated ARE sequence did. These results indicated that the promoter
region of the RIL3 gene did not have any mutant elements (DNA sequences) in it which
resulted in abrogation of cytokine-dependency and the 3´R region of the RIL3 gene was re-
sponsible for abrogation of cytokine-dependency. The DNA sequence of the RIL3 promoter
region was determined and confirmed that there were no differences in the promoter regions of
the GIL3 and RIL3 genes.
To determine whether deletion of the ARE region of the IL-3 gene was sufficient for
abrogation of cytokine-dependency, an IL-3 construct was made lacking the AUUUA region.
Transfection of cells with the GIL3 + ∆AUUUA construct did not result in the frequent isola-
tion of factor-independent cells. To determine if an LTR region was also required to abrogate
cytokine-dependency, an IAP-LTR was added to the gIL3 + ∆AUUUA construct. Transfection
of cells with this construct resulted in the isolation of factor-independent cells. These results
indicated that addition of the IAP-LTR was necessary for the transcription of the IL-3 gene.
Additional IL-3 constructs were made containing the various LTRs inserted in different
positions. The exogenous Moloney Murine Leukemia Virus (Mo-MuLV) LTR was more effec-
tive in inducing the expression of the IL-3 gene than the endogenous IAP-LTR. As a control,
bacterial DNA was inserted where the various LTRs were positioned. Transfection of IL-3
dependent cells with this control construct did not result in the isolation of factor-independent
cells. LTR-CAT transient transfection experiments indicated that the LS-IAP-LTR contained
in the rIL3 gene was weaker than other LTRs and enhancer regions, thus IAP transpositions
involving this class of IAP-LTR may require additional mutagenic events to stimulate sufficient
gene transcription to induce malignant transformation.
The effects of the retroviral LTRs and the presence of the ARE on the levels of IL-3
expression in the transfected factor-independent cells are illustrated in Figure 5. This was de-
termined by purifying supernatants from the various cell lines and then titering them on the
factor-dependent parental cell line. The activity in the supernatants was determined to be IL-3
as treatment of the supernatants with an α-IL-3 Ab inhibited their abilities to stimulate
12 Cell Cycle Checkpoints and Cancer

Fig. 5. Effects of LTRs on the levels of IL-3 secretion. The levels of IL-3 secreted in the various transfected
cell lines were determined by preparing supernatants from some factor-independent cells transfected with
some of the IL-3 constructs presented in Figure 4. The level of [3H]-thymidine incorporation is a measure
of DNA synthesis and a marker of proliferation. The WEHI-3B supernatant is a control since it is prepared
from the WEHI-3B cell line which produces a large amount of murine IL-3 and is a source of IL-3 for the
growth of murine IL-3 dependent cells. The gIL3 supernatant was prepared from a rare factor-independent
FL5.12 cell line transfected with the germline IL-3.59-61

[3H]-thymidine incorporation. In contrast, when the supernatants were incubated with an

α-GM-CSF Ab, there was no inhibition of [3H]-thymidine incorporation. Transfection of the
parental FL5.12 cells with a germline IL-3 construct ligated to a strong LTR (e.g., Mo-MuLV
LTR) led to the highest level of IL-3 secretion detected. In contrast transfection of FL5.12 cells
with a LTR with a low level of activity (e.g., IAP-LTR) led to a lower level of IL-3 synthesis.
Transfection of FL5.12 cells with a rearranged IL-3 gene which had a deletion of the mRNA
stability region and an IAP LTR resulted in an intermediate amount of IL-3 expression.
These studies indicated that the IAP transposition stabilized IL-3 mRNA. The remaining
two AUUUA motifs could not efficiently destabilize IL-3 mRNA, and hence, the transfected
cells were autocrine-transformed and tumorigenic. Site-directed mutagenesis studies indicated
that destabilization of IL-3 mRNA requires a clustering of either the three 5´ or the distal three
3’ AUUUA motifs present in the IL-3 ARE. However, the cluster of the three 3´ AUUUA
motifs was a stronger destabilizer.63
In order to determine how the IAP transposition altered the binding of proteins to the
IL-3 ARE, EMSAs were performed. Proteins were specifically bound to the wild-type IL-3
mRNA ARE region, whereas no protein binding was detected to the RNA which had only two
AUUUA motifs, nor to an artificial RNA probe which did not contain any AUUUA motifs
(Fig. 3, Panel C).62 Thus, certain IAP transpositions disrupt IL-3 AREs and prevent the bind-
ing of proteins to this region. These mutations result in autocrine growth stimulation leading
to malignant transformation.
Autocrine Trasformation: Cytokine Model 13

Conclusions
This Chapter has examined the mechanisms of regulation of IL-3 expression in normal
and autocrine transformed cells. We have also described therapeutic approaches which might
be effective in treating autocrine tumors. Clearly the aberrant expression of growth promoting
cytokines represents a significant challenge in cancer therapeutics because they can be activated
by diverse mechanisms.

Acknowledgments
We sincerely thank Ms. Catherine Spruill for the outstanding artwork. This work was
supported in part by grants (R01CA51025) from the NCI and the North Carolina Biotechnology
Center (9805-ARG-0006, 2000-ARG-0003) to JAM.

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58. Algate PA, McCubrey JA. Autocrine transformation of hemopoietic cells resulting from cytokine
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59. Mayo MW, Wang X-Y, Algate PA et al. Synergy between AUUUA motif disruption and enhancer
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60. Wang X-Y, McCubrey JA. Malignant transformation induced by cytokine genes: A comparison of
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gene expression and autocrine transformation. Leukemia 1997; 11:1711-1725.
62. Wang X-Y, McCubrey JA. Characterization of proteins binding specifically to the interleukin 3
(IL-3) mRNA 3' untranslated region in IL-3-dependent and autocrine transformed cells. Leukemia
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63. Stoecklin G, Hahn S, Moroni C. Functional hierarchy of AUUUA motifs in mediating rapid
interleukin-3 mRNA decay. J Biol Chem 1994; 269:28591-28597.
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66. Brewer G. An A+U-rich element RNA-binding factor regulates c-myc mRNA stability in vitro.
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and C proteins with reiterated AUUUA sequences. J Biol Chem 1993; 268:8881-8887.
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71. Henics T, Sanfridson A, Hamilton BJ et al. Enhanced stability of interleukin-2 mRNA in MLA
144 cells: Possible role of cytoplasmic AU-rich sequence-binding proteins. J Biol Chem 1994;
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81. Hoyle PE, Moye PW, Steelman LS, et al. Differential abilities of the Raf family of protein kinases
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CHAPTER 2

Signal Transduction Pathways:

Cytokine Model
James A. McCubrey, William L. Blalock, Fumin Chang, Linda S. Steelman,
Steven C. Pohnert, Patrick M. Navolanic, John G. Shelton,
Paul E. Hoyle, Phillip W. Moye, Stephanie M. Oberhaus,
Martyn K. White, John T. Lee and Richard A. Franklin

Abstract

G
rowth factors (GF) initiate and maintain transition through G1 to S phase. GF-
dependence ends with phosphorylation of Rb by cyclin-dependent kinases (CDKs),
enabling cells to pass through the restriction (R) point and to complete the remaining
phases of the cell cycle. Cyclin D-dependent kinase phosphorylates Rb leading to induction of
cyclin E which in turn activates CDK2 and collaborates with cyclin D-CDKs to complete Rb
phosphorylation. GF simultaneously induce cyclins and CDK inhibitors. Not only their ratio
but also cellular context determines response: proliferation vs arrest. R-point, a prototype of
cell cycle checkpoints, is usually lost in cancer. Loss of R-point can be exploited for selective
killing of cancer cells by cycle-dependent chemotherapy.

Cytokine-Induced Signal Transduction Resulting in Growth

and the Prevention of Apoptosis
In the previous Chapter, we discussed the mechanisms by which IL-3 is synthesized after
T-cell activation, mitogen stimulation, chromosomal translocations, and retroviral infection.
Next, it is logical to consider the effects of the synthesized IL-3 on signal transduction path-
ways leading to growth and the prevention of apoptosis. The intracellular signal transducing
machinery induced by cytokines, such as IL-3 and granulocyte/macrophage colony stimulat-
ing factor (GM-CSF), represents a promising area to exploit for the therapy of leukemia. The
ultimate goal of many of these studies described below is the development of specific com-
pounds or therapies, which will modulate key intermediates in signal transduction pathways.
An overview of some of the growth and anti-apoptotic pathways induced by IL-3 is presented
in Figure 1.
Neither the α nor the β chains of the specific receptors for IL-3 (IL-3R) has any obvious
homology to known signaling molecules, such as kinases, phosphatases, nucleotide binding
proteins, or src homology (SH)-containing proteins.1-36 However, the IL-3 βc-chain functions
in the activation of signal transduction pathways by recruiting the necessary kinases.10-36 An
immediate response of cells upon IL-3 activation is the tyrosine phosphorylation of Jak and
STAT proteins1,2,8,10-32 and the activation of Ras, Raf, MEK, and MAPK (mitogen-activated
protein kinase, ERK1/2). MAP kinase is a generic name referring to a group of three serine-
threonine MAP kinases (ERK, p38, and JNK).1,2,15-20 Subsequently, these signals are

Cell Cycle Checkpoints and Cancer, edited by Mikhail V. Blagosklonny. ©2001 Eurekah.com.
18 Cell Cycle Checkpoints and Cancer

Fig. 1. Proliferative and apoptotic pathways regulated by IL-3. This diagram is an overview of the different
effects which IL-3 has on cell growth and the prevention of apoptosis. IL-3 can stimulate Jak kinases, which
activate gene expression through STAT proteins. Some of the genes that are induced by STAT stimulate
proliferation (e.g., cyclins) or prevent apoptosis (e.g., Bcl-XL), whereas others (e.g., Cis and Socs), serve to
inhibit the Jak/STAT signal transduction pathway or regulate cell cycle progression (e.g., p21CIP1). IL-3 can
also induce anti-apoptotic pathways by stimulating the Ras, or PI3K pathways, which can result in the
phosphorylation of the pro-apoptotic Bad, Gsk-3, FKHR and caspase 9 proteins. Also shown are the
negative effects of phosphatases, which can dampen IL-3 mediated signal transduction.
Signal Transduction Pathways: Cytokine Model 19

transduced to the nucleus resulting in the transcriptional induction of proto-oncogenes such as

c-myc and c-fos.21-29

Adaptor Proteins that Couple Receptors with Downstream Pathways

Upon IL-3 stimulation, the adapter molecule Shc is also rapidly phosphorylated and asso-
ciates with the phosphorylated βc chain.30,36-40 Shc contains two domains that are capable of
interacting with tyrosine-phosphorylated proteins: an N-terminal phosphotyrosine-binding
(PTB) domain and a C-terminal SH2 domain.40 The PTB domain is responsible for the physical
association of the Shc protein to the receptor βc chain.40
Phosphorylated Shc protein binds to another adapter protein, Grb2 (growth-factor-
receptor-bound protein-2), which in turn associates with the GTP exchange factor, mSos (mam-
malian son of sevenless homologue), to activate Ras.38,39 The protein tyrosine kinases respon-
sible for the phosphorylation of Shc have been suggested, but not exclusively identified. The
kinase which phosphorylates Shc is proposed to be Jak2.35,38
IL-3 stimulation also results in tyrosine phosphorylation of an SH2-containing inositol
phosphatase (SHIP), which forms a complex with Shc, Grb2, and SOS and may act to regulate
this pathway.41 Phosphorylation of SHIP does not appear to be necessary for its IP3-phos-
phatase activity; rather, it may be involved in the binding of proteins necessary for targeting
SHIP to its correct subcellular component where its catalytic activity is necessary. Indeed,
phosphorylated SHIP is found predominantely in the membrane fraction of cells.41
The Vav protein is yet another adaptor/signaling molecule activated by IL-3/GM-CSF
stimulation.42,45 Vav contains a single SH2 domain and two SH3 domains.44,45 The SH2
domain mediates the interaction of Vav with Jak2, which has been proposed to be responsible
for the phosphorylation and activation of Vav.43 Once phosphorylated, Vav can interact with
the Tec protein kinase through Tec’s SH2 domains. Tec can then bind phosphatidylinositol
3-kinase (PI3K) and initiate additional signal transduction cascades. In addition, PKC can also
activate Vav leading to Ras/Raf or potentially Ras/PI3K activation.45

The Jak-STAT Pathway

Upon binding of IL-3 to the IL-3 receptor, the IL-3 receptor α and β chains heterodimerize,
and the entire receptor oligomerizes with other IL-3 receptors.1,30-32 The association of Jak2
with the cytoplasmic membrane-proximal region of the βc chain allows for the subsequent
oligomerization, phosphorylation, and activation of Jak2 upon IL-3 receptor aggregation.32 A
diagram of IL-3 mediated signal transduction pathways is presented in Figure 2.
Jak2 is a member of a multi-gene family including Jak1, Jak2, Jak3, and Tyk2.14,32-34 A
unique characteristic of Jak family members is that they contain two tyrosine kinase domains:
a carboxy-terminal catalytic domain and an amino-terminal pseudo-kinase domain.32-34 Jak2
has been demonstrated to be the molecule responsible for some of the immediate responses of
IL-3 stimulation and is required for mitogenesis.14,32,46
Jak activity is necessary for STAT activation by non-tyrosine kinase receptors, such as the
IL-3 receptor.12-32,36,40,46-63 IL-3 receptor binding leads to the activation of Jak2 and the
recruitment and subsequent tyrosine phosphorylation of STAT5 (Y694 of STAT5a and Y699
of STAT5b).32 Tyrosine phosphorylation of STATs leads to their dimerization and activation.32
These STAT dimers then translocate to the nucleus where they act as transcription factors by
binding regulatory sites within the promoter region of immediate-early genes such as c-myc, β-
casein, Osm, and Bcl-XL,32,47 as well as feedback inhibitors of the JAK/STAT pathway (e.g.,
Cis).47 Although STAT activation requires tyrosine phosphorylation by Jak kinases, STAT
translocation to the nucleus is enhanced by threonine phosphorylation via Raf/MEK/ERK
activation.2,64-66 The consequences of activation of this pathway will be discussed later.
Constitutive activation of members of the Jak-STAT pathway has been associated with
the onset of HTLV-induced adult T-cell leukemia67 as well as v-Abl,68 BCR-ABL69 and
v-Src70-72 mediated transformation of various hematopoietic cells. Mutant Jak proteins have
20 Cell Cycle Checkpoints and Cancer

Fig. 2. IL-3 mediated signal transduction resulting in proliferation and the prevention of apoptosis. IL-3
mediates activation of the Jak/STAT and Ras/Raf/MEK/ERK signal transduction pathways. IL-3 can also
affect apoptosis by inducing the PI3K pathway which can be regulated by the PTEN tumor suppressor gene
which functions as a phosphatase. Also shown are the activation of PKC and kinase suppressor of Ras (KSR),
which can also activate the Raf pathway. Inactivated proteins are depicted in clear ovals whereas the activated
forms are depicted in grey ovals. Proteins which have a negative role on cell growth are indicated in black
ovals. ER = endoplasmic reticulum.
Signal Transduction Pathways: Cytokine Model 21

Fig. 3 Activation of Akt by IL-3. A) In cytokine-deprived cells, Akt is not localized to plasma membrane.
Also the phosphatase encoded by the PTEN tumor suppressor can result in the inactivation of PI3K. The
LY294002 drug inhibits the catalytic activity of the p110 kinase. B) When IL-3 binds the receptor, phos-
phorylation of a tryosine residue on the IL-3β chain occurs creating a binding site for the PI3K p85
regulatory subunit. This results in the recruitment of p85 via an Sh2 domain. P85 in turn activates the PI3K
p110 catalytic site. p110 PI3K then phosphorylates certain membrane lipids which result in the activation
of PDK-1 and PDK-2 which occurs via their plextrin homology domains. C) PDK-1 and PDK-2 phos-
phorylate Akt on two different serine/threonine residues which results in activation of Akt. D) Akt
can phosphorylate many downstream targets which result in their activation/inactivation and the
prevention of apoptosis.
22 Cell Cycle Checkpoints and Cancer

also been observed in certain patients with immunodeficiencies.73-79 Thus, modulation of Jak
and STAT activities may be a method of therapeutic intervention in HTLV-I-induced leuke-
mias, chronic myelogenous leukemia, immunodeficiency and other diseases which rely upon
Jak/STAT mediated signal transduction.

The PI3K/Akt Pathway

The stimulation of appropriate target cells by IL-3 also leads to the rapid activation of
PI3K.2,80 PI3K is a heterodimeric protein consisting of an 85 kDa regulatory subunit, which
contains SH2 and SH3 domains and a 110 kDa catalytic subunit.2,80-85 IL-3 stimulation leads
to the creation of a binding site for PI3K on the IL-3R. The SH2 domain of the p85 subunit
associates with this site on the receptor βc chain.51,83 The p85 subunit is then phosphorylated
which subsequently leads to the activation of the p110 catalytic subunit that in turn activates
the downstream targets p70 S6 kinase (p70S6K) and protein kinase B (PKB), also known as
Akt.86-90
The kinase which phosphorylates PI3K may be a member of the Src tyrosine kinase fam-
ily, which includes Fgr, Fyn, Hck, Lyn, Src, Syk, Tec, and Yes in hematopoietic cells.91-94 In
addition, Ras, as well as other Rac and Rho family proteins, can activate or enhance PI3K
activity.2,80-95
Activated PI3K phosphorylates certain membrane lipids which serve to activate the
phosphoinositide kinase dependent kinases (PDK-1 and PDK-2). PDK-1 and PDK-2 then
phosphorylate the Akt kinase (aka protein kinase B, PKB) on a serine and a threonine residue.2,
96
A model of IL-3 induced Akt activity and the subsequent effects on the prevention of apoptosis
is presented in Figure 3.
Activated Akt can further transduce the signal to other targets (e.g., glycogen synthase-3
[Gsk-3] and Tec family kinases) and mediate anti-apoptotic functions by phosphorylating the
pro-apoptotic Bad protein and the regulatory caspase, caspase 9 (See below).2,9,88,89,95-107 In
contrast to the inactivation of the previous molecules by Akt phosphorylation, Akt can also
phosphorylate I-κK, which phosphorylates I-κB, resulting in its ubiquitination and subse-
quent degradation in the proteasome.101-108 Since I-κB is disassociated from NF-κB, NF-κB
can then enter the nucleus and transactivate gene expression. NF-κB can promote gene expression
that, under certain circumstances, stimulates growth as well as prevents apoptosis.101-108,181-187,121
Akt can also phosphorylate certain transcription factors such as the Forkhead family of transcrip-
tion factors (FKHR).97,98 Phosphorylation of the FKHR family of transcription factors prevents
their ability to transactivate the expression of certain pro-apoptotic genes including Fas.
The PI3K pathway can also result in the activation of ribosomal protein kinases. The
p70S6K is an S6 ribosomal protein kinase that phosphorylates S6 in vitro and enhances pro-
tein translation of certain mRNAs.109 The inhibitors wortmannin and LY294002 suppress the
activity of PI3K and rapamycin can inhibit the activity of p70S6K (see Fig. 7). Alternatively,
p70S6K can be activated by PI3K-independent means as well.
The PI3K pathway is also regulated by phosphatases which serve to decrease the activity of
PI3K. The phosphatase PTEN (phosphatase and tensin homologue deleted on chromosome
ten, aka MMAC1 mutated in multiple advanced cancers) has been proposed as a tumor sup-
pressor gene. PTEN is a dual specificity lipid and protein phosphatase that can remove the
phosphates on PI3K-phosphorylated substrates. PTEN downregulates events catalyzed in re-
sponse to Shc, Ras and ERK activation.5

The Ras/Raf/MEK/ERK Signal Transduction Pathway

The Ras/Raf/MEK/ERK cascade is perhaps one of the best-studied signal transduction
pathways. It is centrally involved in the transmission of mitogenic and anti-apoptotic signals as it
couples information initiating from membrane receptors to transcription factors which control
gene expression. Many of the members of this pathway (e.g., Ras, Raf, MEK), as well as additional
downstream targets (e.g., c-Fos, c-Jun, and Ets) are proto-oncogenes. One important reason why
Signal Transduction Pathways: Cytokine Model 23

this pathway was one of the better studied cascades is that certain transforming retroviruses
contained activated oncogenes encoding viral homologues of some of these genes. In contrast,
only the Akt gene and the downstream NF-κB gene have been shown to have viral counter-
parts in the PI3K/Akt cascade and no viral counterparts have been detected in the Jak/STAT
pathway. The Ras/Raf/MEK/ERK pathway is often aberrantly regulated in transformed cells.
Thus, elucidation of the regulation of this pathway may aid in the development of drugs which
will be useful in the treatment of various malignancies.
Ras is a small monomeric GTP-binding protein whose GTP-bound form can associate
with its downstream target, which in some cases is Raf.110-123 Because Ras is ubiquitously
expressed and often mutated in human cancer, Ras was one of the first oncogenes identified as
a potential chemotherapeutic target by pharmaceutical companies.117-122 For Ras to be func-
tional, it must be farnesylated by the enzyme farnesyltransferase (FT) which attaches a 15-
chain fatty acid to Ras. This modification allows Ras to be tethered to the plasma membrane.
There is a large family of Ras-related proteins, including Rho and Rac.117 The roles of these
Ras-related proteins in the growth and transformation of hematopoietic cells remains unde-
fined, but they may serve as potential targets for the FT inhibitors as well.
Ras frequently passes its mitogenic signal onto the Raf proteins, a family of three serine/
threonine kinases (Raf-1, A-Raf and B-Raf ) which contain binding sites for interaction with
Ras.110-116 Activated Ras will induce the translocation of Raf from the cytosol to the plasma
membrane.110-116 Thus, mutations which alter Ras activity may also perturb the actions of Raf
and the downstream cascade. Evidence suggests that this pathway is intimately associated with
the control of apoptotic machinery in myelo-monocytic cells.124
Activation of the Raf-1 pathway is essential for growth factor-induced proliferation dur-
ing hematopoiesis.125 The events that lead to activation of Raf-1 at the plasma membrane are
not fully understood. However Raf-1 activation often occurs in the presence of GTP-Ras.
Inactive Raf-1 proteins are present in the cytosol bound to 14-3-3 chaperonin proteins. The
14-3-3 proteins may bind to a cysteine-rich domain (CRD) present in Raf.126 Cytosolic Raf-1
is translocated to the plasma membrane through interactions with GTP-Ras. This occurs be-
tween the Ras binding domain (RBD) on Raf-1 (aa 55 to 131) and the switch region of GTP-
Ras.116, 127 Once Raf-1 is localized to the plasma membrane, Ras can interact with the Raf-
CRD via the Ras switch-2 region.116,127 These interactions between Ras and the Raf-CRD
serve to displace the 14-3-3 proteins from Raf-1 and uncover its kinase domain, allowing the
phosphorylation of two regulatory tyrosine residues (Y340 and Y341 on Raf-1) by a Src-related
protein-tyrosine kinase.127-145
Displacement of the 14-3-3 proteins from the Raf-CRD also permits the dephosphoryla-
tion of two regulatory serine residues on Raf-1 (S259 and S621). Once all of these changes in
phosphorylation have occurred, Raf-1 is fully activated. It has also been noted that partial
activation of Raf-1 can also be achieved through phosphorylation by other membrane-associ-
ated kinases. There is some evidence for direct activation of Raf-1 by certain protein kinase C
(PKC) isotypes.208,214-217 The α, δ, and ε isoforms of PKC will lead to phosphorylation of Raf-
1; however, only PKC e may functionally activate Raf-1.130,137-139 Alternatively, different PKC
isoforms may stimulate autocrine growth factor loops that, in turn, activate Raf-1.146
There is evidence for crosstalk between the Jak/STAT and the Ras/Raf pathways. Activa-
tion of Raf-1 by Jak is dependent upon recruitment of Raf-1 to the plasma membrane by Ras
and occurs by phosphorylation of Raf-1 at Y340 and Y341.67,71,130,140 Moreover, Ras may
activate both Raf and PI3K.
Certain Raf proteins appear to promote cell cycle arrest. High levels of B-Raf and Raf-1
induce p21Cip1 expression, which inhibit the kinase activities of CDK4/6 and CDK2, thereby
preventing cell cycle progression.147-150 p21Cip1 functions by binding CDK/cyclin And block-
ing the phosphorylation of inhibitor pocket proteins, such as Rb. Many of the effects of the Raf
and downstream MEK1 proteins have been elucidated by conditionally-active DRaf:ER and
∆MEK1:ER constructs.2-9,150-152 These constructs have been developed by Dr. Martin McMahon
24 Cell Cycle Checkpoints and Cancer

Fig. 4. Activation of the ∆Raf:ER and ∆MEK1:ER constructs. The ∆Raf:ER and ∆MEK1:ER retroviruses
have been used to evaluate the interactions between different signaling pathways in the abrogation of the
cytokine-dependency of hematopoietic cells. A) In the absence of either β-estradiol or 4-HT, the ∆Raf:ER,
and ∆MEK1:ER proteins molecules are believed to be complexed with heat shock proteins and present in
monomeric forms. B) Upon addition of β-estradiol or the estrogen receptor antagonist, 4-HT, dimerization
of the ∆Raf:ER or ∆MEK:ER constructs occurs as well as disassociation of the heat shock proteins. C) The
MP-1 scaffolding protein may interact with the dimerized ∆Raf:ER and ∆MEK1:ER constructs creating
a more efficient signalling complex within the cell. One means to activate Raf in cells is by cross-linking two
Raf proteins together. Thus the dimerization of the ∆Raf:ER molecules by either β-estradiol or 4HT may
resemble a natural mechanism by which Raf molecules are activated. Activated Raf and MEK are then
believed to phosphorylate their respective targets and induce gene expression in the nucleus.
Signal Transduction Pathways: Cytokine Model 25

Fig. 5. Effects of activated Raf and MEK1 on the cytokine-dependency of FDC-P1 and TF-1 cells. An outline
of the effects of activated Raf and MEK1 expression on the cytokine—dependency of the FDC—P1 and TF-
1 cell lines is presented in this figure. Factor-independent FDC-P1 and TF-1 cells can be isolated after infection
with retroviruses encoding activated forms of Raf (∆A-Raf:ER, ∆B-Raf:ER or ∆Raf-1:ER) or MEK-1
(∆MEK1:ER). This results in the activation of downstream ERK activity and the secretion of an autocrine
growth factor (GM-CSF) as well as other cytokines (e.g., IL-5, IL-6). In the presence of ∆Raf:ER or ∆MEK1:ER
and autocrine cytokines, a decrease in the mitochondrial membrane potential does not occur and neither
downstream activation of the caspases nor fragmentation of cellular DNA occurs. Active kinases, transcription
factors and anti-apoptotic molecules are shown in gray ovals, whereas pro-apoptotic molecules are shown in
black ovals. IL-3 and the ∆Raf-1:ER and ∆MEK1:ER are shown in clear ovals.
26 Cell Cycle Checkpoints and Cancer

(University of California San Francisco) and can be related by the addition of β-estradiol or the
estrogen receptor antagonist 4-hydroxy-tamoxifen (4HT). A model for the activation of the
∆Raf:ER and ∆MEK1:ER constructs is presented in Figure 4.
We have observed that the introduction of activated Raf and MEK1 oncogenes into the
FDC-P1 and TF-1 hematopoietic cells resulted in the abrogation of the dependency upon
exogenous growth factors in some of the cells. There was a hierarchy in terms of the ability of
the different Raf genes to abrogate cytokine-dependency as the ∆A-Raf:ER was more efficient
than the ∆Raf-1:ER which in turn was more efficient than either ∆MEK1:ER or ∆B-
Raf:ER.151,152 Thus the weakest Raf kinase, ∆A-Raf:ER was more efficient in abrogating the
cytokine-dependency of these hematopoietic cells. In contrast, ∆B-Raf:ER, the strongest Raf
isoform, was more efficient in relieving contact inhibition in fibroblast NIH-3T3 cells. The
hematopoietic cells which grew in response to Raf and MEK1 activation synthesized
sufficient GM-CSF to promote autocrine growth. A model for the effects of Raf and
MEK1 on the downstream signal transduction and apoptotic pathways in FDC-P1 and
TF-1 cells is presented in Figure 5.
It is conceivable that there are specific interactions between certain Raf and 14-3-3 family
members.153-163 These interactions may modulate the activity of Raf proteins and regulate
their ability to lead, either directly or indirectly to the phosphorylation of the pro-apoptotic
Bad protein. Phosphorylation of Bad, which leads to sequestering of Bad by 14-3-3, can also
inhibit apoptosis (see below). We have shown that the B-Raf oncoprotein was the least efficient
Raf oncoprotein in abrogating the cytokine-dependency of human hematopoietic cells.151,152
This may be a reflection of the enhanced capacity of the B-Raf oncoprotein to induce the
expression of cell cycle inhibitory proteins or prevent the phosphorylation of the Bad protein.
In summary, there may be a delicate balance between inducing cell growth and inducing
cell cycle arrest in hematopoietic cells. A Raf oncoprotein with high kinase activity may
actually be the least efficient protein in terms of abrogating cytokine dependency. These studies
indicate that results obtained with fibroblastic models may not always be relevant for other cell
systems (e.g., hematopoietic cells).

The Ras/Raf/MEK/ERK Pathway: Downstream Kinase Activation

Raf activates the dual specific serine/threonine and tyrosine kinase, MEK1, which, in
turn, activates the MAP kinases ERK1 and ERK2 (p42/p44). In cells expressing normal MEK1,
the kinase appears as a 45-kDa protein.164-173 The amino terminal end of the kinase has a
negative-regulatory domain, since deletion of these residues results in constitutive activation of
MEK1, while the catalytic activity is localized to the carboxyl terminus of the protein.174,178
Proline-rich sequences between kinase domains IX and X of MEK1 are required for Raf-1
binding and subsequent activation of MEK1.164
Raf-1-mediated activation of human MEK1 requires the phosphorylation of MEK1 serine
residues 218 and 222.165-168 Substitution of either of these residues with aspartic or glutamic
acid results in a 10- to 50-fold increase in MEK1 activity.177,178 When both serines are replaced
with aspartic acid or aspartic and glutamic acid (218 & 222), MEK1 activity was 400-to 6000-
fold greater.164-168,177,178 These substitutions are believed to confer to the MEK1 protein a
configuration that is constitutively active.167,168 When these MEK1 mutants were transfected
into NIH3T3 cells, constitutive activation of p42/p44 ERKs occurred as well as foci forma-
tion.164-168 This suggests ERK activation through Raf requires MEK1. In support of this hy-
pothesis, PD98059, a MEK1 inhibitor developed by Parke-Davis, prevents ERK activation
mediated by activated Raf constructs.151,152,179,180
In addition to the three Raf kinases, several other kinases influence MEK activity. One
such kinase is the oocyte-expressed proto-oncogene, Mos.169,170 Constitutively active forms of
Mos (v-Mos) transform fibroblasts via a MEK1-ERK-dependent pathway implicating Mos as a
MEK kinase.170 Mos preferentially phosphorylates MEK1 on serine 222.169,170 In contrast, the
MEK kinase-1 (MEKK1), which is associated with stress-activated pathways (SAPK) and whose
Signal Transduction Pathways: Cytokine Model 27

Fig. 6. Synergy between Raf/MEK and PI3K/Akt in the abrogation of cytokine-dependency of FL5.12 cells.
The stimulation of the Raf/MEK/ERK and Akt/PI3K pathways can lead to autocrine transformation of
FL5.12 cells. Inactive kinases are depicted in black ovals. Activated kinases, phosphatases and transcription
factors are depicted in clear ovals. Induced expression of ∆Raf:ER and Akt:ER may lead to GM-CSF
expression transcription and autocrine transformation. The steps after MEK1 activation, which result in
GM-CSF transcription, are not known at the present time, although a plausible pathway is indicated.
Potential effects of Raf and Akt on p38MAPK activation are indicated in dotted lines. Also shown are
additional pathways regulated by Akt which may be activated (TCF Responsive gene transcription) or
inactivated (Fas).
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