Supplementary Sidebars Collection

Supplementary Sidebars
Supplementary Sidebar 1.1






Each female cell can access information only from a single X chromosome
Reproductive cloning demonstrates the extraordinary efficiency of the DNA repair apparatus
The network of miRNA-controlled genes
Knocking down gene expression with shRNAs and siRNAs
Gene cloning strategies
Commonly used histopathological techniques
The complicated conventions for classifying and naming tumors
Is SV40 responsible for the mesothelioma plague?
Maintenance of KSHV and HPV genomes in episomal and chromosomal form
Re-engineering the retrovirus genome for gene therapy
Classic Kaposi’s sarcoma appears to be a familial disease
Viruses like RSV have very short lives
Endogenous viruses can explain tumor development in the absence of infectious viral spread
Boveri and Hansemann independently hypothesized genetic abnormality as the cause of
cancer cells’ malignant behavior
Southern and Northern blotting
Genes undergo amplification for a variety of reasons
Making anti-Src antibodies presented a major challenge
The protozoan roots of metazoan signaling
Lateral interactions of cell surface receptors
Systematic surveys of phosphotyrosine and SH2 interactions
The complexities of understanding RTK signaling
The rationale for multi-kinase signaling cascades
Non-canonical Wnt signaling
The Hippo pathway and control of stem cell proliferation
Heterozygosity in the human gene pool
Which is more likely—LOH or secondary, independent mutations?
The polymerase chain reaction makes it possible to genetically map tumor suppressor genes
rapidly
The MSP reaction makes it possible to gauge methylation status of promoters
Ubiquitylation tags cellular proteins for destruction in proteasomes
Krebs cycle enzymes and cancer development
Homologous recombination allows restructuring of the mouse germ line
The origins of embryonic stem cells
Plasticity of the cell cycle clock
Chromatin immunoprecipitation (ChIP)
Some tumors increase Id concentrations by de-ubiquitylating them
Specific targeting of cell cycle regulators by E3 ubiquitin ligases
The major puzzle surrounding the RB gene: retinoblastomas
UV-B radiation, HPV, and cutaneous squamous cell carcinomas
The TUNEL assay
Dominant-negative functions of mutant p53 alleles: functional interactions between p53 and
its p63 and p73 cousins
Some mutant p53 alleles cause highly specific tumors
Autophagy is critical to post-fertilization development
The use of the TRAP assay and adaptations thereof permits the rapid and quantitative assessment
of the levels of the catalytic activity of telomerase enzyme in eukaryotic cells
Monoclonal antibodies and fluorescence-activated cell sorting (FACS)
How does multi-step tumor progression actually take place?
Symbiosis between distinct subpopulations within a tumor
Comparative genomic hybridization
Are rodent carcinogen tests reliable indicators of danger to humans?
Does saccharin cause cancer?
How does diet affect colon cancer incidence?
Hematopoiesis as a model for the organization of many kinds of tissues
Stem cell pools may explain the protective effects of pregnancy
The conserved-strand mechanism and protection of the stem cell genome
Oxidation products in urine provide an estimate of the rate of ongoing damage to the cellular
genome
How does red meat cause colon cancer?
A convergence of bacterial, yeast, and human genetics led to the discovery of hereditary
non-polyposis colon cancer genes

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Homology-directed repair
Localization of growth factors is important for proper heterotypic interactions
Ongoing heterotypic signaling in carcinomas
Certain highly advanced tumors provide exceptions to the generally observed dependence
of carcinoma cells on stroma
Myofibroblasts predict clinical progression of cancer
A technique for separating stromal from epithelial cells
Microvessel leakiness dooms many forms of anti-cancer therapy: optimizing anti-angiogenic
treatments
The temporary nature of vessel regression created by anti-angiogenesis therapy
Kaposi’s sarcoma cells hold the record for the number of documented heterotypic signals they
receive
Effects of an anti-VEGF-R monoclonal antibody on the growth of a human tumor xenograft
Fibroblasts are heterogeneous and can change dynamically in response to signals
Visualization of the dynamics of pathfinding fibroblasts followed by squamous cell carcinoma
cells
Metastasizing cancer cells often take on hitchhikers while traveling through the blood
Instruments for detecting circulating tumor cells
Hidden micrometastases are revealed through organ transplantation
Wolves in sheep’s clothing: when carcinoma cells invade the stroma
TGF-β works in conflicting ways during tumor progression
Dynamics of EMT induction: the EMT may be controlled in some cancer cells exclusively by
their own genomes
An example of an EMT relatively late in embryonic development
Relatively rapid metastatic dissemination of advanced primary tumor cells
Our cells devote an enormous number of genes to regulating protein degradation
Peritoneovenous shunts provide dramatic support for the seed and soil hypothesis
Tooth extractions may occasionally become exceedingly painful
Tumor stem cells further complicate our understanding of the metastatic process
Does Darwinian evolution accommodate metastasis-specific alleles?
Rearrangements of chromosomal DNA segments generate a vast array of antigen-binding
domains in antibodies and T-cell receptors
Virus-infected cells may not always be recognized by the immune system
Bizarre tumors reveal how cancer cells can become infectious agents
Mice have proven to be far more useful for tumor biologists than chickens
An HPV vaccine protects against many cervical carcinomas
An unexpected type of anti-p53 reactivity is often found in cancer patients
Immune recognition of tumors may be delayed until relatively late in tumor progression
Some paraneoplastic syndromes reveal defective tolerance and overly successful immune
responses to tumors
TSTAs can arise as by-products of chemical and physical carcinogenesis
Are melanomas more antigenic than other tumors?
Strategies for cloning genes encoding melanoma TATAs
Anti-CD47 therapies hold promise in treating lymphomas and other hematopoietic
malignancies
Cancer cells may thwart extravasation by circulating T cells
Herceptin can be modified to potentiate cancer cell killing
Bone marrow transplantation and the treatment of hematopoietic malignancies
Whole genome sequencing allows a new attack on tumor cells
Modern cancer therapies have had only a minor effect on the overall death rate from the disease
Prostate cancers usually do not require aggressive intervention—a tale of two countries
Clinical practice and our understanding of disease pathogenesis have often been poorly
aligned, leading to sub-optimal, often tragic outcomes
The ability to assign tumors to specific disease subtypes is critical to the success of drug
development
p53 germ-line polymorphisms and somatic mutations can complicate the induction of
apoptosis by drugs
Ras function can be inhibited by interfering with the enzymes responsible for the
maturation of the Ras protein
Chemical synthesis, compound libraries, and high-throughput screening
Evolution can generate huge collections of structurally similar proteins
Large-scale screen of the inhibitory effects of a drug on various kinases
Epidermal growth factor receptor expression levels predict little about a tumor’s
susceptibility to receptor antagonists
Akt/PKB function is controlled by multiple upstream signals

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Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg
Supplementary Sidebar 1.1 Each female cell can access
information only from a single X chromosome In the simplest
depiction of sex chromosome behavior, each male cell relies
on the information carried by its single X chromosome, while
each female cell is able to consult both of its X chromosomes.
Given the large number of genes present on the X chromosome,
this disparity would create substantial physiologic differences
between male and female cells, since female cells would, in general, express twice as much of each of the products specified by
the genes on their X chromosomes. This problem is solved by

© 2014 Garland Science

the mechanism of X-inactivation. Early in embryogenesis, one
of the two X chromosomes is randomly inactivated in each of
the cells of a female embryo. This inactivation silences almost
all of the genes on this chromosome and causes this chromosome to shrink into a small particle termed the Barr body
(Figure S1.1). Subsequently, all descendants of that cell will
inherit this pattern of chromosomal inactivation and will therefore continue to carry the same inactivated X chromosome.
Accordingly, the female advantage of carrying redundant copies
of X chromosome–associated genes is only a partial one.

Figure S1.1 X chromosomes as Barr bodies One of the two
X chromosomes in each cell of an early female embryo is
randomly inactivated and remains inactivated in all lineal
descendant cells. It is visible in the interphase nuclei of these cells,
where it remains condensed and associated with the nuclear
membrane (white arrow). (From B.P. Chadwick et al., Semin. Cell
Dev. Biol. 14:359–367, 2003.)

TBoC2 b1.10/s1.01

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Supplementary Sidebar 1.2 Reproductive cloning demonstrates the extraordinary efficiency of the DNA repair apparatus The efficiency of the complex apparatus that repairs DNA
(see Chapter 12), the resulting suppression of somatic mutations, and the consequent integrity of the genomes of somatic
cells have all been illustrated dramatically by the successes of
animal cloning in recent years. In the much-celebrated case
of the sheep Dolly, the nuclei from cells taken from the breast
tissue of her “mother” were removed and implanted in egg
cells (oocytes) whose own nuclei had previously been eliminated. The resulting cells, containing donor nuclei and recipient egg cytoplasm, were then induced to proliferate and to form
embryos (Figure S1.2). The fact that an essentially normal sheep
(Dolly) was born many months later, having developed from
one of these embryos, demonstrated that the genome in one of


her mother’s mammary gland cells carried no mutations that
compromised normal embryologic development.
The implied intactness of the genome in the breast cells of
Dolly’s mother indicates a high degree of integrity in DNA replication. During embryonic development, the genome of the fertilized egg that was destined to develop into Dolly’s mother was
copied and recopied dozens of times during the cycles of cell
growth and division that intervened between the initial formation of the fertilized egg and the formation, many months later,
of the mother’s breast tissue. This recopying process, involving
DNA replication and a repair apparatus that operated to weed
out mutant DNA sequences, was highly effective in preserving
an intact somatic cell genome that was capable of programming
normal organismic development.

Dolly’s “mother”
cells prepared
from mammary gland

single cell prepared
cells fuse by
electric shock

unfertilized
sheep egg

haploid
nucleus
removed

egg
induced
to divide

early
embryo

embryo
transferred
to womb
of second
sheep

Dolly
is born

enucleated
egg

Figure S1.2 Reproductive cloning and genomic integrity
In the initially reported reproductive cloning procedure, the nucleus
of an unfertilized egg was removed and the nucleus from a
somatic cell—a mammary gland cell from Dolly’s “mother”—was
introduced into the enucleated egg. The resulting cell was diploid,
having received its entire genetic complement from the mammary
gland cell originating with Dolly’s “mother.” Being diploid, it was
genetically equivalent to a fertilized egg. This reconstituted egg
cell was induced to divide by chemical means and proceeded to
generate an embryo that could then be implanted in the womb
of a foster “pseudo-pregnant” ewe—a ewe whose physiologic

state was comparable to that of a naturally pregnant ewe but had
been induced in this case by hormonal treatment. This implanted
embryo could then develop into a newborn, albeit at low efficiency.
Successful generation of a healthy newborn revealed that the
donor somatic cell from Dolly’s “mother’s” mammary gland carried
the genome of the species in an essentially intact, functional form.
This donor mammary gland cell initially arose after many successive
cell divisions during the development of Dolly’s “mother” from a
natural fertilized egg. The ability of this cell’s genome to generate
Dolly indicated that these many successive cell divisions did not
affect its integrity.

TBoC2 b1.13/s1.02

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Supplementary Sidebar 1.3 The network of miRNA-controlled genes Research into the functions of microRNAs makes it
increasingly apparent that the molecules are key components
of myriad regulatory circuits within cells. Indeed, it is plausible
that almost all regulatory circuits rely on one if not several miRNAs in order to modulate or fine tune the signals that they are


processing. The image presented here (Figure S1.3) presents the
state-of-the-art in miRNA research in 2008 that focused on miRNAs that affect signaling pathways relevant to cancer pathogenesis. Subsequently reported research has generated a far more
elaborate network of interactions.

been demonstrated by 2008 to play a role in modulating various
Figure S1.3 OncomiRs and cancer pathogenesis
cancer-related cell-biological phenotypes; this class of microRNAs
Characterizations of microRNAs have revealed that they play
have been dubbed oncomiRs. The complexity of this microRNAimportant roles in modulating the expression of proteins in a
TBoC2 n1.108/s1.03
regulated circuitry is growing progressively. (From Melanoma
wide variety of subcellular regulatory circuits, including those that
Molecular Map Project; see http://www.mmmp.org/MMMP/public/
are deregulated during the course of cancer development. This
biomap/viewBioMapImage.mmmp)
map illustrates the involvement of a series of microRNAs that had

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Supplementary Sidebar 1.4 Knocking down gene expression
with shRNAs and siRNAs The discovery of microRNAs (miRNAs), as described in Section 1.10, led to an understanding of
the complex molecular machinery that allows their processing
from primary transcripts in the nucleus and their dispatch into
the cytoplasm. There they inhibit the functioning of thousands of
cellular mRNA species. At the same time, these discoveries enabled the development of novel experimental strategies in which
exogenous miRNAs could be introduced into cells through the
use of viral vectors or transfection, whereupon these introduced
RNAs exploit the cellular miRNA-processing machinery to produce mimics of endogenously synthesized miRNAs; the mimics then proceed to shut down the operations of targeted mRNA
species, partly by blocking translation and partly by driving the
degradation of these mRNA molecules (Figure S1.4).
This ability to shut down the expression of proteins of
expressed genes within cells has become an invaluable tool for
cancer researchers who wish to demonstrate the essential role
that one or another protein plays in a cell-biological process
under study. Extending the earlier nomenclature that described
the inactivation of genes in the mouse germline—termed knockout (see Supplementary Sidebar 7.7)—the new procedure has
come to be called “knockdown.”
For example, knockdown of the expression of a certain


pro-apoptotic protein may protect a cell from apoptosis, demonstrating the essential role that the protein plays in orchestrating apoptosis in such a cell. Similarly, knockdown of the expression of a growth factor receptor may render a cell unresponsive
to that factor, demonstrating the receptor’s key role in conferring such responsiveness.
While highly useful in principle, in practice the application
of this technique has proven challenging in the case of certain
genes being targeted for knockdown. Thus, a targeted mRNA
may not respond significantly for reasons that are unclear,
necessitating the screening of multiple vectors, each targeting a
distinct nucleotide segment in the 3ʹ untranslated region of an
mRNA under study; in some cases, four, five, or even six alternative siRNAs or shRNAs must be tested before an experimenter
discovers one that can reduce protein expression by 90% or
more.
Independent of these difficulties in achieving significant
knockdown is the opposite problem: some siRNAs and shRNA
have been found to affect far more mRNAs than their intended
target—the phenomenon of “off-target effects.” Off-target effects
often make it impossible to draw rigorous conclusions from the
results of these experiments because of the confounding effects
on other mRNAs and proteins operating in unrelated signaling
pathways.

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lentiviral or
retroviral
vector

CYTOPLASM

NUCLEUS
polIII

integrated provirus

Drosha
pre-shRNA


Figure S1.4 Suppression of RNA function by RNA
interference The process of RNA interference (RNAi) operates
normally in cells to produce microRNAs (miRNAs) that
suppress mRNA function, as described in Section 1.10 and
Figure 1.20. This process can be exploited experimentally to
suppress targeted mRNAs. Two major strategies can be used.
In the first, a retrovirus or lentivirus vector (see Supplementary
Sidebar 3.3) is constructed that uses an RNA polymerase III
promoter to drive expression of a transcript that serves as
precursor of an shRNA (small hairpin RNA). The resulting preshRNA is processed by the Dicer enzyme into an siRNA (small
interfering RNA) of 19–21 nucleotides, which is reduced to a
single-stranded RNA (ssRNA). The ssRNA associates with the
Argonaut protein (Ago2) and other proteins to form a RISC
(RNA-induced silencing complex), which anneals to partially
homologous target sequences that are usually located in
the 3ʹ untranslated region (3’UTR) of certain mRNAs. This
results either in the cleavage of the mRNA or inhibition of its
translation, blocking in both cases expression of the mRNA and
thus the gene encoding this mRNA. Because the retroviral or
lentiviral provirus is integrated into the cell chromosome and is
transmitted heritably to the progeny of an initially infected cell,
the expression of a targeted mRNA can be suppressed stably
over many successive cell generations.
As an alternative strategy, an siRNA of a particular
configuration can be chemically synthesized and transfected
into a cell. While this is easier than the procedure described
above, the targeted mRNA will be suppressed only transiently,
since there is no mechanism to ensure the continued synthesis
of the siRNA in the descendants of the initially transfected cell.

Dicer

RISC

inhibition of translation

mRNA cleavage

s1.04

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Supplementary Sidebar 1.5 Gene cloning strategies Gene
cloning strategies first became practical in the late 1970s, and
since that time a diverse array of strategies has been developed.


Figure S1.5 illustrates the logic of gene cloning that is common
to many of these strategies.

fragment genome

introduce fragments separately into vector
arms of vector

amplify each fragment separately in a
bacterial colony

identify and amplify
inserted fragment
of interest

genome segment
(carrying gene of interest)
has been cloned

Figure S1.5 Molecular cloning of
genes Many versions of the gene cloning
procedure have been developed since
this technology was first invented in
the 1970s. Pictured is an outline of one
version. The genome from an organism
(e.g., humans) is fragmented into
relatively small segments, often several
tens of kilobases long (top); each DNA
segment is linked to the DNA of a vector
(gray arms), yielding a recombinant vector.
Each recombinant vector is then inserted
independently into a bacterium, which
is expanded into a colony containing
many thousands of bacteria. The bacterial
colony bearing the vector with the gene
of interest is identified and the vector is
retrieved, yielding millions of copies of this
genomic fragment (bottom right).

TBoC2 b1.21/s1.05

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Supplementary Sidebar 2.1 Commonly used histopathological techniques Several techniques are used frequently to mark
structures in the fields viewed through a light microscope in
order to resolve tissue architecture, the subcellular structures

Figure S2.1 Some frequently used techniques for staining
histopathological sections While certain modern microscopic
techniques allow visualization of tissues at various depths of
focus (sometimes termed the “Z axis”), the great majority are
limited to viewing at a single depth, either in cell monolayers
or in tissue sections. The latter are prepared by sectioning
tissues that have been stabilized by certain fixatives, such as
formaldehyde, glutaraldehyde, or ethanol, which permeabilize
cells and cross-link molecules in the biological sample prior
to its being embedded in paraffin wax and sectioned; this
embedding increases the mechanical strength of the sample,
thereby facilitating sectioning. Alternatively, a tissue sample
may be frozen and sectioned. In both cases, the resulting tissue
sections are then deposited on glass slides. The magnification
of the images (e.g., 20×) is then represented as the size of an
object in an image of a microscopic field divided by its actual
size; this factor may then be further increased or decreased as
the initially viewed image is printed or presented in digital form.
(A) Hematoxylin and eosin (H&E) staining reveals complex tissue
architecture in this section of a human basal-like carcinoma of
the breast (40×). Hematoxylin stains nuclear structures, notably
the histones in the chromatin, blue; in contrast, eosin stains
cytoplasm, connective tissue, and extracellular structures, such
as extracellular matrix, pink. Note the two major types of tissue
organization here: the carcinoma cells have large dark purple
nuclei and are assembled in continous aggregates while the nonneoplastic cells forming the loose, fibrous stroma have small very
dark nuclei. (B) Use of Masson’s trichrome stain allows a variety
of structures to be visualized with a single staining procedure.
In this micrograph (20×) of experimentally transformed human
mammary epithelial cells growing as a tumor xenograft in a
mouse, erythrocytes (red blood cells) appear bright red, collagen
appears blue, cytoplasms appear light pink, and cell nuclei
appear dark pink (C) Immunohistochemistry (IHC) depends on
the ability to conjugate (link) an antigen-specific antibody with
an enzyme, usually horseradish peroxidase; this enzyme uses
added hydrogen peroxide (H2O2) to oxidize an added chemical
substrate, usually generating a dark brown or black product. In
this image (40×), the section of a human basal-like breast tumor
has been immunostained using an antibody that binds the p53
tumor suppressor protein, which, in mutant form, accumulates
in high concentrations in cell nuclei, revealing them here as dark
ovals. The section was then stained with hematoxylin which was
used here as a counterstain to reveal remaining structures in the
tumor, seen here as light blue. Nuclei stained with hematoxylin
but not with the anti-p53 peroxidase-conjugated antibody are
mostly normal, non-neoplastic cells of the tumor-associated
stroma. (D) Normal human mammary gland tissue has been


within individual cells, proteins that form these various structures, and even specific genes borne by chromosomal DNA
(Figure S2.1).

stained here using the technique of immunofluorescence (IF),
in which antigen-specific antibodies have been conjugated to
fluorescent dyes (sometimes termed fluorophores) that emit light
at various wavelengths after being excited with light of different,
specific wavelengths. This allows the staining with multiple
distinct antigen-specific antibodies (in this case three) that enables
the concomitant visualization of multiple distinct antigens and
thus the cells that express them. In this micrograph (63×), cells
expressing the CD44 antigen have been stained with a conjugated
antibody that generates a pink/purple signal, cells expressing the
CD24 antigen have been stained with an antibody that generates
a blue signal, while cells expressing α-smooth muscle actin have
been stained with an antibody that generates a green signal. The
blue mammary epithelial cells are arrayed as the lining of ducts
that have a dark lumen (cavity) in their center. (E) An alternative
combination of antibodies allows this visualization of human
basal-like breast cancer at 40× magnification. In this case, the
anti-p53 antibody has been conjugated to a fluorescent dye
emitting a blue signal, an antibody recognizing the BRCA1 protein
has been conjugated with a dye emitting a red signal (yielding
small red dots in cell nuclei), while an antibody recognizing the
PTEN tumor suppressor protein has been stained with an antibody
that emits green light. This image indicates that the neoplastic
cells with pinkish blue nuclei generally have lower levels of the
BRCA1 protein than do the non-neoplastic cells of the tumor
stroma, which lack mutant p53 protein and thus the pinkish blue
nuclei. (F) In this micrograph (63×), immunofluorescence staining
has been combined with fluorescence in situ hybridization (FISH),
which allows the detection of DNA sequences in the section
by annealing (hybridizing) sequence-specific DNA probes that
have been coupled with specific fluorescent dyes; the combined
staining is sometimes termed immunoFISH. Here, an anti-p53
antibody stains carcinoma cell nuclei dark blue, while specific
DNA probes have detected DNA sequences associated with the
centromeres of Chromosome 10 (CEP10, red) and Chromosome
17 (CEP17, green), as well as the genes encoding the PTEN
(yellow) and BRCA1 (light blue) proteins. (G) Monkey kidney cells
growing in monolayer culture were forced to express a protein
that caused them to undergo cell–cell fusion, generating a multinucleated cell, sometimes termed a polykaryon or syncytium
(center). The nuclei were stained with DAPI (4’,6’-diamidino-2phenylindole), a dye that emits blue fluorescence when it binds to
dsDNA (double-stranded DNA), while the actin cytoskeleton was
stained with phalloidin, which binds actin filaments and in this
case has been linked to the green fluorescent dye FITC (fluorescein
isothiocyanate). (A,C–F, courtesy of F. Martins and K. Polyak.
B, courtesy of S. McAllister. G, courtesy of S. Rozenblatt.)

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(A)

(B)

(C)

(D)

(F)


(E)

(G)

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Supplementary Sidebar 2.2 The complicated conventions
for classifying and naming tumors There are several hundred distinct cell types forming the specialized tissues in the
human body. Human tumors that arise in the cells of these
various tissues exhibit a wide spectrum of phenotypes that
defy categorization by any single classification scheme that utilizes either purely morphological or purely molecular criteria.
Consequently, human tumors are currently classified by a combination of their histological, clinical, and molecular features.
A purely molecular taxonomy may well emerge in the future,
but for the moment we are forced to deal with the complex classification schemes that have developed over the past century.
The rationale behind the currently used approaches is outlined
below.
The great majority of human tumors arise from epithelial
tissues. While the term cancer is generally used to refer to all
malignant human tumors, the term carcinoma specifically
refers to human tumors that arise from epithelia. The term adenocarcinoma is a further subdivision of epithelial cancers that
arise from single-cell-layer or two-cell-layer epithelia that share
key features in their form and function. For example, these epithelia form glands in which the epithelial cells exhibit an apical–
basal polarity, with the apical end oriented toward the lumen
(the central space enclosed by the gland) and the basal end oriented away from the lumen. In general, these cells secrete substances into the lumen and some may also absorb substances
from it.
This explains why the prefix adeno- (which is derived from
the Greek word for gland) is used to distinguish the above
tumors from other carcinomas that arise in epithelia—such as
those of the skin and bladder—that act as a protective barrier
rather than facilitator of molecular transport. Benign epithelial
tumors are in general referred to as adenomas. It is notable that
the majority of human cancers are adenocarcinomas arising
from lung, breast, prostate, pancreas, liver, colon, uterus, ovary,
and so forth. Precisely why adenocarcinomas account for such a
disproportionate share of human tumors is not easily explained
by invoking either the proportion of the human body that these
epithelia represent or the proliferation rate of their constituent
cells.
Nonepithelial tumors are dramatically different from epithelial tumors in several important aspects. Thus, when referring to


these other tumors, a uniform terminology, comparable to the
adenoma and adenocarcinoma terminology used for epithelial tumors, is often replaced by a differentiation state-based
system, that is, one that is based on the degree of differentiation and tumor progression. For example, benign nonepithelial
tumors that arise from the mesenchymal cells of the stroma are
designated by adding the suffix -oma to the name of the normal
differentiated tissue that they resemble; hence, lipoma is the
term for fat-cell tumors. The suffix -sarcoma is used for the corresponding malignant tumors—liposarcoma, in this example—
that appear to arise in the same tissue.
The hematopoietic neoplasms are classified based upon
their physical appearance, specifically, lymphomas (solid
masses) or leukemias (individual cells suspended in the blood),
regardless of their benign or malignant behavior. Much of this
terminology developed independently for each tissue type and
reflects the history of pioneering research for each neoplasm.
By now, the terms in common use are so embedded in clinical
oncology that they are essentially impossible to change.
Regardless of these quirks, these broad categories can teach
important lessons about the nature of tumors that arise in different tissue types. For example, there is a great difference between
benign epithelial and stromal tumors, both of which may be
called “-omas.” Epithelial adenomas are often intermediaries
in a multi-step progression that begins with fully normal cells
and ends with malignant adenocarcinomas, as is discussed in
Chapter 11. In contrast, certain benign stromal “-omas” may
persist stably in a benign state without further progression and
may thus behave very differently from malignant sarcomas arising in the same tissues.
In addition, there is a great difference in the types of genetic
lesions that drive malignancy in these two broad classes. Most
nonepithelial tumors are typically associated with a single,
dominant genetic event, such as a specific gene translocation.
Often, these translocations are so uniform (“recurrent”) among
different patients and so distinctive that their presence is determined in order to help make a definitive diagnosis of the specific type of tumor being examined. Examples of these tumors
are listed in the accompanying Table S2.1 below.
In contrast, it is generally very difficult to find any single
mutation or translocation that is uniformly present in most
cases of a given epithelial cancer type, with the K-ras mutations

Table S2.1 Examples of malignant nonepithelial tumors and their characteristic genetic lesions
Tumor

Translocation

Involved genes

CML

t(9;22)(q34;q11)

Bcr-Abl

Ewing’s sarcoma

t(11;22)(q24;q12)

EWS-FLI1

Clear-cell sarcoma

t(11;22)(p13;q12)

EWS-WT1

Myxoid chondrosarcoma

t(9;22)(q22;q11-12)

EWS-ATF1

Alveolar rhabdomyosarcoma

t(2;13)(q35;q14)
t(1;13)(q35;q14)

PAX3-FKHR
PAX7-FKHR

Synovial sarcoma

t(X;18)(p11.2;q11.2)

SYT-SSX1; SSX2

Myxoid liposarcoma

t(12;16)(q13;p11)

CHOP-TLS(FUS)

Congenital fibrosarcoma

t(12;15)(q13;q25)

ETV6-NTRK3

Text and table courtesy of Tan A. Ince.

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in pancreatic carcinomas and HER2/neu amplifications in a
subset of breast cancers being rare exceptions. Epithelial malignancies are typically driven by multiple, accumulated genetic
events that are present in different combinations in different
tumors and follow the well known “multi-hit” model of stepwise
progression to cancer (see Chapter 11).
Tumors that arise in the nervous system represent yet
another special category. The cells of the brain and peripheral
nerves are derived from the specialized neural ectoderm of the
early embryo (see Figure 2.5) and thus differ from non-neural
epithelia and mesodermal derivatives (that is, stromal and
hematopoietic cells). Due to their distinct embryologic origin, the neuroectodermal tumors that arise in the central and


peripheral nervous system are considered to be a fourth category of tumors. Nature knows no fixed rules, however, and there
are some tumors that are difficult to classify from this simple
perspective. For example, choroid plexus cells that line the ventricles of the brain are also derived from the neural ectoderm,
but they give rise to tumors with an epithelial phenotype.
Finally, some tumors can develop into subpopulations of
neoplastic cells exhibiting distinct cell phenotypes, yielding
admixtures of epithelial and even stromal cell types. The prototypical example of this class of tumors is provided by teratomas,
which are composed of tissues from multiple germ cell layers
and hence are suspected to arise from germ or stem cells.

10

Supplementary Sidebar 3.1 Is SV40 responsible for the
mesothelioma plague? The mesothelium is the membranous
outer lining of many internal organs and derives directly from
the embryonic mesoderm. Mesotheliomas—tumors of various mesothelial surfaces, notably the pleural membranes of the
chest—were virtually unknown in the United States in the first
half of the twentieth century. Beginning in about 1960, however,
their incidence began to climb steeply. By the end of the century,
the annual incidence in the United States approached 2500 and
was attributed mostly to asbestos exposure, specifically a subtype termed crocidolite. The disease is seen frequently among
those who have worked with asbestos, which was used as a heatresistant insulating material until being banned in the 1980s.
However, 20% of mesothelioma patients have no documented
exposure to asbestos, a fact that has provoked a search for other
etiologic (causative) agents.
SV40 is a plausible etiologic agent of mesothelioma. By 2003,
forty-one laboratories across the world had reported SV40 DNA,
RNA, or protein in mesothelioma cells. Traces of the virus are
otherwise rarely found in human tumors, with the exception of
certain types of brain tumors. Cultured human mesothelial cells
are readily infected by SV40, and this infection leads rapidly to
their immortalization, that is, to the ability of these cells, which
usually have limited proliferative potential in culture, to multiply
indefinitely.


The presence of SV40 contamination in poliovirus vaccine
stocks has raised concerns that mesothelioma may have been
induced in many individuals as an unintended side effect of
poliovirus vaccination. However, there are many individuals with
mesotheliomas who are highly unlikely to have been exposed
to these vaccines. Moreover, the viral T-antigen protein, whose
presence is invariably observed in SV40-transformed cells (see
Section 3.6), is rarely detected in mesothelioma tumors. Also,
attempts at demonstrating viral DNA have usually yielded DNA
segments that are indicative of laboratory artifacts, for example,
contamination of mesothelioma tumor samples by laboratory
stocks of SV40 or by recombinant DNA plasmids engineered to
include segments of the viral genome. The discoveries of such
contaminations provide increasing ammunition for those who
are skeptical of SV40’s role in mesothelioma pathogenesis.
As many as 85% of humans are known to be infected with two
viruses that are closely related to SV40—JC and BK—and antisera
that recognize the capsids of these viruses cross-react with the
capsid of SV40, explaining many of the claims that SV40 is often
present in human tissues. In immunosuppressed individuals,
notably AIDS patients, JC virus can cause a fatal brain degeneration—progressive multifocal leukoencephalopathy (PML). And
in patients who are immunosuppressed because they are organ
transplant recipients, BK virus has been implicated in the causation of certain carcinomas (see, for example, Figure 3.14B).

11

Supplementary Sidebar 3.2 Maintenance of KSHV and HPV
genomes in episomal and chromosomal form Both herpesvirus and papillomavirus genomes are able to persist for years in
a virus-infected tissue in an episomal form, that is, as molecules
that are not integrated into the host-cell chromosomal DNA. In
the case of Kaposi’s sarcoma herpesvirus (KSHV), this is achieved
through the actions of its LANA protein (Figure S3.1A), which
binds to terminally repeated sequences in the viral genome as
well as to the histone molecules forming the core nucleosome
(see Figure 1.19). This enables maintenance of the viral genome
even though direct covalent linkage between viral and host
DNAs is absent.
A similar dynamic operates in human papillomavirus (HPV)infected tissues. Following initial infection, HPV-infected cells
and viral genomes in general are cleared from the infected tissue within a period of weeks or months by the immune system.
However, in a minority of infected individuals, the HPVs persist,
establishing chronic infections in epithelial tissues and maintaining their genomes, once again, in an episomal state, apparently for years if not decades. These viral infections sometimes
trigger low- and then high-grade squamous intraepithelial
lesions (LSILs, HSILs) of the cervix; the number of episomal HPV
genomes per cell generally increases during this progression
and, depending on the subtype of HPV virus, may reach more
than several hundred copies per infected cell.
On rare occasion, these chronic infections may trigger the
onset of frank malignancies—cervical carcinomas; squamous
cell carcinomas triggered by HPV can also arise in the oropharynx. In the genomes of the cervical carcinoma cells, oncogenic
fragments of the HPV genomes are invariably found in an integrated state, while the episomal HPV genomes are generally no


longer detectable. This integration is achieved by mechanisms
similar to those used by SV40 to establish its genome in cells that
it has transformed (see Figure 3.16/n3.103) and involves nonhomologous recombination between viral genomes and hostcell chromosomal sequences. This chromosomal integration,
as was the case with SV40, ensures the perpetuation of the HPV
genome fragments in descendant cells. The HPV genomes seem
to be integrated preferentially in chromosomal “common fragile
sites”—chromosomal regions (>100) scattered throughout normal genomes and exhibiting relatively high genetic instability,
including susceptibility to dsDNA breakage.
This integration has a second functional consequence: the
molecular steps that lead to integration usually result in the
deletion or inactivation of certain viral genes that are needed for
viral replication of episomal viral DNA, and of other genes whose
products operate to repress expression of the virus-encoded
oncoproteins (Chapters 7 and 8). Without this repression, the
viral oncoproteins are expressed by the integrated viral genomes
at far higher levels, thus driving the progression of pre-neoplastic
virus-infected cells into carcinoma cells.
The repression of HPV oncoprotein expression is the task of
the viral E2 protein, which has a second, ostensibly related function: it acts to link the unintegrated viral genome to a cellular
protein that is bound to chromatin during mitosis (Figure S3.1B).
This ensures the tethering of the viral genome to chromosomes
during mitosis (Figure S3.1C). When this tethering is blocked
experimentally, the viral genomes are unable to “hitchhike” with
the chromsomes during mitosis and consequently are largely
left in the cytoplasmic portion of the mitotic cells, which leads
to their rapid loss following multiple successive cellular growthand-division cycles.

12

(A)


(B)
DNA

N-terminal
domain of E2

C-terminal
domain of E2

H3
Brd4
chromatin
protein

H2A

C

N
hinge

H4
LANA

N

α1

α2

α3

binding to
viral DNA

H2B
(C)

E2 functional

~175 on chromosomes
E2 blocked

Figure S3.1 Tethering of episomal viral genomes to
host-cell chromatin Certain DNA viruses maintain chronic
infections through their ability to maintain their genomes stably
but in an unintegrated (episomal) configuration over many
successive cell generations. They do so by synthesizing viral
proteins that are able to physically link the viral nucleoprotein
to the chromatin of the infected host cell when that cell passes
through mitosis, ensuring transmission of the viral episomal
genomes to both daughter cells. (A) In the case of KSHV/
HHV-8, the agent of Kaposi sarcoma, the virus makes a protein
termed LANA (latency-associated nuclear antigen; blue), that
is able, on the one hand, to associate with a nucleosome at
the interface between its H2A and H2B histones (see Figure
1.19) and, on the other, to bind terminal repeat sequences
in the viral DNA genome. (B) Human papilloma virus type 16
(HPV-16) uses the N-terminal domain of its E2 protein (light
green) to tether the episomal viral DNA genome to the Brd4
chromatin-remodeling protein (blue), which is bound, in turn,
to cellular chromatin during mitosis, enabling the viral genome
(tethered to the C-terminal domain of E2) to “hitchhike” with
the cellular chromosomes during mitosis. (C) The association
of HPV-16 genomes with metaphase chromosomes can be
gauged using HPV-16-specific DNA probes in FISH (fluorescence
in situ hybridization) analyses. The chromosomes are stained
here in red, while the viral genomes are detected as yellow dots
if they are located above the metaphase chromosomes and as
green dots if they are not located above the chromosomes.
In the upper panel, the E2 protein is functioning normally in
chromosomal tethering, whereas in the lower panel, its ability
to bind to the Brd4 chromatin-associated protein has been
blocked. As indicated, enumeration of the viral genomes above
chromosomes reveals a drastic decrease in the metaphase
chromosome-associated viral genomes when E2 tethering
function is blocked. (A, from A.J. Barbera et al. Science
311:856–861, 2006. B and C, upper, from E.A. Abbate,
C. Voitenleitner, and M.R. Botchan. Mol. Cell 24:877–889,
2006. C, lower, courtesy of C. Voitenleitner and M.R. Botchan.)

~5 on chromosomes

TBoC2 s3.01

13

Supplementary Sidebar 3.3 Re-engineering the retrovirus
genome for gene therapy The technology of gene therapy has
depended on the ability to insert genes into cells or tissues that
lack functional versions of those genes, with the intent of reversing the genetic and thus phenotypic defects. The technique of
transfection, in which naked DNA is introduced into cells via a
variety of techniques (for example, see Figure 4.1) is relatively
inefficient and the introduced DNA is integrated into the chromosomal DNA of the recipient cell in a haphazard fashion,
resulting in multiple copies of introduced DNA, some of which
Figure S3.2 Construction of a retroviral vector While a variety
of retroviral-like genomes have been used to construct viral
vectors, the basic schemes used in all of these constructions are
quite similar. (A) The dsDNA provirus of a retrovirus—the product
of reverse transcription—is introduced into a cloning plasmid and
modified subsequently by the standard techniques of recombinant
DNA. Note that the provirus contains two identical copies of a
segment (red) termed the LTR (long terminal repeat) at its ends—
products of the reverse transcription process. The presence of
these LTRs is critical for the transcription of the provirus by the
cellular RNA polymerase II.
Restriction enzymes are then used to cleave out viral genes
(dashed lines) and to replace them with a gene-of-interest (orange)
that will be transduced by the future retroviral vector. In addition,
a gene encoding a selectable marker (purple) is introduced. Once
expressed in a vector-infected cell, this marker gene can be used
to ensure that this cell will be protected from the toxic effects of
an applied drug (for example, neomycin), while uninfected cells
lacking this marker gene (and thus the remainder of the vector’s
genome) will be killed off. This strategy can guarantee that the
only surviving cells in a large population of cells that have been
exposed to this re-engineered virus will be those cells that have
been successfully infected by the viral vector. (B) The introduction
of two reading frames into this vector—that encoding the geneof-interest—creates a problem, since in general, the translation of
a mammalian mRNA can only be initiated at one site near its 5ʹ
end. Hence, when this bicistronic provirus (carrying two genes) is
transcribed into an mRNA, ribosomes may only be able to translate
the 5ʹ-proximal reading frame of this mRNA.
This problem can be addressed in at least two ways. Thus, the
selectable marker can be introduced in place of the viral env gene;
following transcription of a normal retroviral provirus, a certain
proportion of the primary pre-mRNA transcripts are routinely
processed by splicing in a fashion that causes the viral gag and pol
genes to be excised, thereby juxtaposing the env gene (or a gene
introduced into its place) with the 5ʹ end of the resulting mRNA.
By replacing the viral env gene with an introduced gene-ofinterest, spliced mRNA can then serve as a template for translation
by ribosomes, since this gene has now been juxtaposed with the
5ʹ end of the mRNA.
A more frequently used strategy, shown here, is to introduce
an IRES (internal ribosome entry site; blue) between the two
introduced genes just upstream of the (in this case) selectable
marker. This IRES segment overrides the normal mechanisms
preventing ribosomes from initiating translation at more than one
site of an mRNA template; instead, in the presence of an IRES
segment, a ribosome can now initiate translation in the middle of
an mRNA in addition to its normal 5ʹ-proximal site of initiation.
In principle, if this provirus is introduced into a cell (for example,
via gene transfer/transfection, see Figure 4.2), it can be transcribed
by the cellular RNA polymerase II and thus create a polyadenylated
RNA molecule that is exported into the cytoplasm. However, this


are fragmented at random sites during the integration process.
Many of these problems are addressed by the use of retrovirus genomes as vectors to transduce (carry) cloned DNA
sequences into recipient cells (Figure S3.2). These vectors take
advantage of the high efficiency of integration of retroviral DNA
and the resulting stable establishment in the recipient cell of
integrated proviruses that carry intact copies of the introduced
DNA. This technology is limited, however, by the carrying capacity of a retroviral vector, which is only able to accommodate four
to five kilobases in introduced sequences in its genome.
RNA cannot leave the cell because it cannot be packaged by viral
proteins, since some and perhaps all of the viral protein-encoding
genes have been deleted during the construction of this viral
vector. (The strategy of leaving these genes present and intact in
the re-engineered viral genome is not viable, since retroviral virions
can only package an RNA molecule of ~10 kb in length. Hence,
viral genes must be excised to make room for inserted genes-ofinterest and selectable marker genes.)
This problem necessitates co-transfecting the re-engineered
vector DNA together with wild-type proviral DNA, which expresses
the requisite virion proteins. Such co-transfection can ensure that
many of the cells in a population exposed to these two DNAs
will take up and express both DNAs. In such co-transfected cells,
the viral proteins expressed by the wild-type viral genome can
now package the RNA transcribed from the re-engineered vector
DNA, resulting in infectious virions that can then be used to
infect a variety of target cells, thereby transducing (introducing)
the gene-of-interest into these cells. (C) The above strategy is
effective in transducing genes-of-interest into cells that previously
did not express them. However, it has a major drawback: these
initially infected cells may become co-infected with wild-type viral
genomes, and the resulting co-infected cells may then release
progeny particles that can spread further in a population or in
the body of an infected animal during a second infectious cycle.
This creates a major problem if the transduced gene-of-interest
happens to be a gene that may be toxic or oncogenic, since
subsequently occurring, endless rounds of infection might, in
principle, spread this gene in an uncontrollable fashion, thereby
creating a biohazard. Alternatively, the infectious spread of the
wild-type virus may also create a hazard, since nontransforming
retroviruses may be tumorigenic if they create large numbers of
infected cells and thereby inadvertently activate by insertional
mutagenesis infected proto-oncogenes (see Section 3.11).
In order to prevent these possibilities, a strategy has been
devised that allows the viral vector to be introduced into an initially
infected cell but precludes further infectious spread from that cell
to yet other cells in second and subsequent rounds of infection.
This strategy depends on the creation of “packaging cells,” which
express the three viral mRNAs (encoding the viral gag, pol, and
env proteins) from three unlinked viral genes, none of which is
part of a provirus. Such cells can package the re-engineered viral
vector RNA, because they synthesize all the proteins required for
assembly of an intact virion and thus for virion infectivity. However,
when another cell is subsequently exposed to the infectious virions
leaving this cell, this cell will acquire the re-engineered vector
genome and will express its genes, but this cell will be unable to
generate a new generation of infectious virions, since no intact
viral genomes were ever co-introduced into such cells. Therefore
this infected cell, although genetically altered, will not become
the source of infectious virus particles that can launch subsequent
rounds of infection.

14

(A)

gag

MLV RNA 5′

pol

MLV RNA

(B)

env

AAAAAA...3′

dsDNA
LTR

reverse transcription

LTR
introduce IRES
segment

dsDNAs
LTR


LTR
gene of interest

excise viral genes

selectable marker

LTR
LTR

LTR
IRES

LTR
introduce gene
of interest

+
viral vector with
introduced sequences

gene of interest
LTR

wild-type proviral DNA
co-transfect engineered vector
with wild-type proviral DNA

LTR

transcription of transfected
proviral DNA

introduce selectable
marker gene
selectable marker gene
LTR

packaging of both RNAs
into infectious virions

LTR

(C)
engineered viral vector DNA

mRNAs expressed by packaging cells
5′

transfect vector DNA
into packaging cells that
express wild-type viral
mRNAs from three
unlinked genes

5′

env

gag

5′

AAAAAA...3′
pol
AAAAAA...3′
AAAAAA...3′

infectious virions that can transduce
vector into cells

secondary spread from initially
infected cells cannot occur
because these virions
lack the gag, pol and env genes

TBoC2 s3.02

15

Supplementary Sidebar 3.4 Classic Kaposi’s sarcoma appears
to be a familial disease Some virus-induced malignancies are
limited largely to small subpopulations and thus resemble
familial cancer syndromes. We will encounter many of these
syndromes in Chapters 7, 8, 9, and 12. There, we will learn that
inheritance of mutant alleles of tumor suppressor genes or DNA
repair genes can create a strong, inborn cancer predisposition.
Prior to the onset of the AIDS epidemic, the disease of
Kaposi’s sarcoma (KS)—a malignancy of cells related to those
forming the endothelial lining of lymph ducts—was confined
largely to small subpopulations, notably men of Mediterranean
and Jewish descent. This resembled a familial cancer, in which
cancer-predisposing alleles were present only in the gene pools
of certain ethnic subpopulations. After the onset of the AIDS
epidemic, however, KS became 1000-fold more common, and
at least this form of KS could be associated with an infectious
agent—human herpesvirus-8 (HHV-8), also known as KSHV.
This virus, along with a number of other infectious agents, is an
opportunistic pathogen that thrives in the bodies of those lacking a functional immune system. Because of the AIDS epidemic
in Africa, KS has now become the fourth most common infection-induced cancer worldwide.
The virology of HHV-8 failed to explain how the “classic,”
pre-AIDS KS is transmitted in immunocompetent populations
even though these tumors could also be associated with HHV-8
infections. Moreover, it was unclear why this disease should be
confined to small populations rather than spreading, like other
viral infections, through large populations. Recent research
has revealed that different substrains of HHV-8 (as defined by
sequence polymorphisms in viral DNAs) are present in different subpopulations of Jews; one substrain predominates among
Ashkenazic Jews (of recent European descent), while a second
is common among Sephardic Jews (of North African and Middle
Eastern descent). Both populations have infection rates that are


as much as 10 to 20 times higher than in non-AIDS Western
populations. Provocatively, within these subgroups, the transmission of specific HHV-8 substrains is correlated with inheritance of certain mitochondrial DNA polymorphisms far more
strongly than with inheritance of Y-chromosome polymorphic
markers. Mitochondrial DNA is transmitted maternally, indicating that maternal transmission of virus (occurring possibly via
saliva) has played a major role in creating pockets of disease in
family lineages that are likely to extend back to founder populations that existed more than 2000 years ago. [Another maternal transmission route may explain the high incidence of adult
T-cell leukemia (caused by the HTLV-I retrovirus) in southern
Japan (see Section 3.12).] Unexplained by these findings is the
fact that KS is confined in the non-AIDS populations to males—
a mystery that remains unresolved.
Hence, certain geographically and ethnically localized
malignancies, such as classic KS and adult T-cell leukemia, are
actually due to viruses that spread poorly “horizontally” (that is,
from one adult to another) but can be transmitted “vertically”
(between parent and offspring) through long-term, intimate
contact. This echoes the behavior of certain strains of mice that
have high rates of breast cancer. As first shown in Bar Harbor,
Maine, in 1933, transmission of disease from parent to offspring
occurred when females of high-incidence strains were mated
to males of low-incidence strains, but not following the reverse
matings. Also, when female pups of high-incidence strains were
transferred to low-incidence foster nursing mothers within 24
hours of birth, only 8% eventually developed breast cancer,
compared with a 92% incidence exhibited by mice that had
been nursed by mothers from a high-incidence strain. This
led to the conclusion that this breast cancer susceptibility was
transmitted from one generation to the next by a milk-borne
infectious agent, which was later identified as mouse mammary
tumor virus (MMTV).

16

Supplementary Sidebar 3.5 Viruses like
RSV have very short lives Circumstantial
evidence indicates that infection of a
chicken cell by a virus like avian leukosis virus (ALV) resulted in the introduction of a copy of the chicken c-src gene
into the ALV genome, creating the hybrid
virus that we term RSV. This recombination event seems to have occurred only
once in the twentieth century; that is,
all src-containing chicken retroviruses
appear to be the descendants of the RSV
that arose on one occasion in a chicken
coop in about 1909. The discovery of RSV
depended on a fortuitous circumstance:
Peyton Rous was able to study the virus
only because a Long Island, New York,
chicken farmer brought him a prized,
tumor-bearing hen with the hope that
Rous would be able to cure the bird of its
cancer (see Figure 3.1B).


The presence of the acquired src gene
in the viral genome confers no obvious
growth advantage on this virus, and RSV
is not naturally spread from animal to
animal. Moreover, similarly configured
retroviruses, which also carry both viral
and cellular sequences in their genomes,
spread poorly from one host animal to
another, often because they have deleted
essential viral replication genes from
their genomes in order to accommodate
acquired oncogenes of cellular origin. For
these reasons, it seems clear that hybrid
retroviruses such as RSV arise through
rare genetic recombination events within
infected animals, create tumors in these
animals, and usually disappear when
these animals die, unless an alert chicken
farmer or slaughterer happens to discover the tumors and pass them on to an
interested virologist.

17

Supplementary Sidebar 4.1 Endogenous viruses can explain
tumor development in the absence of infectious viral spread
The exposure of a mouse or chicken to a retrovirus often results
in the infection of a wide variety of cell types in the body including, on occasion, the cells in the gonads—ovaries or testes.
Infections of cells in these organs can result in the integration
of a retroviral provirus (see Section 3.7) into the chromosomes
of cells that serve as precursors to either sperm or egg. Such
proviruses become established in a genetic configuration that

m.m.dom.

m.m.mol.

transmission of
infectious virus
particles

is equivalent to that of the cellular genes carried by the sperm
or egg. Therefore, when these gametes participate in fertilization, the provirus can be transmitted to a fertilized egg and thus
to all of the cells of a resulting embryo and adult (Figure S4.1).
This provirus might now become ensconced in the genome of
all animals that descend from the initially infected animal. Such
a provirus would be termed an endogenous virus to distinguish
it from viruses that are transmissible from one individual to the
next via infection.

Mus musculus
outbred subspecies
Mus
musculus
laboratory
strains

(B)

m.m.cas.
m.m.mus.

(A)

a b c d e f g h i j k

kb
9.4

infection of germ
cells, e.g., in testes

6.6

4.4
production of sperm
carrying a provirus

production of a zygote
carrying a provirus

2.3
2.0

development of animal
carrying a provirus in all
of its cells

(C)
5′ LTR

3′ LTR

K10 probe
transmission via germ
line to descendants

1 2 3 4 5 6 7 8 9 10

12q14
transcriptional activation
of an endogenous virus in
one cell

11q22

3q24

spread of virus via
infection of cells
throughout the body

108b
109
115


Figure S4.1 Origin of endogenous
retroviruses (A) These viral genomes
arise when retroviruses (red dots) succeed
in establishing a systemic infection in an
organism (for example, a mouse) and
infect, among other cells, a precursor
cell of gametes—sperm or eggs. Once
a resulting provirus (green rectangle)
becomes integrated in the genome of
a gamete (in this case sperm), it may be
introduced into the genome of a fertilized
egg and thereafter be distributed to all
cells (green dots) of the organism arising
from this zygote. This organism can in turn
transmit the provirus to its descendants via
the normal route of sexual reproduction.
Activation of expression of the endogenous
provirus in an animal (red dot) can lead
to infectious spread throughout the body,
viremia, and eventually leukemia (bottom).
(B) The presence of endogenous retrovirus
(ERV) genomes can be detected by probing
the genomic DNA of a species using the
DNA of an infectious retrovirus as the probe.
Shown is a Southern blot (see Figure S4.3)
of the ERV genomes present in the DNAs of
a variety of mouse strains and subspecies. In
this case, only the subclass of ERV genomes
related to xenotropic murine retroviruses is
being probed. Each band in a gel channel
represents a restriction fragment in a cell
genome that carries part or all of an ERV
genome. The variability of ERV integration
sites from one lab strain to another indicates
that numerous novel ERVs have been
integrated into the mouse germ line since
the speciation of Mus musculus, from which
all these strains derive. (C) In contrast to the
mouse ERV genomes, those detected (using
as a probe a fragment of the clone of a
human ERV, above) in a collection of human
DNAs show rather similar integration sites
across the species, indicating their germ-line
integration before the emergence of the
human species; the polymorphic differences
(black arrows) are largely the results of
recombination between the pair of long
terminal repeat (LTR) sequences at the ends
of individual ERV proviruses and resulting
deletion of the intervening stretches of
proviral DNA. (B, from K. Tomonaga and
J.M. Coffin, J. Virol. 73:4327–4340, 1999.
C, from J.F. Hughes and J.M. Coffin, Proc.
Natl. Acad. Sci. USA 101:1668–1672, 2004.)

18

Endogenous proviruses that are actively transcribed in an
animal’s tissues may create a viremia and, following millions of
replication cycles, may induce cancer in that animal. Such disease-inducing endogenous proviruses are therefore disadvantageous and, during the course of evolution, will be eliminated
from a species’ gene pool. This explains why the endogenous
proviruses found in the germ lines of most species are, with rare
exception, either transcriptionally silent or incapable of encoding infectious virus particles.
Careful examinations of the genomes of a variety of mammalian and avian species demonstrate the presence of numerous endogenous retroviral genomes, most of which are clearly
relics of germ-line infections that occurred in the distant evolutionary past. Having resided for millions of years in a species’
germ line, most have suffered so many mutations that they are
no longer able to specify infectious virus particles. However, a
small subset of endogenous viral genomes, notably those that
have been recently inserted into a species’ germ line, may
remain genetically intact. Given the proper stimulus, these previously latent proviruses may suddenly be transcribed in one or
another cell, generating virus particles that spread via infection
from this cell throughout the body and eventually trigger some
type of malignancy, usually of hematopoietic cells (see Section


3.11). For example, the high rate of leukemia in mice of the AKR
strain is attributable to the frequent spontaneous activation
of an endogenous murine leukemia provirus, the subsequent
infectious spread of virus throughout the mouse, viremia, and,
finally, via insertional mutagenesis, the activation of a protooncogene and the eruption of a leukemia.
Such germ-line transmission of viral genomes was thought
to be nothing more than a laboratory curiosity until reports,
beginning in 2006, revealed that certain people carry loads of
human herpesvirus type 6 (HHV-6) in their peripheral mononuclear cells (for example, lymphocytes, monocytes, macrophages) that are 1000 to 10,000 times higher than the average
person carries. (Latent HHV-6 infections are widespread in the
general population.) Subsequent studies revealed that these
high loads of HHV-6 were often present in multiple members of
a family and that in these families, HHV-6 genomes were inherited in the germ line integrated in certain chromosomes, specifically in telomeric DNA. Thus, a mechanism that HHV-6 usually
employs in order to enter into a latent state (that is, by integrating its genome into one or another chromosome; see Section
3.6) occasionally results in germ-line transmission. The clinical
consequences of inherited HHV-6 genomes remain unclear.

19

Supplementary Sidebar 4.2 Boveri and Hansemann independently hypothesized genetic abnormality as the cause
of cancer cells’ malignant behavior The discovery that cancers
arise as a consequence of aberrations in genetic material is widely
attributed to the work of Theodor Boveri (1862–1915), a dominant
figure in early twentieth-century German biology. This story, like
many accounts of scientific history, is actually a bit more complicated and more interesting than conventional accounts depict. The
roots of this notion actually reach back to an earlier set of observations by Boveri’s contemporary David Paul von Hansemann (1858–
1920) (Figure S4.2A). In 1890, Hansemann, a pathologist, first
reported his observations that the mitotic figures (for example, see
Figure 12.38) of cancer cells contained a variety of abnormal chromosomal configurations, including multipolar mitotic spindles—
unusual arrays of chromosomes at anaphase, and asymmetric
mitoses. He also proposed that the cells within tumors underwent
partial dedifferentiation, another theme that anticipated twentiethcentury histopathological studies of tumors. Hansemann continued to report his observations in the decade that followed, but at
the time had little insight into the cell-biological consequences of
abnormal chromosome number and configuration.
In 1900, the work of Mendel, long forgotten, was rediscovered
by three geneticists. They popularized the idea that cells are essentially diploid, with pairs of genetic determinants being separated
from one another during meiosis. In the several years that followed,
the behaviors of Mendel’s genetic determinants were realized to
parallel that of chromosomes, leading many to conclude that the
chromosomes were the seat of heredity. Hence, cells with abnormal
(A)


numbers of chromosomes were perceived as being genetically
mutant, though the term “mutant” had not yet come into currency.
Unlike Hansemann, Boveri (see Figure S4.2B) was a zoologist and worked largely with early sea urchin embryos. He
hardly acknowledged the work of Mendel and minimally that of
Hansemann. Instead, his own work in manipulating metaphase figures in early sea urchin embryos led him to propose that the karyotypic aberrations observed occasionally in otherwise normal cells
represented genetic abnormalities, that each chromosome carries a
distinct subset of the genetic information present in the genome as
a whole, and that the karyotypic abnormalities within cancer cells
were responsible, in some fashion, for the aberrant behavior of cancer cells. The corrupted information content present in one cell was,
he proposed, heritable from one cell generation to the next.
Boveri’s posthumously published 1916 monograph, in which
he laid out these ideas, was followed 13 years later by an English
translation, carried out by his doting widow, the former Marcella
O’Grady of Boston, Massachusetts. Hansemann lacked a loyal
widow and translator, and so awareness of his pioneering contributions in connecting an abnormal genetic constitution of cells
with the development of tumors receded. Boveri’s personality also
may have helped impress on the scientific community his unique
role in many of these advances; an otherwise uncritical colleague
described him as “vehement, inflexible, and [a] relentless assailant.”
Today, few remember that Hansemann began the charge that led to
the now widely embraced notion that cancer cells are genetically
abnormal, and that these cells’ chromosomes are responsible for
their malignant behavior.

(B)

Figure S4.2 The first clues to the genetic
origins of human tumor development
(A) David Paul von Hansemann described the
abnormal mitotic figures and chromosomes
in human cancer cells without knowing
their significance. (B) Theodor Boveri drew
on Hansemann’s observations and his own
to propose that abnormal chromosomes
somehow were responsible for the abnormal
behaviors of cancer cells. (A, courtesy of
Wolfgang von Hansemann. B, courtesy of
the Biocenter, University of Wüzburg.)

TBoC2 s4.02

20

Supplementary Sidebar 4.3 Southern and Northern blotting
Southern blotting was invented in 1975 by the British biologist
Edwin Southern as a means of detecting specific DNA fragments
that are generated by restriction enzyme cleavage of DNA; these
fragments may be present amid a millionfold excess of other
concomitantly generated DNA fragments, revealing the sensitivity of this procedure (Figure S4.3). Within several years, the
procedure was adapted to detect specific mRNA molecules,
once again present amid a vast excess of unrelated molecules,
in this case other mRNA species; for want of a better name, this

(A)

s

resi

pho

tro
elec

labeled RNA or
DNA of known sizes
as size markers

unlabeled
RNA or DNA


newer procedure was dubbed Northern blotting. Thereafter, a
further adaptation of the procedure was developed in order to
detect individual proteins amid a vast excess of unrelated proteins and termed the Western blotting procedure (not shown
here). While the Southern and Northern blots use radiolabeled
nucleic acid probes, the Western blotting procedure uses specific antibodies, usually monoclonal antibodies, to detect proteins that have been adsorbed to filters and for this reason is also
termed immunoblotting.

stack of absorptive paper towels
(B)

REMOVE NITROCELLULOSE
PAPER WITH TIGHTLY BOUND
NUCLEIC ACIDS

(C)

gel

nitrocellulose
paper

agarose
gel

NUCLEIC ACIDS SEPARATED
ACCORDING TO SIZE BY AGAROSE
GEL ELECTROPHORESIS

sponge
buffer
SEPARATION OF NUCLEIC ACIDS BLOTTED
ONTO NITROCELLULOSE PAPER BY SUCTION
OF BUFFER THROUGH GEL AND PAPER

Figure S4.3 Southern and Northern blotting procedures Use of these blotting
procedures makes possible the detection of specific fragments of the cell genome
(or specific RNA transcripts) if an appropriate radiolabeled DNA probe is available.
(A) Either DNA fragments that have been generated by restriction enzyme cleavage
of genomic DNA (in a Southern blot) or a mixture of cellular RNAs (in a Northern
blot) are resolved by gel electrophoresis. (B) The gel slab is then placed beneath a
nitrocellulose filter, and paper towels (or other absorptive material) are used to wick
fluid through the gel, allowing the DNA (or RNA) molecules to adsorb to the filter
paper, creating a replica of their previous positions in the gel. (C) The filter paper is
peeled away from the gel and (D) placed in a plastic bag together with a solution of
radiolabeled probe (pink). (E) Hybridization of the probe with complementary DNA
(or RNA) molecules adsorbed on the filter and subsequent autoradiography with a
photographic emulsion allow the detection of DNA fragments (or RNA molecules)
that were present in the initial DNA (or RNA) preparation; these are manifested by
bands of silver grains on the film. (From B. Alberts et al., Molecular Biology of the
Cell, 5th ed. New York: Garland Science, 2008.)

(D)

LABELED PROBE HYBRIDIZED TO
ADSORBED DNA OR RNA
sealed
plastic
bag

labeled
probe
in buffer
LABELED PROBE HYBRIDIZED TO
COMPLEMENTARY DNA OR RNA;
BANDS VISUALIZED BY
AUTORADIOGRAPHY
(E)

positions
of
labeled
markers

labeled
bands

TBoC2 b4.04/s4.03

21

Supplementary Sidebar 4.4 Genes undergo amplification
for a variety of reasons The process of gene amplification is
defined as a preferential increase in the copy number of a discrete chromosomal region (an amplicon), rather than increases
in the ploidy of an entire genome, as may occur in certain specialized mammalian cells. In mammalian cells, gene amplification seems to occur only as a pathological process accompanying and driving tumor promotion (Figure S4.4). In other phyla,

however, it may be part of a normal developmental program
that responds to the need to express large amounts of a certain
protein or group of proteins; this is achieved by directed, localized gene amplification, which results in proportional increases
of the corresponding mRNA and protein. For example, in
Drosophila, genes specifying chorion proteins undergo amplification in order to enable the synthesis of massive amounts of
these proteins during oogenesis.

ss nick or DSB

(A)

Alu elements

MYB

MYB

(B)

strand invasion and homologous recombination
with upstream repeat on sister chromatid
MYB

MYB

(C)
MYB

MYB

(D)
MYB

MYB

MYB


Figure S4.4 Mechanism of generating
tandemly duplicated genes A number of
molecular mechanisms have been proposed
to explain the generation of tandemly
duplicated genes, such as those that form
the homogeneously staining regions (HSRs)
that create oncogene amplification. In the
example presented here, the mechanism
of amplification of the MYB oncogene via
tandem duplication was studied in a human
T-cell acute lymphoblastic leukemia. The
amplification mechanism was traced, with
high likelihood, to the presence of Alu
elements, which are present in about one
million copies scattered throughout the
human genome (see Figure 4.7). In this
case, the normal human MYB gene is
flanked by two Alu repeat sequences.
(A) When the MYB gene underwent
replication during the S phase of the cell
cycle, a single-strand (ss) nick or doublestrand break (DSB) in the newly synthesized
upper sister chromatid allowed the resulting
free end (which contains an Alu sequence,
empty rectangle) to invade the doublestrand helix of the lower sister chromatid
and anneal to another Alu sequence (dark
red rectangle) that lies upstream of the
MYB gene on the lower sister chromatid
(B). The 3ʹ end of the newly synthesized,
annealed strand could then be elongated
using as a template this lower sister
chromatid (light blue). (C). Subsequently,
this elongating strand could jump back to
the upper sister chromatid (dark green)
and continue elongation, generating two
head-to-tail tandem copies (D) of the MYB
gene. This process might then be repeated,
leading to an exponential expansion of this
chromosomal region. (From J. O’Neil et al.,
J. Exp. Med. 204:3059–3066, 2007.)

TBoC2 s4.04

22

Supplementary Sidebar 5.1 Making anti-Src antibodies
presented a major challenge Thorough biochemical analyses of thousands of cellular proteins have depended upon the
ability to detect and isolate a protein of interest (for example,
Src) amid the complex soup of proteins present in cells. This
detection frequently relies on the use of antibodies that specifically recognize and bind a protein such as Src while ignoring all
other cellular proteins around it. The production of such antibodies involves the injection of an intact protein (for example,
the entire Src protein) or a chemically synthesized oligopeptide (for example, a short protein whose amino acid sequence
reflects the sequence of a small segment of the Src protein) into
an animal, usually a mouse, rabbit, or goat. The hope is that the
injected material will be immunogenic (will elicit an immune
response) in the animal, causing it to form antibodies that
specifically bind and precipitate this protein from a cell lysate
(a preparation of disrupted cells).
The immune systems of mammals often react vigorously to
a foreign antigen, that is, a protein carrying peptide sequences
that are unfamiliar. Conversely, the immune system often
mounts no immune response at all (and thus fails to make antibody) against an injected protein that closely resembles one of
the proteins normally present in the animal. This lack of immunoreactivity against familiar proteins is termed immunological
tolerance, a phenomenon that we will return to in Chapter 15.
Those who first produced anti-Src antibodies used a strain
of RSV that can infect and transform mammalian cells and can
therefore induce tumors in mammals. They anticipated that the
resulting tumors would express the chicken Src protein, and
that dying tumor cells would expose this protein to the immune
systems of the tumor-bearing mammalian hosts and thus provoke an immune response. In fact, the Src protein proved to
be poorly immunogenic in most animals. The reason for this
was discovered much later: the structure of the Src protein is
highly conserved evolutionarily. Accordingly, the chicken Src
protein is almost identical to the corresponding protein of most
mammalian species and is therefore not readily recognized
as a foreign protein by these species’ immune systems. In the
end, these investigators succeeded (for unknown reasons) in
producing anti-Src antiserum only from rabbits bearing RSVinduced tumors. The factors that determine the immunogenicity of a protein such as Src in one or another species are complex and often obscure. Once in hand, the anti-Src antiserum
could be used to precipitate (that is, immunoprecipitate) Src
protein from lysates of a wide variety of RSV-transformed cells
and from normal, untransformed cells (Figure S5.1). As in many
such analyses, these lysates were prepared from cells that had


been incubated in the presence of radiolabeled amino acids,
resulting in the incorporation of radiolabel in the cells’ proteins.
Following immunoprecipitation, the resulting proteins were
resolved by gel electrophoresis, and the presence of radiolabeled proteins was detected by autoradiography. In the present
case, the long-sought Src protein was detected as a polypeptide
migrating as a 60-kD protein.

150,000

88,000

Pr76
Pr66
60,000
Pr53

35,000
p27
(daltons
mol. wt.)

p19
T7

1

2

3

4

5

6

7

8

9

p12, 15

Figure S5.1 Immunoprecipate of Src protein Initially, the
only means of studying the Src protein depended on first
immunoprecipitating it from the mixture of tens of thousands
of other cellular proteins. Many attempts at making an anti-Src
serum finally resulted in an antibody preparation that could be
used to immunoprecipitate this protein from cells. Molecular
weight markers are in the leftmost channel T7; channels 1–3:
lysate of normal, uninfected cells; channels 4–6: lysate of cells
infected with an RSV mutant with a deleted src gene; channels
TBoC2 b5.06/s5.01
7–9: lysate of cells infected with wild-type RSV. Channel 7:
normal rabbit serum; channel 8: tumor-bearing rabbit serum;
channel 9: same as channel 8 except pre-incubated with
lysate of RSV particles. The band at 60 kD in channels 8 and 9
(arrow) represented the first detection of the RSV oncoprotein.
(p12, p15, p19, p27, Pr53, Pr66, and Pr76 are RSV virion
proteins.) (From A.F. Purchio et al., Proc. Natl. Acad. Sci. USA
75:1567–1571, 1978.)

23

Supplementary Sidebar 5.2 The protozoan roots of metazoan signaling In the Cambrian era, ~540 million years ago,
a diverse array of complex multicellular organisms evolved;
many of these were the ancestors of contemporary metazoan
phyla and already had recognizable features of modern organisms. Examination of contemporary animal phyla leads to a
key insight into our origins: a metazoan organization plan was
invented only once, and all modern metazoa descend from
a common pre-metazoan ancestor that somehow solved the
problem of cell–cell communication and the organization of
complex tissues. This organization depended in significant part
on the evolution of intercellular signaling systems, including the
growth factors, receptors, and cell–cell adhesion molecules that
are found in all modern metazoa. The fossil record indicates that
during the earlier Vendian/Ediacaran era, which spanned from
650 to 540 million years ago, a variety of soft-bodied metazoa
were already present, pushing the evolutionary origins of metazoa back another ~100 million years.
The nature of the common pre-metazoan ancestor has been
obscure until recently. However, accumulating evidence now
implicates choanoflagellates as being the closest living relatives
of metazoans. Each single-cell form of a choanoflagellate has a

(A)


single flagellum (which provides propulsion) that is surrounded
by a large collar (assembled from 30–40 microvilli) that enables
these protozoans to trap the bacteria that they consume for food
(Figure S5.2). Large multicellular choanoflagellate colonies
contain flagellated cells on the outside and possibly other flagellum-free cell types on the inside. The evolution of these multicellular colonies presumably depended on the development of
cell–cell signaling and adhesion molecules.
Importantly, sequencing of a choanoflagellate genome
indicates that these protozoans make proteins that are found
in higher metazoa, including receptor tyrosine kinases, EGFlike proteins, G-protein–coupled receptors, a TNF receptor-like
molecule (Chapter 9), SH2 groups (Chapter 6), and cadherins
(which bind metazoan epithelial cells together in two-dimensional sheets; Chapter 13). Once evolved to enable the assembly
of these primitive multicellular colonies, these proteins could
then be adapted in various ways to permit the formation and
explosive diversification of far more complex multicellular animals during the early Cambrian period. At the same time, the
signaling systems that were assembled from these proteins laid
the foundations for the disease that, half a billion years later, we
call cancer.

(B)

5 µm

Figure S5.2 Origins of cell–cell communication The tyrosine kinase receptors, along with
a series of other important signaling molecules, were clearly developed long before the
Cambrian explosion generated the ancestors of the various modern metazoan phyla. The
choanoflagellate genome encodes many of these critical metazoan signaling molecules.
(A) The choanoflagellates received their name from the distinctive collar that surrounds their
single flagellum and is composed of an array of actin-rich microvilli (red). The single flagella,
like the microtubules of the cytoplasm, are revealed here by an anti-tubulin antibody (green),
while the nuclei are stained blue. (B) This scanning electron micrograph reveals that each
solitary cell extends a single long flagellum with a surrounding collar that, in this case, has
been disrupted into its individual component microvilli. Bacteria, which are trapped by these
microvilli and used as food by the choanoflagellates, are seen nearby. (A, courtesy of Melissa
TBoC2 n5.108,9/s5.02
Mott; from N. King, Curr. Biol. 16:R113–114, 2004. B, courtesy of M.J. Dayel, K. McDonald,
and N. King.)

24

Supplementary Sidebar 5.3 Lateral interactions of cell
surface receptors While GF receptors, such as those responding to EGF or PDGF, and integrins have been presented here as
independently acting signaling proteins, it is becoming increasingly apparent that these cell surface receptors often form lateral complexes with one another and regulate each other’s
signaling. In addition, integrin:RTK complexes are formed
through association of these proteins with components of the
ECM (Figure S5.3). For example, after binding its extracellular
matrix (ECM) ligands, the αvβ1 integrin can cause phosphorylation of the EGF receptor even in the absence of EGF or EGFrelated ligands. This cross-phosphorylation appears to require
physical association between the integrin and the EGF-R and
depends, as well, on the complex of signaling molecules that the
integrin attracts to its C-terminal tail in the cytoplasm. The Src
tyrosine kinase, an important component of these integrin signaling complexes (see Figure 6.24) is activated following αvβ1
integrin binding to the ECM and is required for the phosphorylation of tyrosine residues in the C-terminal tail of the closely
apposed EGF-R. This phosphorylation occurs independently of
the EGF-R’s binding its normal ligands and results in a different
spectrum of C-terminal phosphotyrosines than when the EGF-R
binds its ligands. In addition, the amount of EGF-R present on
the cell surface increases after the αvβ1 integrin binds ECM
ligands. Indeed, efficient EGF-R signaling may require integrin
association and integrin-mediated signaling.
This example is only one of many. Thus, integrin-mediated
modulation of tyrosine kinase receptor (RTK) signaling has been
described for the platelet-derived growth factor-β (PDGF-β)
receptor; a vascular endothelial growth factor (VEGF) receptor;
the hepatocyte growth factor (HGF) receptor, termed Met, and

the Met-related Ron RTK, which binds HGFL (HGF-like) ligand;
the insulin receptor (IR); and the EGF-R–related ErbB2/HER2
receptor. And not unexpectedly, interactions between integrins
and RTKs can be reciprocal: activation of RTKs by their respective ligands can stimulate signaling by the proteins associated
with the C-terminal tails of integrins. Such reciprocal signaling
suggests that RTKs signal in part by activating integrin-associated signaling proteins, while integrins signal in part by activating RTK-associated proteins.
As receptor biochemistry is studied in increasing detail, it
is also apparent that cross-phosphorylation between receptors
occurs commonly and may represent a critical mechanism for
controlling cell type–specific signaling. Within the EGF-R family of receptors, for example, HER2 can form heterodimeric
complexes with HER3 and phosphorylate the C-terminal tail
of the latter; in fact, HER3 itself lacks a catalytically functional
kinase. Perhaps more unexpected are the signals exchanged
between unrelated receptors. Thus, the mutant truncated
EGF-R present in glioblastomas (Section 5.4) can drive phosphorylation of the activation loop of the tyrosine kinase domain
of the HGF-R (termed Met); such phosphorylation causes the
activation loop to swing out of the way of the catalytic cleft of
this kinase, permitting its active signaling. In lung carcinomas
that have become resistant to a low–molecular-weight inhibitor
of the EGF-R tyrosine kinase, a new signaling channel is erected
in which the Met receptor can drive HER3 tyrosine phosphorylation—a biochemical function that is usually mediated by the
HER2 receptor–associated kinase. The formation of physical
complexes between these various cross-talking receptors has
yet to be directly demonstrated.

ﬁbrillin
LTBP


ﬁbronectin

LAP

syndecan

TGF-β

HGF

VEGF

Met

αvβ6
integrin

P

P

P

P

α5β1
integrin

α4β1
integrin
P

II I
TGFβR

VEGFR2

P

P

P

P

P

P

P

P

Figure S5.3 Association of integrins and growth factor
receptors in the form of immobilized ligands. In addition, TGF-β
receptors with extracellular matrix components and with
is synthesized in an inactive form and is activated in association
one another Many growth factors do not function as diffusible
with LAP (dark blue), LTBP (yellow), and fibrillin (purple) , and
signaling molecules. Instead, they are frequently attached to
αvβ6 integrin. Moreover, the αvβ6, α5β1, and α4β1 integrins
components of the extracellular matrix (ECM) following initial
provide physical anchorage to the ECM via their association with
synthesis and secretion by a signal-emitting cell and are later
fibronectin and syndecan, a third ECM protein that binds, via a
mobilized from the ECM and activated by proteases produced by
specialized domain (heparan sulfate, purple, blue), to fibronectin.
TBoC2 n5.111/s5.03
a second, signal-receiving cell or presented as solid-phase ligands
These various associations with common ECM components serve
attached to the matrix. Fibronectin is a key component of the
also to nucleate multi-protein complexes containing both integrins
ECM (green and pink). The HGF, TGF-β, VEGF growth factors are
and RTKs. (Adapted from R.O. Hynes, Science 326:1216–1219,
bound to the ECM and are apparently presented to their cognate
2009.)

25

Supplementary Sidebar 6.1 Systematic surveys of phosphotyrosine and SH2 interactions As illustrated in Figure 6.9, different tyrosine kinase receptors (RTKs) display distinct sets of phosphotyrosine residues following ligand-mediated binding and
transphosphorylation. These phosphotyrosines serve as binding sites for a variety of SH2-domain–containing proteins. The
identity of each phosphotyrosine recognized and bound by an
SH2 is defined by its sequence context, more specifically, by the
sequence of the three amino acid residues that flank the phosphotyrosine (pY) on its C-terminal side. In fact, there is a second
distinct type of protein domain, termed PTB (phosphotyrosinebinding), that also functions to recognize and bind pYs; in contrast to SH2 domains, the recognition of a pY by a PTB domain is
determined by the amino acid residues that flank the pY on its
N-terminal side. All told, the human genome encodes more than
140 proteins that possess either an SH2 domain, a PTB domain,
or both domains.
While the RTKs possess tyrosine kinase catalytic domains
that are very similar to one another, these receptors induce very
distinct types of biochemical and thus cell-biological changes
following receptor activation by growth factor ligands. These
differences in responses to RTK activation are likely due to the
fact that the various RTKs bind and thereby signal through distinct but overlapping sets of SH2- and PTB-containing partner
proteins.

In order to study in greater detail the interactions of a single
receptor with its signaling partners, the binding affinities between
a receptor’s array of pYs and the cell’s complement of SH2 and
PTB domains have been measured. In the case of the EGF, FGF,
and IGF1 RTKs, there are a large number of binding partners (39
are indicated in Figure S6.1) that are shared in common; many
of these are involved in activating the signaling pathways, such as
the Ras → Raf → MAPK pathway, that are described in detail in
this chapter. Other interactions with binding partners are unique
to individual receptors. For example, there are 11 SH2- or PTBcontaining binding partners that associate only with the EGF-R
but not with the other two RTKs. These types of analyses should
reveal why different RTKs evoke different responses from cells.
The details of these interactions for a given RTK—in this case
the EGF-R—are illustrated in (see Figure S6.1B). This figure indicates that the binding affinities of the individual SH2 and PTB
domains with the pY-containing phosphopeptides of the EGF-R
vary greatly, there being as much as 20-fold differences in binding constants. These differences in binding are likely to affect the
strength of the signaling that results following binding of various
SH2- or PTB-containing signaling proteins to the receptor. These
binding interactions have also been surveyed for a number of
other RTKs. The complexity of these interactions illustrates the
difficulty that confronts biochemists in understanding with precision how ligand-activated receptors affect the cells into which
they are releasing signals.

(B)
FGF-R1

IGF1-R

IGF1-R &
FGF-R1

891

992
974

1068
1045

1101

PLCG2

GRB2

N

C

GRB7
GRB10
GRB14
GRAP2
CRK
CRKL
NCK1
NCK2
APS
SH3BP2
LNK
SH2B

C

1148
1114

1086

PLCG1
N

C

N

1173

C
N
C

C

N

C

N

C

N

C

SH2 domain
PTB domain
Phosphopeptide

<100 nM 500 nM

PTPN6
PTPN11
E18941
HSH2D
CTEN
E109111
E138606
E169291
E105251
SHB

N

CHN2
SH2D3A
RIN1
BLNK
LCP2
BRDG1
EAT2
SH2D1A
TENS1
TENC1
TNS

EGF-R &
IGF1-R

954

N

RASA1

3

845

C

RABGAP1L
CBL
STAT2
STAT1
JAK3
JAK2
SHC3
SHC1
VAV3
VAV2
VAV1
SH2D3C
SH2D2A
BCAR3
INPPL1

4
2

N

APBB1

39

APBB2

11

C

SYK

N

C

ZAP70

N

C

APBB3

7

IGF1-R,
FGF-R1 &
EGF-R

EB-1
CCM2
EPS8L2
GULP1
APPL
ANKS1
RABGAP1
FRS3
IRS4
IRS1
DOK5L
DOK5
DOK4
DOK2
DOK1
E129946
NUMB
NUMBL
DAB2
DAB1
APBA3
APBA2
APBA1

BMX

PI3KR3

N

11

DAPP1
ABL1
ABL2
FGR
SRC
SLA2

EGFR
PI3KR2

EGF-R &
FGF-R1

PI3KR1

EGF-R

TEC
LYN
BLK
ITK
BTK
TXK
FER
HCK
PTK6
LCK
FES
YES1
MATK

(A)


1 µM

KD

1.5 µM

2 µM

stronger
weaker
binding

26

Figure S6.1 Phosphotyrosine-binding signal-transducing
proteins (A) Three widely studied RTKs—the EGF, IGF1, and FGF
receptors—induce quite different cellular responses because they
bind distinct sets of SH2- and PTB-containing signaling proteins.
In the work illustrated here, the oligopeptides of the SH2 and
PTB domains of more than 100 proteins thought to bind to the
phosphotyrosines (pYs) of ligand-activated RTKs were synthesized,
along with the pY-containing oligopeptides of these three
receptors. The binding affinities of the phosphopeptides to the
individual SH2 and PTB domains were then studied in vitro. Those
binding affinities above a certain threshold are represented in the
Venn diagram shown here. They reveal which of the SH2 or PTB
domains are able to bind phosphopeptides that are present in
the cytoplasmic tails of one, two, or all three receptors of these
RTKs. As illustrated, a group of 39 SH2 and PTB domains is able to
bind to pYs displayed by all three receptors, 11 domains (yellow)
are able to bind pYs generated by activation of the EGF and FGF
RTKs (but unable to bind to a pY on the IGF1-R), and 4 domains
(light blue) are able to bind pYs generated by activation of the
IGF1 and FGF RTKs (but unable to bind to a pY on the EGF-R).
Finally, 11 domains (red) bind only to the EGF-R, 3 exclusively to
the IGF-R (dark blue), and 7 to the FGF-R1 (green). (B) The EGF-R
has 12 pY sites (only 7 of which are illustrated in Figure 6.9).


Each of the associated phosphopeptides (red circles) is identified
here by the residue number of its tyrosine in the overall EGF-R
amino acid sequence. Individual SH2-containing domains (green
circles) and PTB domains (blue circles) are arrayed around the
edge of the rectangle and are identified according to the protein
that carries them. Each binding interaction is indicated by a line
connecting a given SH2- or PTB-containing domain with a specific
phosphopeptide. For example, the GRB2 (= Grb2) protein (top
right) has a single SH2 domain whose affinities for each of the
12 pY-containing phosphopeptides of the EGF-R were measured
separately. The strength of binding is indicated by the color of
the line, with strong interactions indicated by red lines and much
weaker interactions indicated by blue lines. In the case of GRB2,
it associates significantly with only 2 of the 12 phosphopeptides
of the EGF-R. It has a relatively strong (red) interaction with the
phosphopeptide-containing pY 1068 and a second, slightly weaker
(orange) binding to pY 1114. Conversely, the phosphopeptidecontaining pY 1173 can bind both SHC1 (= Shc1) and ABL2, both
with slightly weaker binding affinities. (A, from A. Kaushansky et
al., Mol. Biosyst. 4:643–653, 2008. B, from R.B. Jones et al., Nature
439:168–174, 2006; A. Kaushansky et al., Chem. Biol. 15:808–
817, 2008; and courtesy of G. MacBeath.)

27

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