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Chapter 1

Cell and Tissue Organisation

Emma Marshman, Catherine Booth and Christopher S. Potten
Paterson Institute for Cancer Research, Manchester, UK

CONTENTS
 Tissue Classification
 Cell Proliferation and its Control
 Cellular Hierarchies
 Cell Organisation in Specific Tissues
 Control of Tissue Organisation in Development
 Cancer Development and Tissue Organisation
 Conclusions

To understand the changes that take place during cancer and oxygen-supplying cells of the body and are mostly
development, it is important to understand the basic prin- derived from bone marrow precursor cells. The reticulo-
ciples of cell and tissue organisation and the mechanisms endothelial cells or haematopoietic cells are distributed
that control growth and structure. throughout the body as free cells in blood and lymph or
make up organs such as the spleen and lymph nodes.

Muscle Tissue
TISSUE CLASSIFICATION
Muscle tissue is responsible for movement, such as ske-
Groups of cells that are similar in structure, function and letal movement, but also movement of food, blood and
embryonic origin are referred to as tissues. The tissues of secretions. To carry out this function, muscle cells pos-
the body can be divided into four main groups as follows: sess organelles and properties distinct from those of other
cells which makes them capable of powerful contractions
Epithelial Tissue that shorten the cell along the longitudinal axis. There are
three types of muscle tissue: skeletal, cardiac and smooth
Epithelial tissue covers most of the free surfaces of the muscle. The contraction mechanism is similar in all three,
body, both internal and external, and often invaginates to but they differ in their internal organisation.
form specialized structures such as glands. For example, it
forms the outer layer of skin and the lining of the gastro- Nervous Tissue
intestinal tract and breast ducts. In addition to providing
physical protection, epithelial cells control permeability, Nervous tissue is specialized for the conduction of elec-
provide sensation and produce specialized secretions from trical impulses from one region of the body to another.
glands, e.g. mucus, hormones and enzymes. Taking all the Neural tissue consists of two basic cell types, neurons and
surface linings and their associated glands and structures supporting cells called glial cells. About 98% of the neural
together, epithelial tissues make up the major part of total tissue in the body is concentrated in the brain and spinal
body mass. chord with the rest making up the peripheral nervous
system.
Connective Tissue Since each tissue is made up of a number of specia-
lized cell types that maintain tissue structure and function,
Connective tissue, or mesenchyme, protects and supports there must be exquisite control over cell numbers to
the body and its organs. Types of mesenchymal tissue maintain the integrity of the tissue. The ability to respond
include cartilage, bone and adipose tissue. The reticu- to cell loss (via damage or senescence) varies in the dif-
loendothelial system is often considered a type of con- ferent tissues, since not all cells have the same capacity
nective tissue. Reticuloendothelial cells are the defensive for regeneration. Tissues can therefore be classified into
4 The Molecular Basis of Cell and Tissue Organisation

three groups depending on this cell replacement cap- Systemic Paracrine

ability, as follows:
Autocrine
Rapidly Self-renewing Tissues
Epithelial cell
In tissues such as the skin, the intestine and the haemato- Extracellular matrix
poietic system, there is continuous cell loss either by sur-
face abrasion, by damage or because the cell has aged. This Epithelial–mesenchymal
cell loss has to be compensated for by cell production interactions Fibroblast
(proliferation), otherwise the tissue would begin to shrink
(or expand if proliferation exceeds cell loss). Thus, the Figure 1 Signal mechanisms involved in cellular
number of cells produced by cell division precisely bal- communication.
ances cell loss in order for the tissue to maintain its size
and mass. apoptosis. Apoptosis will be discussed in detail in the
chapter Apoptosis.
Conditionally Renewing Tissues
In tissues such as the liver, breast, prostate and connective Malfunctions of Control
tissue, there is little or no replacement under normal cir-
The first stages of cancer formation are associated with
cumstances. However, there is potential for regenerative
malfunctions in the control mechanisms described above,
proliferation under conditions in which the tissue's integ-
in such a way that the critical balance between cell pro-
rity is significantly compromised, e.g. damage or disease,
liferation and cell loss by differentiation or apoptosis is
or in response to hormonal influences.
disturbed or deregulated. If this balance is shifted in favour
of proliferation, the tissue will expand in a progressive and
Essentially Non-renewing Tissues eventually uncontrolled manner, distorting the tissue
In some tissues, e.g. the female germ line and the central structure and function. The balance may only need to be
nervous system, there is little or no cell replacement or shifted slightly in favour of proliferation for a cancer to
capacity for regeneration in the adult. develop. Cancer development will be further described
towards the end of this chapter.

CELL PROLIFERATION AND CELLULAR HIERARCHIES

ITS CONTROL
At the bottom of all the hierarchies in the body are the
Control of cell division within a tissue is particularly embryonic stem cells. Embryonic stem cells are referred to
important in rapidly self-renewing tissues when pro- as totipotent, i.e. they are capable of differentiating into all
liferation must balance cell loss. Although the exact types of tissue. Embryonic cells may separate and each
mechanisms used by tissues to sense the need to increase or form complete embryos, e.g. twins. Human embryonic
decrease cell division are unclear, it is obvious that pro- stem cells have recently been isolated from embryonic
liferation must be regulated by a complex network of tissue and can be maintained as undifferentiated cells in
signals and messages including growth factors, cytokines laboratory cultures under certain carefully controlled
and hormones. These messages can be produced by the conditions. Even after 4--5 months in culture, these cells
cells themselves (autocrine regulation), may be produced are still able to form types of cells from all three embryonic
by neighbouring cells of either similar or unrelated cell germ layers; including gut epithelium (endoderm), cartil-
types (paracrine regulation, e.g. epithelial--mesenchymal age, bone, smooth muscle and striated muscle (mesoderm)
interactions), and by circulating hormones (systemic and neural epithelium and embryonic ganglia (ectoderm)
endocrine regulation) as illustrated in Figure 1. (Thomson et al., 1998). Studies with embryonic stem cells
Some of the network of signals that control tissue will give valuable information about the mechanisms
homeostasis may prevent overproduction where necessary controlling differentiation and organisation and may ultim-
or arrest the cell cycle if a cell is damaged. In the latter ately allow us to grow replacements for tissues or even
scenario DNA damage is detected and the cell cycle organs that have been damaged by disease.
arrested as it reaches specific checkpoints. These check- In development, these embryonic stem cells are abun-
points will be discussed in more detail in the chapter dant; however, as the animal ages the cellular potency
Regulation of the Cell Cycle. At the checkpoint, the becomes more and more restricted (the capability for wide
defective DNA can either be repaired or, if too severe, gene expression becomes more restricted) until ultimately
the cell may commit suicide in a process referred to as stem cells only remain in tissues capable of regeneration.
 Cell and Tissue Organisation 5

Such stem cells are found at the point of origin of cell their stem cell properties and acquire a more mature
production within an adult tissue and can produce a steady phenotype until, after a given number of divisions, they
stream of cells (Potten, 1992). These daughter cells, are fully differentiated cells. These transit amplifying cells
termed dividing transit cells, can expand their numbers via are therefore generally a short-lived phenotype, although
further cell divisions and mature into functional differ- during the early cell generations they may be called upon
entiated cells, called simple transit cells. Simple transit to behave as stem cells in a trauma situation in some
cells are eventually lost from the tissue at the end of their tissues. With successive divisions they eventually lose this
functional lifespan. The linear evolution in the adult ability. The later-generation differentiated cells then per-
animal tissue is therefore organized into a hierarchy or form the function for which they were generated, gradu-
`family tree' with the cells responsible for cell production ally senesce and die. This is also therefore a form of
at the bottom and the functional cells at the top. The spe- programmed cell death.
cialization process involved in the progression from the The advantage of such an organisation is that only a few
bottom to the top of the hierarchy, termed differentiation, stem cells are needed to maintain a whole tissue. Generally,
represents a change in the pattern of gene expression which these stem cells have a slow cell cycle time which allows
may be the consequence of changes either in the internal for genetic housekeeping, i.e. time to repair any genetic
programming of the cell or of the external stimuli that damage. Small numbers of stem cells followed by around
affect the cell. five generations of transit amplifying cells create an envir-
At the bottom of the adult hierarchy, and ultimately onment in which the greatest risk of introducing a mutation
responsible for cell replacement in renewing tissues, are (during division) is in the transit cells (which are ultimately
the pluripotent stem cells (capable of producing many but lost from the tissue) rather than in the long-lived stem cells.
not all differentiated cell lineages, i.e. they are not toti- In conditionally renewing tissues the organisation is less
potent). In many cases, these cells cannot be identified by clear. Although stem cells must exist, it is possible that they
a common marker or a single property. Instead, cells are are normally quiescent or are cycling very slowly, and are
classed as stem cells if they exhibit or have the potential to only activated by trauma or hormonal stimuli.
exhibit the following properties: The progression from stem cell to differentiated cell
could be preprogrammed but is more likely to be con-
1. stem cells are undifferentiated (relative to the cells in
trolled by extrinsic factors. An organized hierarchy
the tissue);
obviously experiences (and/or is able to respond to) dif-
2. stem cells are capable of proliferation;
ferent control signals at different stages. This can be aided
3. stem cells are capable of self-maintenance;
by a physical organisation, such that there is a spatial
4. stem cells can produce differentiated progeny;
distribution within the hierarchy controlled by a series of
5. stem cells can regenerate the tissue after damage.
microenvironments or niches. A gradient of controlling
When a stem cell divides, under normal circumstances, factors probably exists along the maturation axis.
it is thought to generate a daughter that is another stem cell Particularly important in the microenvironment is the
(thereby maintaining itself) and one daughter that will basement membrane upon which epithelial cells sit. This
move up the hierarchy towards differentiation. Although basement membrane is a highly organized extracellular
this situation remains to be conclusively proven, it is cer- matrix (ECM) made up of proteins such as collagen and
tainly the average situation that must occur in an adult laminins. The effects of the matrix are primarily mediated
tissue. Whether the determinants of such division are by cell adhesion molecules such as integrins and cad-
intrinsic to the stem cell itself or are influenced by the herins which are families of cell surface receptors. Cell
surrounding environment also remains to be determined. adhesion molecules help to connect the exterior of the cell
If stem cell numbers need to increase or decrease in with the interior of the cell in two ways: by transducing
response to external stimuli, this asymmetric form of cell signals initiating from the extracellular interactions and
division will switch to symmetrical division in which by mediating structural linkages between the cytoskeleton
either two stem cell daughter or two nonstem cell daugh- and the ECM of other cells (Horwitz and Werb, 1998).
ters are produced. Stem cell expansion will inevitably These processes will be further described in the chapters
increase cellular production (i.e. speed up regeneration, Wnt Signal Transduction and Extracellular Matrix: The
generate hyperplasia), whereas stem cell removal will Networking Solution.
reduce or remove cellular production (depending on how
many stem cells remain in the tissue), e.g. generate aplasia
or hypoplasia. CELL ORGANISATION IN
The next steps in the life of a nonstem cell daughter, SPECIFIC TISSUES
particularly in a rapidly renewing tissue, are the ampli-
fication of cell numbers. The daughter cells divide a To illustrate the points made in the previous section, the
number of times and are known as transit amplifying stem cells and hierarchies of a number of tissues will be
cells. During this time the cells gradually appear to lose described in more detail.
6 The Molecular Basis of Cell and Tissue Organisation

Haematopoietic System 0.01--0.1% of total bone marrow cells (Heyworth et al.,

1997). Functional assays have been devised, the first of
The hierarchical organisation of the continually renewing which was described by Till and McCullogh (1961). This
cells in the bone marrow has been extensively studied. All method involves transplantation of some healthy bone
mature blood cells in the body are derived from a small marrow cells into mice whose own bone marrow has been
number of stem cells that reside in the bone marrow in destroyed by irradiation. The transplanted cells produce
a process called haematopoiesis. Over 1011 new cells are colonies of differentiated haematopoietic cells in the spleen
produced daily to maintain homeostasis since the majority which can be counted. In addition to functional assays,
of mature blood cells are short-lived. In addition, normal external markers have been used to identify progenitor
daily cell replacement must also be sporadically increased cells. Myeloid and lymphoid stem cells and early pro-
to fight infection or to compensate for blood loss. genitor cells can be separated from blood by antibodies that
The haematopoietic lineage is shown in Figure 2. The react to specific antigens only present on these cell types,
most primitive stem cell of the bone marrow is the pluri- e.g. CD34 antigen which is expressed on 0.5--5% of human
potent stem cell which has the capability to produce all the bone marrow cells. Methods for separation of pluripotent
different cell types of the blood. To add another level of stem cells using specific markers are under development.
complexity, this pluripotent stem cell may itself be part of In the bone marrow, stem cells and their progeny
a stem cell hierarchy. Myeloid and lymphoid stem cells are are exposed to a number of different stimuli including
produced from the pluripotent stem cell population. The physical interactions with other cells mediated by cell
myeloid stem cell then goes on to produce a number of adhesion molecules, interactions with extracellular matrix
progenitor cells which are the precursors of the six types of molecules such as collagen and fibronectin and exposure
mature functional myeloid cells: erythrocytes, thrombo- to growth-stimulatory and growth-inhibitory chemicals
cytes, eosinophils, macrophages, mast cells and neu- called cytokines. There are over 15 cytokines involved in
trophils. These cells have different functions within the haematopoiesis and these are produced by a number of
immune system and in the blood. There may be further as cell types including the mature cells themselves, e.g.
yet unknown subdivisions in the stem cell hierarchy. The neutrophils, B and T cells, as well as by fibroblasts and
lymphoid stem cell produces a number of lymphoid pro- bone marrow stromal cells providing autocrine and para-
genitors which mature into B and T lymphocytes to pro- crine regulation (Heyworth et al., 1997). All these signals
vide defence against pathogens or toxins. coordinate the self-renewal and differentiation of the stem
Although mature blood cells can be distinguished from cells and the formation of the mature cell types.
each other, stem cells and progenitor cells have no specific The role of cytokines in determining which type of cell
distinguishing features under the microscope. Identification (e.g. mast cell or neutrophil) an early progenitor cell dif-
of early progenitor cells and stem cells is also made diffi- ferentiates into is highly complex. Some cytokines have
cult by the low incidence of these cells in blood. For many target cells, whereas others are much more restricted.
example, pluripotent stem cells are thought to make up only Interleukin (IL-3), for example, can stimulate stem cells

Pluripotent stem cell

Myeloid stem cell

Lymphoid stem cell

BFU-E Meg-CFU Bas-CFU Eos-CFU GM-CFU Pre-B cell Pre-T cell

CFU-E

Erythrocytes Megakaryocytes Basophils Eosinophils Neutrophils Macrophages B cell T cell

(Mast cells)

Figure 2 Haematopoietic cell lineage.

Abbreviations: BFU-E, erythroid burst-forming unit; CFU-E, erythroid colony-forming unit; Meg-CFU, megakaryocytic
colony-forming unit; Bas-CFU, basophilic colony-forming unit; Eos-CFU, eosinophilic colony-forming unit; GM-CFU,
granulocyte macrophage colony-forming unit.
 Cell and Tissue Organisation 7

to produce myeloid progenitor cells and can also stimulate 1400 cells/villus/day
myeloid progenitor cells to produce a number of mature VILLUS
cell types (Dexter, 1993). Another example is granulo- 3500 cells
cyte--macrophage colony-stimulating factor (GM-CSF)
which acts on the granulocyte--macrophage progenitor cell
and the eosinophil progenitor cell to produce neutrophils,
macrophages and eosinophils. In contrast, some growth
factors have direct effects on only one cell population, e.g.
erythropoietin, which acts only on the erythroid progenitor
cell to produce erythrocytes. Other cytokines mainly influ- 6–10 crypts/villus
ence the maturation of cells rather than the proliferation of 300 cells
progenitor cells, e.g. IL-5 and eosinophil development.
Originally it was thought that haematopoiesis was
regulated solely by modulation of the production of these CRYPT Dividing transit cells
directly acting cytokines, e.g. stem cells would be acted 250 cells
Stem cell zone
upon by IL-3 to produce progenitor cells and then specific Paneth cells
cytokines would be made to induce maturation of the
progenitor cells into whichever specific cells were required Figure 3 Organisation of small intestinal epithelium.
by the bone marrow. It is now known that control is exerted
at a more complex level such that a certain growth factor
alone will not have effects on a particular cell type; how-
ever, when it is combined with another factor proliferation
or maturation can be induced. For example, lymphoid stem
cells will not respond to macrophage colony-stimulating Dividing
factor or IL-1 alone, but are stimulated in the presence of transit
a combination of these two growth factors. cells
The haematopoietic cell lineage has illustrated the
complex communication network required for the differ-
Clonogenic
entiation of relatively unknown stem cells into the specific Stem cell
potential hierarchy
cells of the blood. stem cells

Small Intestine Stem cells

The epithelium of the small intestine provides another

example of a self-renewing tissue which has been studied Figure 4 Proposed stem cell model for the small
for many years. The tissue organisation of the small intestine.
intestine is different from that described for the haemato-
poietic system, being highly polarized and structured. Replacement of cells shed at the villus tip must be
However, the regulation mechanisms are equally compli- balanced by cell production in the crypt, at a rate of about
cated and largely unknown at present. 1010 cells per day in humans (Figure 3) (Potten, 1992).
In the small intestine, epithelium covers finger-like Cell replacement is achieved by stem cells located
projections called villi and flask-shaped crypts located at amongst or just above the Paneth cells at the base of the
the base of the villi which are embedded in the connective crypt. Unfortunately, there are no markers for intestinal
tissue (see Figure 3). Epithelial cells are produced in the stem cells and at present, characterization studies can only
lower part of the crypt and migrate up the crypt on to the be carried out by disturbing the system and observing the
villi and are continuously shed from the villus tip. In outcome. A stem cell model has been proposed based on
common with the haematopoietic system, migration from clonal regeneration studies following radiation or drug
the early precursor cells is accompanied by differentiation exposure (Figure 4). The proposed model suggests that
and specialization. Cells differentiate into three functional there are 4--6 ancestor or functioning stem cells per crypt
cell types as they move up the crypt: the predominant (Potten, 1998). These stem cells are very sensitive to toxic
enterocyte, the mucus-secreting goblet cell and the peptide insults (e.g. radiation and some chemotherapeutic agents)
hormone-secreting enteroendocrine cells. In addition, a and are unable to repair damaged DNA. If damaged they
number of cells migrate down to the base of the crypt to readily initiate apoptosis and die. This sensitivity may
become the fourth cell type, the Paneth cells. Paneth cells reflect the need to avoid repopulation of the crypt with
secrete a number of proteins including lysozyme, which is cells containing damaged DNA, and thereby preserves the
thought to play a role in fighting bacterial infection. integrity of the tissue. Stem cells that die, however, are
8 The Molecular Basis of Cell and Tissue Organisation

easily replaced by the other surviving stem cell members predominantly made up of keratinocytes (about 80% of the
or by their immediate daughter cells, which make up the total). Other epidermal cell types exist with specific
second tier of the hierarchy. The second tier stem cells functions: melanocytes give the skin its pigmentation and
have a better repair capacity and, if not required to afford some protection against ultraviolet light, Merkel
regenerate the first tier (such as in a normal situation), they cells sense fine mechanical events and Langerhans cells
are displaced into the transit compartment. If this second form part of the body's immune system.
tier is destroyed, a third tier may also exist that contains The outer surface of the epidermis is called the stratum
about 20 even more resistant stem cells with the best repair corneum and is composed of a layer of thin, dead kerati-
capacity. These three tiers therefore make up a population nocytes. These cells bear little resemblance to normal
of around 30--40 potential stem cells--cells that are acting keratinocytes, since by the time they reach the surface their
as stem cells or retain the ability to act as a stem cell if nucleus and internal organelles have disappeared and they
required. Since each of these cells can regenerate a clonal are reduced to thin plates of keratin. Keratins are a family
population (a crypt), they are also termed clonogenic cells. of insoluble proteins that form intermediate filaments
Above the level of clonogenic stem cells there are about within cells and confer mechanical strength. Surface ker-
124 dividing transit cells which have no stem-cell atinocytes represent the final mature functional differ-
attributes. These proliferative cells move or are displaced entiated cells of the skin. These cells are continually being
at a rate of 1--2 cell positions per hour from the crypt on to shed or lost and therefore perpetual cell replacement is
the villus (Potten, 1992, 1998). required to maintain epidermal function.
Regulation of cell proliferation in the gut is not fully Below the stratum corneum are three other epidermal
understood. However, a large number of factors are known cell layers: the granular layer, the spinous layer and the
to be involved, including growth factors, cytokines and basal layer. These epidermal regions are depicted in
ECM molecules. The epidermal growth factor (EGF) Figure 5, although there are many more layers of cells
family is one group of substances known to stimulate than are shown in the diagram. In common with the small
proliferation and includes epidermal growth factor itself intestine and the haematopoietic system, stem cells are
and TGF- (Potten et al., 1997). In contrast, the TGF- responsible for the regenerative potential of skin. These
family of growth factors have been associated with nega- stem cells are located within the basal layer (Lavkar and
tive regulation or inhibition of crypt cell proliferation (see Sun, 1983). Studies carried out on mouse epidermis
also the chapter Signalling by TGF- ). In common with suggest that 5--12% of cells in the basal layer are stem cells
growth factors, in vitro studies suggest that some inter- (Potten, 1992). Transitory dividing cells produced from
leukins have stimulatory effects (e.g. IL-4) and some have these stem cells make up about 50% of the basal layer with
inhibitory effects (e.g. IL-11 and IL-6). the remaining basal layer cells being postmitotic and
The ECM underlying the epithelium plays a role in having no proliferative characteristics. These cells are
a number of key processes, one of which is cell migration. committed to terminal differentiation and achieve this as
The process of migration is not fully understood and it was they slip out of the basal layer and migrate into the spinous
initially thought that cells moved in tandem with under- layer, where they flatten. From the spinous layer, cells
lying connective tissue. More recent studies suggest that progress up into the granular layer until they reach the
cells `walk' over stationary ECM which contains a number stratum corneum where they are eventually shed. The
of adhesion molecules such as E-cadherin, laminin, stem-cell progeny generate a discrete column of cells, from
fibronectin, tenascin and collagen. Migration is thought to basal cell to keratinized cell, arranged in a hexagonal
involve decreased cell attachment to one or more of these pattern and called an epidermal proliferative unit (Potten,
adhesion molecules, since adhesion molecule expression 1981). It has been estimated that it takes the human kera-
patterns vary along the crypt/villus axis. The stationary tinocyte between 26 and 42 days to travel from the basal
nature of stem cells may be due to their strong anchorage layer to the outermost cornified layer and therefore it takes
to the stroma. For example, fibronectin, which is a parti- 1--2 months for the epidermis to replace itself completely.
cularly `sticky' adhesion molecule, is abundant in the crypt
whereas tenascin which is less adhesive is predominant on
the villus. In addition, movement is controlled by the
expression/availability of integrins, epithelial cell recep-
} Stratum corneum
tors for these adhesion molecules -- a cell can only be
} Granular layer
influenced by adhesion molecule levels within the base-
ment membrane if it expresses the appropriate receptors. } Spinous layer
} Basal layer
Epidermis Stem cell Melanocyte Basal membrane containing
Langerhans cell extracellular matrix, integrins etc.
The outermost layer of the skin, the epidermis, is another
example of a self-renewing tissue. The epidermis is Figure 5 The murine epidermal proliferative unit.
 Cell and Tissue Organisation 9

In common with the small intestine, the underlying The third phase of mammary development occurs during
ECM plays a key role in basal layer processes. It has been pregnancy and lactation and at this stage the breast can be
suggested that the ECM mediates adhesion, regulates considered to be morphologically mature and functionally
terminal differentiation and aids cell movement upward active. The alveolar buds and lobes subdivide further,
from the basal layer. When basal keratinocytes become giving rise to large clusters of alveolar lobes. During lac-
committed to undergo terminal differentiation, their ability tation, the clusters of alveolar lobes become distended and
to adhere to components of the ECM decreases and upward form secretory alveoli lined with alveolar cells which
cell migration occurs (Jones and Watt, 1993). Populations produce milk. After cessation of lactation, involution of
of putative stem cells that are greater than 90% pure have the breast occurs where the secretory cells of the alveoli
been isolated on the basis of their adhesive properties. degenerate and disappear. Similarly, after the menopause,
The epithelial cells of the skin, and indeed other sites of there is progressive involution of the ductal and glandular
the body, are able to form a barrier due to a number of components of the breast. The connective tissue supporting
functionally and structurally distinct epithelial cell junc- the breast also degenerates with loss of stromal cells and
tions, including tight junctions, gap junctions, desmosomes collagen fibres.
and hemidesmosomes. Tight junctions seal neighbouring In the normal breast, the ducts and lobes of the mammary
cells together to stop water-soluble molecules leaking gland are separated from the stroma by a basement mem-
between the cells and confine transport proteins either to the brane. This basement membrane is lined with two cell
outward-facing membrane (apical) or to the inner mem- types, an outer lining of myoepithelial cells containing
branes (basolateral) to control the passage of certain che- myofilaments and an inner lining of epithelial cells. As
micals (e.g. glucose transport in the small intestine). In described in the section regarding the epidermis, the epi-
contrast, gap junctions are involved in cell--cell signalling. thelial cells of the breast are connected together with
Gap junctions are intercellular channels made up of con- desmosomes whereas myoepithelial cells connect to the
nexin proteins that allow inorganic ions and other small basement membrane with hemidesmosomes.
water-soluble molecules to pass directly from the cyto- Studies in rodent mammary glands indicate that epi-
plasm of one cell to the cytoplasm of another, thereby thelial cell types and alveolar cells arise from stem cell
coupling the cells both metabolically and electrically. To populations capable of generating the fully differentiated
maintain mechanical strength, cells are linked together lactating mammary gland. These stem cells are thought
with desmosomes. Desmosomes consist of a dense plaque to be present in the basal cell layer of ducts and end
of intracellular attachment proteins (including plakoglobin buds, although little more is known about their identity
and desmoplakins) which are associated with rope-like (Rudland et al., 1997). It has been suggested that stem
intermediate keratin filaments that form a continuous net- cells can give rise to either ductal epithelial cells in a
work throughout the tissue. Hemidesmosomes, or half- reversible manner or myoepithelial cells in an irreversible
desmosomes, connect the basal surface of epithelial cells manner. Alveolar cells are thought to be derived from ductal
to the underlying basement membrane such as that which epithelial cells.
separates the epidermis and the dermis. In addition to aiding As with the other tissues described, the differentiation of
attachment, hemidesmosomes have also been found to cells produced by breast stem cells is strictly controlled.
be important in modulating the organisation of the cyto- Unlike the haematopoietic system where differentiation is
skeleton, proliferation and differentiation. These effects are controlled mainly by paracrine and autocrine secretions, the
mediated by integrins which transduce signals from the breast is also subject to control by circulating hormones
ECM to the interior of the cell as described earlier. Absence secreted by the pituitary, ovary and adrenal glands. For
or defects of hemidesmosomal proteins can result in example, during each menstrual cycle at about the time of
devastating blistering skin diseases. ovulation, there is an increase in lobular size and epithelial
cell vacuolization under the influence of oestrogens and
Breast rising progesterone. When menstruation occurs, the fall in
hormone levels causes lobular regression. Similarly in
The breast or mammary gland is an example of a condi- pregnancy, oestrogens and progesterone stimulate pro-
tionally renewing tissue in that cell replacement is generally liferation and development, and prolactin released by the
limited except under certain conditions, e.g. pregnancy. pituitary gland activates the production of alveolar cells.
The organisation of the breast changes during three Additionally, lactation is triggered by the release of oxy-
developmental phases. The first stage occurs in the foetus tocin, which causes contraction of the smooth muscle
where mammary glands arise as buds from the epidermis components of the myoepithelial cells surrounding the
which elongate to form simple, branched ducts. At puberty, alveoli leading to milk expulsion.
there is rapid extension and branching of the ducts which Local growth hormones are also important since the
terminate in globular structures called terminal end buds. growth promoting effects of oestrogen are believed to
These terminal end buds and terminal ducts then go on to be mediated by TGF- and insulin-like growth factor-1
form lobules of alveolar buds. (IGF-1) which increase epithelial cell growth and inhibit
10 The Molecular Basis of Cell and Tissue Organisation

myoepithelial cell differentiation (Rudland et al., 1997). CONTROL OF TISSUE ORGANISATION

Additionally, production of basic fibroblast growth factor IN DEVELOPMENT
(bFGF) by breast stem cells may regulate their own growth
and that of myoepithelial cells in an autocrine/paracrine Regulation of tissue organisation in development is likely
fashion. to involve a number of highly complicated mechanisms;
however, one group of genes called homeobox genes has
Liver already been found to play a significant role in this process.
Homeobox genes are a family of regulatory genes encod-
The liver is another example of a conditionally renewing ing transcription factors (homeoproteins) that can activate
tissue. Epithelial cells make up the majority of the liver, or repress the expression of a large number of target genes
particularly hepatocytes and also biliary duct epithelium. and so determine cell fate and general pattern formation.
The hierarchial organisation of the liver, however, is not One major family of homeobox genes termed Hox genes
fully understood. Unlike the tissues mentioned above, control the identity of various regions along the body axis.
where one population of stem cells is responsible for cell These Hox genes are activated in sequence such that early
replacement, it seems likely that a two-compartment Hox genes that control hindbrain development, followed
system is operative in this tissue (Alison, 1998). First, in by activation of Hox genes that control the thoracic region
the event of damage, hepatocytes are able to regenerate and late genes that control the lumbo-sacral region. Per-
themselves very efficiently. This is unusual because the turbing the expression of these factors can induce gross
ability to regenerate is normally characteristic of undif- changes in tissue, organ and even limb development. For
ferentiated stem cells, and hepatocytes are thought to be example, synpolydactyly, an inherited disease character-
fully differentiated. A second compartment is activated ized by hand and foot malformation, is caused by expan-
if the function of surviving hepatocytes is drastically sions of the HOXD13 gene.
impaired and involves generation of new hepatocytes In addition to developmental regulation, certain homeo-
from stem cells. Liver stem cells, believed to be located box genes are also involved in inducing differentiation in
in biliary ductules, produce oval cells. Oval cells are renewing tissues. For example, the cdx genes are involved in
then thought to differentiate into the functional cell types, controlling intestinal epithelial cell differentiation, possibly
e.g. hepatocytes. The proposed model is illustrated in by transducing signals from laminin-1 in the underlying
Figure 6. mesenchyme, and HOXA9 and PBX1 are some of the many
genes involved in the control of haematopoietic differ-
Others entiation. Altered expression of any of these can suppress
differentiation and ultimately lead to tumour formation.
As indicated above, knowledge about the organisation of
tissues ranges from the haematopoietic system and small
intestine, which have been extensively studied, to the liver
and breast, where information is more limited. Recently, CANCER DEVELOPMENT AND
a number of developments have been made in under- TISSUE ORGANISATION
standing the hierarchy of other tissue types. For example,
mesenchymal stem cells have been isolated from human The chapter Overview of Oncogenesis will discuss the
bone marrow. These cells replicate as undifferentiated mechanisms involved in carcinogenesis in detail, but
cells and have the potential to differentiate into lineages of briefly the process is thought to involve a number of steps.
mesenchymal tissues including bone, cartilage, fat, tendon, First, a cell experiences a mutation that may or may not
muscle and marrow stroma (Pittenger et al., 1999). Neu- influence its immediate behaviour. This cell may then be
ronal stem cells have also been discovered in foetal brain more susceptible to subsequent mutations and, over time,
which can develop into neurons and glial cells if certain gradually accumulate enough damage such that the normal
growth factors are present in vitro (Vogel, 1999). control or `braking' mechanisms is perturbed. This gradual
accumulation of mutations is therefore known as the
multistage model of carcinogenesis and explains why
cancer is generally a disease of old age unless, for example,
Stem cells
the primary mutation is an inherited disorder.
Within a tissue experiencing this process, the first
Oval cells
observable histological stage is hyperplasia or cellular
Biliary duct epithelium Hepatocytes overgrowth, although this term must be used carefully
Other differentiated epithelial cells since tissue regeneration in response to wounding is itself
a form of hyperplasia. Hyperplasia can therefore be benign
Figure 6 A proposed model for liver cell generation in addition to cancerous. Since in the adult hyperplasia can
and regeneration. only occur in proliferating tissues it is not surprising that
 Cell and Tissue Organisation 11

almost all cancers arise in rapidly renewing or conditionally Heyworth, C. M., et al. (1997). Growth factors and the regulation
renewing tissues. In each case there is a malfunction in of haematopoietic stem cells. In: Potten, C. S. (ed.), Stem
cellular homeostasis and cell production exceeds cell loss. Cells. 423--446 (Academic Press, London).
The origin of cell production, and the only permanent Horwitz, A. R. and Werb, Z. (1998). Cell adhesion and the
resident of a renewing tissue, is the stem cell. Cancers can extracellular matrix: recent progress and emerging themes.
therefore be thought of as stem cell diseases (transformation Current Opinion in Cell Biology, 10, 563--565.
of a maturing cell would have no long-term effect since even Jones, P. H. and Watt, F. M. (1993). Separation of human epi-
if it divides a few times, each cell is ultimately lost from the dermal stem cells from transit amplifying cells on the basis
tissue in a relatively short time frame). An expansion of stem of differences in integrin function and expression. Cell, 73,
cell numbers can therefore lead to hyperplasia. Normally 713--724.
such an expansion would be detected by the tissue and the Lavker, R. M. and Sun, T.-T. (1983). Epidermal stem cells.
excess stem cell removed, via apoptosis. However, if this Journal of Investigative Dermatology, 81(1S), 121--127.
does not occur, cellular output will be dramatically Pittenger, M. F., et al. (1999). Multilineage potential of adult
increased. For example, in the colon expression of the human mesenchymal stem cells. Science, 284, 143--147.
anti-apoptotic gene bcl-2 may allow the survival of a Potten, C. S. (1981). Cell replacement in epidermis (keratopoi-
single extra stem cell in an intestinal crypt (Potten et al., esis) via discrete units of proliferation. International Review
1997). This alone can lead to 128 extra cells being produced of Cytology, 69, 271--317.
by that one crypt (owing to the expansion by the transit Potten, C. S. (1992). Cell lineages. In: McGee, J. O'D., et al.
amplifying cells). As the animal ages these excess stem (eds), Oxford Textbook of Pathology, Vol. 1, 43--52 (Oxford
cells persist and may experience further mutations (e.g. in University Press, Oxford).
apoptosis regulation such as by p53, growth factor signal Potten, C. S. (1998). Stem cells in gastrointestinal epithelium:
transduction such as in SMAD and ras, DNA repair by numbers, characteristics and death. Philosophical Transac-
mismatch repair enzymes such as MSH2, or in cellular tions of the Royal Society of London, 353, 821--830.
adhesion such as changed integrin or E-cadherin expres- Potten, C. S., et al. (1997). The intestinal epithelial stem cell: the
sion), thereby increasing cancer risk. These mutations gen- mucosal governor. International Journal of Experimental
erally occur in three vital areas -- regulation of cell division Pathology, 78, 219--243.
in the renewing population (restraint), DNA repair (such Rudland, P. S., et al. (1997). Mammary stem cells in normal
that the normal DNA is not maintained) and interactions development and cancer. In: Potten, C. S. (ed.), Stem Cells.
with the extracellular environment (cells or matrix). Toge- 147--232 (Academic Press, London).
ther these will subvert the normal differentiation process Thomson, J. A., et al. (1998). Embryonic stem cell lines derived
and allow unrestrained tissue growth without the accom- from human blastocysts. Science, 282, 1145--1147.
panying levels of cell death, followed by invasion and Till, J. E. and McCulloch, E. A (1961). A direct measurement of
metastasis into other tissue sites. the radiation sensitivity of normal mouse bone marrow cells.
Radiation Research, 14, 213--222.
Vogel, G. (1999). Harnessing the power of stem cells. Science,
CONCLUSIONS
283, 1432--1134.
The organisation of cells and tissues has been discussed in
development and in the normal adult and we have
attempted to highlight the complex nature of the regulation FURTHER READING
processes that control cell proliferation, differentiation and
regeneration. Cancer development provides us with an
Cillo, C., Faiella, A., Cantile, M., and Boncinelli, E. (1999).
excellent example of the devastating effects observed Homeobox genes and cancer. Experimental Cell Research,
when these processes are subverted and emphasizes the
248(1), 1--9.
need for such exquisitely controlled mechanisms.
D'Andrea, A. D. (1994). Haematopoietic growth factors and the
regulation of differentiative decisions. Current Opinion in
REFERENCES Cell Biology, 6, 804--808.
Loeffler, M. and Potten, C. S. (1997). Stem cells and cellular
Alison, M. (1998). Liver stem cells: a two compartment system. pedigrees -- a conceptual introduction. In: Potten, C. S. (ed.),
Current Opinion in Cell Biology, 10, 710--715. Stem Cells. 423--446 (Academic Press Ltd., London).
Dexter, T. M. (1993). Synergistic interactions in haemopoiesis: Schwarzbauer, J. (1999). Basement membrane: putting up the
biological implications and clinical use. European Journal of barrier. Current Biology, 9, R242--244.
Cancer, 29A, S6--S9. Stappenbeck, T. S., Wong, M. H., Saam, J. R., Mysorekar,
Graham, G. J. and Pagnell, I. B. (1992). The haematopoietic stem I. U. and Gordon, J. I. (1998). Notes from some crypt
cell: properties and control mechanisms. Seminars in Cellular watchers: regulation of renewal in the mouse intestinal epi-
Biology, 3, 423--434. thelium. Current Opinion in Cell Biology, 10, 702--709.
Chapter 2

Regulation of the Cell Cycle

Arthur B. Pardee
Dana-Farber Cancer Institute, Boston, MA, USA

CONTENTS
 Overview
 The Normal Cell Cycle
 Molecular Biology of the Cycle
 Regulation of Cycle Phases
 Checkpoints, Mutations and Cancer
 Cancer Therapy and the Cycle
 Acknowledgement

OVERVIEW two cells. Until about 50 years ago, no changes could be

observed during most of this interval, until about 1 hour
In each of us are about 50 trillion living cells, all of which before division when chromosomes which contain the
originated from only one cell, a fertilized egg. As we hereditary material become visible and are equally parti-
developed into adults this cell divided into two cells, these tioned between the two daughter cells, a complex process
into four, and so forth, at least 45 times. The orderly pro- termed mitosis that takes place through M phase.
cess by which one cell becomes two is named the cell Production of two cells from one requires duplication of
cycle. This cycle is fundamental not only for under- all of the myriad molecules that compose each cell. The
standing cell growth, but also for replacement of cells lost most evidently duplicated molecules are deoxyribonucleic
by damage, as in wound healing and from the normal wear acid (DNA), the heredity-containing material in chromo-
and tear of our bodies. The cell cycle is evidently tightly somes. DNA does not duplicate throughout the cycle, but
regulated, because we usually make new cells only when only during several hours in mid-cycle. This period is
they are needed. Indeed, cancers arise when cell growth named the S phase, for DNA synthesis. Other molecules
control is defective. `Cancer is a wound that does not heal.' are duplicated at different times throughout the cycle.
One should remember that cells in most tissues are not These findings about DNA synthesis (Howard and Pelc,
usually progressing through the cycle, but are at rest, 1951) showed that the cycle is organized as a series of
happily performing their specialized functions in support events, and created the present framework of its four
of the whole organism. But as exceptions, bone marrow, phases: a `gap' (G1 phase) during which a cell prepares for
intestinal epithelial and some other cells are constantly DNA synthesis, DNA synthesis (S phase), preparation for
dividing. A cell has a life cycle. It is formed, eventually mitosis (G2 phase) and the mitotic M phase, after which the
becomes worn and dies by a programmed cell-death cell divides and two new cycles commence (Figure 1).
mechanism called apoptosis. Thereafter, nearby cells grow For a historical summary of biology of the cycle, see
and divide to replace it. Cell numbers are balanced by Baserga (1985).
proliferation versus apoptosis. After a cell becomes can-
cerous the balance is perturbed in favour of proliferation.
These facts can be overlooked because much research is Quiescence
performed with cells put into culture and under conditions
that permit proliferation. Commencing by considering normal animal cells, most of
the cells within us are in a quiescent state (G0 phase). They
have left their cycling during the G1 state, so in quiescent
THE NORMAL CELL CYCLE cells DNA has not yet duplicated. But quiescent cells differ
from G1 cells in many other properties, in particular
History of Cell Cycle Biology lacking molecules required for growth. This fact told us
that the molecular switch that controls growth versus
Before the cell cycle, microscopy revealed an interval of quiescence, and that is defective in cancers, is to be found
about 1 day between successive divisions of one cell into in G1 phase (Pardee, 1989).
14 The Molecular Basis of Cell and Tissue Organisation

Quiescence Growth produce needed enzymatic machinery. Many G2 products

R are unknown; a terminal one is the maturation promoting
factor (MPF).

S
M Phase and Cell Division
G0 G1 Cycle
Mitosis requires less than 1 h, and is subdivided into four
main stages, in which the duplicate chromosomes pair and
G2
M condense, and a mitotic `machinery' consisting mainly of
microtubule proteins segregates them equally between the
two daughter cells. At completion of M phase, proteins of
Figure 1 The basic cell cycle. The consecutive phases
the mitotic apparatus are destroyed. The daughter cells
of the cycle, entry from G0 and exit from G1 to quies-
then become separated, and each can repeat the cycle
cence and differentiation are indicated.
processes.

G1 Phase
When cells are activated to proliferate they advance MOLECULAR BIOLOGY OF THE CYCLE
from G0 to G1 phase, during several hours after several
growth factor proteins are provided in their environ- Signalling Molecules
ment. These include epidermal growth factor (EGF) and
insulin-like growth factor (IGF-1), which must over- Comparisons of growth of mammalian cancer and normal
come inhibitions by crowding of cells and the negative cells in culture revealed in 1974 that the basis of cancer's
factor TGF- . Growth factors and nutrients must be deranged growth control is located in G1 phase, shortly
supplied from the blood in an organism. To grow cells before initiation of DNA synthesis (Pardee, 1989). In
outside the body, in tissue culture, a nutrient medium the same year, genetic studies of the cycle were intiated;
is required, in which growth factors are usually supplied research with cycle-controlling yeast mutants led to the
by adding blood serum. Cells complete their cycle and discovery of numerous cycle-regulatory genes (Hartwell
then become quiescent after growth factors have been and Kastan, 1994). Biochemistry and molecular biology
removed. soon followed, with the identification of new genes and
The length of time that cells in a culture spend in G1 key enzymes; in particular proteins named cyclins that
phase is highly variable, e.g. from 6 to 24 h, unlike the activate these kinases were discovered by Hunt and
fairly uniform time they spend in the other phases. Many Ruderman (see review by Murray and Hunt). These rise
other synthetic biochemical processes take place in G1 and fall during the cycle because of periodic changes
phase (see below). in their synthesis and destruction (Minshull et al.,
1989). Cyclin-dependent kinases (cdks) that phospho-
rylate proteins required for cell cycle progression were
S Phase identified (Nurse et al., 1998). Several proteins that
The requirement for growth factors to pass through G1 inhibit these kinases and that vary during the cycle were
phase is lost at the restriction point (R), located shortly discovered later. This involvement of both positively
before cells start to synthesize DNA. At the beginning of and negatively acting molecules illustrates the Ying--
S phase, enzymes involved in DNA duplication increase, Yang principle of dynamic opposing actions, frequently
and they move into the nucleus where DNA is dupli- seen in biology.
cated, from the surrounding cytoplasm where proteins
are synthesized. Then at specific times during the next G1 Phase Kinases, Cyclins and Inhibitors
6--8 h the DNAs of the perhaps 40 000 genes located on
23 pairs of chromosomes are replicated, each according We will outline the main steps of growth activation and
to a timed program. For example the dihyrofolate control in G1 phase, but this process is too complex to
reductase gene replicates quickly in very early S phase, describe here fully (Figure 2) (see Murray and Hunt;
but other genes are duplicated at other specific times Andreef). In summary, a biochemical network regulates
throughout S phase. the critical process of controlling cell growth during G1
phase. Numerous nutrients including sugars, salts, vita-
G2 Phase mins and essential amino acids are required for cell
growth (Baserga, 1985). Externally supplied growth fac-
After DNA synthesis is completed, several hours are tors start the cell cycle, from G0 into G1 phase. They
required before initiation of mitosis, presumably to initiate a multi-step cascade of signals that ultimately
 Regulation of the Cell Cycle 15

G0
Growth
Apoptosis M factors
p21
p Signal transduction molecules
CycB cdc2 Transcription
p
factors
CycB cdc2 X CycD cdk4/6
X
G2 pp
G1
INKs (p15, p16,
p18, p19)
CycA cdc2
Transcription P P Bcl2
S-phase genes E2F Rb
P P Rb
p21 Active replication complexes
CycE cdk2 Bax
CycA cdk2
X
Apoptosis
p57 CIPs (p21)
p21 KIPs (p27, p57) p53
S
Cell cycle arrest
DNA damage

Figure 2 Cell-cycle control molecules. Some of the many molecules that provide growth-regulating signals
throughout the cycle are shown, and are discussed throughout this chapter. (Adapted from Ford and Pardee, 1999.)

activates genes to produce their messenger RNAs and Phosphorylations are also regulatory, in addition to the
proteins, and which culminates in the starting up of DNA synthesis of cyclins. Yet another kinase, CAK, activates
synthesis. the cyclin-dependent kinases. Furthermore, a major role is
The growth factors bind extracellulary to their specific played by relocalization of cyclin--cdk to the active nuclear
receptor proteins that traverse the membrane surrounding compartment within a cell during the cell cycle.
each cell (Figure 3). These receptors conduct the external Further investigations revealed yet other proteins whose
signal to the interior of the cell, and there they activate role is to block activities by binding to cyclin--cdk com-
the receptor's special kinase. These then turn on a cas- plexes. These are a family named inhibitors of kinases
cade of signals involving other proteins including Ras, (INKs). They counterbalance the cyclin's activation of
Fos, Myc and MAP kinases. The Ying--Yang principle cdks, to affect cycling, development and tumorigenesis
is again involved, as illustrated by phosphorylations (Sherr, 1996). The inhibitory proteins block cyclin D--cdk
catalysed by PI-3 kinase that are balanced by dephos- activities. p27 blocks cell progression, is high in quiescent
phorylations catalysed by the PTEN phosphatase enzyme. cells and decreases during late G1 to release cdk--cyclin
The activation of G1 phase results in expression of at least activities. Inhibition of cyclins by the cdk inhibitor p21 has
100 genes. often been demonstrated to be induced under various
The discovery of cyclins, which are the key proteins growth-arresting conditions.
regulating transition through the cycle (Roberts, 1999), In the next step, activated cdks phosphorylate proteins
was soon followed by discoveries of multiple cdks. Their that are essential for progression of the cell cycle. The
complexes with cyclins catalyse stages of cell cycle pro- retinoblastoma tumour suppressor (pRb), absent in reti-
gression (Figure 4). As cells proceed through the cycle, noblastomas, releases a gene-activating protein named
four major cyclins are produced sequentially (D, E, A and E2F when it is phosphorylated. If this is prevented, E2F is
B), and they activate several cyclin-dependent kinases. not active, cyclin E is not synthesized, and cells cannot
Central is cyclin D which increases in early to mid G1 pass through the R point. Additionally, proteasomes'
phase and regulates cyclin-dependent kinases cdk4 and activity of destruction of key inhibitory proteins is vital for
cdk6 (Sherr, 1996). Cyclin D/cdk triggers the synthesis of passing each checkpoint in the cycle (Koepp et al., 1999).
cyclin E in late G1 phase, which in turn activates cdk2, The proteasome is a biochemical machine, composed of
cyclin A production and DNA synthesis. protein subunits, that chews up proteins including cyclins
16 The Molecular Basis of Cell and Tissue Organisation

1. Growth factors
G0
2. Receptors

3. 2nd Messengers 4. Genes

G1

5. mRNAs
7. DNA replication
S

6. Proteins G2

8. Mitosis
M

9. Division
Figure 3 The path to cell proliferation. Growth factors initiate a signalling cascade that takes a cell through the
cycle, indicated at the right.

Active

Inactive
Inactive
cdk Cyclin
cdk Cyclin
cdk
Inhibitor

Protein Phospho-protein
+

Cyclin
Proteasome

Figure 4 Interactions of cdk with cyclin and inhibitory protein. A cyclin-dependent kinase (cdk) acquires the
enzymatic activity to phosphorylate substrate proteins when it binds a cyclin, and this protein complex is inactivated
by binding of an inhibitory protein. These reactions are readily reversible and they depend upon phosphorylation of
cdk. Cyclins are irreversibly degraded by proteasomes after they are no longer needed.
 Regulation of the Cell Cycle 17

after they become chemically labelled and targeted for (wee1) activity is greater than that of the phosphatase
removal. cdc25, and this imbalance keeps cyclin B--cdc2 inactive.
Unlike peptide growth factors, steroid hormones do not At the G2--M boundary, wee1 is degraded, allowing cdc25
initiate cytoplasmic signalling pathways, but move directly to activate the complex. Furthermore, during G2 the cyclin
to the nucleus where they activate genes. The sex hormone B--cdc2 complex resides in the cytoplasm, and at the G2--M
oestrogen binds to its receptor protein in the nucleus of boundary it is rapidly relocated to the nucleus, where it
breast cells, and this in turn binds to and activates growth- phosphorylates the nuclear membrane protein laminin,
stimulating target genes. which causes the nuclear membrane to break down.
Thereafter, chromosomes condense and mitosis proceeds.
Entry into S Phase These many phosphorylations are important for the mas-
sive morphological changes that are necessary for a cell to
Increased cyclin D and E overcome inhibition of cdk divide.
activity, and pRb is phosphorylated. This releases E2F and
activates genes involved in initiating S phase, including
The Next Cycle -- Licensing for
enzymes of DNA synthesis. An example is DNA poly-
merase, whose transcription is regulated at G1/S phase by DNA Synthesis
a complex of proteins that contains pRb-like p107, cyclin
A process named licensing permits only one DNA repli-
A and kinase. It is worth noting that most major cell cycle
cation per cycle. DNA synthesis cannot be reinitiated until
processes are catalysed by large complexes composed of
after mitosis is completed. pRb is a critical determinant in
many proteins.
preventing DNA reduplication. Perhaps related is the
Progression through S phase depends upon cyclin A
breakdown and reformation during mitosis of the mem-
kinase. Early in S phase, cyclins D and E are degraded by brane around the nucleus. This permits interaction of
proteasomes. Degradation also removes E2F, which is
molecules from the nucleus and cytoplasm. Degradation of
necessary to prevent programmed cell death (apoptosis) of
cyclin B by proteasomes is necessary for the start of S phase
S phase cells (Lees and Weinberg, 1999).
in the following cycle. Licensing can be disrupted: cells
that have lost the cdk inhibitor p21 undergo multiple rounds
G2 Phase and Entry into Mitosis of DNA synthesis without mitosis, and this process is also
activated by anticancer agents. Staurosporin can eliminate
Mitosis depends upon completion of S phase, and events in
the dependence of DNA synthesis on the prior M phase.
G2 phase are preparatory for it. The complex molecular
basis for onset of mitosis was explosively discovered in the
early 1980s. Ruderman found that fertilization of oocytes Cell Ageing
triggers activation of cyclin mRNAs. Hunt discovered that
the amounts of cyclin proteins oscillate during the cell The normal cell cycle outlined above is modified by var-
cycle, rising during DNA replication and early mitosis and ious conditions. One of these is cell age. The cycle in early
falling at the end of mitosis. Injection of isolated cyclin A embryo cells is very rapid. It lacks G1 phase and the cor-
into quiescent oocytes drove the cells into M phase. At this responding growth-controlling G1 checkpoint. Mature
time also, Nurse identified the cdk kinase cdc2 as essential human cells slow their cycle as they become older, and
for entry into M phase. Unbound cdc2 by itself was in- they cease growing, in G0 or G1 phase, after about 50
active. Newly formed cyclin B was shown to bind to and cycles, as initially shown by Hayflick (Baserga). A cdk
activate cdc2, establishing the first molecular mechanism inhibitor was first discovered in ageing cells by its increase
to explain cell-cycle progression. Then destruction of before final arrest of cycling. A progressive shortening of
cyclin B, involving a specialized multi-subunit anaphase- the telomeric DNA, located at the ends of chromosomes,
promoting complex, is essential for completion of the after each cycle is proposed to provide a biological `clock'
cycle. for cell ageing. (See chapter on Telomerase.)
Research with cell free systems has permitted detailed
biochemical investigations of mitosis, showing for ex-
ample that cyclin B binds to cdc2 (Figure 2). This acti- REGULATION OF CYCLE PHASES
vation of cdc2 kinase is necessary for progression into and
through mitosis. The kinase is regulated by a variety of Checkpoints
proteins that include cyclin B, phosphatases and kinases
and by its subcellular localization. Cyclin B1 begins to Entry into and exit from S and M phases are very carefully
accumulate in S phase and increases through G2. It forms regulated events. Checkpoint is a name given (Hartwell
a complex with cdc2, which primes cdc2 phosphorylation. and Kastan, 1994) to the set of identified cycle-regulatory
The complex is, however, still inactive, owing to other steps: G1 restriction point (and the similar START in yeast)
phosphorylations on cdc2. During G2 phase, a kinase's and the G1/S and G2/M blocks resulting from DNA
18 The Molecular Basis of Cell and Tissue Organisation

damage. Cell-cycle checkpoints are based upon pathways DNA damage

and feedback mechanisms ensuring that a phase of cell
cycle does not begin until the preceding phase has been

nt
completed with high fidelity. If a checkpoint fails, pro-

de
grammed cell death (apoptosis) or genomic instability

en
ATM

ep
ensues. Such failures are important steps in the progression

ind
from normal to cancerous cells.

3-
A surveillance system is engaged to make the choice

p5
between cell growth and quiescence (Pardee, 1989). When p53
extracellular stimulation by growth factors or nutrients is
inadequate, cells cannot pass beyond a specific point in
late G1 phase, in mammalian cells named the restriction
Bax p21
point (R). Instead they revert to quiescence (G0). The
final steps that are needed to pass R require synthesis of
an unstable protein, proposed to be cyclin E. Under
inadequate conditions, synthesis does not keep up with G1 and G2 arrest
loss, and so this protein cannot be accumulated to an
amount sufficient to move the cell into S phase. Once Apoptosis
beyond the R point, cells are committed to divide and Figure 5 A molecular sequence from DNA damage
they no longer require the extracellular growth factors to apoptosis. A variety of conditions that make a cell
during the remainder of the cell cycle. Restriction point unnecessary, such as irreversible damage to it, initiate
control is defective in cancer cells, and this indepen- p53-dependent and independent signalling pathways that
dence releases cancer cells to continue growing under lead to apoptosis.
conditions that keep normal cells in the quiescent state
(Pardee, 1989).

for DNA repair before the cell goes through mitosis. If

The DNA Damage-induced repair is not completed in this interval, the cells progress
G1 Checkpoint into mitosis without repairing all the DNA damage
(Fingert et al., 1988), and this results in death or mutations
After DNA is damaged, other checkpoint controls delay of surviving daughter cells which can thereby become
entry into the next phase of the cell cycle. One such major cancerous. This molecular G2--M checkpoint mechanism
checkpoint is at the G1 to S transition, which prevents cells is a complex network of phosphorylations and depho-
from beginning DNA synthesis until the damaged DNA is sphorylations catalysed by several enzymes and proteins
repaired. Several proteins, in particular p53, have been that are moved between cytoplasm and nucleus. Basically,
implicated in this checkpoint mechanism (Figure 5). a block in activation of cyclin B--cdc2 prevents the
Individuals who are mutated in the ataxia telangiectasia movement of cells into mitosis.
gene, ATM, are very sensitive to X-rays and have a high
incidence of tumorigenesis. In response to DNA damage,
ATM phosphorylates and increases the level of the p53
protein, a tumour suppressor that is mutated in more than
M phase Checkpoints
50% of cancers (Levine, 1997). p53 causes cells to arrest Mitosis properly segregates chromosomes into the daughter
at the G1--S boundary, which is at least partly due to its cells. Accurate segregation depends on proper chromo-
production of p21, one of the proteins that inhibits cyclin-- some alignments on and attachment to the mitotic spin-
cdk complexes. dle, which is composed of microtubule proteins. A
checkpoint ensures that this segregation process occurs
The DNA Damage-induced S and correctly. As little as one double strand break in DNA,
G2--M Checkpoints or depletion of deoxynucleotide building blocks, activates
the checkpoint control and stops cells at the G2--M
Within several minutes of exposure to DNA-damaging boundary. This control mechanism delays completion of
agents, such as X-rays, mammalian cells in S phase exhibit mitosis until all the chromosomes are attached to the mitotic
a dose-dependent reduction in DNA synthesis. Less is spindle. The mechanism blocks progression through
known about the mechanism of this S phase checkpoint mitosis if chromosomes are misaligned, and assembly of
than about those in G1 and G2. the microtubules that guide the chromosomes can be
DNA damage also induces a G2--M checkpoint, as inhibited by anticancer drugs such as taxol. Mutations of
described by Tolmach. This checkpoint delay gives time mitotic checkpoint genes are found in human cancers.
 Regulation of the Cell Cycle 19

Checkpoints and Programmed Mutations are causal for cancer; the disease is based
Cell Death (Apoptosis) upon them. Mutations are found in many genes in advanced
cancers. Some of these change cell-cycle controls, includ-
Apoptosis is a highly regulated process that eliminates ing creating a supply of nutrients through angiogenesis,
physiologically unneeded cells and those that are damaged modulating DNA repair, apoptosis, immortalization and
beyond repair (see the chapter Apoptosis). Activated metastatic capability (see Hanahan and Weinberg).
checkpoints give time for a cell to repair its damaged The minority of cancer-prone mutations are hereditary.
DNA, but if the damage is not soon corrected the cells will In these cases, a mutated gene on one of a pair of chromo-
initiate apoptosis. This mechanism therefore may prevent somes is inherited. If, later in life, a mutation occurs of this
the mutations that cause cancer (Sellers and Fisher, 1999). gene in its partner chromosome, a cancer cell can be pro-
Checkpoint genes, including p53, called `the guardian of duced. Several inherited diseases that are associated with
the genome,' are involved in causing apoptosis, as is bax cancer susceptibility have defective checkpoint control. Li--
and other members of the bcl-2 family (Figure 5). Dif- Fraumeni syndrome is a hereditary disease characterized by
ferent cells show various responses to damage and drugs, cancers arising in close relatives. It is a result of a germline
partly because they express different members of the Bcl-2 mutation in the p53 gene that abrogates the G1 checkpoint.
family (see the chapter Apoptosis). Ataxia telangiectasia is characterized by acute cancer pre-
The cyclin A--kinase complex necessary for S phase disposition and also other major dysfunctions. Cells from AT
progression is inhibited when cells are treated with X-rays, patients in culture exhibit severely impaired G1, S and G2
and this can result in apoptosis because of the inability of checkpoint functions. As mentioned, the ATM gene is acti-
this complex to remove the apoptotic G1--S factor E2F vated in response to DNA damage and is necessary for
(Lees and Weinberg, 1999). activating p53. Another cancer, retinoblastoma, involves
mutations in the Rb gene, and produces childhood retinal
Cell Ageing and the Cycle tumours. Survivors have a high risk of developing secondary
cancers, particularly osteosarcoma. The BRCA-1 and -2
The elimination of cell ageing is named immortalization. It mutations are associated with hereditary breast cancer; they
is an important step in cancer progression, although it does modify cell cycling and DNA repair. Several other genetic
not cause cancer-associated changes (see Hanahan and diseases, including Bloom's syndrome, Fanconi anaemia
Weinberg). One way in which human cells can be and Nijmegen breakage syndrome, are associated with
immortalized is by inserting the gene for the enzyme tel- defects in cell cycle checkpoints and cancer susceptibility.
omerase, which restores the ageing cell's telomere lengths. The majority of cancer-related mutations arise through-
Telomerase is also involved in the G2--M checkpoint. out life. For example, cyclin A levels often become
Cancer is a major cause of death in the elderly. Its abnormally high in cancer cells, and contribute to tumori-
incidence increases rapidly, killing about 10% of people genesis. The cyclin D1 and E genes are amplified and
between ages 75 and 85 versus 1% between ages 45 and over-expressed in many human cancers.
55. Yet in spite of these epidemiological facts, there is 60% Carcinogenesis can also be caused by viruses such as
under-representation of cancer patients 65 years or older in SV40 and papillomavirus. They introduce their genes that
treatment trials, few studies have specifically focused on produce proteins that bind to and eliminate the functions of
persons over age 65 and many pathological and molecular p53 and Rb, thereby bypassing G1--S, and to a lesser extent
investigations do not include age as a determinant variable. G2, checkpoint controls.
Cancers are often associated with environmental muta-
gens, such as are produced by smoking. Repeated expo-
sures can produce the several different mutations that are
CHECKPOINTS, MUTATIONS required to cause a cancer. Master mutations can activate
AND CANCER growth-promoting oncogenes or loss or inactivation of the
tumour-suppressor genes that limit growth. As an example,
The general sequential organisation and duration of the many cancers have lost or mutated the p53 gene. One
cycle are preserved in cancer, but checkpoint controls are consequence of this mutation is survival of the cancer cell,
defective (Pardee, 1989; Hartwell and Kastan, 1994). because p53-dependent checkpoints are eliminated and the
Modifications in cancers are found at many levels of programmed cell death mechanism is diminished. Another
growth regulation, some of which have already been consequence is that the mutation rate is increased, termed
mentioned. The main defect is misregulation of growth genomic instability (see the chapter Genomic Instability
initiation at the R point. Furthermore, since checkpoints and DNA Repair).
ensure that mutations are kept low in normal cells, The mutations of a half-dozen or more cellular genes is
defective checkpoints increase the mutation rate in cancer required for tumour formation (Kinzler and Vogelstein,
cells and result in progressive loss of control and emer- 1996). This number of events is very unlikely in normal
gence of neoplastic disease. cells, whose rate of mutation is approximately 10 7 per gene
20 The Molecular Basis of Cell and Tissue Organisation

per cell duplication. Therefore, mutations of genes that the cancer reappears within a few years. One drug provided
increase the overall mutation rate are frequent early events in alone is generally ineffective, because some cancer cells
tumorigenesis. Defects of checkpoint controls in cancers, survive this treatment and so the cancer reappears. Multi-
including hereditary ones, create mutations which are likely ple drugs are necessary for effectiveness, but this multi-
to be misrepaired and are progressive because of error-prone targeting is limited by toxicity to normal cells.
repair mechanisms in cancer cells. For example, mutations of Molecular differences between cancer and normal cells
BRCA1 or BRCA2 cause p53 abnormality, which leads to are subtle (see Hanahan and Weinberg). They are mainly
breast cancer (Tseng et al., 1997). Other good examples are related to defective controls of cell growth and survival.
mutations in colon cancer that cause defective repair of Many clinically applied drugs preferentially kill the
damaged DNA and thereby create genomic instability. cycling cancer cells relative to the generally quiescent
Substances that modify checkpoint controls can change normal cells, which are essential for survival of the indi-
the rate of appearance of mutations, and therefore the vidual. But some kinds of normal cells are cycling, and so
progression of cancer. For cells in tissue culture, very high the drugs are toxic to the patient. Several cell cycle events
doses of caffeine or related compounds bypass the G2--M provide targets for therapy (Figure 6). (See the section on
checkpoint, and as a consequence most damaged cells die. The Treatment of Human Cancer.)
These results demonstrate the protective role of the
G2--M checkpoint against damage-induced chromosomal Antagonists of Growth Factors
aberrations (Fingert et al., 1988). However, chromosomal
abnormalities may appear in the few surviving cells. Sex hormones stimulate the growth of some breast, ovarian
and prostate cancer cells. Blocking these hormones' action
can kill these cells. Tamoxifen is chemically related to the
sex hormone oestrogen, with which it competes for binding
CANCER THERAPY AND THE CYCLE to oestrogen receptors in a cell. Since tamoxifen blocks the
stimulation by oestrogen and does not activate growth, it is
Classical Chemotherapies in fact inhibitory.
Currently applied therapies are aimed at killing cancer
Blocking S Phase
cells with cytotoxic agents that are applied in combinations.
They can prolong the lives of patient with some kinds of Cornerstones of standard chemotherapy are inhibitors of
cancer but have little effect against others, and all too often DNA synthesis. These are small molecule antagonists

Mitotic inhibitors
M (iv)
G2 checkpoint
G0

G2

Herceptin
(i)

DNA
damage
(iii)
G1

S
G1 checkpoint, R

Antimetabolite
(ii)

Figure 6 Current therapeutic approaches. Various therapeutic methods that are discussed in the text are
summarized here.
 Regulation of the Cell Cycle 21

structurally similar to metabolic compounds required in of blood vessels from their assembly. Both antiangiogenic
the synthesis of DNA and cell survival. As examples, drugs and also antibodies that neutralize VEGF are being
fluorouracil is structurally very similar to uracil, which is developed as anticancer agents (Boehm-Viswanathan,
needed for DNA synthesis, and methotrexate is an ana- 2000).
logue of the vitamin folic acid, also essential for DNA About one third of breast tumour cells lack oestrogen
synthesis. receptors. Unlike those discussed above these are not stimu-
lated by oestrogen or inhibited by tamoxifen, and so they
DNA-damaging Agents are generally treated with classical anticancer agents fol-
Agents that damage DNA are lethal. Examples are X-rays lowing surgery. They are stimulated to grow by EGF,
and clinically applied alkylating compounds such as because too many EGF family receptors are on their sur-
cytoxan and cisplatin. They are more effective against face. The monoclonal antibody (herceptin) made against
cancer versus noncancer cells because the latter generally these receptors is effective against some of these cancers,
are not growing. Also, the normal cells more effectively especially when applied in combination with the drugs
repair damage during checkpoint delays, before the lethal taxol and doxorubicin.
event of passage of the damaged cell through mitosis and
Targets in the G1 Phase Signal
consequent partitioning of damaged DNA between the
daughter cells.
Transduction Pathway
The molecules that transmit growth signals from a cell's
Mitotic Inhibitors membrane receptors to its nucleus during G1 phase
Several clinically applied drugs upset the mitotic (Figure 2) provide numerous targets for cancer treatment,
mechanism in cycling cells, and thereby are lethal. These now under investigation (Adams and Kaelin, 1998; Kaelin,
include taxol from the yew tree and alkaloid toxins from 1999). One major participant is Ras, a small protein that
the vinca plant. These currently used compounds, and also must be positioned against the inner surface of the cell
experimental epothilones derived from microorganisms, membrane to interact with growth factor receptors. En-
cause lethal mitotic arrest of cycling cells. Their targets zymes must chemically modify Ras for it to occupy this
are the microtubule proteins, which guide chromosomes position, and so drugs are being developed that prevent this
through their mitotic separation. Purified plant and micro- modification and thereby block the signalling pathway.
bial products such as these very frequently are starting Signalling events require numerous kinases that modify
points for finding anticancer drugs. the activities of other proteins by addition of phosphates to
A novel drug such as epothilone enters the clinic every them. Kinase inhibitors can arrest cell growth and cause
few years. Another current example is the antimetabolite death of tumour cells (Shapiro and Harper, 1999). Specific
gemcytabine, which during S phase is incorporated into inhibitors of critical cyclin-dependent kinases are being
newly forming DNA where it arrests continuation of developed. An inhibitor has already demonstrated high
lengthening of the molecule. Difficulties in introducing efficacy in the treatment of chronic myelogenous leukae-
novel drugs have roots not only in drug discovery, but to mia, a malignancy characterized by the activation of Abl
a great extent in complex legal requirements for meeting kinase (Drucker and Lydon, 2000).
safety standards. These require extremely extensive clin-
S Phase Lethality
ical trials, which with the many costs of doing business,
require hundreds of millions of dollars to develop one drug. A cell initiates a sequence of molecular events culminating
in apoptosis during S phase unless certain molecules that
Cycle Activators as New Targets initiated DNA synthesis are first inactivated. Applying
a molecular analogue of part of the G1--S factor E2F blocks
Discovery in cancer cells of over-activated growth- the degradation of E2F and causes apoptosis (Lees and
signalling pathways provides possibilities for chemothe- Weinberg, 1999).
rapy at every step. Drugs targeted against these reactions
are being applied clinically and are in clinical trials. Modulating Checkpoints
Tuning Down External Stimuli Several novel potential therapies are being developed
Some tumours secrete self-stimulating growth factors into (Figure 7).
their environment, which also can affect nearby cells.
Mitotic catastrophe
A fascinating example is stimulation by a tumour of the
production of new blood vessels, angiogenesis. This pro- The loss of G2 cell cycle checkpoints can increase tumour-
cess creates the blood supply essential for nourishment of cell sensitivity to chemotherapy. Furthermore, these cells
the tumour. Secretion by the tumour of a growth factor often cannot take refuge at the G1 checkpoint owing to the
VEGF stimulates this production of blood vessel cells, and loss of p53 or other G1 checkpoint molecules, whereas
22 The Molecular Basis of Cell and Tissue Organisation

Taxol Mitotic catastrophy

G2 Inhibitors (i)
(iv) M
G0

G2 Caffeine Antiangiogenesis
(i)

Kinase inhibitors
Apoptotic
clash
(ii)
G1

Differentiating agents
S (iii)

Differentiation

Figure 7 Potential therapies. Several potential therapies described in the text are illustrated.

cells with the normal checkpoint may still recover. Fol- The mechanism of this powerful synergistic and
lowing DNA damage and the arrest at the G2 checkpoint, tumour-specific lethality is being investigated. One
some compounds can cause cycling cancer cells to move hypothesis is based upon the proposal of `clashing'
on through a lethal mitotic cell division. This process was checkpoint signals; apoptosis is caused by the production
recently aptly renamed `mitotic catastrophe'. Post-treat- of two simultaneous molecular checkpoint signals created
ment with a caffeine analogue enhanced cytotoxicity of by growth conditions (Evan and Littlewood, 1998;
drugs to cancer cells implanted in mice, but not to the mice Blagosklonny, 1999). Since -lapachone causes G1 arrest
(Fingert et al., 1988). These agents, however, proved to be whereas taxol gives a G2--M arrest, such clashes of con-
toxic to humans at doses that abrogate the G2 checkpoint, flicting molecular signals might also be created by this
which limits their therapeutic use. Other inhibitors that can combination of drugs. They could selectively cause
eliminate the G2 checkpoint such as the inhibitors of apoptotic death of cancer cells which already have defect-
cyclin-dependent kinases, flavopiridol and UCN-0101, are ive checkpoint and apoptotic mechanisms.
currently undergoing clinical trials (Shapiro and Harper,
1999).
Differentiation Therapy
An alternative to toxic cancer therapies is to restore the
Clash Hypothesis
normal cell's properties. Cells in the blood are created by
Remarkably effective synergistic killing of a variety of growth and then differentiation of precursor stem cells,
human cancer cells was found with the combined appli- followed by R point arrest of the matured cells and their
cation together of two molecules derived from plant eventual death. Leukaemic cells are mutated blood cells
sources, -lapachone and taxol (Li et al., 1999). These that do not undergo this terminal differentiation and death,
combined drugs killed nearly all of several kinds of cancer and instead they continue to proliferate. Drugs have been
cells in culture, at concentrations that did not show major discovered that recreate this differentiation-growth arrest
lethality when the drugs were applied singly. Several kinds process. Retinoids (vitamin A derivatives) are used in this
of human cancer cells growing in mice were destroyed way to treat promyelocytic leukaemia.
when the drugs were applied together but not if they were Newer drugs such as Saha show potential to inhibit
applied separately. The tumours did not reappear for at leukaemias through differentiation therapy (Richon et al.,
least 2 months. Very importantly, the mice showed no 1999). This approach is being generalized to make solid
signs of toxicity under these conditions, so there was a very tumours differentiate as well. The drugs function by
high therapeutic index. Hence there are strong indications turning on differentiation-related genes, a process that
of clinical utility of this drug combination. is activated by addition of acetyl groups to the histone
 Regulation of the Cell Cycle 23

Inactive Active arrest causes them to enter S phase, where they are killed
while they are synthesizing DNA. This idea, proposed
Histone acetylase DNA
DNA in 1975, has been revived in a new form. Low doses of
Acetylated doxorubicin or etoposide induce p53- and p21-dependent
Histone
Histone deacetylase histone growth arrest of normal cells without cell death, but these
drugs do not arrest cancer cells. This pretreatment thereby
mRNA abolished the cytotoxicity otherwise caused by later addition
of microtubule-active drugs (paclitaxel, vincristine, epothi-
Figure 8 Control by histone acetylation. Histones that
lones). Protection of cells with normal checkpoint was
surround the DNA in chromatin block activities of genes.
achieved, whereas no protection was observed in cancer
Acetylation of the histones permits them to produce their
cells lacking p53 or p21 (see Blagosklonny and Pardee).
messenger RNAs.
Novel inhibitors of the cell cycle are being developed
as lethal drugs against solid cancers and leukaemia cells.
However, these are active also against normal cells; pro-
liferating bone marrow and epithelial cells are parti-
proteins associated with DNA in chromatin (Figure 8).
cularly vulnerable. Thus, searches for compounds that
This process is catalysed by the enzyme histone acetylase,
reversibly inhibit proliferation of these normal cells will be
and it is reversed by deacetylase enzymes, which produce
especially valuable to protect the individual. For example,
an inactive structure. These changes from the acetylated to
although two compounds similarly inhibited protein kinase
the deacetylated state function as an on--off switch for
C, UCN-01 was selected as the drug to develop because it
regulation of gene expression. The differentiating drugs
had higher cytotoxicity to cancer. For a selective growth
shift this balance by blocking deacetylase, thereby the
arrest of normal cells, dependent on protein kinase C, one
activating acetylation dominates. Thus, drugs that permit
would choose the less toxic inhibitor GF109203X. Other
histone acetylation, or that decrease the closely connected
strategies utilizing the retention of checkpoints in normal
DNA methylation, are approaches for re-expression of
cells to protect them versus tumour cells are discussed (see
tumour-suppressor genes such as BRCA1, p16 and p21 that
Blagosklonny and Pardee). Since proliferation of normal
are silenced in cancers by these processes.
cells is highly regulated, the search for such inhibitors
should produce surprises.
Selective Protection of Normal Cells
In summary, defects in cancers of various molecular
Chemotherapy of cancer is limited by toxicity to normal mechanisms that control cell growth, differentiation and
cells. With traditional chemotherapy, dose-limiting side apoptosis have recently been discovered. These differ-
effects emerge, including toxicity to bone marrow and ences from normal cells provide novel targets for therapy,
gastrointestinal tract, dermatological toxicity and cardio- some of which are being developed and tested.
toxicity. Therefore, selective protection of normal cells
against chemotherapeutic drugs could improve the thera-
peutic index (the ratio of doses that affect cancer versus
host), permitting the application of higher drug con-
ACKNOWLEDGEMENT
centrations (see Blagosklonny and Pardee). Defective
This work was supported by NIH Grant CA RO1 61253.
checkpoint mechanisms in cancer cells can be the basis of
such a selective survival of normal cells (Figure 6).
Until recently, the mainstream approach for cancer
treatment was directed to finding synergistic combinations REFERENCES
in which all the drugs are toxic against cancer cells, and so
combinations with an independently inactive drug were Adams, P. D. and Kaelin, W. G., Jr (1998). Negative control
considered inappropriate. However, a high therapeutic elements of the cell cycle in human tumors. Current Opinion
index, with less toxicity to normal cells, was found in in Cell Biology, 10, 791--797.
clinical trials when drugs that blocked entry of normal cells Blagosklonny, M. V. (1999). A node between proliferation,
into M phase were administered before subsequently apoptosis, and growth arrest. Bioessays, 21, 704--709.
adding taxol. This prevented taxol lethality in M phase. Boehm-Viswanathan, T. (2000). Is angiogenesis inhibition the
This antagonism was translated in the clinic as a decrease Holy Grail of cancer therapy? Current Opinion in Oncology,
in side effects to normal cells. 12, 89--94.
G1 checkpoint arrest in normal but not in cancer cells is Druker, B. J. and Lydon, N. B. (2000). Lessons learned from the
produced by low, nonlethal concentrations of compounds development of an Abl tyrosine inhibitor for chronic myelo-
such as cycloheximide that slow protein synthesis. Later genous leukemia. Journal of Clinical Investigation, 105, 3--7.
addition of a toxic S-phase-specific agent cannot kill them. Evan, G. and Littlewood, T. (1998). A matter of life and cell
In contrast, independence of cancer cells from this cycle death. Science, 281, 1317--1322.
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