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Cytoskeleton, Centrosome Concluding Remarks
Overview
Cells are the basic structural and functional units of all liv-
ing organisms. The estimations about the total cell count of a 
human body vary widely; a number as large as 1014 seems con-
ceivable. Although the principal components of all cells are very 
similar, the differentiation of cells results in a wide variation of 
cellular morphology and function.
The smallest human cells by diameter are spermatozoa 
with a size of ~3 μm, followed by the anucleate erythrocytes 
(8 μm). The largest cells are female oocytes that can be as large 
as 35–40 μm and are visible to the naked eye. Motor neurons are 
extremely long cells, with their axons reaching from the spine to 
the distal extremities (up to 1.4 m length).
Most cells can only be functional in large united structures, 
such as organs or suborganic structures. Other cells, mainly 
of hemato- or lymphopoietic origin, are mobile and active 
as single cells, although in many cases, their functionality is 
dependent on interaction with other cells.
Cytopathology studies diseases on the cellular level. While in 
histopathology, cells are assessed in the spatial context, in vir-
Apart from the loss of the spatial information, cells can have 
considerably altered morphology when taken out of the united 
structures. Cell–cell contacts are important features that build 
the shape of a cell. Many structural elements within a cell are 
connected to proteins that are attached to other cells or the 
basal membrane. This must be taken into account when cells 
are compared in histological and cytological assessments.
Another important difference between histology and cytol-
ogy is based on the fact that in histology, plain two-dimensional 
tissue sections are assessed, while in most cytology applications, 
complete cells that still have some three-dimensional features, 
although they might appear flat in the microscope, are analyzed. 
Based on these facts, the transfer of histological morphology to 
the picture seen in cytology is limited.
In this section, an overview of the most important cellular 
structures and functions relevant for cytopathology are pre-
sented (Fig. 1.1). We have assembled the most important infor-
mation on cellular structures by describing the regular function 
in brief, the relevance for cytopathology, and the morphology 
in normal and abnormal cells. For more detailed information 
PART I: BASIC STRUCTURE AND FUNCTION OF MAMMALIAN CELLS
C h a p t e r
The Cell: Basic Structure and Function
Magnus von Knebel Doeberitz and Nicolas Wentzensen
1
Contents
PART I: BASIC STRUCTURE AND FUNCTION OF MAMMALIAN CELLS
Overview
Nucleus
Contents of the Nucleus
Nuclear Morphology
hematoxylin
Nucleoli
Nuclear envelope and Nuclear Shape
Cytoplasm and Plasmalemma
Cytoplasmic Stain
Endoplasmic Reticulum
Golgi Apparatus
Mitochondria
Lysosomes
Cell Membrane, Receptors, and Signal Transduction
Cell Junctions
Cell Growth and Division
PART II: THE MOLECULAR BASIS OF NEOPLASIA
Overview
Principles of Malignant Transformation
Cancer-related Genes
The Major Pathways of Carcinogenesis
Carcinogenesis induced by Papillomavirus Infections
Basic Structure of the Virus and Its Genome
epidemiology of hpV Infections
the role of the hr-hpV e6 and e7 Genes
progression of hpV-Infected epithelial Cells to Invasive Cancer Cells
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tually all cytological applications, they are removed from their 
spatial context and must be assessed isolated or as cell sheets.
about cellular structures and functions, a cell biology textbook 
is recommended.
General CytologyPART ONE
Nucleus
The nucleus contains the genomic DNA, histones, and several 
proteins that are responsible for DNA replication, repair, and 
transcription of genetic information (Fig. 1.2A).
The assessment of a cell’s nucleus is one of the most impor-
tant tasks in cytopathology. The size of the normal nucleus is 
highly variable, depending on the underlying cell type. In many 
malignant cells, nuclei are considerably enlarged. Apart from 
nuclear size, the chromatin density, the nuclear membrane, 
and the presence of nucleoli are important features of nuclear 
 morphology and will be described in detail.
The nucleus contains about 25% dry substance, of which 18% 
is DNA plus a similar amount of histone proteins. The rest of the 
dry substance contains the non-histone proteins, nucleoli, and 
the nuclear membrane.
Contents of the Nucleus
DNA
The genetic information of organisms is coded in deoxyribo-
nucleic acid (DNA). DNA is a long stretch of single nucleotides 
connected by a sugar–phosphate backbone (Fig. 1.2C). The 
genetic information is stored in specific sequences consisting 
of four different bases: adenine, guanine, thymine, and cytosine 
(A, G, T, C). A triplet of bases is coding for an amino acid that 
constitutes the basic component of proteins. Although in prin-
ciple the triplet code allows for 64 different variations, only 20 
protein-building amino acids exist. Many amino acids are coded 
by multiple base triplets. The genetic code is degenerate, thereby 
tolerating errors in the base sequence to some degree. Two DNA 
stretches are combined as a double helix; one complete turn 
is reached after 10 bases. The DNA stretches are not covalently 
bound, but attached via hydrogen bonds between complemen-
tary bases A–T and C–G.
DNA is a very robust and stable molecule, since it must pro-
tect the genetic code of an organism. The genetic information is 
transferred to the ribosomes (the protein production machinery) 
by ribonucleic acid (RNA) that has 3 important features differ-
ent from DNA: RNA is based on a ribose backbone, it contains 
uracil instead of thymidine, and it is usually single-stranded. 
Compared to DNA, RNA is a rather unstable molecule.
Cytoskeleton
Mitochondria
Endoplasmic
reticulum
Nucleus
Lysosomes
Cell junctions
Golgi
apparatus
Cell
membrane
Fig. 1.1 Schematic presentation of an epithelial cell displaying the most 
important structures discussed in this chapter.
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The total DNA of a cell is separated on chromosomes. In 
total, 22 different chromosomes and two sex chromosomes 
exist. The chromosomes vary in size and in the content of 
coding sequences, they are numbered in decreasing order of 
their size. During the metaphase of mitosis, chromosomes 
are condensed and can be identified in light microscopy. In 
transcriptionally active cells, DNA is decondensed and takes 
up the room of the complete nucleus. When metaphase chro-
mosomes are stained according to Giemsa, a heterogenous 
pattern of regions with strong staining (G-bands) and regions 
without staining (R-bands) can be observed. R-bands contain 
more genes than G-bands and are replicated early during cell 
division. The banding pattern of chromosomes has been used 
to determine chromosomal regions by indicating the chromo-
some number, the position with reference to the centrosome 
(p = short arm, q = long arm), and the position of the chromo-
somal banding (e.g. 3q26). In total, the human genome con-
sists of ~3.2 billion bases, coding for approximately 25,000 
genes.1
Nuclear Proteins
Histones are basic proteins that build a structural unit together 
with the DNA, called the nucleosome (Fig. 1.2B). In the nucleo-
some, 146 base pairs (bp) are coiled around different histone 
subunits. The main function of the nucleosome is the high-
 density packing of DNA inside the nucleus, leading to a 50,000-
fold increased compactness of DNA as compared to unpacked 
DNA.
Histone acetylation reduces the affinity between the DNA 
molecule and histones, leading to increased accessibility of DNA 
for transcription machinery components such as RNA polymer-
ase and transcription factors. In general, for gene transcription,the DNA needs to be unpacked from the histones.
Besides histones, nuclear non-histone proteins build the 
nuclear scaffold structure and are involved in DNA transcription 
and replication.
Nuclear Morphology
Chromatin
Chromatin represents the complex structure of proteins and 
DNA in the nucleus of non-mitotic cells. There is usually twice 
as much protein as DNA in a nucleus. Since most cells in the 
human body are non-mitotic, chromatin is the morphological 
appearance of most cell nuclei assessed in cytology. The chro-
matin distribution and organization depends on many different 
factors, such as cell type, differentiation, metabolism, prolifera-
tion status, and, most important in cytopathology, neoplastic 
transformation.
Two conformations of chromatin are discriminated: euchro-
matin and heterochromatin. Euchromatin contains transcrip-
tionally active protein-coding DNA regions. Heterochromatin 
represents the complex of DNA that is densely packed on 
histones. DNA sections not transcribed are usually stored in 
heterochromatin. Heterochromatin is further differentiated 
into constitutional, facultative, and functional heterochro-
matin. Constitutional heterochromatin consists mainly of 
highly repetitive DNA stretches in the centromeric region that 
are supposed to have structural functions but have also been 
found to express microRNAs that do not code for proteins but 
are involved in gene regulation. Facultative heterochromatin 
The Cell: Basic Structure and Function
1
hematoxylin is a very weak stain. Different mordants, such as 
and are reconstituted in the daughter cells. Shortly after cell 
division, a larger number of nucleoli that fuse gradually can be 
N
potassium alum, are used to generate the typical dark blue or 
purple staining. Hematoxylin strongly binds to acidic com-
ponents of a cell, most importantly to the phosphate groups 
of nuclear DNA; the stained structures are therefore called 
“basophilic” (Fig. 1.3A).
Based on the nuclear stain, a wide variation of chromatin 
alterations can be observed, both alterations of structure and 
staining intensity. Structural aberrations include chromatin 
margination, i.e. aggregation of chromatin to the nuclear mem-
brane, which is a sign of cell degeneration. Other chromatin 
alterations are coarsening and clumping that is usually accom-
panied by chromatin thinning in other regions.
Hyperchromasia, i.e. increased staining intensity, can result 
from increased chromatin loads or from decreased nuclear vol-
ume, which inversely applies to hypochromasia. In addition, 
chemical modifications of the chromatin (e.g. during specific 
stains or cell treatments) can increase the stain uptake, simulat-
ing hyperchromasia.
Nucleoli
Nucleoli are small basophilic spherical bodies located in the 
nucleus. Usually they can be found in the central nuclear region 
but may also be close to the nuclear membrane. A nucleolus is 
observed.
Depending on the cell type, the presence of nucleoli is physi-
ological or can indicate malignant processes: liver cells that regu-
larly produce a lot of protein can frequently exhibit nucleoli. In 
reactive or regenerative cells, nucleoli can become more promi-
nent. In hepatocellular carcinoma, usually more than 50% of 
the cells show prominent, frequently multiple nucleoli. Intesti-
nal epithelial cells also regularly show single nucleoli. In ageing 
and starving cells, a shrinking of nucleoli can be observed. In 
cancer cells, nucleoli can vary substantially with regard to size 
and shape.
In many malignant cells, multiple nucleoli that appear dis-
joint, odd-shaped, and spiculated can be observed. Proteins 
associated with nucleolar organizer regions can be visualized 
by a simple argyrophilic staining method. The structures high-
lighted by this method are called “argyrophilic nucleolar organ-
izer regions” (AgNORs). Different distributions of AgNORs have 
been described between normal, dysplastic, and malignant tis-
sues. In several cancer entities, AgNOR aberrations were found 
to have independent prognostic significance with respect to 
patient survival.2 Increased NOR counts have been explained 
by increased metabolism with a high demand of ribosomes, 
but also by aneuploidy leading to increasing numbers of NOR 
regions in cancer cells.
 designates inactivated DNA regions that usually code for pro-
teins but are not necessary in the respective cell, e.g. inacti-
vation of the second X chromosome (Barr body). Functional 
heterochromatin requires DNA regions not necessary for the 
respective differentiation of a cell.
Hematoxylin
In many cytological applications, the chromatin is stained with 
hematoxylin. Hematoxylin is a basic dye extracted from the 
heartwood of the tree Haematoxylum campechianum. By itself, 
Nuclear
membrane
Nucleoli
Euchromatin
P P
G
C
A
T
T
A
C
G
G
C
A
T
T
A
P
P
Sugar-phosphate backbone
P P P P P P
P P P P P
Heterochromatin
Histone DNA
A
C
B
built by a nucleolus organizing region (NOR) of a specific chro-
mosome. These regions contain the genes for ribosomal RNA 
subunits that build the protein synthesis machinery. Since in a 
diploid human cell, in total 10 chromosomes containing NORs 
exist, in principal 10 nucleoli per nucleus could be present. 
Usually, only one or two nucleoli are found, since NORs from 
several chromosomes build a common nucleolus. Nucleoli have 
two distinctive regions, the pars fibrosa that contains the proteins 
required for transcription and the pars granulosa that contains 
the ribosomal precursors. During mitosis, nucleoli disappear 
C
G
P
ucleosome
Organic bases
Fig. 1.2 Contents of the nucleus, DNA. 
(a) a nucleus displaying nucleoli, euchromatin, 
and heterochromatin. (B) two nucleosomes 
 consisting of DNa coiled around histone 
 proteins. (C) the structure of double-stranded 
DNa. Organic bases are connected to a 
sugar–phosphate backbone. Complementary 
bases (a-t, C–G) are held together by hydrogen 
bonds.
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General CytologyPART ONE
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The Cell: Basic Structure and Function
1
Nuclear Envelope and Nuclear Shape
The nuclear envelope (NE) consists of two lipid membranes. 
The inner membrane is associated with the telomeres and 
anchors the chromosomes, while the outer membrane is part 
of the endoplasmic reticulum. The space between the two lipid 
layers is called perinuclear cisterna. The nuclear envelope con-
stitutes the nucleus and separates the genomic material from the 
cytosol. During cell division, the nucleus disappears; the nuclear 
envelope is broken down to vesicles and is reassembled during 
telophase. The nuclear envelope builds a strong barrier between 
nucleus and cytosol; a number of nuclear pore complexes regu-
late the traffic between both compartments. There can be passive 
diffusion or active transport; in general, proteins synthesized in 
the cytoplasm require a specific nuclear signal in order to have 
access to the nucleus.
Inside the nuclear envelope is a network of chromatin fibrils 
and a nuclear lamina built from laminins. The nuclear envelope 
can be visible in light microscopy.
The regular nuclear shape is that of a smooth sphere or sphe-
roid, based on the orderlyarrangement of the chromosomes 
and the nuclear lamina. Many factors can affect the shape of 
the nucleus: stress, transcriptional, and synthetic activities can 
disturb the arrangement of interphase chromosomes; DNA 
amplifications can lead to uneven distribution of the nuclear 
material and to nuclear enlargements. At the same time, aber-
rations of the nuclear envelope can lead to alterations of the 
nuclear skeleton, resulting in altered chromosomal distribu-
tions. It has been assumed that changes of the nuclear envelope 
occur mainly after mitosis, when the nuclear envelope is reas-
sembled. Alterations of the nuclear envelope have been directly 
linked to oncogene activity: Six hours after transfection with the 
ret oncogene, increasing cell counts with NE alterations were 
observed in human thyroid cells, indicating that nuclear altera-
tions may occur even independent of postmitotic re-assembly.3 
NE alterations and the respective nuclear shape are an impor-
tant diagnostic feature of many malignancies, especially papil-
lary thyroid cancers and different types of leukemias.
Cytoplasm and Plasmalemma
The cytoplasm consists mainly of water (80–85%). The remain-
ing constituents are proteins (10–15%), lipids (2–4%), polysac-
charides (1%), and nucleic acids (1%). The cytoplasm is confined 
to the outside by the plasma membrane, a lipid bilayer, and to 
the inside by the nuclear membrane. In most cytology applica-
tions, normal cells have a homogenous cytoplasm with occasional 
granules or inclusions.
Cytoplasmic Stain
Eosin is the most common dye to stain the cytoplasm in histol-
ogy. It is an acidic dye that binds to basic components of a cell, 
mainly proteins located in the cytoplasm. It gives a bright pink 
color that contrasts the dark blue nuclear hematoxylin staining 
(Fig. 1.3B). A combination of hematoxylin and eosin is the most 
frequently used dye in histology. In cytology, frequently, a Pap 
stain is performed. It consists of a hematoxylin-based nuclear 
stain and a polychromatic cytoplasmic stain, including Orange 
G and two polychromic dyes, EA36 and EA50. The cytoplasmic 
stain results in highly transparent cells, making it possible to 
assess superimposed cells in a Pap smear. Based on the cell type 
and the differentiation status, the cytoplasm can be pink–light 
red (e.g. superficial cervical cells) or light blue–green (e.g. cer-
vical parabasal and intermediate cells) with all variations in 
between. The nuclei are dark brown or dark blue/violet and the 
nucleolus appears bright red (Fig. 1.3C).
Endoplasmic Reticulum
The endoplasmic reticulum (ER) consists of a single membrane 
making up for more than half of all internal membranes of the 
cell (Fig. 1.4A). The part of the membrane that faces the cytosol 
is studded with ribosomes. This part of the ER is called rough 
ER; the regions without ribosomes are called smooth ER.
The main function of the ER is the packaging and delivery of 
newly synthesized proteins.
Golgi Apparatus
The Golgi apparatus is part of the membrane system that also 
contains the ER. It consists of stacked membrane-coated cavities, 
called dictyosomes (Fig. 1.4B). The Golgi apparatus is located 
close to the nucleus and can be very large in secretory cells, where 
it fills almost the complete cytoplasm. The convex side facing 
the ER/nucleus is called cis-Golgi; the concave side facing the 
cytoplasm is called trans-Golgi. From the Golgi apparatus, small 
vesicles transport products to other cellular sites or the exterior. 
Inside the structure, complex biochemical operations are being 
performed, most of them resulting in post-translational modi-
fications of synthesized proteins. Several secretory mammalian 
cell types are characterized by a prominent polarized Golgi 
apparatus located between the nucleus and the luminal surface: 
Goblet cells in the respiratory and digestive tract produce large 
Cytosol
Nucleus
Trans-Golgi
Dictyosome Outer
membrane
Inner
membrane
Crista Lumen
Cis-Golgi
Ribosomes
ER membrane
ER lumen
MitochondrionGolgi Apparatus
Rough Endoplasmic Reticulum (RER)A
B C
Fig. 1.4 Membranous organelles. (a) the rough endoplasmic reticulum. 
(B) the Golgi apparatus. (C) a mitochondrion.
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General CytologyPART ONE
amounts of glycoproteins, pancreatic cells secrete enzymes such 
as zymogen, and breast cells produce milk droplets.
Mitochondria
Mitochondria produce ATP, the universal fuel of living organ-
isms, by oxidative processing of nutrients. They are located in 
the cytoplasm and separated from it by a double membrane 
(Fig. 1.4C). On average, an eukaryotic cell contains about 2000 
mitochondria. Depending on age and cell type, mitochondrial 
size can vary between 0.5 and 10 μm. The highest mitochon-
drial counts can be found in cells with high energy demand, 
such as muscle cells, nerve cells, or metabolically active cells in 
the liver. Mitochondria are inherited in non-mendelian fashion 
via the cytosol of oocytes. During cell division, mitochondria 
are divided between the two daughter cells. They are genetically 
semi-autonomous since they possess their own circular genome, 
but are dependent on a number of proteins encoded by the 
nuclear DNA.
The Pap stain does not color mitochondria, but iron hema-
toxylin or acid fuchsin does. A more specific stain for mito-
chondria is rhodamine 123. Stained mitochondria appear as 
single spheres or long, branching structures, up to 7 × 0.5 μm 
in size. Mitochondria can be found in large numbers in 
hepatocellular carcinoma, resulting in a granular appearance 
of the cytoplasm. There are many other causes of granular 
 cytoplasm; the underlying cellular components can only be 
visualized by ultrastructural methods. Since mitochondria 
represent the energy system of living cells, they are very impor-
tant in the malignant development. Multiple functional and 
structural alterations during carcinogenic processes have been 
described.4
Lysosomes
Lysosomes are small vesicles derived from the Golgi apparatus; 
they contain up to 40 acidic enzymes (hydrolases) at a pH 5. 
The membrane prevents the aggressive enzymes from destroying 
cellular structures. Although the contents can vary substantially, 
there are basically no morphological differences between func-
tionally different lysosomes. The main function of lysosomes 
is the digestion of internal (non-functional cell organelles) and 
external (nutrients, bacteria, leukocytes, debris) material. The 
processed material is either released to the cytoplasm, secreted, 
or stored in lysosomes.
Several storage diseases (e.g. Hunter-Hurler-Syndrome) are 
characterized by a deficiency of lysosomal enzymes. These disor-
ders lead to accumulation of incompletely digested mucopoly-
saccharides in the lysosomes.
Cytoskeleton, Centrosome
The cytoskeleton is a complex lattice of various filaments 
building the cellular structure and shape; it is responsible for 
dynamic activities such as movement in growth and differen-
tiation (Fig. 1.5). Although is has been thought for a long time 
that the cytoskeleton is a special feature of eukaryotic cells only, 
it is becoming more and more clear that also prokaryotes have 
cytoskeleton-like structures.5
The filaments are required for cell movement (cytokinesis), 
transport of material across the cell surface, muscle contraction, 
intracellular transport, and sorting and dividing of replicated 
chromosomes by the mitotic spindle.
Three main classes of cytoskeletal filaments are distin-
guished: actin filaments, intermediate filaments, and micro-
tubules (Fig. 1.5).
Actin filaments have a diameter of 7 nm and are built from 
six different actin types; in muscle cells, actin is functionally 
linked to myosin. They can be found in all cells, with especially 
high numbers in fibroblasts and the highestconcentrations 
in muscle cells, since actin is part of the contractile structures. 
Lamellipodia (bulges of the cell surface for cell motility) and 
filopodia (enhanced cell surface for absorption) are built from 
bundled actin. Several glandular tissues such as breast and pros-
tate have contractile myoepithelial cells that can forcibly express 
the glandular contents.
Intermediate filaments have a diameter of 10 nm, con-
sist of one or more of 19 different cytokeratin molecules, and 
are the strongest fibers among the cellular filaments. They are 
mainly responsible for the structural framework of a cell and 
determine the cell’s tensile strength. They build rope-like poly-
mers. Keratins belong to the group of intermediate filaments. 
In keratinizing epithelial cells, keratin filaments accumulate 
and are cross-linked by other proteins and disulfide bonds. The 
keratinizing process starts at the periphery and progresses to the 
nuclear area. In fully differentiated cells, the nucleus becomes 
more and more pyknotic and finally dissolves.
Other examples for intermediate filaments are desmin in 
skeletal muscle, glial filaments in astrocytes, and neurofilaments 
in axons.
Microtubules are long, hollow tubes with 25 nm diameter 
assembled from microtubule oligomers originating in a mem-
braneless body, the centrosome. The centrosome is the main 
microtubule organizing center (MTOC) of a cell and functions as 
an important regulator of the cell cycle. The centrosome consists 
of two orthogonally arranged centrioles surrounded by peri-
centriolar material and is situated between the nucleus and the 
Golgi apparatus. Upon cell division, each daughter cell receives 
one centriole. Although in most model organisms, a proper cell 
division can be achieved without a functional centrosome, an 
organism requires functional centrosomes to survive in the long 
Actin
filaments
Intermediate
filaments
(Cytokeratins)
Tight
junction
Adherence
junction
Gap
junction
Desmosome
Centrosome
Hemidesmosome
Basal membrane
Fig. 1.5 Display of an epithelial cell with cytoskeleton and cell–cell 
contacts.
The Cell: Basic Structure and Function
1
term. Aberrant centrosomes are a hallmark of chromosomally 
instable cancer cells. Because of aberrant formation of the 
mitotic spindle apparatus, these cells acquire more and more 
chromosomal aberrations.
Two classes of substances can interfere with the microtubular 
network: Colchicine prevents the polymerization of microtu-
bules, while paclitaxel interferes with their depolymerization. 
In a living cell, the microtubular network is continuously 
 polymerized and depolymerized. Therefore, both agents lead to 
a non-functional spindle apparatus abrogating the cell division 
and are used as cytotoxins in cancer therapy.
Cell Membrane, Receptors, and Signal 
Transduction
The basic structure of the cell membrane is a semi-permeable 
lipid bilayer built from phospholipids, glycolipids, and steroids 
that contains various proteins floating on one side of or reaching 
through the complete membrane. The lipid bilayer has a gauge 
of 6 to 10 nm and is barely visible in light microscopy. The cell 
membrane separates all cellular components from the environ-
ment and it assures the spatial and functional entity of a single 
cell. It allows the cell to persist in environments that would be 
harmful to the cellular components, such as extreme pH condi-
tions and ion concentrations different from the cytoplasm. The 
cell membrane controls what is going into and out of a cell; 
thereby it regulates the import of nutrients and the export of cel-
lular products. The transport is organized by passive (transport 
via a gradient that does not require energy) and active (transport 
against a gradient that requires energy) protein channels.
There are different types of membrane proteins. Peripheral 
membrane proteins only temporarily adhere to the respective 
cell membrane; they usually interact with integral membrane 
proteins. Many regulatory subunits of ion channel and trans-
membrane receptors, as well as enzymes and hormones, are 
peripheral membrane proteins. In contrast, integral membrane 
proteins are permanently attached to the membrane. Trans-
membrane proteins are integral proteins that span both lipid 
layers; they must contain a hydrophobic part that is placed in 
the lipid section and hydrophilic intra- and extracellular parts. 
Typical representatives are ion channels, proton pumps, and 
G-protein coupled receptors. Lipid-anchored proteins are cova-
lently linked to lipids in the cell membrane; the most common 
are G-proteins.
The communication of a cell with the environment (i.e. other 
cells or the extracellular matrix) is mediated by a wide varia-
tion of interacting molecules, usually designated as receptors. 
The functional principle of receptors is based on the key–lock 
principle; i.e. a specific receptor requires the binding of a specific 
ligand (either cell based or freely circulating) to be activated. 
The transfer of information between cells may be mediated 
by two different classes of receptors located in the cell mem-
brane: Ionotropic receptors are based on specific ion channels 
that change the electric potential of cells upon activation (Fig. 
1.6A). Non-ionotropic receptors have no pores, but are based 
on transmembrane proteins that stimulate intracellular proteins 
linked to the receptors and thereby modulate intracellular signal 
cascades.
The two most important non-ionotropic receptor types are 
G-protein coupled receptors and tyrosine kinase receptors. 
G-protein coupled receptors consist of seven transmembrane 
domains, an extracellular receptor region, and an intracellular 
part that binds the G-protein (Fig. 1.6B). Upon activation by 
an outside signal, the receptor changes its conformation and 
releases the G-protein in a not yet fully resolved mechanism. 
G-proteins, short for guanine nucleotide binding proteins, are 
the most important proteins involved in second messenger 
cascades, responsible for many central nervous (vision, olfac-
tory system, neurotransmitters) and immune system func-
tions. Receptor tyrosine kinases activate downstream targets 
by adding phosphate groups to intracellular proteins (Fig. 
1.6C). They are frequently promoting cell growth and cell 
division (e.g. receptors for insulin-like growth factor, epider-
mal growth factor, EGF). Consequently, many malignant pro-
cesses are linked to aberrations of G-protein coupled receptors 
and especially tyrosine kinase receptors. In about 25% of the 
breast cancers, a subtype of EGF receptors (ErbB2/Her2neu) is 
overexpressed, which leads to increased signaling of the EGF 
pathway, resulting in increased cell proliferation. Meanwhile, 
inactivating antibodies directed against Her2neu are success-
fully used in breast cancer therapy. Similarly, cetuximab is a 
monoclonal antibody downregulating ErbB1 signaling that 
has been approved for special types of colorectal cancer.
Cell Junctions
As complex organisms are built from billions of cells, the cell-
to-cell contact is a very important parameter. Depending on the 
organ or the function of a tissue, cell contacts establish adherence 
of functionally connected cells, build a barrier against a lumen, 
and are involved in intercellular communication (Fig. 1.5).
Adherence is based mainly on spot desmosomes (macula 
adhaerens) consisting of keratin filaments that connect the 
cytoskeleton of individual cells. A different form of adherence 
is the adhesion belt (zonula adherens) that connects the apical 
part of an epithelial cell to another epithelial cell. Hemidesmo-
somes are located at the basal pole of the cell and attach the cell 
to the basal membrane.
Tight junctions (zonula occludens) build an impermeable 
barrier between cells and are typicalfor structures that have a 
lumen.
Gap junctions are required for communication; small inor-
ganic molecules and electric signals are exchanged via 1.5-nm 
pores in the cell membrane. Gap junctions can be quickly estab-
lished from precursors floating in plasma membrane.
The terminal bar describes a light microscopic structure that 
represents the sum of the adhesion belt and actin filament bun-
dles as well as other protein filaments at the apical end of a cell.
In general, malignancy causes loss of cell-to-cell adherence. 
It has been shown that the cell adhesion molecule E-cadherin is 
lost during the formation of some epithelial cancers. The loss of 
E-cadherin is frequently accompanied by an overexpression of 
other cadherins, an effect called the “cadherin switch.” Besides 
the cell adhesion, signal transduction pathways are altered, 
inducing malignant transformation of the respective cell.
Cell Growth and Division
In mammalian organisms, some cells are created early in embry-
ogenesis and remain unchanged throughout the whole life 
(e.g. lens of eyes, cells of CNS, heart muscle cells, auditory cells 
�
General CytologyPART ONE
AC AC
s s
of ear). However, many epithelial tissues, as well as hemato-
lymphopoietic cells, depend on a continuous replenishment 
of their cell pools. Other tissues have retained a regenerative 
capacity, e.g. liver stem cells, that make it possible to regener-
ate the organ after tissue damage. The detailed mechanisms of 
cell division can be recapitulated in a cell biology textbook. In 
brief, the cell cycle consists of four phases, the G1, S, G2, and 
M phases. Resting cells are in a constant G0 phase that has a 
direct transition from the G1 phase. G phases are gap phases, 
S indicates the synthesis phase in that the genomic material 
is doubled, and M indicates the phase of mitosis, in that two 
daughter cells are created (Fig. 1.7A). In order to assure a proper 
cell division with equal distribution of the genomic material in 
the daughter cells, several checkpoints (after G1, S, and G2) 
must be passed to allow for a continuation of the cell cycle. If 
the requirements at a checkpoint are not met, the cell is not 
allowed to continue; it might even go into apoptosis.
In light microscopy, mitotic figures represent the M phase, i.e. 
the distribution of chromosomes into the opposite poles of a cell 
(Figs. 1.7B, C). Mitotic figures are rare in normal tissue; in nor-
mal liver, about 1–2 mitoses can be observed per 10,000 cells. 
In rapidly dividing cancer tissue, the frequency of mitotic figures 
can be much higher, depending on the underlying cell type.
The regulation of the cell cycle is very complex. In general, 
uncontrolled activation of the cell cycle is a basic feature of all 
tumors. The reasons for uncontrolled cell cycle activation can be 
either the loss of inhibitory gene functions (inactivated tumor 
suppressor genes) or the activation of cell cycle promoting gene 
functions (activated oncogenes). The detailed mechanisms of 
carcinogenesis are described in Part II of this chapter.
GTP GTPGDP
EGF EGFCR1CR1
CR2 CR2
L1
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C
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STAT 3Src
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P P
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esaniK
esaniK
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Fig. 1.6 Important membrane proteins. (a) Different calcium ion channels controlling the intracellular calcium concentration. (B) G-protein receptors; 
the example shows adrenergic activation of the adenylate cyclase (aC). (C) tyrosine kinase receptors; the example shows eGF-mediated phosphorylation of 
intracellular downstream targets.
10
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1
The Cell: Basic Structure and Function
1
1
sarcomas, hematopoietic tissues that may be transformed into 
lymphoma and leukemia, and finally even germ cells that may 
be transformed into benign teratomas and malignant terato-
carcinomas.
Benign lesions are partially growth restricted: They do not 
invade, do not metastasize, but do grow locally. In contrast, 
malignant tumors usually acquire more autonomous growth 
properties, and develop complex strategies to invade other 
organs, to disseminate their cells to distant anatomical sites, to 
evade immunological attack of the host’s immune system, and 
to initiate from disseminated cells again autonomous prolifera-
tive metastatic lesions. Following the concept that all these fea-
tures are mediated by a complex network of genes, the question 
arises what genes are regulating the process of growth control in 
cancer cells and why are these genes activated or inactivated in 
distinct somatic cells that thus acquire the capacity to grow out 
as cancer cell. Evidently the modifications required to induce 
benign tumors are by far less complex than those required 
for an invasive cancer. They usually can be achieved by minor 
 modifications of the genome of the affected cells. In many cases 
benign tumors are therefore precursors of malignant tumors 
that require a substantially more complex pattern of genetic 
modifications (Fig. 1.8).
The biological phenotype of cancer cells is defined by 
the expression of certain genes, whereas other genes are not 
expressed. These phenotype-specific gene expression patterns 
are referred to as “gene signatures” that differentiate a distinct 
Severe alterations of the gene expression
profile induced by the accumulating 
mutations finally allow for invasive
metastatic growth
Normal cell
Transformed cell
Fig. 1.8 Transformation of epithelial cells.
G2
s
G1
G0
M
G0
A
B
C
Fig. 1.7 The cell cycle and its morpholgical 
appearance. (a) Schematic presentation of the cell 
cycle. (B) typical anaphase during mitosis of plant 
cells. (C) Mitosis of transformed epithelial cells in 
immunohistochemistry.
PART II: THE MOLECULAR BASIS OF NEOPLASIA
Overview
Single cells in complex organisms work in a hierarchical order 
determined by their differentiation state. Each single cell ful-
fills its function through biochemical processes that are highly 
regulated and act in well-orchestrated pathways. The outgrowth 
of tumors occurs if the cells have lost the capability to follow 
these predetermined rules. Growth restriction, a typical feature 
of normal tissues, is lost; the capability to commit cellular sui-
cide (apoptosis) once certain death signals have been activated 
is lost. Differentiation pathways that permit the cell to enter irre-
versible cell cycle arrest become disconnected. Mechanisms that 
under normal conditions are required to maintain the normal 
histological architecture of an organ are erroneously activated to 
feed the outgrowing tumor with essential nutrients.
Tumors may be derived from more or less all human tis-
sues, including epithelium that may be transformed into benign 
adenomas and malignant carcinomas, mesenchymal tissues 
that may be transformed into benign fibromas and malignant 
Several early mutations lead to
epithelial cell expansion and
initial transformation
Normal epithelium
Transformation
Defects in genes that control the
genetic homeostasis results in rapid
accumulation of many mutations
(genetic instability). Emergence of
cell clones thatdisplay genetic
instability is the key event required for
malignant transformation
General CytologyPART ONE
cell (e.g. cancer cells) from cells with other biological properties 
(e.g. a normal stem cell that is not transformed). Cancer cells of 
the same organ origin, e.g. gastric cancers, may display substan-
tially different biological properties that reflect different gene 
signatures.6 These changes of the gene expression pattern of cells 
or tissues can be monitored by gene expression arrays (Fig. 1.9). 
To convert a normal somatic cell into a full-blown cancer cell 
many distinct steps are required, during which the gene expres-
sion pattern of the respective cell is gradually modified and the 
phenotype of the cell is transformed into increasingly neoplastic 
B
C
D
E
F
Fig. 1.9 analysis of the gene expression signature of a panel of gastric cancer cells with different biological phenotypes. Genes expressed at high levels are 
indicated in red, those expressed at lower levels are indicated in green. By comparing the expression level of distinct genes (or c-DNas) among different cell 
lines, complex differences among the expression of many genes can be monitored in a large number of tissues of cell lines. Using distinct software algorithms 
tissues or cell lines can be clustered in an hierarchical order that reflects their biological phenotype. the figure shows an example of various gastric cancer cell 
lines of which some display similar phenotypes: (B) epithelial cell cluster; (C) B lymphocytes cluster; (D) t lymphocytes cluster; (e) fibroblast cell cluster; 
(F) endothelial cell cluster. the results were visualized and analyzed with treeView (M eisen; http://rana.lbl.gov). Data and images were taken from Ji et al.6
12
A
The Cell: Basic Structure and Function
1
cells that are selected in an ongoing Darwinian selection 
process. Specific modifications of the gene expression signature 
trigger the next step of selection.
Given the complex alterations required to achieve the signa-
ture of a full-blown cancer cell and given the many individual 
selection steps required to transform a normal cell into a can-
cer cell, transformation cannot be achieved by a linear selection 
process but depends on higher level mechanisms that allow 
for major modifications of the genetic code in a relatively brief 
period of time or restricted number of cell divisions. The integ-
rity of the number and structure of chromosomes, for example, 
is one particularly important aspect herein. If the mechanisms 
that maintain the integrity of the chromosomes fail, major 
genomic modifications may rapidly occur. Since most of these 
are non-viable, most cells that experience these failures will die 
in a process called genomic catastrophe. However, some cells 
may survive the complex modifications induced by malfunction 
of the mechanisms that preserve chromosomal homeostasis. 
If the gene signatures of the surviving cells allow for continu-
ous autonomous growth eventually even at distant anatomical 
sites, the respective cell clones may be selected and their sus-
tained growth may then clinically manifest itself as metastasiz-
ing cancer.
To preserve the ordered function of cells in higher order 
organisms a number of redundant genome protective mecha-
nisms that prevent consequences of genetic catastrophes have 
evolved. They primarily constitute organized suicide mecha-
nisms called apoptosis that become activated once major 
modification of the cellular genome in distinct genetically 
damaged cells occur. They assure that most cells that undergo 
genetic catastrophes undergo apoptosis before they can grow 
out as transformed cancer cell. Thus, outgrowth of a cancer 
cell still is the rare exception in view of the many billions 
of proliferating cells that constitute the human body and the 
many events that trigger genomic catastrophes in damaged 
cells.
Principles of Malignant Transformation
The development of a cancer cell from a normal cell goes 
through three basic steps:
 (1) Immortalization: In contrast to normal cells, immor-
talized cells can divide indefinitely, as long as they 
are supported with nutrients. They still have the 
same shape as normal cells, and they stop growing 
when they reach other cells (contact inhibition).
 (2) Transformation: Transformed cells are independent of 
tissue-specific growth factors; they lose their contact 
inhibition and may grow invasively. Their shape is 
altered; the specific differentiation is lost.
 (3) Metastasis: Metastasizing cells acquire the potential 
to migrate to distant sites and grow out to tumors.
These paramount changes occur on the level of the individ-
ual cancer cell. Subsequently, the establishment of tumors larger 
than 1–2 mm3 requires the development of a vascular system 
that can support the growing tumor with nutrients. In order to 
achieve this, tumors induce angiogenesis via angiogenic factors 
such as vascular angiogenic growth factor (VEGF), fibroblast 
growth factor (FGF), and platelet-derived endothelial growth 
factor (PDGF).
Tumor growth is based on a complex interplay between the 
transformed cells and the surrounding tissue: invasive growth 
is associated with the expression of proteolytic enzymes that 
degrade the peritumoral stroma, most importantly matrix-
 metallo-proteinases (MMPs). Furthermore, the immune system 
is involved in the local control of growing cancers. It is esti-
mated that the majority of malignant cell clones that develop in 
the human body are eliminated by immune system components 
directed against the transformed cells, a process called immune-
surveillance. Invasive tumor development needs to evade these 
immune control mechanisms. A number of immune evasion 
strategies, including loss of antigen presentation machinery 
components, induction of suppressive T cells, and induction of 
apoptosis in attacking lymphocytes, have been analyzed.
Cancer-related Genes
Three major groups of genes are involved in carcinogenesis: 
oncogenes, tumor suppressor genes, and genes that are respon-
sible for DNA repair and stability.7 Oncogenes are mostly activa-
tors of the cell cycle that are strictly controlled in non-malignant 
cells. Activation can occur by chromosomal translocations that 
bring an active promoter close to an oncogene that is usually 
not expressed (e.g. BCR-ABL translocation in leukemia). Gene 
amplifications frequently lead to overexpression of oncogenes, 
as it is the case for the MYC gene. In addition, point mutations 
can lead to continuous activation of oncogenes, e.g. activating 
BRAF or RET mutations. In general, mono-allelic activation of 
oncogenes is sufficient for malignant transformation. In con-
trast, tumor suppressor genes (TSGs) usually require two hits, 
since the unaffected allele can substitute in part for the mutated 
allele. Still, in some cases, a partial effect (haploinsufficiency) 
conferred by the loss of one allele has been described. TSGs can 
be altered by point mutations or by larger chromosomal losses. 
Typically genes involved in cell cycle control, regulation of pre-
programmed cellular suicide (apoptosis), or the maintenance 
of genomic integrity may serve as TSGs. Repair/stability genes 
comprise a specific subgroup of TSGs in that they maintain the 
genetic integrity of the cell. Their loss of function is a prerequi-
site for the rapid acquisition of the critical mutations required 
for neoplastic transformation. To the latter group belong among 
others the mismatch repair genes (MMR), nucleotide excision 
repair genes, base excision repair genes, and genes involved in 
chromosomal recombination and segregation, such as BRCA1 
and ATM. Germ line mutations of many of the TSGs have 
been identified as the cause of inherited cancer predisposition 
genes in familial cancer syndromes (e.g. hereditarynonpoly-
posis colon cancer (HNPCC), familiar adenomatous polyposis 
(FAP), multiple endocrine neoplasia II (MENII), BRCA1 and 2 
for familial forms of breast cancer, and p53 for the Li-Fraumeni 
syndrome). In hereditary cancer syndromes, a tumor suppressor 
gene is inactivated in the germ line and a second hit is necessary 
to abrogate the function of the respective tumor suppressor gene 
in individual somatic cells (two-hit-hypothesis). Frequently, the 
initial gene alteration induces uncontrolled proliferation of the 
affected cells. In the course of accelerated cell divisions, genetic 
errors are accumulated and finally lead to malignant transfor-
mation of the cells. For many cancer entities, specific pathways 
of consecutive gene alterations have been described. Two cen-
tral tumor suppressor genes are affected in many cancers: p53 
is a central protein in the control of programmed cell death. 
1�
General CytologyPART ONE
if central pathways that maintain the genetic integrity of a M phase of the cell cycle the chromosomes line up in a plane, 
cell are hit. If such cells succeed to survive, they may rapidly 
accumulate a sufficient number of mutations that permit the 
neoplastic growth properties. Thus, carcinogenesis can for-
mally be subdivided into three major pathways depending 
on the molecular mechanism that makes it possible for a 
sufficient number of mutations or other genetic alterations 
of oncogenes and tumor suppressor genes that initiate and 
maintain the neoplastic conversion and progression to be 
accumulated.
The Major Pathways of Carcinogenesis
Alterations of mechanisms that maintain the genetic integrity of 
the cell’s genome thus constitute the least common denomina-
tor. Consequently, neoplasia emerges as the failure of genetic 
functions that control either the composition of whole chromo-
somes, their number, structure, and distribution during mitosis 
or, alternatively, the integrity of the genetic information encom-
passed in the chromosome even if they do not undergo gross 
numerical or structural alterations. Consequently, cancer is the 
result of three major mechanisms that destroy the integrity of 
the genetic information:
A B
1�
the metaphase plate, and associate with spindle fibers of micro-
tubule proteins. The fibers together form a metaphase spindle. 
They are connected to the kinetochores on the chromosomes, 
i.e. nucleoprotein bodies associated with the centromeric 
DNA of the chromosomes, and the centrosomes at the poles 
of the mitotic cell. In a normal dividing cell the spindle fibers 
pull each sister chromatid apart toward the centrosomes. This 
ensures that each emerging daughter cell will get exactly one 
copy of the sister chromatids to form exactly one new copy of 
the respective chromosomes in the emerging new daughter cells. 
This complex mechanism is controlled by various checkpoints 
that monitor that before the process proceeds to the next step 
exactly two centrosomes and microtubules spindle apparatus 
have been formed, each chromatid in a pair associates with its 
own distinct half of the spindle. Chromatid separation is not 
allowed to proceed until all pairs of chromatids are lined up in 
the metaphase plate. If these checkpoint controls fail, chromo-
somal segregation may fail and both chromatids may be pulled 
to one chromosome (non-disjunction). As consequence one of 
the daughter cells may become haploid for the respective chro-
mosome and the other triploid. Alternatively, one chromosome 
is completely lost, if the attachment between microtubules and 
kinetochores fails.
Fig. 1.10 Influence of centrosome aberrations 
on chromosomal instability. (a) Normal 
centrosomal distribution with two spindle poles 
results in equal distribution of the chromosomal 
material to the daughter cells. (B) aberrant spindle 
pole formation leads to unequal distribution of the 
chromosomal material; as a result, most cells die 
while some can acquire genetic alterations that 
lead to a growth advantage and the development 
of malignant cell clones.
Inactivation of p53 seems to be very important for malignant 
progression to be possible. pRb is a key regulator of cell cycle 
progression by controlling the E2F protein. The majority of 
 cancers show inactivation of these TGSs themselves. However, 
specific cellular functions can be abrogated by attacking dif-
ferent components of the respective functional pathway. Thus 
functional pathways that represent a complex network of differ-
ent gene functions may be hit on various levels in order to pro-
mote tumor development. Many different functional pathways 
that explain the heterogeneity of the genes affected in sporadic 
cancers have been described.7
The accumulation of gene mutations necessary for 
malignant progression cannot be achieved at the standard 
mutation rates (1 mutation/million bases) observed in pro-
liferating cells. It seems clear now that this baseline mutation 
rate is not sufficient for carcinogenesis and that some kind of 
genetic instability must occur in order to allow for the nec-
essary mutations in cancer cells. This can only be achieved 
 1. Chromosomal instability (CI)—Induced by failure of 
mechanisms that guarantee the even distribution 
of chromosomes to the daughter cells that emerge 
during mitosis;
 2. Microsatellite instability (MSI)—Induced by failure of 
DNA mismatch repair enzymes that proofread and 
repair errors that occur during the de novo synthesis 
of DNA in the S-phase of the cell cycle; and finally
 3. CpG island methylator phenotype (CIMP)—Induced by 
failure of the epigenetic control of genes that regulate 
critical steps in these processes and is often associated 
with the later development of MSI-induced cancers.
The vast majority of cancers occur via the CIN pathway. The 
major underlying mechanisms of carcinogenesis mediated 
by CIN is induced by disturbances of the bipolar character of 
the mitotic spindle during mitosis and the desegregation of 
chromosomes during mitosis (Fig. 1.10).8 During the normal 
The Cell: Basic Structure and Function
1
ular mechanisms for evading this cellular suicide mechanism. 
In the case of papillomavirus-associated cancers it is the HPV 
E6 protein that binds to and inactivates p53. In other cancers 
not induced by oncogenic HPVs, p53 functions are usually abro-
gated by an inactivating mutation or deletion of the p53 gene 
itself or related genes within the same functional pathway.
Cells that achieve to evade the suicide control may survive the 
genomic catastrophe and form the initial cells of an outgrowing 
cancer. The emerging disproportionate distribution of chromo-
somal material in these transformed cell clones induce various 
important morphological alterations of the affected cells’ nuclei 
that are the cornerstones of cytopathology (Fig. 1.11). Aneuploid 
nuclei, coarser texture of the chromatin, changes in the size and 
shape of the nuclei, hyper- and hypochromasia, and altered 
shape and number of nucleoli are all immanent consequences 
of the desegregation of chromosomes during mitosis of cells that 
A
C D
B
proofreads and fixes the mutations. If distinct components of 
this repair complex are lost, the proofreading capacity decreases 
and mutations particularly in thermodynamically sensitive 
DNA sequences occur. These lead to the rapid accumulation of 
mutations particularly in repetitive DNA sequences that con-
sist of longer stretches of mononucleotides (mononucleotide 
repeats). Since these sequences are also commonly referred to 
as microsatellites, this latter mechanism of carcinogenesis is 
referred to as microsatellite instability. MSI is observed in up 
to 15% of colorectal cancers, a subset of endometrial cancers, 
and a number of urinary tract cancers. But it is also found in 
a number of endometrial and bladdercancers, leukemias and 
lymphomas, and skin cancers. It is the hallmark of an inher-
ited cancer predisposition syndrome referred to as hereditary 
non-polyposis colon cancer syndrome. HNPCC syndrome is 
characterized by inherited mutations of defect copies of genes 
Fig. 1.11 Comparative genomic 
hybridization (CGH) and spectral karyotype 
hybridization (SKY). the average CGh ratio 
profiles for the diploid cell line DLD-1 and the 
aneuploid cell line ht29 are presented in (a) 
and (B). Note the remarkably stable genome of 
DLD-1 with only three copy number variations 
(chromosomes 2, 6, and 11). ht29 shows a 
highly aberrant ratio profile, with copy number 
alterations occurring on 13 chromosomes. 
the gains of 7, 8q, 13, and 20q are common 
aberrations in colorectal carcinomas. SKY of 
metaphase chromosomes prepared from these 
cell lines is shown in (C) and (D). No numerical 
aberrations were identified in the diploid cell 
line (C), whereas trisomies were common in 
the aneuploid cell line ht29 (D). all aberrations 
detected by SKY were also seen by CGh analysis. 
this indicates that no reciprocal, balanced 
chromosomal translocations have occurred. Data 
and images were taken from Ghadimi et al.11
More severe, however, is the impact of failing checkpoints 
that control the centrosomes themselves. Cancer cells that 
arise through the CIN pathway are characterized by an uneven 
number and uneven distribution of centrosomes during mitosis. 
This results in a total disorder of the normally bipolar spindle 
apparatus and leads to complex multipolar spindle structures 
that during mitosis result in disruption of the chromatids and 
a complex uneven distribution of the chromosomal material to 
the emerging daughter cells (Fig. 1.10).
The major checkpoints that control these processes appear 
to act at the transition of the G1 phase of the cell cycle to the 
S phase and are controlled by cyclins and cyclin-dependent 
kinase complexes. Interestingly, these processes are targeted by 
two important viral oncoproteins encoded by high-risk human 
papillomaviruses (HPV), the HPV E6 and E7 proteins, that 
induce and maintain transformation of cervical cells as we will 
learn later in this chapter. This results in failing control of the 
mitotic processes and in particular distribution of chromosomes 
during mitosis and severe numerical and structural alterations 
of the chromosomes of the emerging daughter cells. The affected 
cells usually undergo apoptosis, induced primarily by p53 gene 
or related genes. Thus most emerging cancer cells develop molec-
have lost control over the strictly bipolar mitotic figures, result-
ing in chaotic multipolar mitotic spindle complexes (Fig. 1.10).
Alternatively, cancer cells may arise through the MSI path-
way.9,10 Cancers that emerge through this pathway are char-
acterized by substantially different biological properties. They 
usually do not display numerical or structural changes of the 
chromosomes or the mitotic figures.11 Cells of these tumors 
 usually divide normally. Consequently, these tumor cells do not 
display aneuploidy, aberrant mitotic figures or gross alterations 
of their chromosomes. They usually remain diploid without 
major morphological alteration of their nuclei. However, errors 
emerge through a more subtle, superficially less brutal mecha-
nism that in its clinical consequences may end as disastrously 
as the chromosomal instable cancers. In these cases the cancer 
cells acquire increasing mutations of the DNA sequence itself. 
After each round of DNA replication in the S-phase of the cell 
cycle usually hundreds and thousands of mutations, which 
need to be checked and repaired before the cell cycle proceeds 
to avoid very high accumulation, occur in the replicated genetic 
code due to misannealing and mispairing. Hence, all cells in 
nature develop a sophisticated proofreading mechanism medi-
ated by the DNA-MMR complex, a multiprotein complex that 
1�
1�
General CytologyPART ONE
that encode components of the DNA-MMR complex. MSI may, 
however, also occur in sporadic cancers without distinct inher-
ited background. In most of these cases accidental failure of the 
epigenetic regulation of the genes that encode the components 
of the MMR complex may fail usually due to altered methyla-
tion of the respective promoter sequences.
The third emerging major mechanism of carcinogenesis is 
referred to as CpG island methylator phenotype.12 Cancer cells that 
emerge in frame of this molecular phenotype initially experi-
ence alterations of the epigenetic control mechanisms that tell 
genes of specific cells where and when they should be active 
or silent. In many instances this is regulated by methylation of 
specific sites within the DNA by the addition of methyl groups 
particularly to CpG sites frequently found throughout the whole 
genome. The addition of these methyl groups either completely 
blocks the expression of the respective gene by condensing it 
chromatin structure or may just modify the binding proper-
ties of activating or inhibitory factors that activate or repress 
 transcription of the respective gene. The CIMP phenotype is 
clearly the less well-characterized cancer phenotype so far. 
Because of the basic mechanism it may end up finally in cancer 
cells that impress as CIN phenotype due to the transcriptional 
inactivation of genes involved in chromosomal homeostasis or 
alternatively as MSI cancers due to the inactivation of genes that 
maintain the DNA-MMR functions; here the most pronounced 
example is the hMLH1 gene frequently inactivated via the CIMP 
phenotype in MSI-positive sporadic colorectal cancers.
Carcinogenesis Induced by 
Papillomavirus Infections
Human papillomavirus infections play the predominant carci-
nogenic role for cancers of the lower female genital tract, and 
in particular cervical cancer.13 Molecular pathways of how these 
viruses contribute to neoplastic transformation of epithelial cells 
have to a large extent been clarified and thus will be considered 
in more detail in the following paragraphs.
Published reports on the concept that infectious agents may 
be involved in the pathogenesis of cancers of the female lower 
genital tract dates back to the middle of the nineteenth century. 
Domenico Rigoni Stern described his observation that women 
with frequent sexual contacts with various partners are at sub-
stantially higher risk to develop cervical cancer than women 
who did not have sexual contacts. Since then sexually transmit-
table agents that may explain this peculiar epidemiological fea-
ture of these cancers have been extensively investigated. It lasted 
until the end of the 1970s when the first truly important clues 
that made it possible to delineate the formal molecular patho-
genesis of cervical cancers were put forward. In 1976 Harald zur 
Hausen published his hypothesis that cervical cancer and its 
precursor lesions may be caused by agents similar to those that 
cause hyperproliferative lesions in the genital tract, the condy-
lomata acuminata or genital warts.14 This hypothesis initiated 
an intense chase to track down the putative agents. Genomes 
and viral particles of a new type of human papillomavirus were 
identified in biopsies of genital warts and labeled as HPV 6.15 
Shortly later a related HPV type was cloned from DNA samples 
isolated from laryngeal papillomas and referred to as HPV 11.16 
This HPV type showed substantial homologies with HPV 6 and 
in subsequent studies both HPV types were found in laryngeal 
as well as in genital papillomas.17
Both HPV types were used as probes to look for related DNA 
sequences in DNA extracted from tumor biopsies and cell lines 
derived from cervical cancers in further hybridization experi-
ments. These experiments led to the identification of related 
but clearly distinct HPVsequences in cervical cancer cells. Sub-
sequent cloning and detailed characterization has revealed that 
these sequences are indeed new types of the HPV group that 
have since then been referred to as HPV 16 and 18.18, 19
Basic Structure of the Virus and Its Genome
Human papillomaviruses are small viral particles that constitute 
viral capsids built up by self-assembling proteins encoded by the 
L1 and L2 genes of the virus (Fig. 1.12). They lack an envelope 
and are thus relatively resistant toward environmental hazards. 
Viral capsids measure about 55 nm in diameter and include an 
about 8,000-bp-long circular episomal genome that is highly 
twisted (supercoiled circular genomes). This circular genome 
encompasses three major genetic and functional regions:
 • Early region E, which includes about eight different 
genes (E1–E8);
 • Late region L, which includes the two genes that encode 
the proteins that make up the capsids (L1 and L2); and
 • Upstream regulatory regions of the early region (URR), 
which includes the important regulatory sequences 
of the early promoter and enhancer that mediates the 
highly complex regulation of the viral gene expression 
pattern in their host cells that is so important for all 
processes related to HPV-related carcinogenesis and 
that we will discuss later.
A second regulatory element that regulates the expression of 
the late genes is included in sequences that are part of the E7 
gene (late promoter).
Papillomavirus types are differentiated in HPV genotypes 
based on distinct variations of their nucleic acid sequences. 
A certain stretch within the L1 gene is used as a reference to 
 differentiate types according to an international agreement on 
the classification of HPV types.20
Meanwhile more than 120 different HPV types have been 
characterized, but it is estimated that the true number of pap-
illomaviruses that may infect humans exceed 200. Papilloma-
viruses are strict epitheliotropic viruses that exclusively infect 
epithelial cells of the outer surfaces of the human body. Most 
of the HPV types infect the cutaneous parts of the skin (skin 
types), whereas about 40 HPV types are preferentially found 
in lesions in the mucosal surfaces in the lower anogenital 
tract (mucosa types).21 Among the mucosa types two different 
classes of HPV types are distinguished (Table 1.1): (a) the low-
risk HPV (LR-HPV) types that cause massive exophytic, hyper-
plastic wart-like lesions, and (b) the high-risk HPV (HR-HPV) 
types associated with cancer particularly of the uterine cervix 
(Fig. 1.13). The latter, however, usually cause only very minor 
lesions without massive hyperplasia. These lesions rarely exceed 
the surface of the epithelium in the early stages of the infection. 
They commonly occur and regress without the infected person 
realizing the infection. Thus although these infections occur in 
men and women apparently with the same incidence, clinical 
consequences that would trigger follow-up usually only occur 
in women who have developed cervical lesions as part of a car-
cinogenic process, whereas in men the infections usually occur 
and regress unnoticed.
The Cell: Basic Structure and Function
1
Papillomaviruses infect the basal cells of the epithelium via 
binding to certain cell-surface glycosaminoglycans expressed 
on the basal cells of the epithelium (Fig. 1.14). Once they have 
entered the cell, the viral capsids are broken down and the 
 episomal viral genome is released in the nucleus. Viral early 
genes are expressed at this stage on a very low and highly con-
trolled level that allows for low copy replication of the genomes 
on the order of 10 to 50 genomes per infected basal cell; how-
ever, massive amplification of the viral genome or even repli-
cation of the virus does not occur at this stage of infection in 
basal cells. Only in certain instances not yet characterized in 
detail does high-level gene expression of the viral early genes 
occur in differentiated cells of the intermediate layers of the epi-
thelium. This triggers amplification of the viral genome. Once 
the cells have reached the superficial cell layers the early genes 
are shut off and high-level expression of the late genes (L1 and 
L2) occurs (early–late switch). This results in expression of the 
HPV16
7000 1000
E6 E7
E1
E4
E2
E5
L1
L2
6000 2030
5000
4000
9004m
3000
ori
Regulation of
virus gene expression
and virus replication
Disruption of 
cell growth cycle
p53
Telomerase pRb
URR
Capsid
proteins
Assembly
and release
Gene
expression
DNA
replication
Membrane
signaling protein
Fig. 1.12 Human papillomaviruses. top: electron microscopy pictures of 
viral particles and viral DNa. Bottom: Schematic diagram of the viral genome 
indicating the most viral genes and most important functions.
respective proteins, packaging, and self-assembly of new mature 
viral particles that are finally released from the cells once dur-
ing the normal differentiation process the infected and virus-
 producing keratinocytes decay into keratin fragments. The 
replication strategy of the human papillomaviruses is therefore 
tightly linked to natural differentiation processes of their host 
cells. For the virus this has two advantages.
First, the biology of the primary host cells at the bottom of 
the epithelium that retain the capacity to multiply and gener-
ate new cells is only marginally altered by the infection. HPV 
infections induce no cytolysis, no inflammation, or other tissue 
damage in the basal cell compartment of the epithelium. Acute 
LR-HPVs induce more proliferation of the infected basal cells 
and cause exophytic lesions that may clinically impress as warts 
or condylomata seen with comparable incidence in men and 
women. Proliferation of basal cells is induced by acute HR-HPV 
types to much less extent since a simple infection almost never 
causes exophytic lesions. Usually these infections regress spon-
taneously and clinically unrecognized.
Secondly, due to the fact that the virus is only produced on 
the very superficial surfaces of the infected epithelium there is 
very limited contact between viral antigens and the immune sys-
tem of the infected host. Thus the acute infection induces very 
little humoral immune responses and serum antibodies are only 
observed in low titers in some of the infected individuals. They 
do not induce a protective humoral immunity. Cellular immune 
responses are only weakly activated, which usually takes a long 
time during which the virus can multiply and spread until reli-
able cytotoxic immune functions have been activated to defeat 
the virus and the lesions it has caused.
The tight association of the replication strategy of the papillo-
maviruses with the differentiation status of their host cells further 
allows the virus to multiply and spread with a highly restricted 
amount of their own genetic information. Complex genetic fea-
tures that control the restricted expression pattern of the virus dur-
ing the different differentiation stages are contributed apparently 
by the host cell and not by the virus; thus the virus has no need to 
retain genes that may mediate these functions by themselves.
Epidemiology of HPV Infections
HR- and LR-HPV infections are usually acquired in the early 
years upon uptake of sexual activity. Most studies have been 
 performed in women, but it can be extrapolated that the 
Phylogenetic 
 classification
 
Epidemiologic classification
High-risk HPV types Low-risk HPV types
high-risk hpV 
types
16, 18, 31, 33, 35, 39, 45, 
51, 52, 56, 58, 68, 82, 26*, 
53*, 66*
70
Low-risk hpV 
types
73 6, 11, 40, 42, 43, 44, 61, 
72, 81, Cp6108
*putative high risk types.
the epidemiological classification of these types as probable high risk is based onzero controls and one to three positive cases.21
 Table 1.1 Correlation between phylogenetic and epidemiologic 
classification of mucosal hpV types
1�
General CytologyPART ONE
The HPV types 16 and 18 are found in about 70% of all 
cervical carcinomas, whereas among healthy women the 
remaining HR-HPV types are more common. This observa-
tion suggests that women infected with the two types, HPV 
16 and 18, may have a higher risk to developing high-grade 
precursor lesions or even invasive carcinomas than women 
who are infected with the other HR-HPV types.23 This notion 
is strongly supported by a variety of studies that indicate 
that women infected with HPV 16 and 18 are more likely to 
develop high-grade lesions (cervical intraepithelial neoplasia 
(CIN) 3) in less time than women infected with other HR-
HPV types. Moreover, the time of progression of high-grade 
lesions to invasive carcinomas appears also to depend on 
the HPV type that causes the high-grade lesions.23a
HR-HPV infections usually last for several months and in 
most instances regress spontaneously without causing relevant 
clinical lesions.24 The acute HPV infection may impress as CIN 
1 lesions in histological sections in that the typical koilocytes 
indicate the acute replication of the virus in intermediate and 
superficial cells of the infected epithelium. These acute infec-
tions strictly adhere to the regulatory gene expression pat-
tern outlined earlier in that the cellular differentiation stage 
determines the expression of the viral genes in the epithelium. 
 1. In more than 99% of cervical cancers genomes of 
HR-HPV types can be found;
 2. Two viral genes, E6 and 7, are in all cervical cancer 
preserved and expressed;
 3. The E6 and E7 genes can transform primary epithelial 
cells into neoplastic cell clones; and
 4. If the expression of these genes is inhibited cervical 
cancer cells stop to proliferate and lose the neoplastic 
growth properties.
These facts clearly underline the central role of the viral 
E6 and E7 genes for the HR-HPV mediated transformation 
 processes.
During the past 20 years basic research on the biochemical 
activities of both viral proteins have revealed three major aspects 
that underline their oncogenic activities: the E6 protein of the 
oncogenic HPV types induces premature degradation of the p53 
tumor suppressor gene and thus interfere with its proapoptotic 
functions25; the E7 protein induces destabilization of the retino-
blastoma protein complex and thus allows the cell to evade cell 
cycle control at the G1/S phase transition thorough the pRB 
pathway26; and both genes interfere with centrosome synthesis 
and function that results in desegregation of the chromosomes 
during mitosis and numerical and structural chromosomal 
1�
LR-HPV
HR-HPV
Infection First lesion
Incubation
1 to 6 months
Active growth
3 to 6 months
Immune
response
>9
<1
Fig. 1.13 Different types of HPV infection. (1) a state of very low viral activit
(“latent phase”). (2) the replicative infection characterized by strong viral gene e
layers. (3) the transforming infection characterized by deregulated oncogene ex
neoplasia, grade 1–3.
 infection pattern in men is not substantially different from that 
in young women. The highest infection rates are seen in young 
women at 18 to 25 years of age.22 Over time the incidence of 
HPV infections decreases, but there seems to be a second peak 
of HPV infections in older women of more than 45 years of age. 
The infection clearly shows a typical sexually transmitted pat-
tern and depends on personal lifestyle habits, for example, the 
number of sexual partners, and the frequency of sexual contact 
with partners who have themselves sexual contact with other 
partners. Several studies have shown that the cross-sectional 
incidence of HR-HPV infections ranges between 3 and 25% 
of the female population. The cumulative infection in average 
women is calculated to be more than 50% once in their lifetime. 
Thus a tremendous amount of women (and hence also men) are 
infected with these agents.
 
0%
Complete
regression
Persisting
lesion
0%
y shortly after initial infection through microlesions of the epithelium 
xpression and viral capsid formation in the upper differentiated epithelial 
pression in the replication competent basal cells. CIN, cervical intraepithelial 
According to this, limited and highly regulated gene expression 
is found in basal and parabasal cells of the epithelium. By this 
replication strategy, HPVs successfully avoid expression of 
viral genes in proliferation competent epithelial cells that may 
become transformed stem cells for a cancer. Under “normal” 
conditions viral genes are only expressed in cells that are irre-
versibly cell cycle arrested. HPVs thereby can multiply almost 
unnoticed by the host and spread to many other individuals 
(Fig. 1.14).
The Role of the HR-HPV E� and E� Genes
The causal relationship of HR-HPV types and cervical cancer is 
based on four major experimental observations:
The Cell: Basic Structure and Function
1
 layers document the massive production and release of HPV. itself. In normal cells this would have been counteracted by the 
1�
In CIN 2 lesions the proliferating cells extend to the two lower 
thirds of the epithelium. In these lesions the number of koilo-
cytes decreases gradually. CIN 3 lesions are characterized by 
proliferating cells that extend now into the upper third of the 
epithelium or in case of the carcinomata in situ lesions (CIS) 
extend through the full thickness of the epithelium. These diag-
nostic categories are very useful in clinical practice since they 
make it possible to subdivide precancerous stages according to 
their likelihood to progress to invasive cancer. However, since 
they do not make it possible to visualize the molecular events 
induced by the deregulated expression of the viral oncogenes 
in these cells, they do not formally make it possible to subdi-
vide the preneoplastic lesions according to the molecular events 
involved in the carcinogenic processes.
As discussed above, the expression of HPVs is tightly regu-
lated in basal and parabasal cells of the epithelium. Thus, the 
initiation of the carcinogenic process is not the infection of basal 
epithelial cells by HR-HPVs but rather the emergence of epithe-
lial cell clones that fail to downregulate the expression of the 
activation primarily of p53 mediated control mechanisms that 
either would have resulted in stoppage of cell cycle progression 
or, if the genomic damage is already too severe, induction of 
the cellular suicide mechanisms (apoptosis) that prevent sur-
vival and expansion of cells with damaged genomes. During 
the normal life cycle this never occurs since the expression of 
the viral early genes is restricted to terminal differentiated cells 
of the intermediate or superficial cell layers (Fig. 1.14).
Cells that display chromosomal instability under the influ-
ence of the papillomavirus genes E6 and E7 rapidly accumulate 
numerous morphological alterations. First aberrant mitotic fig-
ures are seen in dividing cells, then enlarged nuclei with altered 
chromatin structures appear, and finally severe changes of the 
DNA content of the cells results in aneuploidy and anisonu-
cleosis. All these criteria are classical features that build up the 
 cytological classification system to score the degree of abnor-
malities induced by HPV infections in cervical cells.
One important aspect in the pathogenesis of HPV-induced 
cancers in the female anogenital tract is its typical anatomical 
aberrations.27 As a result of chromosomal instability induced by 
E6 and E7, HPV genomes may become integrated into the host 
genome.28 Both viral oncoproteins interact with many more 
 proteins of epithelial