Download General Definitions and Basic Concepts Describing Cancer

General Definitions and Basic Concepts Describing Cancer
There are also some terms describing phenomena and processes that can be defined at this point
to avoid later confusion. Throughout the semester we will use the terms “transformed” and
“malignant”, to describe the end point of genotoxic damage so it will be very helpful to define
exactly what they mean, as a way of correlating experimental results in vitro and in vivo. Also
important is to have some definition for cancer, which is not always obvious. To begin with, all
of these terms are applicable to eukaryotic cells only- eukaryotic = cells of higher organisms,
distinguished by existence of nuclei and organelles separated from the cytoplasm by trilaminar
membranes.
The term transformation defines a set of characteristics acquired by cells in vitro, and the
overhead is a list of some characteristics that by general agreement are associated with
transformed cells. It is important to keep in mind that no one characteristic is sufficient to define
a cell as transformed and also that not all characteristics need to be present for a cell to be
considered as transformed.
(1) Common to all transformed cells is the characteristic of immortality. Normal cells cannot be
maintained indefinitely in culture, but die after a number of passages; however, transformed cells
considered to be immortal because they can be maintained indefinitely in culture as long as a
hospitable environment is maintained. We will discuss immortality again in describing the role
that oncogenes play in cell transformation. It is important to point out that immortalization, in
and of itself, does not constitute transformation. For example established cell lines, which are
derived from cells that have survived the crisis occurring after primary cells are passaged
numerous times, have acquired the trait of immortality, but do not have the additional
characteristics by which transformed cells are defined. Nevertheless, both transformed and
established cell lines share the property that neither is truly diploid, which means that they do not
have a normal complement of chromosomes.
(2) Transformed cells grow in an unrestricted manner. Unlike normal cells, growth is not orderly
and does not stop when cells reach confluence, which exists at the point when a mono-layer
covers the surface in which cells are touching but not overlapping. Growth continues beyond this
point with the creation of disorganized masses called "foci". This behavior indicates that the
transformed cells are no longer subject to "contact inhibition" or density dependent regulation.
Signals normally halting growth at confluence are either absent or disregarded.
(3) Transformed cells no longer require a firm surface to which to attach in order to grow. This
change is defined as "loss of anchorage dependence".
(4) The requirement for growth factor-containing serum to sustain growth is reduced or absent in
transformed cells.
(5) Characteristic cytoskeletal changes appear. Normal cells have distinctive shapes: for
example, hepatocytes have a typical hexagonal shape. Transformed cells, instead of lying flat
and extended on a growth surface, appear rounded as cells in mitosis. The morphological
similarity to mitotic cells is directly correlated with the absence or diminished concentration of
certain plasma membrane proteins (known as microfilaments) and the consequent increased
fluidity of the plasma membrane. This observation is an example of the incomplete
understanding of carcinogenesis is. There is a biochemical explanation for this morphological
change, but the cause of the biochemical change and its significance with respect to the overall
pathway of transformation is not yet apparent.
(6) Accompanying the change in morphology is a loss of cell function referred to as
dedifferentiation. Normal cells have very specific functions. Hepatocytes, as you learned, are
rich in mixed function oxidases and are involved primarily in metabolism (which of course
includes the metabolic activation of carcinogens). Transformed hepatocytes may lose the ability
to metabolize; another example is epithelial cells, which are normally programmed to keratinize,
will not follow the normal programmed cell cycle when transformed.
(7) Transformed cells often yield tumors when inoculated into a syngenetic host -syngenetic
meaning the same species from which the transformed cell line was derived. It turns out that this
characteristic can be problematic, because contrary to what may intuitively make sense, cells
from transformed foci do not necessarily become tumorigenic when inoculated into a host. Even
in a situation where cells transformed by known carcinogenic chemicals are tested in this way,
only ~83% of hosts will develop tumors. This observation serves as a generally applicable
warning that there is not a 1: 1 correspondence between experimental results in vitro and
behavior in vivo.
Begin 01/15/09
We will take, as an operational definition of cancer, that it is an in vivo process related to cell
transformation. In fact, one source of transformed cell lines is to culture tumor cells. A tumor
might be considered as an in vivo analogue of a focus, and the other characteristics of
transformed cells apply to cancer cells as well. Thus, tumor formation is related to loss of
density-dependent growth regulation, while loss of anchorage dependence is the characteristic
conferring metastatic properties, which means the ability of tumors to spread beyond the site of
origin. Metastasis also involves acquisition of capabilities not described in defining the in vitro
process of transformation. Important examples are: (1) the ability to penetrate blood vessel walls
by special proteins (called matrix metalloproteinases) and (2) the ability to develop vascular
network for blood supply to tumors (called angiogenesis). About 10 years ago there was a
tremendous amount of excitement surrounding the clinical trials of the angiogenesis inhibitor
proteins angiostatin and endostatin. These are naturally occurring proteins that inhibit
angiogenesis, and the in mice, the murine proteins essentially cured solid tumors. Unfortunately,
the human analogues exerted only small and temporary effects.
An important characteristic common to transformed cells and tumor cells is that the changes just
described are heritable. By implication, this involves alteration of genetic information; i.e.,
changes in DNA. Hence, DNA must be a target for oncogenic agents, including chemicals, and
therefore DNA is a major focus for studies of carcinogenesis on a molecular level, including
chemical carcinogenesis. Very recently, epigenetic processes, particularly the switching on or off
of critical genes by environmentally induced changes in methylation state of 5′-GpC-3′ doublets
have become a focus of attention. This area of research is new and rapidly evolving, but will not
be a focus of this course, as it doesn’t directly involve exposure to chemicals.
Before proceeding further, it is important to point out that discussion of chemical carcinogenesis,
like any other area of molecular biology is replete with its own specialized vocabulary. While the
introduction of terminology may at first not seem to be overwhelming, it accumulates rapidly,
and it is wise to be conscientious in learning definitions and keeping them straight. (An example
is the nucleobase, nucleoside, and nucleotide series from ENVR 430.)
OVERVIEW OF CHEMISTRY
I will quickly review the chemistry which I think that you should be familiar with to feel at ease
with the course material.
The presence of carbon defines organic molecules. Carbon requires 4 electrons to fill the K shell,
and does this by forming bonds with 2 – 4 other atoms. When bonded to 4 atoms, carbon is
tetravalent, and the bonds are oriented towards the vertices of a tetrahedron. In an undistorted
structure, the bonds make internal angles of 109 o. This angle is important to us because it
determines 3-dimensional structure, which is critical in determining function of biomolecules.
Furthermore, any distortion from optimum geometry introduces strain which destabilizes
structure. Another property of tetrahedral carbon is that when molecular groups at the apices are
all different, as indicated in the overhead, property of chirality is introduced. Reflection of the
tetrahedron in a mirror gives an image that cannot be superimposed on the original model. The
images are called enantiomers. Molecules with chiral centers rotate plane-polarized light and
therefore have optical activity. The importance of chirality will become evident when we discuss
metabolism, because metabolizing enzymes have chiral active sites and therefore selectively
generate one out of several possible enantiomers. Since macromolecular targets of activated
metabolites, including DNA are also chiral, chirality plays a crucial role in determining reaction
pathways and hence in biological activity. When carbon is bonded to three atoms – tri-valent
carbon – it must share two electrons with one substituent in a “double” bond to satisfy valence
requirements. The overhead also shows the geometry of trivalent carbon, with bonds distributed
in a plane at angles of 120o. Sharing two electrons in the double bond is accomplished by
forming a σ bond and a π bond to one of the atoms. Because of the π bond, the geometry around
the double bond is fixed, since rotation around the bond axis would require breaking the π bond.
The resulting geometric relationship between the substituents of the π-bonded atoms defined as
cis when they are on the same side of the perpendicular to the plane through the bonded atoms
and trans when they are on opposite sides.
Approximate bond energies, i.e., the energy in Kcal /mole required to break a bond, or
conversely the energy released in bond formation is:
83 Kcal/mole for C-C σ bonds
150 Kcal/mole for C=C double bonds
Bond energies are of interest to us because energies required to assemble molecules provide a
measure of relative stability of starting compounds and products and for reversible reactions, will
determine the direction in which reactions will proceed spontaneously.
In addition to valence and geometry, the idea of functional groups will be important from the
standpoint of our discussions. Functional groups are arrangements of atoms that are not complete
molecules, but occur frequently and are usually associated with specific properties when they are
incorporated into molecules. Functional groups that we most commonly encounter are shown in
the next overhead.
The interaction of biomolecules with their environment will also be an important for us, and will
depend on the physico-chemical properties of the molecules. Chemical compounds may be
divided into two classes based on physico-chemical properties: polar and non-polar. Polar
molecules have higher solubility than non-polar molecules in water because the dipoles are
attracted to and fit into the lattice created by polar water molecules, illustrated on the next
overhead. Although water is not solid at ambient temperatures, this slide shows that it is
nevertheless highly ordered.
Polarity can be actual separation of charge, as in the case of salts, such as Na+Cl- or the
zwitterionic form if amino acids, or may be the result of unequal sharing of electrons in covalent
bonds between atoms of different electornegativities. Generally, compounds in which carbon is
bonded to the electronegative atoms O, N and S are polar. Polar molecules can be accommodated
efficiently into the water lattice and therefore tend to be water-soluble.
Non-polar molecules have structures in which there is little or no charge separation. Non-polar
molecules are not soluble in water because they are excluded from the water lattice by lack of
interaction with water dipoles. However, non-polar molecules are soluble in non-polar media,
such as oils and fats. Compounds containing only C, H are non-polar, but lack of polarity may
also be determined by symmetry of distribution of polar bonds. The overhead shows CCl4 as an
example.
In addition to electrostatic attractions, polar compounds may enhance interactions with water or
other polar molecules by hydrogen bonding, which is very important in biochemistry. When H is
bonded to an electronegative atom such as N or O, it loses most of its valence electron and thus
can partially share an electron pair with an electron donor if one is within a short distance. Hbonds are stabilizing but weak, amounting to ~5 Kcal/mole, compared to 83 Kcal/mole of C-C
bonds. Nevertheless, the energy of stabilization gained from H-bonding often determines 3-D
structure of large, flexible biomolecules in aqueous medium, and so is very important in
structure-function relationships. A critical property of H-bonds is that they are directional –
atoms involved must be collinear for optimum overlap of the atomic orbitals involved in the
sharing. The significance of directionality is that it imposes geometric constraints on H-bond
formation.
Enzymes and other proteins are made up of amino acids, which have the general structure shown
on the right side of the next overhead:
The group R is called a “side chain” and in amino acids of physiological importance, can be any
of 20 groups. The overhead gives the side chains for the 20 “essential” amino acids, along with
their three-letter abbreviations and one-letter codes. Table is a convenient reference, because
single letter code often used to represent amino acid sequences in large proteins, and some of the
letters are not obvious. Starting with Genes VIII, this table has been omitted.
Remembering the description of a chiral molecule, you can see that all amino acids, except for
glycine (R = H) are chiral and naturally occur in the L (=S) configuration. We shall discuss
conventions associated with the terminology of chirality later, so for now accept that L and S
indicate enantiomers (i.e., mirror images). Proteins are formed by linking amino acids together
through condensation of the carboxyl group of one aa with the α amino group on a second aa
through the elimination of water. The bond formed in this manner is a peptide bond, and proteins
are comprised of polymeric structures of 100 - 500 amino acids linked by such peptide bonds.
Proteins are also referred to as polypeptides. Amino acids determine protein conformation in two
ways. Through the primary structure, bends are introduced in the protein backbone at the site of
the cyclic amino acid proline, as shown in the overhead:
Cross link formation through oxidative coupling of sulfhydryl groups of cysteines may result in
juxtaposition of two regions of a protein that are widely separated in the linear representation.
Oxidative coupling can be described by oxidation of the –SH groups by one electron to give a
thiyl radical, followed by coupling with a second thiyl radical to form a disulfide bond. Formally,
H2 is released. Secondary structural features are introduced by non-covalent interactions between
the side chains of the amino acids and between the side chains and the protein environment.
There are three fundamental structures that are recognized: α-helices, β-sheets and spherical
globules, illustrated in the structure of horseradish peroxidase shown as a ribbon diagram on the
next overhead. Cys 11 and Cys 91, separated by 80 aa in the linear sequence are brought into
juxtaposition by the Cys-Cys bond. This structure and the use of conventions such as ribbons and
stick bonds is an example of how crystsal structures available from the Rutgers Protein Data
Bank can be manipulated to illustrate various characteristics.
Structures features may involve repetitive patterns, which may be recognizable in linear
representations of proteins, which is the type of information available from sequencing. Such
repeated patterns are called motifs. One area of bioinformatics currently receiving considerable
attention is to scan sequences of newly isolated protein for recognition of motifs as a means to
relate the new molecule to known proteins and to infer possible function through alignment of
recognizable functional domains. An important point regarding the interactions just described is
that they illustrate why changes in only one amino acid at a critical site can drastically affect the
3-D structure of a protein and can thus change or abolish function. This will be important in
considering the differences between normal gene products and oncogene products when we
discuss the effects of chemically induced mutations of genes.