Download Background information (includes references for the draft literature

Background project information
Mutations in the Tumour Suppressor
Gene p53
Background Information
Cancer and the Cell Cycle
The cell cycle represents the normal progression of cells through the division cycle to produce two
identical daughter cells. It consists of four discrete stages (See Figure 1):
G2/M Phase
Checkpoint
G2
Anaphase
Checkpoint
S
The Cell
Cycle
G1
G1/S Phase
Checkpoint
M
Cytokinesi
s
G0
Figure 1 – The Cell Cycle

G1 (Gap 1) phase occurs just after division. It is where the cell carries out normal metabolism
and begins to grow in size and duplicate its organelles. Some cells arrest in G1 phase, staying
at a stage called G0 phase and ceasing the cycle of cell division.

S (Synthesis) phase represents the time where the genomic DNA is duplicated.

G2 (Gap 2) phase is the time where the cell prepares for division.

M (Mitosis) phase is the time when the replicated chromosomes are divided (Prophase,
Prometaphase, Metaphase, Anaphase and Telophase and the cell splits into two identical
daughter cells (Cytokinesis).
Collectively, G1, S and G2 phases are known as Interphase.
The normal state for most mature and differentiated cells is to remain within G0 phase and to carry
out normal cellular function. When a cell is given the signal to divide, normal function is suspended
and all of the cell’s resources are directed to replication of DNA and duplication of organelles. When
this occurs, the cells cannot carry out their normal role until division is complete. As a result, most
cells do not continuously divide, as this would interfere with normal body function. Therefore in all
cells, progression through the cell cycle is strictly controlled.
Progression through the cell cycle is controlled by a series of checkpoints. If a cell cannot meet the
conditions needed at each checkpoint, the cycle arrests at that point. This prevents cells being
duplicated with significant errors. The major checkpoints occur at the G1/S phase interface (to ensure
that the cell is ready to start DNA duplication), the G2/M phase interface (to ensure that DNA has been
copied without major errors) and at the end of mitosis before the daughter cells divide (to ensure that
chromosome separation has occurred correctly).
If something occurs which interferes with the regulation of the cell cycle, cells may enter into a state
of continuous division. This not only increases the number of cells present, the cells that are formed
cannot carry out their normal function. This hyperproliferation is one of the hallmarks of the range of
conditions called cancer.
The relationship between cancer and cell cycle regulation is complex. On the one hand, cancers may
arise when a breakdown of the regulatory roles of the checkpoints allow cells to enter mitosis
containing significant errors which are passed on to the daughter cells. On the other hand, functional
checkpoints may act to protect cancer cells, delaying division long enough for the repair of errors
which would normally result in the death of the cells. In either case, a deeper understanding of the
mechanisms of checkpoint regulation can help us understand the factors which lead to the
development and progression of cancer, as well as indicating potential targets for chemotherapy.
p53 as a Tumour Suppressor
The progression of cells through the cell cycle is governed by a complex array of interlinked proteins.
Some of these proteins respond to triggers caused by a need for more cells and stimulate cells to
proceed into mitosis. The effects of these proteins are in turn kept in check by other proteins which
prevent the progression into mitosis. Other proteins respond to other stimuli such as damage to the
DNA and prevent progression through the cell cycle until the damage can be repaired. Each of these
pathways interact with others, with many proteins in the chains having multiple effects.
p53 is a protein involved in the regulation of the cell cycle. If the DNA is damaged by radiation (such
as UV radiation from sunlight) or a chemical agent, the protein ATM (Ataxia Telangiectasia Mutated)
is expressed. One of ATM’s roles is to attach phosphate groups to p53, creating an active form which
is protected from degradation by the cell’s normal processes. This allows levels of p53 to accumulate
in the cell. Once the levels of p53 reach a critical level, it binds to the DNA and activates the expression
of the protein p21 which causes cell cycle arrest by inhibiting the activity of CDK-cyclin complexes. This
pauses progression through the cell cycle until the DNA repair mechanisms in the cell can fix the
problem and prevent the cell from entering into mitosis with significant DNA damage or mutations. In
addition, if the damage to DNA is so severe that it cannot be repaired, p53 will bind to the DNA and
activate key genes (eg. PUMA) which trigger apoptosis (programmed cell death). In this way, in the
presence of dangerous damage or mutations to the DNA, p53 acts both as a “brake” on progression
through the cell cycle and cell division, and as a promoter of the removal of cells with damaged DNA
through apoptosis (Figure 2). As a result, p53 plays an important role in preventing the
hyperproliferation which is a hallmark of cancer. It is not surprising therefore that mutations in the
p53 gene which interfere with its normal function are among the most common in cancer and are
associated with a wide range of cancers.
DNA Damage
ATM
ATP
ADP
P
Inactive p53
broken down
P
Phosphorylated
p53 (stable &
active)
P
P
p21
Inhibits
CDK-Cyclin
P
P
PUMA
Inhibits
Bcl2
G1 phase arrest while
DNA is repaired
Apoptosis
Figure 2 – The role of p53 in cell cycle arrest and initiation of apoptosis
Like all proteins, p53 consists of a number of regions or domains, each of which carries out a particular
function (Figure 3).
NLS
AD1
1
4
3
AD
2
6
3
Pr
o
DBD
Olig
9 10
2 2
29
2
32
0
Reg
35
5
39
3
Figure 3 - Domain Structure of p53
Transactivation Domains (AD1 and AD2) – amino acids 1-43 and 44-63 – These parts of the
protein activate the transcription factors which leads to the expression of genes such as p21.
AD2 specifically activates transcription factors which cause the expression of genes related to
apoptosis.
Proline Rich Domain (Pro) – amino acids 64-92 - involved in apoptotic activity
DNA Binding Domain (DBD) – amino acids 102-292 - the part of the protein which binds it to
the DNA strand upstream of the genes which are activated by p53
Nuclear Localisation Sequence (NLS) – a part of the protein which ensures that p53 localises
to the nucleus
Oligomerisation Domain (Olig) – amino acids 320-355 - functional p53 consists of four
identical p53 proteins bound together to form a homotetramer. This part of the protein binds
to other p53 molecules to form the tetramer.
Regulatory Domain (Reg) – amino acids 356-393 – involved in the downregulation of DNA
binding
A mutation is any change to the sequence of nucleotides in a DNA strand. The order of nucleotides in
a gene determines the sequence of amino acids in the protein encoded by that gene, so any change
in the nucleotides may change the amino acids which make up the protein. Since it is the amino acids
which determine the secondary and tertiary structure of the protein, a simple change to one or two
nucleotides in the gene may have significant consequences for the function of that protein in the cell.
Some mutations are silent. For example, the part of the p53 gene which encodes for the 248th amino
acid reads CGG. This places an arginine residue at location 248 in the protein. A change to the third
nucleotide in this sequence will have no consequences on the structure of the protein, as codons CGA,
CGT and CGC all code for arginine as well. However, changing the first or second nucleotide in this
sequence will change the amino acid.
eg.
GGG encodes for the amino acid Glycine (R248G)
TGG encodes for the amino acid Tryptophan (R248W)
CTG encodes for the amino acid Leucine (R248L)
CAG encodes for the amino acid Alanine (R248A)
CCG encodes for the amino acid Proline (R248P)
Note that the convention for describing mutations which result in amino acid substitutions is as
follows :
R248G
Single letter code for
original amino acid
(in this case “R” is for
“Arginine”)
Position in
peptide chain
(ie. the 248th
amino acid)
Single letter code for
the substituted
amino acid (in this
case “G” is for
Glycine)
The mutations to p53 listed above are all associated with an increased risk of a wide range of cancers,
including colon, breast, head and neck, lung and leukaemia. The reason for this may be explained by
examining the location of the mutation in the protein expressed. Location 248 is part of the DNA
Binding domain (see Figure 3). A mutation which results in change of an arginine residue to another
amino acid may have two possible effects. Firstly, at position 248, the side chain of the arginine residue
binds directly to the DNA strand. If that side chain is not present, the ability of p53 to bind to DNA is
severely diminished. Secondly, substituting out amino acids is likely to change the three-dimensional
shape or conformation of the p53 protein in the region which allows it to attach to the DNA and affects
its ability to bind - it may no longer have the correct shape.
p53 performs its regulatory role by binding to the DNA upstream of genes which encode proteins
which arrest the cell cycle. Once bound, p53 activates transcription factors which transcribe the target
genes to mRNA and this leads to the expression of the proteins. If p53 can not bind to the DNA, these
transcription factors will not induce the expression of proteins like p21 and cell division will be allowed
to progress unchecked (Figure 4).
p53 binds to
DNA P
P
p21 is
expressed
p21
G1 phase arrest
while DNA is
repaired
Mutated p53
cannot bind to
P
P
No p21 expressed
Mitosis
Figure 4 - Effect of mutations in the DNA binding domain on the function of
p53
Residue 248 is just one location in the p53 protein in which mutations have been identified which
correlate with an increased risk of cancer. In this project, you will investigate the effects of mutations
within the DNA binding domain of p53 on the expression of protein in a model system, comparing it
to the effects of mutations in another region of the protein.
Pre-reading and useful References:
Essential readings:1. Cell and Molecular Biology techniques- booklet on SPARQ-ed website
2. Essential knowledge- booklet on SPARQ-ed website
3. Celli, J., Duijf, P., Hamel, B.C., Bamshad, M., Kramer, B., Smits, A.P., Newbury-Ecob, R.,
Hennekam, R.C., Van Buggenhout, G., van Haeringen, A., Woods, C.G., van Essen, A.J., de
Waal, R., Vriend, G., Haber, D.A., Yang, A., McKeon, F., Brunner, H.G. and van Bokhoven, H.
(1999) Heterozygous germline mutations in the p53 homolog p63 are the cause of EEC
syndrome. Cell 99(2),143-53. [PMID:10535733]
4. Brady, C. A. and L. D. Attardi (2010). "p53 at a glance." Journal of Cell Science 123(15): 25272532.
5. Vogelstein, B., Sur, S. & Prives, C. (2010) p53: The Most Frequently Altered Gene in Human
Cancers. Nature Education 3(9):6
6. Lasky, T. and Silbergeld, E. (1996) p53 mutations associated with breast, colorectal, liver, lung,
and ovarian cancers. Environmental Health Perspectives 104(12), 1324-31 [PMID:9118874]
Useful websites:
Biology info: http://guides.library.uq.edu.au/c.php?g=210303&p=1388496
The TP53 Website : http://p53.free.fr/Database/p53_cancer/all_cancer.html
Optional readings:
Muller, P.A.J. and Vousden, K.H. (2013) p53 mutations in cancer. Nature Cell Biology 15, 2–8 [PMID:
23263379]
Soussi, T. and Béroud, C. (2003) Significance of TP53 mutations in human cancer: a critical analysis of
mutations at CpG dinucleotides. Human Mutation 21(3), 192-200 [PMID:12619105]