ONCOLOGY exam 1 summary (AB_1184); study: Health and Life/Biomedical Sciences; VU Amsterdam
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Course
Oncology (AB_1184)
Institution
Vrije Universiteit Amsterdam (VU)
Book
Molecular Biology of Cancer
This document contains a summary for exam 1 of the course 'Oncology' of chapter 2 till 6 from the book 'Molecular biology of Cancer' 4th edition, from Lauren Pecorino. I decided not to include chapter 1, because it is a lot of recap from previous years. I included information obtained from the lect...
Summary Oncology Molecular Biology of Cancer chapter 7-14
Oncologie Tentamenstof DT 1 – alle colleges (incl. plaatjes)
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2.1 Gene structure – two parts of a gene: the regulatory region and the coding region
There are two distinct functional parts to a gene. A 5’ end and a 3’ end. On these ends, the following
parts are placed.
Promotor region: region involved in regulating the expression of the gene
TATA box: located near the start site of transcription, and one of the most important regulatory
elements for most genes. Binding of the TATA box-binding protein (TBP) is crucial for the initiation of
transcription.
Response element (RE): a short sequence of DNA within a promoter that is recognized by a specific
protein and contributes to the regulation of the gene.
Enhancer elements: additional regulatory DNA sequences that are position- and orientation
independent relative to a promoter and are important for tissue-specific and stage-specific expression.
2.2 Mutations
Most carcinogens are mutagens. These induce mutations either by modifying DNA or by causing
chromosomal damage. There are different types of mutations:
- Transitions (base substitution)
- Transversions (base substitution)
- Insertions
- Deletions
- Chromosomal translocations
These mutations can cause misreading of the DNA template by DNA polymerase, because the genetic
code is a triplet code read in a sequential, but non-overlapping manner. When the reading frame is
altered, a non-functional or truncated protein product can be made. Also gene amplification can occur,
that means that the copy number of a gene increases and more products than should are being made.
Driver mutations: located in cancer genes and confer a growth advantage on cells
Passenger mutations: do not confer a growth advantage but instead ‘are there for a ride’.
Some mutations may give rise to a cell that has an increased rate of mutation as a result of (f.e.)
defective DNA repair, and these cells are said to have a ‘mutator phenotype’. The consequence of a
mutation in a gene is determined by its location with respect to the two functional parts of a gene.
*Its important to remember that there could be a possible link between mitochondrial DNA and cancer.
Because mitochondrial DNA lacks histones and has a lower capacity for DNA repair, it makes itself
susceptible to mutation.*
2.3 Carcinogenic agents
Carcinogens are the responsible agents for cancer-causing mutations. There are several classes of
carcinogens that will now be discussed.
1
,Radiation
You have electromagnetic radiation, this is naturally occurring radiation with possesses a broad range
of energies. Examples are X-rays, and UV radiation. When radiation reaches the cells, the particular
energy it contains affects the mechanism and extent of damage to DNA. The amount of biological
damage caused by a particular source of radiation is measured in sieverts (Sv). Two classes of
radiation have been demonstrated to act as carcinogens and damage DNA
Ionizing radiation
This type of radiation includes α, β particles and γ rays. When this radiation strikes molecules,
electrons may be displaced from atoms within the molecule. The loss of electrons, can ionize the
molecule. ROS can contribute too with destructing the DNA.
- The most frequent ionizing radiation induced cancer is leukaemia
Ultraviolet radiation
This is the principal of skin cancer. There is UVA,B and C light and UVB is the most effective
carcinogen. The conjugated double bond in the rings of the bases of DNA absorb UV radiation.
Chemicals
The common mechanism of action of chemical carcinogens is that an electrophilic form reacts with
nucleophilic sites in the purine and pyrimidine rings of nucleic acids. Some can act directly on DNA,
but others become active only after they are metabolized in the body. The major classes of chemical
carcinogens are:
- Polycyclic aromatic hydrocarbons (PAHs): carcinogen in cigarette smoke
- Aromatic amines: carcinogens produced by cooking meat
- Nitrosamines and nitrosamides: carcinogens found in tobacco and can be also formed when
nitrites react with amines in fish and meats during smoking.
- Alkylating agents: mustard gas (WWI)
- Fibrous minerals: asbestos and erionite
Infectious pathogens (ch. 4)
Viruses that are oncogenic can be classified as DNA tumour viruses or RNA tumour
viruses(retroviruses), depending on the nucleic acid that defines their genome. Their mechanisms
differ.
DNA tumour viruses
Encode viral proteins that block tumour suppressor genes, often by protein-protein interactions
Retroviruses
Encode mutated forms of normal genes that have a dominant effect in host cells.
Endogenous carcinogenic reactions
Endogenous cellular reactions generate mutations. Oxidative respiration and lipid peroxidation,
produce ROS that can react with DNA and lipids to produce oxidized products also seen by exposure
to radiation.
2.4 DNA repair and predispositions to cancer
DNA repair mechanisms are essential for preventing cancer development. There are five types of DNA
repair systems:
One-step repair
This involves the direct reversal of DNA damage.
Nucleotide excision repair (NER)
NER is specific for helix-distorting lesions such as pyrimidine dimers and bulky DNA adducts induced
by environmental agents. The damage interfers with transcription and replication.
Base excision repair (BER)
BER targets chemically altered bases (8-oxoguanine) induced mostly by endogenous mechanisms; in
the absence of such repair, the damage will cause a point mutation.
Mismatch repair
This corrects replication errors that have escaped editing by polymerases. It includes repair of
insertions and deletions produced as a result of slippage during the replication of repetitive
sequences, as well as nucleotide mismatches.
2
,Recombinational repair
Homologous recombination & non-homologous end-joining are two types of recombinational repair
that mend double-strand DNA breaks. Homologous recombination depends on the presence of sister
chromatids formed during DNA synthesis as a template for recombining severed ends.
2.5 conventional therapies: chemotherapy and radiation therapy
Chemotherapy
This therapy can also be divided in three groups
Alkylating agents and platinum-based drugs
These work by a similar mode of action. Alkylating agents have the ability to form DNA adducts by
covalents bonds via an alkyl group. Some form intra and inter-strand cross-links in DNA that alter the
conformation of the double helix or prevent separation of the DNA strands and interfere with DNA
replication. Platinum-based drugs can induce apoptosis by their caused DNA damage. This can help
beat some cancers, but can also be associated with irreversible damage of earlier healthy tissues.
Antimetabolites
These are compounds that are structurally similar to endogenous molecules, and therefore can mimic
their role and inhibit nucleic acid synthesis.
Organic drugs
f.e. Doxorubicin is a microbial antibiotic that inhibits topoisomerase II, an enzyme that releases
torsional stress during DNA replication, by trapping single-strand and double-strand DNA
intermediates.
Radiation therapy
Radiation reacts with water inside cells to generate ROS that damage DNA. Apoptosis will be induced
in cells that contain large amounts of DNA damage. The supply of oxygen affects the potency of
ionizing radiation and is thought to be caused by the generation of ROS.
Heterogeneous cell sensitivity and drug resistance: obstacles to these treatments
A major obstacle in achieving long-term effects with chemotherapy is drug resistance.
Cancer cells will receive different doses of treatment, depending on the location of
individual cells within the mass. Cells deep within the tumour will receive lower doses than
cells on the surface of the tumour. Cells within the same tumour may have acquired
different mutations, some that lead to drug resistance. Cells within the tumour that are
classified as cancer stem cells are intrinsically resistant to the therapies.
2.6 strategies that target DNA repair pathways
Synthetic lethal strategies
In involves interactions whereby inhibiting the function of one gene is cytotoxic only in the presence of
an additional mutation. So two mutations are necessary. When both target genes are mutated, the cell
dies. When (f.e.) gene A is mutated, it isn’t lethal, but with gene B also mutated, this leads to lethality.
3.1 Transcription factors and transcriptional regulation
➔ Process in which an RNA copy is made from a specific part of the genome (gene)
Promoter: site for initiation of transcription
Steps:
1. Transcription factor binds to response element
2. RNA polymerase II binds to TATA-box
3. Transcription starts
3
,Transcription factors (TF)
~3000 in humans that regulate 20,000 genes.
~ DNA-binding proteins
~ recognize specific DNA sequence (response element)
Transcription factors are a family of proteins that are mostly only devoted to regulating transcription.
Co-activators/suppressors: accessory/supportive molecules that interact with the DNA-binding proteins
to promote/suppress RNA transcription
Other regulatory elements
These can be activating (enhancers) and repressing (silencers). They’re located outside the promoter
region, up- and downstream of gene and can be bound by regulatory proteins and interacting co-
factors.
TF domains
To function as a TF multiple domains are needed with specific functions:
All TFs need:
1) DNA binding domain
o Zinc finger
o Helix-loop-helix
o Helix-turn-helix
o Leucine zipper
These domains are characteristic protein
conformations that enable a transcription factor to bind DNA.
They have in common that they bind to DNA and have tails that
interact with certain nucleotides.
2) Transcriptional activation domain (binding of other components
of the transcription machinery (pol II)
Some TFs (domains to control activity of TF) have:
3) Dimerization domain
Mechanism of TF activity regulation -> example (hypothetical): 3
factors can give rise to 6 different dimers with different
transcriptional activation properties. So f.e. AP-1; there are 18
possible combinations that can regulate transcription. The Jun
and Fos family are able to transform normal cells in culture to
cancer cells and are frequently overexpressed in tumour cells.
4) Ligand binding domain
Mechanism of TF activity regulation -> example vit. A (retinoic acid) receptor (RAR). This
receptor represses transcription of retinoic acid gene, in the absence of RA.
Somatic mutations in the promoter region can lead to altered gene regulation of oncogenes!
For example, somatic mutations upstream of the TAL1 oncogene, which codes for a basic helix-loop-
helix transcription factor, have been shown to create a super-enhancer that upregulates the
expression of this oncogene in T-cell leukaemia.
4
, Examination of TF binding
EMSA (Electrophoretic mobility shift assay)
You take a part of DNA that has the response element
to your transcription factor. This part of the DNA (called
an oligoduplex) is radioactively labelled. When put on a
gel and run a current through it, it will run to a positive
pool. The speed depends on the size and the affinity of
other proteins to the response element. If they bind, the
DNA runs slower through the gel. With this method,
you can determine if your TF is active in the nuclear
extract you want to examine.
DNAse footprinting
Its more trying to find the location of your response element. The enzyme DNAse is
used and breaks down DNA. But it will only be able to break down DNA that is not
obscured by complexes. So you incubate the DNA with a nuclear extract and DNAse. In
the picture depicted right you can see that at the places ‘4 and 5’ some bands are
missing. This indicates that DNAse wasn’t able to break down these bands and this also
indicates where the TFs are.
ChIP-seq overview
ChIP-seq → chromatin immuno presentation sequencing. You start with
DNA and bound protein. The protein is cross-linked to the DNA. This is
covalently done, so they can be taken down with specific antibodies. After
taking down, the DNA is released and prepared for sequencing. At the
analysis you can see which sequences are enriched.
Reporter assays
Here we are interested in figuring out how a certain promoter of a gene
works. We can put the promoter in front of a reporter. This reporter usually
is an enzyme called Luciferase that lights up when given the right substrate
(a GFP can also be used). Now, you can do all these type of manipulations on this promoter and see
which of these manipulations increases or decreases the reporter expression.
3.2 Chromatin structure
DNA packaging
- Human genome: 3.2 x 109 bp of DNA (~20,000 genes)
- 1-2 meters of DNA if stretched from end to end
- Nucleus of a human cell is only 6μM in diameter
*How is DNA packaged into the cells and how are genes accessible to the transcriptional regulatory
proteins that control their expression?*
5
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