Cancer 6: DNA damage Flashcards
How is p53 different to other tumour supressor genes
A single mutation can make it cancer-driving
Examples of possible carcinoogens
dietary lifestyle environmental occupational medical endogenous
Examples of radiation which can cause cancer
ionizing
solar
cosmic
Why does DNA damage lead to cancer
DNA damage can lead to mutation
Mutation may lead to cancer
Outline types of damage from carcinogens
DNA adducts (DNA adduct is a segment of DNA bound to a cancer-causing chemical) & alkylation
Base hydroxylations
& abasic sites formed (removed by DNA damage repair enzymes, and might not be replaced, leading to abasic sites))
Base dimers &
chemical cross-links
Double & single
strand breaks
T/f environmental pollutants etc pose the biggest risk of cancer
F….. this is very overplayed…. things like food and alcohol are much more important
Outline mammalian metabolism
Phase 1
Phase II
Outline phase 1 metabolism
addition of functional groups
e.g. oxidations, reductions, hydrolysis
mainly cytochrome p450-mediated
Outline phase II metabolism
conjugation of Phase I functional groups
e.g. sulphation, glucuronidation, acetylation, methylation, amino acid and glutathione conjugation
Generates polar (water soluble) metabolites.
What are polycyclic aromatic hydrocarbons
Common environmental pollutants
Formed from combustion of fossil fuels
Formed from combustion of tobacco (i,e smoking)
ELECTROPHILIC
Benzene ring structure
Give an important example of a polycyclic aromatic hydrocarbons. How many rings does it have
Benzo(a)pyrene…. 5 rings
What type of DNA damage does benzo(a)pyrene (as polycyclic aromatic hydrocarbons) cause, and how
DNA adducts
2 STEP EPOXIDATION OF B[a]P…. the labels make sense
CP450 oxidises benzo(a)pyrene, which forms an epoxide, which is a 3 membered ring (i.e. the carbon ring with the oxygen group is 3 membered)
= [Benzo(a)pyrene-7,8-oxide]
Epoxide hydrolase then splits the 3 membered ring up to make two OH [Benzo(a)pyrene-7,8-dihydrodiol]
But then p450 adds another epoxide to make [Benzo(a)pyrene-7,8-dihydrodiol-9,10-oxide]
Now it has two hydroxyl groups and an epoxide group, which is extrodinarily reactive,
then this spontaneously degrades (the added epoxide forms a another OH group), to form a charged carbon atom, which then adducts DNA
Covalently binds to DNA at GUANINE
What is epoxidation
The 3 carbon ring with -O- attached, which p450 leaves
Give an example of a carcinogenic biological matter
Aspergillus flavus mould creates aflatoxin B1
How does aflatoxin B1 lead to carcinogenesis.
Where does it affect
Epoxidation of aflatoxin B1 [to aflatoxin B1. 2, 3-epoxide)
Generates positively charged carbon atom
which can then adduct DNA (guanine) (covalent)
Potent liver carcionogen
What is the most damaging type of DNA damage
Double strand breaks
Where is aspergillus flavus mould often found
Common on poorly stored grains and peanuts
Where is incidence of liver carcingogenesis highest and why
Africa and Far-East:
Hepatits
AND
Aflatoxin B1 is a potent human liver carcinogen (can’t test for the moud)
Why was 2-naphthylamine used
Past components of dye-stuffs (German dye industry)
Where is aflatoxin damage focused. How is it converted to cause cancer
Aflaxtoxin in the liver that’s where the p450 is and the product of the p450 reactions is extremely dangerous
Aflatxoin B1 metabolised by p450 and has epoxide added (Aflatoxin b1, 2, 3-epoxide) which is reactive
It then becomes a positively charged molecule which covalently DNA at guanine
SAME MECHANISM AS BENZO(A)PYRENE!
Why does 2 napthylamine cause damage to the bladder
Different mechanism to the other 2
The amine part of 2-napthylamine is firstly hydroxylated (NOT oxidised, like aflatoxin B1/benzo(a)pyrene) by the cP450-1A2, (to make hydroxylamine, which is reactive) and then has glucoronide group added in a glucuronyl transferase mediated phase II reaction
This product is less damaging
But when it gets to the bladder, the acidic (low) urinary pH, glucoronide is hydrolysed, releasing the previous, reactive hydroxylamine derivative.
It then rearranges to form positively charged nitrenium ion which is an electrophile, adn adducts to DNA.
2-naphthylamine is a potent human bladder carcinogen
What molecule is like 2-naphthlamine
AND where do each of the key chemicals act (benzopyrene, aflatoxin and naphthylamine)
benzidine
benzopyrene: all over body,
aflatoxin: liver and
naphthylamine: bladder
What is the effect of solar (UV) radiation on DNA
What type of DNA repair is used
Pyrimidine (thymine) dimers between BASES
COVALENT bonds
The cell tries to repair this, but induces mutations
Skin cancer
REPAIR: DIRECT REVERSAL OF DNA DAMAGE
How does ionising radiation cause damage to DNA
All ionising radiation generates free radicals in cells
Includes oxygen free radicals
- super oxide radical: O2•
- hydroxyl radical: HO• (more reactive)
These possess unpaired electrons so are electrophilic and therefore seek out electron-rich DNA
T/F oxygen free radicals are electrophilic and adduct to DNA
F! Polycyclic aromatic hydrocarbons will adduct but
Oxygen free radicals cause double and single strand breaks
(double strand hard to repair)
What is the effect of oxygen free radicals on DNA
(remember this is due to ionising radiation)
- Single/double strand breaks (single fine, double much worse)
- Apurinic & apyrimidinic sites result from DNA removal
- Base modifications
- ring-opened guanine & adenine
- thymine and cytosine glycols
- 8-hydroxyadenine &
- 8-hydroxyguanine (mutagenic)
What enzyme system is most frequently involved in actvation of chemicals to metabolites than can damage DNA
C
Outline p53 normal state. as well as what happens in response to stimuli
What are its effects.
How is it different to other genes of its kind
p53 bound p to MDM2
Responds to oxidative stress/nitric oxide, hypoxia, mitotic apparatus dysfunction/oncogene activation/DNA replication stress/double strand breaks
In response, MDM2 unbinds, activating p53. p53 then forms a dimer with othjer p53 molecule
p53 can then act as TF and upregulate genes….. It is a tumour suppressor gene
It can upregulate DNA repair mechanisms in teh case of mild stress, or can induce apoptosis under severe stress.
Different to other TSGs because it only requires one mutation to potentially cuase cancer
Outline the types of repair
Direct reversal of DNA damage
Base excision repair
Nucleotide excision repair
During- or post-replication repair
When doees Base excision repair and Nucleotide excision repair occur
Base excision repair (mainly for apurinic/apyrimidinic damage)
Nucleotide repair mainly for bulky DNA adducts (where you need more than just one base removed!!)
Give 2 examples of direct reversal of of DNA damage
- Photolyase splits cyclobutane pyrimidine-dimers (from UV light!)
- methyltransferases & alkyltransferases remove alkyl groups from bases (and become degraded)
What is the function of
excision repair
Outline base excision repair. Which proteins are involved
DNA glycosylases (cut sugar part of molecule) & apurinic/apyrimidinic endonucleases + other enzyme partners
A repair polymerase (e.g. Polb) fills the gap and DNA ligase completes the repair.
Outline nucleotide excision repair
Xeroderma pigmentosum proteins (XP proteins) assemble at the damage. A stretch of nucleotides either side of the damage are excised. Repair polymerases (e.g. Pold/b) fill the gap and DNA ligase completes the repair.
Outline during- or post- replication repair
mismatch repair (i,e, mistakes during replication)
recombinational repair (due to crossing over, mistakes can be made)
Outline the base excision repair pathway
Mutagen exposure/DNA adduct (the adduct can cause this method of repair OR abasic sites)
- Glycosylase cuts the base and the adduct out of the strand (but not the backbone)
- AP- endonuclease comes in and cuts the DNA strand
- DNA polymerase comes in and inserts the correct base (reads complimentary base to the other side of the strand)
- DNA ligase then seals the strand
Outline the nucleotide excision repair pathway
- Endonuclease chops either side of the problem (can be hundreds of nucleotides)… long or short patches can be removed
- Helicase unwinds the DNA and allows access to the large gap, and allows the loss of the cut DNA
- There is now a large gap, DNA polymerase then reads opposite strand and puts in appropratie bases
- DNA ligase then seals
How comes even though DNA damage occurs all the time, the cell is okay
Because the damage per hour per cell is less than the max repair rate of the cell (i.e. cells have high repair capacity)
BUT
The greater the persistence of damage then the greater the chance of a mutagenic event (such as if you smoke then you’re constantly inhaling chemicals that are damaging the DNA)
Outline the consequence of carcingogen damage leading to altered DNA
CARCINOGENIC DAMAGE LEADING TO ALTERED DNA leads to either:
- Efficient repair –> normal cell
- Apoptosis –> cell death
- Incorrect repair/altered primary sequence –> DNA replication and cell division (fixed mutations)
leads to
TRANSCRIPTION/TRANSLATION GIVING ABBERANT PROTEINS
CARCINOGENESIS IF CRITICAL TARETS ARE MUTATED (oncogenes,
tumour suppressor genes)
How do we test for DNA damaging products
Structural alerts/SAR
In vitro BACTERIAL gene mutation assay e.g. Ames test with S. typhimurium
In vitro MAMMALIAN CELL assay
e.g. chromosome aberration,
TK mutation in mouse lymphoma cell
Micronucleus assay
In vivo MAMMALIAN assay
e.g. Bone marrow micronucleus test
transgenic rodent mutation assay
Investigative in vivo MAMMALIAN assays
What is the difference when testing mammalian cells and bacteria
Chromosomes not present in bacteria but not mammal (chromosomes provide a level of protection)
What is the difference between mammal cells in vitro and the in vivo test
In vivo, using a rodent, it has enzyme systems and membranes etc….. as we’ve seen enzyme systems can convert substances into carcinogens so this is important
What is the Ames test for mutagenicity of chemicals
Place the chemical to be tested in test tube with some rat liver enzyme S9 (gives metabolic capability to the solution, to see if any of the metabolites are cancer causing either)
Then you put in some bacteria e.g. Salmonella typhimurium, which have been GE not to produce histine, yet need it to grow.
Then, you take the cells out and put them on a HISTIDINE-FREE plate. The bacteria should not form into colonies. However, if they have acquired mutations that allow them to produce histidine, then will colonise, this means a carcinogen was present in the test tube.
Outline what colonies show in the bacterial ames test
The bacteria should not be able to produce histidine and thus not be able to grow
However, if there has been a mutation (due to the chemical in question) then the bacteria may acquire ability to produce histidine and thus grow and form colonies
t/f in a non-carcinogenic sibstance you might get basal level of colonies in the ames test
How can you work out the capability of the chemical in question to damage DNA
true, a few because there is a background rate of mutation
The capability for the chemical to cause damage is proportional to the number of colonies formed
Outlien chromosome tests to detect DNA damage
Treat mammalian cells with chemical in question in the presence of liver S9. Look for chromosomal damage
chromatid exchange chromosome gap double minutes chromosome interchanges acentric ring chromosome break
Outline the in vitro mammalian micronucleus assay
Cells treated with chemical and allowed to divide
Binucleate cells assessed for presence of micronuclei (which would occur if the chemical could break up the DNA i.e. cancer causing)
Can stain the kinetochore proteins to determine if chemical treatment caused:
- clastgenicity (chromosomal breakage) or
- aneuploidy (chromosomal loss)
Give the example of a bone marrow micronucleus assay in mice and rats
You are using the pluripotent nature of the bone marrow in producing blood cells
The animals are treated with the chemical and the bone marrow cells or peripheral erythrocytes are examined for the presence of micronuclei
The erythrocytes normally remove the nucleus during development but it CAN NOT remove small fragments of DNA (e.g. a micronucleus)
So if the chemical can generate small fragments of DNA as the erythrocytes are formed from the pluripotent stem cells, these fragments will persist
The presence of micronuclei in the erythrocytes indicates DNA damage
Why is guanine most commonly adducted, and what kind of attachment forms between them
The most electron-rich base is guanine (adenine is also very electron rich).
Covalent bond
