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Canc > Cancer 6: DNA damage > Flashcards

Cancer 6: DNA damage Flashcards

1
Q

How is p53 different to other tumour supressor genes

A

A single mutation can make it cancer-driving

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2
Q

Examples of possible carcinoogens

A 
dietary
lifestyle
environmental
occupational
medical
endogenous
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3
Q

Examples of radiation which can cause cancer

A

ionizing
solar
cosmic

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4
Q

Why does DNA damage lead to cancer

A

DNA damage can lead to mutation

Mutation may lead to cancer

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5
Q

Outline types of damage from carcinogens

A

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

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6
Q

T/f environmental pollutants etc pose the biggest risk of cancer

A

F….. this is very overplayed…. things like food and alcohol are much more important

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7
Q

Outline mammalian metabolism

A

Phase 1

Phase II

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8
Q

Outline phase 1 metabolism

A

addition of functional groups
e.g. oxidations, reductions, hydrolysis

mainly cytochrome p450-mediated

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9
Q

Outline phase II metabolism

A

conjugation of Phase I functional groups

e.g. sulphation, glucuronidation, acetylation, methylation, amino acid and glutathione conjugation

Generates polar (water soluble) metabolites.

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10
Q

What are polycyclic aromatic hydrocarbons

A

Common environmental pollutants

Formed from combustion of fossil fuels

Formed from combustion of tobacco (i,e smoking)

ELECTROPHILIC

Benzene ring structure

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11
Q

Give an important example of a polycyclic aromatic hydrocarbons. How many rings does it have

A

Benzo(a)pyrene…. 5 rings

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12
Q

What type of DNA damage does benzo(a)pyrene (as polycyclic aromatic hydrocarbons) cause, and how

A

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

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13
Q

What is epoxidation

A

The 3 carbon ring with -O- attached, which p450 leaves

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14
Q

Give an example of a carcinogenic biological matter

A

Aspergillus flavus mould creates aflatoxin B1

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15
Q

How does aflatoxin B1 lead to carcinogenesis.

Where does it affect

A

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

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16
Q

What is the most damaging type of DNA damage

A

Double strand breaks

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17
Q

Where is aspergillus flavus mould often found

A

Common on poorly stored grains and peanuts

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18
Q

Where is incidence of liver carcingogenesis highest and why

A

Africa and Far-East:

Hepatits

AND

Aflatoxin B1 is a potent human liver carcinogen (can’t test for the moud)

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19
Q

Why was 2-naphthylamine used

A

Past components of dye-stuffs (German dye industry)

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20
Q

Where is aflatoxin damage focused. How is it converted to cause cancer

A

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!

21
Q

Why does 2 napthylamine cause damage to the bladder

A

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

22
Q

What molecule is like 2-naphthlamine

AND where do each of the key chemicals act (benzopyrene, aflatoxin and naphthylamine)

A

benzidine

benzopyrene: all over body,
aflatoxin: liver and
naphthylamine: bladder

23
Q

What is the effect of solar (UV) radiation on DNA

What type of DNA repair is used

A

Pyrimidine (thymine) dimers between BASES

COVALENT bonds

The cell tries to repair this, but induces mutations

Skin cancer

REPAIR: DIRECT REVERSAL OF DNA DAMAGE

24
Q

How does ionising radiation cause damage to DNA

A

All ionising radiation generates free radicals in cells

Includes oxygen free radicals

  1. super oxide radical: O2•
  2. hydroxyl radical: HO• (more reactive)

These possess unpaired electrons so are electrophilic and therefore seek out electron-rich DNA

25
Q

T/F oxygen free radicals are electrophilic and adduct to DNA

A

F! Polycyclic aromatic hydrocarbons will adduct but

Oxygen free radicals cause double and single strand breaks

(double strand hard to repair)

26
Q

What is the effect of oxygen free radicals on DNA

A

(remember this is due to ionising radiation)

  1. Single/double strand breaks (single fine, double much worse)
  2. Apurinic & apyrimidinic sites result from DNA removal
  3. Base modifications
    - ring-opened guanine & adenine
    - thymine and cytosine glycols
    - 8-hydroxyadenine &
    - 8-hydroxyguanine (mutagenic)
27
Q

What enzyme system is most frequently involved in actvation of chemicals to metabolites than can damage DNA

A

C

28
Q

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

A

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

29
Q

Outline the types of repair

A

Direct reversal of DNA damage

Base excision repair

Nucleotide excision repair

During- or post-replication repair

30
Q

When doees Base excision repair and Nucleotide excision repair occur

A

Base excision repair (mainly for apurinic/apyrimidinic damage)

Nucleotide repair mainly for bulky DNA adducts (where you need more than just one base removed!!)

31
Q

Give 2 examples of direct reversal of of DNA damage

A
  • Photolyase splits cyclobutane pyrimidine-dimers (from UV light!)
  • methyltransferases & alkyltransferases remove alkyl groups from bases (and become degraded)
32
Q

What is the function of

A

excision repair

33
Q

Outline base excision repair. Which proteins are involved

A

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.

34
Q

Outline nucleotide excision repair

A 
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.
35
Q

Outline during- or post- replication repair

A

mismatch repair (i,e, mistakes during replication)

recombinational repair (due to crossing over, mistakes can be made)

36
Q

Outline the base excision repair pathway

A

Mutagen exposure/DNA adduct (the adduct can cause this method of repair OR abasic sites)

  1. Glycosylase cuts the base and the adduct out of the strand (but not the backbone)
  2. AP- endonuclease comes in and cuts the DNA strand
  3. DNA polymerase comes in and inserts the correct base (reads complimentary base to the other side of the strand)
  4. DNA ligase then seals the strand
37
Q

Outline the nucleotide excision repair pathway

A
  1. Endonuclease chops either side of the problem (can be hundreds of nucleotides)… long or short patches can be removed
  2. Helicase unwinds the DNA and allows access to the large gap, and allows the loss of the cut DNA
  3. There is now a large gap, DNA polymerase then reads opposite strand and puts in appropratie bases
  4. DNA ligase then seals
38
Q

How comes even though DNA damage occurs all the time, the cell is okay

A

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)

39
Q

Outline the consequence of carcingogen damage leading to altered DNA

A

CARCINOGENIC DAMAGE LEADING TO ALTERED DNA leads to either:

  1. Efficient repair –> normal cell
  2. Apoptosis –> cell death
  3. 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)

40
Q

How do we test for DNA damaging products

A

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

41
Q

What is the difference when testing mammalian cells and bacteria

A

Chromosomes not present in bacteria but not mammal (chromosomes provide a level of protection)

42
Q

What is the difference between mammal cells in vitro and the in vivo test

A

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

43
Q

What is the Ames test for mutagenicity of chemicals

A

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.

44
Q

Outline what colonies show in the bacterial ames test

A

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

45
Q

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

A

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

46
Q

Outlien chromosome tests to detect DNA damage

A

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
47
Q

Outline the in vitro mammalian micronucleus assay

A

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:

  1. clastgenicity (chromosomal breakage) or
  2. aneuploidy (chromosomal loss)
48
Q

Give the example of a bone marrow micronucleus assay in mice and rats

A

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

49
Q

Why is guanine most commonly adducted, and what kind of attachment forms between them

A

The most electron-rich base is guanine (adenine is also very electron rich).

Covalent bond

Canc (16 decks)

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