Cancer 5 Flashcards
1
Q
what is the nature of the DNA bases?
A
- they are flat carbon rings
- they are chemically reactive species able to react with other molecules
- they contain double bonds
- they are easily mutated
2
Q
what is deamination?
A
- The primary amino groups of nucleic acid bases are somewhat unstable
- deamination is the removal of an amine group
- They can be converted to keto-groups
- Other deamination reactions include conversion of adenine to hypoxanthine, guanine to xanthine, and 5-methyl cytosine to thymine.
3
Q
what causes hyper-reactive oxygen to be formed?
A
bases can undergo reactions which produce hyper-reactive oxygen as by-products
eg. singlet oxygen, peroxide radicals, hydrogen peroxide, and hydroxyl radicals
4
Q
what causes modifications of DNA to be formed?
A
- ionising radiation
- hyper- reactive oxygen species
5
Q
what is a common oxidation reaction?
A
- A common product of thymine oxidation is thymine glycol
6
Q
what happens to the glycol?
A
- they react with chemical species which favor it
- these reactants can be carcinogens
- the addition of a larger molecule produces what we call an ‘adduct’
7
Q
What is photodamage to the DNA?
A
- Ultraviolet light is absorbed by the nucleic acid bases
- the resulting influx of energy can induce chemical changes
- photoproducts are formed as a consequence of bond formation between adjacent pyrimidines within one strand.
8
Q
what are the types of DNA damage?
A
- base pair mismatch
this is when one base is normal and the matching base is abnormal it causes a slight bulge
- thymine dimer
- radioactive damage
9
Q
what can cause damage to DNA?
A
- endogenous means from within the body
eg mitochondria produce reactive oxygen species that have the ability to damage DNA
- Diet is strongly associated with cancer (about 40=45% of human cancers)
- Medical treatments, such as radiotherapy, can also damage DNA and increase the risk of cancer
10
Q
why is DNA damage important?
A
- DNA damage can lead to mutation
- mutation can lead to cancer
- •Damaging DNA is an important strategy in cancer therapy
11
Q
how can carcinogens cause DNA damage?
A
- Base Dimers and Chemical Cross=Links
- Base Hydroxylations
- Abasic Sites
- Single Strand Breaks
- Double Strand Breaks
- DNA Adducts and Alkylation
12
Q
explain base dimers and chemical cross-links ?
A
- This is where the DNA molecules are being chemically linked up
13
Q
explain base hydroxylations?
A
- An oxidative reaction occurring on one of the DNA bases and this can cause problems
- This could mean that the DNA has to get repaired and during the repair process, it could become mutated
14
Q
explain abasic sites?
A
- during the repair process the entire DNA base has been removed so the sugar backbone is maintained but we have removed the base from the mutagenic molecule
- During replication, the missing base will cause problems
15
Q
explain single-strand breaks?
A
- These are very common and can be very useful
- There are physiological enzymes that are responsible for making single-strand breaks
- Topoisomerase is involved in the relaxing and unwinding of DNA
- it works by chopping the strand of DNA and allowing the strand to unwind and we can gain access to the DNA as the strand is re- annealed
- So we can deal with single strand breaks in DNA
16
Q
explain double-strand breaks?
A
- These are a bit of a disaster
- After the double-strand breaks, there is a tendency for the two bits of DNA to drift apart and this is intolerable from the cell’s point of view
- There are a number of DNA repair mechanisms that attempt to amend this, but sometimes the DNA repair can go wrong and introduce DNA damage
17
Q
explain DNA adducts and alkylation?
A
- This is generally the type of damage that is caused by chemicals
- Some chemicals tend to be metabolically activated into electrophiles
- DNA is very rich in electrons because of all the nitrogens in the bases
- The electrophiles bind to the DNA and form a covalent bond
- this causes issues in DNA replication due to the binding of the big bulky chemical
- In short, DNA polymerase cannot recognise the base because of the chemical adduct
18
Q
what are polycyclic aromatic hydrocarbons?
A
- Polycyclic aromatic hydrocarbons are common environmental pollutants
- they are present in smoke
19
Q
overview drug metabolism in the body?
A
- Phase 1: introduce or unmask functional groups that can be used in Phase 2
- Phase 2: we use the functional groups (made available by phase 1) to conjugate it with an endogenous molecule to make it water-soluble so that it can be excreted in the urine
overall purpose is to make something that is lipophilic more polar to get rid of it
20
Q
what are the main enzymes in phase 1?
A
- Cyrochrome P450 enzymes
- responsible for oxidising chemicals
21
Q
what is the two-step Oxidation of Benzo[a]pyrene (B[a]P)?
A
22
Q
explain why the two-step oxidation of Benzo[a]pyrene (B[a]P) is dangerous?
A
- B[a]P is a substrate for CYP450, which oxidizes it to form an oxide (Benzo[a]pyrene-7,8-oxide) (toxic)
- This oxide is reactive and wants to find electrons (it is an electrophile)
- There is a defence mechanism in the body = epoxide hydroxylase cleaves the three membered strained ring of the oxide to form a dihydrodiol (this is NOT toxic)
- the non-toxic dihydrodiol metabolite is also a substrate for P450
- So P450 converts this non-toxic metabolite into another oxide
- This is very reactive and is desperate to find some electrons to react with
- the best source of electrons is DNA, so DNA adducts are formed
23
Q
explain why the metabolism of aflatoxin B1 is dangerous ?
A
- aflatoxins are produced from fungi aspergillus fungi
- grow on foodstuffs that are not maintained well and peanuts
- aflatoxin B1 is not a carcinogen
- cytochrome P450 converts it to an apoxite
- this apoxite is very reactive with DNA
- it reacts with the guanine in DNA at the N7 position
- we end up with an adduct formation on the guanine that will cause mutation
- this is very carcinogenic for hepatic cancers
24
Q
explain why the metabolism of 2-naphthylamine is dangerous?
A
- 2- naphthylamine is a part component of dye-stuffs
- Benzidine is another important past component of dye- stuffs
- Both benzidine and 2- naphthylamine are potent BLADDER carcinogens
- 2- naphthylamine is a substrate for CYP450, which converts the amino group to form a hydroxylamine
- Hydroxylamines are reactive
- in the liver when this reactive hydroxylamine is formed it is glucuronidated ( detoxifying reaction)
- This glucuronidation is done by glucuronyl transferase
- The inactive metabolite is excreted by the liver and it goes into the bladder and mixes with the urine
- Urine is ACIDIC, and, under acidic conditions, the glucuronides are hydrolysed
- This releases the hydroxylamine derivative which in acidic conditions rearranges to form a positively charged nitrogen
- The nitrenium ion is an electrophile, which then goes and binds to the DNA and forms adducts
- The bladder isn’t as capable of detoxifying the hydroxylamine derivative as the liver
25
Q
how does solar radiation cause cancer?
A
- UV radiation can lead to the formation of pyrimidine dimers
- if there are two pyrimidines next to each other in the presence of UV radiation they can covalently link
- this might form skin cancer
26
Q
how does ionizing radiation cause cancer?
A
- Examples of ionising radiation: gamma, X=ray, beta particles
- They can all generate free radicals
- Oxygen- free radicals are produced by normal biochemistry and there are good defence mechanisms for dealing with them = however, ionising radiation can overwhelm the defence mechanisms
27
Q
what is a superoxide radical?
A
- a superoxide radical is very powerful
- this is a molecule of oxygen that has an extra electron so it is very reactive
28
Q
what is a hydroxyl radical?
A
- a hydroxyl group that has grabbed an extra electron
- this is even more reactive than the superoxide radical
- these radicals are electrophiles
- DNA is electron-rich
29
Q
how do oxygen free radicals attack DNA?
A
- single strand breaks are not very damaging
- double-strand breaks are more dangerous
- when the base has been oxidized by an oxygen free radical and the DNA repair enzymes come and cut out the base itself it leaves behind the sugar- phosphate backbone with gaps
- these gaps are called apurinic / apyrimidic sites
- You can also get base modifications:
Ring- opened guanine + adenine
Thymine + cytosine glycols
8- hydroxyadenine + 8- hydroxyguanine (mutagenic)
30
Q
what is the role of P53 in dealing with cellular stress?
A
- P53 is a crucial tumour suppressor gene
- it is normally tied to MDM2 which keeps the P53 inactive
- when it is released from MDM2 it forms a dimer which activates many different pathways
- If we have mild physiological stress = p53 carries out a transcriptional series of events and activates proteins that help repair the problem
- if there is severe stress = p53 can activate an apoptotic pathway by directly interacting with apoptosis proteins
31
Q
what are the 4 main types of DNA repair?
A
- direct reversal of DN damage
- base excision repair
- nucleotide excision repair
- during or post replication repair
32
Q
explain direct reversal of DNA damage?
A
Photolyases
- specific group of enzymes called photolyases can reverse cytlobutane- pyrimidine dimers
- Photolyase looks specifically for cytlobutane=pyrimidine dimers and cuts them to restore the normal sequence
- this allows repair of the DNA to occur
Methyltransferases and alkyltransferases
- Methyltransferases and alkyltransferase remove alkyl and methyl groups from bases to restore the identity of the base
- these enzymes then destroy themselves
33
Q
what is DNA mismatch repair?
A
- mismatch repair involves scrutinisation of of DNA for apposed bases that do not pair properly
- Mismatches that arise during replication are corrected by comparing the old and new strands (proofreading)
- this is in place as there is a large rate of error that takes place in DNA replication
34
Q
why does guanine often form adducts?
A
- The most electron=rich base is guanine
- if we introduce an electrophile it will probably target guanine and form a covalent bond, this is toxic and the cell must remove this
35
Q
what is the base excision repair pathway?
A
- DNA glycosylase hydrolyses between the sugar and the DNA base
- Then an AP- endonuclease splits the DNA strand so there is a gap in the backbone
- DNA polymerase then fills in the missing base
- DNA Ligase then seals the DNA to form intact DNA
36
Q
what is nucleotide excision repair?
A
- Endonuclease makes two cuts in the DNA on either side of the site of damage
- These patches can be long (100 - 200 nucleotides) or short (10 - 20 nucleotides)
- Helicase will then remove this patch, leaving the double stranded DNA with a patch missing
- DNA Polymerase then replaces the bases that have been removed using the complementary strand as a template
- DNA Ligase then joins the DNA up again
- this process is energy demanding and requires a lot of proteins
37
Q
what is Xeroderma Pigmentosum?
this is the main disease to remember
A
- this is a disease in which nucleotide excision repair does not work properly
- causes severe light sensitivity
- severe pigmentation irregularities
- early onset of skin cancer and GI cancer at high incidence
- frequent neurological defects
- this disease is not curable - only managed, must AVOID UV night
38
Q
what is Trichothiodystrophy?
A
this is a disease in which nucleotide excision repair does not work properly
- sulphur deficient brittle hair
- facial abnormalities
- short stature
- ichthyosis (fish-like scales on the skin)
- light sensitivity in some cases
39
Q
what is cockaynes syndrome?
A
this is a disease in which nucleotide excision repair does not work properly
- dwarfism
- light sensitivity in some cases
- facial and limb abnormalities
- neurological abnormalities
- early death due to neurodegeneration
40
Q
when do DN A double-strand break repairs happen?
A
- Under physiological conditions during somatic recombination and transposition
- During Homologous Recombination.
- As a result of ionizing radiation and oxidative stress induced DNA damage.
41
Q
how to join DNA after DN double-strand break repair?
A
there are two main methods
homologous recombination and nonhomologous DNA end-joining
homologous recombination =
- Direct joining of the broken ends.
- This requires proteins that recognize and bind to the exposed ends and bring them together for ligating.
- this happens with complementary nucleotides
non homologous recombination = (more complicated)
- 3 DNA ends are picked up by KU proteins
- KU holds the DNA together and you force the DNA to link up
42
Q
what are the estimated rates of endogenous damage and repair?
A
- It appears that human cells have plenty of spare capacity to deal with both endogenous and exogenous damage
- errors occur, especially with greater age
- If the damage is poorly repaired, then there is greater risk of carcinogenesis
43
Q
what happens if the cell has too much damage?
A
If the DNA damage is excessive, the cells will commit to apoptosis
44
Q
at what point do most issues occur resulting in cancer?
A
- However, most problems occur between excessive damage and small amounts of damage
- This could lead to incorrect repair
- this results in fixed damage in the daughter cells (permanent mutations)
45
Q
how to test for DNA damage in cells?
A
- You firstly look at the structure of the chemical to see if there are any functional groups that could cause problems
- The simplest way to see whether an agent can cause mutation is to introduce it to bacteria and see whether it causes mutation
- if it damages bacteria DNA it might damage human DNA
- Then you test it on Mammalian Cells
- then you test it on mammals using bone marrow micronucleus tests
- bone marrow is used because it contains pleuripotent stem cells
- This means that you can look at the formed elements of the blood as a mechanism of what is happening in the bone marrow
46
Q
what is the bacterial Ames test for mutagenicity of chemicals?
A
- Normally, Salmonella typhimurium is used for this assay
- we take chemical and make it metabolically active
- to do this we incubate the chemical with a preparation of rat liver enzymes (containing P450 enzymes) and that generates the reactive chemical
- The bacteria are genetically engineered so that they can NOT produce histidine (amino acid)
- This means that these bacteria require exogenous histidine to be able to grow
- However, using the chemical mutagens, these cells can be mutated so that they regain the ability to produce histidine, thus meaning that it can grow in the absence of histidine
- So you mix the bacteria with the activated chemical and put them on a plate (which doesn’t have any histidine on it)
- Anything that has NOT been mutated will need exogenous histidine to grow and hence will die on the plate
- So the more the DNA damaging capability of the chemical, the more colonies will grow in the absence of histidine
47
Q
how to detect DNA damage in mammalian cells?
A
- We can look at what is happening to the chromosomes themselves
- Chemicals can cause double strand breaks that leads to fragmentation of chromosomes
- This is very labour intensive investigation
48
Q
how to use in vitro micronucleus Assays?
A
- Mammalian cells are treated with the chemical in vitro and allowed to divide
- We are trying to measure the ability of the chemical to break up DNA into fragments,
- then we can count the fragments
- We need the cell to go through one replication cycle and then stop it when a binucleus is formed
- Cytochalasin=B is used to block cytokinesis
- Then the binucleate cells are assessed for the presence of micronuclei
- The kinetochores of the chromosomes can be stained to determine if chemical
treatment caused:
o Clastogenicity = chromosomal breakage
o Aneuploidy = chromosomal loss/change in the number of chromosomes
- both of these cause cancer
- The cell on the left has NOT been damaged by the chemical
- The cell on the right has extra nuclear material
49
Q
how to carry put bone marrow micronucleus in mice or 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 (micronucleus)
- so presence of micronucleus indicates DNA damage which could cause cancer
