carcinogenesis 1: DNA damage & repair

Question 1

why do bases undergo so many reactions?

Answer 1

planar carbon ring structures = very chemically reactive

Question 2

what is deamination?

Answer 2

removal of amine groups from bases 
eg cytosine -> uracil    
adenine -> hypoxanthine    
guanine -> xanthine     
5-methyl cytosine -> thymine

Question 3

what oxidation reactions are common?

Answer 3

thymine -> thymine glycol

Question 4

how can hyper-reactive oxygen species be generated?

Answer 4

ionising radiation

byproducts of normal oxidative metabolism

Question 5

what is adduction?

Answer 5

linking of bases to other chemical entities in cell via covalent bonds

Question 6

what does photodamage occur?

Answer 6

UV light absorbed by bases

• > chemical changes induced
• > most common products are thymine dimers (bond formation between adjacent pyrimidines within one strand)

Question 7

how can UV radiation damage DNA?

Answer 7

breaks phosphodiester bonds in backbone -> nicks formed -> lots of nicks can form a gap

Question 8

what are some causes of DNA damage?

Answer 8

chemicals: dietary, lifestyle, environmental, occupational, medical, endogenous
radiation: ionising, solar, cosmic

Question 9

what is an abasic site?

Answer 9

place where base is sufficiently destroyed that it effectively isn’t there anymore
- predominant mutation type seen in active cells

Question 10

what are the 2 phases of mammalian metabolism?

Answer 10

phase I: addition of functional groups (eg oxidation, reduction, hydrolysis) - mainly cytochrome p450 mediated
phase II: conjugation of phase I functional groups (eg sulphation, glucuronidation, acetylation) - generates polar metabolites

Question 11

why can BAP be dangerous?

Answer 11

metabolism processes convert BAP into carcinogenic compound by formation of DNA adducts

Question 12

why is aflatoxin B1 dangerous?

Answer 12

gets metabolised into carcinogen that reacts with guanine bases -> adduct formation on guanine

Question 13

why can 2-naphthylamine be dangerous?

Answer 13

joins to glucoronide in phase II metabolism -> becomes soluble -> pH of urine breaks down compound -> reacts with DNA

Question 14

how can ionising radiation cause cancer?

Answer 14

generates free radicals in cells
- this includes oxygen free radicals (eg super oxide, hydroxyl) which are electrophilic therefore react easily with electron rich DNA

Question 15

how can oxygen free radicals cause cancer?

Answer 15

structure of base lost -> abasic

• purine bases converted to 8-hydroxy purines (more reactive)
• > adduct formation

Question 16

what is p53?

Answer 16

tumour suppressor gene

- changes in gene expression -> activation of DNA repair pathways

Question 17

which factors lead to activation of p53?

Answer 17

mitotic apparatus dysfunction

• dna replication stress
• double strand breaks

Question 18

what happens during the activation of p53?

Answer 18

mdm2 lost -> p53 activated

- p53 is a transription factor -> activates pathways including dna repair pathways

Question 19

what is direct dna repair?

Answer 19

reversal or simple removal of the damage using proteins that carry out specific enzymatic reactions
eg methyltransferases & alkyltransferases remove alkyl groups from bases

Question 20

how do photolyases work?

Answer 20

recognise thymine dimers -> (activated by normal light ->) excise thymine dimers

Question 21

how do MGMTs work?

Answer 21

remove bases that have undergone methylation by recognising specific methylated bases -> remove methyl groups
- suicidal enzymes

Question 22

what is dna mismatch repair?

Answer 22

scrutinisation of dna for opposed bases that do not pair properly

Question 23

how can mismatches arise?

Answer 23

during dna replication

- by base conversions eg those that are a result of deamination

Question 24

how are mismatches that arise during replication repaired?

Answer 24

proofreading (old & new strands compared)

- msh & mlh wrap around mismatch -> enuclease cuts out stretch of dna with mismatch -> dna polymerase replaces bases

Question 25

what is base excision repair?

Answer 25

(mutagenic exposure to base -> damaged base but not phosphodiester backbone ->)
dna glycosylases remove base of nucleotide ->
ap-endonuclease cuts open dna strand ->
dna polymerase inserts correct base ->
dna ligase seals gap in phosphodiester backbone

Question 26

what is nucleotide excision repair?

Answer 26

(mutagenic expsosure to nucleotide -> damage to whole nucleotide ->)
endonuclease removes patch of dna including affected nucleotide ->
dna polymerase (δ/β) fills gap ->
dna ligase seals phosphodiester backbone

Question 27

what are some diseases that result from NER pathway defects?

Answer 27

• xeroderma pigmentosum
• trichothiodystrophy
• Cockayne’s syndrome

Question 28

what is xeroderma pigmentosum?

Answer 28

severe UV light sensitivity -> early onset of skin cancer at high incidence + elevated frequency of other cancer

Question 29

what are the symptoms of trichothiodystrophy?

Answer 29

• sulphur deficient brittle hair
• facial abnormalities
• short stature
• ichthyosis (fish-like scales on skin)
  ( - light sensitivity in some cases)

Question 30

what are the symptoms of Cockayne’s syndrome?

Answer 30

• dwarfism
• facial & limb abnormalities
• neurological abnormalities
• early death due to neurodegeneration
  ( - light sensitivity in some cases)

Question 31

how can double strand breaks arise?

Answer 31

under physiological conditions during somatic recombination & transposition eg VDJ recombination

• during homologous recombination
• as a result of ionising radiation & oxidative stress

Question 32

what happens if there is incorrect repair to damaged dna?

Answer 32

impaired repair ->
altered primary sequence ->
fixed mutations ->
can give rise to abberant proteins OR carcinogenesis if oncogenes / tumour suppressor genes affected

ΟR apoptosis

Question 33

what are some examples of therapeutic agents that cause DNA damage?

Answer 33

alkylating agents

• agents that make bulky adducts (eg cisplatin)
• agents that induce double strand breaks (radiotherapy)

Question 34

how can you test for DNA damage?

Answer 34

structural alerts ->
in vitro bacterial gene mutation assay ->
in vitro mammalian cell assay (eg chromosome aberration) ->
in vivo mammalian assay (eg bone marrow micronucleus test) ->
investigative in vivo mammalian assay

Question 35

what is the ames test?

Answer 35

bacterial test for mutagenicity of chemicals

salmonella strains unable to make histidine because of mutation mixed with rat liver extract ->
bacteria exposed to carcinogenic agents ->
causes mutations ->
corrects mutation so salmonella can make histidine ->
bacteria can grow on media without histidine

Question 36

how can dna damage be detected in mammalian cells in vitro?

Answer 36

chromosomal aberrations treat mammalian cells in presence of liver S9 ->
exposed to carcinogen ->
chromosomal aberrations

micronucleus assay: cells treated with chemical ->
divide ->
cells assessed for presence of micronuclei (can stain kinetochore proteins to determine if chemical treatment caused clastigenicity (breakage) or aneuploidy)

Question 37

what is the murine bone marrow micronucleus assay?

Answer 37

treat animals with chemical ->

examine bone marrow cells / peripheral blood erythrocytes for micronuclei