carcinogenesis 1: DNA damage & repair Flashcards
why do bases undergo so many reactions?
planar carbon ring structures = very chemically reactive
what is deamination?
removal of amine groups from bases eg cytosine -> uracil adenine -> hypoxanthine guanine -> xanthine 5-methyl cytosine -> thymine
what oxidation reactions are common?
thymine -> thymine glycol
how can hyper-reactive oxygen species be generated?
ionising radiation
byproducts of normal oxidative metabolism
what is adduction?
linking of bases to other chemical entities in cell via covalent bonds
what does photodamage occur?
UV light absorbed by bases
- > chemical changes induced
- > most common products are thymine dimers (bond formation between adjacent pyrimidines within one strand)
how can UV radiation damage DNA?
breaks phosphodiester bonds in backbone -> nicks formed -> lots of nicks can form a gap
what are some causes of DNA damage?
chemicals: dietary, lifestyle, environmental, occupational, medical, endogenous
radiation: ionising, solar, cosmic
what is an abasic site?
place where base is sufficiently destroyed that it effectively isn’t there anymore
- predominant mutation type seen in active cells
what are the 2 phases of mammalian metabolism?
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
why can BAP be dangerous?
metabolism processes convert BAP into carcinogenic compound by formation of DNA adducts
why is aflatoxin B1 dangerous?
gets metabolised into carcinogen that reacts with guanine bases -> adduct formation on guanine
why can 2-naphthylamine be dangerous?
joins to glucoronide in phase II metabolism -> becomes soluble -> pH of urine breaks down compound -> reacts with DNA
how can ionising radiation cause cancer?
generates free radicals in cells
- this includes oxygen free radicals (eg super oxide, hydroxyl) which are electrophilic therefore react easily with electron rich DNA
how can oxygen free radicals cause cancer?
structure of base lost -> abasic
- purine bases converted to 8-hydroxy purines (more reactive)
- > adduct formation
what is p53?
tumour suppressor gene
- changes in gene expression -> activation of DNA repair pathways
which factors lead to activation of p53?
mitotic apparatus dysfunction
- dna replication stress
- double strand breaks
what happens during the activation of p53?
mdm2 lost -> p53 activated
- p53 is a transription factor -> activates pathways including dna repair pathways
what is direct dna repair?
reversal or simple removal of the damage using proteins that carry out specific enzymatic reactions
eg methyltransferases & alkyltransferases remove alkyl groups from bases
how do photolyases work?
recognise thymine dimers -> (activated by normal light ->) excise thymine dimers
how do MGMTs work?
remove bases that have undergone methylation by recognising specific methylated bases -> remove methyl groups
- suicidal enzymes
what is dna mismatch repair?
scrutinisation of dna for opposed bases that do not pair properly
how can mismatches arise?
during dna replication
- by base conversions eg those that are a result of deamination
how are mismatches that arise during replication repaired?
proofreading (old & new strands compared)
- msh & mlh wrap around mismatch -> enuclease cuts out stretch of dna with mismatch -> dna polymerase replaces bases
what is base excision repair?
(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
what is nucleotide excision repair?
(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
what are some diseases that result from NER pathway defects?
- xeroderma pigmentosum
- trichothiodystrophy
- Cockayne’s syndrome
what is xeroderma pigmentosum?
severe UV light sensitivity -> early onset of skin cancer at high incidence + elevated frequency of other cancer
what are the symptoms of trichothiodystrophy?
- sulphur deficient brittle hair
- facial abnormalities
- short stature
- ichthyosis (fish-like scales on skin)
( - light sensitivity in some cases)
what are the symptoms of Cockayne’s syndrome?
- dwarfism
- facial & limb abnormalities
- neurological abnormalities
- early death due to neurodegeneration
( - light sensitivity in some cases)
how can double strand breaks arise?
under physiological conditions during somatic recombination & transposition eg VDJ recombination
- during homologous recombination
- as a result of ionising radiation & oxidative stress
what happens if there is incorrect repair to damaged dna?
impaired repair ->
altered primary sequence ->
fixed mutations ->
can give rise to abberant proteins OR carcinogenesis if oncogenes / tumour suppressor genes affected
ΟR apoptosis
what are some examples of therapeutic agents that cause DNA damage?
alkylating agents
- agents that make bulky adducts (eg cisplatin)
- agents that induce double strand breaks (radiotherapy)
how can you test for DNA damage?
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
what is the ames test?
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
how can dna damage be detected in mammalian cells in vitro?
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)
what is the murine bone marrow micronucleus assay?
treat animals with chemical ->
examine bone marrow cells / peripheral blood erythrocytes for micronuclei
