Lecture 10- Genomic instability Flashcards
Inherited loss of what causes cancer??
DNA damage response/repair proteins causes cancer
Cancer cells aquire…
a “mutator “ phenotype during progression from normality to neoplasia
What characterises a mutator phenotype ?
characterised by gross chromsomal instability and increased mutation
Checkpoints are extrinsic control pathways that:
i) ensure dependency events within the cell cycle
ii) ensure viability (if possible) when cells experience genomic stress
How were checkpoint genes identified ?
Checkpoint genes were largely identified by synthetic lethality screens in yeast
(homologues found in higher organisms)
How was the existence of checkpoint genes demonstrated ??
Existence of checkpoint pathways in metazoan cells demonstrated
by the existence of small molecules that uncouple later cell cycle stages
from earlier ones
Why are checkpoints a good target point for treatment ?
Because many cancers have acquired mutator phenotype by losing some checkpoint
gene function, we can judiciously target checkpoints to selectively kill cancer cells
What is genomic instability ??
An elevated rate of genetic alterations ??
series events that occur as a result of genomic instability..,
Normal epithelium
> Hyperplastic epithelium > early > intermediate adenomas > late
> Carcinoma > invasion and metastasis
APC
critically involved in beta catenin signalling
Wood, et al.
20,857 transcripts were sequenced for 18,191 genes
11 colon cancers were analyzed vs. normal tissue
Mutated genes were analyzed in up to 120 additional tumours to aid bioinformatic analysis
~80 mutations/cancer cell
<15 “drivers”
Analysis of gene transcripts
colon cancer
Freq of transcripts and no of mutations- common set of 15 genes < likely to be the driver mutations
Lung mutations mapped
- Point mutation in lung tumour 1000s
- Arrows indicate translocation have occurred
Does cancer occur along a set pathway ?
Many alternative routes to neoplasia
Multiple ways to go from normal to invasive carcinoma
Every tissue niche has a different profile of genes and orders required for cancer
What is important about mutations within cancer?
Mutation doesn’t necessarily mean cancer – order in which mutations occur is important
What is the mutator hypothesis?
Tumour cells acquire a mutator gene that increases the rate of mutation as an early event in development.
In tumour cells this instability takes two forms…
CIN-chromosomal instability- gross chromosomal change
MIN-microsatellite instability- concept of point mutations – base in specific seq may change
Microsatellite identified in the genome as long segments of repeated bases
- Read out of point mutations
CIN
- chromosomal aberrations-rearrangements
- loss of heterozygosity (LOH)
- aneuploidy/polyploidy
- gene amplification
MIN
• Microsatellite instability
•point mutation-base substitution,
microdeletions or insertions
CIN/MIN events
Usually very rare bu tHowever the rate of
such mutations in tumour cells may be several thousand
times higher.
Where do mutations come from and how do cells normal defend themselves ??
-DNA replication or processing errors
> Precursor control proof-reading mismatch repair
- DNA damage from exogenous agents
> nucelotide excision repair
> Base excision and double strand break repair
- DNA damage from endogenous agents - Hydrolysis, oxidation, methylation
> Base excision repair direct reversal
inherited forms of disease
- ability of cells to repair double stranded breaks is essential and significantly affect the stability – leads to genome instability
- same for Homologous recombination- involved in genomic instability
Large tomatoes
Induce repeat of S phase
Meiosis
No M phase
RAD9: a paradigm for S/M
checkpoint control in
S cerevisiae
Cells lacking RAD9 are healthy in the absence
of extrinsic interference
CDC9 encode
DNA ligase- bonds okazaki fragment essentially you get fragment
Lack of CDC9
- lee hartwell
Restrictive- DNA ligase not allow in synthesis> cells arrest in G2 therefore not entering mitosis
Entry into mitosis dependent on S phase – not inviable just stuck – reduce temp again they will grow
RAD9 mutants viable all the time
RAD9 crossed with CDC9 – loss of viability – occurs by exposing to stress e.g high temp
Thus viability in cells experiencing stress in S phase requires the
gene RAD 9
What can you use to screen for checkpoint mutants ?
Can use loss of viability as a screen for checkpoint mutants
PCC
a phenotype associated with loss of S/M checkpoint
Premature condensation
Checkpoints:
abrogated by small molecular inhibitor
Example of integral dependency
bacteriophage assembly
Lee Hartwell
identified a mutant in the RAD 9 gene in cerevisiae
Schlegel and Pardee
in mammalian tissue culture cells if you block DNa replication cells dont go into mitosis
- DNA replication Block + caffeine resulted in PCC phenotype
Carry on and try mitosis anyway even though no DNA replication
***Wasn’t an absolute dependence of S phase occurring first, you can abrogate it and get rid of the dependency by adding caffeine
cell free system
Mush of frog eggs that artificially go through the cell cycle
- creates a nuclei in a test tube
- mitotic spindle
- add inhibitor into this system cyclin levels go up and no activation of kinase
- system allow recapitulation of schlegel and pardee experiment
Carl Smythe and J Newport experiment
** coupling of mitosis to the completion of Sphasein xenopus occurs via modulation of the tyrosine kinase that phosphorylates P34 cc2 ***
Carl Smythe and J Newport experiment
- coupling of mitosis to the completion of Sphasein xenopus occurs via modulation of the tyrosine kinase that phosphorylates P34 cc2 ***
- cell free system
- adding caffeine restores entry to mitosis in the absence of DNA replication
- Even in synthetic systems you can show dependency because you can abrogate it
Break down of control elements
- sense incomplete DNA replication
- DNA damage
- Transducers that signal to effectors
- ordinarily the pathways block cell cycle transitions
- decide > apoptosis or Activate DNA repair
Two components of the pathway - transducers
ATR or ATM both protein kinases
- ATR activated when incomplete DNA replication phosphorylates and activates chk1 which acts on cell cycle regulators
ATR
Target of caffeine
ATm also caffeine sensitive
Caffeine
inhibits ATR pathway - no switch on of pathway > catastrophic mitosis
Replication stress is identified by
ssDNA- Single stranded DNA – sensor complex assembly activates Chk 1 which blocks further replication and inhibit cell cycle progression
- stabilise rest of replication fork
To acquire a mutator phenotype
many cancers have lost some checkpoint controls
inherited loss of checkpoint component proteins
causes cancer
many cancers who have lost their check point controls provide
Therapeutic opportunity
cancer cell may have lost a relevant checkpoint so if you inflict DNA damage or replication stress in normal cell normal cell cycle response - induce DNA damage response> resolved
- in cancer cells
wont be cell cycle delay and wont induce DNA damage response
you get cell death >. basis of much chemotherapy (differential checkpoint response)
DNA damaging agents differentially cause cell death in cancer cells vs normal cells but in normal cells they repair themselves whereas cancer cells cannot
