Lecture 10- Genomic instability

Question 1

Inherited loss of what causes cancer??

Answer 1

DNA damage response/repair proteins causes cancer

Question 2

Cancer cells aquire…

Answer 2

a “mutator “ phenotype during progression from normality to neoplasia

Question 3

What characterises a mutator phenotype ?

Answer 3

characterised by gross chromsomal instability and increased mutation

Question 4

Checkpoints are extrinsic control pathways that:

Answer 4

i) ensure dependency events within the cell cycle

ii) ensure viability (if possible) when cells experience genomic stress

Question 5

How were checkpoint genes identified ?

Answer 5

Checkpoint genes were largely identified by synthetic lethality screens in yeast
(homologues found in higher organisms)

Question 6

How was the existence of checkpoint genes demonstrated ??

Answer 6

Existence of checkpoint pathways in metazoan cells demonstrated
by the existence of small molecules that uncouple later cell cycle stages
from earlier ones

Question 7

Why are checkpoints a good target point for treatment ?

Answer 7

Because many cancers have acquired mutator phenotype by losing some checkpoint
gene function, we can judiciously target checkpoints to selectively kill cancer cells

Question 8

What is genomic instability ??

Answer 8

An elevated rate of genetic alterations ??

Question 9

series events that occur as a result of genomic instability..,

Answer 9

Normal epithelium
> Hyperplastic epithelium > early > intermediate adenomas > late
> Carcinoma > invasion and metastasis

Question 10

APC

Answer 10

critically involved in beta catenin signalling

Question 11

Wood, et al.

Answer 11

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”

Question 12

Analysis of gene transcripts

Answer 12

colon cancer

Freq of transcripts and no of mutations- common set of 15 genes < likely to be the driver mutations

Question 13

Lung mutations mapped

Answer 13

• Point mutation in lung tumour 1000s

- Arrows indicate translocation have occurred

Question 14

Does cancer occur along a set pathway ?

Answer 14

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

Question 15

What is important about mutations within cancer?

Answer 15

Mutation doesn’t necessarily mean cancer – order in which mutations occur is important

Question 16

What is the mutator hypothesis?

Answer 16

Tumour cells acquire a mutator gene that increases the rate of mutation as an early event in development.

Question 17

In tumour cells this instability takes two forms…

Answer 17

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

Question 18

CIN

Answer 18

• chromosomal aberrations-rearrangements
• loss of heterozygosity (LOH)
• aneuploidy/polyploidy
• gene amplification

Question 19

MIN

Answer 19

• Microsatellite instability
•point mutation-base substitution,
microdeletions or insertions

Question 20

CIN/MIN events

Answer 20

Usually very rare bu tHowever the rate of
such mutations in tumour cells may be several thousand
times higher.

Question 21

Where do mutations come from and how do cells normal defend themselves ??

Answer 21

-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

Question 22

inherited forms of disease

Answer 22

• 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

Question 23

Large tomatoes

Answer 23

Induce repeat of S phase

Question 24

Meiosis

Answer 24

No M phase

Question 25

RAD9: a paradigm for S/M

Answer 25

checkpoint control in
S cerevisiae
Cells lacking RAD9 are healthy in the absence
of extrinsic interference

Question 26

CDC9 encode

Answer 26

DNA ligase- bonds okazaki fragment essentially you get fragment

Question 27

Lack of CDC9

- lee hartwell

Answer 27

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

Question 28

What can you use to screen for checkpoint mutants ?

Answer 28

Can use loss of viability as a screen for checkpoint mutants

Question 29

PCC

Answer 29

a phenotype associated with loss of S/M checkpoint

Premature condensation

Question 30

Checkpoints:

Answer 30

abrogated by small molecular inhibitor

Question 31

Example of integral dependency

Answer 31

bacteriophage assembly

Question 32

Lee Hartwell

Answer 32

identified a mutant in the RAD 9 gene in cerevisiae

Question 33

Schlegel and Pardee

Answer 33

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

Question 34

cell free system

Answer 34

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

Question 35

Carl Smythe and J Newport experiment

Answer 35

** coupling of mitosis to the completion of Sphasein xenopus occurs via modulation of the tyrosine kinase that phosphorylates P34 cc2 ***

Question 36

Carl Smythe and J Newport experiment

Answer 36

• • 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

Question 37

Break down of control elements

Answer 37

• sense incomplete DNA replication
• DNA damage
• Transducers that signal to effectors
• ordinarily the pathways block cell cycle transitions
• decide > apoptosis or Activate DNA repair

Question 38

Two components of the pathway - transducers

Answer 38

ATR or ATM both protein kinases

- ATR activated when incomplete DNA replication phosphorylates and activates chk1 which acts on cell cycle regulators

Question 39

ATR

Answer 39

Target of caffeine

ATm also caffeine sensitive

Question 40

Caffeine

Answer 40

inhibits ATR pathway - no switch on of pathway > catastrophic mitosis

Question 41

Replication stress is identified by

Answer 41

ssDNA- Single stranded DNA – sensor complex assembly activates Chk 1 which blocks further replication and inhibit cell cycle progression
- stabilise rest of replication fork

Question 42

To acquire a mutator phenotype

Answer 42

many cancers have lost some checkpoint controls

Question 43

inherited loss of checkpoint component proteins

Answer 43

causes cancer

Question 44

many cancers who have lost their check point controls provide

Answer 44

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