Oncogenes and Tumour Suppresor Genes

Question 1

What are the major functional changes in cancer ?

Answer 1

1. Increased growth -loss of growth regulation , stimulation of environment promoting growth e.g. -angiogenesis
2. Failure to undergo programmed cell death (apoptosis or senescence )
3. Loss of differentiation (including alterations in cell migration and adhesion)
4. Failure to repair DNA damage (chromosomal instability )

Question 2

What is an oncogene mutation and what can it lead to ?

Answer 2

Gain of function mutation-

• An altered gene whose product can act in a dominant fashion to help make a cell cancerous
• Oncogene is a mutant form of a normal gene (proto-oncogene) involved in the control of cell growth or division

A single mutation - Activates cell proliferation

Question 3

What is a tumour suppressor mutation and what can it lead to ?

Answer 3

This is loss of function -

A gene whose normal activity prevents formation of a cancer

Both genes for the tumour suppressor must be mutated

Loss of this function by mutation enhances the likelihood that a cell can become cancerous (a normal process to maintain control of cell division is lost )

Question 4

What was Rous’s protocol for inducing sacroma in chickens ?

Answer 4

1. Chicken with sarcoma in breast muscle
2. Broke the sarcoma into small tissue chunks
3. Ground with sand
4. Filtrate
5. Inject the filtrate into young chicken
6. Observe sarcoma in injected chicken

He concluded that a virus must be responsible for the induction of tumour formation.

Rous sarcoma virus

Question 5

How were retroviruses important experimentally ?

Answer 5

Technological advances
Funcing
Improved tissue culture techniques
The discovery of reverse transcriptase , RNA genome , replicates via DNA intermediate and they are enveloped

Question 6

What is V-Src

Answer 6

This was an (extra gene ) oncogene found in the genome.

V-src= proto oncogene altered from transduced by retroviruses.

It was conclused that the Rous sarcoma viral gene was a host gene which was ‘ kidnapped ‘ and transformed into an oncogene.

An oncogene is any cellular gene that upon activation can transform cells.

Question 7

Describe the process of capture of c-src by retrovirus

Answer 7

During evolution , the virus can acquire fragments of genes from the host at integration sites and this process results in the creation of oncogenes.

The oncogene product was characterised as a 60kDa intracellular tyrosine kinase.

Can phosphorylate cellular proteins and effect growth

Exception to central dogma DNA-RNA protein

Question 8

Describe the typical retroviral life cycle

Answer 8

There is infection of the host cell. It will then undergo reverse transcription which produces dsDNA provirus .Following this there is accidental integration next to host c-src. The consequence is a fusion which gets packaging into capsid.
This will form a RSV virion which carries the src sequences

Question 9

What does the v-src oncogene cause ?

Answer 9

It was identified that v-src oncogene is responsible for causing cancer

Through hybridisation experiments , they found that the c-src gene was present in the genome of many species.

They then showed that the host cell c-src gene was normally involved in the positive regulation of cell growth and cell division.

Following infection , the v-src oncogene was expressed at high levels in the host cell , leading to uncontrolled host cell growth, unrestricted host cell division and cancer

Proto-oncogenes are normal genes that can control growth.They become active oncogenes following mutation

Various agents including radiation, chemical carcinogens and exogenously added viruses may transform cells by switching on the endogenous oncogenic information.

Question 10

Describe Viral oncogenesis and how it can be transmitted

Answer 10

Around 15-20% of human cancers are caused by oncoviruses.

Viral oncogenes can be transmitted by either DNA /RNA viruses.

DNA viruses can cause lytic infection which can lead to death of the cellular host or can replicate their DNA along with that of the host and promote neoplastic transformation.

Question 11

Describe DNA viruses

Answer 11

DNA viruses- These encode various proteins along with environmental factors can initiate and maintain tumours.

Question 12

Describe RNA viruses

Answer 12

RNA viruses -Integrate DNA copies of their genomes into the genome of the host cell and as these contain transforming oncogenes they can induce cancerous transformation of the host.

Question 13

Describe the process of oncogene activation

Answer 13

Oncogenes for every type of protein involved in growth factor signal transduction pathway

Activation is usually through altering the genetic sequence of the gene.

(Mutations , insertions, amplifications and translocations)

=Loss of response to growth regulatory factors
-One allele needs to be altered.

Proto-oncogene becomes an oncogene

Question 14

What are different ways in which oncogenes can be activated ?

Answer 14

There is a protein encoded by proto oncogene :

a. There can be a mutation/deletion which causes an encoded protein with altered structure/function
b. Gene duplication which can cause increased synthesis of encoded protein
c. DNA regulatory sequence translocated from distant site alters expression of downstream gene which causes increased synthesis of encoded proteins , synthesis of protein containing portions encoded by different genes, protein-coding gene translocated from distant site fuses with portion of gene causing formation of a fusion gene

Question 15

What are the four types of proteins which are involved in the transduction of growth signals?

Answer 15

Proto-oncogenes encode components of the growth factor signal transduction pathways and can be one of the four following proteins:

Growth factors e.g.EGF
Growth factor receptors ErbB
Intracellular signal transducers 
Intracellular signalling molecules e.g. Ras/Raf
Nuclear transcription factors

Question 16

How do oncogenes act as growth factors ?

Answer 16

Majority of oncogenes proteins function as elements of the signalling pathways that regulates cell proliferation and survival in response to growth factor stimulation.

Oncogene proteins act as growth factors e.g.EGF
,growth

Question 17

What are the functions of Ras/Raf ?

Answer 17

Activates the ERK MAP kinase pathway , leadings to the induction of additional genes (fos) that encode potentially oncogenic transcriptional regulatory proteins.

Question 18

Give an example of an intracellular signal transducer

Answer 18

RAs proteins are small GTPases that are normally bound to GDP in a neutral state.

Oncogenic activation of ras is seen in around 30% of human cancers.

Activated through mutations.
Codons 12,13,61
Glycine to valine -Bladder carcinoma (codon 12)

Glycine to cysteine -Lung cancer

Question 19

What is the normal function of RAS.Outline its mechanim .

Answer 19

Intracellular signal transducers

1. Binding of extracellular growth factor signal
2. Promotes recruitment of RAs proteins to the receptor complex
3. Recruitment promotes Ras to exchange GDP (inactive Ras ) with GTP (active RAs)
4. Activated Ras then initieates the remainder of the sigballing cascade (mitogen activated protein kinases)
5. These kinases ultimately phosphorylate targets such as transcription factor to promote expression of genes important for growth and survival.

Ras hydrolyses GTP to GDP quickly which turns itself off

Question 20

Describe the difference in this mechanism in ‘hyperactive ‘ Ras

Answer 20

There is a point mutation in codons 12,13 and 61

The consequence of each of these mutations is a loss of GTPase activity of the RAS protein normally required to return active RAS to the inactive RAS GDP.

This leads to constitutive activation

Hyperactive RAs protein (product of oncogene ) issues signals on its own

Question 21

Outline the MYC oncogene family and what its function is

Answer 21

The MYC oncogene family consists of 3 members :
C-MYC, MYCN and MYCL which encodes c-Myc, N-MYC and L-Myc

• Originally identified in avian myelocytomatosis virus (AMV)
• The MYC oncoproteins belong to a family of transcription factors that regulate the transcription of at least 15% of the entire genome .
• Major downstream effectors of MYC include those involved in ribosome biogenesis , protein translation , cell cycle progression and metabolism , orchestrating a broad range of biological functions such as cell proliferation , differentiation ,survival and immune surveillance

Question 22

Outline all the functions of the MYC family of genes

Answer 22

Signal transduction 
Cell cycle 
Metabolism 
Translation 
DNA repair 
Cell adhesion /cytoskeleton 
MicroRNAs
Protein biosynthesis 
Transcription

Question 23

Outline the mechanism through which MYC is activated

Answer 23

The Myc oncogene is overexpressed in majority of human cancers and can causes at least 40% of cancers

Encoded a helix-loop-helix leucine zipper transcription factor that dimerises with its partner protein -Max= transactivates gene expression

Activated when it comes under control of foreign transcriptional promoters.
Leads to deregulation of the oncogene which drives proliferation.

This is a result of chromosomal translocation

Question 24

Outline the mechanism of activation of MYC in Burkitt’s Lymphoma

Answer 24

Epstein Barr virus is associated with BL

BL cases carry one of three characteristic chromosomal translocations that place the MYC gene under the regulation of the Ig heavy chain.
c-myc expression is deregulated

In BL three chromosomal translocations occur involving 2,14,22

A region from three chromosomes is fused to a section of chromosome 8
This loses control of Myc

Question 25

Give an additional example of chromosomal translocation as a method of activating oncogenes

Answer 25

Chronic myelogenous leukaemia which accounts for 15-20% of all leukaemias.

95% of CML patients carrying the philadeliphia chromosome which is the product of the chromosomal translocation t(9;22)(q34;q11).
This causes a BCR-ABL fusion protein

Result:
Tyrosine kinase activity of the oncogene ABL is constitutive leading to abnormal proliferation.

Therapeutic strategies for CML include imatinib (Gleevac) a tyrosine kinase inhibitor

Question 26

What are tumour suppresor gene s?

Answer 26

These are used to control the processes which regulate cell numbers

Tumour suppressor gene products act as stop signals to uncontrolled proliferation .
Regulators of cell cycle checkpoints e.g. RB1, differentiation (e.g. APC) or DNA repair (e.g.BRCA1)

Loss of tumour suppressor gene function requires inactivation of both alleles of the gene

Anti-oncogenes/

Question 27

What is Retinoblastoma ?

Answer 27

A rare childhood cancer which develops when immature retinoblasts continue to grow fast and do not turn into mature retinal cells.

Leukocoria

Types:
Familial (40%) /Sporadic (60%)

Mutation is on chromosome 13 (13q14) the retinoblastoma 1 (Rb1) gene

Question 28

How was Retinoblastoma discovered?

Answer 28

Proposed :
Development of retinoblastoma requires two mutations which cause loss of the functional copies of the Rb gene =Two hit hypothesis .

Question 29

What is loss of heterozygosity. Define this

Answer 29

Used to describe the process that leads to the inactivation of the second copy of a tumour suppressor gene , a heterozygous cell receives a second hit in its remaining functional copy of the tumour suppressor gene =becomes homozygous for the mutated gene

Mutations that cause this are called loss-of-function mutations.
Point mutations/small deletions which disrupt the function of the protein that is encoded by the gene

Question 30

Outline the Retinoblastoma protein structure

Answer 30

The Rb gene group includes : Rb/(p105/110), p107 and Rb2/p130
(These are called pocket proteins).

pRb is a multifunctional protein (110kDa).
(A transcriptional co-factor which can bind to transcription factors )

RB’s main binding partner is the E2F transcription factor , interacting with the large pocket.

Question 31

How is Rb involved in the cell cycle ?

Answer 31

The main function of Rb is to regulate the cell cycle through inhibiting the G1 to S phase transition .

2 important proteins involved in the cell cycle are :
cyclins and their associated cyclin dependent kinase (cdks)

Passage of a cell through the cell cycle is regulated cyclins and cyclin dependant kinases (cdks)

Question 32

What is the first cylcin which is synthesised in the cell cycle ?

Answer 32

Cyclin D is the first cyclin to be synthesised and drive progression through G1 together with cdks4/6.

The G1 checkpoint leads to the arrest of the cell cycle in response to DNA damage .

Key substrate for cyclin D is RB protein

Cyclin D + E families and their cdks phosphorylate RB

Question 33

What are the consequences of phosphorylated RB?

Answer 33

Rb protein regulates the activity of the E2F transcription factor crucial for the expression of genes required for the S phase.

Rb activity is regulated by phosphorylation

When the Rb tumour suppressor is active it can inhibit cell proliferation.

When Rb is dephosphorylated /hypo phosphorylated it is active and binds to E2F

When Rb is active it blocks the progression to the S phase.

When Rb is hyperphosphoryted , in response to extracellular physiological signals it is inactive

Question 34

What other methods inactivate Rb leading to loss of function ?

Answer 34

Through mutation or viral oncoprotein binding.

In retinoblastoma , pRb is functionally inactivated by mutations or partial deletions

Viral inactivation found in small DNA tumour viruses mainly by disrupting E2F binding or destabilisatiation of Rb
Adenovirus-E1A
Papilloma-E7
Polyoma-Large T antigen

In cancer cells RB phosphorylation is deregulated throughout cell cycle .E2F transcription factors can induce deregulation of the cell cycle .
Cells will therefore move through G1- S without further checks

Question 35

What is p53 and what is its function ?

Answer 35

The p53 gene was the first tumour suppressor gene to be identified.

The p53 protein isthe ‘guardian of the genome’

Senses DNA damage and regultes cell death/apoptosis

p53 is mutated in 30-50% of cancers

Prevents appearance of abnormal cells

Question 36

What is the structure of p53?

Answer 36

It is a transcription factor

Has a amino transactivation domain , central DNA binding domain , tetramerization domain and a carboxy regulatory domain.

Can bind to around 300 gene promoter regions - Transcription factor

Question 37

What is the normal regulation of p53 ?

Answer 37

Normally low levels of p53 proteins

Kept low by MDM2 protein -ubiquitin ligase

In unstressed normal cells both p53 and MDM2 move between nucleus and cytosol.

MDM” binds p535 to form a complex in the nucleus where MDM modifies the carboxyl terminus of p53 and targets it for degradation by the proteasome .

WT p53has a short 20 min half life

Question 38

How is p53 activated ?

Answer 38

Stress signals are able to activate p53

Signals are sensed by kinases which will phosphorylate the p53.

This will disrupt the interaction between it and MDM”.

Ionising radiation signals through two kinases ATM/ATR activate oncogenes such as ras induce activity of p15arf responsible for sequestering MDM2.

p53 can therefore regulate genes involved in DNA Damage repair apoptosis and cell cycle arrest

Question 39

Outline the therapeutic strategies which have been used to treat p53 mutations

Answer 39

Retroviruses integrate in a stable form into the gnome of infected cells .

Retrovirus-mediated gene transfer of the wild-type TP53 gene into human lung tumour cell lines and xenograft models could lead to inhibition of tumour cell growth.

1. PRIMA-1 restores mutant p53 by modifying the thiol groups in the core domain of the protein
   2.Nutlin-potenti MDM2 antagonist (enhances half life)
   3.RITA binds to p53 and can restore mutp53 activity
   4.Inhibitors of CRM1 result in nuclear accumulation of p53
   (inhibits export)