Oncogenes and Tumour Suppresor Genes Flashcards
What are the major functional changes in cancer ?
- Increased growth -loss of growth regulation , stimulation of environment promoting growth e.g. -angiogenesis
- Failure to undergo programmed cell death (apoptosis or senescence )
- Loss of differentiation (including alterations in cell migration and adhesion)
- Failure to repair DNA damage (chromosomal instability )
What is an oncogene mutation and what can it lead to ?
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
What is a tumour suppressor mutation and what can it lead to ?
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 )
What was Rous’s protocol for inducing sacroma in chickens ?
- Chicken with sarcoma in breast muscle
- Broke the sarcoma into small tissue chunks
- Ground with sand
- Filtrate
- Inject the filtrate into young chicken
- Observe sarcoma in injected chicken
He concluded that a virus must be responsible for the induction of tumour formation.
Rous sarcoma virus
How were retroviruses important experimentally ?
Technological advances
Funcing
Improved tissue culture techniques
The discovery of reverse transcriptase , RNA genome , replicates via DNA intermediate and they are enveloped
What is V-Src
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.
Describe the process of capture of c-src by retrovirus
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
Describe the typical retroviral life cycle
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
What does the v-src oncogene cause ?
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.
Describe Viral oncogenesis and how it can be transmitted
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.
Describe DNA viruses
DNA viruses- These encode various proteins along with environmental factors can initiate and maintain tumours.
Describe RNA viruses
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.
Describe the process of oncogene activation
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
What are different ways in which oncogenes can be activated ?
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
What are the four types of proteins which are involved in the transduction of growth signals?
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
How do oncogenes act as growth factors ?
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
What are the functions of Ras/Raf ?
Activates the ERK MAP kinase pathway , leadings to the induction of additional genes (fos) that encode potentially oncogenic transcriptional regulatory proteins.
Give an example of an intracellular signal transducer
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
What is the normal function of RAS.Outline its mechanim .
Intracellular signal transducers
- Binding of extracellular growth factor signal
- Promotes recruitment of RAs proteins to the receptor complex
- Recruitment promotes Ras to exchange GDP (inactive Ras ) with GTP (active RAs)
- Activated Ras then initieates the remainder of the sigballing cascade (mitogen activated protein kinases)
- 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
Describe the difference in this mechanism in ‘hyperactive ‘ Ras
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
Outline the MYC oncogene family and what its function is
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
Outline all the functions of the MYC family of genes
Signal transduction Cell cycle Metabolism Translation DNA repair Cell adhesion /cytoskeleton MicroRNAs Protein biosynthesis Transcription
Outline the mechanism through which MYC is activated
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
Outline the mechanism of activation of MYC in Burkitt’s Lymphoma
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
Give an additional example of chromosomal translocation as a method of activating oncogenes
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
What are tumour suppresor gene s?
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/
What is Retinoblastoma ?
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
How was Retinoblastoma discovered?
Proposed :
Development of retinoblastoma requires two mutations which cause loss of the functional copies of the Rb gene =Two hit hypothesis .
What is loss of heterozygosity. Define this
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
Outline the Retinoblastoma protein structure
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.
How is Rb involved in the cell cycle ?
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)
What is the first cylcin which is synthesised in the cell cycle ?
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
What are the consequences of phosphorylated RB?
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
What other methods inactivate Rb leading to loss of function ?
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
What is p53 and what is its function ?
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
What is the structure of p53?
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
What is the normal regulation of p53 ?
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
How is p53 activated ?
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
Outline the therapeutic strategies which have been used to treat p53 mutations
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.
- 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)
