Oncogenes and Tumour Supressor Genes Flashcards
- List the four major functional changes in cancer?
- Inc Growth ( loss of growth regulation–> Promote growth eg angiogenesis)
- Failure to undergo apoptosis/Senescence (loss of cell growth/division)
- Loss of differentiation (alternations in cell migration and adhesion)
- Failure to repair DNA damage and chromosomal instability
- What are normal oncogenes components of? What is the clinical significance of this?
Normally components of growth factor signalling pathways
When these pathways are mutated they produce products in higher quantities or there altered products have increased activity and so act in a dominant matter
- What kind of signal do tumour suppressor genes give?
What is the response to this signal
A stop signal to uncontrolled growth
May inhibit cell cycle or trigger apoptosis
- Compare the function of oncogenes in their normal orientation to their mutated orientation?
Normally: Drive cell divison . Called proto-oncogene
Cancer: Mutations that make it permanently active . Continuous dividing
- Compare the function of tumour suppressor genes in their normal orientation to their mutated orientation?
Normally: They counteract the oncogene, they stop cell division
Cancer: Mutations that switch the gene off so there is no stopping cell division
- Out of oncogenes and tumour suppressor genes , which one when mutated experiences loss of function, and which one experiences gain of function?
Oncogene = Gain of function
Tumour Suppressor Gene = Loss of function
- For an proto-oncogene to become an oncogene only ONE mutation event in necessary. Is this the same for tumour Suppressor genes?
NO
Two inactivating mutations are required to eliminate the tumour suppressor gene
If one- no effect
- Who is Rous?
He basically discovered the Rous sarcoma virus (RSV)
Discovered this using the cell free filtrate from a chicken sarcoma
- Explain Rous’s protocol for inducing sarcoma in chickens?
- Get a chicken with sarcoma in the breast muscle
- Remove the sarcoma and break it up into small chunks of tissue
- Grind up the sarcoma with sand
- Put this through a fine-pore filter and collect the filtrate
- Inject this filtrate into a young chicken
- Observe the sarcoma in the injected chicken
- The following questions refer to Rous protocol
- How long did tumours take to develop after injection?
- How many times could the cycle be repeated
- How could Rous conclude that a virus must be responsible for the tumour formation?
-Tumours developed weeks later
-Cycles could be repeated indefinitely
-Filter used excluded bacteria but didn’t exclude virus - so he knew it was a virus
Thats why its called Rous Sarcoma Virus
- What gene does Rous Sarcoma have?
SARC gene
Viral SARC is a mutated gene
- Why were retroviruses important experimentally?
- Technological advances
- Funding
- Improved tissue culture techniques
- The discovery of reverse transcriptase, RNA genome, replicates via DNA intermediate and that they are enveloped.
13.So how was Rous Sarcoma Virus caused?
Basically there was oncogenic transformation of a typical retrovirus - this was caused by an extra gene an ‘oncogene’ called v-SRC
- What is the difference between c-src and v-src ?
c-src = cellular oncogene v-src = proto oncogene altered form transduced by retroviruses
- Is it possible to have some genes of cancer causing viruses that are mutated forms of the cellular gene and not viral genes?
What is the case for the rous sarcoma viral gene?
Yes it is possible
The rous sarcoma viral gene was a host gene (C-SRC) that had been ‘kidnapped’ by the virus and transformed into an oncogene (V-SRC)
- Explain the capture of C-SRC by retrovirus?
- During evolution the virus acquires fragments of genes from the host cell at integration sites= creating of oncogenes
1. The virus infects and reverse transcription
2. Created dsDNA provirus
3. Accidentally integrated next to c-src
4. Creates v-src
5. Packaged into capsid
6. Rous Sarcoma virus carrying SRC sequence
- What is the v-SRC oncogene characterised as? What does it do?
In this case the oncogene was characterised as a 60kDa intracellular tyrosine kinase , it can phosphorylate cellular proteins and effect growth.
- How did Bishop and Varmus discover the v-src oncogene as responsible for cancer?
- Using hybridizations experiments they found the c-src gene present in many genomes of species
- Then showed that the c-src gene was used in positive regulation of cell growth and cell division
- After infection the v-src gene was expressed in high levels -> uncontrolled cell growth , unrestricted host cell division and cancer
- Give some examples of agents that transform cells by switching on the oncogenic info?
Radiation
Chemical Carcinogens
Exogenously added viruses
- Approximately what % of all human cancers are caused by oncoviruses?
15-20%
- How can viral oncogenes be transmitted?
What kind of infection can DNA viruses cause?
- DNA or RNA viruses
- DNA can cause lytic infection—> death of the cellular host or replicate their DNA along the host and promote neoplastic (tumour) formation
- Compare DNA viruses and RNA viruses?
DNA Viruses:
-Encode various proteins and along with environmental factors —> Initiate and maintain tumours
RNA Viruses:
- Incorporate DNA copies of their genome into the genome of host cells - these will contain transforming oncogenes —> Cancerous transformation of the host
- Finish off the sentence:
“There are examples of oncogenes for every type of protein involved in a …….
growth factor signal transduction pathway
- How are oncogenes captured and activated?
Oncogenes are captures by animal retroviruses and are altered in human cancer
Activation can involve mutations , insertions, amplifications and translocations. These lead to loss of response to growth regulatory factor, remember only one allele needs to be altered.
- What are the 4 types of proteins involved in the transduction of growth signals?
- Growth Factors
- Growth factor receptors
- Intracellular signal transducers
- Nuclear transcription factors
- Give an example of an oncogene protein that acts as a:
growth factor
growth factor receptor
intracellular signalling molecules
Growth factor = EGF
Growth factor receptor = ErbB
Intracellular signalling molecules= Ras and Raf
- Which pathway do Ras and Raf activate and briefly explain this process
Activate the ERK MAP Kinase pathway which leads to the induction of additional genes (eg fos) that encode potentially oncogenic transcriptional regulatory proteins
- The following questions refer to the RAS oncogene family:
- How were Ras genes identified
- What kind of proteins are RAS proteins?
- What are they normally bound to in their neutral state?
- What % of human cancers are due to oncogenic activation of ras- is this common?
-ras genes identified from 2 studies of cancer-causing viruses ( The Harvey sarcoma virus and Kirsten sarcoma virus) discovered in rats hence the name ( RAtSarcoma= RAS)
-RAS proteins are small GTPases
-Normally bound to GDP
-30%
Most commonly mutated oncogene
- In the RAS gene:
- Which codons do the point mutations occur in?
- What tranformations does this cause
- What cancer does this transformation cause?
-Point mutations in Codon 12,13,61
Glycine–>Valine = Bladder Carcinoma
Glycine –> Cysteine = Lung cancer
- Explain in steps a detailed list of how the RAS protein acts
- Binding of extracellular growth factor signals promotes recruitment of RAS proteins to the receptor complex
- This promotes Ras to exchange GDP (inactive ras) with GTP (active ras)
- Activated ras then initiates the remainder of the signalling cascade - mitogen activated protein kinase
- These kinases ultimately phosphorylate targets - eg TF to promote expression of genes
- Ras hydrolyses GTP–> GSP, and then “turns off”
- Explain what happens in the Ras protein after the mutations?
-Loss of GTPase activity , it usually required to turn active RAS-> inactive RAS GDP
and so constant activation
- List the 3 members of the MYC oncogene family and the proteins they encode
- C-MYC = C-Myc
- MYCN = N-Myc
- MYCL-LMyc
- How was the MYC oncogene originally identified?
In the avian myelocytomatosis virus (AMV)
34.What family do the MYC oncoprotein belong to?, what % of the genome does this family regulate the transcription of?
Transcription factors
15%
- List some major downstream effectors of MYC?
- Ribosome Biogenesis
- Protein translation
- Cell-cycle progression and metabolism
- Orchestrating a broad range of biological functions( eg cell prolif, differentiation, survival and immune surveillance)
- How does the MYC gene act normally and how does it act in cancer?
Normally:
Encodes a helix-loop-helix leucine zipper transcription factor that dimerizes with its partner protein (Max) to transactivate gene expression
Cancer:
MYC oncogene is over-expressed in the most cancers ( 40% tumours due to this). Generally activated when it comes under the control of foreign transcriptional promoters–> deregulation of oncogene—> Relentless proliferation
- The following questions refer to Burkitts lymphoma (BL):
- Which virus is associated with BL?
- What type of lymphoma is BL?
- At what age would you get BL?
- Who has reduced resistance to BL?
- Epstein Barr Virus is associated with BL
- It is a high grade lymphoma
- Effect children from 2-16 yrs
- In central Africa, children with chronic malaria infections have reduced resistance to the virus- known as classical african or endemic BL
- Explain how MYC oncogene is activated in BL?
So in all BL cases, there are 1 of three characteristic chromosomal translocations( could involve chromosome 2,14, or 22) that place the MYC gene under the regulation of the Ig heavy chain and so c-myc expression is deregulated
All translocations, a region from the chromosome (2,14 or 22) is fused to section of chromosome 8
- The following questions refer to Chronic myelogenous leukaemia (CML)
- What % of leukemia’s does it account for?
- 95% of CML patients carry which chromosome?
- What is this chromosome a product of?
- What is the result of the BCR-ABL fusion protein?
- What is a therapeutic strategy for CML?
- 15%
- Philadelphia Chromosome
- Product of chromosomal translocation t(9;22)(q34;q11) generating the BCR-ABL fusion protein
- Tyrosine kinase activity of the oncogene ABL is constitutive –> abnormal cell proliferation
- Imatinib (Gleevac) is a tyrosine kinase inhibitor ( 96% remission in early stage patients)
- What did Henry Harris discover after performing somatic cell hybridization experiments?
Fusion of normal cells + tumour cells = Hybrid cells containing chromosomes from both parents.
Cells not capable of forming tumours –> Genes from normal parent inhibited/suppressed tumour development (tumour suppressor genes)
- The following questions refer to tumour suppressor genes:
- What are they usually regulators of?
- What does loss of tumour suppressor gene function require?
- What can inactivation be result of?
- What type of genes are tumour suppressor genes defined as?
- What are they sometimes referred to as?
- Cell cycle checkpoints (eg RB1), Differentiation (eg APC) or DNA repair ( eg BRCA1)
- Inactivation of both alleles of the gene
- Mutation or deletion
- Recessive genes
- anti-oncogenes
- How many oncogenes and tumour suppressor genes identified?
At least 100 oncogenes
At least 15 tumour suppressor genes
- The following questions refer to retinoblastoma (RB):
- At what age are you likely to get it?
- How does it develop?
- What will an eye that contains a tumour be able to do?
- What is the technical term for “Cat eye’s appearance”
- What are the two forms of this disease?
- What chromosome is the RB1 gene on?
- Rare CHILDHOOD cancer
- Develops when immature retinoblasts continue to grow very fast and don’t turn into mature retinal cells
- An eye that contains a tumour will reflect light back in a white colour- cat eye appearance
- Technical term in leukocoria
- Two forms of the disease :Familial(40%) and Sporadic (60%)
- The hereditary mutation is on chromosome 13, the retinoblastoma 1 (rb1) gene
- What does the term “ loss of heterozygosity” mean in reference to tumour suppressor genes?
Used to describe the process that leads to inactivation of the 2nd copy of a tumour suppressor gene A heterozygous ( one mutated , one not) receives a 2nd hit in its remaining functional copy of the gene --> homozygous ( both mutated )
- Mutations that inactivate tumour suppressor genes are called “loss of function mutation”, what kind of mutations do they cause?
Often point mutations or small deletions –> Disrupt function of protein
- The following questions refer to the structure of RB
- List the three members of the RB family
- What are the members collectively known as?
- What cellular pathways does RB function in?
- How does RB regulate these processes?
- What is RB’s main binding partner?
- What does this interact with
- What else can bind to RB?
- Rb/(p105/110), p107 and Rb2/p130
- Known as pocket proteins
- Diverse cellular pathways such as apoptosis and the cell cycle
- regulates via stimulation or inhibition of the activity of interacting proteins
- Main binding protein is E2F transcription factor
- Interacts with the large pocket
- Other viral oncoprotein can bind to Rb
- What is the main function of the retinoblastoma protein?
Why is Cyclin D important?
How is RB used in Cyclin D and E families?
-Main function is to regulate the cell cycle by inhibiting G1 to S phase transition
The important proteins involved in the cell cycle are : Cyclins and CDK’s
Cyclin D is the first cyclin to be synthesized and drive progression through G1 together with CDKs4/6
G1 checkpoint leads to arrest of the cell cycle in response to DNA damage
A key substrate for Cyclin D is RB protein
Cyclin D and E families and their cdk’s phosphorylate RB
48.Explain the function, phosphorylation and activation of RB?
RB protein regulates the activity of E2F transcription factor - needed for expression of genes required for S phase
RB activity is regulated by phosphorylation
Hypophosphorylated form: This is how RB exists No phosphate groups When bound to E2F, prevents progression into S phase --> Triggers cell death Active-Inhibit cell prolif Remains bound to E2F
Hyperphosphorylated:
-Becomes hyperphosphorylated in response to extracellular physiological signals
-Inactive
-E2F is released and migrates to nucleus–> Induces transcription
Cell cycle progression from G1 to S occurs
- The following questions refer to the inactivation of RB:
- How can RB be inactivated?
- How is pRb inactivated in Retinoblastoma?
- What is viral inactivation found in?
- Give 3 examples
- What happens in cancer cells?
- Rb can be inactivated by phosphorylation, mutation or viral oncoprotein binding
- In retinoblastoma, pRb is inactivated by mutations or partial deletions
- Viral inactivation found in small DNA tumour viruses mainly by disrupting E2F binding or destabilisation of Rb:
1. Adenovirus-E1A
2. Papilloma-E7
3. Polyma-Large T antigen - In cancer cells RB phosphorylation is deregulated throughout the cell cycle –> E2F TF can induce deregulation of cell cycle –> Cell move through G1 into S phase without proper checks from RB
- The following questions refer to p53 tumour suppressor gene:
- What was the 1st tumour suppressor gene identified?
- What has p53 been nicknamed?
- What is it involved in?
- In what % of cancers is it mutated in?
- What does the freq mutation of p53 in tumours suggest?
- what does p53 specialise in?
- First tumour suppressor gene to be identified in P53
- “Guardian of the genome”
- Involved in sensing DNA damage and regulating cell death/apoptosis
- 30-50%
- Tumour cells try to eliminate p53 function before they can thrive
- Specialize’s in preventing the appearance of abnormal cells
- What is the structure of p53 tumour suppressor gene? ( list the 4 domains, how many gene promoter regions can it bind to, what is its main role as?)
-Protein has an amino transactivation domain, a central DNA binding domain, a tetramerization domain and a carboxyl regulatory domain
o Can bind to around 300 different gene promoter regions
main role as a transcription factor
- Explain the regulation of P53 by MDM2?
MDM2 is a ubiquitin ligase
Adds ubiquitin to lycine residues of molecule so is targeted to proteasome for proteolytic
degradation
Normally levels of p53 protein are low in cells
These levels are kept low by MDM2 protein, a ubiquitin ligase (also an oncogene)
In unstressed normal cells both p53 and MDM2 move between the nucleus and cytosol
MDM2 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 p53 has a short 20 min half life
- How is P53 activated?
tress signals are able to activate p53
o Signals are sensed by mainly kinases that then phosphorylate p53
o Phosphorylation of p53 disrupts the interaction between it and MDM2
e.g. ionizing radiation signals through two kinases ATM/ATR activate oncogenes such as ras induce activity of p14arf responsible for sequestering MDM2
o P53 can thus regulate genes involved in DNA damage repair, apoptosis and cell cycle arrest o Need p53 to be able to keep on top of DNA damage mechanisms
- What happens when p53 is mutated?
o Mutational inactivation is considered to be one of the most common molecular mechanisms behind the dysfunction of p53
o Extensive mutation search revealed that more than half of human cancers carry loss of function mutations of p53
o Among them, 95% of mutations were detectable within the DNA-binding domain
- How can P53 be used as a promising therapeutic target?
Role of p53 a s star player in suppressing tumourgeneisis makes it a promising therapeutic target o Different strategies aimed at:
Correcting p53 mutation and restoring wild-type p53 function by targeting its regulators
- What are some therapeutic strategies against P53 damage?
Gene therapy obvious approach
o Many vectors and retroviruses have been examined
o Retroviruses integrate in a stable form into the genome of infected cells. It has been demonstrated that o retrovirus-mediated gene transfer of the wild-type TP53 gene into both human lung tumour cell lines
and xenograft models could lead to the inhibition of tumour cell growth o Alternative strategies- use of inhibitors
Inhibitors can be used in two ways:
1. Refold mutant p53 by modifying the thiol groups in the core domain of the protein e.g. PRIMA 1
2. Regulate the regulators of p53
- i.e. MDM2; the half life would therefore be greater so more ability to function as a tumour
suppressor
- Nutlin (MDM2 inhibitor) prevents the ubiquitination of the molecule
- What is genetic analysis and personalised medicine?
o A detailed readout of the molecular faults in a patient’s tumour, and new generation of drugs that precisely target them
o Classifies tumours according to their genetic make-up instead of where they grow in the body
o People with the ‘same’ cancer can have different forms of the disease so responses to treatment vary o Cancers growing in different parts of the body may also share the same genetic faults so respond to
similar treatments
