Cancer Lectures Flashcards
Metastasis
-When the cancer spreads from the primary tumour
-Invades other parts of your body
When do cancer symptoms usually occur?
-Usually symptoms don’t occur until after the primary tumour metastasizes
Radioactive Glucose scan?
-Used to show a bunch of tumours along the body
-Cancer cells are driven by glycolysis for energy which is why a glucose scan is used
Benign vs Malignant tumours
Benign:
- Cells are immobile
-Cancer can normally be removed
-Tumour is localized and surrounded by an extracellular shell
-Cancer at this stage mostly goes undetected
Malignant:
-Cells are mobile and no longer have an extracellular shell around them
-Cells acquire mobility through random mutations
Five most common types of cancers?
-Lung
-Stomach
-Breast
-Colon
-Uterine cervix
Growth of a tumour?
Can take years for the tumour to become visible via X-ray and even longer for it to be palpable and even longer for it to cause death
Why can cancer take a long time?
-Cancer starts from one mutated cell this cell must then proliferate/grow and acquire even more mutations to become cancerous
Sometimes cellls can stop proliferating and then start again this results in it taking a long time to even become cancer
Cancer is a disease of age?
If you cut open an old person they would be filled with benign tumours because overtime they will have accumulated a bunch of mutations. Additionally, as you age your immune system weakens which allows tumours more time to accumulate
How many mutations are needed in one cell to cause cancer?
At least 10
The philadelphia chromosome?
-Found in many patients with chronic myelogenous leukemia
-It is translocation between chromosomes 9 and 22 which creates a longer chromosome 22(philadelphia chromosome)
T/F: The older you are the more at rick you are for developing cancer?
True, since with aging you accumulate mutations increasing your risk of developing cancer
How would cancer be independent of age?
If only a single mutation were required to trigger cancer and this mutation had an equal chance of occuring at any time during life.
Carcinogens?
-Ex. UV, X-rays, chemicals
-Agents, substances or factors that can cause cancer
Delayed onset of cancer following exposure to a carcinogen?
The longer you are exposed to a carcinogen the earlier the cancer will start
Low grade intraepiethelial neoplasia?
Proliferating epithelial cells are found throughout the lower third of the epithelium. The ECM is intact preventing the cells from entrain the bloodstream/tissue
-This a benign tumour
High-grade intraepithelial neoplasia?
-Cells in all the epithelial layers are proliferating and show no sign of differentiation
-No normal cells all of them have been replaced by cancer cells
Invasive Carcinoma?
-Cells gain the ability to destory the basal lamina and invade the underlying CT
-No longer localized and leads to secondary tumours
Pap smear
-Cells taken from the cervix are put onto a slide and stained, just by looking at the shape of the cells you can check for cancer
Normal cerivcal cells?
-Big cells with tiny nuclei(highly condensed_ and well differentiated
Benign tumour cervical cells?
-Cells have larger nuclei, different shape, lymphocytes invade everyhere
-Differentiation and proliferation are abnormal(cells are in various stages, some quite immature)
Invasive carcinoma cervical cells?
-Huge nuclei
-Strange shape
-Lymphocytes present
-Cell all appear undifferentiated
What is clonal evolution?
Somatic cells that are normal and dividing at a certain rate and one of the cells acquires a mutation that provides proliferative advantage. That cell will then start to divide more rapidly and make clones of itself with the same advantage. Eventually, one of these clones can acquire a secondary mutation that provides additional proliferative advantage. Those cells will then also make clones of themselves and then may acquire a third, fourth and fifth mutation. Eventually, it results in cancer cells with multiple mutations that allow the cells to have cancer-like characteristics.
Genetic instability in normal cells?
Normal cells have a low level of genetic instability
-When normal cells hit a selection barrier(ie. low levels of O2 or scarcity of proliferation signals), they are very unlikely to be mutable enough to produce a cell that continues proliferating
Cells with high levels of genetic instability?
-Cells with high levels of genetic instability may suffer deleterious mutation and either proliferate much more slowly than their neighbours or are eliminated by cell death
-These cell lineages can be extinct
Tumour cell precursor genetic instability?
-These cells have the perfect amount of genetic instability. When they encounter a selection barrier such as low O2 most of the time at least one of their cells will contain a requisite genetic alteration allowing them to pass the selection barrier and continue the process of tumour progression
How might tumour cells get past a selection barrier of low O2 levels?
These cells may change their metabolism to glycolysis or recruit more blood vesels
How might tumour cells get past a selection barrier of low proliferation signals?
These cells may develop cells that are growth factor independent
Normal stem cell proliferation?
- Stem cell gives rise to a non-stem cell daughter cell which has limited proliferation capacity
- Stem cell is self-renewing
What happens when a stem cell fails to give rise to a non-stem cell daughter?
The stem cell only gives rise to stem cell daughter cells which don’t have a proliferation limit and thus can proliferate forever leading to a tumour
What happens in the stem cell daughter cell does not have limited proliferation?
The daughter cell will proliferate continously leading to tumour formation
The process of metastasis?
- Cells grow and form a benign tumour in the epithelium(proliferate cancer cells)
- Cells aquire the ability to break through the basal lamina and into the CT
- More mutations in the cells allow them to invade the capillary or lymphatic vessel
- Cells will then adhere to the blood vessel wall
- Cells then acquire a mutation allowing them to cross the blood vessel wall and enter into the tissue of the liver
6.Cells will then proliferate to form another tumour in the liver
How do cells that enter the lymphatic vessels reach other tissues?
Cells enter the lymphatic vessel which eventually drains into the bloodstream then enter the tissue. Often cells in the lymphatic vessels become trapped in the lymph nodes causing metastasis there.
1/1000 malignant tumour cells that enter the bloodstream will survive to produce a tumour at a new site?
Yes it is hard for tumours cells to survive this
Functionof the Ames Test?
Used to test whether a drug/substance is mutagenic and carcinogenic
What bacteria are used for the Ames test?
A strain of salmonella bacteria that have mutation in the gene that allows them to synthesize the essential amino acid histidine
-Without this gene the bacteria are unable to grow unless they are supplied histidine in their environment
How does the Ames test work?
- Expose the salmonella lacking histidine to the substance being tested for mutagenicity alonng with liver extracts
- If the substance causes mutations in the bacteria’s DNA, these mutations could restore the bacteria’s ability to synthesize histidine. The bacteria with these mutations can now grow in a histidine free environment
- If the test substance causes a high number of bacteria to grow on the histidine-free medium, the substance increases the mutation rate and can thus be classified as mutagenic and carcinogenic
- If the test substance does not cause many bacteria to grow on the histidine-free medium, the substance can be classified a non-mutagenic
Why are liver extracts added to the Ames Test?
To replicate how the substance would be metabolized in a living organism, some substances don’t become mutagenic until they are metabolized
How can the sensitivity of the Ames test be increased?
By having the bcateria also have a defect in their DNA repair machinery. This makes them even more susceptible to agents that damage DNA
Aflatoxin B1 and metabolic transformation?
Aflatoxin B1 is an example of a substance that must be metabolized before it becomes mutagenic and causes DNA mutations
THe compound is found in mold that grows of peanuts and grains when stored in humid conditions. It does not become mutagenic/toxic to humans until it is oxidized by the liver and then it causes DNA mutations
How do retroviruses cause cancer?
They incorporate into the genome and suppress the immune system
Immunodeficiency?
When the ability of the immune system to fight disease is reduced/imparied, now viruses that don’t normally affect people can cause cancer
Why has there been a large increase in lung cancer?
due to tobacco smoking and industrialization
Why has there been a decrease in stomach cancer ?
In the early 1900s not many people had a fridge and so they relied on preserved foods rather than fresh foods
Why has colon cancer stayed relatively constant?
It has decreased due to colonoscopies but is still prevalent due to the poor western diet
Why are you more likely to develop breast cancer if you have children later in life?
It is thought that the first-full term pregnancy results in permanent changes in the state of differentiation of the cells of the breast, altering their response to hormones. The later this change occurs the more likely you are to develop breast cancer.
T/F: Exposure to certain reproductive hormones such as estrogen have been shown to cause breast cancer?
True
Two types of cancer causing genes?
- Tumour suppressor genes/loss of function genes
- Oncogenes/gain of function genes
Characteristics of tumour suppressor genes?
- There are two copies in the cell
- These genes function to stop cell proliferation
What happens if only one of your tumour suppressor genes is mutated?
You are normally fine since you still have another gene expressing the protein (haplosufficient)
If the gene is haploinsufficient then the mutation in one allele will cause you to have a phenotype
T/F: Heterozygotes of tumour suppressor genes tend to get tumours more frequently than normal wild-type people?
True
T/F: Tumour suppressor genes are recessive?
True, both alleles of the cancer critical gene must be lost to drive a cell toward cancer
Characteristics of oncogenes?
-Genes have two copies in the cell
-These genes push cells into proliferation and prevent cell death
-These are dominant/gain of function mutations where only a single allele needs to be mutated to drive cancer
Normmal function of oncogenes?
They are turned on/off in cells when needed (tightly regulated)
Mutated oncogene?
Makes the gene permanently active which causes unregulated cell proliferation
T/F: A mutation in a single oncogene will cause a tumour?
False, there needs to be multiple mutations
Oncogenes used by viruses?
Oncogenes can be used by viruses who pick up oncogenes from the host cell and then express the oncogene resulting in unregulated expression
What was discovered first tumour-suppressor genes or oncogenes?
Oncogenes since they are dominant
What happens when you place cancer cells onto a Petri dish?
Cancer cells will proliferate and pile up onto each other since they lack contact inhibition
What happens when you put oncogenes in normal cells?
Since the oncogenes are dominant they will override the endogenous proteins and cause multilayered uninhibited cancer cells
What happens when you put a mutant tumour suppressor gene in normal cells?
Nothing happens since the cell already has two healthy copies of the tumour suppressor gene and since they act in a recessive manner the wild-type copies mask the mutant one
What happens in nonhereditary retinoblastoma?
- All cells initially contain two functional copies of the Rb gene
- Ocasionally, a cell can inactivate one of its two functional Rb genes. (does not cause loss of function since still has one copy of the gene)
- If the second copy in the same cell is inactivated then the gene loses function of the Rb gene and the cell will proliferate excessively and give rise to a tumour
Hereditary Retinoblastoma?
- In these patients, they only inherit one unfunctional copy of the Rb gene in their cells(heterozygous).
- If any of these cells gain a second mutation in the second copy of the Rb gene then the Rb gene becomes fully unfunctional.
- Now tumours may occur
Proto-oncogene converted to oncogene via deletion/mutation?
A deletion/mutation in the proto-oncogene sequence can lead to the hyperactive protein(oncogene protein) being made in normal amounts.
Proto-oncogene converted to oncogene via gene amplification?
This is when the normal proto-oncogene is over transcribed resulting in the protein being overproduced. Could be caused by the mechanism that down regulates the gene being overwhelmed
Proto-oncogene converted to oncogene via chromosome rearrangement?
A proto-oncogene can be fused to a strong promoter/regulatory sequence, causing the normal protein to be overproduced or the fused protein could make a hybrid that is produced in high amounts or a fusion protein that is hyperactive
Double minute chromosomes?
Miniature chromosomes that all contain an oncogene resulting in its amplification in the cell
T/F: A cell could contain multiple chromosomes with the same oncogene?
True, this is seen with the Myc gene amplification
7 ways of losing the remaining good copy of a tumour suppressor gene?
- Nondisjuction: loss of the chromosome
- Nondisjunction and duplication: loss of good copy and duplication of mutated
- Mitotic recombination
- Gene conversion: converts to mutant gene
- Deletion: Normal gene is deleted
- Point mutation: makes the functional gene inactive
- Silencing: gene is no longer expressed, no DNA alteration
How do you know how the remaining tumour suppressor gene was lost?
Must sequence the chromosomes, cells with the that do not die in liquid suspension and are able to proliferate contain the oncogene
How were transgenic mice created containing Ras and Myc?
Myc and Ras are both proto-oncogenes that were made into oncogenes in the mice by fusing the proto-oncogenes with the mammary tumour virus regulatory DNA. This drives the overexpression of the proto-oncogenes in the mammary tissue.
Latency?
Delay between when the oncogene was inserted and when it started development of tumour
What happened in mice that only contained the Myc oncogene and what can we interpret?
These mice have a long latency and then tumours develop
Interpretation: Myc overexpression activation is not sufficient to cause a tumour, you have to have other things going on
What happened in mice that only contained the Ras oncogene and what can we interpret?
These mice have a shorter latency suggesting that the Ras mutation is more aggressive. If you make transgenic Ras mice in another tissue the tumour may take more time to form.
What happend in mice that contained both Myc and Ras oncogenes and what can we interpret?
Tumour formation was much faster in these mice
The two genes work together to cause tumour formation
Still need other mutations to cause cancer but these two genes together have a powerful effect
What did the Myc and Ras mice experiment show?
In all three cases the tumours arise only after a delay and only from a small proportion of the cells in the tissues where the two genes are expressed. Some further accidental changes, in addition to the two oncogenes is most likely required for the development of cancer.
T/F: Products of both oncogenes and tumour suppressor genes often occur within the same pathways?
True, certain genes drive the pathway(oncogene) and other genes block the pathway(tumour suppressor genes)
How do scientist study the signalling pathways in cancer?
Scientist will find the gene responsible for the cancer and then study what is upstream/downstrwam of it. To find things that are potentially easier to target.
What is typically upstream in a signalling pathway?
-Receptors
What is typically downstream in signalling pathways?
Signalling molecules
-Ex. kinase, phosphatases
At the end of the pathway is typically a transcription factor
Signalling pathways in cancer cells?
Multiple of these pathways are typically altered in cancer cells(5-10), depending on where the cancer cell is
Why dont cancer drugs work for every type of cancer?
Because different cancers turn on/off different signalling pathways in the cell
Most common signalling pathway mutated in cancer cells?
- p16(Cdk inhibitor), inhibit 2
- Cyclin D1/Cdk4 complex(G1-Cdk), inhibits Rb
- Rb, inhibits E2F
- E2F leads to activation of genes that control entry into S phase
What molecules of the pathway are tumour suppressor genes ?
- p16 when mutated causes increased G1-Cdk , normally represses proliferation by inhibiting G1-Cdk
- Rb protein, normally represses proliferation by inhibiting E2F
What happens if Rb is mutated and you treat the cell with a drug that inhibits G1-Cdk?
Nothing will happen since Rb is further downstream from G1-Cdk. E2F needs to be regulated by a drug.
What kind of drug could help a mutation in p16?
If p16 is mutated, you need to find a drug that can inhibit G1-Cdk
Why are kinase inhibitory drugs “dirty”?
Because they tend to inhibit many other kinases and not just the one we want
Why are viruses easier to target than oncogenes/tumour-suppressor genes?
Because viruses have their own genome and don’t have the same proteins as us
T/F: most of the time patients must take a combination of drugs?
True, since in most patients multiple pathways are typically affected
How can we target a mutated tumour suppresor gene with a drug?
-It is not possible since they are loss of function mutations which means protein is not present
How the Rb signalling pathway controls cell cycling?
- The Rb protein inhibits entry of the cell into S phase when it is unphosphorylated by binding E2F and preventing it from transcribing the S phase entry genes.
- The complex Cdk4-cyclin D1 phosphorylate the Rb, encouraging cell proliferation
- When a cell is stressed p16 will bind the Cdk4-cyclin D1 complex and prevent cell proliferation
- Inactivation of p16 or Rb by mutation encourages cell division while oceractivity of Cdk4 or cyclin D1 encourages cell division
The antiproliferation pathway?
- An anti growth factor binds to TGFB receptor which causes the phosphorylation of Smad3 and causes Smad3 to form a complex with Smad4 which is a transcription factor.
- The Mad complex enters the nucleus and induces transcription of anti-proliferative genes such as p15 and PAI-1
p15?
Gene that encodes proteins that inhibit the cell cycle
PAI-1?
Gene that encodes proteins that inhibit proteases that degrade ECM proteins
-Prevents proliferation
What happens if you have a loss of function in the Smad3/4 receptor?
Now there is no anti-proliferative signal which can lead to increased proliferation and metastasis
Decreased production in p15 or PAI-1?
Increases proliferation and risk of metastasis
How replication of damages DNA can lead to chromosomes abnormalities, gene amplification and gene loss?
- Accidental DNA damage occurs in a cell that lacks p53 protein, instead of halting at the p53 checkpoint the cell enters S phase.
- The DNA damage caused a strand break, resulting in a chromatid with no telomere.
3.When the cell goes into S-phase it will replicate it DNA and since both chromatids are missing telomeres their ends will fuse together. - Now when the chromatids are pulled apart during anaphase another break will occur which can cause one of the chromatids to have two copies of a proto-oncogene.
- If this happens over and over again you can end up with a chromosome with many copies of the oncogene.
- Selection in favor of cells with increased numbers of copies of a gene in the affected chromosomal region will lead to mutants in which the gene is amplified to high copy number
- The chromosomal disorder can also lead to loss of genes, with selection in favour of cells that have lost tumour suppressors
Breakage-fusion bridge cycle?
When chromosomes undergo repeated rounds of chromatid fuion, unequal breakage and replication
Papillomavirus DNA?
Double stranded circular DNA
Benign papillomavirus wart/tumour?
The papillomavirus chromosomes are stably maintained in the basal cells of the epithelium as plasmids whose replication is regulated so as to keep regulated with the chromosomes of the host
How does the papillomavirus generate cancer?
Rare accidents cause the viral plasmid to integrate into a host chromosome. This disrupts the control of the viral gene expression. Unregulated production of the viral replication proteins interferes with the control of cell division thereby promoting cancer
HPV benign vs Malignant?
Benign: The virus gets into the cell and drive proliferation of its own genome resulting in benign warts
Malignant: When the virus has been in the cell for a while it will integrate into the host DNA which can affect proliferation of the host genome
Activation of cell rpoliferation by papillomavirus?
The papillomavirus uses its own viral protein, E6 and E7, to sequester the p53 and Rb respectively. E6 binds p53 leading to its degradation via Ub and E7 binds Rb inactivating it.
SV40 virus
Infects monkeys and uses a single dual purpose protein called T antigen to bind and sequester both p53 and Rb to promote cell proliferation
How do cells that lack telomerase stop dividng?
Most human cells lack telomerase therefore as cells divide the telomeres shrink. When the cell no longer has functional telomeres it will produce an intracellular signal that triggers activation of p53 and cell division arrest.
What happens to cells lacking telomerase and that have mutated p53?
- Telomeres will shorten and the cell will continue to divide due to lack of p53.
- This will cause the cell to enter a breakage-fusion-bridge cycle that causes massive chromosomal damage.
- Some cells may survive this period of genetic disruption by reactivating telomerase, which halts the catastrophic cycle and restored enough chromosomal stability for cell survival
- These cells can then accumulate additional mutations needed to produce cancer
Steps of metastasis that are difficult?
- Escaping from the parrent tissue
- Ability to survive and grow in foreign tissue
How are those steps of metastasis difficult?
They are difficult in the sense that the step results in a large number of cells that fail or are lost
-It is in these steps that highly metastatic cells are observed to have much greater success than nonmetastatic cells
Rare tumour syndromes?
When individuals are more susceptible to developing one or more types of tumours, often due to inherited mutations in tumour suppressor genes
Exampe of a rare tumour syndrome?
Familial Adenomatous polposis coli
Familial adenomatous polyposis coli?
-Disease that causes polyps to form on the colon which are benign but become malignant when mutated
-Colonoscopy can detect
-Caused by a mutation in APC
-Signalling pathway involved is the Wnt signalling pathway
MLH1?
Genes that correct point mutations in the DNA
-When you lose one of these genes you get more point mutations
-99% of these mutations are silent(harmless)
Karyotypes in colon cancers?
-Abnormalities in chromosome number and structure
Sequence of events that causes development of colorectal carcinoma?
- Loss of APC causes the epithelium to become hyperproliferative
- Increased genetic instability/loss of p54 causes early adenoma
- Activation of K-Ras results in intermediate adenoma
- Loss of Smad4 and other tumour suppressors results in late adenoma
- Carcinoma and metastasis
T/F: p53 is lost in 50% of cancers?
True
Tumour heterogeneity?
Every tumour in every patient is different
T/F: Two tumours can look identical and look to be in the same phase but have two completely different genetic compositions?
True
What does tumour heterogeneity mean in terms of treatment of tumours?
The same treatment might not work on two tumours that appear to be the same
T/F: Tumours that arise from different tissues are generally more different in their genetic abnormalities than tumours of similar origin?
True
Effects of ionizing radiation on normal cells?
Ionizing radiation causes DNA damage which leads to the activation of p53 which causes cell cycle arrest, if the damage is mild it will be repaired but if the damage is too extensive the cell will apoptose
Effects of too much ionizing radiation on cancer cells?
Cells with DNA damage will continue to divide since they lack p53. Too much radiation damage can causes mitotic failure and cell death leading to patient death.
Effects of too little ionizing radiation on cancer cells?
Too little radiation causes cells to become more mutated and can make the cancer cells even more aggressive.
Abl gene?
Gene that encodes a kinase that regulates cell proliferation
Translocation in CML?
- Chromosome 22 contains a Bcr gene and chromosome 9 contains the Abl gene.
- In CML, there is often a translocation between these two chromosomes resulting in the philadelphia chromosome which creates the fusion protein Bcr-Abl
- The fusion protein has the N-terminus of Bcr joined to the C-terminus of Abl
- This fusion causes the Abl kinase domain to be hyperactive which drives excessive proliferation of a clone of hematopoietic cells in the bone marrow resulting in leukemia
Gleevec?
Oral drug that blocks the activity of the Bcr-Abl fusion protein and halts CML
Active Bcr-Abl?
Bcr-Abl binds its substrate and phosphorylates it causing proliferation in the bone marrow leading to leukaemia
Bcr-Abl blocked by Gleevec?
- Gleevec sits in the ATP binding pocket of the kinase domain of the Bcr-Abl and prevents it from transferring a phophate from ATP onto the susbtrate protein
- This blocks the signal for cell proliferation in the bone marrow
Problem with Gleevec and how it is fixed?
-Stem cells develop resistance to Gleevec
This is fixed by giving the patient another drug that targets the mutated resistant Bcr-Abl protein
Angiogenesis?
Endothelial cells which face the lumen of the artery grow/proliferate to make new blood vessels
Why does angiogeneiss occur?
When the cells needs more O2 or nutrients, capillaries will signal secretion of angiogenic factors and GF then the signal will turn, cancer cells abuse this process by always producing GF and angiogenic factors
Capillary formation in response to wound healing?
-Angiogenesis creates new blood vessels to feed damged tissues and repair it
New capillaries begin to sprout toward the site of injury
HIF?
Transcription factor that senses O2 levels in the blood and stimulates release of angiogenic factors in low O2 conditions
Angiogenesis in high O2?
In high O2 concentrations we do not need new capillaries so HIF concentrations are low and new blood vessels are not being made
Angiogenesis in low O2?
In low O2 we need more blood vessels to supply O2 and there is a high concentration of HIF which secreted VEGF and other angiogenic factors which then make blood vessels
How do tumours use angiogeneis to their advantage?
-Normally large tumours die due to a lack of blood supply
-Tumours will switch on an angiogenic switch to release VEGF
-VEGF allows the tumours to recruit blood vessels which supply the tumour with O2 and nutrients allowing them to grow
VHL(Von Hippel-Lindau disease)?
-Rare cancer syndrome
-Patients are typically born with a mutation in the VHL gene that leads to many tumours
How many tumours can be in the brain/kidney in VHL and what can you interpret from this?
-Up to 50 brain tumours
-Up to 500 tumours per kidney
Interpretation: this pathway most likely is directly linked to the proliferation(due to many tumours) and blood vessel formation(due to most of the tumours being filled with blood/hypovascular(full of blood vessels)
Renal carcinoma in VHL?
The most dangerous tumours resulting from VHL because they are completely resistant to therapy and can metastasize
Brainstem tumours in VHL?
-Can cause neurological problems or paralysis if they block CSF fluid flow
Knudson’s 2 hit hypothesis?
Both alleles of a tumour suppressor gene such as VHL must be mutated for tumorigenesis
Can uninherited people develp VHL?
VHL mutations can occur sporadiacally in people and lead to CNS hemangioblastoma and kidney cancer
786-0(v) cells vs 786-0(wt) cells
786-0(v) cells:
-Renal carcinoma cells that have a deleted VHL gene
786-0(wt) cells:
-Renal cells that have functional VHL gene
What happens when 786-0(v) cells and 786-0(wt) cells are grown in 10% serum?
786-0(v) cells: Knockout genes look like normal proliferating cells and look identical to the 786-0(wt) cells
Interpretation:
-This is expected since the mutated gene is a tumour-suppressor gene that doesn’t affect proliferation
What happens when the 786-0(v) cells and 786-0(wt) cells are grown in 0.1% serum?
786-0(v) cells: keep proliferating
786-0(wt) cells: arrested in G1 phase after 48 hours
Hypothesis from the 0.1 % serum experiment ?
Factors in the serum must be sensed by the VHL pathway, to sense whether the cells need to stop or not
Cell were then given different growth factors/mitogens, the best were?
TGR-alpha and EGF
-This tells us the VHL absent cells most likely produce their own EGF/TGR-alphha growth factors which allows them to proliferate in 0.1% serum
How did we determine VHL interacting proteins?
1.Take the gene encoding your protein of interest(VHL) and put it on a plasmid so you can introduce it into a cell
2. Put an epitope tag on(small aa sequence that produces a region where an antibody can bind)
3.Introduce the plasmid into the cell
4. Add the antibody and beads then centrifuge. Then in the pellet is the protein along with its friends that it interacts with. Now you know all of the associated proteins
What proteins interact with the VHL protein?
- Hs-CUL-2: E3 ubiquitin ligase
- Elongin B
- Elongin C
The pVHL complex?
Similar to the SCF/Skp1and cullin complex in yeast
HCul-2 associates the the VHL protein via adaptor proteins elongin B/C
pVHL?
The substrate recognition unit of the complex
When mutates the complex falls apart because it cannot bind to its substrate since its lacking specificity
T/F: Both pVHL complex and the F-box complex bind specific target proteins and add unbiquitins to target them for degradation via the proteasome?
true
What is the target of the pVHL complex?
HIF
How does the pVHL target HIF in the absence of oxygen?
- In low O2 concentration pVHL is inactive
- HIF thus accumulates and drives transcription of VEGF(blood vessel formation), PDGF-beta, TGF-alpha(proliferation of cells), EPO(RBC formation)
- This helps cells recruit more blood vessels and increase O2 concentration and nutrient concentration
How does the pVHL target HIF in the presence of oxygen?
- pVHL is activated
- Enzymes are active in high concentrations of O2 and cause the two prolines on HIF to be hydroxylated this then allows pVHL to bind to the HIF and add ubiquitous
- The ubiquitins then target the HIF for degradation via the proteasome
What happens in VHL mutant genes?
In people with two mutant copies of the VHL gene. The VHL gene is never activated thus HIF is always present even in the presence of O2 and this allows tumours to grow due to angiogenesis and proliferate.
Cells lacking VHL create their own growth factors?
Cells lacking VHL create their own TGF-alpha and VEGF which have a positive feedback loop and stimulate increased secretion due to receptors for the GFs on the cell
What happens when EGF-R is added to VHL negative cells?
It results in growth inhibition
