BMS379 Cancer Biology Flashcards
What are the main hallmarks of cancer?
- Sustaining proliferative signalling
- Evading growth suppressors
- Resisting cell death
- Activating invasion and metastasis
- Inducing angiogenesis
- Enabling replicative
What are the two elements that underpin cancer development?
Genome instability and mutation and Avoiding immune destruction are the two underpinning elements that allow the other cells to express the other hallmarks
How common is cancer?
- 50% of the population will be diagnosed with cancer
- 75% of 75 year olds who die from something that was not cancer is shown to have a tumour
What is the main risk factor of cancer?
Age
Is cancer increasing?
Cancer incidence is increasing because life span is increasing
Give a study that shows that risks to certain types of cancers are due to environment
Migration studies
- On two occasions there are accounts of large amounts of migration from japan to US
- In japan have an increased risk of stomach cancer and a low risk of prostate and breast cancer
- Caucasian people in the US have a large risk of prostate and breast cancer but low risk of stomach cancer
- Japanese American people (who have lived in America) have profiles that resemble Caucasians, implying that a major factor for risks to types of cancer is based on the environment and not genetics
What is the relationship between the log of cancer incidence and the log of Age?
Linear
Why is the relationship between the log of cancer incidence and the log of Age linear?
- The probability of a change associated with cancer P(C1)
- The chance that the change will have happened goes up with age: P(C1) A
- Several changes needed (n) before a tumour (T) develops: P(T)=P(C1). P(C2). P(C2) ….P(Cn) x A^n
- This can be rearranged to LogP(T) = nLogA + Constant which is the equation of a straight line
How many mutations are required for a tumour to form?
Can use LogP(T) = nLogA + Constant to calculate the slope (n)
= 6
Give evidence for the genetic basis of cancer
- Some families are susceptive to some types of cancer e.g. retinoblastoma, Wilms Tumour
- This provides evidence for a genetic component e.g. the existence of tumour suppressor genes
What were the first experiments that showed an environmental component in cancer?
Percival Pott
- He undertook an examination of chimney sweeps and noticed that they were prone to scrotal cancer suggesting an environmental influence
- Lead to the Chimney Sweepers Act of 1788
John Hill
- Found that people who took snuff were prone to nasopharyngeal cancer
How did Richard Doll look find a link between smoking and cancer?
- Looked at data in relation to cancer and found that an increase in smoking in men and women resulted in an increase in counts of deaths due to lung cancer with a 20-year lag
- Lung cancer is a rare form of cancer unless a smoker
- Link between number of smokers in a country and lung cancer prevalence in men
Give the first experimental evidence for link between smoking and tumour formation
Katsusaburo Yamagiwa 1915
- His experiment was the first experimental method to induce cancer
- Took an extract from coal tar and treated the ears of rabbits, forming carcinomas. This proved that a substance found in cigarettes directly induced cancer
In the 1950s what origins of cancer had been identified?
- Benzopyrene is the substance found in coal tar
- Also found that x rays could induce cancer
- Chicken virus could cancer
- At the time, it was not clear what the relationships were between these things but it did eventually lead to the mutagenesis theory of cancer
What are the two types of mouse models used to model cancer?
OncoMouse
- Genetically engineer mice where genetic changes are introduced
- This looks at endogenous cancers in that organism
Nude mice
- Xenograft model of human cancer
- Introduce human cells into the immuno-compromised mice
- These mice will develop tumours derived from the human cells
What are oncomice used for?
Useful for looking and understanding mouse cancer, as well as cancer development
What are nude mice used for?
This is useful when studying treatments for human cancers – essential before human clinical trials
What are the problems with oncomice?
- Charcateristics of mouse cancer
- Time of onset
- Nature and behaviour of primary and metastatic tumour
What are the problems with nude mice?
- Some characteristics of human cancer
- Time of onset
- Nature and behaviour of primary and metastatic tumour
What model of cancer dominated in the 1970s?
The viral model
What and when was the first tumour virus discovered?
In 1910, Peyton Rous discovered the first tumour virus: Rous Sarcoma Virus (RSV)
How did Peyton Rous shown that RSV induced cancer?
He removed the sarcoma from the muscle of the chicken and filtered through fine pore filter. Too small to bacteria so must be virus. He then injected this filtrate into chickens and they all developed sarcomas. This suggested that there was an infectious agent causing cancers
How did Howard Temin and Harry Rubin contribute to understanding of viral cancers?
- They found that RSV could continuously infect cells and have a productive lifecycle in cultured cells. Infected tissue culture cells displayed traits that were similar to cancerous cells
- Primary cells morphology changes if infected with RSV - increased thickness of cell layer, loss of contact inhibition and became rounded
- Anchorage independent growth - If suspend cells in a semi solid medium and infect them with RSV, they will grow in the medium unlike primary cells. Primary cells need to attach to something to grow.
- They took fibroblasts (primary cells) that were previously infected with RSV into immune compromised mouse (nude. This resulted in tumour formation
What kind of virus is RSV?
Retrovirus
How many genes do retrovirus’s have?
Three genes: gag, pol (polymerase), env
How many genes does RSV have?
RSV has an extra gene to other retrovirus’s: src
Who gave the first theory to how an RNA virus can persist through successive cell growth cycles?
Temin first suggested that the viral genome could be reverse transcribed into a DNA intermediate
How was the theory of reverse transcription proved?
David Baltimore discovered the enzyme reverse transcriptase in 1970, proving this theory. This provides the basis for molecular biology
How does an RNA virus persist in the cell through successive cell growth cycles?
- The virus converts RNA into DNA using reverse transcriptase which is then integrated into the host genome. This DNA is then transcribed by the host cell machinery making lots of RNA copies of the virus
- This suggested away that mutations are caused by these viruses: potential explanation for causing cancer
Why did the viral theory of cancer dominate?
- Due to experiments where cells, that are treated with a nucleic analogue, were added into a culture medium. This resulted in the retroviral sequences, that were embedded in DNA, excising from DNA causing the virus to propagate, producing viral particles
- Theory was that these insults to nucleic acids would cause the nucleic acids reactivation in some way. This could induce viral production
- This might lead to an infectious and transforming virus under certain circumstances
Why was the viral origin of cancer wrong?
Would expect that there would be
- clusters of infectious outbreaks
- that you could isolate virus particles from all human tumour cells
These are not true showing that virus is not the only mechanism to cause cancer
Give examples of some cancer causing viruses?
- Epstein Barr Virus (EBV) - associated with lymphoma and nasopharyngeal cancers.
- Human Cytomegalovirus (HCMV) – associated with malignant glioma, colon, and prostate cancer.
- Hepatitus C Virus (HCV) – associated with hepatocellular carcinoma.
- Human Immunodeficiency Virus (HIV) – associated with Non-Hodgkin’s Lymphoma and HIV.
After the viral theory of cancer was disproved, what was discovered?
Mutagenesis theory of cancer
What was the mutagenesis theory of cancer?
The theory was that If something happens to a gene then it would cause the cell to change
What is contact inhibition?
Usually, when cells touch other cells they stop growing
What do transformed cells do in culture?
Cells that show transformation do not show this contact inhibition meaning cells overgrow.
Give evidence for mutated cells resulting in tumour formation?
- Took transformed mice fibroblasts and expose them to 3 methylcholanthrene (DNA damaging drugs)
- Under appropriate conditions you could get normal fibroblasts to take up the DNA fragments (transfection)
- Some of these fragments would be integrated into the genome
- The transfected cells (foci) were picked and injected into a mouse (xenograft)
- This resulted in a tumour. If you don’t expose to the DNA mutating agent then no tumour forms
What is a foci?
Colony of cells that aggregate together and do not show contact inhibition
Why was it concluded that only one mutated gene caused tumour formation?
They could calculate how much of the genome was taken up into the cells by transfection. They roughly found that 30 genes were taken up and concluded that it was unlikely that more than one gene is mutated. This disagreed with the idea that 6 mutations are required for tumour formation
Who discovered the first cellular oncogene?
Michael Wigler
How did Micheal Wigler discover the first cellular oncogene?
- Extracted DNA from the transformed cells. There would many pieces of DNA but only one, when introduced into a new cell, would cause the cells to transform (the oncogene)
- He attached an essential bacterial gene to the extracted DNA
- And purified through biochemical separation techniques
How did Micheal Wigler purity the oncogene?
- Put the DNA with bacterial gene into the fibroblasts. When the fragment with the oncogene were transfected into a cell then foci would form.
- He then selected the foci and then extracted the DNA out of those cells.
- There would be a mixture of DNA that was transfected in (with the essential bacterial gene) and DNA from the original cell
- Added this mixture to bacteria that lack the bacterial essential gene and plate out the bacteria, only bacteria containing the tagged DNA will survive. This tagged DNA will contain the oncogene.
- Can then extract the DNA from the living bacteria and transfect it into more fibroblasts. Find Foci, which will have taken up the gene of interest and extract the DNA. Put back into bacteria lacking essential gene to select for tagged DNA.
- Repeat this multiple times. Each time this happens, the fraction of the oncogene in the mixture will increase
- Will eventually result in a pure mixture of oncogene attached to the bacterial gene
What is southern blotting?
- Involves taking an agarose gene and inserting fragmented DNA/RNA into the gel. The fragments will move through the gel and organise based on size in response to an electric charge
- Then put a sheet of nitrocellulose on top which binds macromolecules. Put buffer liquid under the gel and weighed down with paper towels. This causes the macromolecules to move up the gel and stick to the nitrocellulose
- Can then integrate these molecules using radioactivity. If the probe is complementary to the fragment, then will anneal. Wash away inactivity and will be left with a radioactively labelled fragment
How did Micheal Wigler show that Viruses and mammals are related?
Michael Wigler took transformed cells with fragmented DNA and probed using H-Ras viral oncogene. This labelled transformed cell DNA but not untransformed cell DNA
- This implied that mammals had a similar gene to virus so when mutated it could bind to the viral gene. This showed that mammals and virus are related
What is c-Ras?
Not mutated H-Ras - cellular Ras
What was the first oncogene discovered?
c-Ras
How does the oncogene of Ras differ from normal Ras?
Used sequencing
- There was a single base change between c-Ras and oncogenic Ras. Changed code from glycine residue to a valine residue. This told us that mutating a single base in a single gene can transform cells to grow a tumour
Give evidence for a single base change being capable of tumorigenesis
Experiment where pure H-Ras or C=Ras was transfected into cells and injected into mouse, resulted when H-Ras was used there is tumour formation but when C-Ras is used then there is no tumour formation. This is evidence that a single mutation in c-Ras is capable of tumorigenesis
What is Src?
The extra gene in the rous sarcoma virus - found to be a protein kinase
Who discovered that Src was a protein kinase?
Collett and Erikson (1978)
How did Collett and Erikson (1978) discover that Src was a protein kinase?
- Developed an Src anti-serum by injecting into the Src protein into rabbits
- They did immunoprecipitation in transformed and untransformed cells when Src had been incubated with radioactive ATP
- Ran the products on a gel and did an autoradiogram and saw that in the transformed cells there was a band present – the phosphate.
- Src appeared to be a protein kinase transferring the phosphate from ATP to the antibody
- This is unusual because protein kinases are usually specific with their targets but by chance the antibody mimicked the target
What is an immunoprecipitation experiment?
Antibodies are attached to beads which was passed down a centrifuge tube. The control serum removes non-specific binding and the antibody specifically binds.
What is the role of a protein kinase?
Catalyses the transfer of phosphate from ATP to another protein
What is responsible for the phosphorylation of Polyoma Large T?
Src
- Polymoma Large T is a viral protein that disrupts multiple regulatory pathways in transformed cells
What amino acids can phosphorylation occur on?
Usually phosphorylation occurs on threonine or serine due to their hydroxyl group but can also occur on tyrosine (only 0.1% of phosphoamino acids)
How was it discovered that phosphorylation can occur on tyrosine?
- Took Polyoma large T, which was radioactively phosphorylated, and incubate in large concentrations of HCl.
- This will hydrolyse everything down to single amino acids. Can then do 2D separation of the amino acids using chromatography and electrophoresis. This allows them to determine where the protein was phosphorylated
- Found that it was on tyrosine
- This lead to the discovery of tyrosine kinases
Why was the discovery of Src being a tyrosine kinase important?
- it provided a mechanism of transformation. Tyrosine kinases were signalling devices which induces a changes in function through phosphorylation.
- Therefore, cells infected with a virus which contains a novel tyrosine kinase could have a change in protein functionality
- When cells were transformed by Scr oncogene the phosphotyrosine levels increased from 0.1% to 1% of the total phosphoamino acids. However, when transformed with different oncogene (H-Ras) it did not increase showing that the tyrosine kinase activity is specific to Scr and not a mechanism involved in all cell transformation
Why is Src an unusual tyrosine kinase?
It is quite promiscuous – will phosphorylate many things – not specific
How were growth factors discovered?
Proliferation of cells in culture requires serum. Growth factors can fulfil the roll of serum - found to be mitogenic. Growth factors were therefore purified
What did Stan Cohen investigate?
Investigated EGF and what it can bind to
How did Stan Cohen find the EGF receptor?
- He attached EGF to a solid support and passed Hela cell extracts through the bound EGF. The theory of this was that the EGF would bind specifically to target in the cell – didn’t account for non-specific binding
- Then eluted the excess using salt concentrations and look at the contents of the tube to investigate if it was homogeneous. They found that one protein was present – huge purification
How did Stan Cohen identify the EGF receptor sequence?
Identified the protein by digesting it with a specific protease and then isolated various peptides. Then used an alternating peptide sequencing and used sanger sequencing to determine the sequence. Used the sequence to estimate the DNA that coded for it and screened libraries for this gene.
How did Stan Cohen identify the structure of the EGF receptor?
- Used a specific protease to chop up the protein to generate three large fragments. They ran these fragments on gel and incubated it with radioactive EGF (125I-EGF). There was a 50Kd fragment which contained this radioactive EGF meaning this fragment is the part that binds the growth factor. Termed its ectodomain
- Used bioinformatics – Plotted a graph of how hydrophobic each amino acid of the protein is. Hydrophobic parts of the protein tend to be either intracellular or in the plasma membrane. Therefore, the large hydrophobic area in the middle of the protein was the transmembrane domain
How did they discover that the EGF receptor was a tyrosine kinase?
Found that there was high homology between EGF receptor and Src showing that EGF was a receptor tyrosine kinase
What does the discovery of EGF receptor being a tyrosine kinase suggest about viral oncogenes?
Suggests that viral oncogenes which transform cells through signalling mechanisms were related to things involved in the growth of these cells
What two explanations are suggested about EGF receptors being a tyrosine kinase involved in cancer?
- Growth factors induce their cellular effects by triggering a tyrosine kinase signalling pathway
- Oncogenes might work by triggering signalling pathways in the absence of appropriate extracellular cues (making the signalling overactive)
Give evidence for EGF exerting its effects by phosphorylation
- Radioactive labelling experiment where they did amino acid analysis using chromatography and electrophoresis.
- They separated the essential amino acids and looked at phosphothreonine, phosphoserine and phosphotyrosine.
- They took petri dishes with cells and medium and added 32Phosphate and asking whether it is incorporated into proteins which it did.
- The amount of phosphotyrosine in response to EGF also increases. This is evidence that EGF works by phosphorylation
How to tyrosine kinases cause signal transduction?
Tyrosine kinases have a transmembrane domain and a receptor which, upon growth factor binding, brings together multiple tyrosine kinases and phosphorylate themselves
What did Offtrdinger et al do?
- Did an immunofluorescence experiment which allowed them to look what happens at the plasma membrane when cells are triggered with EGF. A phosphotyrosine binding protein labelled in a fluorophore was used to see if there was a phosphorylation on the plasma membrane occurring in response to EGF
- They found that there was initially little phosphorylation but significantly increases after 60 seconds
What are growth factor receptors in relation to cancer?
Each of these growth factor receptors are proto oncogenes and if mutated could become oncogenes by triggering signalling pathways in the absence of extracellular cues
Outline receptor tyrosine kinase signalling
- Receptor in off state are often monomeric
- Dimer ligand binds to monomeric receptor which induces dimerization. The other part of the ligand that is unbound binds to the other monomeric receptor forming a cross-link between the two receptors.
- Increased local concentration of active receptor kinase and receptor substrate results in trans-phosphorylation
- This phospho receptor then acts as a platform for signalling events
Does it always have to be a tyrosine kinase to be an oncogene?
- It doesn’t have to be a tyrosine kinase to be working in this way e.g could be a serine/threonine kinase
- It doesn’t even have to be a kinase, it is only important that it generates a signal and if mutated will increase this
What did Ed Skolnick do?
Radioactive displacement experiment to find what Ras binds to
Outline Ed Skolnick experiment
- Immunoprecipitation of Ras and incubate it with radioactive guanine nucleotides (3H-GDP).
- This was a radioactive displacement experiment. They incubated this radio-precipitation with other nucleotides that were not radioactive.
- If the correct thing is found (specifically binds) then you should be able to displace a radioactive ligand with another ligand. This would compete away radioactivity to see if Ras binds to these nucleotides
What did Ed Skolnick find?
RAS binds GTP and GDP
- Found that GDP addition removed the radioactivity showing it binds to Ras
- Cyclic GMP and cAMP doesn’t compete with the radioactive ligand because Ras doesn’t bind cGMP or cAMP.
What did Gross et al, 1984 do?
- Expressed and purified recombinant Ras in bacteria and incubated it with radioactive GDP (radioactive in the guanine not the phosphate)
- Incubated Ras with GTP for different amounts of time. They used both c-Ras and a viral Ras from a rat sarcoma: Ha-Ras
- Expose to film which goes black with radioactivity.
What is the difference between c-Rad and Ha-Ras?
c-Ras is cellular as found in normal tissue
Ha-Ras is a viral Rat which has a mutation causing glycine 12 to be swapped to valine
What did Gross et al, 1984 find?
In a time dependant manner, in the presence of Ras GDP appears implying GTPase activity.
- It is an enzyme that can hydrolyse GTP. Ha-Ras however is not a GTPase
What did the results of Gross et al, 1984 tell us about the difference between c-Ras and the oncogenic viral Ha-Ras?
- This first told us that Ras is a GTPase and that viral Ras does not work.
- This means that viral Ras is capable of binding GTP but not hydrolysing it
How were GAP proteins identified?
McCormack et al, 1988
- Used recombinant Ras. Took cell extracts and asked if these extracts contained anything that could stimulate the GTPase activity. There was something that could stimulate this activity
- They used this assay as a biochemical purification to see what was causing this
- This lead to the identification of a GAP protein which stimulate GTPase activity of Ras and Ras like molecules
How were GEF proteins discovered?
Macara and Wolfson (1990)
- Pre-incubate Ras with GDP and then want to remove this GDP molecule. Incubate GDP with Ras and nothing would happen and would not displace. Want to know if there was a biochemical way to remove it
- Brain cytosol contains an activity that was concentration dependant and could facilitate dissociation of GDP.
- Tried to identify what was causing GDP to be removed from Ras. Identified GEFs
What was the first GEF identified?
The first GEF identified was homologous to CDC25 in yeast giving a function to the gene Wigler et al was investigating
Why does oncogenic viral Ras or mutated Ras make it non functional?
Glycine 12 is involved in the catalysis of hydrolysis of GTP. If this glycine is mutated then this won’t occur. The oncogenic viral Ras has a mutated glycine12 and changed to valine making it non-functional
What animal model was used to study the relationship between Ras and tyrosine kinases?
Drosophila
What did Gerry Rubin do?
Gerry Rubin investigated the stricture of the drosophila eye. Their eyes are made up of ommatidia which consist of seven cell. A mutant which missed the seventh cell was called sevenless. They identified using genetic enhancer repressor screens the epistasis of sevenless signalling. Bride of sevenless (BOS) -> Sevenless -> Son of sevenless (SOS)
What did Bob Horvitz do?
Bob Horvitz used C. elegans to look at the genes involved in vulval formation. He identified several genes, Lin-3 -> Let-23 -> SEM-5 -> Let-60. He discovered the pathway order of these genes
What did the work of Gerry Rubin and Bob Horvitz lead to?
That the pathways they investigated involved a growth factor binding to a tyrosine kinase which lead to Ras activation
e.g. BOS binds to sevenless (EGF-R) which activates Ras
They were unsure of the role of SEM-5 or SOS so investigated it
What was SOS found to be?
SOS was found to be CDC25 so was a GEF.
How was the role of SEM-5 discovered?
Expression cloning strategy
- Took mammalian mRNA, converted to cDNA and put in expression factor for bacteria and plate out the bacteria Each gene product will be expressed in at least one bacterium in the plate
- Receptor tyrosine kinases auto phosphorylate in the presence of growth factors
- They therefore created a probe by attaching a fluorophore to something that has phosphorylated domains
- They then transferred bacterial gene products to nitrocellulose and washed with a solution of the probe
- Was there anything that bound the probe tightly to the nitrocellulose filter so was still bound after washing
- Want to know what bacteria colony it is and therefore what gene it is. You see what bacterial colony that is the bound protein came from, extract the DNA and sequence and find out the gene that bound it
What was STEM-5 found to be?
GRB2
What is the model of tyrosine kinase receptor signalling?
Tyrosine kinase receptors bind growth factor and phosphorylates itself. GrB2 binds to the phosphorylated version and interacts with SOS (GEF). This causes Ras to go from GDP bound state to GTP bound state. Ras can then bind GTP making a two-state system
Why is Ras referred to as a two state switch system?
It is useful to think of Ras as a two state switch system. The on state of Ras (bound to GTP) will then hyndrolyse GTP to GDP to the off state. It is switched off more quickly if there is a GAP present.
How do most of effectors bind to Ras?
Every effector of Ras has a Ras binding domain which interacts with Ras but only in a GTP bound form.
Why has the discovery of the way effectors bind to Ras shown why Ras is oncogenic?
Many pathways involved in cell growth, gene expression and cell morphology and movement were discovered. All of these pathways have single entities at the top of the pathways which turn them on which interact directly with oncogenic Ras. E.g. PI3K, Raf, Ral. This is why Ras is oncogenic because when it is constitutively active these pathways are overactive and cause transformation
How many EGFR are there?
EGFR
HER2
HER3
HER4
What mutations in EGFR have been found in human cancer?
- Many EGFR mutations have been associated with human cancers and result in an increased catalytic tyrosine kinase activity of the receptor. The most prevalent of the mutations in tumours were found to be a deletion of exons 2-7.
- Mutations that lead to a reduction in receptor downregulation also cause tumours. For example, oncogenic forms of ubiquitin ligase CBL prevent CBL from negatively regulating RTKs
What type of cancer are HER2 mutations related too?
The Ullrich laboratory found that HER2 was amplified in 30% of invasive breast cancers and found a correlation between HER2 expression in tumours and reduced patient survival
How did the discovery of HER2 mutations in breast cancer lead to target specific cancer treatments?
This discovery gave a target for therapeutic treatments. Specific monoclonal antibodies for HER2 were therefore developed which bind to HER2 on tumour cells and induce receptor internalisation, inhibiting the cell cycle progression
How many genes have been identified that when mutated are capable of driving cancer formation and progression?1.
140
How many of the 140 genes in the cancer genome are kinases?
18%
Give examples of kinase inhibitors as treatments for cancers
EGFR kinase inhibitors, such as gefitinib, have been approved for non small cell lung cancer.
Similarly BRAF inhibitors were approved for V600W BRAF melanomas
Are cancer phenotypes dominant or recessive?
Most cancer cell phenotypes are recessive but Viral oncogenes exert a dominant effect
What is interesting about the sendai virus?
Can cause cells to merge due to their fusagenic nature
After cells fuse, all the chromosomes are in the same image
How did Rao and Johnson come up with the idea of tumour suppressor genes?
- They used the sendai virus to fuse monkey kidney cells with NIH3T3 cells. Asked if take a cancer cell and non-cancer cell and ask what happens if form these
- Introduce these hybrid cells into mice and see if tumours formed. Found that tumour didn’t form meaning that cancer must be recessive
- This lead to the idea of tumour suppressor genes that would require the loss of two alleles for cancer to form. This appeared very unlikely
What is the only current treatment for retinoblastoma?
Remove the organ
What are the two types of retinoblastoma?
- Sporadic retinoblastoma appears to be unilateral and only affects one eye. If the tumour is removed then the patient has no further risk of developing a tumour there or elsewhere in the body.
- Familial appears to be bilateral effecting both eyes and occurs when the parent of the patient also suffered with the disease. Removal of the tumours does not protect the patient from the risk of tumour development in other locations around the body
When is familial retinoblastoma diagnosed?
Diagnosed very early in life. By age 10, often lost one eye
What is the 1-hit/2-hit hypothesis?
The genome must take 1 hit in bilateral case and 2 hits in unilateral. This refers to the loss of one or two alleles. This is why unilateral occurs more slowly over time as two hits are required. It is surprising that unilateral, that require two hits, is happening at all as it is unlikely two genes will be lost
How was the 1-hit/2-hit hypothesis found?
Knudson
- Took data from patients presenting with unilateral and bilateral diseases
- Created a log scale of the percentage of people not diagnosed at certain ages to find out whether a reaction involves one or two reactants. If one thing involved is involved then a semi log plot leads to a straight line. If there are two things involved then there is a curve
- Therefore shows that in bilateral cases, familial, only one thing must go wrong whereas unilateral, sporadical, two things must go wrong
- That there was a gene, Rb, which in familial cancer is mutated but in sporadical, a mutation in one allele of Rb is acquired followed by a second one
The likelihood of losing both cooped if Rb seems unlikely so why is the incidence of sporadical retinoblastoma so high?
This is explained by homologous recombination resulting in the loss of heterozygosity
- There could be one random somatic mutation in Rb that causes the chromosome to be heterozygous (one mutant and one Wt Rb) but because it is recessive it will have a Wt phenotype.
- The chromosomes are duplicated during replication meaning that there are two chromatids carrying the mutant Rb and two carrying the Wt allele. If homologous recombination occurs so that the mutant Rb alleles are on opposite sister chromosome, then it results in the issue of segregation.
- Depending on how these chromosomes line up on the mitotic spindle and are separated, it may end up with a daughter cell which has both copies of the mutated Rb (DNA is replicated in cell division) in the same cell while the other daughter cell doesn’t have either of the mutated copies due to this homologous recombination.
- This results in the loss of heterozygosity resulting in the lack of a critical tumour suppressor gene in those cells
- Draw diagram
What is hemizygosity?
Loss of heterozygosity can also occur by breaking and loss of chromosome arm carrying the Wt copy of Rb. This will result in the mutant Rb copy being the only copy of the gene and therefore dominant
What is homologous recombination?
This process allows sister chromosomes to be recombined so that arms of chromosomes can swap over. This occurs in cells after replication. Once S phase has occurred, then there is the opportunity for cells to undergo this.
What is the critical chromosomal region in retinoblastoma?
Cytogenetic analysis revealed that a band is missing on chromosome 13 on people with retinoblastoma
This is the critical region in retinoblastoma
What is D esterase?
- Gene encoding the enzyme known as D esterase, which hydrolyses esters, is well characterised and located at 13q14. The region lost in retinoblastoma.
- There are two distinct alleles of this gene that is present in the population and they encode proteins of slightly different lengths so run at different rates in electropherisis.
What is zymography?
Zymography is a form of gel electrophoresis but instead the proteins aren’t denatured and the gel is impregnated with an enzyme that can detect products with a colour change on the gel at the size and molecular weight of the protein involved
How did Sparkes et al, 1981 give evidence for the loss of heterozygosity in sporadical retinoblastoma?
- Used zymography to tell if someone is homozygous or heterozygous for the two D esterase alleles which are located close to Rb. If heterozygous, there will be both sizes of the D esterase alleles, have one band for each allele
- In people with retinoblastoma, in tumour tissue, there is always only one or the other of the isoforms of D esterase despite being heterozygous for D esterase in normal tissue (should be two bands). This shows that there has been a loss of heterozygosity and recombination has occurred
How did Stephan Friend et al, 1986 find mutations in the Rb gene?
- Sequenced along an entire chromosome of people with and without retinoblastoma to try and find the Rb gene
- This lead to a northern blot which is when the fragmented RNA from a patient is probed with a radiolabelled probe
- Found a DNA segment missing from patients with RB but present in people with RB
- None of the people had a functioning gene corresponding to its locus and there were different mutations along the Rb gene
What are the two ways to find tumour suppressor genes?
- Restriction fragment polymorphism (RFLP analysis)
- Single nucleotide polymorphism (SNP)
How is RFLP analysis used to find tumour suppressor genes?
- Cleave the DNA of a persons normal tissue with specific restriction enzyme (EcoRi) that you know how it should fragment.
- Then use a probe to see the lengths of the fragments and if it differs from what is the normal pattern then this person naturally has a polymorphism in the cleavage site on one of the chromosomes because the restriction enzyme can’t cleave it (heterozygosity).
- Then do the same in the tumour tissue and if the same change in fragmentation isn’t seen then loss of heterozygosity has occurred and its likely to be by a tumour suppressor.
What is the original way of finding tumour suppressor genes?
- Restriction fragment polymorphism (RFLP analysis) but there are now more efficient ways
- This is analogous to Sparkes experiment and how we search for tumour suppressor genes in the whole genome
How SNPs used to find tumour suppressor genes?
- Two thirds of a persons genome have SNPs in a heterozygous state resulting in an SNP marker. There are much more SNPs then RFLPs allowing loss of heterozygosity to be minimised to smaller areas.
- Sequence entire genome and see if people with genomes have lost an area of heterozygosity compared to the normal genome
- Can see single base pair mutations
- PCR based approach. Primers used to generate PCR product. If there is a base change then the primer will not allow a product to be made showing that there is clearly heterozygosity at that site
How many are SNPs are there in the human genome?
There are 3 million SNPs identified in the human genome so far
What is synthetic lethality?
When drugs targets two genes whose action together is essential for cell survival causing cell death
This technique is often used in anti-cancer drugs
How is synthetic lethality used in anti-cancer drugs?
The presence of a mutated version of one of these genes in cancer cells but not in normal cells can create opportunities to selectively kill cancer cells while leaving normal tissue unaffected. This is achieved by mimicking the effect of the second genetic mutation with targeted therapy
- loss of function mutations like those found in DNA repair genes or tumour suppressor genes could be exploited in this way
How could gene interactions that could be targeted by synthetic lethality investigated?
- RNAi-based screens allow for discovery of unknown gene-gene interactions and pathways.
- There are multiple approaches to RNAi screens such as using reverse transfection, plasmid vectors and retroviruses.
- Despite RNAi based screens allowing for direct discovery of gene interactions, it does not necessarily lead to potential therapeutic targets
Give an example of anti-cancer drugs that utilise synthetic lethality?
PARP inhibitors and BRCA1/2 mutations
What is the function of BRCA1 and BRCA2?
BRCA1 and BRCA2 are tumour suppressor genes involved in the repair of double stranded DNA breaks through homologous recombination and mutations in these genes are related to hereditary forms of breast and ovarian cancers
What is the function of PARP?
PARP functions to recruit DNA repair proteins to the sites of single stranded DNA break
How do PARP inhibitors target tumours with BRCA1/2 mutations
Synthetic lethality
- PARP inhibitors would stop the repair of these single stranded breaks and without homologous recombination ability in tumour tissue, due to the BRCA mutation, would result in the death of the cancer cells.
How do we know how long the cell cycle phases take?
- See the amount of time required for cells to double
- Functional in vivo assays or morphological markers are required to see what stage the cell is in
Pulse chase experiment - Cells will take up the radioactive phosphate and incorporate into the cell’s DNA If a cell is undertaking DNA replication
- Wash away excess and expose to film which will cause the film to go black and can see some cells are incorporating phosphate and some aren’t
- Can then work out the proportion of cells in the population which are undergoing DNA replication
What percentage of cells are undergoing DNA replication?
This experiment revealed that 35% of cells are replicating DNA. If you multiply this faction by the doubling time, it tells how long that phase it must last for
How long is the mammalian cell cycle?
20 hours
How long are cells in mitosis?
1 hour
What percentage of cells are in mitosis?
Cells spend one hour in mitosis. This suggests that 5% of the cells will be mitotic at any one time as mammalian cells takes 20 hours to replicate
How can you investigate whether DNA is replicating?
Used halogenated derivatives of deoxyuridine to mimic thymidine. This means that wherever there is a thymidine in the DNA sequence, halogenated deoxyuridine can take its place. Then do a microscopy experiment and use antibodies to detect the halogenated derivatives
If DNA is replicating then this deoxyuridine will be present in the DNA
How does early embryo drosophila cell cycle differ to mammalian cell cycle?
- Drosophila embryo has an unusual cell cycle. Very fast and only occurs S and M and no G1 or G2. This occurs in one big cell – syncytium.
- This occurs every 15 minutes in the early drosophila where is it is 20 hours in a mammalian cell
How does DNA content vary through the cell cycle?
- When cells are not proliferating they are in G1 they have a 2n copy of the number of haploid genes when cells are not proliferating.
- When undergoing replication then the DNA content increases meaning that it G2 has twice the amount of DNA. The profile will show the S phase to M phase proportions
How can you tell what stage of the cell cycle cells are in?
- Can use FACS to allow us to analyse the population of cells to see whether cells are in G1 (only one copy of DNA, less fluorescence) or G2.
- Can plot a profile to see which phase the cells are in
- The fluorophore will give out light as it moves down the tube and can be detected. The amount for fluorescence given off is proportional to the amount of DNA in each cell
How can FACS be used to investigate cell size?
This works by funnelling cells into a channel of liquid that allows one cell to pass. There are a series of lasers which detect the size of the cell to see how long the laser is blocked for.
What is the problem with using FACS to see what stage of the cell cycle cells are in?
It is not precise
- When does S phase start and end? S phase must start when the first new nucleotides are taken into a cell.
How can the limitations of using FACS to investigate cell cycle stage be overcome?
Bivariate FACS analysis
- 3D plot
- Can plot the cell, DNA content and the rate of incorporation (using the halogenated deoxyuridine). This is shown in a graph. When BRDU is incorporated, cells move up the Y axis (S phase). The colour shows the density of cells – most in G1. The x axis shows the DNA content, increases from G1 to G2
- See graph
How can investigate what stage of the cell cycle has been arrested?
- Can interrupt DNA replication with a drug and see how cell cycle is affected. Would expect cells that are not in replication would migrate away while the others will be stuck in the compartment
- Using the bivariate FACS analysis can determine when the cell cycle has arrested e.g. if there are many cells in S phase then they cycle has been arrested there.
What model system was used to genetically dissect the cell cycle?
Yeast
What two types of yeast were used to study the cell cycle?
Fission and Budding
Why are budding yeast useful when studying the cell cycle?
Became clear that mutants in budding yeast could be detected because could see what cell cycle they were in e.g. if could see a bud then it is in S phase, if in mitosis can see the reorganisation of mitotic spindle
Why are fission yeast useful when studying the cell cycle?
Fission yeast are interesting because it only grows laterally, never another direction. By knowing the length of the yeast, can tell where they are in the cell cycle
What are temperature sensitive mutants?
Select mutants that are temperature sensitive by looking for things at a high temperature don’t grow and arrest at point where the morphology tells us what part of the cell cycle they are in
How can yeast temperature sensitive mutants be used for investigate the roles of genes in the cell cycle?
If isolated a particular temperature sensitive mutant then it is possible to introduce a library of plasmids into the population of this mutant. Cells that receive the correct gene will recuse this phenotype and be able to enter the cell cycle. This will allow identification of the gene
Why are frog oocytes useful when studying the cell cycle?
- The oocytes in the frog grow without cell division for many months which is then deposited in response to hormones
- When this occurs the oocyte undergoes meiosis to form a haploid egg. In the early stage of the cell cycle, the cells get smaller after every cell division – isn’t any time to create more proteins as cell division is very fast until 4000 cell stage
- This is useful because these initial large frog eggs have enough material to make 4000 cells worth of DNA and nuclei – stores ahead of time
How can the protein and nucleotide rich frog oocyte be used to study the cell cycle?
- Can take these frog eggs and put them in a test tube and centrifuge to generate a highly-concentrated extract of the cytoplasm
- If add DNA to extract then it will form a nucleus in vitro in the test tube. Aim for a nucleus per microliter.
- This will then recapitulate the cell cycle in vitro allowing experiments to be conducted
What are cyclins?
Cyclins are proteins that are expressed in different levels of the cell cycle and when present bind to specific cyclin dependant kinase, Cdks to activate them
How is the directionality of the cell cycle controlled?
- The directionality of the cell cycle, the order, is controlled by the sequential appearance and destruction of cyclin proteins is what targets the kinases towards different substrates at different phases of the cell cycle.
- Each cyclin allows the transition of a cell cycle phase
What are CDKs?
- CDKs turn on or turn off the functionality that is required at each transition of the cell cycle. The activation of these kinases requires the binding of the cell cycle
What categories of CDKs are there in mammalian cells?
In mammalian cells, there are three categories of CDKs
- Active in G1/S transition
- Active throughout S phase
- Active through G2/M transition
To end mitosis, don’t need a specific CDK just need destruction of them all
What four main experiments proved the cell cycle was controlled by cyclins and CDKs?
- Rao and Johnson 1970
- Masui 1970
- Hunt et al, 1983
- Nurse et al, 1983
What did Rao and Johnson 1970 do and what did it show?
- Used sendai virus to fuse to types of cells together
- They fused cells together that were in different phases of the cell cycle. Can see the chromosomes and can compare them. E.g. In M and G1 fusion, there is still chromosome condensation. This doesn’t usually occur at G1. When the fusing has occurred, mitosis is dominant resulting in chromosome condensation of all chromosomes.
- Fusion of interphase and mitotic cells cause interphase cells to enter mitosis. Mitosis is dominant
How did Masui 1970 use frog oocytes to investigate the cell cycle?
- When an egg is laid they have a white spot on the top, oocytes do not. The white spot is indicative of where the mitotic spindle is as the dark pigment of the egg is actively removed from where the spindle is.
- If take a mature egg, laid by the frog which are in meiotic metaphase, and inject into an oocyte, that are arrested in G2 of the cell cycle, induces the oocyte to develop into a mature egg and produce this white spot
- Must be a factor in egg cytoplasm capable of doing this
What is MPF?
Maturating promoting factor
- Component present in the cytoplasm of a mature egg that when injected into an oocyte results in maturation
How was MPF investigated?
- Purified MPF to try and separate the proteins that were causing this maturation. He would inject these purified MPF proteins into oocytes to see if it induced maturation. whichever proteins worked, then purified further.
- Concluded that the factor must be a kinase because it can phosphorylate histone H1. This was thought to be responsible, in part, for chromosome condensation
- Tried to figure out what proteins were in these purified fractions by digesting the proteins and obtaining the protein sequence
What did Hunt 1983 do?
- Took sea urchin eggs and incubated it radioactive methionine. Cells that take this up will incorporate into proteins and electrophoresis will reveal which are radioactive
- He noticed that there are a couple of proteins where synthesis is mainly occurring but then they would be destroyed
- These oscillations correlated well with when the eggs divided. This lead to the discovery of cyclins, made and destroyed in synchronous with the cell cycle
- He purified and sequenced the proteins
What did Nurse et al, 1983 do?
- Used Pomb yeast to identify cell division mutants
- At restrictive temperature, the Cdc2 mutant yeast are very long. Cdc25 mutant was also very long whereas Wee1 resulted in very small cells.
- He hypothesised that cell division cycle mutants slow down the timing that induced mitosis e.g. the longer they spend in G2, the longer the cells will be – Cdc2 and Cdc25 whereas less time in G2, smaller cells – Wee1.
What did Nurse et al, 1983 discover about the relationship between Cdc2, Cdc25 and Wee1?
He cloned Cdc2, Cdc25 and Wee1. It became clear that Cdc25 is a positive regulator of Cdc2 while Wee1 is a negative regulator of Cdc2 which is a regulator of mitosis. Cdc2 was renamed to CDK1
How did the four principle experiments into cell cycle lead to the discovery pf cyclins?
The genes isolated in yeast, cdc25, were almost identical to band sequenced from MPF from frogs. Cyclins discovered by Hunt also had a similar sequence to another band in MPF suggesting that this theory is right. This discovery gives the cell cycle directionality
How does the expression of cyclins and MPF vary?
Cyclins gradually increase and then are destroyed but MPF is much more sudden and it was not understood why this occurred
What are the four main cyclins in vertebrates?
- G1 - Cyclin D to activate CDK4,6
- G1/S phase transition – Cyclin E to activate CDK2
- S – Cyclin A to Cdk2/Cdk1
- M – Cyclin B to Cdk1
How are CDKs regulated?
CDK regulators include activators, mainly the cyclins, and inhibitors, generically known as CKIs (CDK kinase inhibitors).
Where do CKIs mainly work?
Inhibitors mainly operate in G1 and S
How are CDKs regulated by phosphorylation?
- They must be phosphorylated on a threonine residue located in their T LOOP for proper catalytic activity.
- This is carried out by the CDK7–cyclin H complex (also known as CAK; CDK-activating kinase)
- A serine/threonine kinase that is also involved in transcription and DNA repair.
Give an example of Cdks being regulated by phosphorylation
- Inhibitory phosphorylation of adjacent threonine and tyrosine residues is mediated by dual-specificity kinases such as WEE1. This inhibition is relieved when the CDC25 phosphatases dephosphorylate these residues.
- Wee1 is a kinase that phosphorylated and inactivates CdK1 whereas Cdc25 is a phosphatase that activates Cdk1 by removing the phosphate. First Wee1 dominates meaning CDK1 is off and then Cdc25 predominates so CDK1 is off
What can indue CKI expression?
Anti-proliferative signals such as TGFbeta
How can the loss of CKI lead to cancer?
- TGFbeta induces the transcription of P15 mRNA (a CKI) which inhibits CDK4/6 which are responsible for G1 activation
- This is how contact inhibition occurs, it is expressed by one cell which stops the neighbouring cells from growing
- If this mechanism is lost then contact inhibition will not occur – cancer
What affect do mitogens have on CKI?
- Mitogens control the cell cycle partly though regulating CKI function. It inhibits P21 and P27 (CKIs) and allows progression through the cycle. P21 is regulated by downstream AKT. AKT inhibits P21 preventing it from entering the nucleus stopping its activation and allowing cell cycle progression
How can misregulation of CKIs have clinical consequences?
- P27 (CKI) functionally should be in the nucleus (to inhibit cell cycle progression).
- Can look where P27 is in a. tumour – cytoplasmic or nuclear
- Breast carcinoma P27 tumour revealed that those with only nuclear P27 have qa good prognosis whereas those where it is nuclear and cytoplasmic have less – 50%.
What is meant by restriction points in the cell cycle?
Restriction point blocks cell cycle progression unless the correct nutrients and mitogens are continuously present
When crossed the restriction point, cells are committing to a round of the cell cycle
What controls the restriction point in the cell cycle?
Rb is involved in the control of this restriction point that when passed causes a round of the cell cycle
How does Rb regulate the restriction point of the cell cycle?
- Rb is a DNA binding protein and binds and inactivates the transcription factor E2F which is a principle transcription factor involves in S phase entry – recruits the RNA polymerase allowing transcription of S phase genes
- RB regulates E2F and stops its activation.
- Genes downstream of this therefore are kept off in the presence of Rb stopping cell proliferation.
- Without Rb, cells will always be in the cell cycle and proliferate regardless of the environment
What controls Rb function?
Cyclin D controls Rb function by phosphorylation. When it is hyper phosphorylated it is no longer able to bind to E2F meaning E2F is switched on
How can interfering with the regulation of the restriction point lead to cancer?
- Interfering with the regulation of this system would result in no restriction point so cells would continue to enter the cell cycle.
- This is the same effect of not having any Rb.
- Mutations in Ras and receptor kinases also result in this as Cyclin D is trandcribed and stablised in repsonse to the RAS pathway
What is SV40?
A virus that when infected with cells induces proliferation and DNA replication
What is Large T and how was it discovered?
Discovery
- When cells are transformed with SV40 the cells are very immunogenic so it is easy to produce proteins against them so SV40 proteins are easy to detect.
Viral protein
- SV40 is a transforming virus. The protein identified of being responsible was Large T which is multifunctional protein which works to perturb multiple distinct regulatory circuits in transformed cells.
- Large T can also bind to Rb and inactivate it
