BMS379 Cancer Biology Flashcards

Question

How many genes do retrovirus's have?

Answer 1

Three genes: gag, pol (polymerase), env

Answer 2

RSV has an extra gene to other retrovirus's: src

Answer 3

Temin first suggested that the viral genome could be reverse transcribed into a DNA intermediate

Answer 4

David Baltimore discovered the enzyme reverse transcriptase in 1970, proving this theory. This provides the basis for molecular biology

Answer 5

- 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

Answer 6

- 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

Answer 7

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

Answer 8

- 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.

Answer 9

Mutagenesis theory of cancer

Answer 10

The theory was that If something happens to a gene then it would cause the cell to change

Answer 11

Usually, when cells touch other cells they stop growing

Answer 12

Cells that show transformation do not show this contact inhibition meaning cells overgrow.

Answer 13

- 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

Answer 14

Colony of cells that aggregate together and do not show contact inhibition

Answer 15

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

Answer 16

Michael Wigler

Answer 17

- 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

Answer 18

- 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

Answer 19

- 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

Answer 20

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

Answer 21

Not mutated H-Ras - cellular Ras

Answer 22

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

Answer 23

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

Answer 24

The extra gene in the rous sarcoma virus - found to be a protein kinase

Answer 25

Collett and Erikson (1978)

Answer 26

- 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

Answer 27

Antibodies are attached to beads which was passed down a centrifuge tube. The control serum removes non-specific binding and the antibody specifically binds.

Answer 28

Catalyses the transfer of phosphate from ATP to another protein

Answer 29

Src | - Polymoma Large T is a viral protein that disrupts multiple regulatory pathways in transformed cells

Answer 30

Usually phosphorylation occurs on threonine or serine due to their hydroxyl group but can also occur on tyrosine (only 0.1% of phosphoamino acids)

Answer 31

- 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

Answer 32

- 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

Answer 33

It is quite promiscuous – will phosphorylate many things – not specific

Answer 34

Proliferation of cells in culture requires serum. Growth factors can fulfil the roll of serum - found to be mitogenic. Growth factors were therefore purified

Answer 35

Investigated EGF and what it can bind to

Answer 36

- 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

Answer 37

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.

Answer 38

- 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

Answer 39

Found that there was high homology between EGF receptor and Src showing that EGF was a receptor tyrosine kinase

Answer 40

Suggests that viral oncogenes which transform cells through signalling mechanisms were related to things involved in the growth of these cells

Answer 41

- 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)

Answer 42

- 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

Answer 43

Tyrosine kinases have a transmembrane domain and a receptor which, upon growth factor binding, brings together multiple tyrosine kinases and phosphorylate themselves

Answer 44

- 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

Answer 45

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

Answer 46

- 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

Answer 47

- 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

Answer 48

Radioactive displacement experiment to find what Ras binds to

Answer 49

- 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

Answer 50

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.

Answer 51

- 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.

Answer 52

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

Answer 53

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

Answer 54

- 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

Answer 55

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

Answer 56

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

Answer 57

The first GEF identified was homologous to CDC25 in yeast giving a function to the gene Wigler et al was investigating

Answer 58

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

Answer 59

Drosophila

Answer 60

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)

Answer 61

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

Answer 62

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

Answer 63

SOS was found to be CDC25 so was a GEF.

Answer 64

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

Answer 65

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

Answer 66

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.

Answer 67

Every effector of Ras has a Ras binding domain which interacts with Ras but only in a GTP bound form.

Answer 68

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

Answer 69

EGFR HER2 HER3 HER4

Answer 70

- 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

Answer 71

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

Answer 72

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

Answer 73

EGFR kinase inhibitors, such as gefitinib, have been approved for non small cell lung cancer. Similarly BRAF inhibitors were approved for V600W BRAF melanomas

Answer 74

Most cancer cell phenotypes are recessive but Viral oncogenes exert a dominant effect

Answer 75

Can cause cells to merge due to their fusagenic nature | After cells fuse, all the chromosomes are in the same image

Answer 76

- 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

Answer 77

Remove the organ

Answer 78

- 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

Answer 79

Diagnosed very early in life. By age 10, often lost one eye

Answer 80

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

Answer 81

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

Answer 82

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

Answer 83

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

Answer 84

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.

Answer 85

Cytogenetic analysis revealed that a band is missing on chromosome 13 on people with retinoblastoma This is the critical region in retinoblastoma

Answer 86

- 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.

Answer 87

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

Answer 88

- 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

Answer 89

- 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

Answer 90

- Restriction fragment polymorphism (RFLP analysis) | - Single nucleotide polymorphism (SNP)

Answer 91

- 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.

Answer 92

- 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

Answer 93

- 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

Answer 94

There are 3 million SNPs identified in the human genome so far

Answer 95

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

Answer 96

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

Answer 97

- 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

Answer 98

PARP inhibitors and BRCA1/2 mutations

Answer 99

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

Answer 100

PARP functions to recruit DNA repair proteins to the sites of single stranded DNA break

Answer 101

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.

Answer 102

- 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

Answer 103

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

Answer 104

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

Answer 105

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

Answer 106

- 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

Answer 107

- 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

Answer 108

- 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

Answer 109

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.

Answer 110

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.

Answer 111

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

Answer 112

- 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.

Answer 113

Fission and Budding

Answer 114

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

Answer 115

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

Answer 116

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

Answer 117

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

Answer 118

- 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

Answer 119

- 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

Answer 120

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

Answer 121

- 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

Answer 122

- 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

Answer 123

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

Answer 124

- Rao and Johnson 1970 - Masui 1970 - Hunt et al, 1983 - Nurse et al, 1983

Answer 125

- 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

Answer 126

- 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

Answer 127

Maturating promoting factor | - Component present in the cytoplasm of a mature egg that when injected into an oocyte results in maturation

Answer 128

- 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

Answer 129

- 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

Answer 130

- 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.

Answer 131

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

Answer 132

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

Answer 133

Cyclins gradually increase and then are destroyed but MPF is much more sudden and it was not understood why this occurred

Answer 134

- 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

Answer 135

CDK regulators include activators, mainly the cyclins, and inhibitors, generically known as CKIs (CDK kinase inhibitors).

Answer 136

Inhibitors mainly operate in G1 and S

Answer 137

- 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.

Answer 138

- 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

Answer 139

Anti-proliferative signals such as TGFbeta

Answer 140

- 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

Answer 141

- 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

Answer 142

- 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%.

Answer 143

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

Answer 144

Rb is involved in the control of this restriction point that when passed causes a round of the cell cycle

Answer 145

- 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

Answer 146

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

Answer 147

- 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

Answer 148

A virus that when infected with cells induces proliferation and DNA replication

Answer 149

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

Answer 150

To infect cell culture and immunoprecipitate Large T. Made the medium of the cells radioactive with radioactive methionine. If take an extract from the cells, do electrophoresis and expose to film and the film will turn black wherever there is radioactive band. There seemed to be a protein that specifically associated with SV40 Large T.

Answer 151

Asked if this gene was a tumour suppressor gene or an oncogene and if an oncogene what would happen if in combination with another oncogene

Answer 152

- Cloned p53 and looked at anchorage dependant growth (a) in the presence of oncogenic Ras and p53 deletion (b) in the presence of oncogenic Ras and p53 cloned from tissue culture. - In (b) they found that more fibroblastic foci formed, suggesting p53 is an oncogene. - However, p53 from tissue culture has a val135 mutation, so the experiment was repeated with WT p53, and p53 acted as a tumour suppressor gene, removing the oncogenic Ras function. - WT p53 is a tumour suppressor, mutant p53 is not.

Answer 153

Huge numbers of tumour tissue banks around the world. Used this to ask the question what is the stasis of p53 in these different tumours. There is huge amount of p53 mutations in at least 50% of every tumour. It is the most commonly mutated gene in cancer

Answer 154

Donehower LA et al (1992) - Most are difficult to experiment with. When the knockout ability became widespread, most tumour suppressor genes resulted in embryonic lethality in animal models. Because most are negative regulators of cell numbers then they are important in development. Without either, there are no cell progeny at all. This is true for most tumour suppressor cells but not p53

Answer 155

If follow p53 null, heterozygous and Wt mice and plot a curve of survival, most mice that lack p53 are dead before 1 year due to tumour formations. There is also increased incidence in heterozygote mutants due to the loss of heterozygosity during life

Answer 156

These are antibodies that are clonally produced and are made by taking the spleen of animal that is showing an immune response to protein of interest, dissociating the spleen and fusing the cells to a cancer cells line. This immortalises the antibody producing cells so that there can be an endless supply of these antibodies. This is normally done in mice

Answer 157

- They made a whole series of monoclonal antibodies against p53. Mapped where a particular antibody bound to the sequence of interest and saw which part they bind to. They mapped used recombinant techniques by creating p53 truncation mutants. - He then labelled fibroblasts with radioactive methionine for one hour. Will only see cells on the autoradiogram cells that have just been made in that hour. Previous cells and new cells will not be labelled - Harvest cells at different intervals after the one hour and immunoprecipitate p53 with the monoclonal antibody. As time increases, the radioactive band is going away.

Answer 158

This is telling us that the radioactive p53 synthesised in the previous hour has gone by 40 minutes. This tells us that this protein has a half-life of 20 minutes. This is a relatively short half life

Answer 159

- You can’t see p53 with Pab421 when large T is bound. Pab246 can detect p53 in a manner that was selective for if large T antigen was present or not. This showed that a certain subset of mABs (246) were conformation specific and can recognise p53 in a normal conformation, but not if it adopts a differently folded alternative - Performed western blot with all the other antibodies in these two conditions and the results were the same. Couldn’t see the proteins but could detect on a western blot. There is a subset of monoclonal antibodies that are conformational specific – cant detect if folded differently

Answer 160

Most the mutations in p53 in cancer (75%) are missense mutations (substitution of amino acids)

Answer 161

- Scientists set up yeast transcriptional reporter system using the gal4 reporter in yeast and looking at the output after this protein is added. Add various promotors and ask what impact this has on the downstream. - Remove parts of gal4 and replace with parts of p53. If p53 is in the system then there is an increase in transcriptional activation. - The n terminal part of p53 has this ability

Answer 162

It is a tetramer

Answer 163

Prives et al - Determine the molecule by using sucrose density centrifugation. Have a density gradient and centrifuge and molecules migrate down the gradient slowly, the bigger the molecule the slower it will migrate. - Can then take out layers to see where in the tube the cells are and compare to known proteins to known molecular weights. - This revealed that four molecules of P53 come together to form a tetramer.

Answer 164

Where there are multimers, then it is possible that mutants of this protein could be affectively dominant as you need all four parts of the multimer to be functional for the it to work

Answer 165

- If there a mixture of Wt and mutant p53 subunits then there are many combinations of tetramers that can be formed. To retain functionality all subunits, need to my Wt therefore only one of a possible 16 combinations would be functional. - This is the reason why p53 is a dominant negative. Even in the presence of wild type the mutant have a profound effect on fucntion

Answer 166

Thought to look after the integrity of chromosomes. This is lost in cancer

Answer 167

- Became clear that the distinction between wildtype and mutant p53 could be established by looking at monoclonal antibodies and determining whether they can be seen as AB 246 is conformational specific so will not detect the mutant. - If wildtype p53 interacts with SV40 or acquires missense mutations then it could not be detected by these antibodies

Answer 168

- Exposed human skin to UV and then extract and blot for actin and p53. At various time points after exposure, there is an increase in p53. It becomes stabilised in response to UV - This stabilisation effect occurs in response to many things and not just UV: lack of nucleotides, UV radiation, ionising radiation, oncogene signalling, hypoxia. All of these things are cellular stresses

Answer 169

A CDK inhibitor p21 Cip1. Binds to CDK and directly inhibit them meaning progression through the cell cycle is arrested It is activated by p53

Answer 170

- Made a p53 null cell line and added a Wt p53 whose expression is inducible by small molecule dexamethasone The principle was that p53 is a transcription factor so can compare the output of the mRNAs between the two lines. - This was done using subtractive hybrisation by converting the mRNA into cDNA and hybridise to find nucleic acids which are expressed in one cell line and not the other. Resulted in one gene product which did not hybridise and isolated it

Answer 171

- Target genes were discovered which were involved in DNA repair, blocking angiogenesis and pro-apoptotic - If there are mutations, these things are required to stop propagation of the mutated gene – to arrest the cell cycle, repair the DNA or cell death - One of the hallmarks of cancer is losing the ability of cell death – this is why p53 is lost in so many cancers, they need to lose the ability to die

Answer 172

The rate of synthesis and degradation are both high.

Answer 173

Ubiquitination of proteins is a way of tagging for destruction and MDM2 is a ubiquitin ligase and targets p53 subunits for destruction in a proteasome. This can be shown using a proteasome inhibitor

Answer 174

- In mouse sarcomas, there was a small set of gene products that were responsible for a phenotype in the sarcomas called double minute chromosomes. This is a standard observation in this sarcoma and the gene responsible was names MDM2. It is directly involved in regulating p53 levels – it is a p53 binding protein

Answer 175

MDM2 is a target for p53 binding meaning that the destruction of p53 is itself regulated by p53 so p53 has a feedback loop to ensure that its levels are maintained in a certain range. - If high levels of p53 it will activate the transcription of genes including MDM2. MDM2 will then bind to the P53 protein and ubiquitinate it and target for degradation by the proteasome

Answer 176

- P14 Arf regulates the MDM2 system by binding to MDM2 and sequestered it into the nucleolus stopping it from binding to p53

Answer 177

- p14 Arf is downstream of Ras, C-myc etc. and can result in deregulation of the cell cycle and apoptosis making it a tumour suppressor

Answer 178

Found that when there was no genotoxic stress, p53 is ubiquitinated. In the presence of genotoxic stress, pair of protein kinases (ATM/ATR) are activated, phosphorylating p53. This form of p53 cannot be ubiquitinated and can activate transcription of genes involved in cell cycle arrest, DNA repair and even apoptosis. ATM/ATR are critical to switching on p53, and ATM mutant children are prone to cancer

Answer 179

ATM is turned in response to double stranded DNA breaks and then ATR arrests DNA replication. DNA replication fork slows, ATR is turned on and p53 is activated. They are protein kinases and have kinase domains on the C terminus but these domains look like PIP3 - a lipid kinase. This lead to people looking for a lipid substrate despite it being a protein

Answer 180

Inactive forms of p53 enables oncogene activation without apoptosis, higher tolerance of anoxia and sloppy oversite of chromosome integrity due to the lack of induction of DNA pathways

Answer 181

Can look for small molecules that can interact with p53 and change its shape to move the mutant p53 back to Wt p53 PRIMA1 - Cell line expressed a mutant p53 and treat cells with PRIMA1, the cells accumulate more frequently in G2 meaning DNA is being repaired and population increases below G1. This is the apoptotic group of cells meaning that p53 functionality is restablished by PRIMA1 Nutlins - Nutlins are compounds that interact with the P53 binding pocket of the MDM2 molecule and dislodge P53. This was found to shrink tumours in experimental animals

Answer 182

Recent genetic studies indicate that CDK2, CDK4 and CDK6 are not essential for the mammalian cell cycle. Instead, they are only required for the proliferation of specific cell types. By contrast, CDK1 is essential for cell division in the embryo. Moreover, CDK1 is sufficient among the cell cycle CDKs for driving the cell cycle in all cell types

Answer 183

Constitutive and deregulated CDK activation may contribute not only to unscheduled proliferation but also to genomic and chromosomal instability in cancer cells. The alteration of the DNA damage and mitotic checkpoints frequently results in increased CDK activity that drives tumour cell cycles

Answer 184

Cancer cells grow at one specific site due to uncontrolled proliferation. This is the primary tumour.

Answer 185

10% | Most are due to a metastasis resulting in a secondary site

Answer 186

Organs are surrounded by the basement membrane which separates the organ from surrounding stroma. When found within the basal membrane they are benign as they are localised

Answer 187

Localised invasion Intravasation Extravasation Epithelial to mesenchymal transition

Answer 188

Localised invasion | - Cancer cells have the capability to dissolved the basement membrane and invade the nearby stroma

Answer 189

- Which invasion starts, the cancer cells can reach the blood and lymph. This mechanism is not as understood. Thought that the interaction between cancer cells and macrophages are involved in breast cancer progression into the lumen of the capillaries

Answer 190

Intraversion - Once inside the vessels, can progress around the body - Only small number of cancer cells can survive in the blood and reach different organs. Through mechanism that are not fully understood, cancer cells preferentially migrate to different organs Extraversion - Cancer cells leave the blood vessel and enter the new tissue through extravasation. Intravasation between cancer cells and macrophages has been shown to facilitate extraversion

Answer 191

Cancer cells arrive at the new environment and start growing. Initially only micro metastasis. Then grow and form macro metastasis. Colonisation is the most difficult step because the growing environment is very different to their original environment. For this to occur, cancer cells need to change drastically. This is way the efficiency of metastasis is so low. Eventually, many cancer cells can lead to metastasis

Answer 192

- The organisation of normal epithelial cells in normal tissues is incompatible with motility - They therefore need transition so that they can move. This is known as epithelial to mesenchymal transition (EMT) - This involves the loss of epithelial cell polarity and gene expression, loss of tight junction and loss of cytokeratin expression. Also involves in the acquisition of mobility, protease secretion and mesenchymal gene expression e.g. fibronectin

Answer 193

Cancer cells can move in a variety of ways as they can adapt to different environmental conditions and assume different morphologies to stay motile

Answer 194

Single cell migration | Collective cell migration

Answer 195

- Lack of cell-cell interaction during migration - Low correlation in the migration pattern between a cell and its neighbours - Cells can display different phenotypes: in amoeboid-like (round cell body with short or blebbing protrusions), or mesenchymal-like (elongated cell body and longer protrusions)

Answer 196

- Group of cells retaining cell-cell adhesions for long period of time - Cells move either as narrow linear strands lead by one leader cell or as broad, irregularly shaped sheets, which are multiple cells in diameter and lead by several leader cells - Leader cells acquire partial mesenchymal characteristics but also retain adhesion to cells at the back

Answer 197

- Can switch between every migration mode that is known - This causes a problem when developing drugs to try and block migration as the cancer cells can simply switch to use another mode that is not being blocked - Therefore, trying to understand the master regulators that control the switching between migration modes. Can then force the cancer cells to use a specific mode and then block that pathway

Answer 198

- Loss of p53 function influences cell cycle checkpoint controls and apoptosis but p53 also regulates other key stages of cancer progression, such as cell migration and invasion - Expression of mutant p53 is not equivalent of p53 loss. Mutant p53 can acquire new functions to drive cell migration, invasion and metastasis

Answer 199

When p53 is present, it can inhibit cell migration and EMT. It also inhibits the activity of proteins involved in cell migration resulting in a reduction in the ability to migrate using different migration modes

Answer 200

Morton et al - Used a PDAC mouse model to answer this question. Two transgenic mice: K-Ras G12D active mutant with no p53 and K-Ras G12D active mutant with mutant p53. These mice were generated using cre: lox. Upon Cre activation stop codon will be lost and K-Ras will be expressed and p53 will be lost or substituted with a mutant p53 - There was no difference in the survival of these mice, both died from pancreatic cancer at a similar rare. The was however a difference in metastasis in the liver. Mutant p53 mice showed liver metastasis whereas no p53 did not. Suggests that it is the mutant p53 that drives metastasis

Answer 201

- Mutant p53 expression promotes the recycling of integrins and growth factor receptors from the endosomes to the plasma membrane - The detail molecular mechanisms mediating this have not been elucidated yet. The increased recycling sustains downstream Akt signalling, promoting cancer cell invasion and metastasis

Answer 202

- Two p53 mutants that are commonly found in human cancer and that have been extensively used to study p53’s role in cell migration are R273H (R270H in mice), which directly compromises DNA binding, and R175H (R172H in mice), which causes a global conformational distortion of p53 - These mutations inhibit p53’s ability to act as a transcription factor, accounting for their reduced ability to function as tumour suppressors

Answer 203

- RhoA and RhoGDI are found to be upregulated by mutant p53s leading to the acquisition of amoeboid-type migration in cancer. - p53 is also known to impose upon the function of other transcription factors, including the well-studied p53 family members, p63 and p73. loss of p63 is seen in a range of tumour types correlating closely with the invasiveness of these cancers

Answer 204

Wood et al, 2007 - Looked at the genomic transcripts of colon cancer and found that there were around 80 mutations in every cancer cell. There are some mutations that were common to all tumours but some which were different - The driver mutations are the ones that occur earliest and present in all tumours - There is an enormous amount of mutations in cancer cells. This shows the degree of evolution of mutations in cancer

Answer 205

In epithelia, a Ras mutation alone is not sufficient to cause cancer. If the mutation occurs early, they will attempt to over proliferate and die rather than form a tumour The basic reason why cells become successful in becoming genes is that they acquire a mutator phenotype in the early stages

Answer 206

``` Chromosomal instability (CIN) Microsatellite instability (MIN) ```

Answer 207

Gross changes in chromosomal structure: aneuploidy (loss of chorsmomes or arm of chromosome)

Answer 208

- Point mutations - Microsatellites are regions of repeating bases in the genome. Molecular biology techniques could be developed quickly to detect these and the variations in lengths. The detection of point mutations are more easily determined in areas where microsatellite lengths will change

Answer 209

These events are usually very rare in normal cells. Usually occur once very 10 million divisions. The error rate in replication is extremely low. The error rate in cancer cells is several thousand times higher

Answer 210

A mutation that occurs early and leads to more mutations that cause genetic instability and over proliferation

Answer 211

DNA polymerases have a precursor proofreading mismatch repair. Nucleotide excision repair and base excision repair occur to deal with microsatellite instability. However, mutations in these mechanisms can occur. If this occurs, then push towards a mutator phenotype

Answer 212

That each stage in the cell cycle can't start until the previous on is complete

Answer 213

This doesn’t occur in meiosis, two M phases without S phase to produce a haploid cell or in the salivary glands of drosophila there are rounds if S phase but no M phase

Answer 214

Evidence for checkpoints: Lee Hartwell - Identified a mutant in RAD9. RAD9 where temperature sensitive mutants (not essential). Cells lacking RAD9 where healthy in the absence of extrinsic interference - Crossed these yeast with mutant CDC9. This is a cell division cycle temperature sensitive mutant. - This system can be analysed by looking at permissive and restrictive temperatures - Take cells and FACS analyse. Happily replicating at the permissive temperature. At the restrictive temperature, the cells arrest at G2 – not going into mitosis. This suggests that mitosis entry is dependent on S phase

Answer 215

CDC9 is a DNA ligase – glues okazaki fragments together. Without it, there is fragmented DNA on one side

Answer 216

- Yeast strains of Cdc9 and Rad9 mutants are 100% viable, but if you cross these strains, the viability goes away- this is known as synthetic lethality. is not needed usually - Loss of viability occurs as a consequence of exposing cells to stress- tells us that viability in cells experiencing stress requires Rad9.

Answer 217

There are two genes which each on their own are not essential for life but the combination of them is

Answer 218

If lose checkpoint where mitosis is no longer dependant on completion of S phase then it results in the mitotic spindle trying to segregate chromosomes that have not been replicated - premature condensation phenotype. These cells will not be viable

Answer 219

Robert Schlegel and Arthur Pardee - High levels of caffeine resulted in premature condensation phenotype. S phase does not occur and mitosis is attempted – fails and leads to cell death

Answer 220

- Crush DNA and the DNA is replicated and mitotic spindle form - If add DNA replication inhibitor, cyclin levels are increased and not destroyed and doesn’t enter M phase If add caffeine, then entry to mitosis is restored in the absence of DNA replication. This suggests that there are genes in a pathway that ensures that mitosis will only happen if DNA replication happens

Answer 221

Smyth et al - ATR (also ATM) is a kinase upstream of Chk1 which activates Wee1 and downregulates Cdc25 which acts on cdk1 - Identified ATM an ATR as damage sensors- DNA-activated protein kinases. They are both targets of caffeine, meaning that in the presence of caffeine, even if DNA damage is happening, the pathways don’t turn on, leading to catastrophic mitosis. - Chk1 is phosphorylated and activated by ATM/ATR, and has a role as an intra-S phase checkpoint. - In an arrested replication fork, ATR binds to this. This complex activates Chk1 and blocks any further replication from starting and inhibits cell cycle progression. It is also stabilising the replication forks to allow them to restart later. This pathway has a number of functions as well as preventing cell cycle progression

Answer 222

The genes involved in cell cycle checkpoints are involved in ensuring DNA can be repaired. Therefore, many cancers have lost the checkpoint control. The genes involved in this pathway are either DNA repair genes or checkpoint controls. These checkpoint pathways are therefore important in cancer susceptibility

Answer 223

- If a cancer cell may have lost a relative checkpoint then inflict DNA damage on both sets of cells, normal cells will repair the DNA through the DNA damage response. - However, in cancer cells with a poor checkpoint response, there will not be an arrest in cell cycle or DNA damage response causing the cells to die. - This is the basis for many chemotherapies. The damage in normal cells will be repaired but not in cancer cells

Answer 224

The immune system is a network of cells and tissues that is constantly patrolling our body for invaders. It is spread out in the body involved many types of cells organs and proteins. It distinguishes self from non-self (pathogens) and clears away dying cells

Answer 225

- Viruses - Intracellular bacteria, protozoa - Extracellular bacteria, parasites - Parasitic worms

Answer 226

- When the body is affected by an infection - Kicks in after a couple of hours in a very local area - Upon entry of bacteria, the infection is established and the innate immune system recognises that something is happening. It is not specific but instead recognises parts of the bacteria that is different from our bodies e.g. double stranded RNA - There is no memory response. - If these cannot fight the infection, the adaptive immune response kicks in

Answer 227

- Neutrophils - Mast cells - Macrophages - Dendritic cells - Basophils - Eosinophils

Answer 228

- Occurs after the innate response so takes around seven days. These cells are scattered around the body so must migrate to the lymph node closest to the infection - Produce specific antibodies to fight the pathogen - These two responses work together to fight the pathogen - Memory phase: remembers the specificity of the pathogen and cells produced to fight it – quicker response next time infected

Answer 229

- B cells | - T cells

Answer 230

The primary lymphoid organs are the thymus (T cells develop here), foetal lover and bone marrow

Answer 231

The secondary lymphoid organs are the lymph nodes, spleen, tonsils, appendix, Payer’s patches (at mucous surface to recognise presence of pathogens)

Answer 232

- A cascade of proteins circling in the blood triggered by different stimuli - There are three different pathways which are specific to different pathogens. It is not as specific as antibodies. There is the classical, MB lectin and alternative pathway - The classical pathway is a connection between the innate and adaptive. It requires antibodies - All three pathways work together to activate C3 convertase - The aim is to form a membrane attack complex which punches a whole in the membrane of the pathogen and then lyses the cells

Answer 233

- Cytokines are peptides that have. Fundamental role in communication within the immune system and in allowing the immune system and host tissue cells to exchange information - Cells stimulated by a pathogen secrete cytokines to the surrounding system. They shape the microenvironment around the cells and can be inflammatory or anti-inflammatory

Answer 234

IL-1beta. TNFalpha, IL-6

Answer 235

- They act on the vascular endothelia to increase adhesion molecules, increase permeability, decrease floe rate and increase chemokines - Also have systemic effects in the liver to increase acute phase proteins, the hypothalamus and the bone marrow to increase neutrophil mobilisation

Answer 236

In a tumour specific response, antibodies aren’t in a key area of research as T cells are more involved in the tumour response

Answer 237

- Phagocytes mediating the earliest response to pathogens - Different granules containing different enzymes - Neutrophil extracellular trap (NET) – DNA released to trap pathogen - Short half-life but produced very quickly. Only survive fro about 6 hours in the blood but if needed and enter the tissue they survive from 1-2 days

Answer 238

Eosinophils - The granular content is slightly different and can be involved in phagocytosis - Important in the histamine response – allergic reactions Basophils - Mainly circulating cells - Granules like mast cells - FceR1 expression

Answer 239

Monocytes circulate the blood and enter the tissue and differentiate into macrophages

Answer 240

- Phagocytic cells and ingest and clear pathogens - Express pattern recognition receptors and complementary receptors (e.g. FC receptors) - These cells clear up the apoptotic debris - Also secrete cytokines to recruit other effector cells

Answer 241

MHCI is expressed on most cells and is very general but MHCII is specific to Antigen Presenting Cells. This antigen presentation can help already differentiated T helper cells to act

Answer 242

The most important APCs as they are the only cells which can activate naïve T cells

Answer 243

- They can phagocytose and take antigens up and carry into the lymph node. They can present these pathogens - Have pattern recognition receptors. Upon recognition, they produce cytokines which help the T cells differentiate into difference cell fates depending on the type of response needed

Answer 244

Part of the innate response | and spontaneously kill cells

Answer 245

Spontaneously kill cells via perforin (punches hole) and granzyme B which cleaves the enzymatic pathways in the target cell. This is a dual process so they are activated by certain molecules on the cell surface. This target molecule needs to be able to produce an inhibitory receptor to stop the activation of the NK cell to allow its survival. If both events occur then the NK cell will not kill the target cell.

Answer 246

A tumour cell can downregulate the MHCII expression. Without this, it cannot escape the T cell response. NK cells would still recognise these cells however as they cannot produce the inhibitory receptor to stop NK cell action

Answer 247

- Mediators of cellular immunity - Express antigen specific TCF repertoire - CD4+ (helper) and CD8+ (cytotoxic) - Produced in the thymus - Produce memory cells

Answer 248

Th1 cells - Proinflammatory cells – beneficial in a tumour environment Th2 - Anti-parasitic Th17 - Recruit neutrophils and become activated by environment at the site of the infection TFh - Cells that stay in the lymph and help B cells Treg - Borderline autoreactive – recognise their antigens but can be differentiate into a suppressor state to reduce the cytotoxic state. These are recruited into a tumour state

Answer 249

Jeremy M.R. Lambert et al.(2009)

Answer 250

PRIMA-1 - A p53-reactivating small molecule - Modifies thiol groups in mutant p53 PRIMA-1 conversion products is sufficient to restore its tumour suppressor activity

Answer 251

- Treated recombinant mutant p53 proteins with PRIMA-1 for 20 minutes. This protein was introduced into p53 null Saos-2 cells. This reduced cell survival by 28%. Mutant p53 which had not been incubated with PRIMA-1 did not induce cell death - After 24 hours, there was a considerable increase in the fraction of cells with G2/M content indicating that there was G2 cell cycle arrest and apoptosis

Answer 252

- The ability of PRIMA-1 to restore the tumour suppressor ability of p53 might open possibilities for the design of more potent mutant p53-specific compounds based on PRIMA-1, and eventually the development of efficient anticancer drugs.

Answer 253

- Tumour growth is self sufficient - Can avoid apoptosis - Ignore anti-proliferative singles - Limitless replication potential - Sustained angiogenesis - Invade tissues - Escape immune surveillance

Answer 254

- They have low immunogenicity as they do not have many antigens to activate the immune system. - They are treated as a self-antigen. They arise from our own cells so are treated as part of the body. It isn’t a foreign marker - Antigenic modulation. An antibody against a tumour surface antigen can induce degradation. This however is not typical in cancers. - Tumour induced immune suppression – tumour cells secrete TGFbeta and IL-10 to supress immune cells - Factors secreted by tumour cells which create a physical barrier against the immune system

Answer 255

The ability to attack the tumour | - The natural killer cells and cytotoxic T cells act at this stage in response to IL-12

Answer 256

Cytokines released from the tumour which supresses the immune response

Answer 257

Can be antitumor can also support tumour progression and metastasis There are some macrophages with antitumor activity but these arise early in tumour development and are over taken by TAMs

Answer 258

Tumour-associated macrophages

Answer 259

- If give chemotherapy to patients then more TAMs form. We know this because there are many of these TAMs found in tumours - For example, in breast tumours there are 50-80% of the tumour being TAMs.

Answer 260

They supress the immune system, promote angiogenesis and promote T cells to stop anti-tumor activity.

Answer 261

- In the 1850’s, doctors in Germany noticed that patients would occasionally shrink if their tumour became infected with another disease. This lead to the idea that the body’s immune system could be harnessed to fight cancer - William Poley noticed that TB vaccine could kill cancer. Now used as a drug for bladder cancer

Answer 262

Harness the immune system to kill tumours without killing normal cells

Answer 263

Monoclonal antibodies - Over 75 drugs - Immune system can recognise cancer cells Immune checkpoint inhibitors - 6 drugs - Targets checkpoints in the cell cycle and allow immune cells to work Cancer vaccines - Preventative e.g. cervical - Possibility for personalised medicine Adoptive cell transfer - Only two drugs approved - Used gene editing Cytokines - Man-made versions of some protein that boost immune system - Took a while to get to work in cancer

Answer 264

- Vaccination | - Helps stimulate the immune response

Answer 265

- Killer tumour vaccine - Purified tumour antigens - Professional APC based - Cytokine and costimulatory enhanced vancines - DNA vaccines - Viral vaccines

Answer 266

Transfer of T Cells or antibody therapies that are specific to a particular receptor or cell type

Answer 267

- Cellular therapies can be used to activate a patient’s immune system to attack cancer - They do not act directly on cancer calls but instead work systemically to activate the bodies immune system - They can also be used as delivery vehicle to target therapeutic genes to attack the tumour

Answer 268

- Found throughout the body | - Detect and chew up foreign invader proteins and then present a piece of the invaders on their surface

Answer 269

- To make a dendritic cell vaccine, the blood of cancer patients is collected and enriched to increase the population of DC - Isolate monocytes, add cytokines and activate dendritic cells using peptides - Take cell therapy and put back in the patient to activate the immune cell

Answer 270

- Needed to find better ways to get into the tumour cells and destroy them - Used macrophages to deliver a cancer killing virus

Answer 271

- Monocytes differentiate into macrophages in culture and are incubated with the cancer killing virus - Put back the macrophages into a patient, go into the hypoxic area and produce lots of virus - Usually hypoxic tumours grow even more when given radiotherapy or chemotherapy. Even though this is usually bad, this is increases hypoxia causing more of the macrophages to enter and attack the tumours

Answer 272

Used mice and implant human tumours - Inject macrophages and remove tumour 24 hours later to see if the macrophages have entered the tumour and produced the virus - When given normal therapy and then the macrophages, the tumour shrinks. This stops the extra metastasis that is usually seen - Now want to enter the clinical trials. However, funding was dropped due to expense. Now being funded by an American company - Now looking in melanoma, breast cancer and lymphoma

Answer 273

When inject macrophages into the mice only 4% were entering the tumour

Answer 274

Magnetise the therapy by adding iron ions to drag the therapy to the tumour - Grow a tumour in mice, prepare macrophages and transfect with reporter line and add to mice, apply magnet to top of the mice - When looked at tumours four hours later, 15% of the macrophages reached the tumour showing that drug delivery could be increased. However, this was done on a surface tumour

Answer 275

Used MRI - MRI locate the tumour and then programme the magnetic field to focus on it and pulsed the field. Then removed the therapy to see if the delivery improved from the 4%. Nearly half of the macrophages reached the tumour. Then used MRI for conventional therapy to see if the tumour had the therapy – saw iron was there

Answer 276

Genetics | Epigenetics

Answer 277

Mutations in DNA sequence, change the genes function altering repertoire of proteins expression. This in turn alters the phenotype

Answer 278

Epigenetic modifications cause stable alterations to chromatin structure and gene expression but do not change nucleotide sequence of DNA - Histone modifications – acetylation, methylation - DNA methylation - non-coding RNAs

Answer 279

- Different cell fates during developing are the end results of distinct journey through an epigenetic landscape. This can include encountering signalling pathways, cell to cell contact etc. - Early in development, cells lie in the high reaches of the epigenetic landscape where there is not much signalling. Cells then transition through this landscape depending on what signals they receive resulting in a differentiated cell

Answer 280

Cancer cells arise because they become stuck in the upper regions of this landscape and unable to adopt the post mitotic characteristics that differentiated have

Answer 281

The histone octamers are covalently modified on lysine, serine or arginine side chains to the N terminal tails of the core histones by histone modifying enzymes. These enzymes either: - acetylate of lysine - Mono, di or tri methylated

Answer 282

Acetylation and methylation occur on the lysine side chains in a mutually competitive way. If a lysine is acetylated it cannot be methylated. The only way it can be changed if a specific enzyme is recruited to remove the modification

Answer 283

- Methylation of lysine 4 activates gene expression - Lysine 27 and lysine 9 methylation is associated with transcriptionally silent chromatin - Acetylation on any domain leads to transcriptional activation

Answer 284

Methylation creates binding sites for either repressors (lysine 9 and 27) or activators (lysine 4). Lysine 27 recruits a protein with a chromodomain whereas lysine 4 recruits a protein with a PHD finger domain activator

Answer 285

Acetylation of proteins recruits bromodomain which activates transciption

Answer 286

Bithorax complex and Antennapedia complex

Answer 287

In the Antennapedia complex, loss of function of Scr results in inhibition of sex comb development on the first pair of legs

Answer 288

Jones and Gelbart, 1990 - Discovered a class of genes in which the sex combs were present on the second third legs as well when usually only present on first. Names these Polycomb group Enhancer of zeste. This shows opposite to Scr mutation - The enhancer of zeste encodes histone H3 methyltransferase and is involved in converting the homeotic complex to a heavily transcription repressed locus apart from on the first leg. In animals that don’t have Ezh2 then this can’t be repressed and the sex comb was present on every leg

Answer 289

- The two complexes of Polycomb Group of proteins (Polycomb Repressive Complexes; PRC) includes proteins that can either generate or recognise repressive chromatin modifications: Histone Code Writers and Readers - The chromodomain protein that recognises chromatin modification is Polycomb. Polycomb was the first protein retrieved through genetic screens. - PRC2 - makes the mark - triggers transcriptional repression - PRC1 - recognises the mark - maintains repressed state

Answer 290

- The Polycomb family of proteins are highly expressed in many cancers - Immunostaining for EzH2 in prostate cancer tumour showed overexpression - Increased expression of Ezh2 is accompanied by increased H3K27 methylation and decreased transcription of many genes in prostate cancer cells. This was shown through the RT PCR of the tissues

Answer 291

Bryant et al, 2007 - Inactivated EZH2 using siRNA, and studied metastasis using Matrigel (culture cells in presence of these tiny holes and If the cells are invasive, they will squeeze through these holes and adhere to the other side). When they knocked down an irrelevant gene (luciferase), the prostate cancer cells are still highly invasive. However, the EZH2 knockdowns have a heavily compromised ability to migrate. EZH2 is required for metastasis. - Infected cells with control siRNA or EZH2 siRNA and measured rates of cell proliferation. The EZH2 knockdowns have much lower proliferation rates

Answer 292

Found that there are mutations in Ezh2 in lymphoma

Answer 293

Y641F and A677G

Answer 294

- Mutated forms of EZH2 were discovered to be hypersensitive to a set of small molecules. These act as selective inhibitors of mutant EZH2 with little effect of WT. - These EZH2 mutant proteins are hyperactive and only catalyse trimethylation of H3K27 (not able to cause mono- or di-methylation, unlike wild-type EZH2) - Small molecule EPZ005687 can specifically block H3K27 trimethylation in a dose dependant manner. - Methylation on histone H3 lysine 27 in the presence of this mutation specific small molecules is reduced. This is a cancer specific inhibitor of proliferation. This therefore will not have the lethal effects on normal cells

Answer 295

Chromosomal translocations due to aberrant cross-overs between non-homologous chromosomes can disrupt genes to create hybrid genes encoding hybrid proteins

Answer 296

- Translocation between chromosome 16 and chromosome 8 that creates a fusion protein between two histone acetyl transferases: MOZ and CREBBP. Also found to occur between chromosome 11 and chromosome 16 fusing proteins MLL and CREBBP - MLL fusion proteins drive the rise of a leukemic stem cell population by preventing differentiation of pluripotent haemopoietic stem cells and more committed progenitors. This is leukemia

Answer 297

- MLL contains a catalytic domain that allows it to make histone modifications. Its role is to modify histone lysine 4 modifications. It has a PHD finger allowing it to bind to lysine 4 when methylated: can methylate lysine 4 and recognise these and bind to them. This leads to methylation on adjacent nucleosomes. This hyper methylates the domain causing transcriptional activation. - When MLL is fused, the domains involving lysine 4 methylation and recognition are missing. This suggests that MLL is involved in leukemia by exerting its dominant negative action on its wild type counterpart stopping the activation of target genes

Answer 298

- Gain understanding of disease mechanisms which could lead to potential treatments e.g. BRCA1 - Can identify who are high risk people so they can be screened - Identify which people might respond best to particular drugs - To identify which people might have bad reactions to drugs

Answer 299

- Familial adenomatous polyposis – mutation in APC - Breast cancer – BRCA1, BRCA2 - Retinoblastoma – RB - Many of these genes are involved in the cell cycle

Answer 300

Proposed the two-hit hypothesis which required to turn a normal cell into a cancer cell and that in the strongly familial cancers, the first hit was inherited

Answer 301

Often the inherited hit is a point mutations causing to premature termination of the protein whereas the second hit is often a loss of larger chromosomal region including the wild type allele on the other chromosome

Answer 302

- Positional cloning: Genetic linkage analysis in families to identify the region of the chromosomes the gene is on - Loss of heterozygosity in tumours - Testing tumours for mutations of genes known to be involved in cell cycle regulation - Sequencing of candidate genes in people with a family history

Answer 303

- The extent to which a particular gene results in a specific phenotype apparent in the individual - For example, if a mutation in the gene responsible for a particular autosomal dominant disorder has 95% penetrance, then 95% of those with the mutation will develop the disease, while 5% will not

Answer 304

Highly penetrant genes cause rare syndromes. If you have these gene mutations then 1000 times increase in getting syndrome – do not usually occur without this.

Answer 305

Example of mutations in this are RB1, APC, XP

Answer 306

Familial forms of common cancers e.g. breast and colon cancer. These genes still have a high penetrance and cause a 10-100 fold increase in cancer incidence but these cancers still occur in the general population

Answer 307

An allele with low penetrance will only occasionally produce the trait with which it is associated

Answer 308

Low penetrance genes can be involved but each individual mutation only confers a low risk

Answer 309

An allele with low penetrance will only occasionally produce the trait with which it is associated. It can therefore be difficult to distinguish environmental from genetic factors.

Answer 310

- Assemble a population of families affected by the gene of interest - Sequence their genome using microsatellite markers that span the whole genome - Look to see whether it co-segregates with markers on a particular chromosome – this is done by linkage analysis - Fine-map the region of linkage using more markers - Identify likely candidate genes in the region of interest and look for mutations carried by people with the disease but not by healthy family members

Answer 311

Most cases are sporadic but 4-5% are familial | - Familial differs and is often a bilateral disease and occurs earlier in life

Answer 312

Newman et al, 1988 - Segregation analysis to study a large number of families - Found a dominant disease allele linked to breast cancer. The frequency is 0.01 and if a carrier the risk of cancer is 0.36 by age 40 whereas risk is 0.004 when not a carrier - This could then be used in linkage analysis to localise the disease gene on a chromosome

Answer 313

Can be used to localise a disease gene on a chromosome

Answer 314

Morton 1955

Answer 315

- Can genotype markers and observe whether or not particular marker alleles segregate with disease in familial in which there are multiple cases - Can use this data to work out the likelihood that out marker is linked to the disease gene - A common pattern of inheritance between the disease and the marker suggests that the gene is located closely on the same chromosome. If located far away, would suggest the inheritance of the marker and gene not to be so common

Answer 316

The answer is in the form of a lod score – this is the measure f how likely it is that the disease gene and marker are linked. Lod score above 3 shows significance

Answer 317

Hall et al, 1990 - Identification of BRCA1 using linkage in breast cancer. Used genotype markers and observed whether or not particular marker alleles segregate with disease in families where there are multiple cases. Can then use this data to work out the likelihood that the marker is linked to a disease gene. - Breast cancer and chromosome 17 marker had a lod score of 5.98. Chromosome 17 is linked to breast cancer. Miki et al, 1994 - Further studies used linkage analysis with more families and more markers. Narrowed down the region to 1-2 megabases, then down even further to 600kb. They then cloned the gene for BRCA1 Wooster et al, 1994 - Gene was localised on chromosome 13 and the gene was clones. Found to be BRCA2 - Genetic heterogeneity - different association found in different families - different gene causing it

Answer 318

- These genes do not share sequence similarity but have some common structural features. Large with other 20 exons, about 60% homologous with mice - Large multifunctional proteins and both have a domain that binds RAD51 as well as transcriptional activation domains

Answer 319

One of the most important roles in terms of cancer is their role in DNA damage and repair signalling

Answer 320

Ataxia telangiectasia (AT) is autosomal recessive and caused by a mutation in ATM. Gives a predisposition to cancer

Answer 321

Cells from patients with AT show increase sensitivity to ionising radiation, failure to induce p53 at G1/S checkpoint and fail to stop DNA synthesis in response to ionising radiation. They are failing to signal DNA damage. This is why people with his mutation are predisposed to cancer

Answer 322

ATM phosphorylates MRE11 and NBS1 in response to DNA damage. The RAD50, NRE11, NBS1 complex binds at sites of DNA double strand breaks and initiates DNA repair. Mutation in these genes causes familial syndromes with genomic instability

Answer 323

Caused by mutation in NBS1 autosomal recessive and leads to microcephaly, immunodeficiency, predisposition to haematopoietic malignancies

Answer 324

Cortez et al, 1999 - Showed that ATM also phosphorylates BRCA1. - They took wild-type and AT-mutant fibroblasts and g-irradiated them, then showed by Western blotting using anti-BRCA1 antibodies that BRCA1 was phosphorylated in the wild-type and not in the mutant cells. - A mutated BRCA1 lacking the 2 phosphorylation sites failed to rescue the radiation hypersensitivity of a BRCA1 deficient cell line; thus this pathway is important to protect cells against DNA damage.

Answer 325

ATM mutations have been identified in breast cancer families and heterozygote ATM mutation carriers are at a 2-fold increased risk of breast cancer A variant of the cell-cycle arrest gene CHEK2 also contributes to familial breast cancer as do mutations in PALB2

Answer 326

ATM phosphorylates BRCA1 and BRCA2 which are together in a complex which regulate DNA repair

Answer 327

- Sporadic cases of cancer are caused by a combination of genes and environment - The genes have weaker effects and act in combination with each other and environmental influences - The genetic variation in this case is polymorphisms rather than mutations; i.e. common variation such as single nucleotide changes (SNPs)

Answer 328

Polymorphisms present in the population at a reasonable frequency. We can identify SNPs affecting breast cancer risk in genome-wide association studies

Answer 329

The distribution of the risk between the general population and the cancer population has a large overlap – many people have cancer who carry low risk alleles or high risk alleles and do not have cancer. These genes are low penetrance as many other factors affect the risk

Answer 330

We can compare allele or genotype frequencies in cases and controls; if the marker allele we are looking at is associated with the disease allele, we will see an increase in the frequency of that allele in cases compared to controls

Answer 331

- Can assay thousands of SNPs at once | - Need large sample size due to these genes low effect

Answer 332

- Used 400 UK controls and 400 high risk breast cancer cases - Compared the SNPs in both groups and genotyped - In stage 2, the top hits were identified and genotyped again - Final stage took the most significant hits and geneotypes with more cases and controls to identify the SNPs - Many stages and controls are required as there will be many false positive results due to the high incidence of SNPs and their low effects - The top hit was a mutation in FGFR2 on chromosome 10. There were 6 genes all together.

Answer 333

The Oncoarray Network genotyped 400,000 samples across 5 cancers: colon, breast, lung, prostate, ovarian

Answer 334

Oncoarray: 570K SNP markers custom bead-chip array manufactured by Illumina. The array includes a backbone of approximately 260,000 tag SNPs that provide genome-wide coverage of most common variants. Markers of interest for each of the five diseases were identified through: genome-wide association studies (GWAS), fine-mapping of known susceptibility regions, sequencing studies

Answer 335

- Found that 20 genes did overlap: these genes are associated with breast cancer in a GWAS and found mutated in breast cancer tumours. Most of these genes are not coding but are regulatory effecting expression e.g. enhancer or activator

Answer 336

Cytotoxic chemotherapy is when the treatment selectively treats cancer cells based on their loss of checkpoints in the cell cycle

Answer 337

- Induce differentiation - Discourage proliferative signalling - Promote pro-apoptotic signalling - Discourage anti-apoptotic signalling - Exploit checkpoint vulnerability - Identify the relevant population for specific strategy

Answer 338

It is important to identify the type of cancer because there will always be people that do not benefit from the treatment. It is important to include these people in the data to improve for people who do benefit from it

Answer 339

- Due to ensuring food quality and appropriate storage (avoid food that leads to cancer e.g. smoked food) and cervical and colorectal cancer screening

Answer 340

Using MRI’s which can detect very small growths (1.7mm)

Answer 341

People can be over treated

Answer 342

- Indolent tumours: low invasive, metastatic potential - Aggressive tumours: high metastatic potential - Tumours of intermediate grade: treatable, resectable

Answer 343

- Patient age - Tumour size - Number of axillary lymph nodes (under arm in breast cancer) - Histology (antibody staining)

Answer 344

Based on the specific histology of the tumour - how should it be treated

Answer 345

Specific treatments only work when receptors are present | - e.g. oestrogen receptor antagonists can be used but only if oestrogen receptors present

Answer 346

- Only 19% of the tumours go into show a life-threatening disease. - This is overtreatment as 80% of people with breast cancer likely do not need to be treated - However, we do not know which 80% they are

Answer 347

- Cost of a referral to a consultant for a test for cancer is around £200. - If the tests were cheaper more people would be screened - This would stop over treatment

Answer 348

- For each patient do a microarray screening for genes involved in breast cancer and look to see if up or down regulated - Compare this to a gene expression profile of other breast cancer patients and their prognoses - Can then use a computer tooling these results to see if the patients gene expression is a good (high percentage that survive) or a bad signature (low survival rate) - The people with a bad gene expression signature should then receive treatment

Answer 349

Can’t tell which are aggressive and relatively benign and therefore the patients do not know their prognoses

Answer 350

- There are three different lymphomas which have a common microscopic appearance but show different gene expression - Therefore, need microarray analysis to identify the type of lymphoma present. Different changes in gene expression drive different forms in myeloma - Gene expression profiling can then be used and a computer can read what type the patient has. This will tell us the prognosis of the patient and the treatments they can have

Answer 351

- Can look specifically at different genomic changes and the patterns are different in each type - The NF-kB signalling pathway is constitutively active in two of the types of lymphoma but not the other - This pathway can be blocked with drugs using specific inhibitors - There is no benefit to treating the myeloma with no active NF-kB signalling with that inhibitor - Therefore, the type of cancer and genes expressed changes the type of treatment

Answer 352

As tumour cell populations evolve to greater degrees of malignancy they usually lose increasing numbers of differentiation markers

Answer 353

- RARa is a receptor for retinoic acid and when it is bound, proliferation is down regulated in the progenitors and they differentiate. This often goes wrong in cancer - Predictable chromosomal rearrangement occurs in cancer cells and causes breaks in RA receptor meaning that it fuses to PML gene (Pro-myelocytic leukemia). RA then can't bind to RARa and prevents differentiation - This results in a protein that is a mixture of the PML gene and RA receptor that sits in the nucleus (PML bodies). This is leukemia

Answer 354

- This can be treated with a derivative of RA called all trans RA (different structure) then it binds to this fusion protein. Can also use arsenic trioxide – it is selective in this disease - It works by promoting a specific ubiquitin ligase called RNF4 to target the fusion protein and drive to the proteasome for destruction. All trans RA does the same thing. This is a way of destroying the gene product involved in the Pro-myelocytic leukemia without destroying the Wt protein - The Wt RARa is then able to bind RA and reinstate differentiation

Answer 355

Cells have been engineered to express CFP or GFP to mark Wt PML or the fusion PML with RARa, respectively. After arsenic trioxide the fusion gene product is disappearing while normal PML is still present

Answer 356

- DNA replication will be arrested and will have a normal checkpoint response and wait until the damage or stress has been removed - Part of the checkpoint response is to block where future DNA replication could occur in the genome. Block in S phase – all the replication forks stop and inhibits future activation of replication forks (all replication forks don’t start at the same time during S phase).

Answer 357

- In cancer cells that have lost the checkpoint response, DNA replication is attempted but is not doing anything. Comes out in G2 without replicated DNA. - This will result in the cell to attempt mitosis and undergo mitotic catastrophe and die

Answer 358

- By knowing tumours may have lost replication checkpoints, can inform us what kind of DNA damage should be done to treat the tumour - e.g. Hepatoma cells lack G2 checkpoint so can treat with doxorubicin to damage DNA. Normal cells will repair this damage by cell cycle arrest but will not be repaired in hepatoma cells due to the lack of the checkpoint. This results in a treatment specific for this type of tumour

Answer 359

Drugs are usually small molecular weight compounds that inhibit rather than enhance the biochemical functions. When both copies of tumour suppressor genes have been lost in a tumour, due to loss of heterozygosity, we want to enhance its function not inhibit it. It is therefore difficult to target

Answer 360

PRIMA-1 - Can promote pro-apoptotic signalling - Most p53 mutations are missense mutations that cause conformational changes. FACS shows that cells with these mutations do not undergo apoptosis. Adding PRIMA-1 restores apoptosis of the cell by restoring the original shape of p53 by covalent binding to the core domain

Answer 361

MDM2 is a ubiquitin ligase which promotes the destruction of p53 It binds to p53 by the alpha helix of p53 sitting on MDM2 – this is not enzymatic.

Answer 362

MDM2 is a ubiquitin ligase which promotes the destruction of p53. Tumours with elevated MDM2 or have low levels of p53 could be treated by targeting MDM2 to increase the levels of p53.

Answer 363

Nutlin-2 is a protein-protein interaction inhibitor which can mimic the structure of p53 and sits in the MDM2 valley. This will disrupt the binding of p53 and stop its destruction meaning there will be more apoptosis

Answer 364

- RB is required for G1/S checkpoint and is therefore antiproliferative. When RB is phosphorylated the cells will not have this checkpoint - CDKIs such as p21 and p27 prevent phosphorylate RB and stop cells entering S phase

Answer 365

Cisplatin based drugs but cancer cells develop resist to them

Answer 366

- VR54 molecule blocks the proliferation of cells by increasing the expression level of p27 (inhibitor of CDK) in a concentration dependant manner, blocking the phosphorylation of RB. This therefore blocks entry into S phase - This is a molecule that is inducing a gain of function and restoration of the G1 checkpoint in cells that largely ignore it

Answer 367

- A defined structure that will specifically bind an appropriate low molecular weight chemical entity - The small molecule when then inhibit the function of the cavity

Answer 368

As it will give more of an effect for a given concentration of drug

Answer 369

e. g. Hydrogen bonds and Van Der Waals - The more points of contact, the higher the specificity and potency of the drug. - This will mean it will not bind elsewhere and can use less concentration of drugs as it will bind tightly – less side effects

Answer 370

Gleebac - There are many interaction sites where there are hydrogen bonds and van der Waals between atoms in the Gleebac and Abl protein. - Gleebac will sit in the catalytic cavity of the Abl protein which can now be used to treat cancers where there is an increase in Abl.

Answer 371

So you can then visualise where the drug will sit within the protein

Answer 372

Whether the protein can be targetted specifically for a drug

Answer 373

Transcription factor onogenes

Answer 374

- Transcription factors have some degree if specificity (DNA binding) but there are not many compounds known to inhibit a specific transcription factor – only all TFs.

Answer 375

- Many kinases are oncogenes (often constitutively active) - 518 (and counting) kinases in the genome - Very druggable (ATP substrate binding cleft)

Answer 376

- Protein kinases have derived from a common ancestor meaning that the structure is very similar - However, usually the ATP binding site is different enough to produce specific competitive inhibitors for this binding site - These are often used as treatments for cancer.

Answer 377

Leukemia can be either caused by block in maturation (results in excess blasts in marrow, and hence not enough mature cell types), or over proliferation (too many cells in marrow)

Answer 378

Lymphoid | Myeloid

Answer 379

- This lineage gives rise to lymphocytes and is found in senior children. - The earlier the cells stage of maturation the more serious the cancer - It results in a block of maturation of that cell type which can result in problems with the immune system and an accumulation of immature cell types – body tries to make more lymphocytes as there are no mature ones but these can also not mature

Answer 380

CLL | - Present more in elderly people rather then children

Answer 381

- More common and involve more cell types (red blood cells, basophils etc.). - There are two types of this: problems with maturating (do not have functioning cell type) and proliferation syndrome (cells are mature but over proliferation due to signalling errors).

Answer 382

- AML (acute myeloid leukaemia) is a maturation disorder | - MDS is when there is less then 20% impaired blasts. If its over 20% then it is AML

Answer 383

The detection of structural abnormalities that lead to a fusion of two genes, creating chimeric gene. FISH will also detect genes juxtaposed to new gene controls, causing altered gene expression.

Answer 384

Cytogenetics is the term used to generally describe analysis of genetics at the chromosomal level ie the cellular level

Answer 385

Banding the chromosomes involves exposing them to trypsin for a few seconds and then staining using Leishmans stain

Answer 386

- If AT rich, late replicating or heterochromatic then it can remove more proteins meaning that the stain is stringer resulting in dark bands - Light bands are GC rich, early replicating and euchromatic

Answer 387

- Pairing the chromosmes in size order and examining the banding pattern for abnormalities for each pair of chromosomes.

Answer 388

- Fusion genes: Breakpoints can occur within the two genes or next to the genes which give rise to a hybrid gene which gives rise to a chimeric protein e.g CML - Dysregulation is the juxtaposition of a gene to a regulation gene. Altered regulation can result in increased transcription e.g. Burketts lymphoma

Answer 389

A breakapart probe consists of 2 probes which hybridise at opposite ends of the same gene. In a normal cell the red and green signals are fused producing a yellow signal, in a abnormal cell with a gene rearrangement there will be one yellow fusion signal representing the normal chromsome and a separate red and green signal reprsentiing the break in the gene

Answer 390

FISH can also be used to detect fusions resulting from a known translocation. This can be done using dual colour dual fusion probes. Unlike breakapart probes, there is a probe for each chromosome involved in the translocation and gives us speacific information on both chromosomes involved in the translocation.

Answer 391

RQ-PCR fluorescent labelling allows quantification of amplified DNA molecules as PCR progresses through the reverse transcription of mRNA present in the cell

Answer 392

ddPCR is a highly sensitive mutation detection technique. To less than 0.01% detection

Answer 393

NGS is a powerful method of gene sequencing. This can be done for either single genes to whole genomes Much greater depth and accuracy than sanger sequencing

Answer 394

Diagnostic Prognostic Treatment stratification

Answer 395

Acute Myeloid leukemia - 55% patients chromosome abnormality - Of these 80% are non-random, and have specific correlations

Answer 396

- Patients with APML, are at high risk of DIC, this is Disseminated intravascular coagulation and is a condition in which blood clots form throughout the body. - It has a very high mortality rate, however if treated with retinioic acid this type of leukaemia is categorised in the good risk group - Samples where a 15;17 translocation is a possibility are treated urgently with an overnight FISH result to confirm the rearrangement

Answer 397

Acute pro-myelocytic leukaemia - Affects promyelocytes – platelets stop coagulating - PML/RARa gene fusion and results in transcriptional activation - Required fast treatment – ATRA (all trans retinoic acid)

Answer 398

- Caused by the Philadelphia chromosomes is the derivative chromosome 22 from the t(9;22) - the translocation results in the fusion of two genes. - At diagnosis 90% of patients show a 9;22 translocaton. - This results in increased tyrosine kinase activity and neoplastic growth.

Answer 399

- ABL1 is a cellular oncogene and encodes a nuclear tyrosine kinase protein - BCR/ABL1 gives an abnormal fusion protein with increased tyrosine kinase activity Is a positive regulator of cell growth and leads to increased proliferation and malignant growth - Alone, is sufficient to cause CML

Answer 400

``` FISH - To confirm rearrangement - To pick up cryptic rearrangements - To determine deletion status - To allow for follow up FISH Full cytogenetics - To pick up further abnormalities Request EDTA sample for molecular testing - BCR-ABL1 screen Baseline quantitation by RQ-PCR ```

Answer 401

Imatinib Specific inhibitor of of tyrosine kinase domain of ABL1 Blocks the binding site for ATP and inhibits phosphorylation Prevents activation of the signal transduction pathways

Answer 402

100,000 genomes project reveal new genetic alterations - Identify genes involved in tumourigenesis - Allow greater understanding of their roles in tumourigenesis - Allow development of more specific and effective forms of therapy - Increasing personalised medicine - Wider range of targeted therapies

Answer 403

Erlotinib is specific for the EGF-R kinase that is often upregulated in cancers

Answer 404

- Need to be able to measure the protein kinase activity that is upregulated in the cancer easily and cheaply. - Then purify significant amounts of it so it can be used in drug screenings with a compound library. - This can be done with a robot and look for compounds that cause some inhibition. - Investigate the structure of the compounds that inhibit and then use this for rational drug design. - Then ask how they can build on these structures to increase specificity based on the structure of the kinase

Answer 405

Altering the codons of the monoclonal antibodies produced in mice to make them more specific for the human homologue. This is less likely to be rejected.

Answer 406

Cetuximab - Now used as a treatment for carcinomas - Specific for human EGF-R

Answer 407

Patients have different mutations in these receptors that lead to cancer - Some might lead to ligand independent binding so blocking binding with a drug will not treat this - Therefore need to know what kind of mutations the patient must give an appropriate and effective treatment

Answer 408

There are many tumours now which have become resistant to treatments

Answer 409

- Gleevac treats ABL dependant tumours - There are multiple mutations that emerge in tumours which results in a resistant form of BCR-ABL. The mutation stops gleevac from binding but is still catalytically active

Answer 410

The addition of a drug that wills electively kill these cells will result in natural selection and the emergence of resistant mutations. These mutations are predictable so trying to develop inhibitors that have a secondary function so will still work after the primary inhibition has become ineffective after tumour mutation

Answer 411

- Need clinical trials which include people with different types of resistance to different treatments to see if a new treatment works for them. - However, there are very few people in each group meaning that the statically significance will be very low. - This is major problem that needs to be addressed going forward

Answer 412

HRAS KRAS NRAS

Answer 413

20% | - 80% of these being in KRAS

Answer 414

- Receptor tyrosine kinases (e.g. EGFR) are activated and auto phosphorylated. This then binds to adaptor protein GRB2 which is bound to SOS by which RAS is also localised. SOS then catalysis the nucleotide exchange of RAS with GDP being swapped for GTP - GTP bound RAS then binds and activates effector enzymes which control cell proliferation and survival. RAF is one of these effectors which then activates the MAP kinase signalling pathways. Also found to activate PI3kinases and phospholipase C.

Answer 415

- RAS proteins are active if they are bound to GTP and inactive if bound to GDP. - In vitro, RAS GTPase activity is low but this is catalysed in vivo by GEFs and GAPS. Several GEFs have been characterised including SOS1 and SOS2 which are the mammalian homologies of son of sevenless

Answer 416

In tumours, the mutations in RAS compromise the GTPase activity of RAS preventing GAPs from preventing hydrolysis of GTP causing it to remain in its active form. - leads to over proliferation and survival

Answer 417

Farnesyltransferase inhibitors - The covalent attachment of the farnesyl isoprenoid group to RAS proteins is responsible for maintaining RAS to the plasma membrane. This is therefore a good target for therapeutics - Some FTIs have now been identified through the screening of compound libraries. Saw success when treating HRAS expressing transgenic mice but this success was not repeated in humans

BMS379 Cancer Biology Flashcards

(451 cards)