1
Q
What is the purpose of primary cell culture techniques?
A
Grow cells directly from body, in vitro, to recreate invivo environment as closely as possible.
2
Q
Describe primary cell culture technique
A
- From tissues (hematopoietic or non-hematopoietic)
- Cells can be variable
- Finite lifespan, so cell eventually dies
- carry out normal function, including death)
- cells can divide or differentiate
3
Q
Describe cell lines
A
- cells spontaneously arise from tumours or through genetic manipulation
- cells are identical, so there is no variability
- there is an infinite lifespan, as they are placed in liquid nitrogen before and after use
- cells divide only
- may not carry out normal function if abnormal gene expression.
4
Q
Hematopoietic cells
A
Hematopoietic stem cells (HSCs) are responsible for the production of mature blood cells in bone marrow.
They are stem, progenitor cells. Can become T &B cells, monocyte, macrophages, osteoblasts, dendritic cells, neutrophils, eosinophils, basophils, mast cells, erythrocytes, or karyocytes or platelets.
Hematopoietic cells are already in a cell suspension, so we can take them directly from body, do not have to manipulate them.
-cELLS ARE ALREADY IN A CELL SUSPENSION, CAN take them directly from body, do not have to manipulate them
5
Q
Non-hematopoietic cells
A
These non-hematopoietic stem cells make up a small proportion of the stromal cell population in the bone marrow and can generate bone, cartilage, and fat cells that support the formation of blood and fibrous connective tissue.
Liver, muscle, skin, nerves, fibroblasts and endothelial cells.
6
Q
How do we remove the non-hematopoietic cells from tissue they come from?
A
Put the tissue into culture, cells will then grow and migrate out of explant. Use enzymes (break bones between cells, eg trypsin, collagenase, hyaluronidase, protease, DNA ligase) Mechanically-remove them from tissue by pipetting or sieving or mincing.
7
Q
What is haemopoiesis
A
The production of blood cells and platelets, which occurs in the bone marrow.
stem cells–> early progenitors–>late progenitors–> immature precursors (cells become distinguishable from each other)–>mature cell stage (able to distinguish from microscope).
-Growth factors important in every stage
-As we go along, cells become more differentiated, get amplification.
-Stem cells are also self-renewing, so divide into more stem cells, either self renew, or differentiate.
8
Q
What are the ways to distinguish between stem cells/prognitors etc
A
Antigen markers-if positive, cell expresses them
1) CD34–> STEM AND progenitor cells are CD34 positive. Mature cells are cd34 NEGATIVE
2) Lin–> stem cells and progenitors are lin negative, mature cells are lin positive.
3) RH123 dye, fluorescent, only picked up by cells in cycle, otherwise dull
9
Q
How do we get stem cells out of bone marrow?
A
We use different methods, depending on how pure we want the cell to be.
1. Erythrocyte lysis
->produces enriched population of stem cells
2. Density gradient centrifugation
-> bit more pure than erythrocyte lysis
->removes red cells/neutrophils
3. Adherence depletion
->put bone marrow on plastic, some cells will just stick to the plastic, eg fibroblasts, and macrophages
Then harvest the rest for enriched population.
4. Antibody depletion
->remove unwanted cells
->deplete all Lin positive cells
->gives enriched population
5. Antibody selection
->positively select the cells that we want.
->positively select CD34 positive cells, produces pure population. We can use antibodies/antigens with fluorescent markers, or we can use fluorescent cell sorting. Or use magnetic beads attached to antibodies. Pull out the beads with a magnet, and this will give us the purest population.
10
Q
Describe the purpose of colony assays
A
In microbiology, a colony-forming unit (CFU, cfu, Cfu) is a unit used to estimate the number of viable bacteria or fungal cells in a sample.
Look down microscope, scan then count colonies of different types of cells based on their morphology. Then work back and quantitate how many in original cell suspension. We are assaying for cells at progenitor stage. We can assay for colony forming units for all cel types.
Semi-solid medium (agar/methylcellulose)
Growth factors
Incubate for 7-14 days
Individual cells will then form colonies that we can identify through microscope.
We can then quantitate the number of progenitors there were in original suspension that was put into culture.
1. CFU-G ->granulyte progenitor
2. BFU-E burst formng unit
3. CFU-E + BFUE if cells have RBC, they came from CFU-E or BFUE erythroid progenitors
4. CFU-MK megakaryocyte progenitor
5. Some colonies also made of several types of cells -> CFU-GM granulocyte/monocyte progenitors. Progenitor can differentiate down either lineage.
6. CFU-GEMM -> from progenitor much further back in hierarchy/granulocyte/erythroid/monocyte/megakaryocytic progenitor.
7. CFU-bas basophil progenitor
8. CFU-eo ->eosinophil progenitor.
11
Q
What are the applications of primary cell culture
A
Experimental
-research on normal and abnormal cells
Diagnostic
-test toxicity of chemotherapeutic agents and carcinogens
Therapeutic
-generate/amplify cells for stem cell transplantation/manipulation.
12
Q
Stem cells
A
Pluripotent, give rise to all lineages
Self renew
Rare cells
Responsible for engraftment
13
Q
Progenitor cells
A
Undifferentiated
Not distinguished by morphology
Committed to one or more lineages
Detected in colony-forming assays
14
Q
Precursor cells
A
Immature but recognisable
Cells starting to differentiate
Few final divisions before mature cells
15
Q
What are haematopoietic growth factors
A
Polypeptide growth factors (cytokines)
Bind to cell surface transmembrane receptors
Stimulate growth and survival of progenitors.
16
Q
How do you make a cell line?
A
- Isolate cells from solid tissue or blood.
- Produces primary cells
- Transform the cells through transfection and selection
- cause characterization of the cells, by STR profiling/karyotyping.
- Culture the cell systems (cryo-stored cell line)
17
Q
What is the history of cell culture?
A
1882: Sidney Ringer developed solution of salt to maintain frog heart.
1885: Wilhelm Roux cultures embryonic chic tissue
1940-1950: Development of cell culture techniques for growing viruses
1951: George Otto Gey propagates HeLa cells from Henrietta Lacks
1954: Enders, Weller and Robbins receive nobel [rozes
18
Q
How do you isolate stem cells from blood?
A
Blood sample can be easily centrifuged with gradient formed medium. In blood, the different cells are already in isolation and in suspension, as opposed to the close packing of cells found in tissues.
If centrifuge, the different types of cells found in blood will form different layers based on cell density and size,
Layers can then be isolated and purified by using immuno-purification methods, such as mixing cells with antibody coated magnets or using a FACS machine.
19
Q
Describe the immunopurification methods used to isolate stem cells from centrifuge with blood layers
A
- Magnetic beads
-mix the cells with antibody coated magnetic beads
Antibody is a cell surface marker that a specific cell we want to isolate will express
-mix with heterogenous cells
-the magnetic coated beads bind to the specific cells with cell surface marker.
-then isolate the cell with magnet. - Or use a FACS machine
-fluorescence activated cell sorter
-mix cells with antibody that has a fluorescence stand on it.
-Antibody binds to specific cell that has a specific antigen for that.
-Pass the cell through a fast flowing stream of liquid that also vibrates and when it vibrates, each cell is isolated into a single drop, as it passes through laser detector.
As cell passes through lazer detector, the ones that fluoresce can be counted and quantified, so we know how many of these cells are in a sample. fluorescing cells have a positive charge, therefore cells will be sorted into negatively charged container.
20
Q
Describe the isolation of cells from solid tissues
A
Select cells from first trimester placenta (by termination of pregnancy).
Cut placenta and wash it. Use mechanic and enzymatic disruption (dispase, trypsin, collagenase, or mechanical syringe).
When get to layer of cell clumps, use magnetic immuno-purification of CD31 (stem cells are positive)
You can then see the positive (stem cells) cells as they will have magnetic beads when you plate them.
If needing to isolate cells that are on the surface of a tissue, you can do explant for the tissue, and the cells will migrate out and form a monolayer on plate of tissue.
21
Q
Describe growth of cells in culture (out of their natural environment)
A
-cells handled under aseptic conditions
-cells are grown on treated plastic
-cells maintained in a warm, humidified atmosphere
-cells are put in incubators to meet conditions of human body
cells are not arrested (have growth medium with essential amino acids)
-pH has to remain stable and physiological
-cells grow and produce toxic byproducts
->need to change medium regularly by monitoring pH indicator
-cells have classic cell cycle
22
Q
What is the advantage and disadvantage of primary cells
A
Advantage is that they are unmodified, and represent a source of origin as closely as they can
Disadvantages: They have an aberrant expression of some genes-mutation may have happened (not obvious to naked eye)
-Variable contamination (not obvious to naked eye)
-poor growth characteristics (50-100 divisions, have finite lifespan)
-interpatient variation (eg from placenta, cells are unique to that patient-cannot draw conclusion for group of patients, have to do as many samples as possible)
-phenotypic instability
-molecular manipulation is difficult
23
Q
Describe the characteristics of cell lines?
A
Good growth characteristics
Phenotypic stability
Defined population
Molecular manipulation readily achieved
24
Q
How do we make cell lines?
A
Isolate from cancerous tissues (eg HeLa cells)
Derive from primary cultures:
-Spontaneously (from prolonged culture, or through genetic manipulation (where we manipulate cells so they continuously grow and divide, by targeting the tumour suppressor proteins-p53 and retinoblastoma by using viral oncoproteins). Eg, SV40 or HPV virus (simian virus-40, or Human papilloma virus).
SV40s T antigens interact with p53, and pRB, keeps protein bound, causing increased growth without loss of function of these proteins. Cell then continues growing.
E6 targets p53 for degradation & E7 binds to pRB, inactivating it. Cell lines made using E6/E7 oncoproteins are believed to maintain a differentiated phenotype.
25
Describe the p53 protein
-Has checkpoints in cell cycle, R1 checkpoint in g1 phase
-Telomeres bind to telomeric binding protein at ends of chromosomes.
As we age, telomeric repeats get shorter
TBP cannot bind to telomeric repeats anymore.
Therefore p53 (protector of cell) comes and binds to end of chromosome and triggers growth arrest and signals cell to become apoptotic (die)
If p53 is not there, cells continue to divide and there is a greater risk to go into uncontrollable cell division.
26
Describe the retinoblastoma protein
checks whether G1 phase has progressed well, then the cell continues cell cycle.
27
Describe the telomerase enzyme
Ribonucleoprotein
It is a reverse transcriptase (makes DNA from RNA)
maintains and preserves telomeres (repeats at end of chromosomes)
If telomeres not there, machinery of cell will recognize DNA single strands and remove them, so telomeres protect them
As we age, telomeric repeats become shorter. Telomerase not present in every somatic cell, obly present and active in germ cells, adult and stem cells, as well as most cancers (only in cells that have capaciity to divide again and again_.
Having telomerase encourages cells to divide continuously.
28
How do you immortalize cells (eg to make a cell line)
Introduce the telomerase gene (hTERT) into a target primary cell.The telomeres stop getting shorter, and the cell keeps dividing, to make process more efficient, can introduce SV40 large T antigen, and this then produces a tumour cell line by p53 & pRB. Some cells need introduction of telomerase gene, and inactivation of the RB protein for immortalisation.
E6/E7 & telomerase transformations are belived to result in cell lines with a differentiated phenotype.
29
How do we introduce oncogenes, or the telomerase gene into a target primary cell?
Construct plasmids that allow it to be expressed. Apply selection pressures, for instance an antibiotic. If we put the antibiotic on top of cell, cells will die because concentration of antibiotic will die (unless cell is antiobiotic resistant)
Cells create a small colony and express gene wanted to express.
Cells continue to divide further than what was expected. This testifies the method.
30
How do we introduce plasmid DNA into the cells?
1. Efficient transfection method
- CaPO4 co-precipitation, precipitate DNA, cell takes it up by endocytosis. This process is improved by Ca2+ nanoparticles.
- Lipofection: cationic lipid transfection systems
- Electroporation: physically transfecting, using electricity
- Viral transfection: takes advantage of viruses and how they incorporate their gene into gene of infected cell.
- nucleofection
2. Stably incorporate the growth promoting gene into primary cells DNA.
31
What are the disadvantages of using a cell line?
Rapidly dividing cells often lose differentiated function (do not have time to specialize)
32
What is the authentificaton of cell lines
ATCC & ECACC: keep accurate records of all existing cell lnes, keep normal functioning cell lines, no mutations. Cells are constantlychecked by karyotyping and STR profiling.
33
Describe lipofection
A unilamellar liposomal structure. Use a cationic compound (positive head with a hydrophobic tail, connected by linker sequence).
This interacts with DNA/plasmid, to create a net positive charge.
Membrane of cell negative, it interacts with lipoplexes
It is taken up by endocytosis
Released from the endosome
Transported into nucleus
Entry into the nucleus is inefficient, may need mitosis.
34
Describe electroporation
Have a cell in solution with plasmid DNA
Put it into a capacitor of electroporators
Apply electricity onto cell
As you apply small passes of electricity, the cell surface starts to get pores.
The pores allow entrance of plasmid DNA into cell. Rate of pore sealing is dependent on temperature.
35
Describe Nucleofection
Combination of electroporation & lipofection
Increased efficiency particularly of non-dividing cells
Technology is protected under patent
Different solution & protocols are used for each cell type.
36
Describe viral transfection/transduction
Exploits the mechanism of viral infection
High transfection efficiency
Retrovirus, adenovirus, but most commonly lentiviruses are used.
Target the cells needed to express the viral receptor to work.
Put the gene you want to carry, into cells into lentivirus. Make sure the gene is not too big. Transfect into ;ackaging cells, which make virus.
Collect virus particles and isolate them.
Transduce target cells.
Need to work in special labs.
There is overnight incubation, to make sure virus is not dangerous.
Need to work in several labs.
37
Describe the parts of the microscope
Detector (eyes/camera) or PMT,CCD (increases signal and transforms into data)
Objective: magnifying glass, plus/minus immersion medium.
Specimen with cover glass that is normally 0.17-0.18mm, if thicker need to correct the objective lens number as cover glass may have an effect.
Light conditioning system (modifies how light reaches specimen)
-Köhler illumination, phase ring, wallaston prism and polarizers, filter cubes (for fluorescence),
Light source (halogen, XBO, bulb/sunlight)
38
What should be special about a microscope used to observe live imaging?
For life imaging, (viewing alive cells or organisms) need to use incubators box combined with a precision air heater, to create a situation where temperature will not affect objective lens/microscope appliance.
Two boxes, one box prevents temperatures changing a lot, so objective can magnify properly.
The other box, allows correct amount of CO2/O2 for sample to stay alive.
39
Experimental timescales of things to look at in a microscope
For effective timescale, we need to know what we are looking for (processes)
- for development --> hours/days
- microtuble based movement/ polymerisation --> seconds/minutes. (need system that allows you to take images quickly)
- cell motility--> minutes/hours
- cytoskeleton ->seconds/hours
- differentiation-->hours/days
40
wHAT ARE the problems with maintaining certain timescales in microscopy?
Seconds-artefacts in multichannel/4D imaging (lose ability to see process_
Hours/days=stability, viability, difficult to keep maintenance/temp constant--> cannot manage to keep sample in place.
Triangle of frustration (spatial resolution, temporal resolution, sensitivity)
-All detections have their limitations and benefits, what is best depends on the application requirements.
41
Relationship between pixel area and spatial resolution
The more black/white, the higher the resolution/
42
What are the 2 numbers found on objectives?
Largest number is the magnification
| Second number is the coverslip thickness thickness (normal is 0.17-0.18mm)
43
What is the numerical aperture?
Ability of objective to resolve 2 points that are close to each other. Different values give different values of resolution. The higher the NA, the greater the resolution and the more detail.
The numerical aperture (NA) of an optical system is a dimensionless number that characterizes the range of angles over which the system can accept or emit light.
44
What is working distance (WD) of objective?
The length the objective can work at from the sample. The closer, the more inside the sample you can see.
45
What is the immersion medium?
One way of increasing the optical resolving power of the microscope is to use immersion liquids between the front lens of the objective and the cover slip. Most objectives in the magnification range between 60x and 100x (and higher) are designed for use with immersion oil.
46
What are the different types of light microscopy?
In the different types, you are playing with contrast. The more grey, the less contrast, the more black and white, the more definition, but less detail.
Brightfield-> all the light goes through the sample. Sample illumination is transmitted white light, and contrast in the sample is caused by attenuation of the transmitted light in dense areas of the sample.
DIC->Condense a light through a smaller area, creates a 3D like image, because the light allows you to define margins of cell really well. is an optical microscopy technique used to enhance the contrast in unstained, transparent samples. Produces high resolution images by enhancing contrasts. It can accompany confocal microscopy.
Phase contrast phase.-> ring, small circumference light can go through, good to see where cells establish contact with each other. is an optical microscopy technique that converts phase shifts in light passing through a transparent specimen to brightness changes in the image. For transparent, non light absorbing specimens. it is a brightfield microscope.
47
What do we use light microscopy for?
1.Histology, to look at sections of tissue.
-Laser capture microdissection-cuts out specific area and surrounds another to look at localisation of proteins.
Use antibodies against protein of interest.
2. Phase contrast-cell morphology
-reduce amount of greys, make it more black/white, increase contrast.
-can be used to see behaviour of fibroblasts in their substate.
3. Time lapse
-see live events
-eg differentiation of heart cells, or cell migration
-can fix image at diff time events, or make video.
48
Role of electron microscope
Electron source
-electrons travel in beam, reach the specimen.
-energy of electrons turned into image, seen through eyepiece and also in a computer.
TEM: transmission electron microscope: beam of electrons is transmitted through a specimen and focused to produce an image. this is similar to light microscopy. Best resolution with a resolving power of 0.5nm
Scanning electron microscope
-electrons sent at a particular angle, can create images that have 3D dynamity.
scanning electron microscope: a beam of electrons is sent across the surface of a specimen and the reflected electrons are collected. RP: 3-10nm, so resolution is not as good as with transmission electron microscopy but 3D images are produced, giving valuable info about the appearance of diff organisms
49
Fluorescence microscopy
- instead of using whole spectrum of light, you are using a portion of it.
- able to see things in colour
- source of light, mirrors to allow a particular wavelength through, light goes through to specimen, magnified by objective, then goes to eyepiece or camera/computer to allow you to see the image.
50
Describe confocal vs widefield microscopy
Confocal:
- laser emits at a particular wavelength
- laser goes through mirrors, thenobjective
- reaches sample
- when sample receives it, it will excite and emit at a particular wavelength. It will be collected by a photomultiplier.
- Transforms it into image you can see in a screen.
- laser allowed through tiny pinholes: allows you to see a thin layer of the cell. This depends on how much laser is allowed through.
- higher resolution and greater detail, but you can only see a small volume at a time, and requires a lot of power in computers because collecting more data.
Widefield:
- you see whole of the cell
- you cannot focus on a particular area
51
Describe the role of flurofuls in microscopy
It is a cyclic process. Particular wavelengths are able to excite a particular set of protein molecules called flurofuls. When flurofuls absorb particular wavelength, they get excited, increase their energy, and lose it by emitting it in a particular wavelength.
They get excited, they lose energy, and then emit energy at another particular wavelength.
If we use known wavelengths, we can use them to excite flurofuls and then visualise our sample.
Absorption->excitation (generates motion & heat) -> heat is lost (emission).
Excitation and emission have different wavelengths, there is a change in wavelength, and the difference between the two wavelengths is called stokes shift.
Excitation happens at a shorter wavelength than emission. When electrons go from the excited state to the ground state , there is a loss of vibrational energy. As a result, the emission spectrum is shifted to longer wavelengths than the excitation spectrum (wavelength varies inversely to radiation energy). In order to achieve maximum fluorescence intensity, the fluorochrome is usually excited at the wavelength at the peak of the excitation curve, and the emission detection is selected at the peak wavelength (or other wavelengths chosen by the observer) of the emission curve. So excitation wavelength is shorter than emission.
In photobleaching, cycle can be broken.
52
What is stokes shift?
Stokes shift is the change/difference between the excitation wavelength and emission wavelength.
53
What is photobleaching?
loss of colour by a pigment (such as chlorophyll or rhodopsin) when illuminated.
The fluroful cyclic process is broken.
54
How can we see protein of interest for microscopy?
-can use antibodies or protein fusion.
Antibodies specifically recognise epitope or whole protein.
or can use secondary antibody that recognises primary antibody. A secondary antibody has flurofuls that are excited at a particular wavelength and emits at another particular wavelength, so you are able to see protein in colour.
If want to see something live, generate plasmid with sequence of protein linked to sequence of fluroful. When it goes inside cell, you will see it at a particular colour.
55
Describe an MRI scanner
MRI: magnetic resonance imaging.
It is a scan that uses strong magnetic fields and radio waves to produce detailed images of the body.
MRI scanner is a large tube that contains powerful magnets.
You lie inside the tube during the scan
We are picking up signals from protons and fat in water.
Protons align with magnet in scanner, then emit radio waves which are picked up to generate pic.
Need contrast within these signals, if don’t have contrast then you will not be able to detect anatomical variations for pathological changes.
MRI is best for soft tissue
MRIs use radiowaves.
Spatial resolution of MRI -1mm or less.
Signal coming from tissue at 5mm distance.
56
CT scan
Works by x rays and rotation
CT is good for haemorrhage, acute stroke, and brain trauma. It is also best for bone
CT contrast is due to tissue density and dependent on attenuation of x-rays.
The Hounsfield number is a measure of how much the X-Rays are attenuated in passing through any material. The scale goes from air->water->cortical bone. It uses X-rays and a computer to create detailed images of the inside body. CT scans can produce detailed images of many structures inside the body.
You can give a contrast before scan to improve the quality of images. Scanner consists of a ring that rotates around a small section of your body as you pass through it. Unlike MRI, the scanner doesnt surround your whole body at once. The anatomical structure is faint but difference in bone and tissue, in terms of contrast is high. Haemorrhage also shows up brightly. Can also see shadow of oedema, relating to increased water content.
57
Advantage of using explants to produce tissue culture
No need to use enzymes or chemicals that may damage the cells
58
Diff between CT and X-RAY
CT:
CT scans
Radiation – Since CT scans are created by multiple x-rays, there is radiation exposure, although minimal. It is not usually appropriate during pregnancy.
Uses – Excellent for looking at bones, but very good for soft tissues, especially with intravenous contrast dye.
Cost – Usually less expensive than MRI
Time – Very quick. The test only takes about 5 minutes, depending on the area that is being scanned.
Patient comfort – The machine is very open, so concerns about confined space is rarely a problem.
Reactions – The intravenous contrast rarely causes allergic reactions. It does have the potential to injure kidneys, especially with people who already have kidney problems or diabetes or those who are very dehydrated.
Limitations – Patients who weigh more than 300 pounds may have to be sent to a location with a table designed to handle their weight.
59
What is a flurophore?
A flurophore is a fluorescent chemical compound that can re-emit light upon light excitation.
The flurophore absorbs light energy of a specific wavelength and re-emits light at a longer wavelength.
60
What fluorochrome is used for cell cycle analysis
propidium iodide. It requires permeability of the plasma membrane, to allow PI to enter for cell cycle analysis to occur.
PI excitation occurs at 500nm, by common 488 laser, and the emission is at 600nm, in red part of spectrum.
PI allows us to quantitate the proportion of cells at different stages of the cell cycle.
61
What is CT scan used for?
CT is best for bone
62
What is MRI scan used for?
MRI id best for soft tissue, using signals from protons and fats in water
63
What important property is needed to detect changes/pathological variations in images?
contrast
64
What is Hounsfield number?
Measure of how much the x-rays are attenuated in passing through any material. Scale goes from air->water->cortical bone
65
Uses for CT scans vs MRIS
bdominal pain – CT is the preferred test. It is more readily available on an emergency basis and is very accurate. Ultrasound is used for children and pregnant women.
Trauma – CT is present in most emergency departments and is the best at showing bone fractures, blood and organ injury.
Spine – MRI is best at imaging the spinal cord and nerves.
Brain – CT is used when speed is important, as in trauma and stroke. MRI is best when the images need to be very detailed, looking for cancer, causes of dementia or neurological diseases, or looking at places where bone might interfere.
Chest – CT is much better at examining lung tissue and often used for follow up on abnormal chest x-rays. Low dose CT Scans are available and used with high-risk smokers who need to be screened annually.
Joints – MRI is best at showing tendons and ligaments.
66
colour of CSF on T1W imaging vs T2W imaging
CSF is dark on T1-weighted imaging and bright on T2-weighted imaging.
67
what is T1W and T2W
T1 (longitudinal relaxation time) is the time constant which determines the rate at which excited protons return to equilibrium. It is a measure of the time taken for spinning protons to realign with the external magnetic field. T2 (transverse relaxation time) is the time constant which determines the rate at which excited protons reach equilibrium or go out of phase with each other. It is a measure of the time taken for spinning protons to lose phase coherence among the nuclei spinning perpendicular to the main field.
68
WHAT CONTRAST is used foor T1W
Gadolinium
69
What is cyclin D important for?
Acts with cdks4/6 to drive through progression of G1. Also important in regulation and expression of cyclin E
70
What does cyclin E do?
Cyclin E is important for the G1 to S phase transition
71
What determines whether object will show on x ray?
Tissues of high density and/or high atomic number cause more x-ray beam attenuation and are shoqn as lighter grey or white on a radiograph.
72
Cyclin A - cdk2 complex
Important for s phase transition
73
Cyclins A, B-cdk1
directs G2 to the G2 to M phase transition
74
What is carcinogenesis
process where normal cell transformed into cancer cell.
75
What are the major functional changes in cancer?
Increased growth (angiogenesis)
Failure to undergo programmed cell death (apoptosis) or senescence
Loss of differentiation (including alterations in cell migration and adhesion)
Failure to repair DNA damage (including chromosomal instability)
76
What are oncoggenes?
Oncogenes are the normal genes in your cells that can regulate these processes, in response to those signals. They are normaly components of growth factor signalling pathways, that when mutated produce products in higher quantities, or whose altered products have increased activity and therefore act in a dominant manner. Example is the RAS oncogene. Oncogenes gain function, can be caused by a single mutation.
77
What are tumour suppressor gens?
They are genes that lead to cell cycle arrests or checkpoints. They act as a stop signal, to uncontrolled growth, may inhibit the cell cycle, or trigger apoptosis. Tumour suppressor genes lose function, the first mutation will affect one of the alleles, not enough unless econd mutation present, then there is loss of function.
78
What are the different pathways affected by oncogenes and tumour suppressor proteins
Myc
Ras
Rb
P53
79
What are protooncogenes?
They are normal genes, part of various growth factor signal transduction pathways. Can either be:
- Growth factors
- Growth factor receptors
- Intracellular signal transducers
- Nuclear transcription factors,
80
Describe the Ras oncogene family
-Binding of extracellular growth factor signal
-promotes recruitment of Ras proteins to their receptor complex
-Recruitment promotes Ras to exchange GDP (inactive RAS) with GTP (active Ras)
-Activated Ras then initiates remainder of signalling cascade (mitogen activated protein kinase)
-These kinases ultimately phosphorylate targets such as transcription factor to promote expression of genes important for survival.
Ras then hydrolyses GTP to GDP fairly quickly, turning itself off.
81
What is the oncogene hypothesis
Upon finding there was a gene homologous sequence to v-src in uninfected chickens, in 1989 Harold Varmus and J. Michael Bishop received the noble prize for laying down the foundation of mutations in carcinogenesis
Discovered that the some genes of cancer causing viruses were mutated forms of the cellular gene not viral genes
They concluded that the Rous sarcoma viral gene was in fact a host gene that had
been ‘kidnapped’ by the virus (and ‘transformed’ into an oncogene)
An oncogene is any cellular gene that upon activation can transform cells.
Bishop and Varmus used different strains of Rous sarcoma virus in their research, they:
Identified the v-src oncogene as responsible for causing cancer.
Used hybridization experiments, and they found that the c-src gene was present in the genome of many species.
They then showed that the host cell c-src gene was normally involved in the positive regulation of cell growth and cell division.
Following infection, however, the v-src oncogene was expressed at high levels in the host cell, leading to uncontrolled host cell growth, unrestricted host cell division, and cancer.
Proto oncogenes are normal genes that can control growth
Various agents, including radiation, chemical carcinogens, and, perhaps, exogenously added viruses, may transform cells by “switching on” the endogenous oncogenic information.
82
Describe the typical retroviral lifecycle
Rous Sarcoma virus, infects chicken cell,
-it goes through reverse transcription
-produces double stranded DNA provirus
-going from RNA to DNA
-Provirus is accidently integrated next to the host cellular sarc sequence, you then get fusion that is packaged into a capsid, and you end up with a rous sarcoma virus that contains the sarc gene.
This is then a kidnapped gene.
83
Describe the process of viral oncogenesis
Approximately 15%-20% of all human cancers are caused by oncoviruses
Viral oncogenes can be transmitted by either DNA or RNA viruses.
DNA viruses can cause lytic infection leading to the death of the cellular host or can replicate their DNA along with that of the
host and promote neoplastic transformation
DNA Viruses
Encode various proteins along with
environmental factors can initiate
and maintain tumours
RNA Viruses
Integrate DNA copies of their genomes
into the genome of the host cell and as
these contain transforming oncogenes
they induce cancerous transformation
of the host
Approximately 15%-20% of all human cancers are caused by oncoviruses
Viral oncogenes can be transmitted by either DNA or RNA viruses.
DNA viruses can cause lytic infection leading to the death of the cellular host or can replicate their DNA along with that of the
host and promote neoplastic transformation
DNA Viruses
Encode various proteins along with
environmental factors can initiate
and maintain tumours
```
RNA Viruses
Integrate DNA copies of their genomes
into the genome of the host cell and as
these contain transforming oncogenes
they induce cancerous transformation
of the host
```
84
Describe the activation of oncogenes
These genes captured by animal retroviruses are altered in human cancer, activation can involve
mutations, insertions, amplifications and translocations
Loss of response to growth regulatory factors
One allele needs to be altered
85
What are the 4 types of proteins normally involved in the transduction of growth signals?
```
Normally
Growth factors
Growth factor receptors
Intracellular signal transducers
Nuclear transcription factors
```
Growth factors, signal transduction and cancer
The majority of oncogene proteins function as elements of the signalling
pathways that regulate cell proliferation and survival in response to growth
factor stimulation
Oncogene proteins act as growth factors (e.g.EGF),
growth factor receptors (e.g. ErbB) and intracellular signalling molecules (Ras and Raf).
Ras and Raf activate the ERK MAP kinase pathway, leading to the induction of additional
genes (e.g. fos) that encode potentially oncogenic transcriptional regulatory proteins
To date-over 100 identified oncogenes
86
Describe the RAS oncogene family
| -how does it work?
RAS Oncogene Family
ras genes were identified from studies of two cancer-causing viruses the Harvey sarcoma virus and Kirsten sarcoma virus, These viruses were discovered originally in rats hence the name Rat sarcoma
RAS proteins are small GTPases that are normally bound to GDP in a neutral state
Oncogenic activation of ras is seen in about 30% of human cancer
Most commonly mutated oncogene
Point mutations in codons 12, 13 and 61.
RAS Oncogene Family
1. Binding of extracellular growth factor signal
2. Promotes recruitment of RAS proteins to the receptor complex
3. Recruitment promotes Ras to exchange GDP (inactive
Ras) with GTP (active Ras)
```
4. Activated Ras then initiates the remainder of the
signalling cascade (mitogen activated protein kinases)
```
5. These kinases ultimately phosphorylate targets, such as
transcription factor to promote expression of genes
important for growth and survival
Ras hydrolyzes GTP to GDP fairly quickly, turning itself “off”
87
Describe the MYC oncogene family
The MYC oncogene family consists of 3 members,
C-MYC, MYCN, and MYCL, which encode c-Myc, N-Myc,
and L-Myc, respectively
Originally identified in avian myelocytomatosis virus (AMV)
The MYC oncoproteins belong to a family of transcription factors that regulate the transcription of at least 15% of the entire genome
Major downstream effectors of MYC include those involved in ribosome biogenesis, protein translation, cell-cycle progression and metabolism, orchestrating a broad range of biological functions, such as cell proliferation, differentiation, survival, and immune surveillance
The MYC oncogene is overexpressed in the majority of human cancers and contributes to the cause of at least 40% of tumours
It encodes a helix-loop-helix leucine zipper transcription factor that dimerizes with its partner protein, Max, to transactivate gene expression
Generally MYC is activated when it comes under the control of foreign transcriptional promoters. This leads to a deregulation of the oncogene that drives relentless proliferation.
Such activation is a result of chromosomal translocation
88
What is Burkitts lymphoma
Epstein Barr virus is associated with Burkitt’s lymphoma (BL)
BL is a high grade lymphoma that can effect children from the age of 2 to 16 years
In central Africa, children with chronic malaria infections have a reduced resistance to the virus. This is known as classical African or endemic BL
All BL cases carry one of three characteristic chromosomal translocations that place the MYC gene under the regulation of the Ig heavy chain. Therefore c-myc expression is deregulated
In BL three distinct, alternative chromosomal translocations involving chromosomes 2, 14 and 22
In all three translocations a region form one of these three chromosomes is fused to a section of chromosome 8
89
Describe process of chronic myelogenous leukaemia
Chronic myelogenous leukaemia (CML) accounts for 15-20%
of all leukaemias
95% of CML patients carry the Philadelphia chromosome,
that is the product of the chromosomal translocation
t(9;22)(q34;q11) generating the BCR-ABL fusion protein
As a result of this translocation the tyrosine kinase activity
of the oncogene ABL is constitutive leading to abnormal
proliferation
Therapeutic strategies for CML include Imatinib (Gleevac) a tyrosine kinase inhibitor-96% remission in early-stage patients
90
Describe the discovery and identification of tumour suppressor genees
In 1969 Henry Harris and his colleagues performed somatic cell hybridization experiments
Fusion of normal cells with tumour cells yielded hybrid cells containing chromosomes form both parents. These cells were not capable of forming tumours
Genes derived from the normal parent acted to inhibit or suppress tumour development
The first tumour suppressor gene was identified by studies of retinoblastoma, a rare childhood eye tumour
91
Describe the Rb gene
Retinoblastoma is a rare childhood cancer (1 in 20,000) that develops when
immature retinoblasts continue to grow very fast and do not turn into
mature retinal cells.
An eye that contains a tumour will reflect light back in a white colour.
Often called a “cat’s eye appearance,” the technical term for this is leukocoria.
Two forms of the disease, familial (40%) and sporadic (60%)
The hereditary mutation is on chromosome 13 (13q14),
the retinoblastoma 1 (Rb1) gene
The existence of the RB1 gene was predicted in 1971 by Alfred Knudson
Whilst studying the development of retinoblastoma he proposed that the development of retinoblastoma requires two mutations, which are now known to correspond to the loss of both of the functional copies of the Rb gene - "two-hit" hypothesis
"Loss of heterozygosity“ often used to describe
the process that leads to the inactivation of the
second copy of a tumour suppressor gene
a heterozygous cell receives a second hit in
its remaining functional copy of the tumour
suppressor gene, thereby becoming homozygous
for the mutated gene.
Mutations that inactivate tumour suppressor
genes, called loss-of-function mutations, are
often point mutations or small deletions that
disrupt the function of the protein that is
encoded by the gene
92
Describe the retinoblastoma protein RB structure
The Rb gene family includes three members: Rb/(p105/110), p107 and Rb2/p130
-collectively known as pocket proteins
pRb is a multi functional protein (110kDa) with over 100 binding partners
A transcriptional co factor that can bind to transcription factors
RB functions in diverse cellular pathways, such as apoptosis and the
cell cycle, it has also become clear that RB regulates these pathways
through the stimulation or inhibition of the activity of interacting proteins.
Therefore, an important starting point for understanding RB function is its
structure, which acts as a scaffold for these multiple protein interactions
It’s main binding partner is the E2F transcription factor,
interacting with the large pocket
Other viral oncoproteins can bind to Rb
Main function of Rb is to regulate the cell cycle by inhibiting the G1 to S phase transition
2 important proteins involved in the cell cyle are:
Cyclins and their associated cyclin dependent kinases (cdks)
Passage of a cell through the cell cycle is regulated
cyclins and cyclin dependent kinases (cdks)
Cyclin D is the first cyclin to be synthesized and drive progression through G1 together with cdks4/6
The G1 checkpoint leads to the arrest of the cell cycle in response to DNA damage
A key substrate for cyclin D is RB protein
Cyclin D and E families and their cdks phosphorylate RB
93
Describe the RB protein function, phosphorylation and activity
Rb protein regulates the activity of the E2F transcription factor crucial for the expression of genes required for S phase
Rb activity is regulated by phosphorylation
When the Rb tumour suppressor is active it can inhibit cell proliferation
When Rb is dephosphorylated/hypophosphorylated it is active and remains bound to E2F
When Rb is active it blocks the progression of to S phase
When Rb is hyperphosphorylates , in response to extracellular physiological signals it is inactive .
Upon phosphorylation of RB, E2F is released and migrates to the nucleus to induce transcription
When RB is inactive cell cycle progression from G1 to S occurs
Rb can be inactivated by phosphorylation, mutation, or viral oncoprotein binding
In retinoblastoma, pRb is functionally inactivated by mutations
or partial deletions
Viral inactivation found in small DNA tumour viruses
mainly by disrupting E2F binding or destabilisation of Rb
Adenovirus - E1A
Papilloma - E7
Polyoma – Large T antigen
In cancer cells RB phosphorylation is deregulated throughout
cell cycle. As a direct consequence E2F transcription factors can
induce the deregulation of the cell cycle
Without RB on watch , cells move through G1 into S
and are not subjected to usual checks .
94
Describe the role of p53 tumour suppressor
The p53 gene was the first tumour suppressor gene to be identified
The p53 protein is at the heart of the cell’s tumour suppressive mechanism and has been nicknamed the ‘guardian of the genome’
It is involved in sensing DNA damage and regulating cell death/apoptosis
as well as other pathways
p53 is mutated in 30-50% of commonly occurring human cancers
Frequent mutation of p53 in tumour cell genomes suggests that tumour
cells try to eliminate p53 function before they can thrive
p53 specializes in preventing the appearance of abnormal cells.
Protein has an amino transactivation domain, a central DNA binding domain, a tetramerization domain and a carboxyl regulatory domain
Can bind to around 300 different gene promoter regions-main role as a transcription factor.
Normally levels of p53 protein are low in cells
These levels are kept low by MDM2 protein, a ubiquitin ligase (also an oncogene)
In unstressed normal cells both p53 and MDM2 move between the nucleus and cytosol
MDM2 binds p535 to form a complex in the nucleus where MDM modifies the carboxyl terminus of p53 and
targets it for degradation by the proteasome
WT p53 has a short 20 min half life.
Stress signals are able to activate p53
Signals are sensed by mainly kinases that then phosphorylate p53
Phosphorylation of p53 disrupts the interaction between it and
MDM2
e.g. ionizing radiation signals through two kinases ATM/ATR
activate oncogenes such as ras induce activity of p14arf
responsible for sequestering MDM2.
P53 can thus regulate genes involved in DNA damage repair,
apoptosis and cell cycle arrest
95
Describe the therapeutic strategies for tumour suprresor genes and oncogenes
Gene therapy obvious approach
Many vectors and retroviruses have been examined
Retroviruses integrate in a stable form into the genome of infected cells. It has been demonstrated that
retrovirus-mediated gene transfer of the wild-type TP53 gene into both human lung tumour cell lines and xenograft models could lead to the inhibition of tumour cell growth
Alternative strategies- use of inhibitors
PRIMA-1, Restores mutant p53 by
modifying the thiol groups in the core
domain of the protein
Nutlin- is a potent MDM2 antagonist
RITA binds to p53 and can restore
mutp53 activity
Inhibitors of CRM1 result in nuclear
accumulation of p53.
Or we can have genetic analysis and personalised medicine
A detailed readout of the molecular faults in a patient’s tumour, and new generation of drugs that precisely target them
Classifies tumours according to their genetic make-up instead of where they grow in the body
People with the ‘same’ cancer can have different forms of the disease so responses to treatment vary
Cancers growing in different parts of the body may also share the same genetic faults so respond to similar
treatments
96
What causes the lethality of cancers?
Their ability to invade and colonise different sites within the body.
97
What are the characteristics of malignant tumours?
1. Unlimited growth, as long as there is an adequate blood supply to prevent hypoxia, so the cancer can start to proliferate. When they become hypoxic, this then drives blood vessel growth.
2. Invasiveness, migration of tumour cells into the surrounding extracellularmatrix (stroma) where they are free to disseminate via vascular or lymohatic channels to distant organs.
3. Metastasis
Spread of tumour cells from the primary site to form secondary tumours at other sites in the body
98
Describe the sequential process of metastasis
Transformation->angiogenesis->motility and invasion-> arrest in capillary beds->embolism and circulation (transport)->arrest in capillary beds (adherence) -> extrvasation into organ parenchyma ->response to microenvironment -> tumour cell proliferation and angiogenesis ->metastases->metastasis of metastases. Extensive mutagenic and epigenetic changes followed by clonal selection
Angiogenesis (overcomes limitations imposed by hypoxia)
Epithelial to mesenchymal transition (invasive properties allowing intravasation and extravasation)
Colonisation of target organs (ability to expand from micrometastases)
Release of metastatic cells that have acquired the ability to colonize.
99
What is the difference between angiogenesis and vasculogenesis
Angiogenesis is the formation of new blood vessels from pre-existing vessels.
Vasculogenesis is the formation of new blood vessels from progenitors
100
What are the different types of angiogenesis
```
Developmental vasculogenesis (organ growth)
Normal angiogenesis (wound repair, placenta during pregnancy, cycling ovary)
Pathological angiogenesis (tumour angiogenesis ocular and inflammatory disorders)
```
101
Describe the neovascularisation of tumours
Tumours will not grow beyond a size of about 2mm without their own blood supply as cells cannot survive the lack of oxygen (hypoxia)
However, angiogenesis (development of a new blood supply) is promoted by hypoxia
102
Describe tumour hypoxia
Hypoxia is a strong stimulus for tumour angiogenesis
Hypoxia – low oxygen tension <1% O2
Increases with increasing distance from capillaries
Activates transcription of genes involved in angiogenesis, tumour cell migration and metastasis
103
Describe angiogenic factors
Some tumour cells produce factors that stimulate the directional growth of endothelial cells:
Vascular Endothelial Growth Factor (VEGF)
Fibroblast Growth Factor-2 (FGF-2)
Transforming Growth Factor-β (TGF- β)
Hepatocyte growth factor/scatter factor (HGF/SF)
These factors are mainly stored bound to components of the extracellular matrix and may be released by enzymes called matrix metalloproteinases
104
What are the different mechanisms of tumour cell invasion
Increased mechanical pressure caused by rapid cellular proliferation
Increased motility of the malignant cells (epithelial to mesenchymal transition)
Increased production of degradative enzymes by both tumour cells and stromal cells
105
Describe the epithelial-mesenchymal transition
Loss of
Epithelial shape and cell polarity
Cytokeratin intermediate filament expression
Epithelial adherens junction protein (E-cadherin)
Acquisition of
Fibroblast-like shape and motility
Invasiveness
Vimentin intermediate filament expression
Mesenchymal gene expression (fibronectin, PDGF receptor, αvβ6 integrin)
Protease secretion (MMP-2, MMP-9)
106
Describe cell adhesion molecules and invasion
```
E-Cadherins
-Homotypic adhesion molecule (adhesion of cells with the same cadherin)
-Calcium-dependent
-Inhibits invasiveness
-Binds β-catenin
Integrins
-Heterodimers (α and β subunits)
-Heterotypic adhesion molecule
-Adhesion to extracellular matrix (via collagen, fibronectin, laminin)
-Cell migration
```
107
What is the stromal cell contribution to tumour progression
Factors released by stromal cells (macrophages, mast cells, fibroblasts) include angiogenic factors, growth factors, cytokines, proteases
Example: Urokinase-type plasminogen activator (uPA); activated by tumour cells - resulting in plasmin production
Plasmin activates matrix metalloproteinases (MMPs), which permit invasion by degrading extracellular matrix (ECM) thus releasing matrix-bound angiogenic factors
108
What determines the pattern of tumour spread?
Mechanical Hypothesis
Anatomical considerations: Blood and lymphatic systems, entrapment in capillary beds (20-30µm carcinoma cell, ~8µm capillary)
Seed and Soil Hypothesis
Specific adhesions between tumour cells and endothelial cells in the target organ, creating a favourable environment in the target organ for colonisation
Genetic alterations acquired during progression allow tumour cells to metastasize.
109
Who is Judah Folkman
1971 – Angiogenesis hypothesis
Tumour growth dependent on new blood vessel growth
“If a tumor could be held indefinitely in the non-vascularized dormant state….it is possible that metastases will not arise”
Paradigm shift in cancer therapy
Both the tumour and microvascular compartment are valid therapeutic targets
110
What is Avastin
First specific anti-angiogenesis drug
in 2013 was the second biggest selling oncology product
Approved for colorectal, lung, kidney and ovarian cancers and eye diseases
Napoleone Ferrara who developed the drug at Genentech, California won both the Lasker prize in 2010 and the $3 million Breakthrough Prize in Life Sciences in 2013
monoclonal antibody
binds to VEGF
prevents VEGF binding to VEGF receptors on endothelial cells
111
CT
CT contrast is due to tissue density dependent
attenuation of x-rays
-good for haemorrhagic acute stroke, oedema, and haemorrhage.
112
What is MR image contrast?
MR image contrast – i.e. the relative signal intensities between different tissue types and pathologies – depends on physical properties of the tissue such as water and fat content, cellular structure, cell density…..
113
What factors is MRI contrast sensitive to?
MRI contrast is very sensitive to changes in a large variety of the physical properties of tissue water and blood
T1 relaxation paramagnetic blood breakdown products
T2 relaxation tissue fluidity – oedema, deoxyhemoglobin
Water diffusion (micro) cell membrane integrity, cell size
Flow (macro) blood flow
Perfusion blood flow, blood vessel density
114
What is nuclear magnetism
The positive charge of a spinning proton produces a magnetic moment micrometers sign units.
115
What is resonance
In a magnetic field Bo the magnetic moment of a proton precesses at the Larmor frequency VL units.
116
Describe MR imaginge
MR Imaging is formed using a radiofrequency pulse to generate an MR signal from a slice of tissue.
Magnetic field gradients are used to encode the signal in space so that the computer can generate an image
117
What is the MRI signal source
The strong magnetic field creates magnetisation in all the tissue
This magnetisation is from the protons in water and fat in the tissue
The magnetisation can be manipulated by radiofrequency pulses to produce an MRI signal to create an image
The intensity in the image depends on water content, tissue structure, blood flow, perfusion, diffusion, paramagnetics etc
118
Describe the difference between T1W and T2W
Looking more closely at the MR signal we find there are two relaxation times that determine how strong the signal is. These are MR parameters that vary with tissue type e.g. grey or white matter, and with disease. Hence they allow images to be made that demonstrate anatomy and pathology.
Mxy decays according to T2 which affects how long the MR signal lasts
Mz recovers according to T1 which affects how much M there is available to be excited to give the next signal.
T1 and T2 relaxation times vary between different tissues and pathology
The image signal intensity depends on T1 and T2 and provides contrast between tissue in an MR image.
T2 signal decay in xy
T1 signal recovery along z
119
What is a pulse sequence
An MR image is built up from a series of signal acquisations (Radiofrequency pulses).
This acquisition is repeated several hundred times to obtain the data to create a single image. TE and TR determine the image “contrast”
In mri, and slice can be used: coronal, saggital, transverse etc.
Oblique – any slice orientation is possible
Multislice – acquisitions on many different slices within the TR
120
Describe graph of T2W
Exponential decay.
121
Describe scale times and colour of T2
```
T2 is sensitive to water content.
White 90ms
Grey 80ms
CSF >1000ms
The more water, the longer it takes for signal to decay.
```
122
T2 relaxation time
The T2 of tissue determines how quickly the MRI signal decays away after the radiofrequency pulse
T2 is very dependent on how mobile the water is in the tissue and increases with
Oedema, an increase in water content
Demyelination, a loss of brain tissue structure
T2 is reduced by the presence of paramagnetic ions
Fe from blood breakdown products
Gd from contrast agents
123
T1
```
Mz recovery (return to Z axis) with time after RF pulse depends on the T1 relaxation time. Tissue has a shorter T1 than CSF so its Mz recovers more quickly.
CSF has a very long T1.
Signals from tissue and csf are reduced depending on T1
```
124
time scale and colour of T1W images
White 1000ms
Grey 1800ms
CSF >2500ms
FAT has shorter T1, comes out bright because it recovers along Z axis quickly.
CSF will be dark.
T1 has a shorter TE than T2.
125
What can we use 3D T1 images for?
3D T1 images used to determine volume of hippocampus
Neurogenesis proposed to explain need for increased capacity of spatial memory
126
Describe T1 relaxation time
When the repetition time (TR) between pulses is much shorter than T1 the magnetisation that can produce the MRI signal is reduced (“saturated”)
The MR signal is then T1-weighted.
Tissue with long T1 produces a smaller signal than tissue with short T1.
T1 is lower in white matter than grey matter because of myelinated neurones
T1 is also dependent on how mobile the water is in the tissue and T1 increases slightly with oedema
T1 is very dependent on the presence of paramagnetic ions which reduce T1
Fe from blood breakdown products
Gd from contrast agents
127
Give examples of contrast agents
Paramagnetic (unpaired electrons) or superparamagnetic (ferrites)
Chelated to reduce toxicity.
Water in the vicinity of the contrast agent experiences strong fluctuating magnetic fields hence T1 and T2 are reduced.
Using a contrast, the tumour core distinguished as contrast enhancement in areas of BBB breakdown, areas of neo-angiogenesis stimulated by tumour growth.
128
Describe the MRI characteristics of multiple sclerosis lesions
Inflammation, demyelination, BBB leakage.
129
Why/what is MRS and why do we need it?
Water in the vicinity of the contrast agent experiences strong fluctuating magnetic fields hence T1 and T2 are reduced.
A stronger shielded nucleus has a lower resonant frequency
130
What are the chemicals observed by H MRS
NAA (N-acetyl aspartate) found predominantly in neurons, marker for viable neurons, reduced in pathology
tCr (Cr and PCr) cell energy metabolism, marker of viable cell density in some tissue
tCho (Cho, GPC, PC) cell membrane metabolism (growth and degradation), elevated in tumors and gliosis
Glx (glutamate & glutamine) amino acids
mI (myo-Inositol) osmolyte found in glial cells, marker for gliosis inflammation, elevated cell membrane synthesis
Lac (lactate) the end product of anaerobic metabolism, high in tumors & stroke etc.
Ala (alanine) amino acid, marker for meningiomas
Lipids membrane breakdown products, macrophages
Lac, ala and lipids are not visible in normal brain spectra.
Elevated CHO/Cr is a marker of tumours.
High lipids indicates necrotic high-grade tumour.
131
Describe the safety issues of MRI
Underlined are not visible in normal brain spectra
132
Describe the ability to use a combination of fluorochromes
4 laser colour each detected by a different detector.
Emitted light goes through a system of filters and mirrors, so by the time it gets to the detector, the detector is only detecting a narrow range of wavelengths, so we can differentiate between the 4 emissions.
Label the cells with different antibodies of 4 diferent colours, with one laser line. So the cell is emitting signals depending on whether it is positive or negative for that antibody.
But we can restrict what detectors are seeing by differentiating between those 4 fluorochromes.
133
What are the clinical and research applications of flow cytometry
```
Immunophenotyping of leukaemias & lymphomas
Detection of MRD
Stem cell enumeration
CD4/CD8 in HIV
Measurement of intracellular cytokines
Study of cell cycle, viability & apoptosis
Measurement of cell proliferation
Assessment of transfection efficiency
```
134
What are the ways of imaging with Ionising radiation
```
Ionising Radiation
Planar X-ray
CT
Gamma Camera and SPECT
PET
Hybrid Imaging
```
135
What is ionising radiation?
```
Radiation that causes ionisation when it interacts with matter
Types used for medical imaging are:
Gamma rays
X-rays
Why use ionising radiation?
Penetrating
```
136
What are the 2 actions of ionising radiation?
```
Indirect action (majority of body is water, radiation causes water to split into free radicals and hydrogen peroxide).
Direct action, radiation interacts with DNA.
Both actions can cause biological responses, either genetic, cancer, or death.
```
137
Describe the damage and risk of radiation?
Indirect effects
Risk of cancer induction
Risk of genetic change in subsequent population
Effect is proportional to radiation dose, no threshold
--> all radiation has risk
Direct effect
Only at high radiation dose not noticed at usual diagnostic doses
Threshold effect
e.g. Erythema & hair loss
138
units for radiation dose
mSv
139
Types of ionising radiation?
Positrons
Positive electrons interact with matter to create gamma rays, used for PET Scanning.
Gamma rays
Penetrating radiation, used for Gamma camera imaging, eg SPECT.
```
X-rays
Spectrum of electromagnetic
radiation eg.
X-Ray imaging
e.g. radiographs,
CT
Artificially produced in an X‑ray tube
```
140
Describe the attenuation of X rays
Attenuation increases with
Higher atomic number
Higher density
X-Rays are essentially an attenuation map
141
Describe the meaning of emission and transmission imaging
Transmission Imaging
Radiation is directed through the patient
A transmission map collected is essentially an attenuation map
Good at showing structure, especially between tissues of different densities or atomic number
Emission Imaging
The radiation is administered to a patient in the form of a tracer
Emitted radiation is detected outside the patient
142
Describe the structure of the X-ray tube
X-rays only produced when tube is in action i.e. can be switched on/off
We have control over the amount and energy of x-rays produced
High voltage controls the energy of the x-rays
Current control the amount of x-rays
143
What are the detectors for planar X-ray
```
Film Hardcopy
Film processor with tanks of chemicals
High resolution
Computed Radiology computer copy
Phosphor plate
Special laser scanner or CR reader that reads and digitizes the image
Digital enhancement and archiving
Digital Radiology (DR)
Flat panel detector, fully digitised system
```
144
Describe X-ray screening: mammography
High resolution
Compression plate used to reduce breast thickness
Improves resolution
Lowers radiation dose (used as a screening tool)
145
Describe real time X-ray: Fluoroscopy
Real-time imaging
A catheter is fed inside an artery and radio opaque dye is injected
Show blood flow inside vessels and can be used to assist with interventions
146
Describe X-ray intervention: Coronary angiography
Real-time imaging using an image intensifier called fluoroscopy
A cardiac catheter is fed inside the aorta
Radio-opaque contrast agent used to identify areas of occlusion
Treatment may be either balloon angioplasty or insertion of a stent
147
What are the limitations of planar X-ray?
Cannot distinguish between overlying tissues
Tissues other than those being observed reduce contrast in the image
Historically partially solved by moving the film cassette and X-ray relative to the patient to blur out overlying tissues, called “tomography” (from Greek “part/slice” - “write”)
Superseded by Computed Axial Tomography, now abbreviated to CT
148
What is helical scanning and what is it used for?
```
Ct scan,
Also known as spiral scanning
Introduced in late 1980s
Continuous rotation
Continuous table feed
```
149
Helical MSCT
Multi Slice CT
Faster scan
More coverage each rotation
150
Describe the acute diagnosis for CT
Can differentiate between
-haemorrhage or blood clot.
Urgent diagnosis required for treatment
Clot busting drugs may increase bleeding
151
Describe How CT can be used for disease progression
Measurement of the size of the left inguinal lymph node shows progression of disease
Imaging is used for monitoring response to therapy
152
Describe the effect of CT in Treatment planning
External beam radiotherapy irradiates normal tissue as well as tumour
Multiple beams are used to spare normal tissue
CT is used to define area to be treated and the direction of the radiotherapy beams that are used
153
Describe nuclear medicine: emission imaging
Inject radioactive tracer, patient is emitting the gamma rays
Image depends on the metabolism of the tracer: Functional Imaging
For gamma camera and PET
154
Gamma camera
Gamma camera
Uses single photon emitting radionuclides
Can operate in 2D (planar) or 3D (SPECT)
PET
Positron Emission Tomography
Uses positron emitting radionuclides
Always 3D
155
Nuclear Medicine Imaging
We are making an image of the distribution of a radioactive tracer
Nuclear Medicine only shows function
It may reflect anatomy but without metabolism, the tracer will not be taken up
Nuclear Medicine is a functional modality
156
What is half life
Half-life is time taken for the radioactivity to reduce to 50%
157
Describe gamma camera imaging
Gamma cameras have imaging “heads”
For radionuclides that decay with direct emission of gamma rays
Most common radionuclide is Tc-99m (T1/2 = 6 hours)
Tracers used in gamma camera imaging:
Tc-99m MDP (bone scans)
Tc-99m DTPA (kidneys)
Tc-99m White Cells (infection/inflammation)
158
Describe the process of a dynamic renal transplant scan
Camera positioned above the patient
Tc-99m DTPA injected IV
Camera positioned above the patient
Gamma camera records gamma rays and collects image over time
Functional Time –Activity curves are obtained
159
Give examples of ionising radiation imaging and small animal scanners
High resolution
CT
SPECT
PET
160
Give examples of hybrid imaging
PET-CT
SPECT-CT
PET-MR
161
Describe the structure of a PET-CT scanner
patient will undergo transmission scan from CT
Patient is then injected with radioactive tracer and positrons. And they annhilate with body electrons to give of a signal that is detected by the PET scanner.
Fused PET and CT show the exact location of the 'hot spot'.
162
Describe attenuation correction for the PET-CT scan
Gamma rays originating from the centre of the patient will travel through more tissue which mean they are attenuated more
The CT image is used as an attenuation map to correct the PET image
163
What is SPECT-CT used for?
used to localise uptake.
164
PET-MR
```
First WB system
Biograph mMR
3T magnetCrystal
4x4x20mm
64 rings -> axial length 26cm
Very expensive
```
165
what is ultrasound?
Sound waves with frequencies higher than the human audible range,The upper limit is considered to be approximately 20kHz
166
Describe the pulse echo principle
The ultrasound probe has 2 main functions, to first emit a sound wave and then to receive the echoes from the original wave. This is the foundation principle of all Ultrasound applications and technology.
Whenever the ultrasound wave passes through a tissue boundary it can be reflected or will pass through and continue propogating.
Adjacent tissues with varying densities will reflect more of the sound wave, adjacent tissues with similar densities will reflect less..
Eg Air in lungs creates a poor image
167
Describe the basics of an ultrasound image
White-bone,gas
grey (intensity of grey also) reflects soft tissue density.
Black-fluid.
High amplitude: strong reflections.
Low amplitude: poor reflection/no reflections.
left or right
top of image is where the probe is, and bottom is area furthest away from probe.
168
Describe the clinical applications of ultrasound
```
Gynaecology
Abdominal
Urinary
Trauma-POCUS
Testicular
Breast
Head/neck
Vascular
Cardiology
Musculo-skeletal (MSK)
Lungs
Obstetrics
```
169
Why ultrasound
```
No radiation
No documented side effects in humans
Usually non invasive
Well tolerated
"real time" imaging
Results can often be available immediately-bed side
Widely accessible.
```
170
Why not ultrasoung?
No known side effects
Ultrasound image quality is highly dependent on patient habitus
Training is more resource intensive for departments compared to other modalities.
Effectiveness and accuracy are highly operator dependent.
171
Why do we have a selection of types of transducers
Increased choice of technical variables allows for optimisation of your image
Choice over sector width, scan depth (resolution), patient habitus, field of view..
172
What is the major advantage of
Ultrasound over X-ray / CT in obstetric
Imaging?
All women in the UK are offered ultrasound
Screening during pregnancy (12/20 weeks)
-it does not use ionising radiation (not exposing patients to a dose)
-it is mostly non invasive.
-no documented side effects.
-Well tolerated
-Real time imaging.
-widely accessible (can move patient at same time).
-Results can often be available immediately-bedside.
-need to be careful as it is energy and gets converted to heat as moves through body-can create heat increase if done for long time in a focal area.
Can cause gestation to cells if done to foetus in early pregnancy.
173
Describe the 12 week scan for obstetric ultrasound
```
Fetus is approximately 45-84mm in
Length (11+2wks – 14wks)
-First routine scan offered to most low
Risk pregnancies
-Detects ‘Viability’, number of fetus’,
Gross anatomy, detectable major Abnormalities, morphology of ovaries And an accurate gestational age of The foetus.
Anencephaly 1:3300
Omphalocele/exomphalos 1:5386
1:14-30,000
Cystic Hygroma 10% survival rate.
Blighted Ovum/Missed miscarriage 1 in 4.
Molar Pregnancy 1:1000.
Downs syndrome.
```
174
Describe the 12 week scan for Downs syndrome
Down's syndrome is caused by a change in one of the genes in the egg before it is fertilised by the sperm (at the time of conception). This is usually a completely random happening, though it is more common in older mothers. Throughout the world, the frequency of DS is about 3 per 2000 births. 1:1500 at 20 years
1:800 at 30 years
1:270 at 35 years
1:100 at 40 years
1:50 at 45 years and over Fetal nuchal translucency (NT) screening uses
ultrasound to measure the size of the nuchal pad at
the nape of the fetal neck. It should be performed
between 11 weeks and 13 weeks + 6 days
175
Describe obstetric ultrasound -20 week scan
The purpose of the 20 week scan in England is to identify abnormalities which:may indicate the baby has a life-limiting condition
- may benefit from antenatal treatment
- may require early intervention following deliveryOther standard aims;
Placenta localisation
Fetal Biometry
Fibroid Monitoring
Liquor Assessment
-can identify spina bifida.
-identifies achondroplasia
There are many specific types of achondroplasia
-bowing of long bones.
-Frontal bossing
-thickened soft tissue surrounding the long bones.
-identifies low lying placenta
In England at the 20 week scan we measure
The distance from the lowest edge of the
Placenta to the internal OS of the cervix.
If the placenta is within 2.5cm of the cervix
Then future scans are required. If the placenta
Does not raise higher closer to the due date then a C-Section may be required.
```
Identifies Talipes -club foot Ponseti Method
-Unilateral/Bilateral
-Associated findings?
-Mechanical/
Chromosomal?If one parent had the
Condition as a baby their
Own baby would have a
1 in 30 chance of also
Having talipes-Ponseti Method
-Unilateral/Bilateral
-Associated findings?
-Mechanical/
Chromosomal?
```
Clubfoot treatment over 4-6 weeks
176
Describe the weeks that obstetric ultrasound growth scans are done
12 weeks
20 weeks
28 weeks
36 weeks
-Anydraminios/oligohydraminos
Polyhydraminos
Umbilical artery Doppler assessment Can be used to highlight the affects of pre-eclampsia and intrauterine growth restriction (IUGR)
Is used more frequently now as is being suggested we can improve perinatal mortality and morbidity
177
Obstetric ultrasound -Early pregnancy EPU
-Pregnant women can be referred by their GPs in cases of lower abdominal/pelvic pain,
Bleeding, confirmed history of recurrent miscarriage and sometimes due to previous
Obstetric history issues.
At 5 weeksThe fetal pole is detected as an
Area of thickening along the periphery
Of a yolk sac
-Minimum of 1-2mm in length for
Detection (5-6 wks)
-Cardiac Activity should be detected
Routinely from 4-5mm (6wks)
Transabdominal vs Transvaginal
The very nature of the reasons why most
Women would attend EPU clinics unfortunately
Mean that it is a very challenging area to work
With strong counseling skills and empathy required.
When an egg implants outside of the
Uterine cavity it is know as an ectopic pregnancy.
Associated with severe pain and also bleeding
Can be caused by tubal damage (from surgery, PIDS, endometriosis). Treatment depends on the
Individual, Medical or surgical.
178
Obstetric ultrasound -multiple pregnancy
Multiple pregnancy usually caused by delays
In the fertilized egg reaching the womb before Implanting.
-triplets, conjoined twins, twin to twin transfusion syndrome.
179
Describe the obstetric ultrasound-procedure guidance
CVS ultrasound guided
CVS
Amniocentisis
180
What are fibroids
Fibroids consist of fibrous muscular tissue, many eventually grow until the
blood supply they receive can no longer support further growth, but others can
get very large and require surgical interventions ( myomectomy / uterine
embolisation / hysterectomy)
181
Gynaecology -post menopausal bleeding
Uterine Polyps – growths from the inner
Wall of the womb which extend throughout
The cavity and into the cervix and vagina.
Usually benign but on rare occasion some
Can turn cancerous. Surgery would be
Considered.
182
What can an abdominal ultrasound be used for?
```
Liver-cirrhosis/ascites
Kidneys
Aorta
Pancreas
Spleen
Gallbladder / Biliary Tree-gal stones: -Usually caused by an imbalance in the
Chemical make up within the bile in the
Gallbladder (high cholesterol / bilirubin)
```
- Sonographic Murphys Sign
- How do we use gravity in detection?
- Common Bile duct involvement?
183
Abdominal ultrasound -aortic screening
A section of abdominal aorta is defined
As aneurysmal when reaching 3cm in
AP diameter.
AAAs are monitored in specialised clinics
And surgery is often considered once the
Aneuysm meets 5.5cm in AP diameter.
EVAR – Endo Vascular Aortic Repair
184
What can a testicular ultrasound be used for?
Lumps
- Varicocele
- simple cyst -epididymal head
- hydrocele
- testicular cancer
185
Describe a breast ultrasound
```
-Under the age of 35 breast tissue tends to be denser, this leads to difficulty with diagnosing the nature of breast lumps on mammograms as differentiation between solid and fluid filled areas is relatively poor, ultrasound can make the differentiation at an improved rate (about 30% increased)Ultrasound also enables core
Biopsies to be taken of breast
Lumps to allow for histological
Investigation to allow for
Classification of the lump.
```
186
Describe a vascular DVR -Doppler
US is used to exclude or confirm the presence of a deep vein thrombosis in cases of pain
and swelling in the lower limbs. It is often also used as a screening tool for DVT in post
Operative patients and those with known pulmonary embolus (to find the source of the clot)Using colour flow doppler we
Can demonstrate that the Femoral vein is completely
Occluded by the lack of colour
187
Musculo-skeletal ultrasound
Applications :
-Muscle/tendon tears
-Inflammation
-Nerve Entrapments
-Soft tissue lumps
-Cysts
-Hernias
-Paediatric CHD
-Infant Torticollis (neck twisting)
-Early RA
-Joint effusions
-Injection Guidance (contrast and
Therapeutic)
-and many more…
188
POCUS - Fast
| ultrasound
Point of Care Ultrasound – Focused Assessment with Sonography of Trauma
FAST is an ultrasound scan protocol undertaken at the time of presentation of a trauma patient.
Ultrasound can detect as little as 20ml of free fluid, compared to the 200ml required with plain X-Ray
189
Polyhydraminos
a lot of fluid. Indicates gestational diabetes or bp issues with mum.
190
Anhydraminos/oligohydraminos
without/ with less fluid. Poor outcome if at early stage. Can be a chromosomal/genetic issue.
191
Growth of a populaton of cells
Growth of a population of cells
Distinguish between increase in cell numbers (hyperplasia) and increase in cell size (hypertrophy)
Depends on integration of intra- and extracellular signals (checks on cellular physiology, growth and inhibitory factors, cell adhesion etc.)
192
Growth at the cellular level (the cell cycle)
Cell growth = increase in size (sometimes growth refers to this only) and cell division
Cell cycle phases (G1, S, G2, and M)
Progression controlled at three key checkpoints (restriction points)
193
Loss of cells by programmed cell death
A coordinated program of cell dismantling ending in phagocytosis. Distinct from necrosis
Occurs during normal development (e.g. separation of the digits, involution, immune and nervous system development)
And in response to DNA damage and viral infection
194
What are growth factors, cytokines and interleukins?
Proteins that:
stimulate proliferation (called mitogens) and maintain survival
usually named after originally identified target e.g. EGF, FGF, Interleukins (IL2 & IL4), NGF
but see also PDGF (platelet-derived GF) and IGF1 (Insulin-like GF – the main effector of pituitary growth hormone)
stimulate differentiation and inhibit proliferation e.g. TGF
induce apoptosis e.g. TNFα and other members of the TNF family
Three broad classes:
Paracrine: produced locally to stimulate proliferation of a different cell type that has the appropriate cell surface receptor
Autocrine: produced by a cell that also expresses the appropriate cell surface receptor
Endocrine: like conventional hormones, released systemically for distant effects
195
DNA replication
DNA is replicated semiconservatively (daughter cells inherit one parental and one new strand)
New DNA is synthesized in the 5’ to 3’ direction from deoxynucleotide triphosphate precursors at a replication fork by a multienzyme complex (a replication machine)
Fidelity is determined by base pairing (A=T, G≡C) and presence of a proof reading enzyme in DNA polymerase
Synthesis of the new DNA strand uses an RNA primer and occurs continuously on the leading strand and discontinuously on the trailing strand (giving rise to Okazaki fragments, which are ligated together after removal of the RNA primer)
196
Mitosis
```
Interphase
Prophase (1)
Nucleus becomes less definite
Microtubular spindle apparatus assembles
Centrioles (yellow) migrate to poles
Prometaphase
Nuclear membrane breaks down
Kinetochores attach to spindle in nuclear region
Metaphase (2)
Chromosomes (blue) align in equatorial plane
Anaphase (3)
Chromatids separate and migrate to opposite poles
Telophase (4)
Daughter nuclei form
Cytokinesis
Division of cytoplasm
Chromosomes decondense
```
197
Describe drugs acting on cell cycle
S-Phase active
5-Fluorouracil (an analogue of thymidine blocks thymidylate synthesis).
Bromodeoxyuridine (another analogue that may be incorporated into DNA and detected by antibodies to identify cells that have passed through the S-phase).
M-phase active
Colchicine (stabilizes free tubulin, preventing microtubule polymerization and arresting cells in mitosis – used in karyotype analysis)
Vinca alkaloids (similar action to colchicine)
Paclitaxel (Taxol, stabilizes microtubules, preventing de-polymerization)
5-Fluorouracil, paclitaxel, the vinca alkaloids and tamoxifen are used in treatment of cancer
198
What are the cell cycle checkpoints
```
Controls (involving specific protein kinases and phosphatases) ensure the strict alternation of mitosis and DNA replication.
Before M phase: DNA completely replicated,
DNA not damaged
At M phase:
Chromosomes aligned on spindle
Before S phase:
Restriction point:
DNA not damaged, Cell size,
metabolite/nutrient stores,
During G1 phase
Cells responsive to growth factors -
Main site of control for cell growth
```
199
what controls cell cycle progression
Cyclin dependent kinase activity controls cell cycle progression.
Regulation of Cyclin-CDK Activity
Cyclical synthesis (gene expression) and destruction (by proteasome).
Post translational modification by phosphorylation – depending on modification site may result in activation, inhibition or destruction
Dephosphorylation
Binding of cyclin-dependent kinase inhibitors
200
Describe the role of Rb protein
Unphosphorylated RB binds E2F preventing its stimulation of S-phase protein expression
Cyclin D-CDK4
&
Cyclin E-CDK2
Released E2F stimulates expression of more Cyclin E and
S-phase proteins e.g. DNA polymerase, thymidine kinase, PCNA etc.
DNA replication starts.
201
Describe cyclin dependent kinase inhibitors
Two families of CKIs:
CDK Inhibitory Protein/Kinase Inhibitory Protein (CIP/KIP) family (now called CDKN1)
Expression of members of this family stimulated weakly by TGF and strongly by DNA damage (involving TP53)
Inhibit all other CDK-cyclin complexes (late G1, G2 and M)
Are gradually sequestered by G1 CDKs thus allowing activation of later CDKs
Inhibitor of Kinase 4 family (INK4) (now called CDKN2)
Expression stimulated by TGF
Specifically inhibit G1 CDKs (e.g. CDK4 the kinase activated by growth factors)
202
How do growth factors induce cyclin expression
Growth factor
Binds to growth factor receptor
Causes signal transducers.
Travel to nucleus, allows waves of transcription factor activation. Causes expression of mRNA, and then produces proteins.
Growth factor signalling activates early gene expression (transcription factors – FOS, JUN, MYC)
Early gene products stimulate delayed gene expression (includes Cyclin D, CDK2/4 and E2F transcription factors)
E2F sequestered by binding to unphosphorylated retinoblastoma protein (RB)
G1 cyclin-CDK complexes hypophosphorylate RB and then G1/S cyclin-CDK complexes hyperphosphorylate RB releasing E2F
E2F stimulates expression of more Cyclin E and S-phase proteins (e.g. DNA polymerase, thymidine kinase, Proliferating Cell Nuclear Antigen etc.)
S-phase cyclin-CDK and G2/M cyclin-CDK complexes build up in inactive forms. These switches are activated by post-translational modification or removal of inhibitors, driving the cell through S-phase and mitosis.
203
Describe the sequential activities in the cycle
G1 CDKs are activated in response to environmental signals, late CDKs by preceding kinase activities.
Hyperphosphorylated RB is dephosphorylated by protein phosphatase 1. G1 CDKs hypophosphorylate, and late CDKs hyperphosphorylate.
204
What happens when we detect DNA damage
DNA damage detected at check points triggers cell cycle arrest or apoptosis
Stop the cycle
(cyclin dependent kinase inhibitors, CHEK2 etc.)
Causes activation of TP53 by phosphrylation. TP53 then causes DNA repair (excision repair). If repair not possible: apoptosis.
TP53 also causes expression of CKI for cell cycle arrest. TP53 then destroyed by a proteasome.
Attempt DNA repair
(nucleotide or base excision enzymes, mismatch repair etc.)
Programmed Cell Death if repair impossible
(BCL2 family, caspases)
205
Key steps of DNA damage
Growth factor signalling activates early gene expression (transcription factors – FOS, JUN, MYC)
Early gene products stimulate delayed gene expression (includes Cyclin D, CDK2/4 and E2F transcription factors)
E2F sequestered by binding to unphosphorylated retinoblastoma protein (RB)
G1 cyclin-CDK complexes hypophosphorylate RB and then G1/S cyclin-CDK complexes hyperphosphorylate RB releasing E2F
E2F stimulates expression of more Cyclin E and S-phase proteins (e.g. DNA polymerase, thymidine kinase, Proliferating Cell Nuclear Antigen etc.)
S-phase cyclin-CDK and G2/M cyclin-CDK complexes build up in inactive forms. These switches are activated by post-translational modification or removal of inhibitors, driving the cell through S-phase and mitosis.
206
What are the major functional changes in cancer
Increased growth (loss of growth regulation, stimulation of environment promoting growth e.g. angiogenesis)
Failure to undergo programmed cell death (apoptosis) or senescence
Loss of differentiation (including alterations in cell migration and adhesion)
Failure to repair DNA damage (including chromosomal instability
207
What are the 2 major types of mutated gene that contribute to carcinogenesis
Oncogenes Their normal job is to make cells divide, driving cell division forward
In cancer, pick up mutations that mean they are permanently active – a bit like putting a brick on the accelerator. The car approaches the red light and can’t stop
Oncogene: “Gain of function”
an altered gene whose product can act
in a dominant fashion to help make a cell cancerous.
oncogene is a mutant form of a normal gene
(a “proto-oncogene”) involved in the control of cell growth or
division.
Tumour suppressor genes Even if you have a mutation in an oncogene that pushes cell division forward, if your tumour suppressor genes are strong enough, they should still be able to counteract the oncogene
In cancer, pick up mutations that switch the gene off. This is like cutting the brakes in a car. Even if there is no oncogenic brick on the accelerator, without breaks the car definitely can’t stop
Tumour Suppressor gene: “Loss of function”A gene whose normal activity prevents formation of a
cancer.
Both genes for the tumour suppressor must be mutated
Loss of this function by mutation enhances the
likelihood that a cell can become cancerous (a normal
process to maintain control of cell division is lost).
There are many pathways affected by oncogenes and tumour suppressor proteins.
208
The Rous sarcoma virus history
Studies of retroviruses essential in understanding oncogenes
Landmark experiments:
Frances Peyton Rous began his work in 1910 that lead to the discovery of Rous sarcoma virus (RSV).
50 years later he received the Nobel Prize in Medicine in 1986
In 1911 when a farmer brought Rous a prized Plymouth Rock hen that had a large tumour growing in the chest muscle,
he used the cell free filtrate from the chicken sarcoma and was able to induce sarcomas in healthy chickens.
Tumours developed weeks later
Taking the new sarcoma, filtrates produced could also induce tumours in other chickens
The cycles could be repeated indefinitely. Also the carcinogenic agent was small enough to pass through a filter
Although the filter used excluded bacteria it was not small enough to exclude viruses
Rous concluded that a virus must be responsible for the induction of tumour formation
Discovery that this sarcoma was transmissible through viruses- Rous Sarcoma Virus
209
Why are retroviruses important?
Although some human cancers are thought to be transmissible through viruses, most are confined to other animals
The idea that cancer was transmissible was already circulating as early as 1840 when an Italian scientist
Domenico Rigoni-Stern observed that nuns in Verona rarely developed cervical cancer. Papilloma virus was not
Identified until 1983
Retroviruses were important experimentally:
technological advances
funding
improved tissue culture techniques
the discovery of reverse transcriptase, RNA genome, replicates via
DNA intermediate and that they are enveloped.
Between the 1960’s and 70’s we learnt more about these types of viruses.
210
What is the oncogene hypothesis
Decades later oncogenic transformation by this virus was found to be caused by an
extra gene contained in its genome an ‘oncogene’ called v-src
By 1976, homologous sequences were found in uninfected chickens and other organisms-
fruit flies to humans
Fundamental principle: Oncogenes are alerted forms of normal genes or proto-oncogenes
c-src, cellular oncogene
v-src proto oncogene altered form transduced by retroviruses .
Upon finding there was a gene homologous sequence to v-src in uninfected chickens, in 1989 Harold Varmus and J. Michael Bishop received the noble prize for laying down the foundation of mutations in carcinogenesis
Discovered that the some genes of cancer causing viruses were mutated forms of the cellular gene not viral genes
They concluded that the Rous sarcoma viral gene was in fact a host gene that had
been ‘kidnapped’ by the virus (and ‘transformed’ into an oncogene)
An oncogene is any cellular gene that upon activation can transform cells
Bishop and Varmus used different strains of Rous sarcoma virus in their research, they:
Identified the v-src oncogene as responsible for causing cancer.
Used hybridization experiments, and they found that the c-src gene was present in the genome of many species.
They then showed that the host cell c-src gene was normally involved in the positive regulation of cell growth and cell division.
Following infection, however, the v-src oncogene was expressed at high levels in the host cell, leading to uncontrolled host cell growth, unrestricted host cell division, and cancer.
Proto oncogenes are normal genes that can control growth
Various agents, including radiation, chemical carcinogens, and, perhaps, exogenously added viruses, may transform cells by “switching on” the endogenous oncogenic information.
211
How did retroviruses 'kidnap' c-src
During evolution, the virus can acquire fragments of genes from the host at integration sites and this process results in the
creation of oncogenes
The oncogene product was characterised as a 60kDa intracellular tyrosine kinase
Can phosphorylate cellular proteins and effect growth
Virus invades cell, has reverse transcription of RNA, produces DNA provirus. It is accidentlaly integrated next to c-src GENE, AND THERE IS fusion of the sequences and new dna is packaged into a capsid. New RSV virion carries the src sequences.
212
Describe viral oncogenesis
Approximately 15%-20% of all human cancers are caused by oncoviruses
Viral oncogenes can be transmitted by either DNA or RNA viruses.
DNA viruses can cause lytic infection leading to the death of the cellular host or can replicate their DNA along with that of the
host and promote neoplastic transformation
DNA Viruses
Encode various proteins along with
environmental factors can initiate
and maintain tumours
```
RNA Viruses
Integrate DNA copies of their genomes
into the genome of the host cell and as
these contain transforming oncogenes
they induce cancerous transformation
of the host
```
213
Describe the activation of oncogenes
To date-over 100 identified oncogenes
There are examples of oncogenes for every type of protein involved in a growth factor signal transduction pathway
These genes captured by animal retroviruses are altered in human cancer, activation can involve
mutations, insertions, amplifications and translocationsLoss of response to growth regulatory factors
One allele needs to be altered
Activated by mutation, amplification/duplication and translocation.
214
Proto-oncogenes
```
4 types of proteins are involved in the transduction of growth signals
Normally
Growth factors
Growth factor receptors
Intracellular signal transducers
Nuclear transcription factors
```
Growth factors, signal transduction and cancer
The majority of oncogene proteins function as elements of the signalling
pathways that regulate cell proliferation and survival in response to growth
factor stimulation
Oncogene proteins act as growth factors (e.g.EGF),
growth factor receptors (e.g. ErbB) and intracellular signalling molecules (Ras and Raf).
Ras and Raf activate the ERK MAP kinase pathway, leading to the induction of additional
genes (e.g. fos) that encode potentially oncogenic transcriptional regulatory proteins
To date-over 100 identified oncogenes
215
Describe the Ras Oncogene family
RAS Oncogene Family
ras genes were identified from studies of two cancer-causing viruses the Harvey sarcoma virus and Kirsten sarcoma virus, These viruses were discovered originally in rats hence the name Rat sarcoma
RAS proteins are small GTPases that are normally bound to GDP in a neutral state
Oncogenic activation of ras is seen in about 30% of human cancer
Most commonly mutated oncogene
Point mutations in codons 12, 13 and 61
Glycine to valine - bladder carcinoma
Glycine to cysteine - lung cancer
216
Describe the effect of intracellular signalling transducers of RAS oncogene family
1. Binding of extracellular growth factor signal
2. Promotes recruitment of RAS proteins to the receptor complex
3. Recruitment promotes Ras to exchange GDP (inactive
Ras) with GTP (active Ras)
```
4. Activated Ras then initiates the remainder of the
signalling cascade (mitogen activated protein kinases)
```
5. These kinases ultimately phosphorylate targets, such as
transcription factor to promote expression of genes
important for growth and survival
Ras hydrolyzes GTP to GDP fairly quickly, turning itself “off”
In cancer, there are point mutations in codons 12, 13,61 Consequence of each of these mutations is a
loss of GTPase activity of the RAS protein
normally required to return active RAS to
the inactive RAS GDPConstitutive activation
Mutations in codon 12
val gly bladder carcinoma
cys gly lung cancer
217
Describe the MYC oncogene family
The MYC oncogene family consists of 3 members,
C-MYC, MYCN, and MYCL, which encode c-Myc, N-Myc,
and L-Myc, respectively
Originally identified in avian myelocytomatosis virus (AMV)
The MYC oncoproteins belong to a family of transcription factors that regulate the transcription of at least 15% of the entire genome
Major downstream effectors of MYC include those involved in ribosome biogenesis, protein translation, cell-cycle progression and metabolism, orchestrating a broad range of biological functions, such as cell proliferation, differentiation, survival, and immune surveillance The MYC oncogene is overexpressed in the majority of human cancers and contributes to the cause of at least 40% of tumours
It encodes a helix-loop-helix leucine zipper transcription factor that dimerizes with its partner protein, Max, to transactivate gene expression
Generally MYC is activated when it comes under the control of foreign transcriptional promoters. This leads to a deregulation of the oncogene that drives relentless proliferation.
Such activation is a result of chromosomal translocation
218
MYC and Burkitts lymphoma
MYC is activated in Burkitt's Lymphoma Epstein Barr virus is associated with Burkitt’s lymphoma (BL)
BL is a high grade lymphoma that can effect children from the age of 2 to 16 years
In central Africa, children with chronic malaria infections have a reduced resistance to the virus. This is known as classical African or endemic BL
All BL cases carry one of three characteristic chromosomal translocations that place the MYC gene under the regulation of the Ig heavy chain. Therefore c-myc expression is deregulated
In BL three distinct, alternative chromosomal translocations involving chromosomes 2, 14 and 22
In all three translocations a region form one of these three chromosomes is fused to a section of chromosome 8
219
Describe how chromosomal translocation is responsible for the activation of other genes
Chronic myelogenous leukaemia (CML) accounts for 15-20%
of all leukaemias
95% of CML patients carry the Philadelphia chromosome,
that is the product of the chromosomal translocation
t(9;22)(q34;q11) generating the BCR-ABL fusion protein
As a result of this translocation the tyrosine kinase activity
of the oncogene ABL is constitutive leading to abnormal
proliferation
Therapeutic strategies for CML include Imatinib (Gleevac) a tyrosine kinase inhibitor-96% remission in early-stage patients
220
Describe the discovery and identification of tumour suppressor genes
In 1969 Henry Harris and his colleagues performed somatic cell hybridization experiments
Fusion of normal cells with tumour cells yielded hybrid cells containing chromosomes form both parents. These cells were not capable of forming tumours
Genes derived from the normal parent acted to inhibit or suppress tumour development
The first tumour suppressor gene was identified by studies of retinoblastoma, a rare childhood eye tumour
221
Importance of tumour suppressor genes and their balance with oncogenes
Body has mechanisms to ‘police’ processes that regulate cell numbers
Tumour suppressor gene products act as stop signs to uncontrolled growth, promote differentiation or trigger apoptosis
Therefore they are usually regulators of cell cycle checkpoints (e.g. RB1), differentiation (e.g. APC) or DNA repair (e.g. BRCA1)
Loss of tumour suppressor gene function requires inactivation of both alleles of the gene
Inactivation can be a result of mutation or deletion
Tumour suppressor genes are defined as recessive genes
Sometimes referred to as ‘anti-oncogenes’
To date at least 15 tumour suppressor have
been identified
```
Different functions associated with each:
regulators of cell cycle checkpoints (e.g. RB1),
differentiation (e.g. APC)
DNA repair (e.g. BRCA1)
```
222
Describe the retinoblastoma gene Rb
Retinoblastoma is a rare childhood cancer (1 in 20,000) that develops when
immature retinoblasts continue to grow very fast and do not turn into
mature retinal cells.
An eye that contains a tumour will reflect light back in a white colour.
Often called a “cat’s eye appearance,” the technical term for this is leukocoria.
Two forms of the disease, familial (40%) and sporadic (60%)
The hereditary mutation is on chromosome 13 (13q14),
the retinoblastoma 1 (Rb1) gene
223
Describe the discovery of Retinoblastoma
The existence of the RB1 gene was predicted in 1971 by Alfred Knudson
Whilst studying the development of retinoblastoma he proposed that the development of retinoblastoma requires two mutations, which are now known to correspond to the loss of both of the functional copies of the Rb gene - "two-hit" hypothesis
"Loss of heterozygosity“ often used to describe
the process that leads to the inactivation of the
second copy of a tumour suppressor gene
a heterozygous cell receives a second hit in
its remaining functional copy of the tumour
suppressor gene, thereby becoming homozygous
for the mutated gene.
Mutations that inactivate tumour suppressor
genes, called loss-of-function mutations, are
often point mutations or small deletions that
disrupt the function of the protein that is
encoded by the gene
224
Describe the retinoblastoma protein RB structure
The Rb gene family includes three members: Rb/(p105/110), p107 and Rb2/p130
-collectively known as pocket proteins
pRb is a multi functional protein (110kDa) with over 100 binding partners
A transcriptional co factor that can bind to transcription factors
RB functions in diverse cellular pathways, such as apoptosis and the
cell cycle, it has also become clear that RB regulates these pathways
through the stimulation or inhibition of the activity of interacting proteins.
Therefore, an important starting point for understanding RB function is its
structure, which acts as a scaffold for these multiple protein interactions
It’s main binding partner is the E2F transcription factor,
interacting with the large pocket
Other viral oncoproteins can bind to Rb
225
What is the link between RB protein and cell cycle
Main function of Rb is to regulate the cell cycle by inhibiting the G1 to S phase transition.
2 important proteins involved in the cell cyle are:
Cyclins and their associated cyclin dependent kinases (cdks)
Passage of a cell through the cell cycle is regulated
cyclins and cyclin dependent kinases (cdks
Cyclin D is the first cyclin to be synthesized and drive progression through G1 together with cdks4/6
The G1 checkpoint leads to the arrest of the cell cycle in response to DNA damage
A key substrate for cyclin D is RB protein
Cyclin D and E families and their cdks phosphorylate RB
226
Describe RB function, phosphorylation and activity
Rb protein regulates the activity of the E2F transcription factor crucial for the expression of genes required for S phase
Rb activity is regulated by phosphorylation
When the Rb tumour suppressor is active it can inhibit cell proliferation
When Rb is dephosphorylated/hypophosphorylated it is active and remains bound to E2F
When Rb is active it blocks the progression of to S phase
When Rb is hyperphosphorylates , in response to extracellular physiological signals it is inactive.
Upon phosphorylation of RB, E2F is released and migrates to the nucleus to induce transcription
When RB is inactive cell cycle progression from G1 to S occurs
227
Describe the inactivation of Rb-loss of function
Rb can be inactivated by phosphorylation, mutation, or viral oncoprotein binding
In retinoblastoma, pRb is functionally inactivated by mutations
or partial deletions
Viral inactivation found in small DNA tumour viruses
mainly by disrupting E2F binding or destabilisation of Rb
Adenovirus - E1A
Papilloma - E7
Polyoma – Large T antigen
In cancer cells RB phosphorylation is deregulated throughout
cell cycle. As a direct consequence E2F transcription factors can
induce the deregulation of the cell cycle
Without RB on watch , cells move through G1 into S
and are not subjected to usual checks
228
Describe p53 tumour suppressor
The p53 gene was the first tumour suppressor gene to be identified
The p53 protein is at the heart of the cell’s tumour suppressive mechanism and has been nicknamed the ‘guardian of the genome’
It is involved in sensing DNA damage and regulating cell death/apoptosis
as well as other pathways
p53 is mutated in 30-50% of commonly occurring human cancers
Frequent mutation of p53 in tumour cell genomes suggests that tumour
cells try to eliminate p53 function before they can thrive
p53 specializes in preventing the appearance of abnormal cells.
Protein has an amino transactivation domain, a central DNA binding domain, a tetramerization domain and a carboxyl regulatory domain
Can bind to around 300 different gene promoter regions-main role as a transcription factor
229
Describe the regulation of P53 by MMD2
Normally levels of p53 protein are low in cells
These levels are kept low by MDM2 protein, a ubiquitin ligase (also an oncogene)
In unstressed normal cells both p53 and MDM2 move between the nucleus and cytosol
MDM2 binds p535 to form a complex in the nucleus where MDM modifies the carboxyl terminus of p53 and
targets it for degradation by the proteasome
WT p53 has a short 20 min half life
230
Describe the activation of tumour suppressor
Stress signals are able to activate p53
Signals are sensed by mainly kinases that then phosphorylate p53
Phosphorylation of p53 disrupts the interaction between it and
MDM2
e.g. ionizing radiation signals through two kinases ATM/ATR
activate oncogenes such as ras induce activity of p14arf
responsible for sequestering MDM2.
P53 can thus regulate genes involved in DNA damage repair,
apoptosis and cell cycle arrest
231
Describe p53 Mutation/Therapeutic strategies
Mutational inactivation is considered to be one of the most common molecular mechanisms behind the dysfunction of p53.
Extensive mutation search revealed that more than half of human cancers carry loss of function mutations of p53
Among them, 95% of mutations were detectable within the DNA-binding domain
Role of p53 a s star player in suppressing tumorigenesis makes it a promising therapeutic target
Different strategies aimed at:
- Correcting p53 mutation and restoring wild-type p53 function by targeting its regulators.
Gene therapy obvious approach
Many vectors and retroviruses have been examined
Retroviruses integrate in a stable form into the genome of infected cells. It has been demonstrated that
retrovirus-mediated gene transfer of the wild-type TP53 gene into both human lung tumour cell lines and xenograft models could lead to the inhibition of tumour cell growth
Alternative strategies- use of inhibitors.
PRIMA-1, Restores mutant p53 by
modifying the thiol groups in the core
domain of the protein
Nutlin- is a potent MDM2 antagonist
RITA binds to p53 and can restore
mutp53 activity
Inhibitors of CRM1 result in nuclear
accumulation of p53
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Genetic analysis and personalised medicine
A detailed readout of the molecular faults in a patient’s tumour, and new generation of drugs that precisely target them
Classifies tumours according to their genetic make-up instead of where they grow in the body
People with the ‘same’ cancer can have different forms of the disease so responses to treatment vary
Cancers growing in different parts of the body may also share the same genetic faults so respond to similar
treatments
233
Describe the sequence of events triggered by growth factors
Growth factor signalling activates early gene expression (transcription factors – FOS, JUN, MYC)
Early gene products stimulate delayed gene expression (includes Cyclin D, CDK2/4 and E2F transcription factors)
E2F sequestered by binding to unphosphorylated retinoblastoma protein (RB)
G1 cyclin-CDK complexes hypophosphorylate RB and then G1/S cyclin-CDK complexes hyperphosphorylate RB releasing E2F
E2F stimulates expression of more Cyclin E and S-phase proteins (e.g. DNA polymerase, thymidine kinase, Proliferating Cell Nuclear Antigen etc.)
S-phase cyclin-CDK and G2/M cyclin-CDK complexes build up in inactive forms. These switches are activated by post-translational modification or removal of inhibitors, driving the cell through S-phase and mitosis.
234
What are the 2 Cyclin dependent kinase imhibitors
Two families of CKIs:
CDK Inhibitory Protein/Kinase Inhibitory Protein (CIP/KIP) family (now called CDKN1)
Expression of members of this family stimulated weakly by TGF and strongly by DNA damage (involving TP53)
Inhibit all other CDK-cyclin complexes (late G1, G2 and M)
Are gradually sequestered by G1 CDKs thus allowing activation of later CDKs
Inhibitor of Kinase 4 family (INK4) (now called CDKN2)
Expression stimulated by TGF
Specifically inhibit G1 CDKs (e.g. CDK4 the kinase activated by growth factors)
235
Describe how cyclin dependent kinase activity controls cell cycle progression
Cyclical synthesis (gene expression) and destruction (by proteasome).
Post translational modification by phosphorylation – depending on modification site may result in activation, inhibition or destruction
Dephosphorylation
Binding of cyclin-dependent kinase inhibitors
236
What are the different drugs acting on the cell cycle
S-Phase active
5-Fluorouracil (an analogue of thymidine blocks thymidylate synthesis)Bromodeoxyuridine (another analogue that may be incorporated into DNA and detected by antibodies to identify cells that have passed through the S-phase).
M-Phase active
Colchicine (stabilizes free tubulin, preventing microtubule polymerization and arresting cells in mitosis – used in karyotype analysis)
Vinca alkaloids (similar action to colchicine)
Paclitaxel (Taxol, stabilizes microtubules, preventing de-polymerization)
5-Fluorouracil, paclitaxel, the vinca alkaloids and tamoxifen are used in treatment of cancer
237
Describe the causes and mechanisms of cell damage/cell death
Genetic
Abnormal number chromosomes (aneuploidy)
Abnormal chromosomes (deletions/translocations)
Increased fragility (Fanconi's anaemia)
Failure of repair (Xeroderma pigmentosa)
Inborn errors (Storage disorders ie. Tay Sachs disease)
```
Inflammation
Trauma
Thrombo-embolism
Atherosclerosis
Vasculitis
```
```
Physical
Irradiation
Heat
Cold
Barotrauma
```
Traumatic Damage
Interruption of blood supply
Direct rupture of cells
Entry of foreign agents
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Infection
Toxic agents
Competition for nutrients
Intracellular replication
- viruses/mycobacteria provoking
an immune response
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Chemical
Acids/corrosives
Specific actions e.g. enzymes
Interference with metabolism e.g. alcohol
238
Describe necrosis vs apoptosis
1. Necrosis: most common cause of cell death. Occurs after stresses such as ischemia, trauma, chemical injury
2. Apoptosis: programmed cell death. Designed to eliminate unwanted host cells through activation of a co-ordinated, internally programmed series of events effected by a dedicated set of gene products
3. Autophagic cell death: Autophagy is responsible for the degradation of normal proteins involved in cellular remodeling found during metamorphosis, aging and differentiation as well as for the digestion and removal of abnormal proteins that would otherwise accumulate following toxin exposure, cancer, or disease. An example is the death of breast cancer cells induced by Tamoxifen.
239
Causes of necrosis
```
Usually caused by lack of blood supply to cells or tissues, e.g.
injury,
infection,
cancer,
infarction,
inflammation
1. Whole groups of cells are affected.
```
2. Result of an injurious agent or event.
3. Reversible events proceed irreversible
4. Energy deprivation causes changes. (e.g. cells unable to produce ATP because of oxygen deprivation)
5. Cells swell due to influx of water (ATP is required for ion pumps to work).
6. Haphazard destruction of organelles and nuclear material by enzymes from ruptured lysosomes.
7. Cellular debris stimulates an inflammatory cell response
240
Describe the microscopic appearance of necrosis
1. Chromatin condensation/shrinkage.
2. Fragmentation of nucleus.
3. Dissolution of the chromatin by DNAse.
Cytoplasmic changes . Opacification: denaturation of proteins with aggregation.
2. Complete digestion of cells by enzymes causing cell to liquify (liquefactive necrosis).
Biochemical changes . Release of enzymes such as creatine kinase or lactate dehydrogenase
2. Release of proteins such as myoglobin
These biochemical changes are useful in the clinic to measure the extent of tissue damage!
241
Functions of necrosis
Removes damaged cells from an organism
Failure to do so may lead to chronic inflammation.
242
Functions of apoptosis
```
Selective process for the deletion of superfluous, infected or transformed cells.
Involved in:-
Embryogenesis
Metamorphosis
Normal tissue turnover
Endocrine-dependent tissue atrophy
A variety of pathological conditions
```
243
What are the two types of apoptosis
Intrinsic
DNA damage – p53-dependent pathway
Interruption of the cell cycle
Inhibition of protein synthesis
Viral Infection
Change in redox state
Extrinsic Withdrawal of growth factors (e.g. IL-3)
Extracellular signals (e.g. TNF)
T cell or NK (Natural Killer) (e.g. Granzyme).
244
caspases
Cysteine Aspartate-specific proteases
Caspases are Cysteine Proteases that play a central role in the initiation of apoptosis.
Most proteases are synthesised as inactive precursors requiring activation (usually partial digestion by another protease).
Caspase activation leads to characteristic morphological changes of the cell such as shrinkage, chromatin condensation, DNA fragmentation and plasma membrane blebbing
245
Microscopic appearance of apoptosis
Nuclear changes
1. Nuclear chromatin condenses on nuclear membrane.
2. DNA cleavage.
Cytoplasmic changes
1. Shrinkage of cell. Organelles packaged into membrane vesicles.
2. Cell fragmentation. Membrane bound vesicles bud off.
3. Phagocytosis of cell fragments by macrophage and adjacent cell.
4. No leakage of cytosolic components.
Biochemical changes 1. Expression of charged sugar molecules on outer surface of cell
membranes (recognised by macrophages to enhance phagocytosis)
2. Protein cleavage by proteases, caspases
246
How do we activate initiator caspases?
By induced proximity For example:
In response to receptor dimerization upon ligand binding or
Cytochrome C release from the mitochondria
247
Cytochrome C
Mitochondrial matrix protein
Known for many years to be released in response to oxidative stress by a “permeability transition”
Any inducers of the permeability transition also eventually induce apoptosis.
248
Describe p53 and apoptosis
Mutations in the p53 gene are the most common mutations
in cancer. Some mutations destroy the ability of p53 to induce
Apoptosis.
249
Historical background of cancer
Ancient Egypt ( ca. 2500-1600BC) The earliest known descriptions of cancer appear in several papyri from Ancient Egypt. A 2010 extensive study on Egyptian mummies’ tissues showed that only one case among hundreds has been verified as related to cancer and that the ancient Greeks were the first to identify it as a distinct illness
Hippocrates (ca. 460 BC – ca. 370 BC) described several kinds of cancer, referring to them with the Greek word carcinos (meaning crab or crayfish).
This name comes from the appearance of tumour, with “the veins stretched on all sides as the animal the crab has its feet, whence it derives its name”. It was against Greek tradition to open the body, Hippocrates only described and made drawings of outwardly visible tumours on the skin, nose, and breasts.
Celsus (ca. 25 BC – 50 AD) translated carcinos into the Latin cancer, also meaning crab.
Galen (2nd century AD) called benign tumours oncos, Greek for swelling, reserving Hippocrates’ carcinos for malignant tumours. He later added the suffix -oma, Greek for swelling, giving the name carcinoma.
250
Describe the description and early treatments of cancer
Description and Early Treatments
Ancient Egypt Description of a procedure to remove breast tumours by cauterization. It was observed that the disease had no treatment.
Ancient Greece
According to Hippocrates, cancer was the result of an excess in black bile. Physicians of the time described different cancer types. Galen also pointed out in his work, the most common types of cancer were the uterus and breast cancer found in women
Treatment was based on the humor theory of four bodily fluids (black and yellow bile, blood, and phlegm) and treatment consisted of diet, blood-letting, and/or laxatives.
Surgery was undertaken to remove tumours followed by the cauterization of the surrounding vessels to stop excessive haemorrhage.
251
Cancer statistics
Incidence
Every two minutes someone in the UK is diagnosed with cancer
359,960 new cases of cancer in the UK in 2015, that’s 990 cases diagnosed every day
Mortality
Every four minutes someone in the UK dies from cancer
Risk
1 in 2 people in the UK born after 1960 will be diagnosed with some form of cancer during their lifetime
Cancer survival
Half (50%) of people diagnosed with cancer in England and Wales survive their disease for ten years or more (2010-11)
Cancer survival is improving and has doubled in the last 40 years in the UK
Prevention
4 in 10 (42%) of cancer cases in the UK each year are linked to lifestyle
These are cases that can be prevented largely through lifestyle changes
252
Prevention/reducing risks of cancer
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Smoking
Obestiry and weight
Changes in hormone level
Alcohol
Workplaces
Sun and UV
Infection and HPC
Physical activity
Diet and healthy eating
Inherited genes
Air pollution and radon
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Adults aged 50-74 account for more than half (53%)
of all new cancer cases, and elderly people aged 75+
account for more than a third (36%), with slightly
more cases in males than females in both age groups.
There are more people aged 50-74 than aged 75+
in the population overall, hence the number of
cancer cases is higher in 50-74s, but incidence
rates are higher in 75+s.
253
What is cancer
Cancer is the name for a group of diseases characterised by:
Abnormal cell proliferation
Tumour formation
Invasion of neighbouring normal tissue
Metastasis to form new tumours at distant sites
Over 200 different types of cancer have been classified, often according to their origin:
Approximately 85% of cancer occur in epithelial cells-carcinomas
Cancers derived from mesoderm cells (bone and muscle) are sarcomas
Cancers found in glandular tissue are called adenocarcinomas
254
Halmarks of cancer
In 2000, Hanahan and Weinberg defines six hallmarks of most if not all cancers
In 2011, this had been modified to include:
two enabling characteristics: genome instability and tumour inflammation
```
two emerging hallmarks: avoiding immune destruction and reprogramming energy metabolism
Sustaining proliferative signalling
Evading growth suppressors
Avoiding immune destruction
Enabling replicative immortality
Tumour promoting inflammation
Activating invasion and metastasis
Inducing angiogenesis
Genome Instability and mutation
Resisting cell death
Disregulating cellular energetics
```
255
Carcinogens
Carcinogens cause alterations to the DNA - Mutations
DNA from tumours has been shown to contain many alterations from point mutations to deletions
The accumulation of mutations over time represents the multi-step process that underlies carcinogenesis
This accumulation occurs only after the cells defence mechanism of DNA repair have been evaded
In cases if severe damage cell apoptosis is induced
Many mechanisms exist for blocking carcinogenesis but
over burdening the system increases the possibility
that cells will escape surveillance
The longer we live the more time there is for DNA to accumulate
mutations that may lead to cancer
Cancer is more prevalent as lifespan has increased
256
Tumour cells
Tumour cells
Somatic mutations constitute almost all mutations in tumour cells
All cells in a primary tumour arise from a single cell, initiation of the
development of cancer is clonal
Only one of the 1014 cells in body need to be transformed to create a tumour
Continued accumulation of mutations
Tumour cells can ‘evolve’- sub clonal selection allowing a growth advantage and explain and heterogeneity of cells in a tumour
Dependent on interaction with other tumour cells and the tumour microenvironment
257
3 assumptions for multistage carcinogenesis
3 assumptions
Malignant transformation of a single cell is sufficient to give rise to a tumour
Any cell in a tissue is as likely to be transformed as any other of the same type
Once a malignant cell is generated the mean time to tumour detection is generally constant
258
The 5 models of carcingenesis
Cancer is s multi step process that includes initiation, promotion and progression.
1. Chemical carcinogens can alter any of these process to induce their carcinogenic effects
The presence of multiple mutations in critical genes is a distinctive feature of cancer cells and supports that cancer arises through the accumulation of irreversible DNA damage.
In the majority of instances chemical carcinogens can induce this DNA damage and act in a genotoxic manner.
eg
Benzene – an industrial solvent, refined from crude oil
Polycyclic aromatic hydrocarbons – a group of dangerous
DNA-damaging chemicals, including benzo(a)pyrene
Model 2 Genome instability Knudson's Hypothesis for Hereditary Cancers
First proposed by Carl Nordling in 1953 and then formulated by Knudson in 1971
Developed by Knudson for retinoblastoma, which became the basis of the ‘two-hit’ hypothesis
and led to the formulation of the theory of ‘tumour suppressor genes’(TSGs) and then to the
discovery of Rb1, the TSG that causes retinoblastoma when both copies are mutated
Knudson performed statistical analysis on cases of retinoblastoma of which there are two types the inherited type and the sporadic type
Knudson suggested that multiple hits were required to cause cancer. So for example if the first mutated allele was inherited the second mutation would lead to cancer. In the sporadic forms of the tumour both mutations had to take place and hence this could explain the difference of age at diagnosis
At least two events are necessary for carcinogenesis and that the cell with the first event must survive in the tissue long enough to sustain a second event.
Model 3 Non genotoxic
Non-genotoxic is characterized by an emphasis on non-genotoxic effects
Several important modulators of cancer risk (diet, obesity, hormones and insulin resistance) do not seem to act through a structural change in DNA but rather through functional changes including epigenetic events.
There is, however, a group of carcinogens that induce cancer via non-genotoxic mechanisms. Non-genotoxic carcinogens have been shown to act as:
tumour promoters (1,4-dichlorobenzene),
endocrine-modifiers (17β-estradiol),
receptor-mediators (2,3,7,8-tetrachlorodibenzo-p-dioxin),
immunosuppressants (cyclosporine) or
inducers of tissue-specific toxicity and inflammatory responses (metals such as arsenic and beryllium)
Although little is known about this group of carcinogens it is known that in a high proportion of them, multiple pathways need to be altered for cancer induction .
Model 4 Darwinian Carcinogenesis by Mutation and Selection-Model of Clonal Expansion the role of the environment in selecting cells that have some acquired advantage
Model 5 Tissue Organisation
To understand the changes that occur during cancer it is important to understand the principles of cell and tissue organisation and mechanisms that control growth and structure.
Tissues - Groups of cells with similar function are known as tissues: epithelial, connective muscle and nervous
259
What test can we perform to determine whether chemical is carcinogenic
Ames test
260
Heritable carcinogens-syndromes predisposing to cancer
Accounts for 5% of all cancers
An inherited germline mutation, has an increased risk of developing certain tumours but are rarely involved in causing cancer immediately
In most known hereditary malignant syndromes the elevated cancer risk is due to a mutation of a single gene (monogenic hereditary diseases)
The affected genes concerned usually have a controlling function on the cell cycle or the repair of DNA damageA deficiency in DNA repair would cause more DNA damages to accumulate, and increase the risk for cancer
```
syndromes predisposing to cancer:
DNA repair defectsataxia telangiectasia
Bloom’s syndrome
Fanconi’s anaemia
Li-Fraumeni syndrome
Lynch type II
xeroderma pigmentosum
```
Chromosomal abnormalities
Downs syndrome
Klinefelter's syndome
261
DNA Repair defects
Ataxia telangiectasia- neuromotor dysfunction, dilation of blood vessels,
telangiectasia = spider veins
Mutation in ATM gene, codes for a serine/threonine kinase that is recruited and activated by
dsDNA breaks leading to cell cycle arrest, DNA repair and apoptosis -cell cycle arrest
Cancer predisposition: lymphoma, leukaemia and breast cancer.
Bloom’s Syndrome -short stature, rarely exceed 5 feet tall, skin rash that develops
after exposure to the sun
Mutation in BLM gene that provides instructions for coding a member of the RecQ helicase family
that help maintain the structure and integrity of DNA
Cancer predisposition: skin cancer. basal cell carcinoma and squamous cell carcinoma.
Lynch type- LS doesn't cause any symptoms. Sometimes the first sign that a person has LS is when the symptoms of bowel and womb cancer develop.
Mutations in DNA mismatch repair (MMR) genes, notably MLH1, MSH2, MSH6 and PMS2.
Cancer predisposition: colorectal cancer
262
Properties of tumourigenic viruses
```
Stable association with cells
chromosomal integration
episome.
Must not kill cells
non-permissive host (virus cannot replicate)
suppression of viral lytic cycle
viral release by budding.
```
Must evade immune surveillance of infected cells
immune suppression
viral antigens not expressed at cell surface
263
Viruses associated with human cancer
DNA viruses
Epstein-Barr virus Burkitt’s lymphoma, nasopharyngeal carcinoma
papilloma viruses cervical carcinoma, warts
hepatitis B and C hepatoma
RNA retroviruses
HTLV-I Adult T-cell leukaemia, lymphoma
264
What are the different forces driving carcinogenesis
Two drastically different approaches to understanding the forces driving carcinogenesis have crystallized through years of research.
These are the somatic mutation theory (SMT) and the tissue organization field theory (TOFT).
SMT
cancer is derived from a single somatic cell that has successively accumulated multiple DNA mutations
those mutations damage the genes which control cell proliferation and cell cycle
Thus, according to SMT, neoplastic lesions are the results of DNA-level events.
single catastrophic event triggering carcinogenesis
TOFT
Carcinogenesis is primarily a problem of tissue
organization
carcinogenic agents destroy the normal tissue architecture thus disrupting cell-to-cell signaling and compromising genomic integrity
the DNA mutations are randon and the effect, not the cause, of the tissue-level events. carcinogenesis as general deterioration of the tissue microenvironment due to extracellular causes
265
Immune response in cancer
The immune system will:
Protect from virus-induced tumours
Eliminate pathogens
Identify and eliminate tumour cells
This leads to immune surveillance
Despite this tumours can still arise-
Concept of cancer immunoediting
266
Describe cancer immunoediting
The Three Es
Elimination
The immune system is able to
eradicate developing tumours
```
Equilibrium
When incomplete removal is present
tumour cells remain dormant and
enter equilibrium. The immune system
exerts a potent and relentless pressure
that contains the tumour. During this
phase some of the tumour may mutate
or give rise to genetic variants that
survive, grow and enter the next phase
(Longest of the phases, around 20 years)
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Escape
The expanding tumour populations becomes
clinically detectable
SEM4
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