Cellular Pathology Flashcards
What is the purpose of primary cell culture techniques?
Grow cells directly from body, in vitro, to recreate invivo environment as closely as possible.
Describe primary cell culture technique
- 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
Describe cell lines
- 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.
Hematopoietic cells
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
Non-hematopoietic cells
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.
How do we remove the non-hematopoietic cells from tissue they come from?
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.
What is haemopoiesis
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.
What are the ways to distinguish between stem cells/prognitors etc
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
How do we get stem cells out of bone marrow?
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.
Describe the purpose of colony assays
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.
What are the applications of primary cell culture
Experimental
-research on normal and abnormal cells
Diagnostic
-test toxicity of chemotherapeutic agents and carcinogens
Therapeutic
-generate/amplify cells for stem cell transplantation/manipulation.
Stem cells
Pluripotent, give rise to all lineages
Self renew
Rare cells
Responsible for engraftment
Progenitor cells
Undifferentiated
Not distinguished by morphology
Committed to one or more lineages
Detected in colony-forming assays
Precursor cells
Immature but recognisable
Cells starting to differentiate
Few final divisions before mature cells
What are haematopoietic growth factors
Polypeptide growth factors (cytokines)
Bind to cell surface transmembrane receptors
Stimulate growth and survival of progenitors.
How do you make a cell line?
- 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)
What is the history of cell culture?
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
How do you isolate stem cells from blood?
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.
Describe the immunopurification methods used to isolate stem cells from centrifuge with blood layers
- 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.
Describe the isolation of cells from solid tissues
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.
Describe growth of cells in culture (out of their natural environment)
-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
What is the advantage and disadvantage of primary cells
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
Describe the characteristics of cell lines?
Good growth characteristics
Phenotypic stability
Defined population
Molecular manipulation readily achieved
How do we make cell lines?
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.
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.
Describe the retinoblastoma protein
checks whether G1 phase has progressed well, then the cell continues cell cycle.
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.
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.
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.
How do we introduce plasmid DNA into the cells?
- 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 - Stably incorporate the growth promoting gene into primary cells DNA.
What are the disadvantages of using a cell line?
Rapidly dividing cells often lose differentiated function (do not have time to specialize)
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.
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.
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.
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.
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.
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)
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.
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
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.
Relationship between pixel area and spatial resolution
The more black/white, the higher the resolution/
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)
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.
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.
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.
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.
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.
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
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.
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
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.
What is stokes shift?
Stokes shift is the change/difference between the excitation wavelength and emission wavelength.
What is photobleaching?
loss of colour by a pigment (such as chlorophyll or rhodopsin) when illuminated.
The fluroful cyclic process is broken.
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.
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.
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.
Advantage of using explants to produce tissue culture
No need to use enzymes or chemicals that may damage the cells
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.
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.
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.
What is CT scan used for?
CT is best for bone
What is MRI scan used for?
MRI id best for soft tissue, using signals from protons and fats in water
What important property is needed to detect changes/pathological variations in images?
contrast
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
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.
colour of CSF on T1W imaging vs T2W imaging
CSF is dark on T1-weighted imaging and bright on T2-weighted imaging.
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.
WHAT CONTRAST is used foor T1W
Gadolinium
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
What does cyclin E do?
Cyclin E is important for the G1 to S phase transition
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.
Cyclin A - cdk2 complex
Important for s phase transition
Cyclins A, B-cdk1
directs G2 to the G2 to M phase transition
What is carcinogenesis
process where normal cell transformed into cancer cell.
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)
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.
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.
What are the different pathways affected by oncogenes and tumour suppressor proteins
Myc
Ras
Rb
P53
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,
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.
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.
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.
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
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
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
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
- Promotes recruitment of RAS proteins to the receptor complex
- 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)
- 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”
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
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
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
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
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
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
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 .
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
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
What causes the lethality of cancers?
Their ability to invade and colonise different sites within the body.
What are the characteristics of malignant tumours?
- 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.
- Invasiveness, migration of tumour cells into the surrounding extracellularmatrix (stroma) where they are free to disseminate via vascular or lymohatic channels to distant organs.
- Metastasis
Spread of tumour cells from the primary site to form secondary tumours at other sites in the body
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.
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
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)
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
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
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
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
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)
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
