Cellular Pathology Flashcards

Question

Describe the p53 protein

Answer 1

-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.

Answer 2

checks whether G1 phase has progressed well, then the cell continues cell cycle.

Answer 3

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.

Answer 4

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.

Answer 5

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.

Answer 6

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.

Answer 7

Rapidly dividing cells often lose differentiated function (do not have time to specialize)

Answer 8

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.

Answer 9

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.

Answer 10

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.

Answer 11

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.

Answer 12

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.

Answer 13

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)

Answer 14

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.

Answer 15

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

Answer 16

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.

Answer 17

The more black/white, the higher the resolution/

Answer 18

Largest number is the magnification | Second number is the coverslip thickness thickness (normal is 0.17-0.18mm)

Answer 19

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.

Answer 20

The length the objective can work at from the sample. The closer, the more inside the sample you can see.

Answer 21

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.

Answer 22

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.

Answer 23

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.

Answer 24

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

Answer 25

- 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.

Answer 26

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

Answer 27

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.

Answer 28

Stokes shift is the change/difference between the excitation wavelength and emission wavelength.

Answer 29

loss of colour by a pigment (such as chlorophyll or rhodopsin) when illuminated. The fluroful cyclic process is broken.

Answer 30

-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.

Answer 31

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.

Answer 32

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.

Answer 33

No need to use enzymes or chemicals that may damage the cells

Answer 34

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.

Answer 35

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.

Answer 36

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.

Answer 37

CT is best for bone

Answer 38

MRI id best for soft tissue, using signals from protons and fats in water

Answer 39

Measure of how much the x-rays are attenuated in passing through any material. Scale goes from air->water->cortical bone

Answer 40

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.

Answer 41

CSF is dark on T1-weighted imaging and bright on T2-weighted imaging.

Answer 42

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.

Answer 43

Gadolinium

Answer 44

Acts with cdks4/6 to drive through progression of G1. Also important in regulation and expression of cyclin E

Answer 45

Cyclin E is important for the G1 to S phase transition

Answer 46

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.

Answer 47

Important for s phase transition

Answer 48

directs G2 to the G2 to M phase transition

Answer 49

process where normal cell transformed into cancer cell.

Answer 50

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)

Answer 51

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.

Answer 52

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.

Answer 53

Myc Ras Rb P53

Answer 54

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,

Answer 55

-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.

Answer 56

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.

Answer 57

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.

Answer 58

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 ```

Answer 59

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

Answer 60

``` 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

Answer 61

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”

Answer 62

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

Answer 63

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

Answer 64

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

Answer 65

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

Answer 66

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

Answer 67

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

Answer 68

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 .

Answer 69

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

Answer 70

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

Answer 71

Their ability to invade and colonise different sites within the body.

Answer 72

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

Answer 73

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.

Answer 74

Angiogenesis is the formation of new blood vessels from pre-existing vessels. Vasculogenesis is the formation of new blood vessels from progenitors

Answer 75

``` Developmental vasculogenesis (organ growth) Normal angiogenesis (wound repair, placenta during pregnancy, cycling ovary) Pathological angiogenesis (tumour angiogenesis ocular and inflammatory disorders) ```

Answer 76

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

Answer 77

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

Answer 78

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

Answer 79

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

Answer 80

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)

Answer 81

``` 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 ```

Answer 82

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

Answer 83

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.

Answer 84

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

Answer 85

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

Answer 86

CT contrast is due to tissue density dependent attenuation of x-rays -good for haemorrhagic acute stroke, oedema, and haemorrhage.

Answer 87

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…..

Answer 88

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

Answer 89

The positive charge of a spinning proton produces a magnetic moment micrometers sign units.

Answer 90

In a magnetic field Bo the magnetic moment of a proton precesses at the Larmor frequency VL units.

Answer 91

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

Answer 92

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

Answer 93

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

Answer 94

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

Answer 95

Exponential decay.

Answer 96

``` T2 is sensitive to water content. White 90ms Grey 80ms CSF >1000ms The more water, the longer it takes for signal to decay. ```

Answer 97

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

Answer 98

``` 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 ```

Answer 99

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.

Answer 100

3D T1 images used to determine volume of hippocampus Neurogenesis proposed to explain need for increased capacity of spatial memory

Answer 101

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

Answer 102

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.

Answer 103

Inflammation, demyelination, BBB leakage.

Answer 104

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

Answer 105

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.

Answer 106

Underlined are not visible in normal brain spectra

Answer 107

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.

Answer 108

``` 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 ```

Answer 109

``` Ionising Radiation Planar X-ray CT Gamma Camera and SPECT PET Hybrid Imaging ```

Answer 110

``` Radiation that causes ionisation when it interacts with matter Types used for medical imaging are: Gamma rays X-rays Why use ionising radiation? Penetrating ```

Answer 111

``` 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. ```

Answer 112

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

Answer 113

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 ```

Answer 114

Attenuation increases with Higher atomic number Higher density X-Rays are essentially an attenuation map

Answer 115

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

Answer 116

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

Answer 117

``` 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 ```

Answer 118

High resolution Compression plate used to reduce breast thickness Improves resolution Lowers radiation dose (used as a screening tool)

Answer 119

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

Answer 120

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

Answer 121

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

Answer 122

``` Ct scan, Also known as spiral scanning Introduced in late 1980s Continuous rotation Continuous table feed ```

Answer 123

Multi Slice CT Faster scan More coverage each rotation

Answer 124

Can differentiate between -haemorrhage or blood clot. Urgent diagnosis required for treatment Clot busting drugs may increase bleeding

Answer 125

Measurement of the size of the left inguinal lymph node shows progression of disease Imaging is used for monitoring response to therapy

Answer 126

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

Answer 127

Inject radioactive tracer, patient is emitting the gamma rays Image depends on the metabolism of the tracer: Functional Imaging For gamma camera and PET

Answer 128

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

Answer 129

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

Answer 130

Half-life is time taken for the radioactivity to reduce to 50%

Answer 131

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)

Answer 132

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

Answer 133

High resolution CT SPECT PET

Answer 134

PET-CT SPECT-CT PET-MR

Answer 135

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'.

Answer 136

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

Answer 137

used to localise uptake.

Answer 138

``` First WB system Biograph mMR 3T magnetCrystal 4x4x20mm 64 rings -> axial length 26cm Very expensive ```

Answer 139

Sound waves with frequencies higher than the human audible range,The upper limit is considered to be approximately 20kHz

Answer 140

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

Answer 141

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.

Answer 142

``` Gynaecology Abdominal Urinary Trauma-POCUS Testicular Breast Head/neck Vascular Cardiology Musculo-skeletal (MSK) Lungs Obstetrics ```

Answer 143

``` 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. ```

Answer 144

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.

Answer 145

Increased choice of technical variables allows for optimisation of your image Choice over sector width, scan depth (resolution), patient habitus, field of view..

Answer 146

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.

Answer 147

``` 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. ```

Answer 148

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

Answer 149

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

Answer 150

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

Answer 151

-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.

Answer 152

Multiple pregnancy usually caused by delays In the fertilized egg reaching the womb before Implanting. -triplets, conjoined twins, twin to twin transfusion syndrome.

Answer 153

CVS ultrasound guided CVS Amniocentisis

Answer 154

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)

Answer 155

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.

Answer 156

``` 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?

Answer 157

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

Answer 158

Lumps - Varicocele - simple cyst -epididymal head - hydrocele - testicular cancer

Answer 159

``` -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. ```

Answer 160

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

Answer 161

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…

Answer 162

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

Answer 163

a lot of fluid. Indicates gestational diabetes or bp issues with mum.

Answer 164

without/ with less fluid. Poor outcome if at early stage. Can be a chromosomal/genetic issue.

Answer 165

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.)

Answer 166

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)

Answer 167

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

Answer 168

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

Answer 169

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)

Answer 170

``` 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 ```

Answer 171

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

Answer 172

``` 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 ```

Answer 173

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

Answer 174

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.

Answer 175

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)

Answer 176

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.

Answer 177

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.

Answer 178

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)

Answer 179

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.

Answer 180

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

Answer 181

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.

Answer 182

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

Answer 183

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.

Answer 184

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.

Answer 185

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.

Answer 186

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 ```

Answer 187

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.

Answer 188

``` 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

Answer 189

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

Answer 190

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

Answer 191

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

Answer 192

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

Answer 193

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

Answer 194

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

Answer 195

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) ```

Answer 196

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

Answer 197

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

Answer 198

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

Answer 199

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

Answer 200

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

Answer 201

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

Answer 202

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

Answer 203

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

Answer 204

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

Answer 205

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

Answer 206

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

Answer 207

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.

Answer 208

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)

Answer 209

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

Answer 210

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

Answer 211

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 ``` Infection Toxic agents Competition for nutrients Intracellular replication - viruses/mycobacteria provoking an immune response ``` Chemical Acids/corrosives Specific actions e.g. enzymes Interference with metabolism e.g. alcohol

Answer 212

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.

Answer 213

``` 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

Answer 214

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!

Answer 215

Removes damaged cells from an organism Failure to do so may lead to chronic inflammation.

Answer 216

``` 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 ```

Answer 217

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).

Answer 218

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

Answer 219

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

Answer 220

By induced proximity For example: In response to receptor dimerization upon ligand binding or Cytochrome C release from the mitochondria

Answer 221

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.

Answer 222

Mutations in the p53 gene are the most common mutations in cancer. Some mutations destroy the ability of p53 to induce Apoptosis.

Answer 223

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.

Answer 224

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.

Answer 225

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

Answer 226

``` 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 ``` 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.

Answer 227

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

Answer 228

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 ```

Answer 229

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

Answer 230

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

Answer 231

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

Answer 232

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

Answer 233

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

Answer 234

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

Answer 235

``` 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

Answer 236

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

Answer 237

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

Answer 238

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

Answer 239

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) ``` Escape The expanding tumour populations becomes clinically detectable

Cellular Pathology Flashcards

(266 cards)