Pathology Concepts 1 – Types of Cell Injury PDF

Pathology Concepts 1 – Types of Cell Injury BMS 100 Week 8 Overview Introduction to Pathology Reversible and Irreversible Cellular Injury Causes Morphologic Characteristics Necrosis Discrete Mechanisms Convergence of Mechanisms Morphologic Characteristics Apoptosis Intrinsic vs. Extrinsic Pathways I...

Pathology Concepts 1 – Types of Cell Injury BMS 100 Week 8 Overview Introduction to Pathology Reversible and Irreversible Cellular Injury Causes Morphologic Characteristics Necrosis Discrete Mechanisms Convergence of Mechanisms Morphologic Characteristics Apoptosis Intrinsic vs. Extrinsic Pathways Initiation vs. Execution Phases Mechanisms and Signaling Concepts Necroptosis Overview cont… Cellular adaptations to Stress Hypertrophy, Atrophy, Hyperplasia Patterns of Long-Term Extracellular Damage Calcification Cellular Inclusions (post-learning) Definition of Terms Reversible cell injury: ▪ A cell/tissue has been stressed, but overcomes this stress and resumes normal physiologic function No notable long-term morphologic or physiologic changes Irreversible cell injury ▪ A cell/tissue has become damaged and will eventually die due to the severity of the damage Adaptation – there is a change in cellular/tissue structure or function that is almost always due to long-term stresses ▪ Examples: hypertrophy, hyperplasia, atrophy, metaplasia ▪ These changes are usually somewhat reversible Types of Insults to Tissues or Cells Hypoxia and ischemia ▪ Definitions: Infection, inflammation, and immune-mediated disorders Toxins/chemical agents Trauma, compression, thermal injuries Deficiencies in nutrients or growth factors Mechanisms of Cell Injury In general: The cellular response to injury depends on: ▪ Type of injury Ischemic? ROS? Physical? Chemical? Inflammatory? Microbial? ▪ Duration of the injury ▪ Severity of the injury ▪ The adaptability and the metabolism/phenotype of the cell Big difference between cardiac cells and skeletal muscle cells re: vulnerability to ischemia Reversible Cell Injury If the insult/stress is resolved, then the cell resumes normal function and appearance Sometimes the pathophysiologic consequences of the insult is clearly visible under the microscope: ▪ Cellular swelling - why? ▪ Non-specific nuclear changes ▪ Ribosomal detachment, membrane abnormalities due to cytoskeletal disassembly, accumulation of lipids Lipid vacuoles enlarge, collections of damaged membranous components (myelin figures) loss of mRNA → a more eosinophilic cytoplasm Small “blebs” – bubble-like outpouchings in the membrane Sometimes changes are more difficult to observe: ▪ Damage to proteins (including misfolding), DNA, subtle changes in organelle function and size due to damaged membranes Reversible Injury – key features What you can’t see under the light microscope: ▪ Changes in calcium concentrations ▪ Unfolded proteins ▪ Damage to DNA or cytoskeletal elements ▪ Loss of membrane potentials or abnormal distribution of molecules across cell membranes ▪ ATP depletion Kumar et. al., Robbins and Cotran Pathologic Basis of Disease 9th ed. Fig 2-8, p. 41 Irreversible Cellular Injury These changes will be discussed in more depth as we explore necrosis and apoptosis Typical findings under the microscope: ▪ Serious loss of integrity – plasma membrane, lysosomal membranes, mitochondrial membranes, ER membranes ▪ Destruction of cytoskeletal elements ▪ DNA and nuclear “disruption” Karyolysis – chromatin fades Pyknosis – chromatin condenses, more basophilic, nucleus shrinks Karyorrhexis – nucleus fragments (late) Kumar et. al., Robbins and Cotran Pathologic Basis of Disease 9th ed. Fig 2-8, p. 41 Cell Death – two major categories Necrosis ▪ The agents that have injured the physiology/biochemistry of the cell → immediate loss of cellular viability ▪ If cellular signaling is involved in this process, it is disorganized and unregulated Programmed cell death ▪ Cell death is delayed and requires protein synthesis ▪ Cellular signaling is always involved and the cell proceeds through an orderly series of steps → death ▪ This can be due to long-term, irreparable cellular damage or loss of cell use ▪ Best examples – apoptosis and necroptosis Necrosis – mechanisms of injury: ▪ ▪ ▪ ▪ ▪ ▪ ▪ Depletion of ATP Mitochondrial damage Calcium accumulation Oxidative stress / free radicals Membrane damage Denatured proteins DNA damage Kumar et. al., Robbins and Cotran Pathologic Basis of Disease 9th ed. Fig 2-16, p. 45 ATP depletion and cell injury Reduction (5 – 10% of normal) in ATP levels results in: Na+/K+ pump dysfunction and swelling ▪ Eventually leads to membrane damage Anaerobic metabolism decreases pH (lactic acid, inorganic phosphate) Increased production of free radicals Failure of calcium pumps Reduction in protein synthesis, detachment of ribosomes, misfolding of proteins Kumar et. al., Robbins and Cotran Pathologic Basis of Disease 9th ed. Fig 2-17, p. 45 Calcium accumulation in the cytosol Extracellular calcium: 1 – 2 mmol Intracellular calcium – 0.0001 mmol (at rest) High overall levels of cytosolic calcium: ▪ Activate a variety of destructive enzymes ▪ Directly activate caspases ▪ Cause calcium release from mitochondria Kumar et. al., Robbins and Cotran Pathologic Basis of Disease 9th ed. Fig 2-19, p. 47 Calcium accumulation in the cytosol We already know calcium is a “special” ion ▪ Only ion that is a ubiquitous second messenger Interacts with a number of intracellular proteins (i.e. calmodulin) that can activate or inactivate intracellular processes ▪ Enzymes that are particularly relevant to necrosis: Proteases Phospholipases Endonucleases (DNA, chromatin fragmentation) Cytosolic calcium is highly regulated ▪ Loss of regulation → nonspecific overall activation of the enzymes specified above ▪ ATP deficiency disrupts appropriate calcium sequestration ▪ Cytosolic calcium accumulation opens the mitochondrial permeability transition pore (MPTP) Mitochondrial damage Mitochondrial membranes can be damaged by free radical attack if levels of cytosolic calcium increase too high in the cell, the MPTP can open ▪ Loss of mitochondrial membrane potential ▪ Releases H+ ▪ Further increase in cytoplasmic calcium Mitochondrial calcium “dumped” into cytosol ▪ Inability to generate ATP and ultimately necrosis ▪ This channel is poorly understood.. But important Increased cytosolic calcium → MPTP opens → leakage of H+ and calcium Kumar et. al., Robbins and Cotran Pathologic Basis of Disease 9th ed. Fig 2-18, p. 46 Membrane damage Phospholipids are both broken down and not synthesized during ischemic injury ▪ Cytoskeletal damage increases physical stresses on the membrane Lipid breakdown can result in: ▪ Leaky membranes ▪ Lipid breakdown products that can have a detergent effect on cellular membranes Detergent-like effects: unesterified free fatty acids acyl carnitine lysophospholipids Kumar et. al., Robbins and Cotran Pathologic Basis of Disease 9th ed. Fig 2-21, p. 49 Membrane damage and the cytoskeleton Cytoskeletal abnormalities ▪ Activation of proteases by increased cytosolic calcium may cause damage to elements of the cytoskeleton Lose the “anchoring” and stabilizing effect of the cytoskeleton on cell membranes ▪ cell swelling → detachment of the cell membrane from the cytoskeleton → membrane susceptible to stretching and rupture A “broken” cytoskeleton compounds this process Membrane damage and lysosomes Injury to lysosomal membranes results in: ▪ Direct enzymatic damage to cellular components ▪ activation of enzymes by lysosomal enzymes Enzymes include RNases, DNases, proteases, phosphatases, glucosidases and cathepsins Unregeulated enzymatic degradation of cell components → loss of DNA, RNA, glycogen, cytoskeletal proteins → death by necrosis ▪ ?? What needs to happen to the intracellular mileiu before lysosomal enzymes can be activated? Free radicals and cellular damage Generated by: ▪ Normal metabolic processes Oxidation reactions during cellular respiration ▪ In ischemic/low oxygen conditions, free radical production increases ▪ Metabolism of drugs or toxins Acetaminophen, alcohol are good examples ▪ Radiation – UV light, x-ray ▪ Fenton reaction – metals receive or donate electrons (copper, iron) ▪ Leukocytes – to kill pathogens in inflammatory reactions Free radicals damage: ▪ Lipids – leading to membrane damage ▪ Proteins – especially at disulfide bonds ▪ DNA – can cross-link and break strands Free radical formation and effects Kumar et. al., Robbins and Cotran Pathologic Basis of Disease 9th ed. Fig 2-20, p. 48 Useful free radical knowledge Superoxide Hydrogen peroxide Hydroxyl Peroxynitrite O 2- H2O2 OH- ONOO- Formed: -Incomplete reduction of O2 during oxidative phosphorylation; By phagocyte oxidase in leukocytes Generated by SOD from O2- and by oxidases in peroxisomes Generated from H2O by hydrolysis, e.g., by radiation; from H2O2 by Fenton reaction; from O2- Generated by O2and NO synthase in many cell types (endothelial cells, leukocytes, neurons, others) Eliminated Conversion to H2O2 and O2 by SOD Conversion to H2O and O2 by catalase (peroxisomes), glutathione peroxidase (cytosol, mitochondria) Conversion to H2O by glutathione peroxidase Conversion to HNO2 Pathologic effects Production of degradative enzymes in leukocytes and other cells; may directly damage lipids, proteins, DNA; acts close to site of production Can be converted to OHand OCl-, which destroy microbes and cells; can act distant from site of production Most reactive Damages lipids, oxygen-derived proteins, DNA free radical; principal ROS responsible for damaging lipids, proteins, and DNA Free radical generation and elimination The Fenton reaction: ▪ H2O2 + Fe+2 →.OH + OH- + Fe+2 ▪ Because most of the intracellular free iron is in the ferric (Fe+3) state, it must be reduced to the ferrous (Fe+2) form to participate in the Fenton reaction ▪ This reduction can be enhanced by O2 sources of iron and O2- may cooperate in oxidative cell damage Iron accumulation in cells can be toxic Mechanisms to remove free radicals include: ▪ Antioxidants: i.e. Vitamin E, A, C ▪ Enzymes Catalase – breaks down H202 Superoxide dismutase – converts 02- to H202 Glutathione peroxidase – decomposes H202 Apoptosis Overview Tightly regulated intracellular program ▪ Requires synthesis and activation of signaling and effector proteins If you block protein synthesis, apoptosis is blocked ▪ Plasma membrane remains intact However, it is altered → better phagocytosis of cell remnants Dead cell remnants rapidly cleared → no inflammation ▪ Inflammation is prominent after necrotic damage to tissue ▪ Inflammation = Infiltration of immune cells (specifically neutrophils) to either: ▪ Kill pathogens ▪ Clean up dead or dying cells Accompanied by typical vascular changes – vasodilation and increased “leakiness” of capillaries Causes of apoptosis - physiologic Programmed destruction of cells during embryogenesis ▪ The embryo forms many structures that are no longer required in the fetus Hormone-dependent involution in adult ▪ Endometrial cell breakdown during the menstrual cycle ▪ Ovarian follicular atresia in menopause ▪ Regression of lactating breast after weaning ▪ Prostatic atrophy after castration Cell deletion in proliferating cell populations ▪ intestinal crypt epithelia in order to maintain a constant number Causes of apoptosis – apoptosis as an adaptive response to pathology Death of host cells that served their purpose ▪ Neutrophils in acute inflammatory response ▪ Lymphocytes at the end of an immune response Cells undergo apoptosis because deprived of necessary survival signals, e.g. growth factors Elimination of potentially harmful self-reactive lymphocytes ▪ Before or after they have completed their maturation Cell death induced by cytotoxic T cells ▪ Defense mechanism against viruses and tumors - serves to eliminate virus-infected and neoplastic cells Same mechanism responsible for cellular rejection of transplants Causes of apoptosis – apoptosis as an adaptive response to pathology Cell death produced by a variety of injurious stimuli ▪ Radiation and cytotoxic anticancer drugs damage DNA If repair mechanisms cannot cope with injury - cell kills itself by apoptosis Elimination better than risking mutations and traslocations → malignant transformations ▪ Injurious stimuli, heat and hypoxia Induce apoptosis if mild but still irreversible Necrotic cell death if large doses of insult Have ER stress and unfolded proteins triggering apoptosis Apoptosis as the “problem”, not the “solution” Pathological apoptosis occurs during: ▪ Accumulation of misfolded proteins – free radical damage or genetic disease can result in accumulation of misfolded proteins As these proteins accumulate in the ER, this is known as ER stress Proteins can misfold because of free radical damage, ATP depletion, of viral infection ▪ Pathologic atrophy/involution of secretory tissues in parenchymal organs after duct obstruction ▪ (pancreas, parotid, kidney all good examples) Microscopic pathology of apoptosis Cell shrinkage ▪ Cytoplasm dense, organelles tightly packed Chromatin condensation ▪ aggregates peripherally, close to the nuclear membrane, into dense masses ▪ Nucleus eventually fragments Formation of cytoplasmic blebs and apoptotic bodies ▪ Bleb = spherical membrane protrusion → a small body with a membrane containing organelle/organelle fragments = apoptotic body Phagocytosis of apoptotic cells or cell bodies by macrophages ▪ Degraded by lysosomes and adjacent healthy cells migrate or proliferate to replace the space left over Microscopic pathology of apoptosis Very difficult to visualize: ▪ not all cells in a tissue at risk undergo apoptosis at once ▪ no inflammation ▪ apoptotic bodies are quite small Kumar et. al., Robbins and Cotran Pathologic Basis of Disease 9th ed. Fig 2-8, p. 41 Kumar et. al., Robbins and Cotran Pathologic Basis of Disease 9th ed. Fig 2-22, p. 54 Biochemistry and signaling of apoptosis Two major concepts: ▪ There are two basic stages to apoptosis: Initiation – the sequence of events involving recognition of apoptotic signals or cellular damage and activation of intracellular “initiator” caspases Execution – “executioner” caspases are activated by the “initiator” caspases and cause the cellular changes of apoptosis ▪ There are two major types of apoptosis: Intrinsic (mitochondrial) pathway Extrinsic (death-receptor) pathway Pick out the steps and the types! Kumar et. al., Robbins and Cotran Pathologic Basis of Disease 9th ed. Fig 2-13, p. 44 Intrinsic (Mitochondrial) Pathway The intrinsic pathway is results from cellular damage or from lack of growth factors ▪ Results from increased permeability of the mitochondrial outer membrane with consequent release of death-inducing (pro-apoptotic) molecules into the cytoplasm ▪ Release of pro-apoptotic proteins is tightly controlled by the BCL2 family of proteins These proteins are composed of varying numbers of BH4 domains It’s a good name → 4 X “BH” proteins Intrinsic Pathway - initiation Bcl-2, Bcl-X, and Mcl-1 are anti-apoptotic – they are at high levels in the presence of growth factors and other signals that indicate a viable cell ▪ They keep the mitochondria from “spilling its guts” prevent mitochondrial pore formation and leakage of cytochrome c and other pro-apoptotic proteins into the cytosol ▪ BH4 proteins - they are located in the mitochondrial membrane, in the cytosol, and in the ER membrane Bax and Bak are pro-apoptotic - can form channels in the mitochondria and allow leakage of their contents ▪ They’re formed from 3 “BH” domains – called BH1-3 ▪ When activated → pores in the outer mitochondrial membrane Bim, Bad, and Bid, Puma, and Noxa are increase when the cell is “stressed” or should undergo apoptosis ▪ Only one “BH” domain – called BH3 proteins (have the 3rd domain) Intrinsic Pathway - initiation What increases Bim, Bid, and Bad, Puma, Noxa (BH3 only)? ▪ ER stress ▪ Lack of growth signals ▪ DNA damage What activates the mitochondrial leak channel (Bax/Bak)? ▪ Lack of BH4 molecules ▪ BH3-only molecules What happens if you open the mitochondrial leak channel? ▪ Cytochrome C leaks into the cytosol ▪ Cytochrome C directly activates a protein known as apoptosis-activating factor (APAF) → activation of caspases The anti-apoptotic and pro-apoptotic BH families counteract each other – the balance between the two determines whether a cell will pursue apoptosis Kumar et. al., Robbins and Cotran Pathologic Basis of Disease 9th ed. Fig 2-14, p. 45 Remember this? Alberts et. Al., Molecular Biology of the Cell, 6th ed. p. 860, fig 15-53 Apoptosis Activating Factor Key executioner caspases – 3, 6 Yes, it does look like a snowflake. A deadly snowflake. Alberts et. Al., Molecular Biology of the Cell, 6th ed. p. 1026, fig 18-7 Extrinsic Pathway - initiation Initiated by activation of plasma membrane death receptors on a variety of cells ▪ Part of the TNF family of receptors ▪ Fas receptor is best known/studied Intracellular death domains of these receptors then activate caspases 8 and 10 Caspases 8 and 10 then activate other caspases that are involved in the execution phase Extrinsic Pathway Fas is a death receptor expressed on many cell types The Fas ligand (FasL) is expressed on T cells that recognize self antigens and some cytotoxic T cells When FasL binds to Fas, they form a binding site called Fasassociated death domain (FADD), which activates Caspases 8 and 10 Caspases 8 and 10 then activate the executioner caspases Kumar et. al., Robbins and Cotran Pathologic Basis of Disease 9th ed. Fig 2-15, p. 46 Intrinsic and Extrinsic Pathways – Execution Phase Both intrinsic and extrinsic pathways converge in the execution phase Caspases 8, 9, and 10 can all activate executioner caspases, such as caspase 3 and 6 Executioner caspases can: ▪ Cause cleavage of DNA (via DNAse enzyme activation) ▪ Destroy the nuclear matrix ▪ Cause cytoskeletal changes ▪ Activated flippases The end–result of the execution phase DNA is cut into discrete lengths (about the size of nucleosomes) by endonucleases activated by caspases Phosphatidylserine “flips” to the outer envelope of the cell membrane (flippase) ▪ Normally present in the inner envelope of the cell membrane – during apoptosis it flips to the other side ▪ This allows macrophages to recognize and phagocytose apoptotic bodies Phagocytosis of apoptotic cells is so efficient that dead cells often disappear within minutes without leaving a trace ▪ Known as efferocytosis Apoptotic Pathways Growth factors increase synthesis of Bcl-2 and Bcl-x ▪ Lack of growth factor signal (i.e. hormone) triggers apoptosis via intrinsic (mitochondrial) pathway DNA damage leads to the synthesis of p53 ▪ P53 stimulates Bax/Bak (pro-apoptotic) Misfolded proteins that accumulate in the ER (ER stress) cause production of caspases and apoptosis ▪ Feature prominently in alpha-1 antitrypsin deficiency, a number of other disorders Protein misfolding and ER stress Chaperone proteins seem to be ATP-dependent and need to be constantly produced As poorly-folded proteins accumulate, cells usually increase their production of chaperone proteins and increase activity of proteasomes As soon as the misfolded protein “burden” becomes too great → caspase activation Necroptosis Morphologically resembles necrosis: ▪ loss of ATP, cell/organelle swelling, generation of free radicals, etc ▪ Does not involve caspase activation However, unlike necrosis it is triggered by genetically programmed signal transduction ▪ Sometimes called ‘programmed necrosis’ Mechanism of activation is similar to extrinsic pathway, i.e. binding of ligand to a receptor ▪ Example – TNF (tumour necrosis factor) binds to its receptor → RIP-kinase activation → MLKL phosphorylation → pokes holes in the plasma membrane Necroptosis – when does it happen? Seems to be linked to physiologic development (calcification) of the growth plate Cell death in steatohepatitis, pancreatitis, ischemia-reperfusion injury Maybe Parkinson’s disease A backup mechanism for cells infected by viruses that inhibit the activation of caspases i.e. cytomegalovirus (CMV) prevents the activation of caspase 8 by FADD…. but can’t prevent necroptotic pathways Long-term Adaptations to Cellular “Stress” Hypertrophy – increase in size of cells → increase in size of the organ ▪ Can be physiologic or pathologic ▪ Causes: increased functional demand, hormonal stimulation ▪ Example – the heart Mechanical stresses across the cardiomyocyte, growth factors, and constant neuroendocrine stimulation result in: ▪ Induction of different proteins/contractile elements ▪ Increased contractile proteins ▪ (hopefully) increased vascular development Cardiac Hypertrophy Robbins & Cotran (9th ed) – pg 58 Long-term Adaptations to Cellular “Stress” Hyperplasia – increase in the number of cells in an organ ▪ Physiologic or pathologic, again i.e. growth, regeneration, adaptation to mechanical stresses, hormones, growth factors ▪ Long-term, non-physiologic hyperplasia is a known risk factor for many different types of malignancies Can be due to increased division of tissue-resident stem cells or mature cells can be stimulated to enter the cell cycle and divide (more next week) Long-term Adaptations to Cellular “Stress” Atrophy – decrease in the number and/or size of cells ▪ Physiologic or pathologic, again Usually pathologic – disuse, loss of growth factors, compression, loss of nerve supply, reduction of blood supply ▪ Autophagy can be a mechanism for the development of atrophy As the name suggests – cells “eat” or digest unused components Three major types: ▪ Chaperone-mediated lysosomal digestion of old proteins + lysosomes that “surround” old cellular components ▪ Highly regulated “macro-autophagy” Autophagy Figure 2-17 - Autophagy Cellular stresses, such as nutrient deprivation, activate an autophagy pathway that proceeds through several phases: ▪ Initiation ▪ Nucleation ▪ Elongation of isolation membrane Leads to creation of a double-membranebound vacuole (autophagosome) ▪ Surrounds cellular component → fuses with a lysosome → digestion → recycling of metabolites Macroautophagy – the autophagosome The isolation membrane (phagophore) is thought to be derived from the ER ▪ Initiating and nucleating complex cause this phagophore to form and surround cellular components Inhibitory genes mTOR and MAPK prevent the activation of the initiating/nucleating complexes ▪ These are often activated by RTK growth factor receptors Phagophore elongates and surrounds the cell materials destined for digestion Autophagosome Autophagosome docks with a lysosome Autophagy is implicated in: ▪ ▪ ▪ ▪ Destruction and recycling of malfunctioning organelles Both cancer cell death and survival Destruction and rebuilding of muscle in response to activity Destruction of intracellular pathogens Pathologic Calcification Abnormal tissue deposition of calcium salts ▪ Can also include smaller amounts of magnesium and iron salts There are two forms: Calcification in dying tissue = dystrophic calcification ▪ No abnormalities in serum calcium or calcium metabolism Calcification in viable tissue = metastatic calcification ▪ Almost always due to hypercalcemia Dystrophic Calcification Localized in areas of necrosis Usually present in atherosclerotic plaques ▪ Also commonly seen in aging or damaged heart valves that have undergone long periods of stress due to hypertension Whatever the site of deposition, the calcium salts appear macroscopically as fine, white granules or clumps, often felt as gritty deposits Final common pathway is the formation of crystalline calcium phosphate Dystrophic Calcification Dystrophic calcification of aortic valve Dystrophic calcification Calcium is concentrated in membrane-bound vesicles in cells by a process that is initiated by membrane damage and has several steps: (1) Calcium ion binds to the phospholipids present in the vesicle membrane (2) Phosphatases associated with the membrane generate phosphate groups, which bind to the calcium (3) The cycle of calcium and phosphate binding is repeated, raising the local concentrations and producing a deposit near the membrane (4) A microcrystal is formed which can then propagate and lead to more calcium deposition Metastatic Calcification Occurs in normal tissues/cells due to diseases that cause increased levels of serum calcium (hypercalcemia) Tends to affect interstitial tissues of the gastric mucosa, kidneys, lungs, pulmonary veins ▪ Although quite different in location, many of these tissues secrete acid at one surface of the cell (or are acidic at that surface for other reasons) ▪ At the opposite surface of the cell, the fluid is more alkaline → calcium precipitates in a more alkaline environment Exacerbated by high calcium concentrations Usually causes limited clinical dysfunction, relatively rare Morphologic Patterns of Necrosis 1.Coagulative - architecture of dead cells preserved 2.Liquefactive - dead cells digested 3.Caseous - “cheese-like” necrosis, usually seen in TB 4.Fat Necrosis Morphologic Patterns of Necrosis Coagulative Necrosis ▪ Preservation of the basic outline of the coagulated cells Eosinophilic “cell ghosts” with no nuclei may persist for weeks ▪ Affected tissue has a firm (cooked) texture ▪ Injury and increasing intracellular acidosis denature structural proteins and enzymes → blocks proteolysis ▪ Necrotic cells eventually removed by fragmentation and phagocytosis of cellular debris = inflammation ▪ Typical of necrosis in most solid organs, with the exception of the brain ▪ Typical of ischemic damage – i.e. myocardial infarct Coagulative Necrosis Morphologic Patterns of Necrosis Liquefactive necrosis ▪ Characteristic of focal bacteria or fungal infections → accumulate inflammatory cells → completely digests dead cells → liquid viscous mass Microbes stimulate the accumulation of leukocytes and the liberation of enzymes from these cells ▪ Typical of necrosis within the brain… although usually not due to infectious processes In acute inflammation → creamy yellow material accumulates because of dead white blood cells = pus ▪ In some cases, coagulative necrosis may progress to a liquefactive picture over time Morphologic Patterns of Necrosis Caseous Necrosis ▪ Distinctive form of coagulative necrosis ▪ Found in foci of tuberculosis (TB) infection ▪ Cheesy white appearance of area of necrosis, found in the centre of the granuloma Under the microscope, the necrotic area is a structure-less collection of debris enclosed within a distinctive inflammatory border The entire structure is known as a granuloma ▪ Tissue architecture completely obliterated, replaced by the granuloma Figure 1-20 A tuberculous lung with a large area of caseous necrosis. The caseous debris is yellow-white and cheesy. Downloaded from: Robbins & Cotran Pathologic Basis of Disease (on 17 March 2009 07:33 PM) © 2007 Elsevier Caseous Granuloma Morphologic Patterns of Necrosis Fat Necrosis ▪ Not a specific pattern of necrosis, but refers to a focal area of fat destruction ▪ Typically occurs as a result of release of activated pancreatic lipases into the pancreas and the peritoneal cavity i.e. acute pancreatitis ▪ Activated pancreatic enzymes escape from acinar cells and ducts to liquefy fat cell membranes and split the TG esters contained within fat cells ▪ Released FA combine with calcium to produce grossly visible chalky white areas Figure 1-21 Foci of fat necrosis with saponification in the mesentery. The areas of white chalky deposits represent calcium soap formation at sites of lipid breakdown. Downloaded from: Robbins & Cotran Pathologic Basis of Disease (on 17 March 2009 07:33 PM) © 2007 Elsevier Morphologic Patterns of Necrosis Note: Most necrotic cells and debris disappear by a combination process of enzymatic digestion and fragmentation, followed by phagocytosis of debris by leukocytes If not destroyed and reabsorbed, tend to attract calcium salts and other minerals → dystrophic calcification Pathology Concepts 1.04 Carcinogenesis Dr. Hurnik BMS 100 Week 9 Today’s Overview Pre-learning: Definitions & tumour characteristics Risk factors In class: Model of carcinogenesis Tumor progression Oncogenes Ras, PI3 K, Myc, Cdks & cyclins Tumour suppressor genes RB, p53, CKIs, APC APC/Beta-catenin review Hallmarks of cancer Warburg effect Limitless replicative potential Carcinogens Assignment - HPV Molecular basis of cancer Carcinogenesis is a multistep process resulting from the accumulation of multiple mutations § Mutations that results in the attributes of malignant cells - excessive growth, local invasion, distant metastasis Pathologic Basis of Disease(Robbins and Cotran) 10th ed. Figure 7.19. Page 282 Types of mutations Initiating mutation – found in all progeny, begins the process towards malignant transformation § Essentially the first driver mutation § Often include loss-of-function mutations in genes that maintain genomic integrity Leading to genomic instability Driver mutation – mutation that increases malignant potential of the cell Passenger mutation – mutation with low malignant effect Classes of mutated genes Driver mutations fall into 4 main categories: § Proto-oncogenes Gain-of-function mutations => oncogenes § Tumour suppressor genes Generally loss-of-function mutations § Genes regulating apoptosis Can be gain- or loss-of function § Genes responsible for DNA repair Generally loss of function Affected cells acquire mutations at an accelerated rate (aka genomic instability) Tumour progression Once established, tumours evolve genetically based on survival/selection of the fittest § Mutations are acquired at random Resulting in tumour cells being genetically heterogenous Pathologic Basis of Disease(Robbins and Cotran) 10th ed. Figure 7.20. Page 283 Tumour progression - continued Once established, tumours evolve genetically based on survival/selection of the fittest § Tumour subclones compete for access to nutrients with fittest subclones dominating tumour mass As a result , tumour become more aggressive over time (tumour progression) § This also explains changes in tumour behaviours following therapy Tumours that recur after therapy are almost always found to be resistant to the initial treatment Mutation class - Oncogenes Promote excessive cell growth, even in absence of normal growth-promoting signals § created by mutations in proto-oncogenes (unmutated cellular counterparts) § Encode oncoproteins that participate in signaling pathways driving cell proliferation Can include: § growth factors or their receptors, signal transducers, transcription factors, or cell cycle components We will consider the role of the following proto-oncogenes in more details § § § § Ras PI3 K Myc Cyclins and cdks Select Oncogenes All FYI except Ras, Myc, Cyclin D and cdk4 Pathologic Basis of Disease(Robbins and Cotran) 10th ed. Table 7-5. Pg. 285 Oncogenes: Ras Ras § Downstream component of receptor tyrosine kinases signaling pathways § Point mutation of RAS family genes is the single most common abnormality of proto-oncogenes in human tumors FYI - Approximately 15% to 20% of all human tumors contain mutated versions of RAS § Eg. 90% of pancreatic adenocarcinomas and cholangiocarcinomas § 50% of colon, endometrial, and thyroid cancers § 30% of lung adenocarcinomas and myeloid leukemias § Important downstream signaler for lots of growth factors EGF, PDGF, and CSF-1 Review: how were growth factors involved in the cell cycle? Review: Ras pathway Molecular Biology of the Cell (Alberts et al) 6th ed. Figure 15-47. Page 855 Oncogenes: PI3K PI3 family § Also very common in certain cancers FYI - Eg. 30% of breast carcinomas have PI3K gain-offunction mutations Promotes cell proliferation How? Inhibits apoptosis Molecular Biology of the Cell (Alberts et al) 6th ed. Figure 15-53. Page 860 Oncogenes: Myc MYC (transcription factor) § Immediate early response gene Induced by Ras/MAPK signaling (among others) § When activated: Increases cell proliferation & growth § How was Myc involved in the cell cycle? Contributes of other hallmarks of cancer § Warburg effect (eg. can upregulate glycolytic enzymes) § increased telomerase activity (contribute to endless replicative activity) § May also allow more terminally differentiated cells to gain characteristics of stem cells § Implicated in cancers of breast, colon, lung Oncogenes: Cdks & Cyclins Which of the two cell cycle checkpoints regulated by cdk-cyclin complexes do you suppose is more important in cancer? Gain-of-function mutations in cyclin D and Cdk4 How would this affect progression through the G1/S checkpoint FYI - Implicated in melanomas, sarcomas, glioblastomas Mutation class - Tumour suppressor genes Products of tumor suppressor genes apply brakes to cell proliferation § Abnormalities lead to failure of growth inhibition § Many, such as RB and p53 recognize genotoxic stress responds by shutting down proliferation Activation of oncogenes aren’t enough for cancer induction, usually requires loss of tumour suppressor genes as well § We will discuss: RB P53 CKIs Select tumour suppressor genes Know the ones circled in red, rest are FYI Pathologic Basis of Disease(Robbi ns and Cotran) 10th ed. Table 7-7. Pg. 292 Select tumour suppressor genes Know the ones circled in red, rest are FYI Pathologic Basis of Disease(Robbi ns and Cotran) 10th ed. Table 7-7. Pg. 292 Tumour suppressor genes: RB RB: § Functions as a key negative regulator of the G1/S checkpoints How? § What form do we normally find RB in a quiescent cell? § What form is RB in to facilitate passing through the G1/S checkpoint § Directly or indirectly inactivated in most human cancers Directly – loss of function involving both RB alleles Indirectly § Gain of function mutation upregulating CDK4 /cyclin D (slide 32) § Loss of function mutation of CKIs (p16 - coming up) Reminder Pathologic Basis of Disease(Robbins and Cotran) 10th ed. Page 294 Reminder Molecular Biology of the Cell (Alberts et al) 6th ed. Figure 17-61. Page 1013 Tumour suppressor genes: p53 TP53 “ Guardian of the Genome” § Codes for p53 What is p53? § Regulates cell cycle progression, DNA repair, cellular senescence, and apoptosis § Most frequently mutated gene in human cancer Loss of function mutation found in more than 50% of cancers § Including lung, colon, and breast – three leading causes of cancer death § Can include mutations in p53 or Mdm2 Tumour suppressor genes: p53 How can mutated p53 contribute to carcinogenesis? § Hint: think about the functions of p53 Pathologic Basis of Disease(Robbins and Cotran) 10th ed. Figure 7.27. Pg. 296 Tumour suppressor gene: p53 p53 functions in the presence of DNA damage: § Arrests the cell cycle until DNA can be repaired How? § Stimulates DNA repair If DNA repair is successful => cell cycle can resume If DNA repair fails => p53 will activate pro-apoptotic pathways § p53 mutations are commonly responsible for genomic instability, driving tumour progression Tumour suppressor genes: p53 Reminder Molecular Biology of the Cell (Alberts et al) 6th ed. Figure 17-62. Page 1015 Tumour suppressor genes: p53 With loss of p53, DNA damage goes unrepaired & driver mutations accumulate in oncogenes & other cancer genes à malignant transformation Pathologic Basis of Disease(Robbins and Cotran) 10th ed. Figure 7.27. Pg. 296 Tumour suppressor genes: CKIs CDKIs are frequently mutated or otherwise silenced in many human malignancies § p16 Inherited mutations implicated in familial forms of melanoma Acquired mutations detected in many cancers § Eg. Bladder cancers, head and neck tumours, ALL, cholangiocarcinoma p16 can also be silenced by hypermethylation rather than mutation § This is an example of a epigenetic change § Occurs in some cervical cancers Tumour suppressor genes: p16 Reminder of p16 function § Inhibits Cdk4Cyclin D complex (G1cdk complex) needed for progression through the cell cycle Pathologic Basis of Disease(Robbins and Cotran) 10th ed. Figure 1.19. Page27 Thinking Question What do you notice about the common oncogenes and tumour suppressor genes we have discusses? § Hint: where do they all affect the cell cycle? Thinking Question What do you notice about the common oncogenes and tumour suppressor genes we have discusses? § Hint: where do they all affect the cell cycle? Loss of normal cell cycle control is a major contributor to malignant transformation § At least 1 of the 4 key regulators of the cell cycle is dysregulated in the significant majority of all human cancers p16, cyclin D, Cdk4, RB Tumour suppressor genes: other Remember β-catenin? § APC Very commonly mutated in colorectal cancers Part of Wnt-B-catenin pathway § E-cadherin Loss of function mutations can contribute to loss of contact-inhibition in tumours and metastasis Pathologic Basis of Disease(Robbins and Cotran) 10th ed. Figure 7.28. Page 298 Hallmarks of cancer All cancers display 8 fundamental changes in cell physiology: § 1. Self-sufficiency in growth signals § 2. Insensitivity to growth-inhibitory signals § 3. Altered cellular metabolism § 4. Evasion of apoptosis § 5. Limitless replicative potential § 6. Sustained angiogenesis § 7. Ability to invade and metastasize § 8. Ability to invade the host immune system Altered cellular metabolism Warburg effect § Cancer cells take up high levels of glucose and demonstrate increased conversion of glucose to lactate Even in the presence of ample oxygen Also called aerobic glycolysis § Why do you suppose a cancer cell is relying on glycolysis alone for ATP production? Hint: embryonic tissues also use on aerobic glycolysis Altered cellular metabolism Warburg effect § Why do you suppose a cancer cell is relying on glycolysis alone for ATP production? Provides rapidly diving tumour cell with metabolic intermediates needed for synthesis of cellular components § Mitochondrial oxidative phosphorylation does not! Limitless replicative potential Normal human cells divide 60-70 times and then become senescent § Senescent = cell permanently exits the cell cycle & never divides again Cancer cells can evade senescence § Likely due to loss of functions mutations in p53 and p16 § Allows cell to pass through G1/S checkpoint Limitless replicative potential - continued Cancer cells have also demonstrated the ability to express telomerase Remember telomerase is only very minimally expressed in most somatic cells § Allows cancer cells to continue replicating indefinetly Causes of mutations Chemical Carcinogens (FYI) Pathologic Basis of Disease(Robbins and Cotran) 10th ed. Table 710. Page 321 Causes of mutations - Radiation Carcinogenesis Radiation is mutagenic and carcinogenic § UV radiation Associated with squamous cell carcinoma, basal cell carcinoma, and melanoma of the skin § Ionizing radiation Medical X-rays Occupational exposure Nuclear plant accidents Causes of mutations - Microbial Carcinogenesis Many RNA and DNA viruses have been proven to be oncogenic § RNA Viruses: Human T-cell Leukemia Virus Type 1 (HTLV-1) § Associated with Leukemia § DNA Viruses HPV – Human Papillomavirus EBV – Epstein Barr virus HBV (& HCV) – Hepatitis B virus Merkel cell Polyomavirus HHV8 – Human herpesvirus 8 Study questions Build a table comparing oncogenes and tumour suppressor genes § What is the basic difference between the two categories § List the common mutated genes in each category Diagram how they fit into the cell cycle (ie how are they contributing to carcinogenesis References Alberts et al. Molecular Biology of the Cell. Garland Science. Betts et al. Anatomy and Physiology (2ed). OpenStax Pathologic Basis of Disease(Robbins and Cotran) 10th ed. Pathology Concepts III Principles of Genetic Disorders BMS 100 Week 9 Genetic Mutations 1. Gene mutations 1. 2. 3. 4. Frameshift Mutation Point mutation Trinucleotide-repeat mutations Mutations within noncoding sequences 2. Complex multigenic disorders 3. Chromosomal disorders Genes and Disease genotype commonly contributes to disease ▪ Estimated that lifetime frequency is 670/1000 ▪ 50% of spontaneous abortions/miscarriages have a chromosomal abnormality ▪ 5% of those under age 25 develop a disease with a significant genetic component ▪ … and these are just the diseases we understand Classification of mutations Point mutations ▪ 3 types of point mutations: all involving changes at one (or very few) nucleotides SUBSTITUTION ▪ THE FAT CAT ATE THE RAT ▪ THE FAT HAT ATE THE RAT INSERTION ▪ THE FAT CAT ATE THE RAT ▪ THE FAT HCA TAT ETH ERA T DELETION ▪ THE FAT CAT ATE THE RAT ▪ THE FAT ATA TET HER AT C is missing Classification: Substitution Substitutions can be: -Transitions -interchanges of purines (A, G) or of pyrimidines (C, T) - involve bases of similar shape (both one ring or both two ring) -Transversions -interchanges of purine for pyrimidine bases - involve exchange of one-ring and two-ring structures Classification of mutations Insertions or deletions of nucleotide(s) ▪ Insertions or deletions of single nucleotides can lead to frameshift mutations – all of the triplets are off by one These are often called “frame-shifting indels” Often results in total loss of function of the protein: ▪ “O” blood type results from a frameshift mutation and loss of function of the red blood cell antigen ▪ Tay-Sachs disease ▪ If a multiple of three nucleotides are inserted or deleted, then the reading frame is preserved These are often called non-frameshifting “indels” Example – most common cystic fibrosis mutation Nucleotide deletions One nucleotide deletion – frameshift Protein is no longer functional Three nucleotide deletion – non-frameshift, but loss of an amino acid Most common mutation in cystic fibrosis Classification of mutations Point mutations ▪ Silent or conservative missense point mutation = little or no change in function ▪ nonconservative missense point mutation = significant change in function Example – sickle cell anemia ▪ If the nucleotide triplet being changed becomes a stop codon, then premature ending of translation → truncated (shortened) protein = non-sense mutation Example – some types of thalassemia Codon Chart Adenine (A) Cystosine (C) Guamine (G) Thymine (T) Point mutations Non-conservative missense sickle-cell anemia Nonsense beta-thalassemia Mutations aren’t all bad Due to a mutation, some people can’t be infected by the HIV virus ▪ HIV uses a chemokine receptor, CCR5, to enter cells; a deletion in the CCR5 gene thus protects from HIV infection Sickle-cell trait – protects against malaria ▪ RBCs that have some sickle-cell hemoglobin are not good hosts for the parasite that causes sickle cell disease – thus the trait (heterozygote patient) is protective ▪ However, the homozygote (all hemoglobin is sickle-cell hemoglobin) is more vulnerable to the disease than rest of the population Genetic disorders Mendelian Disorders ▪ Autosomal mutations Dominant: structural proteins ▪ Marfan Syndrome (structural protein defect) Recessive: enzyme defects ▪ Lysosomal storage diseases ▪ X-Linked disorders Hemophilia Pedigree Drawing Mendelian disorders Due to mutations in single genes that have large effects ▪ Thought that everyone has 5 – 8 non-beneficial gene mutations Most of these have relatively small effects on phenotype ▪ 80 – 85% familial, rest are new mutations this can differ depending on the type of disorder – 80% of those with achondroplasia (defect in elongation of the bone growth plate) are new mutations ▪ Traits can be dominant, recessive, or codominant Autosomal dominant disorders Manifested in heterozygous or homozygous state Usually have at least one parent with the disorder ▪ Exception is if a spontaneous mutation occurs ▪ New mutations more common when father is older Usually manifests in each generation Autosomal dominant disorders Penetrance = how likely the mutated gene is to be expressed ▪ So, if something is autosomal dominant but has a 50% penetrance, a heterozygote may only have a 50% chance of showing the disease phenotype Expressivity = how “much” the disorder-causing gene is expressed ▪ All heterozygotes still show the trait ▪ The “intensity” of the trait differs from person to person, though Unsure of the molecular mechanisms behind this variability for most conditions Autosomal dominant disorders Most mutations lead to a protein that has reduced function, or is produced less (loss of function mutations) ▪ Autosomal dominant disorders tend to involve genes that are part of metabolic pathways or regulation of these pathways ▪ Some involve defects in structural proteins Disorders due to insufficient production of an enzyme tend to be recessive ▪ Why? Gain of function mutations are rare, but can be autosomal dominant Marfan syndrome - basics disorder of connective tissues, manifested principally by changes in the skeleton, eyes, and cardiovascular system Epidemiology: prevalence of 1 in 5000 Etiology: ▪ Disorder due to a defect in gene for fibrillin-1 75 – 85% are familial; the rest are new mutations Autosomal dominant ▪ chromosome 15 ▪ 600 distinct mutations – most are missense Marfan syndrome Pathophysiology ▪ Fibrillin is an important component of elastic connective tissue, provides a “scaffold” for elastic fibre deposition ▪ Loss of fibrillin-1 explains many findings i.e. aneurysm formation, ligamentous laxity, defects in eye structure Others are more difficult to explain Thought that increased skeletal growth is due to increased bioavailability of TGF-beta, which is affected by fibrillin levels (TGF-beta can also impact smooth muscle development) Marfan syndrome Clinical findings: ▪ Tall, with very long extremities and lax ligaments ▪ Dislocation of the lens ▪ Cardiovascular changes: Mitral valve prolapse – malformed and “weak” heart valve Weakness in the muscular layers of the aorta, which can lead to aortic valvular incompetence and development of serious aneurysms ▪ Variable expressivity – some individuals may be lacking certain clinical findings i.e. skeletal findings with no ocular findings Prognosis: Variable, main cause of mortality and morbidity are aneurysms and valvular defects ▪ Surgical repair of aneurysms, heart valves Autosomal Dominant Disorders: Marfan Syndrome Autosomal recessive disorders Largest category of Mendelian disorders Basic rules of Mendelian inheritance apply As well: ▪ The expression of the defect tends to be more uniform than in autosomal dominant disorders. ▪ Complete penetrance is common. ▪ Onset is frequently early in life. ▪ Although new mutations associated with recessive disorders do occur, they are rarely detected clinically, since the individual with a new mutation is an asymptomatic heterozygote ▪ Many of the mutated genes encode enzymes In heterozygotes, equal amounts of normal and defective enzyme are synthesized Usually the natural “margin of safety” ensures that cells with half the usual complement of the enzyme function normally Consequences of Enzyme Defects Accumulation of a substrate ▪ Sometimes the substrate can be toxic in high concentrations Blockade of a metabolic pathway Failure to inactivate another enzyme or substrate ▪ i.e. alpha-1 anti-trypsin deficiency Lysosomal storage diseases A wide variety, but only Gaucher disease will be discussed today Lysosomal storage disorders can be from a range of problems with lysosomal enzymes: ▪ Lack of the enzyme, leading up to a build-up of a substrate within a cell that is toxic ▪ Misfolding of the lysosomal enzyme ▪ Lack of a protein “activator” that binds to the substrate and improves the ability of the enzyme to act on it Pathophysiogy of lysosomal storage diseases In the example shown, a complex substrate is normally degraded by a series of lysosomal enzymes (A, B, and C) into soluble end products deficiency or malfunction of one of the enzymes (e.g., B) → incomplete catabolism → insoluble intermediates that accumulate in the lysosomes → “primary storage” problem ▪ huge, numerous lysosomes interfere with cellular function secondary storage problem = toxic effects from defective autophagy ▪ autophagy = “cellular housecleaning” FYI Gaucher Disease Most common lysosomal storage disease ▪ Between 1 in 20,000 and 1 in 40,000 live births ▪ Autosomal recessive inheritance Defect in the gene for glucocerebrosidase ▪ Enzyme cleaves the glucose residues from ceramide, found in cell membranes glucosylceramide accumulates in lysosomes ▪ Metabolites accumulate mainly within macrophages and other phagocytic cells as they phagocytose dying cells and metabolize the membranes This can lead to the activation or loss of function of the phagocytes Gaucher Disease Type I – involves organs outside the central nervous system – 99% of cases ▪ Findings are mostly within the spleen and bone Enlargement of the spleen and liver Weakened bones → frequent fractures ▪ Often relatively mild course Type II – involves the CNS as well as other organs ▪ Hepatosplenomegaly and rapid neurological deterioration, with death in early childhood ▪ CNS macrophage activation → production of toxic signals by macrophages → neuronal death X-Linked Disorders All sex-linked disorders are X-linked, and the vast majority are recessive ▪ Males with mutations affecting the Y-linked genes are usually infertile, and hence there is no Y-linked inheritance Features: ▪ An affected male does not transmit the disorder to his sons, but all daughters are carriers. Sons of heterozygous women have a 1 in 2 chance of receiving the mutant gene ▪ The heterozygous female usually does not express the full phenotypic change because of the paired normal allele X-linked disorders Gene carried on the X chromosome and usually only manifests in males ▪ A male with a mutant allele on his single X chromosome = hemizygous for the allele X-linked recessive inheritance: ▪ transmitted by healthy heterozygous female carriers to affected males ▪ affected males to their obligate carrier daughters consequent risk to male grandchildren through these daughters affected males can’t transmit to sons X-linked recessive disorders – a few examples X-linked recessive – Hemophilia A Loss of function of a coagulation factor necessary for clotting ▪ Affects over 20,000 men in North America ▪ Different mutations confer different bleeding risk – thousands of mutations have been identified with variable impacts on coagulation Clinical Features ▪ Bruising and prolonged bleeding with minimal trauma ▪ Mucosal bleeding, hematomas in joint spaces (hemarthrosis) Pedigree drawing – how genetic diseases “appear” in families The person being “examined” (usually the one with a genetic condition) is known as the proband ▪ Position of the proband in the family tree is indicated by an arrow ▪ A complete family history is then taken “centered” around the proband Diagram developed is called a pedigree Pedigree drawing Pedigree – autosomal dominant Frequent appearance of the disease throughout generations ▪ may not show typical 50% chance of transmission (remember reduced penetrance) Affects both males and females Pedigree – autosomal recessive The risk of autosomal recessive disorders manifesting increases if there is consanguinity ▪ if a homozygote has offspring with a heterozygote, can also look deceptively frequent Often parents of affected proband not affected Pedigree – X-linked recessive Only males appear affected Trait is never passed from father to son May see “knight’s move” pattern of transmission