Adaptation, Injury and Death Adaptation, Injury and Death (Part 2)(Part 2)

Tom DavisTom Davis

8-14-148-14-14

Lecture 3 Learning Objectives

• 1. List characteristics of apoptosis and necrosis

• 2. Diagram the intrinsic and extrinsic pathways of apoptosis

• 3. Diagram the mechanism of some anti-neoplastic drugs that exert their effect via apoptosis

• 4. List 4 types of intracellular accumulations and describe the associated pathology

MechanismsMechanisms of Cell of Cell DeathDeath

• NecrosisNecrosis- groups of cells are - groups of cells are killed by injurious agentskilled by injurious agents

• ApoptosisApoptosis- individual cells - individual cells are induced to commit suicideare induced to commit suicide

Apoptosis NecrosisApoptosis Necrosis

• ATPATP-dependent-dependent• Cell membrane intactCell membrane intact• Organelles intactOrganelles intact• No inflammation No inflammation

(macrophages ingest (macrophages ingest apoptotic bodies)apoptotic bodies)

• ATP not requiredATP not required• Cell membrane ruptureCell membrane rupture• Organelles ruptureOrganelles rupture• Inflammation Inflammation

(neutrophils) (neutrophils)

NECROSIS

NECROSIS

APOPTOSIS

APOPTOSIS

Apoptosis MorphologyApoptosis Morphology• Cell shrinkageCell shrinkage• Chromatic condensationChromatic condensation• Plasma membrane wrinkles/blebsPlasma membrane wrinkles/blebs• Fragmentation into apoptotic bodiesFragmentation into apoptotic bodies• Phagocytosis of apoptotic Phagocytosis of apoptotic

cells/bodiescells/bodies

Apoptotic Cells in H & E SectionsApoptotic Cells in H & E Sections

• Oval mass of intensely eosinophilic cytoplasm Oval mass of intensely eosinophilic cytoplasm with dense chromatin fragments; occurs rapidly; with dense chromatin fragments; occurs rapidly; no inflammationno inflammation

Civatte Bodies

CIVATTEBODIES

PHYSIOLOGIC APOPTOSISPHYSIOLOGIC APOPTOSIS• EmbryologyEmbryology- fingers and toes/maleness- fingers and toes/maleness• HormoneHormone-dependent- endometrial cells -dependent- endometrial cells

shed on estrogen withdrawel; breast duct shed on estrogen withdrawel; breast duct regression after weaningregression after weaning

• NeutrophilsNeutrophils (PMNs) disappear in acute (PMNs) disappear in acute inflammationinflammation

• Cytotoxic T cellsCytotoxic T cells eliminate virus-infected eliminate virus-infected cellscells

Apoptosis of Neutrophils

PATHOLOGIC APOPTOSISPATHOLOGIC APOPTOSIS

• Radiation and anticancer drugs damage DNA Radiation and anticancer drugs damage DNA and apoptosis follows ( and apoptosis follows ( p53 p53 expression)expression)

• Hypoxia- apoptosis (if mild) or necrosis if the Hypoxia- apoptosis (if mild) or necrosis if the hypoxia is severe or prolongedhypoxia is severe or prolonged

• Decreased cell death in lymphomas (Decreased cell death in lymphomas (Bcl-2Bcl-2 overexpression)overexpression)

• Misfolded proteinsMisfolded proteins

Apoptosis after Radiation

Pathologic Apoptosis in Viral Pathologic Apoptosis in Viral InfectionsInfections

• HPVHPV- E6 protein inactivates p53- E6 protein inactivates p53

• EBVEBV- Bcl-2-like substance produced- Bcl-2-like substance produced

• HIVHIV- infected cells make high levels of FasL - infected cells make high levels of FasL which will induce apoptosis in HIV- which will induce apoptosis in HIV-uninfected Tcellsuninfected Tcells

Biochemical Events in ApoptosisBiochemical Events in Apoptosis

• CaspasesCaspases (cysteine proteases) cleave the (cysteine proteases) cleave the cytoskeleton and activate DNAses and other cytoskeleton and activate DNAses and other enzymesenzymes

• DNA breaks into 50- to 300-kilobase pieces; further DNA breaks into 50- to 300-kilobase pieces; further broken into multiples of 200 base pairs by broken into multiples of 200 base pairs by endonucleasesendonucleases (Ca++ and Mg++)- demonstrated as (Ca++ and Mg++)- demonstrated as a “ladder pattern” on agarose gel; also a “ladder pattern” on agarose gel; also proteasesproteases..

• PhosphatidylserinePhosphatidylserine is exposed and attracts is exposed and attracts macrophages with little “collateral damage”macrophages with little “collateral damage”

The intrinsic PathwayThe intrinsic Pathway

• Major mechanismMajor mechanism• Increased mitochondrial permeability and Increased mitochondrial permeability and

release of pro-apoptotic molecules release of pro-apoptotic molecules (cytochrome c)(cytochrome c)

• ProPro: 1. membrane- Bim, Bid, Bad and Bax, Bak: 1. membrane- Bim, Bid, Bad and Bax, Bak 2. cytoplasm- Smac/DIABLO2. cytoplasm- Smac/DIABLO• AntiAnti: Bcl-2, Bcl-x: Bcl-2, Bcl-x

Extrinsic PathwayExtrinsic Pathway

• Activation of plasma membrane death receptorsActivation of plasma membrane death receptors

• TNFR1 and FasTNFR1 and Fas

Control and Integration StageControl and Integration Stage

Specific proteins connect the “death Specific proteins connect the “death signals” to proteolytic enzymes in signals” to proteolytic enzymes in the the capasecapase family responsible for family responsible for “the execution phase”.“the execution phase”.

The Execution StageThe Execution Stage

CaspasesCaspases cleave cytoskeletal and cleave cytoskeletal and nuclear matrix proteins and result in nuclear matrix proteins and result in DNA cleavage into fragments giving DNA cleavage into fragments giving “DNA Ladder pattern” by agarose “DNA Ladder pattern” by agarose gel electrophoresis.gel electrophoresis.

A. Normal cellsB. ApoptosisC. Necrosis

Removal of Apoptotic BodiesRemoval of Apoptotic Bodies

Apoptotic cells are coated by Apoptotic cells are coated by Phosphatidyl serine (Phosphatidyl serine (which “flipswhich “flips out”) out”) oror C1q C1q leading to early recognition and leading to early recognition and removal by macrophages. removal by macrophages. Thrombospondin Thrombospondin isis an adhesive an adhesive glycoprotein.glycoprotein.

DNA-damage and ApoptosisDNA-damage and Apoptosis

• Radiation or chemotherapy damages DNARadiation or chemotherapy damages DNA

• p53 accumulatesp53 accumulates

• Cell cycle arrested at G1 (allows repair)Cell cycle arrested at G1 (allows repair)

• If repair fails, p53 triggers apoptosisIf repair fails, p53 triggers apoptosis

Tumor Necrosis Factor and Cytotoxic Tumor Necrosis Factor and Cytotoxic Lymphocytes in ApoptosisLymphocytes in Apoptosis

• FasFas (CD95) –FasL induces apoptosis in (CD95) –FasL induces apoptosis in lymphocytes that recognize “self”; Fas/FasL lymphocytes that recognize “self”; Fas/FasL mutations may cause autoimmune diseasemutations may cause autoimmune disease

• TNFTNF/TNFR1-TRADD-FADD causes caspase /TNFR1-TRADD-FADD causes caspase activation and APOPTOSIS; TNF also activates activation and APOPTOSIS; TNF also activates NF-kB which aids cell SURVIVAL and is NF-kB which aids cell SURVIVAL and is antiapoptoticantiapoptotic

• Foreign Ag-CTLs- lymphocytes produce Foreign Ag-CTLs- lymphocytes produce PERFORMINPERFORMIN which allows entry of which allows entry of GRANZYMEGRANZYME which activates caspases; CTLs kill target cellswhich activates caspases; CTLs kill target cells

Dysregulated ApoptosisDysregulated Apoptosis

““Too little”Too little”

activity diminished in certain cancers activity diminished in certain cancers (p53 mutations in solid tumors)(p53 mutations in solid tumors)

““Too much”Too much”

neurodegenerative diseases, ischemic neurodegenerative diseases, ischemic injury, virus-induced lymphocyte injury, virus-induced lymphocyte depletiondepletion

Defective ApoptosisDefective Apoptosis

• 50% of human cancers have p53 mutations50% of human cancers have p53 mutations• Hormone-dependent tumors (breast, prostate)Hormone-dependent tumors (breast, prostate)• Follicular lymphomas and colon cancers express high Follicular lymphomas and colon cancers express high

levels of Bcl-2 (translocation of bcl-2 gene)levels of Bcl-2 (translocation of bcl-2 gene)• HPV- protein E6 binds and inactivates p53HPV- protein E6 binds and inactivates p53• EBV- proteins that mimic or increase production of EBV- proteins that mimic or increase production of

Bbcl-2Bbcl-2• Autoimmune disordersAutoimmune disorders

Increased ApoptosisIncreased Apoptosis

• Neurodegenerative diseasesNeurodegenerative diseases

• Ischemic injuryIschemic injury

• Death of virus-infected cells (hepatitis)Death of virus-infected cells (hepatitis)

• AIDS (death of uninfected CD4 cells)AIDS (death of uninfected CD4 cells)

• FasL+ tumors are MORE aggressiveFasL+ tumors are MORE aggressive

• Microorganisms induce apoptosisMicroorganisms induce apoptosis

Example of PneumocystisExample of Pneumocystis pneumonia pneumonia

• PneumocystisPneumocystis causes pneumonia in AIDS causes pneumonia in AIDS

• Human macrophages are killed before they can Human macrophages are killed before they can engulf the organismsengulf the organisms

• Apoptosis is triggered in macrophages by Apoptosis is triggered in macrophages by polyaminespolyamines

• PneumocytisPneumocytis is not phagocytosed is not phagocytosed

Apoptosis SummaryApoptosis Summary

• Normal part of the cellular machineryNormal part of the cellular machinery• Pathology results when it is increased or Pathology results when it is increased or

decreaseddecreased• Future Study of Apoptosis: inflammation and Future Study of Apoptosis: inflammation and

repair (cell signaling); immune system; repair (cell signaling); immune system; neoplasia; infectious diseasesneoplasia; infectious diseases

• Future targets for new chemotherapeutic and Future targets for new chemotherapeutic and antimicrobial agentsantimicrobial agents

Clinical ApplicationsClinical Applications

• Velcade (bortezomid)- blocks proteasomes in Velcade (bortezomid)- blocks proteasomes in multiple myeloma; proteins accumulate which multiple myeloma; proteins accumulate which are toxic to myeloma cells; also activates p53 are toxic to myeloma cells; also activates p53 and apoptosis of malignant plasma cellsand apoptosis of malignant plasma cells

• Genasense (oblimersen)- blocks production of Genasense (oblimersen)- blocks production of BCL-2 in lymphomas rendering them more BCL-2 in lymphomas rendering them more susceptible to other anticancer drugssusceptible to other anticancer drugs

Subcellular response to InjurySubcellular response to Injury

• Primary lysosome- hydrolytic enzymesPrimary lysosome- hydrolytic enzymes

• Lysosome/vacuole fusion- secondary lysosome Lysosome/vacuole fusion- secondary lysosome or phagolysosomeor phagolysosome

• HeterophagyHeterophagy

• AutophagyAutophagy

• Others- lipids, proteins, filaments, Ca++Others- lipids, proteins, filaments, Ca++

Cytoskeletal AbnormalitiesCytoskeletal Abnormalities

• Microtubules- 25 nmMicrotubules- 25 nm

• Actin filaments (thin)- 8 nmActin filaments (thin)- 8 nm

• Myosin filaments (thick)- 15 nmMyosin filaments (thick)- 15 nm

• Intermediate- 10 nmIntermediate- 10 nm

Abnormal MicrotubulesAbnormal Microtubules

• Sperm motility deficiency “YBCS syndrome”Sperm motility deficiency “YBCS syndrome”

• Immotile cilia syndrome (Kartagener’s Immotile cilia syndrome (Kartagener’s Syndrome)Syndrome)

• Colchicine- disrupt microtubule formation and Colchicine- disrupt microtubule formation and inhibit PMN migration; gout therapyinhibit PMN migration; gout therapy

• Vinca alkaloids- antitumor; disrupt the mitotic Vinca alkaloids- antitumor; disrupt the mitotic spindlespindle

Intermediate Filaments (IM)Intermediate Filaments (IM)

• Mallory bodies Mallory bodies (alcoholic hyalin)- keratin IM(alcoholic hyalin)- keratin IM

• Neurofibrillary tangles- Neurofibrillary tangles- neurofilament IM seen neurofilament IM seen in Alzheimer’s Diseasein Alzheimer’s Disease

Mallory Hyaline

Neurofibrillary tangles

Intracellular AccumulationsIntracellular Accumulations

• 1. Normal substance that cannot be 1. Normal substance that cannot be metabolized- fatty liver (triglyceride)metabolized- fatty liver (triglyceride)

• 2. Genetic defect in metabolism of a substance 2. Genetic defect in metabolism of a substance (alpha-1-antitrypsin deficiency and “storage (alpha-1-antitrypsin deficiency and “storage diseases” like Gaucher’s)diseases” like Gaucher’s)

• 3. No normal enzymes to degrade an abnormal 3. No normal enzymes to degrade an abnormal substance (silica-silicosis)substance (silica-silicosis)

Fatty Change (Steatosis)Fatty Change (Steatosis)

• Triglycerides accumulates in parenchymal cellsTriglycerides accumulates in parenchymal cells

• Alcoholism, protein malnutrition, anoxiaAlcoholism, protein malnutrition, anoxia

Oil Red O Stain

Cerebroside in Gaucher’s Disease

Gaucher’s Disease in Spleen

Silica in silicosis (polarized view)

Cholesterol AccumulationCholesterol Accumulation

• Atherosclerosis- smooth muscle cells and Atherosclerosis- smooth muscle cells and macrophages (foam cells)macrophages (foam cells)

Foam Cells in Atherosclerosis

Protein AccumulationProtein Accumulation

• Renal failure- reabsorption of filtered protein in Renal failure- reabsorption of filtered protein in the proximal tubule acceleratesthe proximal tubule accelerates

• Vesicles of protein fuse with lysosomes and Vesicles of protein fuse with lysosomes and appear as pink hyalin droplets in the tubulesappear as pink hyalin droplets in the tubules

Protein in Renal tubules

Protein Folding Errors (Protein Folding Errors (failure offailure of chaperoneschaperones))

• Alpha-1-antitrypsin deficiencyAlpha-1-antitrypsin deficiency

• Cystic fibrosisCystic fibrosis

• Familial hypercholesterolemiaFamilial hypercholesterolemia

• Unfolded Protein Response- caspase-12 is Unfolded Protein Response- caspase-12 is activated with apoptosis induction; Alzheimer’s, activated with apoptosis induction; Alzheimer’s, Huntington’s, Parkinson’sHuntington’s, Parkinson’s

• Amyloidosis- amyloid not eliminatedAmyloidosis- amyloid not eliminated

Hyaline changeHyaline change

• Homogenous, glassy, pink appearanceHomogenous, glassy, pink appearance

• Eg. Mallory alcoholic hyalinEg. Mallory alcoholic hyalin

Hyaline Change in Hypertension

PigmentsPigments

• Carbon (exogenous)Carbon (exogenous)

• Lipofuscin- lipid and phospholipid polymers Lipofuscin- lipid and phospholipid polymers complexed with protein; wear-and-tear pigment- complexed with protein; wear-and-tear pigment- liver and heart of aging patientsliver and heart of aging patients

• Melanin- dihydroxyphenylalanine (from Tyr)Melanin- dihydroxyphenylalanine (from Tyr)

• Hemosiderin- iron-ferritin forms hemosiderin Hemosiderin- iron-ferritin forms hemosiderin granules; hemosiderosis (in macrophages); granules; hemosiderosis (in macrophages); hemochromatosis (in parenchymal cells)hemochromatosis (in parenchymal cells)

Lipofuscin

Iron/hemosiderin Prussian blue Stain

Pathologic CalcificationPathologic Calcification

• Dystrophic- necrotic tissue; Dystrophic- necrotic tissue; serum Ca++serum Ca++ normalnormal; atheromas, heart valves; psammoma ; atheromas, heart valves; psammoma body/asbestos bodybody/asbestos body

• Metastatic- Metastatic- hypercalcemiahypercalcemia; parathyroid, ; parathyroid, skeletal metastases, vitamin D, renal failure; skeletal metastases, vitamin D, renal failure; lungs, arterieslungs, arteries

Calcified heart valve

Dystrophic Calcification

Psammoma Body

Asbestos Bodies

Metastatic Calcification in lung

Cellular AgingCellular Aging

• DNA damageDNA damage

• Replicative senescence Replicative senescence – progressive shortening of telomeresprogressive shortening of telomeres

• Defective protein homeostasis Defective protein homeostasis – impaired chaperone and proteasome functionsimpaired chaperone and proteasome functions

• Nutrient sensing system Nutrient sensing system – caloric restriction increases longevitycaloric restriction increases longevity