Adaptation, Injury and Death
(Part 2)
Tom Davis
8-14-14
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 antineoplastic drugs that exert their effect via
apoptosis
• 4. List 4 types of intracellular accumulations
and describe the associated pathology
Mechanisms of Cell
Death
• Necrosis- groups of cells are
killed by injurious agents
• Apoptosis- individual cells
are induced to commit suicide
Apoptosis
•
•
•
•
ATP-dependent
Cell membrane intact
Organelles intact
No inflammation
(macrophages ingest
apoptotic bodies)
Necrosis
•
•
•
•
ATP not required
Cell membrane rupture
Organelles rupture
Inflammation
(neutrophils)
NECROSIS
NECROSIS
APOPTOSIS
APOPTOSIS
Apoptosis Morphology
• Cell shrinkage
• Chromatic condensation
• Plasma membrane
wrinkles/blebs
• Fragmentation into apoptotic
bodies
• Phagocytosis of apoptotic
cells/bodies
Apoptotic Cells in H & E Sections
• Oval mass of intensely eosinophilic cytoplasm
with dense chromatin fragments; occurs rapidly;
no inflammation
CIVATTE
BODIES
Civatte Bodies
PHYSIOLOGIC APOPTOSIS
• Embryology- fingers and
toes/maleness
• Hormone-dependent- endometrial cells
shed on estrogen withdrawel; breast
duct regression after weaning
• Neutrophils (PMNs) disappear in acute
inflammation
• Cytotoxic T cells eliminate virusinfected cells
Apoptosis of Neutrophils
PATHOLOGIC APOPTOSIS
• Radiation and anticancer drugs damage DNA
and apoptosis follows ( p53 expression)
• Hypoxia- apoptosis (if mild) or necrosis if the
hypoxia is severe or prolonged
• Decreased cell death in lymphomas (Bcl-2
overexpression)
• Misfolded proteins
Apoptosis after Radiation
Pathologic Apoptosis in Viral
Infections
• HPV- E6 protein inactivates p53
• EBV- Bcl-2-like substance produced
• HIV- infected cells make high levels of FasL
which will induce apoptosis in HIVuninfected Tcells
Biochemical Events in Apoptosis
• Caspases (cysteine proteases) cleave the
cytoskeleton and activate DNAses and other
enzymes
• DNA breaks into 50- to 300-kilobase pieces;
further broken into multiples of 200 base pairs by
endonucleases (Ca++ and Mg++)- demonstrated
as a “ladder pattern” on agarose gel; also
proteases.
• Phosphatidylserine is exposed and attracts
macrophages with little “collateral damage”
The intrinsic Pathway
• Major mechanism
• Increased mitochondrial permeability and
release of pro-apoptotic molecules
(cytochrome c)
• Pro: 1. membrane- Bim, Bid, Bad and Bax, Bak
2. cytoplasm- Smac/DIABLO
• Anti: Bcl-2, Bcl-x
Extrinsic Pathway
• Activation of plasma membrane death receptors
• TNFR1 and Fas
Control and Integration Stage
Specific proteins connect the “death
signals” to proteolytic enzymes in
the capase family responsible for
“the execution phase”.
The Execution Stage
Caspases cleave cytoskeletal and
nuclear matrix proteins and result in
DNA cleavage into fragments giving
“DNA Ladder pattern” by agarose
gel electrophoresis.
A.
B.
C.
Normal cells
Apoptosis
Necrosis
Removal of Apoptotic Bodies
Apoptotic cells are coated by
Phosphatidyl serine (which “flips out”)
or C1q leading to early recognition and
removal by macrophages.
Thrombospondin is an adhesive
glycoprotein.
DNA-damage and Apoptosis
•
•
•
•
Radiation or chemotherapy damages DNA
p53 accumulates
Cell cycle arrested at G1 (allows repair)
If repair fails, p53 triggers apoptosis
Tumor Necrosis Factor and Cytotoxic
Lymphocytes in Apoptosis
• Fas (CD95) –FasL induces apoptosis in
lymphocytes that recognize “self”; Fas/FasL
mutations may cause autoimmune disease
• TNF/TNFR1-TRADD-FADD causes caspase
activation and APOPTOSIS; TNF also activates
NF-kB which aids cell SURVIVAL and is
antiapoptotic
• Foreign Ag-CTLs- lymphocytes produce
PERFORMIN which allows entry of GRANZYME
which activates caspases; CTLs kill target cells
Dysregulated Apoptosis
“Too little”
activity diminished in certain cancers
(p53 mutations in solid tumors)
“Too much”
neurodegenerative diseases, ischemic
injury, virus-induced lymphocyte
depletion
Defective Apoptosis
• 50% of human cancers have p53 mutations
• Hormone-dependent tumors (breast, prostate)
• Follicular lymphomas and colon cancers express high
levels of Bcl-2 (translocation of bcl-2 gene)
• HPV- protein E6 binds and inactivates p53
• EBV- proteins that mimic or increase production of
Bbcl-2
• Autoimmune disorders
Increased Apoptosis
•
•
•
•
•
•
Neurodegenerative diseases
Ischemic injury
Death of virus-infected cells (hepatitis)
AIDS (death of uninfected CD4 cells)
FasL+ tumors are MORE aggressive
Microorganisms induce apoptosis
Example of Pneumocystis pneumonia
• Pneumocystis causes pneumonia in AIDS
• Human macrophages are killed before they can
engulf the organisms
• Apoptosis is triggered in macrophages by
polyamines
• Pneumocytis is not phagocytosed
Apoptosis Summary
• Normal part of the cellular machinery
• Pathology results when it is increased or
decreased
• Future Study of Apoptosis: inflammation and
repair (cell signaling); immune system;
neoplasia; infectious diseases
• Future targets for new chemotherapeutic and
antimicrobial agents
Clinical Applications
• Velcade (bortezomid)- blocks proteasomes in
multiple myeloma; proteins accumulate which
are toxic to myeloma cells; also activates p53
and apoptosis of malignant plasma cells
• Genasense (oblimersen)- blocks production of
BCL-2 in lymphomas rendering them more
susceptible to other anticancer drugs
Subcellular response to Injury
• Primary lysosome- hydrolytic enzymes
• Lysosome/vacuole fusion- secondary lysosome
or phagolysosome
• Heterophagy
• Autophagy
• Others- lipids, proteins, filaments, Ca++
Cytoskeletal Abnormalities
•
•
•
•
Microtubules- 25 nm
Actin filaments (thin)- 8 nm
Myosin filaments (thick)- 15 nm
Intermediate- 10 nm
Abnormal Microtubules
• Sperm motility deficiency “YBCS syndrome”
• Immotile cilia syndrome (Kartagener’s
Syndrome)
• Colchicine- disrupt microtubule formation and
inhibit PMN migration; gout therapy
• Vinca alkaloids- antitumor; disrupt the mitotic
spindle
Intermediate Filaments (IM)
• Mallory bodies (alcoholic hyalin)- keratin IM
• Neurofibrillary tangles- neurofilament IM seen
in Alzheimer’s Disease
Mallory Hyaline
Neurofibrillary tangles
Intracellular Accumulations
• 1. Normal substance that cannot be
metabolized- fatty liver (triglyceride)
• 2. Genetic defect in metabolism of a substance
(alpha-1-antitrypsin deficiency and “storage
diseases” like Gaucher’s)
• 3. No normal enzymes to degrade an abnormal
substance (silica-silicosis)
Fatty Change (Steatosis)
• Triglycerides accumulates in parenchymal cells
• Alcoholism, protein malnutrition, anoxia
Oil Red O Stain
Gaucher’s Disease in Spleen
Cerebroside in Gaucher’s Disease
Silica in silicosis (polarized view)
Cholesterol Accumulation
• Atherosclerosis- smooth muscle cells and
macrophages (foam cells)
Foam Cells in Atherosclerosis
Protein Accumulation
• Renal failure- reabsorption of filtered protein in
the proximal tubule accelerates
• Vesicles of protein fuse with lysosomes and
appear as pink hyalin droplets in the tubules
Protein in Renal tubules
Protein Folding Errors (failure of
chaperones)
•
•
•
•
Alpha-1-antitrypsin deficiency
Cystic fibrosis
Familial hypercholesterolemia
Unfolded Protein Response- caspase-12 is
activated with apoptosis induction; Alzheimer’s,
Huntington’s, Parkinson’s
• Amyloidosis- amyloid not eliminated
Hyaline change
• Homogenous, glassy, pink appearance
• Eg. Mallory alcoholic hyalin
Hyaline Change in Hypertension
Pigments
• Carbon (exogenous)
• Lipofuscin- lipid and phospholipid polymers
complexed with protein; wear-and-tear pigmentliver and heart of aging patients
• Melanin- dihydroxyphenylalanine (from Tyr)
• Hemosiderin- iron-ferritin forms hemosiderin
granules; hemosiderosis (in macrophages);
hemochromatosis (in parenchymal cells)
Lipofuscin
Iron/hemosiderin
Prussian blue Stain
Pathologic Calcification
• Dystrophic- necrotic tissue; serum Ca++
normal; atheromas, heart valves; psammoma
body/asbestos body
• Metastatic- hypercalcemia; parathyroid,
skeletal metastases, vitamin D, renal failure;
lungs, arteries
Calcified heart valve
Dystrophic Calcification
Psammoma Body
Asbestos Bodies
Metastatic Calcification in lung
Cellular Aging
• DNA damage
• Replicative senescence
– progressive shortening of telomeres
• Defective protein homeostasis
– impaired chaperone and proteasome functions
• Nutrient sensing system
– caloric restriction increases longevity