General Pathology - II Cell Death Jaroslava Dušková Inst. Pathol.,1st Med. Faculty, Charles Univ. Prague

Death irreversible damage of the morphological & functional integrity of organism cells

Cell Death v apoptosis v necrosis

Apoptosis  induced (from outside) or  genetically programmed cell death (cell execution / suicide) v logical and functional contrary to mitosis v a system for the removal of unnecessary, aged, or damaged cells

Necrosis cell death caused from external insult

Apoptosis Biochemistry –expression of genes de novo –production of apoptosis related proteins v inductors of the cell death p53, MTS1 - Multiple Tumor Suppressor, c-myc, Fas v inhibitors of the cell death bcl-2, –activation of endonucleases –fragmentation of DNA –polymerisation of actin

Triggered by a wide range of stimuli. Cell surface receptors like Fas or tumor necrosis factor receptor 1 (TNFR1). Interplay of proapoptotic ( Bax, Bad, Bid, Bik, and Bim) and antiapoptotic (Bcl-2 and Bcl-XL) proteins Apoptosis -1

Oxidative Damage -1 Endogenous production of reactive oxygen by mitochondria. Increased permeability transition (PT) in the mitochondrial membrane

Oxidative damage -2 u Apoptogenic factors leak into the cytoplasm u cytochrome c and apo - inducing factor (AIF) a cascade of proteolytic activity u DNA v fragmentation v mutations v cell death.

Mitochondria in Apoptosis Oxidative damage - 3 u Bcl-2 and Bcl-X –prevent pore formation –block the release of cytochrome c from the mitochondria – prevent activation of the caspase cascade and apoptosis.

Caspase Cascade -1 u Caspases - a class of cysteine proteases involved in apoptosis. u A proteolytic cascade activates enzymes that subsequently degrade cellular targets.

Caspase Cascade - 2 u The mitochondrial stress pathway - release of cytochrome c from mitochondria u Granzyme B and perforin (proteins released by cytotoxic T cells) induce apoptosis in target cells by forming transmembrane pores

Caspase Cascade - 3 u Caspase-activated DNAse (CAD) may be activated through the cleavage of its associated inhibitor ICAD. CAD is then able to interact with components such as topoisomerase II (Topo II) to condense chromatin and lead to DNA fragmentation.

Granzyme B u forming transmembrane pores u cleavage of effector caspases such as caspase-3 u In addition, caspase-independent mechanisms of granzyme B-mediated apoptosis have been suggested. u Caspase-activated DNAse (CAD) is activated through the cleavage of its associated inhibitor ICAD by caspase-3. u CAD interacts with topoisomerase II (Topo II) to condense chromatin – DNA fragmentation and ultimately apoptosis.

Fas Signaling Pathway u Fas/APO-1/CD95 is a member of the tumor necrosis factor (TNF) receptor superfamily –mediator of apoptotic cell death, –involved in inflammation. u Binding of the Fas ligand (Fas-L) induces trimerization of Fas in the target cell membrane. u Activation of Fas causes the recruitment of Fas- associated protein with death domain (FADD) u activation of caspase-8. u Activated caspase-8 cleaves (activates) nine other pro-caspases u a caspase cascade leads to apoptosis.

TNF Signaling Pathway -1 u TNF receptor (TNFR) transduces growth regulatory signals into the cell. u TNF is mitogenic for normal cells u TNF initiates apoptosis in transformed cells causing DNA fragmentation and cytolysis.

TNF Signaling Pathway - 2 u The TNF-induced cell survival pathway is mediated by the transcription factor NF-kB. u cells in which the NF-kB signaling pathway is blocked are more likely to undergo apoptosis in response to TNF. u The availability of NF-kB may play a critical role in the ability of TNF to act as an apoptosis-inducer and anti-tumor agent.

ATM/p53 Signaling Pathway u The ataxia telangiectasia-mutated gene (ATM) encodes a protein kinase that acts as a tumor suppressor. u ATM activation stimulates DNA repair and blocks cell cycle progression. u ATM-dependent phosphorylation of p53 u p53 can cause growth arrest of the cell at a checkpoint to allow for DNA damage repair or can cause the cell to undergo apoptosis if the damage cannot be repaired. u p53 is mutated in over 50% of all human cancers.

Integrin Signaling in Cell Survival and Death u Integrins are heterodimeric transmembrane receptors composed of a- and b-subunits. u Approximately 20 integrins have been identified u Focal adhesion kinase (FAK) is activated via autophosphorylation when cells interact through integrins. u Depending on the integrin interactions, the cell can either survive or undergo apoptosis.

Caspase Activation Intristic Pathway -1 u Cytochrome c release from the mitochondria of pre-apoptotic cells u Binding to Apaf-1 in the presence of dATP/ATP. u Conformational change in Apaf-1 allowing the molecules of Apaf-1 to associate with each other.

Caspase Activation Intristic Pathway - 2 u A wheel-like structure that contains 7 molecules each of Apaf-1, cytochrome c and ATP. u Pro-Caspase-9 autoactivation u Mature caspase-9 remains bound to the apoptosome u Activation of executioner caspases such as caspase-3 and caspase-7.

Apoptosis Morphology – chromatin condensation – cell shrinkage – budding and forming of apoptotic bodies (emission of pseudopodia) –karyorrhexis (not pathognomonic for apoptosis)

Apoptosis Meaning v physiological process necessary for right organ formatting and life course v pathological process leading to organism damage - e.g. atrophy

Apoptosis Ontogenesis intestinal mucose, genit. tract, immune system - T lymphocytes Tissue & organ structures turnover intestinal mucose, blood, endometrium Physiological involution neonatal adrenal cortex, thymus, breast after lactation period Atrophy preassure, hyperplasia regression, slight ischemia

Apoptosis Detection v TUNEL = Terminal deoxynukleotidyl transferase- mediated dUTP Nick End Labeling v silver methenamin impregnation

Necrosis Biochemistry –no expression of genes de novo –energy dependent membrane systems damaged hypoxia, toxins – changes in concentrations of ions – increased water volume (oncosis) – autolysis

Necrosis Morphology – pyknosis, karyorhexis, karyolysis – denaturation of proteins - eosinophilia – cell swelling – cell budding (cytoplasmic protrusions)

Necrosis Meaning pathological process leading to a temporary organism damage or death

Necrosis Classification according to the tissue macroscopy: – simple – liquefaction – coagulation + special types: caseation, Zenker´s of waxy appearance

Necrosis - further development: – no death of organism – gangrene sicca humida emphysematosa (gas g.) – demarcation, sequestration regeneration repair

Necrosis - Causes: u chemical –chlorinated hydrocarbons, heavy metal compounds, ethyl- alcohol, aphlatoxins,... u physical –mechanical trauma, UV light, ionizing radiation, heat, cold, …. u biologic –bacteria, viruses, fungi...

Atrophy diminution of organ or tissue after full development has been attained ( versus hypoplasia, aplasia) v simple (x hypertrophy) v numerical (x hyperplasia)

Atrophy - causes: v vascular v pressure v inactivity v inanition v neurogenic v ionizing radiation v involution v senile v postinflammatory v endocrine v unknown cause

Atrophy - meaning: v may be reversible v loss of specialised structures & hypofunction v clinically silent or unimportant (involution) v clinically apparent v metaplasia, increase of the supportive tissues - pseudohypertrophy