Cell Death It is estimated that cells are born and die each day in a human body (10 14 ). Apoptosis: Greek word meaning “ a falling away”, proteolytic cascade leading to DNA fragmentation, membrane blebbing, and phagocytosis of cell. Necrosis: Greek word meaning “to make dead”. Membrane pumps fall, causing cell to swell, burst and releasing digestive enzymes into ECM Apoptosis is essential for normal growth and development, tissue homeostasis, as well as defense against pathogens. Decreased apoptosis can lead to cancer, autoimmunity, and viral infections. Excessive Apoptosis can lead to AIDS, Neurodegenerative disorders, and Stroke.

Ultrastructural Features of Apoptosis

Cell Lineage in the C. elegans embryo

Nature Structural Biology 8, (2001) Conserved Apoptotic Pathway

The Caspase Family

Science Aug 28;281(5381):

The Integrated Apoptotic Pathways

TNFRI Fas Dr3 Dr4 Dr5 TNFR II CD40 CD30 CD27 LT  R 4-1BB OX40 NGFR HVEM GITR RANK PV-T2 PV-A53R TRID OPG DEATH DOMAIN The Tumor Necrosis Factor Receptor Family Dr6

Type I Type II Death Domains TRAF Proteins Fas Dr4 Dr5 TNF-R1 Dr3 Dr6 DcR1 DcR2 Apoptosis Immune & Inflammatory Responses FADD Caspase-8 RIP TRADD TRAF2 Death Effector Domains Caspase 3 Caspase 7Caspase 6 Survival signals FADD { { mitochondria

RelB c-Rel RelA (p65) NF-  B1 p105 (p50) NF-  B2 p100 (p52) I  B-  Bcl-3 I  B-  I   DNA-Binding Dimerization NLS Rel - Ankyrin Family I  B- 

LPS TNF-  IL-1 IL-2 PHA Calcium ionophore Anti-CD28 Anti-CD3 Anti-  TCR TCR-specific antigen monocyte Rat-2 fibroblast fibrosarcoma hepatoma (Hep G2) T cell lines T-cell lines protein synthesis inhibitors phorbol esters Anti IgM HTLV I tax ds RNA Adenovirus CMV Sendai Herpes simplex 1 Human herpes 6 Inducers of NF-  B InducerCell-TypeInducerCell-Type hepatoma (Hep G2) pre-B splenic B fibrosarcoma monocyte macrophage pre-B (70Z/3) splenic B plasmacytoma T cell lines osteosarcom a HeLa fibrosarcom a fibroblasts UV light H 2 2 Okadaic Acid c-AMP Theilera parva pre-B T cell lines HeLa splenic B O

TIM Domains ASK1 JNK MKK4/7 IkB  p65 p105 IKK  IKK  IKK  NIK? p100 TRAF Proteins Immune and Inflammatory Responses p65 p50 p52 RelB fos Jun ? TRAF-C Zinc Fingers TRAF-N NC Ring Finger Death Domains ? ? p38  PI3K ERK1/2 TRADD RIP type 1type 2 GCK/GCKR

Surface Receptors Ig  light chain IL-2R  chain TCR  chain MHC class I (H-2K ) invariant chain Ii  -2 microglobulin MHC class II (E , E  ) IL-2 IL-6 IL-8 GM-CSF G-CSF TNF-  (cachectin) TNF-  (lymphotoxin)  -interferon proenkephalin Acute Phase angiotensin complement factor B complement C4 Serum amyloid A c-myc HIV SV40 cytomegalovirus adenovirus GGGACTTTCC GGGAATCTCC GGGAGATTCC GGGGATTCCC GGGAATTTCC GGGACTTTCC GGGACTTCCC GGGATTTCAC GGGATTTTCC GGAATTTCCT GGGAACTACC GGGGAATCTC several GGGGCTTCCC GGGAAATTCC GGGACGTCCC GGGATTTCCC GGGAATCCCC GGGAAGTCCC GGGACTTTCC GGGAAAACCC GGGACTTTCC GGGAAGTACC GGGACTTTCC GGGGATTTCC GGGACTTTCC NF-  B regulates a broad range of genes. GGGRNNYYCC b Bcl-x GGGACTGCCC Bfl-1 GGGGATTTAC Cytokines Viruses Apoptosis

TNF-  Tumor Death in some cancers TNF-  Actinomycin D Enhanced Death TNF-  NF-  B Deficient Enhanced Death NF-  B-Regulated Genes Inhibit TNF-  -Mediated Cell Death

TNF-  -Mediated Upregulation of Bcl-x and Bfl-1 Requires NF-  B Activity TNF-  (min) A Bcl-x Bfl-1 BAG-1 GAPDH TNF-  (hrs) A

The Bcl-x NF-  B Cis Element is Capable of RelA Mediated Transcriptional Activation luciferase NF-  B Cis Element Bcl-x WT Promoter luciferase Bcl-x Promoter  B Fold Activation WT Promoter  B- Promoter p

Establishing Cell Lines Expressing Bcl-x or Bfl-1 at Physiological Levels Bcl-x Protein Tubulin TNF-  Stimulation A Bcl-x Bfl-1 Transcript GAPDH TNF-  Stimulation A Bfl-1

% of Control A Bfl-1 Bcl-x MTT ASSAY I  B*-Expressing Cells are Sensitized to TNF-  -Mediated Cell Death, which is Inhibited by Expression of Bcl-x or Bfl-1

I  B* Exogenous Bcl-x CD40 Survival Signaling through NF-  B-Mediated Upregulation of Bcl-x and Bfl-1 Fas Caspase Activity Apoptosis CD40 NF-  B Bcl-x Bfl-1 Survival

Chemotherapy Drugs Differentially Upregulate the Expression of Bcl-x and/or Bfl-1 through an NF-  B-dependent Pathway

A Adriamycin VP MM MM CDDP  g/ml A549 Cells Are Sensitized to Chemotherapy-Mediated Apoptosis Upon Inhibition of NF-  B Signaling Percentage of Cell Survival

The Enhancement of Chemotherapy-Induced Cell Death Through Manipulation of Signaling Pathways Chemotherapy Pro-apoptotic signal Anti-apoptotic signal 1. NF-  B-Mediated Upregulation of Bcl-2 Family Members 2. Other pathways (AP-1?) Death Chemotherapy Pro-apoptotic signal Anti-apoptotic signal 1. NF-  B-Mediated Upregulation of Bcl-2 Family Members 2. Other pathways (AP-1?) Enhanced Death

JNK Activation Death Domains FADD TRADD RIP FADD Caspase 8/10 TRAF Proteins Apoptosis NF-  B Activation Type I FLIP IAPs Bcl-x/A1 Bcl-2/Bcl-X cIAP-1/c-IAP-2 FLIP TIM Domains Ubiquitination Caspase 3/6/7 Type II Activation & Differentiation