1. Cell Death It is estimated that 10 10 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.

2. Ultrastructural Features of Apoptosis

3. Cell Lineage in the C. elegans embryo

4. Nature Structural Biology 8, 394 - 401 (2001) Conserved Apoptotic Pathway

5. The Caspase Family

10. Science. 1998 Aug 28;281(5381):1309-12

13. The Integrated Apoptotic Pathways

14. 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

15. 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

16. 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- 

17. 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

18. 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 ? 12345 TRAF-C Zinc Fingers TRAF-N NC Ring Finger Death Domains ? ? p38  PI3K ERK1/2 TRADD RIP type 1type 2 GCK/GCKR

19. 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

20. 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

21. TNF-  -Mediated Upregulation of Bcl-x and Bfl-1 Requires NF-  B Activity 0 15 30 0 15 30 TNF-  (min) A549 42-7-8 Bcl-x Bfl-1 BAG-1 GAPDH 0 2 4 0 2 4 TNF-  (hrs) A549 42-7-8

22. 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- 0 5 10 15 20 25 30 Fold Activation WT Promoter  B- Promoter p65 - + - +

23. Establishing 42-7-8 Cell Lines Expressing Bcl-x or Bfl-1 at Physiological Levels Bcl-x Protein Tubulin -+ -+ - + TNF-  Stimulation A549 42-7-8 Bcl-x Bfl-1 Transcript GAPDH -+ - + - TNF-  Stimulation A549 42-7-8 Bfl-1

24. 100 80 60 40 20 0 % of Control A549 42-7-8 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

25. 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

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

27. A549 42-7-8 Adriamycin VP-16 010 2030 40 MM MM 0 100 200 300 400 CDDP 0 2.55 7.5 10  g/ml 120 100 80 60 40 20 0 100 80 60 40 20 0 100 80 60 40 20 0 A549 Cells Are Sensitized to Chemotherapy-Mediated Apoptosis Upon Inhibition of NF-  B Signaling Percentage of Cell Survival

28. 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

29. 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