Presented By: Deepanjan Paul M.Sc. (F), MHG.

 The term “PCD” was used in 1965 to describe developmental cell deaths in insect systems by Lockshin & Williams.  Apoptosis (Apo: Away; Ptosis: Falling off) was coined by John F. Kerr, Andrew H. Wyllie & A. R. Currie in  Cell shrinkage, karyorrhexis, blebbing, apoptotic bodies formation, phagocytosis by macrophages.  Autophagy- Portions of cytoplasm or organelles are sequestered into a double-membrane autophagosome and delivered to the lysosomes and autolysosomes for breakdown and recycling.

Cell Death and Differentiation (2002) 9, APOPTOSIS TIMELINE

 Pleiotropic cytokine.  Role in immune and inflammatory responses.  Induction of apoptosis. TNF-  Apoptosis TNF R1 TNF R2 Mitochondrial dysfunction Extrinsic pathway Intrinsic pathway

 Mediator of TNF-  associated cellular response.  Group of dimeric transcription factor.  NF-  Rel family (p50, p52, p65, Rel-B & c-Rel). NF-  pathway Canonical Noncanonical p50 & p65 p52 & Rel-B

I  B NF-  B IKK Kinases I  B Proteosomal degradation Activated NF-  B Nucleus Binds to kB sequence Target genes

NF-kB c-FLIP,Bcl-2,Bcl-X L cIAP2,A1/Bfl-2. Protection from apoptosis. NF-  B p53,Fas,Fas ligand death receptor 4,death receptor 5. Promotes apoptosis

p53- DEPENDENT  radiation Drugs p53 PUMA activation p53- INDEPENDENT Serum starvation,kinase inhibitors, glucocorticoids, ER stress, ischemia/reperfusion. PUMA activation

 PUMA is essential for damage-induced & p53-dependent apoptosis.  Phosphorylation of CREB-binding protein (CBP) by IKK a suppresses p53- mediated gene expression by switching the binding preference of CBP from p53 to NF-kB.  NF-kB can promote thymocyte and T-cell apoptosis, which is critical for shaping the T-cell repertoire during thymocyte ontogeny.  Glucocorticoid dexamethasone induces PUMA dependent apoptosis in mouse thymocytes.  PUMA can complement the function of Bim in controlling T-cell apoptosis in the termination of immune response. Mechanism & function of p53-independent PUMA induction remain unclear.

PUMA is involved in ischemia/reperfusion-induced intestinal injury, inducing inflammatory response, so, it may be regulated by inflammatory cytokines.

 Cell culture and drug treatment.  Transfection and reporter assays.  Western blotting and antibodies.  RT-PCR and CHIP.  Analysis of apoptosis.  Animal experiments.  TUNEL and Immunostaining.  Bioinformatics and statistical analyses.

TNF-  (20ng/ml) HCT116 colon cancer cells  PUMA mRNA and protein were induced my TNF-  within several hours.  Induction of PUMA mRNA and protein was unaffected in p53-KO or BS-KO.  Induction of PUMA by TNF-  was also independent of FOXO3a.  Bad, Bim and Noxa but not Bid were induced by TNF- .

 Potential binding sites of several TF (NF-  B, ATF,IRF & CREB families) known to mediate TNF-  response were identified.  Only transfection of p65 subunit of NF-  B increased PUMA expression in wild-type (WT), p53-KO, and BS-KO HCT116 cells.

p 53-independent induction of PUMA by p 65

 BAY I  B  superrepressor mutant p65 si-RNA (inhibitor of IKK Kinases) (Non-degradable) Suppression of PUMA induction & P 65 nuclear PUMA induction translocation. abrogated.  PUMA induction by TNF-  in mouse embryonic fibroblast (MEF) cells was also found to be p65 dependent, but p53 independent.

p 65 is necessary for PUMA induction by TNF- 

 A luciferase reporter construct containing the putative  B site in the PUMA promoter was constructed.  TNF-  treatment or co-transfection with p65 markedly activated the PUMA reporter.  BAY , I  B  superrepressor mutant & introducing mutations into the kB site abolished the responsiveness of the reporter.  CHIP showed that recruitment of p65 to the PUMA promoter region containing the  B site was enhanced following TNF-  treatment for 6 hrs.

p 65 directly binds to the PUMA promoter to activate PUMA transcription in response to TNF- 

 Knockdown of Bcl-X L (but not cIAP1 or Mcl-1) by siRNA, led to a significant increase in TNF-  -induced apoptosis but apoptosis induction was much reduced in PUMA-KO HCT116 cells.  Caspase 3,8,9 activation, Bid cleavage & cytochrome-c release were also suppressed in PUMA-KO cells.  Dominant-negative (DN) FADD mutant slightly lowered TNF-  -induced apoptosis, but did not affect PUMA-dependent apoptosis following Bcl-X L knockdown. So, NF-  B-mediated PUMA induction is a novel mechanism mediating TNF-  -induced apoptosis.

PUMA-dependent apoptosis induced by TNF-  in colon cancer cells

 TNF-  was injected intravenously at several doses (2, 4, and 10 mg) in WT and PUMA-KO mice.  PUMA, Bcl-X L & Bim were induced by TNF-  in the small intestine.  TNF-  -induced apoptosis was blocked to a large extent (60–90%) in the crypts and villus epithelium in PUMA-KO mice compared to the WT.  Caspase-3 activation was blocked in PUMA-KO mice.

PUMA is necessary for TNF-  -induced apoptosis in the intestinal epithelium

 PUMA (but not other BH3-only proteins) was increased in the liver of WT mice following TNF-  treatment for 8 hr.  TNF-  -induced apoptosis was blocked by 50–70% in the PUMA-KO mice.  Caspase 3 activation induced by TNF-  at different doses was abrogated in the hepatocytes of PUMA-KO mice. So, PUMA mediates TNF-  -induced hepatocyte apoptosis.

PUMA mediates TNF-  -induced hepatocyte apoptosis

 TUNEL staining revealed more than two fold reduction in apoptosis in PUMA- KO thymocytes compared to WT ones.  Primary thymocytes from WT showed spontaneous apoptosis in culture but TNF-  treatment had little effect on PUMA-KO primary thymocytes. So, TNF-  -induced apoptosis in thymocytes is PUMA dependent.

TNF-  -induced and PUMA-dependent apoptosis in thymocytes

 1 st case of direct regulation of a BH-3 only Bcl-2 family by NF-  B in the TNF-  response.  A completely p53 independent PUMA activation, inspite of the well-established cross talk between p53 & NF-  B in regulating gene expression, is quite unexpected.  Phosphorylation of CBP & hence its preferential binding to NF-  B might be directing p65, instead of p53, to the PUMA promoter in response to TNF-   TNF-  -induced apoptosis often rely on using transcription or translation inhibitors, which nonspecifically inhibit gene expression and often complicate data interpretation.  p53 & p65 can cooperatively induce apoptosis in some circumstances, resulting from coordinated induction of PUMA by p53 & p65.  PUMA functions as a novel link between the extrinsic & intrinsic apoptotic pathways & hence necessary for TNF-  -induced apoptosis.

 In vivo analysis of PUMA induction by TNF-  needs to be further examined using more physiological systems including genetic models.  PUMA was required for TNF-  -induced apoptosis in villus epithelial cells but not in villus-inside cells.  Inhibition of NF-  B has been explored as an attractive approach for anticancer therapies.  Again, NF-  B inhibition can compromise PUMA induction by inflammatory cytokines, which may be involved in tumor suppression and be beneficial for anticancer therapies.

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