Synopsis for project:- Abdul Rafay Javed roll no.23

“The rel-a,c-rel, rel-b subfamilies of NFκB are mainly involved in stimuli-induced apoptosis of cell,which are major targets for modulating life and death in cancerous cell”

Introduction:-

NFκB proteins are the key mediators of signaling include transcription of many kinds of genes involving in different cell responses namely Bacteria and Fungi Bacterial or Fungal Products Viruses Viral Products Eukaryotic parasite (Inflammatory) Cytokines Physiological (Stress) Conditions Physical Stress Oxidative Stress Environmental Hazards Therapeutically used drugs Modified Proteins Overexpressed Proteins (ER Overload sometimes) Receptor Ligands Apoptotic Mediators Mitogens, Growth Factors and Hormones Physiological Mediators Chemical Agents.and the genes that regulated are Cytokines/Chemokines and their Modulators, Immunoreceptors Proteins Involved in Antigen Presentation, Cell Adhesion Molecules, Acute Phase Proteins .Stress Response Genes. Cell Surface Receptors Regulators of Apoptosis Growth Factors, Ligands and their Modulators Early Response Genes Transcription Factors and Regulators Viruses Enzymes. So it is too obvious that it has a central role in cell regulatory mechanism, and it all depends upon the regulation of the activation NFκB. Under normal circumstances the NFκB is tightly bound to its inhibitors, the iκB proteins and these inhibitor proteins are further regulated by iκB kinase comples(IKK complex). So in this way cell have systematic mechanism to control the NFκB. The problem arise when due to immune disorders or tumerogenesis the NFκB is dysregulated and it constitutively transcribe oncogenes e.g bcl-2 family oncogenes.This dysregulation leads to the uncontrolled cell proliferation and continous cell survival which shouln’t have been under normal circumstances, so in this way it aids to tumerogenesis by making them escape from immune mediated apoptosis and cytotoxicity. Although there is a lot of data which suggest the role of NFκB as prosurvival transcription factor,but there prevails a solid rationale that upon stimulation of particular stimuli NFκB also transcribe proapoptotic genes.[1-3]

The NFκB Family contains five proteins, RelA(p65),RelB,NFκB1(p50/105),NFκB2(p52/100), c-Rel.These proteins form homo or hetero dimer due to presence of conserved RHD(Rel Homology Regions).These conserved regions comprise of 300 aminoacids. DNA binding takes place at the terminal region of RHD while Carboxy terminal of RHD helps in dimerization and IκBs binding. Normally, NFκB are attached with IκBs and remain non-functional in the cytosol of unstimulated cell. Upon stimulation, the IκBs are degraded by phosphorylation. This process is carried out by specific kinases known as IKKs. In this way translocation of NFκB into nucleus through cytosol to nucleus is made possible. The canonical(p65/p50) NFκB is the most abundant type in all cell types while RelB only dimerize with either p50 or p52, and it also require a leucine zipper for proper activation. P52 and p50 are processed from p100 and p105 by proteosomal activity.In p52 and p50 RHD is also present followed by GRR(glycine rich region) and then come ankyrin repeats.The ankyrin repeats actually gives both p52 and p50, inhibitory effects, as these regions are also found in IκBs.So hom/hetero dimmers of these proteins also inhibit rel related NFκB from DNA binding activity. But when these proteins bind with atypical IκBs they may aid to DNA binding ability of rel/NFκB or help in transactivation of different genes.The inhibition and localization of NFκB in cytosol takes place when NFκB dimers are

bound to any of IκBα,IκBβ,IKBE ,P100 or P105.So we can say tat NFκB’s physiological role is constantly mediated by these inhibitory proteins.The two most common NFκB subsets are canonical and non-cannonical both have different but inter-related mechanism of inhibition. In canonical mechanism the IκBs(IκBα, IκBβ,IKBε) are phosphorylated by the IKKB of IKK(phosphorylation of serine residues 32,36).After phosphorylation IKB proteins are modified by ubiquitin ligases by binding with K-48 linked ubiquitin.These ubiqutin ligases are of SCF family,this family recognize DSpGXXSp motifs of IKB proteins.Now after ubiquitination these IκBs are directly regulated by cytosolic roteosomes. The resulted nuclear localization of NFκB signals resynthesis of IκBs and it again inhibits the transcription mediated byNFκB. The protein of NFκB have NLS(nuclear localization sequence). In Case of canonical NFκB the NLS of p50 is not masked and alongwith NES(nuclear export sequence) of IκBα, helps NFκB to continuously shuttle between nucleus and cytosol.Degradation of this IκBs actually disturb equilibrium and cause nuclear localization of NFκB.The region which give p100 and p105,inhibitory properties, are the ankyrin repeats motifs through which these proteins inhibits NFκB to bind with DNA. However upon activation of IKKs,these p100, p105 are processed upto GRR and only RHD remain. The atypical IκBs i.e IKB£ and Bcl-3 removes the inhibitory p50 or p52 homodimers and help in transactivation of NFκB, however sometimes it remain stabilized with p52 or p50 homodimers and inhibit transactivation by NFκB(rel subunits). The difference in canonical and non-cannonical pathway is that RelA:p52;IKKα:IKKβ:NEMO:IκBs complex form while in non-cannonical only NEMO:IKKα:RelB:p50 forms mediated by NIK(NFκB inducible kinase).(NEMOis NFκB essential modulator or IKKϬ). Termination of NFκB in addition of IκBs, also have a proteosomal activity which directly degrades bound NFκB to DNA[4, 5].

There has always been a controversy about the role of NFκB whether it is pro-apoptotic or anti-apoptotic.Different results has been obtained so far in this regard but how NFκB is mediating, why it tends to be pro survival in one cell line while remaining pro-apoptotic in other is still elusive. NFκB heterodimer or homodimer expression is studied several times in order to study its relation with expressions of oncogenes and tumor repressor.Till now we do know NFκB pro-apoptotic role depends on the nature of apoptotic signal being induced[6]There are many researchers regarding the pro-cell survival role of NFκB or its inhibition enhancing apoptosis.In hodgkin’s disease the constitutive RelA/p50 activation leads to proliferation of (H/RS) hodgkin’s cell and prevents them from apoptosis under stress conditions[7].Similarly NFκB plays a key role in proliferation of multiple myeloma in B cells[8] and strategy made is dual inhibition of canonical and non-cannonical NFκB to potentiate antitumor activities in multiple myeloma cells[9].Similarly these cannonicl and non-cannonical promotes breast cancer tumor initiating cells[10].Apoptosis by TRAIL requires inhibition of NFκB in tumor hepatoma cells[11].TNF induced accumulation of ROS for mediation of MAP kinase and NCD is inhibited by NFκB[12].Migration and invasion of glioblastoma cellsare regulated by Smac-mimetic which is further mediated by non-cannonical NFκB, Smac(second mitochondrian derived activator of caspases) it is an antagonist to inhibitor to IAPsa9inhibition of apoptotic proteins[13].NFκB contributes to apoptosis resistance where Fas and TRAIL mediated apoptosis is being resisted,that happens in ductal pancreatic adrenocarcenoma cells[14].Similarly high gene expression of non-cannonical NFκB are detected in pancreatic ductal adenocarcenoma cells in comparison to non-cannonical NFκB[15].So by all above mentioned examples it is clear that canonical,non-cannonical or even rel proteins could be involved in mediating proliferation and survivalof cells.antagonism of different rel/NFκB,in

which one favors while other inhibit apoptosis is also observed e.g differential role of apoptosis while c-rel and RelA in TNF mediated apoptosis,RelA overexpression leads to inhibition of apoptosis while c-rel overexpression(or knocking out RelA) induces TNF-induced apoptosis[16],sometimes it may also happens that rel proteins protect cell from apoptosis,from one kind of stimuli but not from other e.g c-rel protects Bcell from Ag, corticosteroids and ionizing radiation but not from Fas-mediated Apoptosis[17, 18].NFκB modulates expression of many proapoptotic genes like BAX,BAFF,C-myc e.g rel-related factors mediate C-myc gene transcription in antiserum or receptor mediated apoptosis of murine B cells[19].All above mentioned examples show the pro-survival and proliferation nature of NFκB and its subunits either by mediating survival related genes or by suppressing Poptotic genes.But there are many reported evidences that NFκB, upon specific apoptotic signals of stimuli aid apoptotic gene by transactivating them or suppressing pro-survival genes like high c-rel expression were found associated with PCD in developing avian embryo and bone marrow cell[20] NFκB may not be the good target in the cancer therapy because it may help in apoptosis during tumorigenesis and metastasis[1-3, 21]Drug induced activation of NFκB if disturbed or inhibited e.g RNAi or IκBα Sr then it could disturb drug efficacy of anticancer drugs[22] like doxorubicin,mitoxontone in glioblastoma cells ,similarly in glioblastoma cells the over expression of IκBα Sr reduce or inhibits TRAIL and FAs mediated Apoptosis[23]A novel retinoid related molecule 3Cl-AHCP activates NFκB with subsequent apoptosis through activation via IKKA,RelA is essential for Apoptosis[24]Similarly disturbed or inhibited NFκB abrogated p53 mediated Poptosis involving rel A[25]Uptil now we are able to know that role of NFκB is strikingly dual and it mediates both life and death of cell, but still we don’t know that how it decides to be apoptotic or pro-survival[6, 26]A recent study also shows that PARP mediated,caspase indepentant cell death upon stimulation of Hydrogen peroxide production also activates NFκB, previously senescence of porcine coronary arterial endothelial cells was also observed which was being mediated by Activation of NFκB[27] Study Objective:-Our objective is to study the Rel components of NFKB for their pro-apoptotic abilities and verifying their positive or negative role regarding apoptosis in two different cell lines,we will also study the possible antagonistic behaviour of rel proteins for each other during cell signaling this can be achieved by making knock-out mutants and check their respective levels by RT-PCR,We will use oxidative stress and Fasl as our apoptotic stimuli to induce apoptosis, If rel components are playing their role then proapoptotic genes mRNA level would be higher than pro-survival genes, otherwise,vice versa.

Experimental design:-Materials:-We have selected two cell lines namely MEF (Mouse embryonic fibroblast) and Primary Renal Cortical Epithelial Cells. Both these cell lines are obtained from ATCC.For MEF we will use base medium ,ATCC-formulated Dulbecco's Modified Eagle's Medium, Catalog No. 30-2002. To make the complete growth medium, fetal bovine serum would be added to a final concentration of 15%,and subculturing would be done as method devised by ATCC.FasL is obtained from Mega FasL®.

For RCEC(Renal cortical epithelial cell)

Methods:-

RNAsilencingWe will mutate both cell lines, these mutations would be for RelA,RelB,and C-rel through RNA silencing. Induction of Stimuli.We will separately induce both mutated MEF& RCEC cell lines, one by H2O2 through GO(Glucose Oxidase) treatment and other by FasL treatment.In this way we could verify involvement of NFKB in H2O2 and Fas mediated apoptosis.

Fetal Bovine

Serum (FBS) 2.5 mL 0.5%

Triiodothyronine 0.5 mL 10 nM

rh EGF 1.0 mL 10 ng/mL

Hydrocortisone

Hemisuccinate 0.5 mL 100 ng/mL

rh Insulin 0.5 mL 5 mg/ml

Epinephrine 0.5 mL 1.0 mM

Transferrin 0.5 mL 5 mg/ml

L-Alanyl-L-

Glutamine 6.0 mL 2.4 mM

QuantitativePCRWe could check further quantification of NFκB mediated genes both apoptotic and anti apoptotic.

For MEF and RCECSetA&B, one for MEF and other for RCEC.

Apoptotic Stimuli:-H2O2

We will introduce oxidative stress in one set of sample containing mutated and normal cell lines through Glucose oxidase or treatment with H2O2 to check dependence of H2O2 mediated apoptosis on NFKB and production of ROS will be measured by method used by [28]24 hrs after treatment with GO(Glucose oxidase) we will do Propidium uptake assay[29] to detect apoptosis as used by

Expected Results:-Our Expected result would be that Rel A or C-rel is mediating fas:FasL induced apoptosis.Hydrogenperoxide is being mediated by any of the RelA or c-Rel

.

(1) both normal cell lines(Control)

(2) relA-/- knock out

(3)RelB-/- knockout

(4) c-Rel-/- knockout

(5) RelB-/- knock out

(6) NFKB-/-

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20. Abbadie, C., et al., High levels of< i> c-rel</i> expression are associated with programmed cell death in the developing avian embryo and in bone marrow cells in vitro. Cell, 1993. 75(5): p. 899-912.

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