Indian Journal o f Experi men tal Biology Vol. 42, April 2004 pp. 341-353

Review Article

Nuclear transcription factor NF-kappa B: Role in biology and medicine

Bharat B Aggarwal, Yasunari Takada, Shishir Shishodia, Angelica M G utierrez, Oommen V Oommen, H aruyo Ichikawa, Yuh Baba & Ashok Kumar'

*Cytokine Research Laborato ry , Department of Bioimmunotherapy, The University of Texas M. D. Anderson Cancer Center, Houston, TX 77030, USA. 1Depar tment of Medicine, Bay lor College of Medicine, Hou ston, TX 77030, USA.

The ind ucible transcription factor nuclear factor-kappa B (NF-KB) plays a central role in regulati on of many immune, inflammatory and carcinogenic responses. While normal ac ti vat ion of NF-KB is req uired for cell survival and immunity, ab­errant regul ati on of NF-KB leads to development of many pathological states especially those in volved in acute inflamma­ti on. Recent advances in o ur knowledge of the signaling mechani sms those control the acti vation of NF-KB highli ghts the intr iguing aspect of NF-KB regulation , name ly the ability of many different signal transduction pathways ori gi nating from a wide variety of inducing mechani sms to converge on a single target, the F-KB /IKB complex. In thi s review we summarize our current understanding of the NF-KB signaling pathways, their ro le in various cellular responses and the potential o f us­ing NF-KB as a therapeutic target in modern med icine.

Keywords: NF-KB, T umorigenes is, Inflammation, Immunity, Therapy

IPC Code: Int. Cl7 A61 K 48/00

Beginning with its discovery in 1986 and continuing through the present, transcription factor NF-KB has attracted widespread interest based on its unusual regulation, the variety of stimuli that activates it, the diverse genes and biological responses that it control s, the striking evolution ary conservation of structure and function among famil y members and its apparent in­volvement in a variety of cellul ar responsesl,2. The

NF-KB family contains p50, p52, p65 (Re!A), e-Re! , and Re!B proteins, which form vari ous homo- and heterodimers. Of them, the most common active forms are pSO/RelA or p52/Re!A heterodimer. Di­merization of various NF-KB subunits produces com­plexes )Vith di ffe rent DNA-binding specificiti es and transactivation potential3

.5

. Each member of the NF­KB family has a conserved N-terminal region called Rei-homology domain (RHD), which contains the dimerization, nuclear localizati on, and DNA-binding domains (Fig.l ). The p50 and p52 proteins are formed by the proteolys is of their precursor proteins pl05 and plOO respectively . Among the five members of NF­KB fami ly, proteins p65, RelB and e-Re! contain a C­terminal transactivation domain, which strongly acti­vate the transcripti on of the target genes. Other mem­bers of the family , such as p50 and p52 proteins, lack the transacti vation domain but the homo-dimers of

E-mail: Aggarwa l @mdanderson.o rg

these proteins still bind to the NF-KB consensus si~es in DNA, and hence act as transcriptional repressor by blocking the consensus sites 1• While p50 and p65 proteins are ubiquitously expressed, the expression of other members of NF-KB famil y proteins is mostly restricted to hematopoietic cells and the cell s of i m­mune system. In add iti on the transcription of Re!B , e­Re! and pl05 is regulated by NF-KB3

.

NF -KB activation In most cell types, inactive NF-KB complexes are

seques tered in the cy toplasm via their interaction wi th inhibitory proteins known as lkappaBs (IKB s). In re­sponse to multiple stimuli , including cytokines, viral and bacterial pathogens, and stress-inducing agents, the latent cytoplasmic NF-KB/IKBcx complex is acti-

Abbreviations used:

NF-K B, nuclear factor-kappa B; IKB, IkappaB; IKK, !KB kinase; !L, interleukin; TNF, tumor necrosis factor; NIK, NF-KB- inducing

ki nase; NEMO, NF-KB essential modulator; MEKK, mitogen­act ivated protein kinase kinase kinase; IFN, interfe ron; HI V, hu­man immunodeficiency virus; AIDS, acquired immunodeficiency sy ndro me; !CAM, intra cellular ad hesion molecule; VCAM , vas­cular ce ll adhesion mo lec ules; COX, cyclooxygenase; iNOS. in­ducible nitric ox ide sy nthase; RHO, Rei-homology domain: LTR, long terminal repeat; IP, incontinentia pigme nti ; RA, rheumatoid arthriti s; RANTES, regul ated acti vati on in normal T cells, ex­pressed, and secreted; NSAIDs, non-steroidal anti -infl ammatory drugs .

342 INDIAN J EXP BIOL, APRIL 2004

vated by phosphorylation on conserved serine resi­dues at the N-terminal portion of IKB; this modifica­tion occurs at Ser 32 and Ser 36 in the case of IKBa6

·9

.

Phosphorylation targets IKB <X for ubiquitination by the SCF-ubiquitin ligase complex, which leads to deg-

radation of the inhibitory subunit by the 26S protea­some10'1 1. This process activates NF-KB, which then translocates to the nucleus and binds to its cognate DNA-binding site (5'-GGGRNNYYCC-3') in the promoter or enhancer regions of specific genes (Fig. 2).

551

619

579

969

900

317

356

500

446

-RHO, Rei -homology domain; N, Nuclear Jocolization signal; TAD. Transactivation domain; LZ, leucine-zipper motif; GRR, Glycine-rich region; ANK, Ankyrin repeat domain

• Arowheads indicates proteolytic processing residue. • The size of each human protein is shown on the right (number of amino acids).

Fig. ! - Members of the NF-KB and IKB families of proteins. The arrow points indicate the endoproteolytic cleavage sites of pl00/p52 and p!OS/pSO. The number of am ino acids in each protein is shown on ri ght. [RHD, Rei -homology domain ; N, nuclear locali zation se­quence; TAD, transacti vation domain ; LZ, leucine-zipper motif; GRR, g lyc ine rich region ; ANK, ankyrin repeats].

and their modulat=

Chemokines and their modulat=

Growth facto~ and their modulat=

lmmunorecepto~

Proteins involved in antigen presentation

Cell adhesion molecules

Stress response genes

Cell-surface reoepto~

Regulators of apoptosis

Transcription facto~

Early response genes

Viruses

Enzymes

~\..HI

Inflammatory Cytokines

Growth Facto~

Bacterial & Viral Products

Mrtogens

ROI

Stress

Physical Stress

Parasrte

Environmental Hazards

Hormones

Fig. 2-NF-KB activation pathway. NF-KB heterodimers (pSO!RelA) are sequestered in the cytoplasm by IKB inhibitory proteins (IKBa). Stimulation by divergent agents leads to the act ivation of signaling cascades converging on the IKK complex. Phosphorylation of IKBa by activated IKK is a signal for its ubiquitinat ion and proteasome-dependent degradation. This event unmasks NF-KB, which is then free to translocate to the nucleus, where it binds to KB elements and activates the transcription of a variety of genes involved in various cellu­lar responses.

AGGARWAL et al.: ROLE OF NUCLAR TRANSCRIPTION FACTOR NF-KAPPA B 343

NF-KB activation represents the terminal step in a signal transduction pathway leading from the cell sur­face to nucleus . A seminal event in the activation of NF-KB is the phosphorylation of IKB , which is medi­ated by a multimeric complex; referred to as the IKB kinase (IKK) complex12. The IKK complex consists of two catalytic subunits (IKK.a and lKK.~) 1 3 · 14 and the NF-KB essential modulator alternatively referred to as NEM015 or IKK.y 16. Although IKKy is not itself a kinase per se, it is absolutely essential for NF-KB activation by multiple stimuli7·16. Activation of pre­dominant form of IKK is medi ated via phosphorylation of either IKK.a or IKK~ by the upstream kinases, in­cluding NF-KB-inducing kinase (NIK) and MEKKl of the MAP3K fami li 7-19. The activated IKK complex recruits IKB proteins and phosphorylates them at serine residues.

In the classical pathway , the activation of IKK is considered a major mechan ism of NF-KB activation, however, in certain cases such as in response to short­wave UV light20-22

, pervanadate23-25, H20 226·27

, hy­poxia/reoxygenation28, nerve growth factor29, erythro­poietin30, and HER-2 protein31, the activation of NF­KB does not seem to involve phosphorylation of IKB by IKK or even IKB degradation. A number of NF­KB-regulated target genes involved in immune and inflammatory responses , cellular proliferation, the anti-apoptotic response, and other cellular functions have been identified (Fig. 3). NF-KB has also been shown to regulate the expression of anti-inflammatory genes during the reso luti on of inflammation in vivo32

.

Although, the functionally important NF-KB-binding sites have been located in the promoter/enhancer re­gion of a number of genes, the transcription of indi­vidual genes and the amount of transcribed product after NF-KB activation under specific circumstances depend on many factors , including the composition of NF-KB dimers, the nature of the NF-KB-activating stimulus, and the number of consensus sites in the target gene. Additionally, NF-KB works in coopera­tion with other transcription factors, in particular, activator protein-1 (AP-1)33'34 .

Gene deletion studies of NF -KB signaling proteins The physiological functions of various proteins of

NF-KB signaling pathway have been studied by tar­geted disruption of individual gene (Fig. 4) . These studies have revealed both specific and redundant functions of each member of NF-KB family proteins in the regulation of innate and adaptive immune re-

sponses and in the cell survival. The deletion of RelA (p65) gene in mice causes embryonic lethality due to extensive apoptosis in liver35, which indicates that the function of RelA (p65) cannot be compensated for by other NF-KB family proteins and is indispensable for the survival of the mouse embryo. Despite the essential role of Re!A in prevention of TNF-induced apoptosis in liver, RelA is not involved in the development ofT cells36. However, Re!A-/- T cells showed reduction in the proliferative responses to various stimulators37.

On the other hand, mice lacking p50 or Re!B are . immunodeficient but otherwise develop normall y to adulthood3840. Mice lacking other NF KB proteins,

. including e-Re! and p52, also have defective immune functions41

.42. The knockouts of multiple members of

NF-KB family result in even more severe phenotypes, which suggests that there might be some functional redundancy between the NF-KB family members3.

The gene-targeting experiments have also revealed the importance of other key components of NF-KB signaling pathways in mouse development. Although , both IKKa and IKK.~ are necessary for survival of mouse embryos, their role in embryonic development and survival are quite different4. IKKa has a unique function in skin and skeletal development, as well as in B cell maturation, and its absence cannot be com­pensated for by IKK~43 -45 . In contrast, IKK~ appears to play an indispensable role in inducible NF-KB acti­vation in response to pro-inflammatory and pro­apoptotic stimuli . Lack of IKK~ leads to embryonic lethality and li ver degeneration in knockout mice similar to Re!A knockout mice46

.47. Severe liver de­

generation and early lethality have also been observed in embryos that are deficient in the IKK-y/NEMO subunit of IKK complex48

.49.

Involvement of NF-KB in human diseases The gene knockout studies have clearly established

that NF-KB family proteins are essential for develop­ment of various tissues and to protect the cells from apoptosis. However, the research done in past decade has shown that abnormal activation of NF-KB is in­volved in pathogenesis of a number of human diseases including those related to inflammation, enhanced cel­lular proliferation , viral infection, and genetic diseases (Fig. 5). Although, the complete description of NF-KB­related diseases is beyond the scope of this revi ew, we highlight here the evidences of abnormal activation of NF-KB in some important human diseases.

344

Cytokines BMP-2 G-CSF M-CSF GM-CSF VEGFC EPO IFN-I I IFN-y

LT-n LT-Il TNFn

~L Fas-{jc]and CD401gand

I COS LAG-1 NK-tR NK4 TSP-1 TSP-2 ENA-78 PDGF II chain Proenkephain

IL-1f< IL-10 ll-111 ll-11 IL-2 ll-12 ll-6 ll-13 ll-8 IL-15 IL-9

KC Gro rt MCP-1 MIP-1 ' ' Gro y CINC-1 MIP-111 Gro-1 CXCL6 MIP-2 IP-1 0 CXCL 11 MIP-3u TCA 3 Eolaxin Mob-1 RANTES Siem Cell Factor Angiotcnsinogen

Viruses CMV HIV-1 EBV HSV

IN DIAN J EXP B IOL, APRI L 2004

Acute phase proteins TISSue factor-1 LBP C-rcaclive prolein SAA 1 C4b onding protein SAA 2 Corrpler ncnt factor B PTX 3 Corrfllernent factor C4 lkleferl>;in-2 Urokinase-lype plasrrinogen activator

Early response genes

J"X;2 TlEG B94 221PRG1 Egr-1

Enzymes NGAL y.GCS NOO 1 CRAD1 MMP-9 PTGIS CRAD2 COX-2 PGES CaU1epsin l ADH l -PGDS Collagenase 1 EN02- GSTlP1 -1 Gelilbnase B GAD 6~ iNOS H'-K'-AllPaseo2 PIM-1 POE 7A1 Xanlhinc Oxidase PP 5 RACK 1 5-Upoxygcnasc PKO; Ser1>n 2A 12-Upoxygenasc PLA 2 Lysozyme Hemecxygenase-1 PLC6 1 MAP 4K1 Transgltnan>nase GO 3S MnSOO ABC Transporters MKP- 1 Hyaluronan synlhase TERT lodothyrorinc deiodinase ARFRP 1 Oihydroool dehydrogenase CYP2 Cl 1 Ceran>de glycO>.yl translerase N-acelylg<Jcosamnyltransferase I

ApoJJtosis regulators

Bnt/A t Nr 13 A20 TPAF 1 c-FLIP lEX tl TRAF 2 lAP 1 Bd 2

JCVirus HBV HPV lype 16 SIV Adenovirus SV-40 ~ ------- ._.---~-- ~ Avian Leukosis Vnus Bovine Lcukema VtntS

· Receptors Galt Rece1~or Mdr 1 P AF recep!or 1 Lox -1 Androgen receptor RAGE

lr-q:ioid receptor B 7.1 L-1 re-ceptor antagonist BRL-1 A 1 adenosine reccp!or CCR 5 Glu<.-ocortiooid receptor CCR 7 IL-2 receptor r<-d1arn CD 23 lmmrnoglobuin Cy I CD 40 lrrmunoglob<rin y4 CD 48 MHC dass I (H-2Kb) CD 69 MHC Class I (HLA-97) CD 83 Polymefic lg reccJC.(or CD 95 (Fas) T -cell receptor II chain CD 137 T -cell receptor/CO Jy CD 154 TNF-Receptor. p75'80 Nod 2 Bradikinin B 1-ReceJC.(or EGFR Neuropeplide Y Y1-rece~or IGFBP-1 lrrrrunoglobuin k ighl chain GFBP-2 lrrrrunoglobui n e heavy chain Invariant Chain II NMDA receptor subunit 2A 112 Micruglobuin NMDA receJC.(or subunit NR-1 Arriloride--sensitive sodiurn dlannel

,.

r 'Y Cell adhesion

molecules DC-SIGN MadCAM-1 Cell cycle

I ELAM-1 P.,;clcdin Endogln T enasan-C regulators Fitxonectn VCAM-1 p21 CyclnD I ICAM-1 Gndd 451\.Qycin D3

~ Transcription

factors IRF-1 c-myb IRF-2 c-myc IRF-4 Nurr 1 IRF-7 [LYS Jun B ETH101 l ~o,.Bu. p53 Re!B Slrlt3a e-Re! WT1 NF-,01 E2FJ<, NF-,i32 M"il

Survivin lAP 2 Bel x Caspese-1 1 XIAP F AP-1

Proteins involved in antigen presentation Tapasin LMP 2 Cornplerncnt B TAP 1 ComrJerncnt Rcccpor 2 Complement corll!X>nent 3

Miscellaneous UCP-2 AMH Muon UBE2M GIF GS 3686 Pertorin Mis 1 HMG 14 Vun::ntin ~1 1 K3 Kerabn rnCGM3 ax8 ~~11nylord Ca~cln PCBD glycan Epsi n-Gioon RICK CrtveoLn ·1 rt1 -anti~psin TFPI-2 Clone 68 Feni1rn chain PGKt Clone 156 larrinin 82 Chain Ck>ne 330 Apolipoprotcin C Ill Factor VIII u -1 acid glycofXOiern Galeclin 3 Prostale-speafic antigen Syndecan-4 Wlrn"s Turmr Suppressor Gene Neu~oohl aclatinase associated ipocain

Fig. 3 - NF-KB regulated genes. Genetic and biochemical studies have led to the identificat ion of number of genes wh ich ··are direc tl y

regulated by NF-KB. Many of the NF-KB regulated genes are lis ted here. l t is important to note that activated NF-KB does not lead to

inc reased express ion of al l target genes. The transcriptiona l activity of NF-KB depends o n many factors inc luding the nature of stimulu s,

the compositio 1 of NF-KB comp lex, and the activity of several other accessory transcription factors. [NF-KB , nuclear factor-KB: CINC, Cyro­kine-induced neutrophil chemoattractant ; Gro, Growth regu lated oncogene; !COS, Inducible co-stimulator; IFN, Interfe ron ; IL, lnterleukin ; L T, Lympho­tox in; MCP, Macrophage chemotactic protein; MIP, Macrophage innam matory protein; LAG , Lymphocyte activation gene; RANTES, Regulated upon Activation Normal T lymphocyte Expressed and Secreted; TCA, T-cell activation; TNF, Tumor necrosis factor; TRAIL, TNF-rclatcd-apoptosis-inducing li gand: MHC, Major histocompatibi lity antigen; NOD, Nucleotide-binding oligomerization domain protein; TAP, transporter associated with antigen proc­essing: LMP, Low molecular mass polypeptide: !CAM, Intercellular adhesion molecu le; MadCAM, Mucosal addressin cell adhesion molecule: VCAM. Vascu lar ce ll adhes ion mol ~cule; LBP, Lipopolysaccharide bind ing protein: PTX, Pentraxin ; SAA, Serum amyloid A protein : COX, Cyclooxyge nasc: CYP2C I I, Cytochrome p450 2C II; !NOS, Induc ible nitricoxide-Synthase; MAP4K, mitogen-activated protein kinase ki_nase kinase kinase, MnSOD. Manganese superoxide di smutase; EGFR, Epidermal Growth Factor Receptor; Lox , Receptor for ox idi zed low density lipoprote in ; Mdr, Multiple drug resistance; NMDA, N-me thyi-D-aspartate; PAF, Platelet-act ivating factor ; RAGE, Receptor for Advanced Glycation End products; Bel, B-cell lymphoma; FADD. Fas-associated death domain protein; FLICE, FADD-Iike !L-IB-converti ng enzyme; cFLIP, Cellular FLICE interacting protei n; lAP, Inhibitors of apoptosis; lEX, Radiati on-inducib le immediate-early gene; TRAF, TNF-receptor assoc iated factor; NQO. NAD(P)H quinone oxidoreductase: BMP. Bone morphogen ic protein ; G-CSF, Granulocyte colony st imulating factor; GM -CSF, Granulocyte macrophage colony stimulating fac tor; M-CSF, Macrophage Colony Stimulating Factor: EPO, Erythropoietin: IG FB P, Insuli n-li ke growth factor binding protein ; NK-1 R, Neurokinin-! recep tor; NK4, Natural killer cell transcript 4; PDGF, Platelet-derived growth factor; TSP, Thrombospondin; THBS, Thrombospondin; VEGF, Vascular endothe lial growth factor, Egr, Early growth response; TGF, Transforming growth factor; TIEG, TGF-betal-i nduc ib le early gene; ELYS, Embryonic large molecule derived from yolk sac: IRF, Interferon regu latory factor; IKB , Inhibitor of NF-KB; Mail , Molecule possess ing ankyrin repeats induced by lipopolysaccharide: Nurr, Orphan receptor of the nuclear receptor; STAT, Signal transducer and activator of transcription; WT I, Wilms' tumour gene I ; CMV. Cytomegalovirus: EB V. Epstein-Barr virus; ·HBV, Hepatiti s B virus; HIY, Human immunodeficiency virus; HSV, Herpes simplex virus; HPV, Human Papi lloma vi rus; SlY. Sim­ian immunodeficiency vi rus; SV-40, Simian virus-40; ADH , Alcohol dehydrogenase; ABC transponer, ATP-binding cassette transporter: ARFRP. ADP­ribosylation factor-related protein; CRAD, cis-retinoid/androgen dehydrogenase; ENO, Enolase; GAD67, Glutamic acid decarboxylase 67; GD3S, GD3 synthase; GSTP I- I, Glutathione S-transferase PI-I, HO, Hcmeoxygenase: XO, Xanthine oxidase; LOX, Lipoxygenase; L-PGDS, Lipoca li n-type prosta­g landin D synthase; MKP, MAP kinase phosphatase; MMP, matrix metalloproteinase; PDE7AI, Phosphodiesterase 7Al; PPS, Protein phosphatase 5: PLC, Phospholipase C: PKC, Protein kinase C; PTGIS, Prostagla ndin synthase; PGES, prostaglandin E synt hase; RACK, Receptor for activated C kinase: TERT, Telomerase reve rse transcriptase; AMI-I , Anti-Mu!lerian hormone; Gadd45, Growth arrest and DNA damage; GIF, Growth inhibitory factor: HMGI4, High mobility group 14; Mts l , Multiple tumor suppressor, pi I , Annexi n II li gand ; Pax, Paired box; PCBD, 6-pyruvoyl-tetrah ydrop terin syn­thase; PGK, Phosphoblycerate kinase; rnCGM3, Pregnancy-specific glycoprotein; Rip, receptor-interact ing protein ; RICK, Rip-like interacti ng caspase­like apoptos is-regulatory protein kinase; TFPI , Tissue factor pathway inhibitor: UBE2M, Ubiquitin conjugating enzyme E2M; UCP, Uncoupling protein ]

AGGARWAL eta/.: ROLE OF NUCLAR TRANSCRIPTION FACTOR NF-KAPPA B 345

Cancer -The ability of NF-KB to suppress apop­tosis and to induce express ion of proto-oncogenes such as c-myc and eye! in D I , which direc tly stimulate proliferation, sugges t that NF-KB may' participate in many aspects of oncogenesis2'50. NF-KB also regulates the expression of various molecules such as cell adhe­sion proteins, matrix metalloproteinases, cycloxy­genase-2 (COX-2), iNOS, chemokines, and inflam­matory cytokines, all of which promote tumor cell invasion and angiogenesis51. Indeed , constitutive NF­KB activity has been observed in a number of human cancers, including breast cancer, non-small cell lung carcinoma, thyro id cancer, T- or B-lymphocyte leu­kemia, melanoma, colon cancer, bladder cancer, and several virally-induced tumors, and inhibition of NF­KB abrogates tumor cell pro liferation51 -57. Chromoso­mal alterations of NF-KB family genes provide add i­tional evidence for the role of NF-KB in oncogenesis. It has been shown, for example, that genes encoding

• Embryonic lethality • Survive to adult • TNF-dependent liver apoptosis • Resistance to arthritis

RelA, e-Re!, NF-KBl (p105/p50), and NF-KB2 (pl00/p52) proteins are all located within breakpoint regions of the genome that are involved in oncogenic rearrangements or amplifications55

·58 Although it is

widely accepted that inhibition of NF-KB triggers apoptosis in many tumor cell types59

, there are a few exceptions in which NF-KB activation blocks mali g­nant growth . Inhibition of the NF-KB pathway results in both increased basal frequency of apoptotic cells and the spontaneous development of squamous cell carcinomas60·61. NF-KB and oncogen ic Ras both in­duce cell-cycle arrest in normal human epidermal cells. The cell-cycle anest by oncogenic Ras can be bypassed by inhibition of NF-KB through the overex­pression of IKB cx protein, which results in malignant epidermal tissues resembling squamous cell carci­noma62'63. These fi ndings thus suggest that NF-KB can pl ay a different role in the regulation of cell growth in tissue-context-dependent manner.

• Survive to ad 1 ~

• Abnormal spleen and lymph node architecture

• Defective immune system • Abnormal B cells response • Oefec:Uve B and T cell response

• Die postnatally • Required for development and differentiation of dendritic cells

• Development normally •Impaired T and 8 cell activation • Defect in macrophage functions

• Neonatal lethality • Defects in keratinocyte deifferentiation • Bone and limb develo ment

• Early neonatal lethality • Impaired immune fuctions • Inflammatory dermatitis and granulocytosis

• Embryonic lethal • Liver apoptosis

• Early embryonic lethal in male

• TNF-dependent liver apoptosis

• Sensitivity to TNF • Heterozygous females are model for incontinentia pigmenti

Fi g. 4 - Effect of genetic de leti on o f NF-KB signaling prote ins in mice. Ge nes that encode members of the NF-KB signaling path way have been deleted by homologous recombination in mice. T hese mice models indicate the d istinct roles of the NF-KB signaling protei ns in regulation of innate and adaptive immune responses, lymphocyte functi ons and cell survival.

-346 INDIAN J EXP BIOL, APRIL 2004

AIDS-Although, NF-KB activation during viral infection has been interpreted as a protective response of the host to viral infection, some viruses including HIV have evolved strategies to interfere with NF-KB activation to evade the immune response. The pro­moter/enhancer region of HIV -1 LTR contains two

adjacent NF-KB binding sites that pl ay a central role in inducible HIV gene expression. High levels of viral gene expression and replication result in part from the activation of NF-KB, which in addition to orchestrat­ing the host inflammatory response also activates the HIV -1 long terminal repeat (LTR)64

-66. Indeed, trans­

dominant mutants of lKBa that block NF-KB induc­tion also inhibit de novo HIV-1 infection in T cells by interfering with viral replicati on, suggesting that NF­KB promotes the pathogenesis of HIV - 1 in infected cells67.6s.

Astlzma - A sth!Ila is a chronic inflammation of the bronchial tubes (ai rways) that cause swelling and nar­rowing (constriction) of the airways. The pathogene-

/

/ / Tcell 1 Heart failure f

Atherosclerosis

sis of asthma involves persistent expression of a broad array of genes, such as those encoding pro­inflammatory cytokines, chemokines, adhesion mole­cules, and inflammatory enzymes. Most of these genes contain the consensus NF-KB binding sites within their promoters suggesting that NF-KB plays a major role in the initiation and perpetuation of allergic . fl . 5~ 69 I d d . d NF B . . 111 ammation · . n ee , mcrease -K actiVIty has been observed in the key locations in the airways of asthmatic patients and animal models of asthma8

·70-

74 . Furthermore, agents.such as allergens, ozone, and viral infections, which are assoc iated with exacerba­tion of asthma, stimulate activation of NF-KB. Higher activation of NF-KB has been observed in vitro in as thmatic bronchial epithelial cells on exposure to certain allergens known to cause asthma75 . Treatment of A549 cells (a human type II-like alveolar epithelial cell) with ozone increases the activation of NF-KB and the transcription of the IL-8 chemokine76. About 80% of asthma exacerbations in school-aged children

Fi g. 5-Major phys iological and pathological roles of NF-KB. Different studi es indicate that NF-KB family proteins are essential for normal T and B cell development and their proliferation (indicated in green), but its deregulati on leads to various d iseases (indicated in red) .

AGGARWAL eta/.: ROLE OF NUCLAR TRANSCRJPTION FACTOR NF-KAPPA B 347

and half of all asthma exacerbations . in adults are as­sociated with viral upper respiratory infection, and the majority of viruses isolated are rhinoviruses77

. Rhino­viruses activate NF-KB in various cell types78 and in­duce the expression of ICAM-1 in bronchial epithelial cells79·80. ICAM-1 , besides playing an important role in the recruitment of inflammatory cells, also acts as a receptor for rhinovirus . Respiratory syncytial virus (RSV), which is also involved in the perpetuation of asthma phenotypes, is a potent inducer of NF-KB and expression of IL-8 gene in A549 cells69. The activa­tion of NF-KB also induces the proliferation of airway smooth muscle cells that results in further airway nar­rowing and hyper-responsiveness in asthmatic sub­jects81. Moreover, inhaled glucocorticoids are first­line of treatment of asthma82·83 . Glucocorticoids are potent inhibitors of NF-KB activation in mice and cultured cells84. Glucocorticosteroids have also been shown to inhibit the activation of NF-KB in asthmatic patients85·86. Beside corticosteroids, recent evidence also supports . the beneficial effects of other NF-KB inhibitory molecules in asthmatic animals8·74.

Cardiac diseases-Heart failure is the final con­sequences of many underlying disease states such as hypertension, cardiac hypertrophy, coronary heart disease, arrhythmia, viral myocarditis, and mutation in cytoskeleton protein encoding genes. Strong evi­dence suggests that inflammatory response partici­pates in the development of heart failure87·88 . Aug­mented activation of NF-KB and expression of NF­KB-regulated pro-inflammatory genes such as TNF-a, IL-~, IL-6, IL-8, and iNOS have been reported ih ex­perimental and human heart failure regardless of eti­ology89·90. NF-KB plays an almost exclusive role in ischemialreperfusion90 and in the early phase of myo­cardial infarction91 . In a rat in vivo model of ische­mialreperfusion , NF-KB activity increased biphasi­cally, with peak levels occurring after 15 min and 3 hr92

. Inhibition of J"l'F-KB by introduction of NF-KB decoy cis element in vivo significantly reduced infarct size in ischemia and reperfusion93 . Cardiac hypertro­phy, which involves increase in cardiac protein syn­thesis and cell growth, is a major risk factor for heart failure and death94. NF-KB seems to play an essential role in the induction of cardiac hypertrophy in re­sponse to both biomechanical strain and neurohor­monal stimuli95 . The activation of NF-KB is increased in response to several hypertrophic agonists such as phenylephrine, endothelin-1, angiotension II, and

myotrophin in cultured rat primary neonatal ven­tricular cardiomyocytes, whereas the inhibition of NF­KB reduces cardiac hypertroph/ 6

-99 . These studies

collectively suggest that the NF-KB signaling pathway may play a critical role in induction and/or manifesta­tion of several heart-related ailments.

Incontinentia pigmenti -Until recently, no genetic disease caused by NF-KB dysfunction was known. Ho \vever, emerging reports suggest that mutations in genes of some of the core components of NF-KB sig­naling pathway can cause genetic abnormalities in human 100·101 . Incontinentia pigmenti (IP) is one such diser.se of the skin, hair, teeth and central nervous system. IP is an X-linked, dominantly inherited geno­dermatosis that is antenatally lethal in male 102. Af­fected females with only one copy of the abnormal gene show skin pigmentation abnormality in four characteristic stages that starts with erythematous skin lesion and ends with hypopigmentation and atro­phy102. It has been recently shown that IP in humans is caused by deletion of exons 4-10 of IKK.y/NEMO gene, which prevents the expression of functional IKK.y protein 103 and therefore, the NF-KB response. This leads to death in male fetuses, while female fe­tuses can compensate by selective X-chromosomal inactivation. These findings have been confirmed in male IKKy/NEMO knockout mice, where the NF-KB activation by pro-inflammatory cytokines is com­pletely blocked. Heterozygous females develop skin lesions with hyper-proliferation and increased apopto­sis of keratinocytes48'49 .

Other diseases-Besides the diseases indicated above, the abnormal activation of NF-KB has been linked with several other diseases such as atheroscle­rosis, arthritis, inflammatory bowel disease, muscular dystrophy, bone resorption, multiple sclerosis, Alz­heimer's disease, type I and II diabetes, viral infec­tions and inflammatory bowel diseases such as Crohn's disease and ulcerative colitis. Till recently, it has been suggested that NF-KB might not be involved in the initiation of the pathological state but it plays a major role in the perpetuation of the diseased state. However, we have recently observed that in skeletal muscles of mdx mice (a mouse model of Duchenne muscular dystrophy), the DNA-binding activity of NF-KB and the expression of NF-KB-regulated in­flammatory cytokines such as TNF- a and IL-l~ starts increasing even before the clinical onset of muscular dystrophy9. We have also observed that the activation

34R INDIAN J EXP BIOL, APRIL 2004

of NF-KB in many di seased states such as muscular dystrophy and cardiomyopathy , the activation of NF­

KB is not associated with the degradation of NF-KB

inhibitor protein IKB (our unpublished observations) indicating that different mechanisms might be in­

volved in the acti vation of NF-KB in some human diseases.

Clinical application of inhibition of NF-KB From the observations above, one may think that

inhibitors of NF-KB possess a great therapeutic po­tential in individuals with cancer, HIV-1 infection, and a wide variety of inflam matory diseases. ln addi ­tion to its direct role in tumor development, the acti­

vation of NF-KB by chemotherapeutic drugs and ion­izing radiation provides a strong anti-apoptotic signal that reduces the effic iency of many common cancer therapies 104

• Therefore, such inhibitors could also have a role in the treatment of many cancers in which

aberrant NF-KB activation does not represent one of the underlying causes of the original tumor. A large number of commonly used anti-inflam matory drugs

have been shown capable of inhibiting NF-KB 105, al­

though the efficiency with which this is accompli shed in the clini cal setting is, in most instances, yet to be demonstrated. Relatively well -defined mechanisms exist to deactivate NF-KB after it has been ac tivated in response to di fferent stimuli , thus serving as molecu­

lar 'brakes' to the ongoing NF-KB activation. Both molecular and pharmacological approaches have been used to inhibit the spurious activation of NF-KB in response to inflammatory cytokines and in some dis­ease states.

Molecular strategies - Several molecular level

strategies exist to inhibit NF-KB activation, including transgenic animals, decoy oligonucleotides, and gene transfer strategies. The first evidence that F-KB pathway can be inhibited comes from studies of IKBa mutant, which is not phosphorylated by IKK and is not degraded by proteasome 1

• This IKBa mutant have a domi nant negative phenotype because it sequesters NF-KB in the cytoplasm and therefore, prevent the

induction of specific NF-KB target genes. Adenoviral vectors to deliver this IKBa super-repressor mutant have been effective in RA model systems 106

• Simi­larly, such vecto rs reduce the resistance of tumors to chemotherapy in a mouse model107

• NF-KB has also been shown to be inhibited by intracellular delivery of NF-KB-specific 'decoy' oligodeoxy nucleotide (ODN)

both in in vitro studies and in various animal models of clinical diseases 108

-110

. The concept of .ODN decoy is based on the fundamental principle that transcrip­tion factors are capable of binding specific DNA se­quences in the promoter regions of genes (i.e. , con­sensus binding sites). This specific binding can occur even in absence of surrounding genomic DNA which forms the basis of electrophoretic mobility shift assay. This same property has been applied to study gene expression by intracellular delivery of ' decoy' ODN that can bind specific transcripti on factors in an intact cell and inhibit their respective acti vi t/ 11

.

Pharmacological strategies-A number of phar­

macologic agents are known to inhibit NF-KB at one or multiple activation steps of the signaling pathways (Table 1 ). Our group has contributed signi ficant ly to identi fy ing the pharmacological compounds which are

potent inhibitors of NF-KB and tumor growth. The immunosuppressive and anti-inflammatory actions of g1ucocorticosteroids have been shown to be mediated

at least in part by the induction of IKBa synthesis59.

Non-steroidal anti-inflammatory drugs (NSAIDs) such as aspirin, sodi um salicylate, and leflunomide also inhibit endotoxin- and cytokine-induced nuclear translocati on of NF-KB by preventing IKBa phospho­ryl ation and proteolysis59

. Recently it has been shown that the inhibitory effects of aspirin and sodium sali ­cylate result from the specific inhibition of binding of lKK-~ to ATP 112

• Several pharmaceutical companies are now involved in development of novel specific inhibitors of IKK 113

• Some naturally occurring and sy nthetic inhibitors of ubiquitin-proteasome that can

block NF-KB activation by preventing IKB degrada­tion have also been identified 114

. This includes: (a)-lactacystin, a streptomyces metabolite; (b)-peptide aldehydes, such as carbobenzoxyl -l eucinyl-leuc inyl ­leucin ai-H (MG- 132); and (c)-boronic acid peptides such as PS-341. Among the proteasome inhibitors, PS-341 is gaining increasi ng attention as being su it­able for in vivo admini stration and re latively stable at physiological conditions. In phase I cli nical trials , PS-341 has been shown to produce a significant antitu­mor response in chemo-resis tant multiple myeloma 11 5

.

Concluding remark The NF-KB transcription factor family represents

an important group of regulators of a broad range of genes involved in cellul ar responses to infl ammatory and stress signals. Recently, the knockout mouse studies have revealed the key role for thi s fami ly in

AGGARWAL et al.: ROLE OF NUCLAR TRANSCRIPTION FACTOR NF-KAPPA B 349

Table 1 -A list ofinhibitor~ ofNF-KB* Cytokine & Hormones Anti-oxidants fl·lapachone Stress Dilll)'lhcptanoid 7-(4'-hydroxy-3•-

lntcrlcukin-4' Asl4x01lthin Lovutatin Carbon monoxide methoxyphenyl)-1-pbenylhcpt-4-en-

lnterleukin-10 Butylate<! hydroxy1111isole Luteolin Elcci1"M.atl ~timulation of vagus nerve 3-one 3-ditriv:inc)

lntcrlcukin-11 Ccpharanthinc Manassantins A 1111d B Hypothermia Dimt:thylfurnarate

lnterleukin-13' Camosol Mcaalaminc Metals•• Dioxin'

Growth honnune Carvcdilol Okandrin+ Nitric Oxide Disulfiram

HBEGEF .. Catechol Derivatives Parthenolide Saline (low Na' istonic) E-73 (cycloheximide analog)

hCG .. Dibenzylbutyrolactone lignan• Piceat1rmol' Hypem.<molarity Ecabct sodiwn

Luteirtizin& honnono .. Dietbyldithiocarbamale Quercetin Epoxyquinone A monomer Fibratcs

a-Msu•• Diferox.amine R.,veranor Vitamins F.rythromycin

Somatomammotropin Dihydrolipoic Acid Resinifcratoxin • BTEE"•

Fo~fomycin

VEOF•• Oilazcp + Roco,lamidcs VitaminC

Flunixin me_gluminc

Estrogen Fenofibric acid Sanguinarine+ VitaminD

Gangliosidcs

Glucoeonicoids Dimethyldithiucarbarnat.:s Sauccmcol D and E VitaminE

Gobexote mesilate

PG-1 ~ - deoxy-t.( I 2, I 4)-PGJ(2) •• Dimethylsulfoxide Silibinin .. Nitrosylcobalamin••

Gcldanamycin

Prostaglandin AI Dioulfiram Silymarin+ Glimcpiride

ProsiBglandin E2 Ebselen Tranilut Glucosamine sulfale

EGTA•• Triptolide (PG490) Virus derivatives Herbimycin A

Antiinflammatory llrgolbioncine Uncaria tomento50 Hydroquinone

Ethyl Pyruvate Ursolic acid• Core Protein of Hepatitis C \'irus 4-Hydroxynonenal agents Glutathione Yalruchinone A and 0 EIA Hypochlorite

Acetaminophen Hemalein Epoxyquinol A HIV-1 Vpo protein Hypoothyl sloreh

Aspirin (sodium salicylate) Iron letraki• Plant extracts IKB-Iike proleins lsornollotochromanol K.l protein 1 somBllotochromenc: Flurbiprofen L-cysleinc Apple Kaposi's sarcoma·assoeiatcd herpesvirus Jesterune dimc:r Ibuprofen Lacidipine Aged garlic Pc:rtu.~si!l toxin binding protein Kamebakaurin Leflunamide mcla~lilc•• • Lazamid•

Suliodac o·li(l(liC acid Black raspbeny SspHI and lpaH9.8 .. Lactoferrin

Magnolol Bluebcny YopJ .. LDL (Elctensively oxidized) Oanodenm1lucidum Leptomycin U Cell-signaling Melatonin Ginkgo biloba

N-acctyl-1--cy.tcine Synthetic compounds Mcvinolin, S'·methylthioadcnosinc Inhibitors Ochna macrocalyx bark Monochloramine Atrovastat••

Nacyselyn PC-SPES (8 herb mixture) AS602868 r-.tx781 0609 ..

Nordihydroguaiaritic ocid Phyllanthus omllrUs Decoy oligonucleotides•• Nafamostat mesila1e LY294002 ..

Ortboph011anthroline Qin,kailing DTD•• N-ethyl-maleimidc PDTc•• Shuanghu..,glian E3330•• Quinndrit•• Nicotine Phenolic an1ioxidants•• Stiuging nettle Hydmquinonc ROJJ-11220•• PMc•• Omega 3 fatty acids Tanacetum larvatum Macrolide Mlibiotics Pcrvanadatc • SB203580'• Polysaccharides Une~ria tomentosum MOL 294•• SC236•• Redox faclor I Fungal gliotoxin Pentoxitylline

Petrosaspongiolide M Spbondin Rg(3) (ginseng dcrivalivc) PhenethylifiOthiocyanatc TNP-470 .. Rotenone Polypeptides Phcnylarsim:: oxidt:~

U0126•• Sauchinone and enzymes Others Phenyl-N-tert-butylninone

Tepoxaline Phosphorylation Angiopoietin- I Adenosine Phytic acid IKK inhibitors Ten-butyl hydroquinone Alrial Nalriuretic Peptide 6-aminoquinazolinc derivatives Pnmlukost

AS602868 Phytochemlcals AvrA protein (Solmonella) 6(SH)·pllCIIBnlhridinone + henzaruidc Psychos inc BAY-117082 .. l!·amyloid protein 7·ttmino4·melhylcoumarin Pyrithione BAY-1'17083•• 1 ~-deoxyspergualine

Anethole+ j!-calenin I 5-Deoxyspergualin Raxofelast BMS-34~541

Anetholditbiolthiooe Bovine serum albumin ADP ribosylation inhibitors• • RebliJilipide om .. Baicalcin CaMK.K .. Amentoflavonc Rhein

E3330 .. Betulinic acid• Complcmcnl protein CSa Amrinone Rjbavirin

lF15-019~·· bi.~~ugc:nol Cy1ochalasin n Anandamide Rifnmidcs

MOL294•• D-aminu 1cid peptidt: Anti-thrombin III Rifampic in PSII42 Catalpo1idc APC0576••

Caffeic Aci~ Phenethyl Ester DQ 6~-79•• Rolipram Glucorticoid-induced leucine zipper Artemisinin Rol06-9920 Calagualine

Protease Inhibitors Capsaicin .. prolein AUragaloside IV San~genon C APNE ... Conophylline y-glutamylcy.teine synlbetase Atorva.statin Serotonin derivative •• Boronic Acid Peptide Curcuminf Heat shock protein 72 Aucubin Siah2•• Cyclosporin A Emodin• Hsco•• Azidothymidine SLPJ•• Deoxyspcrgualin Ent.kauranc ditcrpc:noid~ Losanin Oen£01iamine (thiamine derivalive) Statins DCIC'• Epigallocatechin-3-gallate MnSOD••• Bisphenol A Staurosporine DFP .. Epoxyquinol NDPPI (CARD protein) o,o'-bismyristuyi thii!.mine disulfide Sulfasa!azine FK506' 0 Erbstatin• Nf-2 prolcin Cacospongionolide B Surfactant protein A

LLM .. Ergolide NLS cell permeable peptidcs Capsiate Survanta Lacucystine, b-lactone Eugenol p202• .. Caprofin T-614•* MGIOI•• PiogliiAzone (PPARyligand) Chiloaan Taurine + niacine lsoeugenol MG115'• FlavotJiridol' Pituitary adcnylate cyclase-activatin:

Chromcne derivatives Tetnlthiomolybd>te MGI32" Fluorochaloones polypeplide

Clarithmmycin THI ~2" Pefabloc Genistein· Prolcin-bound polysaccharide

Compound 26•• Thalidomide Peptide Aldehydes Glycyrrhizin PTEN

Cycloepoxydon Thiopental PS-341'* Guaianolides Suppressors of cytokine signaling- ( Cyclolinteinone Trin•Jsal TPCK•• Hcmotein Triglyceride·rit.:h lipoproteins Cycloprodigiosin hycrochloride Tyrphostin AG-126 TLCK•• Hypericin Vasoactive intestinal peptide

Dehydroxymethylepoxyquinomicin Wedelolactone Ubiquitin Ligase lnhibilors KT-90'• ZAS3 prolein•• Diamide• Wogonin

*For most references see htlp://pcople.bu.cdu/g ilmorl'lll f-1...1 )/ lah/in,k.\. hlml

**AOP ribosylation inhibitors. nicotinamide and 3-••minobcnz••midc: APC 0576, 5-(((5)-2.2-dimcthylcyc lopropanecarbonyl) amino)-2-(4 -(((5)-2.2-dimethylcyclopropanccarbonyl)amino)­phcoxy)pyri dinc ; APNE, N-accty i-DL-phenylalaninc-b-naphthylcstcr. At rovastata. I-I MG-CoA reducatasc inh ibitor. A 77 1726. Lcnunomide mctaboite; BAY 11 7082, E3((4-mcthylphcnyl)-sulfonyl)·2-propcncnitrile: BAY 11 7083 . E3((4-t-butylphenyl )-sulfonyl)-2-propencni tril c; BTEE N-bcnzoyl L-tyros ine-ethylcstcr; CaMKK. Calc iumlcalmodulin-depcndcnt kinase kinase. Compou nd 26. 2-amino-3-cyano-4-aryl-6-(2-hydroxy-phcnyl)pryridine analog; 0609, phosphat idylcho li nc- phospholip;..~sc C inhibitor, Decoy ol igor.ucleotides. symhctic decoys which ··compete" with transc ript ion factors for bindi ng to the ir consensus sequences; DC lC. 3.4-Ui chloroisocoumarin: OFP, diisopropyl ll uorophosphatcs: DQ 65-79. aa 65-79 of the a hel ix o f the a-chain of the class II HLA molecule OQA 030 11: DTD. (4, 10-dichloropyrido[5.6:4 .5J thieno 13.2-d' -3,2-d] -1 ,2: E3330(2E)-3-]5-(2.3-Dimethoxy-6-methyl- 1,4-benzoquinoyl)]-2-nonyl-2-propenoic ac id; EGTA, Ethylene Glyco l Tetraacct ic Acid; FK506. Tarcolimuc; HBEGEF. Heparin-binding EGF-Iike GF: hCG. human choriogonadOlropine; HSCO. Hepatoma Substratcd-cDNA library clone one: KT-90. syn theti c derivut ive of morphine: LFIS-0195. analog of 15-deoxyspcrgua linc; LLM . N-acetyl-lcucynil -leucynil-methional: LY294002. wortmunnin, PI J-kinasc inhibitor. Metals, examples arc chromium, cadmium. go ld. lead. mercury. zinc. arsenic, and titan ium: MGI OI. also ALLnL. N-ace tyl-lcuc inyl-lcuc inyl-norlcucinal: MG 115, also Z-LlnV, carbobcnzoxyl ~ l cuc iny l -leucynil -norvalinal : MGI 32. al so Z-LLL. carbobcnzoxyl-teucyni t-Jcuoynit-Jeucynal: MOL294. methyl (4 R/S)-4-hydroxy-4(5S.8S)/(5 tl.XR) J-8-mcthyl-1.2-diox -2-pheryt-2.5.5,8-tetrahydro- t H[l ,2,4Jtriazolo[ t ,2.9J-pyridazin-5yl l-2-butynoate: MnSOD, M<mgenese supcrux ide dismutasc: a -MSH, a l pha~Me l anocytc st imu lating hormone: Nitrosylcobal <.m1in, vilam in Bl2 ana log: p202n. IFN-inducible protein: PDTC. Pyrrolned ith iocarbanatc: PG- 15-deox y-6.( 12, 14)-PGI(2).Prostaglandin 15-deoxy-Dclta( 12.14)-PGI(2): Phenolic antioxid;.1nts. example is l lydroquinone; PMC. (2 .2.5.7 .8-pentamcthyl -6-hydroxychromanc); PS34 1. Vclcade, bortezolimb: Quinadril , ACE inhib itor: ROJ I-8220. PKC inhibitor. 58203580. p38 MAPK inhibitor: SC236, COX-2 inhibitOr: Serotonin de rivative, N-(p-coumamyl) serotonin: Siah2, Seven in abstentia homolog2: SLPI. Seercwry leukocyte protease inhibitor: SspHI •md lpaH9.8, Leuc ine-rich effec tor prote ins of Salmonella & Shigella: T-6 14. methoncsulfoanilidc anti- ;;uthri ti s inh ibitor. TH I 52. l·napthylethyl-6.7-dihydrox y-1.2.3.4- tctrahydroisoquinol ine: TLCK, N-a tosy i-L- lysi nc chloromcthyl ke tone: T NP-470. angiogenes is inhibi tor: TPC K. N-a-tosyl- L-phenyla lanine chloromethyl ketone: U01 26. MEK inhi bitor: VEGF, vnseul<.lr endothellial growth factor. YopJ. encoded by Ycrs in ia pseudotuberculosis. ZASJ protein, zinc finge r prote in which binds NF-kB site.

• lnvesti g;lled in author' s laboratory

350 INDIAN J EXP BIOL, APRIL 2004

broad physiological processes, including immune function and metabolism. Further, identification of specific components of the NF-KB signal transduction pathway provides an opportunity to define mecha­nisms at the biochemical level by which specific members of the NF-KB family are activated. Further­more, this may identify specific targets for selective inhibition or promotion of NF-KB functions. Addi­tional studies are required on mechani sms regulating specificity and selectivity of NF-KB function , as well as its role in different diseases , prior to potential clini­cal application .

Acknowledgement This work was supported by Muscular Dystrophy

Association grants (to AK) . Supported partially by the Clayton Foundation for Research (to BBA), Depart­ment of Defence US Army Breast Cancer Research Program grant BC010610 (to BBA), a POl grant (CA91844) from the National Institutes of Health on Lung Cancer Chemoprevention (to BBA) and a PSO Head and Neck SPORE grant from the National In­stitutes of Health (to BBA).

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