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TRANSCRIPTION FACTOR NF-B:A NUCLEAR FACTOR IN B CELLS
AND BEYOND.
Fano, Centro di Biotecnologie04 Aprile 2008
© Linda Palma, 2008
Università degli Studi di Urbino
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● NF-B IDENTITY DOCUMENT
● NF-B REGULATION
● NF-B TARGET GENES
● NF-B FUNCTIONS
● NF-B AND CANCER
● NF-B AS THERAPEUTIC TARGET
© Linda Palma, 2008
Università degli Studi di Urbino
SUMMARY
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© Linda Palma, 2008
Università degli Studi di Urbino
GENE REGULATORY ELEMENTS
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© Linda Palma, 2008
Università degli Studi di Urbino
In early 80’s, research studies on the mechanisms regulating gene transcription led these authors to the discovery of an apparently B cell-specific protein, able to bind to the kappa
light polypeptide gene enhancer. They called it NUCLEAR FACTOR OF LIGHT CHAIN GENE ENHANCER IN B CELLS
DISCOVERING NF-B
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© Linda Palma, 2008
Università degli Studi di Urbino
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© Linda Palma, 2008
Università degli Studi di Urbino
NF-B/Rel AND IB FAMILIES OF PROTEINS
The REL homology domain (RHD) mediates their dimerization and binding to DNA. The RHD also contains, at its carboxyl terminus, a nuclear localization signal (NLS) and is recognized by the IB proteins, thebinding of which to the RHD interferes with the function of the NLS.
All IBs contain 6–7 ankyrin repeats,which mediate their binding to RHDs.
Three family members, p65, cRel and RelB,contain transcriptional activation domains (TAD) at their C-terminus, and are therefore able to directly activate transcription.
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© Linda Palma, 2008
Università degli Studi di Urbino
THE NF-B ACTIVATION PATHWAY (1)
Several stimuli are able to activate NF-B through
the engagement of various cell-surface receptors:
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© Linda Palma, 2008
Università degli Studi di Urbino
THE NF-B ACTIVATION PATHWAY (2)
Several adaptor proteins are recruited to the cytosolic
domains of cell receptors upon their engagement:
IRAKsRIP
RIP MALT
Bcl10
TAB2/3
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© Linda Palma, 2008
Università degli Studi di Urbino
THE NF-B ACTIVATION PATHWAY (3)
IRAKsRIP
RIP MALT
Bcl10
TAB2/3
Transduction of extracellular stimuli converge on either
of two intracellular signaling cascades, referred to as
CLASSICAL NF-B ACTIVATION PATHWAY and ALTERNATIVE NF-B ACTIVATION PATHWAY
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© Linda Palma, 2008
Università degli Studi di Urbino
THE NF-B ACTIVATION PATHWAY (4)
The key regulator of both pathways is the Inhibitor of Kappa B Kinase (IKK),
a high molecular weight complex (900 kDa) composed of three kinds of subunits:
IKK, catalytic
IKK, catalytic
IKK(NEMO), regulatory
Homo- and heterodimers containing the IKK subunit are responsible of activation
of the classical pathway, IKK homodimers are involved in the alternative pathway.
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© Linda Palma, 2008
Università degli Studi di Urbino
THE NF-B ACTIVATION PATHWAY (5)
ALTERNATIVE ACTIVATION PATHWAYCLASSICAL ACTIVATION PATHWAY
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© Linda Palma, 2008
Università degli Studi di Urbino
NF-B TARGET GENES (1)
BAFF *CXCL 11 IL-1b IL-13 *LIX (mouse) ENA-78 (CXCL5) and GCP-2 (CXCL6) *mob-1 BRL-1 CD40 *ICOS MHC Class I HLA-B7 TREM-1 Fibronectin Complement factor B CCL5 Eotaxin IL-1*IL-15 Lymphotoxin a Neutrophil activating peptide-78 CCR5 CD48 IL-2 receptor a-chain b2 Microglobulin Complement B ICAM-1 Complement factor C4 CCL15/Leukotactin/ SCYA15 Fractalkine IL-2 IL-17 Lymphotoxin b RANTES CCR7 *IL-15 Lymphotoxin a Neutrophil activating peptide-78 CCR5 CD48 IL-2 receptor a-chain b2 Microglobulin Complement B ICAM-1 Complement factor C4 CCL15/Leukotactin/ SCYA15 Fractalkine IL-2 IL-17 Lymphotoxin b CCR7 CD83 Immunoglobulin Cgamma1 Nod2 Complement component 3 MadCAM-1 C-reactive protein CCL17 Gro alpha IL-6 IL-23A (p19) MCP-1/JE TCA3 T-cell activation gene 3 CXCR CD86 Immunoglobulin gamma1 Polymeric Ig receptor Complement Receptor 2 NCAM Hepcidin CCL19 Gro beta IL-8 IL-27 (p28) MIG TNFalpha CXCR2 CD98 Immunoglobulin gamma4 PGRP-S Proteasome Subunit LMP2 P-selectin Lipopolysaccharide binding protein CCL20 Gro gamma IL-9 EBI3/IL-27BMIP-1a,b TNFbeta CD137 CD134 Immunoglobulin e heavy chain T-cell receptor b chain Peptide Transporter TAP1 Tenascin-C Pentraxin PTX3 CCL22 Gro-1 IL-10 beta-Interferon TRAIL (aka Apo2 ligand) CD154 *F11-receptor Immunoglobulin k light chain T-cell receptor/CD3gamma Tapasin VCAM-1 Serum amyloid A proteins (SAA1, SAA2, SAA3) CCL23/SYA23 *ICOS IL-11 IP-10 aka (LAG-1) TFF3 (Treefoil factor) CD3gamma FcRn Invariant Chain II TLR-2 DC-SIGN Angiotensinogen (Angiotensin II) Serum amyloid A proteins (SAA1, SAA2, SAA3) CCL28 IFN-g IL-12B (p40) KC MIP-2 VEGI CD21 Fc epsilon receptor II (CD23) Kinin B1 Receptor TLR9 ELAM-1 (CD62E, E-selectin) beta-defensin-2 Serum amyloid A proteins (SAA1, SAA2, SAA3) CINC-1 IL-1a IL-12A (p35) IiGp1 MIP-3alpha/CCL20 B7.1 (CD80) CD38 HLA-G MHC class I (H-2Kb) *TNF-Receptor p75/80 (CD120B) Endoglin C4b binding protein Tissue factor-1 *CYP2E1 CYP2C11 CYP7b COX-2 Ferritin H chain HSP90-alpha *5-Lipoxygenase (guinea pig) 12-Lipoxygenase Inducible NO-Synthase *MAP4K1 Cu/Zn SOD SOD1 SOD2 *Mx1 NAD(P)H quinone oxidoreductase (DT-diaphorase) Phospholipase A2 SEPS1 ABCA1 ABCC6 A1 adenosine receptor A2A *ADAM19 Amiloride-sensitive sodium channel *alpha2B-adrenergic receptor Bradykinin B1-Receptor *CD23 CD69 DOR Epidermal Growth Factor Receptor ErbB2 Gal1 Receptor Lox-1 Ly49 Mdr1 Mu-opioid receptor mGlu2 Neuropeptide Y-Y1 receptor *NMDA receptor subunit 2A (rat) *NMDA receptor subunit NR-1 (GRIN1 gene) Oxytocin receptor PAF receptor 1 P-gp RAGE- receptor ASC Bax Bfl1/A1 Bcl-xL Bcl-2 B7-H1 BNIP3 Caspase-11 Nr13 c-FLIP CD95 (Fas) *Fas-associated phosphatase-1 Fas-Ligand IAPs IEX-1L TRAF-1 TRAF-2 *TRAF-2 binding protein (Carp) XIAP Activin A Angiopoietin BCAP BDNF BLyS BLNK BMP-2 BMP-4 CGRP FGF8 FLRG G-CSF GM-CSF *HGF/SF EPO *IGFBP-1 IGFBP-2 M-CSF (CSF-1) Midkine (neurite growth promoting factor-2) NGF NK-1R NK4 Nrg1 OPN PDGF B chain PlGF Proenkephalin Prolactin Stem Cell Factor *Thrombospondin-1 (TSP-1) *Thrombospondin-2 (THBS2) VEGF C *WNT10B *B94 *Egr-1 p22/PRG1 *p62 *TIEG A20 ABIN-3 Androgen receptor Bcl-3 BMI-1 CDX1 *c-fos (fish gene) c-myb c-myc c-rel C/EBPdelta *DC-SCRIPT Dmp1 E2F3a Elf3 *ELYS *ETR101 Gata-3 *Glucocorticoid receptor HIF-1alphaHOXA9 IRF-1 IRF-2 IRF-4 IRF-7 IkB-a IkB-e junB jmjD3 Lef1 LZIP Mail nfkb2 nfkb1 NLRP2 NURR1 Osterix p53 Progesterone receptor relb Snail Sox9 Stat5a Tfec Twist WT1 YY1 · *ABC Transporters *N-acetylglucosaminyltransferase I (rat gene) ADH AID AMACR ARF-related protein-1 Argininosuccinate synthetase Aromatase (promoter II) ART2.1 alpha 1ACT BACE-1 BtkCathepsin B *Cathepsin L *Ceramide glycosyltransferase Chitinase 3-like protein *cis-retinoid/androgen dehydrogenase type 1 (CRAD1) *cis-retinoid/androgen dehydrogenase type 2 (CRAD2) Collagenase 1*Dihydrodiol dehydrogenase *DYPD DNASIL2 EL *ENO2 *GAD67 GD3-synthase gp91 phox Gelatinase B GSTP1-1 Glutamate-cysteine ligase GCLC *Glutamate-cysteine ligase modifier *gamma glutamylcysteine synthetase *Glucose l-6-phosphate dehydrogenase Glucose-6-phosphatase GnRH II Granzyme B *Soluble Guanylyl cyclase alpha (1) *Heparanase HO-1 Hyaluronan synthase 11bHSD2 *17bHSD H+-K+ATPase alpha2 Iodothyronine deiodinase (type 2) Lipocalin-type prostaglandin D synthase (L-PGDS) Lysozyme Mthfr *MKP-1 MMP-3 matrix metalloproteinaase-3 MMP-9matrix metalloproteinaase-9 MLCK iNOS n-NOS *PDE7A1 PIM-1 Plk3 PIK3CA *PP5 PKCdelta PLCdelta 1 *PTGIS prostaglandin synthase *PGES prostaglandin E synthase PTP1B PTHrP RACK1 *REV3 Slfn-2 Serpin 2A SIAT1 SNARK SSAT *SUV3 TERT Transglutaminase TTG Type II-secreted phospholipase A2 Uridine phosphorylase *Xanthine Dehydrogenase *ABCG5 (bovine) ABCG8 (bovine) AbetaH-J-J alpha-1 acid glycoprotein alpha-fetoprotein AMH *beta-amyloid APOBEC2 Apolipoprotein C III Apolipoprotein D Apolipoprotein E AQP4 *Biglycan BRCA2 Calsarcin-1 *Caveolin-1*Clone 330 *Clone 156 *Clone 68 *p21-CIP1 *Claudin-2 a2(I) collagen *Connexin32 Cyclin D1 Cyclin D2 *Cyclin D3 DIF2 DMT1 Elafin Endothelin 1 Ephrin-A1 Factor VIII Ferritin Heavy Chain Gadd45beta Galpha i2 *GIF Galectin 3 GBP-1 epsilon-Globin zeta-Globin *GS3686 Hair K5 keratin *HCCS1 HMG14 IBABP IMP2 K3 Keratin K6b Keratin K15 Keratin *Lactoferrin Laminin B2 ChainLipocalin-2 Mts1 Mir125b Mir146a, b Mir155 MNE1 Mucin (MUC-2) Myelin basic protein MCT1 Naf1 Neutrophil gelatinase-associated lipocalin NLF1 *p11 PA28 alpha PA28 beta PAI-1 *Pax8 *PCBD Perforin *PGK1 POMC Pregnancy-specific glycoprotein rnCGM3 Prodynorphin Prostate-specific antigen PTEN RAG-1 RAG-2 RbAp48 *RICK SerpinE2 S100A6 (calcyclin) SH3BGRL SK2 channelsSkp2 *Spergen-1 SWS1 Syncytin-1 Syndecan-4 TAUT TASK-2 *Tissue factor pathway inhibitor-2 (TFPI-2) *Transferrin (mosquito) TRIF TRPC1 *UBE2M *UCP-2 Uroplakin Ib 25-hydroxyvtamin D3 1alpha hydroxylase Vimentin a1-antitrypsin Gro-1 Adenovirus (E3 region) Avian Leukosis Virus Bovine Leukemia Virus CMV EBV (Wp promoter) HBV (pregenomic promoter) HIV-1 HSV (ICP90, ICPO) JC Virus HPV type 16 SIV SV-40
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© Linda Palma, 2008
Università degli Studi di Urbino
● Degeneration
● Redundancy
● Sequence-dimer pairs
● Exchange of dimers
NF-B TARGET GENES (2)
B MOTIF: 9-11 mer
GGGRNNYYCC(R = purine; N = any nucleotide; Y = pyrimidine)
B MOTIF: 9-11 mer
GGGRNNYYCC(R = purine; N = any nucleotide; Y = pyrimidine)
● CONSTITUTIVELY AND IMMEDIATELY
ACCESSIBLE GENES (CIA)
● REGULATED LATE ACCESSIBILITY
GENES (RLA)
FINE TUNING OF TARGET GENE EXPRESSION RATES
IN A CELL- AND STIMULUS- SPECIFIC MANNER
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© Linda Palma, 2008
Università degli Studi di Urbino
NF-B TARGET GENES (3)
● Growth factors, ligands and their modulators ● Transcription factors and Regulators ● Enzymes ● Cyclins
● Stress response genes ● Regulators of apoptosis
● Cytokines/Chemokines and their modulators ● Immunoreceptors ● Proteins involved in antigen presentation ● Cell adhesion molecules ● Acute phase proteins
● Viruses● Early response genes ● NF-B proteins● Cell-surface receptors
● IkBs● A20
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© Linda Palma, 2008
Università degli Studi di Urbino
NF-B FUNCTIONS:APOPTOSIS (1)
Fas / DR4 / DR5
FADDPROCASPASE 8
PROCASPASE 10C-FLIP
TRADDTRAF2TRAF5RIP1
TNFR1 / DR3 /DR6 / EDAR
● Death Receptors:
APOPTOTIC RESPONSE
NF-B
FLIPc-IAP1c-IAP2XIAP
Bcl-2 family members
NF-B INDUCES GENES WHOSE FUNCTIONS INTERFERE WITH THOSE OF THE
DEATH-INDUCING SIGNALING COMPLEXES
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© Linda Palma, 2008
Università degli Studi di Urbino
NF-B FUNCTIONS:APOPTOSIS (2)
DEPOLARIZATION
CASPASE-DEPENDENTAND –INDEPENDENT
MECHANISMS
● Mitochondria:
APOPTOTIC RESPONSE
NF-B
NF-B INDUCES GENES WITHANTIOXIDANT FUNCTIONS
REACTIVEOXYGENSPECIES
(ROS)
glutathione-S-transferaseMn SOD
Ferritin heavy chainMetallothionein
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© Linda Palma, 2008
Università degli Studi di Urbino
NF-B FUNCTIONS:APOPTOSIS (3)
NF-B, some members of its activation pathway and some of its target gene products
are themselves substrates of caspases.
The ratio between pro-apoptotic and anti-apoptotic activities can target a given
cell type to life or death in response tovarious stimuli.
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© Linda Palma, 2008
Università degli Studi di Urbino
NF-B FUNCTIONS: IMMUNITY (1)
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 Receptor Ligands Apoptotic Mediators Mitogens, Growth Factors and Hormones Physiological Mediators Chemical Agents
NF-NF-B INDUCERSB INDUCERS
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© Linda Palma, 2008
Università degli Studi di Urbino
NF-B FUNCTIONS: IMMUNITY (2)
Pathogen INNATE IMMUNITYINNATE IMMUNITY
INFLAMMATION AND COMPLEMENT INFLAMMATION AND COMPLEMENT CASCADECASCADE
Epithelial cells from lung, liver and Epithelial cells from lung, liver and intestinal mucosa and professional intestinal mucosa and professional immune cells (macrophages, immune cells (macrophages, neutrophils, dendritic cells, mast cells, neutrophils, dendritic cells, mast cells, eosinophils, basophils and natural eosinophils, basophils and natural killer cells).killer cells).
ADAPTIVE IMMUNITYADAPTIVE IMMUNITY
LYMPHOID ORGANS DEVELOPMENT,LYMPHOID ORGANS DEVELOPMENT,
ANTIGEN PRESENTATION ANDANTIGEN PRESENTATION AND
MAINTENANCE OF MEMORY CELLSMAINTENANCE OF MEMORY CELLS
APCs, T and B lymphocytes.APCs, T and B lymphocytes.
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© Linda Palma, 2008
Università degli Studi di Urbino
NF-B FUNCTIONS: IMMUNITY (3)
NF-B
NF-B
Inflammatory genes
mRNA
Inflammatory genes
mRNA
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© Linda Palma, 2008
Università degli Studi di Urbino
NF-B DYSFUNCTION (1)Ageing Headaches Pain Complex Regional Pain Syndrome
Cardiac Hypertrophy Muscular Dystrophy (type 2A) Muscle wasting Catabolic disorders
Diabetes, Type 1 Diabetes, Type 2 Obesity
Fetal Growth Retardation
Hypercholesterolemia Atherosclerosis Heart Disease Chronic Heart Failure
Ischemia/reperfusion Stroke Cerebral aneurysm Angina Pectoris
Pulmonary Disease Cystic Fibrosis Acid-induced Lung Inury Pulmonary hypertension
Chronic Obstructive Pulmonary Disease (COPD) Hyaline Membrane Disease Kidney Disease
Glomerular Disease Alcoholic Liver Disease Leptospiriosis renal disease Gut Diseases
Peritoneal endometriosis Skin Diseaes Nasal sinusitis Mesothelioma
Anhidrotic Ecodermal Dysplasia-ID Behcet's Disease Incontinentia pigmenti Tuberculosis
Asthma Arthritis Crohn's Disease Colitis (rat)
Ocular Allergy Glaucoma Appendicitis Paget's Disease
Pancreatitis Periodonitis Endometriosis Inflammatory Bowel Disease
Inflammatory Lung isease Sepsis Silica-induced Sleep apnoea
AIDS (HIV-1) Autoimmunity Antiphospholipid Syndrome Lupus
Lupus nephritis Waldenstrom macroglobulinemia Chronic Disease Syndrome Familial Mediterranean Fever
Hereditary Periodic Fever syndrome
Psychosocial stress diseases Neuropathological Diseases Familial amyloidotic polyneuropathy, inflamm neuropathy
Traumatic brain injury Spinal cord injury Parkinson Disease Multiple Sclerosis
Rheumatic Disease Alzheimers Disease Amyotropic lateral sclerosis Huntington's Disease
Retinal Disease Cataracts Hearing loss Cancer
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© Linda Palma, 2008
Università degli Studi di Urbino
NF-B DYSFUNCTION (2)Cancer type
Solid tumors (generally)
Breast Cervix Ovary
Vulva
Prostate Kidney Bladder
Lung Mesothelioma Non small-cell lung Liver
Pancreas Esophygeal/gastric Laryngeal Stomach
Colon Thyroid Parathyroid Melanoma
Endometrial (Uteris) Squamous cell carcinoma Head and neck Hilar Cholangiocarcinoma
Oral carcinoma Cylindromatosis Brooke-Spiegler, Trichoepithelioma
Glioblastoma
Hodgkin’s lymphoma
Astrocytoma/glioblastoma Neuroblastoma Acute T-cell leukemia (+/-HTLV-1)
Acute Non-lymphocytic leukemia
Acute lymphoblastic leukemia Acute myelogenous leukemia Mantle cell lymphoma
Myelodysplastic syndrome
Chronic lymphocytic leukemia Burkitts Lymphoma (EBV) MALT lymphoma
Multiple myeloma
Diffuse large B-cell lymphoma
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© Linda Palma, 2008
Università degli Studi di Urbino
NF-B AND CANCER (1)
Epidemiological data have led to hypothesize an
association between INFLAMMATION and CANCER.
Examples:
silica / asbestos / cigarette smoke and LUNG CANCER
H. pylori infection and GASTRIC CANCER
IBDs and COLON CANCER
HPV and CERVICAL CANCER
EBV and BURKITT LYMPHOMA
viral hepatitis and LIVER CANCER
INFLAMMATION IS INVOLVED IN
TUMOR INITIATION, PROMOTION
AND PROGRESSION
Genetic data exist, linking polymorphisms
of PRRs genes and other inflammatory genes
to increased cancer risk.
Examples:
TLR6 and TLR10 and PROSTATE CANCER
NOD2 locus and COLORECTAL CANCER
IL-1 and IL-1RA and GASTRIC CANCER
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© Linda Palma, 2008
Università degli Studi di Urbino
NF-B AND CANCER (2)
● INADEQUATE PATHOGEN
ERADICATION
● PROLONGED INFLAMMATORY
SIGNALING
● DEFECTIVE ANTI-INFLAMMATORY
MECHANISMS
ENHANCED PROLIFERATION
AND
PROTECTION OF PRE-
NEOPLASTIC /
MALIGNANT CELLS FROM
APOPTOSIS
AND NECROSIS
PERSISTENT NF-B
ACTIVATION
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© Linda Palma, 2008
Università degli Studi di Urbino
NF-B AND CANCER (3)
Tumorigenesis requires six essential
alterations to normal cell physiology:
● self-sufficiency in growth signals ● insensitivity to growth
inhibition
● evasion of apoptosis ● immortalization
● sustained angiogenesis ● tissue invasion and
metastasisChronic infection
IL-8 / VEGF /MMPs
Angiogenesis and metastases
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© Linda Palma, 2008
Università degli Studi di Urbino
NF-B AND CANCER (4)
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© Linda Palma, 2008
Università degli Studi di Urbino
NF-B AS TARGET FORANTICANCER DRUGS (1)
MAJOR ISSUES:
● UPTAKE
● STABILITY
● EFFICACY IN PATIENTS
● SIDE EFFECTS
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© Linda Palma, 2008
Università degli Studi di Urbino
NF-B AS TARGET FORANTICANCER DRUGS (2)