1: Mol Cancer Res. 2005 Jul;3(7):403-12. 

Transforming growth factor-alpha inhibits the intrinsic pathway of c-Myc-induced
apoptosis through activation of nuclear factor-kappaB in murine hepatocellular
carcinomas.

Cavin LG, Wang F, Factor VM, Kaur S, Venkatraman M, Thorgeirsson SS, Arsura M.

Department of Pharmacology, Center for Anticancer Drug Research, College of
Medicine, University of Tennessee Cancer Institute, 874 Union Avenue, Memphis,
TN 38163, USA.

Nuclear factor-kappaB (NF-kappaB) plays an important role during liver
neoplastic development through transcriptional regulation of prosurvival genes,
which then counteract the death-inducing signals elicited by the host immune
response. The c-Myc proto-oncogene is frequently deregulated in liver tumors.
Furthermore, enforced expression of c-Myc in the liver promotes the development
of hepatocellular carcinomas, a process that is accelerated by coexpression with
transforming growth factor-alpha (TGF-alpha). TGF-alpha/c-Myc-derived
hepatocellular carcinomas display reduced apoptotic levels compared with those
of single c-Myc transgenic hepatocellular carcinomas, suggesting that TGF-alpha
provides a survival advantage to c-Myc-transformed hepatocytes. Given that
TGF-alpha/c-Myc hepatocellular carcinomas display constitutive NF-kappaB
activity, here, we have tested the hypothesis that enforced expression of
TGF-alpha results in constitutive NF-kappaB activation and enhanced cell
survival using TGF-alpha/c-Myc-derived hepatocellular carcinoma cell lines. We
show that TGF-alpha induces NF-kappaB through the phosphatidylinositol
3-kinase/Akt axis in these bitransgenic hepatocellular carcinomas. Furthermore,
we found that adenovirus-mediated inhibition of NF-kappaB activity impairs the
ability of TGF-alpha/c-Myc-derived tumor cells to grow in an
anchorage-independent fashion due to sensitization to c-Myc-induced apoptosis.
Lastly, we show that NF-kappaB inhibits c-Myc-induced activation of caspase-9
and caspase-3 through up-regulation of the antiapoptotic target genes Bcl-X(L)
and X-linked inhibitor of apoptosis (XIAP). Overall, these results underscore a
crucial role of NF-kappaB in disabling apoptotic pathways initiated by oncogenic
transformation.

PMID: 16046551 [PubMed - indexed for MEDLINE]


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2: J Immunol. 2005 Jun 1;174(11):7383-92. 

Identification of a novel blocker of I kappa B alpha kinase that enhances
cellular apoptosis and inhibits cellular invasion through suppression of
NF-kappa B-regulated gene products.

Ichikawa H, Takada Y, Murakami A, Aggarwal BB.

Cytokine Research Section, Department of Experimental Therapeutics, University
of Texas M. D. Anderson Cancer Center, Houston, 77030, USA.

1'-Acetoxychavicol acetate (ACA), extracted from rhizomes of the commonly used
ethno-medicinal plant Languas galanga, has been found to suppress chemical- and
virus-induced tumor initiation and promotion through a poorly understood
mechanism. Because several genes that regulate cellular proliferation,
carcinogenesis, metastasis, and survival are regulated by activation of the
transcription factor NF-kappaB, we postulated that ACA might mediate its
activity through modulation of NF-kappaB activation. For this report, we
investigated the effect of ACA on NF-kappaB and NF-kappaB-regulated gene
expression activated by various carcinogens. We found that ACA suppressed
NF-kappaB activation induced by a wide variety of inflammatory and carcinogenic
agents, including TNF, IL-1beta, PMA, LPS, H(2)O(2), doxorubicin, and cigarette
smoke condensate. Suppression was not cell type specific, because both inducible
and constitutive NF-kappaB activations were blocked by ACA. ACA did not
interfere with the binding of NF-kappaB to the DNA, but, rather, inhibited
IkappaBalpha kinase activation, IkappaBalpha phosphorylation, IkappaBalpha
degradation, p65 phosphorylation, and subsequent p65 nuclear translocation. ACA
also inhibited NF-kappaB-dependent reporter gene expression activated by TNF,
TNFR1, TNFR-associated death domain protein, TNFR-associated factor-2, and
IkappaBalpha kinase, but not that activated by p65. Consequently, ACA suppressed
the expression of TNF-induced NF-kappaB-regulated proliferative (e.g., cyclin D1
and c-Myc), antiapoptotic (survivin, inhibitor of apoptosis protein-1 (IAP1),
IAP2, X-chromosome-linked IAP, Bcl-2, Bcl-x(L), Bfl-1/A1, and FLIP), and
metastatic (cyclooxygenase-2, ICAM-1, vascular endothelial growth factor, and
matrix metalloprotease-9) gene products. ACA also enhanced the apoptosis induced
by TNF and chemotherapeutic agents and suppressed invasion. Overall, our results
indicate that ACA inhibits activation of NF-kappaB and NF-kappaB-regulated gene
expression, which may explain the ability of ACA to enhance apoptosis and
inhibit invasion.

PMID: 15905586 [PubMed - indexed for MEDLINE]


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3: J Cereb Blood Flow Metab. 2005 Jan;25(1):30-40. 

Neuronal activation of NF-kappaB contributes to cell death in cerebral ischemia.

Zhang W, Potrovita I, Tarabin V, Herrmann O, Beer V, Weih F, Schneider A,
Schwaninger M.

Department of Neurology, University of Heidelberg, Heidelberg, Germany.

The transcription factor NF-kappaB is a key regulator of inflammation and cell
survival. NF-kappaB is activated by cerebral ischemia in neurons and glia, but
its function is controversial. To inhibit NF-kappaB selectively in neurons and
glial cells, we have generated transgenic mice that express the IkappaBalpha
superrepressor (IkappaBalpha mutated at serine-32 and serine-36,
IkappaBalpha-SR) under transcriptional control of the neuron-specific enolase
(NSE) and the glial fibrillary acidic protein (GFAP) promoter, respectively. In
primary cortical neurons of NSE-IkappaBalpha-SR mice, NF-kappaB activity was
partially inhibited. To assess NF-kappaB activity in vivo after permanent middle
cerebral artery occlusion (MCAO), we measured the expression of NF-kappaB target
genes by real-time polymerase chain reaction (PCR). The induction of c-myc and
transforming growth factor-beta2 by cerebral ischemia was inhibited by neuronal
expression of IkappaBalpha-SR, whereas induction of GFAP by MCAO was reduced by
astrocytic expression of IkappaBalpha-SR. Neuronal, but not astrocytic,
expression of the NF-kappaB inhibitor reduced both infarct size and cell death
48 hours after permanent MCAO. In summary, the data show that NF-kappaB is
activated in neurons and astrocytes during cerebral ischemia and that NF-kappaB
activation in neurons contributes to the ischemic damage.

PMID: 15678110 [PubMed - indexed for MEDLINE]


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4: Cancer Res. 2004 Oct 1;64(19):7030-8. 

Regulation of alpha-fetoprotein by nuclear factor-kappaB protects hepatocytes
from tumor necrosis factor-alpha cytotoxicity during fetal liver development and
hepatic oncogenesis.

Cavin LG, Venkatraman M, Factor VM, Kaur S, Schroeder I, Mercurio F, Beg AA,
Thorgeirsson SS, Arsura M.

Department of Pharmacology, Center for Anticancer Drug Research, University of
Tennessee Cancer Institute, College of Medicine, Memphis, Tennessee 38163, USA.

Nuclear factor-kappaB (NF-kappaB) plays a critical role during fetal liver
development and hepatic oncogenesis. Here, we have assessed whether NF-kappaB
activity is required for murine hepatocellular carcinoma cell survival. We show
that adenoviral-mediated inhibition of inhibitor of NF-kappaB kinase-beta
(IKK-2) activity in hepatocellular carcinomas derived from transforming growth
factor (TGF)-alpha/c-myc bitransgenic mice leads to inhibition of NF-kappaB and
promotes tumor necrosis factor (TNF)-alpha-mediated cell death of malignant
hepatocytes but not the surrounding peritumorous tissue. Induction of apoptosis
is accompanied by inhibition of Bcl-X(L) and XIAP, two pro-survival NF-kappaB
target genes. In addition, we have identified the alpha-fetoprotein (AFP) as a
novel downstream target of NF-kappaB. We show that repression of IKK-2 activity
in hepatocellular carcinomas promotes down-regulation of AFP gene expression.
Likewise, genetic disruption of the RelA subunit results in reduced AFP gene
expression during embryonic liver development, at a time in which fetal
hepatocytes are sensitized to TNF-alpha-mediated cell killing. In this regard,
we show that AFP inhibits TNF-alpha-induced cell death of murine hepatocellular
carcinomas through association with TNF-alpha and inhibition of TNFRI signaling.
Thus, NF-kappaB-mediated regulation of AFP gene expression during liver tumor
formation and embryonic development of the liver constitutes a potential novel
mechanism used by malignant and fetal hepatocytes to evade immune surveillance.

PMID: 15466196 [PubMed - indexed for MEDLINE]


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5: Curr Opin Genet Dev. 2004 Oct;14(5):485-91. 

Transcriptional control of epidermal specification and differentiation.

Dai X, Segre JA.

Department of Biological Chemistry, 234D Med Sci I, University of California,
Irvine, California 92697-1700, USA. xdai@uci.edu

Recent experiments reveal the role of transcription factors in integrating
upstream signals to execute specification and differentiation of epidermal
cells. Based on the skin phenotype observed with misregulation of transcription
factors such as p63, c-Myc, RelA, pRb, Klf4 and others, their function in
controlling proliferation and differentiation is dissected. Understanding the
pathways regulated by these factors and their coordinate interactions remains a
challenge for the future.

Publication Types:
    Review
    Review, Tutorial

PMID: 15380238 [PubMed - indexed for MEDLINE]


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6: Immunity. 2004 Jul;21(1):19-30. 

The mitogen-induced increase in T cell size involves PKC and NFAT activation of
Rel/NF-kappaB-dependent c-myc expression.

Grumont R, Lock P, Mollinari M, Shannon FM, Moore A, Gerondakis S.

The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade,
Parkville, Victoria 3050, Australia.

Cell growth during the G1 stage of the cell cycle is partly controlled by
inducing c-myc expression, which in B cells is regulated by the NF-kappaB1 and
c-Rel transcription factors. Here, we show that c-myc-dependent growth during T
cell activation requires c-Rel and RelA and that blocking this growth by
inhibiting protein kinase C theta (PKCtheta) coincides with a failure to
upregulate c-myc due to impaired RelA nuclear import and inhibition of
NFAT-dependent c-rel transcription. These results demonstrate that different
Rel/NF-kappaB dimers regulate the mitogenic growth of mature T and B cells, with
a signaling pathway incorporating PKCtheta and NFAT controlling
c-Rel/RelA-induced c-myc expression in activated T cells.

PMID: 15345217 [PubMed - indexed for MEDLINE]


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7: World J Gastroenterol. 2004 Feb 15;10(4):491-6. 

Expression of nuclear factor-kappa B and target genes in gastric precancerous
lesions and adenocarcinoma: association with Helicobactor pylori cagA (+)
infection.

Yang GF, Deng CS, Xiong YY, Gong LL, Wang BC, Luo J.

Department of Pathology, Zhongnan Hospital, Wuhan University, Wuhan 430071,
Hubei Province, China. caiyang@public.wh.hb.cn

AIM: To examine the expression of nuclear factor kappaB (NF-kappaB) and its
target genes in intestinal metaplasia (IM), dysplasia (DYS) and gastric
carcinoma (GC) infected with Helicobacter pylori (H pylori) and to investigate
the mechanism underlying H pylori cytotoxin associated gene A (cag A) infection
leading to gastric adenocarcinoma. METHODS: Expressions of NF-kappaB/p65 and its
target genes: c-myc, cyclinD1 and bcl-xl were immunohistochemically examined in
289 cases of gastric biopsy and resection specimens from patients with IM, DYS
and GC infected with H pylori. H pylori in the above mentioned tissues was
detected by Warthin-Starry stain and rapid urease tests. IgG antibody to cagA in
sera of the patients was measured by ELISA. RESULTS: The positive rates of
NF-kappaB/p65 were significantly higher in groups with cagA of IMI-II(28/33), IM
III(48/52), DYSI(27/31), DYS II-III(28/32), GC(35/40) than in groups without
cagA of IMI-II(4/17), IMIII(3/20), DYSI(3/20), DYSII-III(6/21), GC(10/23). The
expressions of c-myc, cyclinD1, and bcl-xl were significantly higher in groups
with cagA of IM III(47/52, 49/52, 46/52), DYSII-III(29/32, 26/32, 25/32) than in
groups without cagA of IM III(8/20, 7/20, 5/20), DYSII-III(10/21, 8/21, 3/21),
which were in conformity with the expression of NF-kappaB in IM III, and
DYSII-III. A significantly higher expression level of NF-kappaB/p65, c-myc,
cyclinD1 and bcl-xl was detected in intestinal type GC(27/28, 18/28, 22/28,
24/28) than in diffuse type GC(8/12, 3/12, 3/12, 6/12), respectively.
CONCLUSION: There may be two different molecular mechanisms in the occurrence of
intestinal and diffuse type gastric carcinomas. Intestinal type gastric
carcinoma is strongly associated with high expression of c-myc, cyclinD1 and
bcl-xl through NF-kappaB/p65 activated by H pylori cagA. Inhibiting the activity
of NF-kappaB is an effective and promising way to prevent intestinal type
gastric carcinoma.

PMID: 14966904 [PubMed - indexed for MEDLINE]


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8: Mol Cell Biol. 2003 Aug;23(16):5738-54. 

Mouse mammary tumor virus c-rel transgenic mice develop mammary tumors.

Romieu-Mourez R, Kim DW, Shin SM, Demicco EG, Landesman-Bollag E, Seldin DC,
Cardiff RD, Sonenshein GE.

Department of Biochemistry, Boston University Medical School, Boston,
Massachusetts 02118, USA.

Amplification, overexpression, or rearrangement of the c-rel gene, encoding the
c-Rel NF-kappaB subunit, has been reported in solid and hematopoietic
malignancies. For example, many primary human breast cancer tissue samples
express high levels of nuclear c-Rel. While the Rev-T oncogene v-rel causes
tumors in birds, the ability of c-Rel to transform in vivo has not been
demonstrated. To directly test the role of c-Rel in breast tumorigenesis, mice
were generated in which overexpression of mouse c-rel cDNA was driven by the
hormone-responsive mouse mammary tumor virus long terminal repeat (MMTV-LTR)
promoter, and four founder lines identified. In the first cycle of pregnancy,
the expression of transgenic c-rel mRNA was observed, and levels of c-Rel
protein were increased in the mammary gland. Importantly, 31.6% of mice
developed one or more mammary tumors at an average age of 19.9 months. Mammary
tumors were of diverse histology and expressed increased levels of nuclear
NF-kappaB. Analysis of the composition of NF-kappaB complexes in the tumors
revealed aberrant nuclear expression of multiple subunits, including c-Rel, p50,
p52, RelA, RelB, and the Bcl-3 protein, as observed previously in human primary
breast cancers. Expression of the cancer-related NF-kappaB target genes cyclin
D1, c-myc, and bcl-xl was significantly increased in grossly normal transgenic
mammary glands starting the first cycle of pregnancy and increased further in
mammary carcinomas compared to mammary glands from wild-type mice or virgin
transgenic mice. In transient transfection analysis in untransformed breast
epithelial cells, c-Rel-p52 or -p50 heterodimers either potently or modestly
induced cyclin D1 promoter activity, respectively. Lastly, stable overexpression
of c-Rel resulted in increased cyclin D1 and NF-kappaB p52 and p50 subunit
protein levels. These results indicate for the first time that dysregulated
expression of c-Rel, as observed in breast cancers, is capable of contributing
to mammary tumorigenesis.

PMID: 12897145 [PubMed - indexed for MEDLINE]


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9: Oncogene. 2002 Oct 3;21(44):6819-28. 

Synergistic and opposing regulation of the stress-responsive gene IEX-1 by p53,
c-Myc, and multiple NF-kappaB/rel complexes.

Huang YH, Wu JY, Zhang Y, Wu MX.

Department of Pathology, Baylor College of Medicine, Houston, Texas, TX 77030,
USA.

NF-kappaB/rel proteins, tumor suppressor p53, and oncogene c-Myc are critical
transcription factors involved in coordinating cellular decision-making events
in response to external stimuli. Consensus sequences for binding these three
transcription factors are found in the promoter region of IEX-1 (Immediate Early
response gene X-1) gene that can either suppress or induce apoptosis in a cell-
and stimulus-dependent manner. Utilizing an electrophoretic mobility shift assay
(EMSA) and a promoter/reporter assay, we show that the NF-kappaB/rel consensus
sequence in the IEX-1 promoter is specifically bound and activated by multiple
NF-kappaB/rel complexes in descending order p65-c-rel-->p65-50-->p50-50.
Interestingly, NF-kappaB/rel-mediated activation of IEX-1 expression was
synergized by p53, but strongly inhibited by c-Myc in a dose-dependent fashion.
Moreover, the ability of c-Myc to inhibit IEX-1 expression requires the presence
of functional p53, which may partially contribute to the varying effects of p53
on IEX-1 expression in different cells. In support of coordinated regulation of
IEX-1 expression by these three transcription factors in vivo, binding of
endogenous p53, c-Myc and NF-kappaB/rel proteins, including p50, p65 and c-rel,
to the IEX-1 promoter was demonstrated in living cells by chromatin
immunoprecipitation using specific antibodies. The study reveals a novel
integrative regulation of specific gene expression by NF-kappaB/rel, p53 and
c-Myc transcription factors.

PMID: 12360408 [PubMed - indexed for MEDLINE]


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10: J Biol Chem. 2002 Sep 27;277(39):36671-7. Epub 2002 Jul 30. 

c-Myc sensitizes cells to tumor necrosis factor-mediated apoptosis by inhibiting
nuclear factor kappa B transactivation.

You Z, Madrid LV, Saims D, Sedivy J, Wang CY.

Laboratory of Molecular Signaling and Apoptosis, Department of Biologic and
Materials Sciences, University of Michigan, Ann Arbor, Michigan 48109, USA.

Nuclear factor kappaB (NF-kappaB) plays a key role in suppression of tumor
necrosis factor (TNF)-mediated apoptosis by inducing a variety of anti-apoptotic
genes. Expression of c-Myc has been shown to sensitize cells to TNF-mediated
apoptosis by inhibiting NF-kappaB activation. However, the precise step in the
NF-kappaB signaling pathway and apoptosis modified by c-Myc has not been
identified. Using the inducible c-MycER system and c-Myc null fibroblasts, we
found that expression of c-Myc inhibited NF-kappaB activation by interfering
with RelA/p65 transactivation but not nuclear translocation of NF-kappaB.
Activation of c-Myc promoted TNF-induced release of cytochrome c from
mitochondria to the cytosol because of the inhibition of NF-kappaB. Furthermore,
we found that NF-kappaB-inducible gene A1 was attenuated by expression of c-Myc
and that the restoration of A1 expression suppressed c-Myc-induced TNF
sensitization. Our results elucidate the molecular mechanisms by which c-Myc
increases cell susceptibility to TNF-mediated apoptosis, indicating that c-Myc
may exhibit its pro-apoptotic activities by repression of cell survival genes.

PMID: 12149248 [PubMed - indexed for MEDLINE]


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11: Biochem J. 2002 Sep 1;366(Pt 2):459-69. 

A novel form of the RelA nuclear factor kappaB subunit is induced by and forms a
complex with the proto-oncogene c-Myc.

Chapman NR, Webster GA, Gillespie PJ, Wilson BJ, Crouch DH, Perkins ND.

Division of Gene Expression and Regulation, School of Life Sciences, University
of Dundee, Dundee DD1 5EH, Scotland, UK.

Members of both Myc and nuclear factor kappaB (NF-kappaB) families of
transcription factors are found overexpressed or inappropriately activated in
many forms of human cancer. Furthermore, NF-kappaB can induce c-Myc gene
expression, suggesting that the activities of these factors are functionally
linked. We have discovered that both c-Myc and v-Myc can induce a previously
undescribed, truncated form of the RelA(p65) NF-kappaB subunit, RelA(p37).
RelA(p37) encodes the N-terminal DNA binding and dimerization domain of
RelA(p65) and would be expected to function as a trans-dominant negative
inhibitor of NF-kappaB. Surprisingly, we found that RelA(p37) no longer binds to
kappaB elements. This result is explained, however, by the observation that
RelA(p37), but not RelA(p65), forms a high-molecular-mass complex with c-Myc.
These results demonstrate a previously unknown functional and physical
interaction between RelA and c-Myc with many significant implications for our
understanding of the role that both proteins play in the molecular events
underlying tumourigenesis.

PMID: 12027803 [PubMed - indexed for MEDLINE]


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12: J Neuroimmunol. 2001 Apr 2;115(1-2):199-202. 

Decreased expression of c-myc family genes in thymuses from myasthenia gravis
patients.

Nagata T, Onodera H, Ohuchi M, Suzuki Y, Tago H, Fujihara K, Ishii N, Sugamura
K, Shoji Y, Handa M, Tabayashi K, Itoyama Y.

Department of Neurology, Tohoku University School of Medicine, 1-1 Seiryo-Machi,
Sendai 980-8574, Aoba, Japan.

The thymus is a critical organ for the elimination of autoreactive T cells by
apoptosis. We studied the expression of apoptosis-associated genes, bcl-xL, bad,
caspase-3, and c-myc family genes in myasthenia gravis (MG) thymuses. We
observed that the mRNA levels of myc family genes, c-myc and max, were markedly
reduced in MG thymuses. These results indicate that c-myc-mediated signaling is
abnormal in MG thymuses. The levels of molecules whose expressions are
associated with myc, such as STAM, prothymosin-alpha, and NFkappaB, were also
analyzed.

Publication Types:
    Clinical Trial

PMID: 11282171 [PubMed - indexed for MEDLINE]


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13: J Immunol. 2001 Jan 15;166(2):1028-40. 

Vasoactive intestinal peptide and pituitary adenylate cyclase-activating
polypeptide inhibit expression of Fas ligand in activated T lymphocytes by
regulating c-Myc, NF-kappa B, NF-AT, and early growth factors 2/3.

Delgado M, Ganea D.

Department of Biological Sciences, Rutgers University, Newark, NJ 07102, USA.

Activation-induced cell death in T cells, a major mechanism for limiting an
ongoing immune response, is initiated by Ag reengagement and mediated through
Fas/Fas ligand interactions. Vasoactive intestinal peptide (VIP) and pituitary
adenylate cyclase-activating polypeptide (PACAP), two multifunctional
neuropeptides, modulate innate and adaptive immunity. We reported previously
that VIP/PACAP protect T cells from activation-induced cell death through
down-regulation of Fas ligand (FasL). In this study, we investigate the
molecular mechanisms involved in the protective effect of VIP and PACAP.
VIP/PACAP reduce in a dose-dependent manner anti-CD3-induced apoptosis in 2B4.11
T cell hybridomas. The protective effect is mediated through the specific type 2
VIP receptor, and the cAMP/protein kinase A pathway. A functional study
demonstrates that VIP/PACAP inhibit activation-induced FasL expression.
VIP/PACAP inhibit the expression and/or DNA-binding activity of several
transcriptional factors involved in FasL expression, i.e., c-myc, NF-kappaB,
NF-ATp, and early growth factors (Egr) 2/3. The inhibition of NF-kappaB binding
is due to the stabilization of I-kappaB (inhibitory protein that dissociates
from NF-kappaB), through the inhibition of I-kappaB kinase alpha activity.
Subsequently, p65 nuclear translocation is significantly reduced. The inhibition
in NF-ATp binding results from a calcineurin-independent reduction in NF-ATp
nuclear translocation. VIP/PACAP inhibit the expression of Egr2 and 3, but not
of Egr1. The effects on the transcriptional factors are mediated through type 2
VIP receptor with cAMP as secondary messenger.

PMID: 11145682 [PubMed - indexed for MEDLINE]


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14: J Virol. 2001 Jan;75(1):215-25. 

Role of NF-kappaB and myc proteins in apoptosis induced by hepatitis B virus HBx
protein.

Su F, Theodosis CN, Schneider RJ.

Department of Microbiology, New York University School of Medicine, New York,
New York 10016, USA.

Chronic infection with hepatitis B virus (HBV) promotes a high level of liver
disease and cancer in humans. The HBV HBx gene encodes a small regulatory
protein that is essential for viral replication and is suspected to play a role
in viral pathogenesis. HBx stimulates cytoplasmic signal transduction pathways,
moderately stimulates a number of transcription factors, including several
nuclear factors, and in certain settings sensitizes cells to apoptosis by
proapoptotic stimuli, including tumor necrosis factor alpha (TNF-alpha) and
etopocide. Paradoxically, HBx activates members of the NF-kappaB transcription
factor family, some of which are antiapoptotic in function. HBx induces
expression of Myc protein family members in certain settings, and Myc can
sensitize cells to killing by TNF-alpha. We therefore examined the roles of
NF-kappaB, c-Myc, and TNF-alpha in apoptotic killing of cells by HBx.
RelA/NF-kappaB is shown to be induced by HBx and to suppress HBx-mediated
apoptosis. HBx also induces c-Rel/NF-kappaB, which can promote apoptotic cell
death in some contexts or block it in others. Induction of c-Rel by HBx was
found to inhibit its ability to directly mediate apoptotic killing of cells.
Thus, HBx induction of NF-kappaB family members masks its ability to directly
mediate apoptosis, whereas ablation of NF-kappaB reveals it. Investigation of
the role of Myc protein demonstrates that overexpression of Myc is essential for
acute sensitization of cells to killing by HBx plus TNF-alpha. This study
therefore defines a specific set of parameters which must be met for HBx to
possibly contribute to HBV pathogenesis.

PMID: 11119591 [PubMed - indexed for MEDLINE]


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15: Oncogene. 2000 Nov 16;19(48):5498-506. 

The RelA NF-kappaB subunit and the aryl hydrocarbon receptor (AhR) cooperate to
transactivate the c-myc promoter in mammary cells.

Kim DW, Gazourian L, Quadri SA, Romieu-Mourez R, Sherr DH, Sonenshein GE.

Department of Biochemistry, Women's Health, Boston University School of
Medicine, Massachusetts 02118, USA.

NF-kappaB/Rel transcription factors regulate many genes involved in control of
cellular proliferation, neoplastic transformation, and apoptosis, including the
c-myc oncogene. Recently, we have observed that levels of NF-kappaB and aryl
hydrocarbon receptor (AhR), which mediates malignant transformation by
environmental carcinogens, are highly elevated and appear constitutively active
in breast cancer cells. Rel factors have been found to functionally interact
with other transcription factors. Here we demonstrate a physical and functional
association between the RelA subunit of NF-kappaB and AhR resulting in the
activation of c-myc gene transcription in breast cancer cells. RelA and AhR
proteins were co-immunoprecipitated from cytoplasmic and nuclear extracts of
non-malignant MCF-10F breast epithelial and malignant Hs578T breast cancer
cells. In transient co-transfection, RelA and AhR gene products demonstrated
cooperation in transactivation of the c-myc promoter, which was dependent on the
NF-kappaB elements, and in induction of endogenous c-Myc protein levels. A novel
AhR/RelA-containing NF-kappaB element binding complex was identified by
electrophoretic mobility shift analysis of nuclear extracts from RelA and AhR
co-transfected Hs578T cells. Thus, the RelA and AhR proteins functionally
cooperate to bind to NF-kappaB elements and induce c-myc gene expression. These
findings suggest a novel signaling mechanism whereby the Ah receptor can
stimulate proliferation and tumorigenesis of mammary cells.

PMID: 11114727 [PubMed - indexed for MEDLINE]


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16: J Biol Chem. 2000 Oct 13;275(41):32338-46. 

NF-kappa B activity is required for the deregulation of c-myc expression by the
immunoglobulin heavy chain enhancer.

Kanda K, Hu HM, Zhang L, Grandchamps J, Boxer LM.

Center for Molecular Biology in Medicine, Veterans Affairs Palo Alto Health Care
System and the Department of Medicine, Stanford University School of Medicine,
Stanford, California 94305, USA.

The c-myc gene is translocated to one of the immunoglobulin genes in Burkitt's
lymphoma resulting in deregulated expression of c-myc. Several enhancers have
been shown to be important for expression of the immunoglobulin heavy chain
gene. Four enhancer regions (murine-hypersensitive sites (MHS) 1, 2, 3, and 4)
located 3' of the murine immunoglobulin heavy chain gene play a role in
activating expression of the translocated c-myc gene. The enhancer regions also
result in a shift in transcriptional initiation from the P2 promoter to P1 that
is characteristic of the translocated c-myc allele. We found that the most 3'
enhancer region (MHS4) activated the c-myc promoter by 46-fold in the Raji
Burkitt's lymphoma cell line, and it was the most active enhancer in these
cells. The addition of enhancer regions MHS1,2 and 3 to MHS4 increased c-myc
transcription by an additional 3-fold and resulted in the full promoter shift
from P2 to P1. By deletion analysis of enhancer region MHS4, we located a region
that was critical for the transcriptional activity of MHS4. Electrophoretic
mobility shift assay analysis revealed that NF-kappaB/Rel family members bound
to this region. Mutation of the NF-kappaB binding site abolished both the
enhancer activity and the promoter shift activity of MHS4. An active NF-kappaB
site was also identified in the human HS4 enhancer. Inhibition of c-myc promoter
activity driven by the immunoglobulin enhancers was observed with expression of
a super-repressor IkappaBalpha construct. These results indicate that the
NF-kappaB/Rel transcription factors play an important role in the deregulation
of the translocated c-myc gene in Burkitt's lymphoma and suggest that
interference with NF-kappaB function may represent a new approach to the
treatment of Burkitt's lymphoma.

PMID: 10931834 [PubMed - indexed for MEDLINE]


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17: Nucleic Acids Res. 2000 Feb 1;28(3):800-8. 

Activation of c-myc promoter P1 by immunoglobulin kappa gene enhancers in
Burkitt lymphoma: functional characterization of the intron enhancer motifs
kappaB, E box 1 and E box 2, and of the 3' enhancer motif PU.

Wittekindt NE, Hortnagel K, Geltinger C, Polack A.

GSF-National Research Center for Environment and Health, Institute of Clinical
Molecular Biology and Tumor Genetics, Marchioninistrasse 25, D-81377 Munich,
Germany. wittekin@biochem.mpg.de

Deregulated expression of the proto-oncogene c- myc in Burkitt lymphoma (BL)
cells carrying a t(2;8) translocation is mediated by a synergistic interaction
of the translocated immunoglobulin (Ig) kappa gene intron (kappaEi) and 3'
(kappaE3') enhancers and characterized by a strong activation of the promoter
P1. We have investigated the functional role of distinct kappa enhancer sequence
motifs in P1 activation on both mini-chromosomes and reporter gene constructs.
Stable and transient transfections of BL cells revealed critical roles of the
kappaEi and kappaE3' elements kappaB and PU, respectively. Joint mutation of
kappaB and PU completely abolished P1 activity, implying that an interaction of
kappaB- and PU-binding factors is essential for the enhancer synergism. Mutation
of the E box 1 and E box 2 motifs markedly decreased P1 activity in transient
but not in stable transfection experiments. Co-expression of the NF-kappaB
subunit p65(RelA) and Sp1, an essential factor for P1 transcription, in
Drosophila melanogaster SL2 cells synergistically enhanced promoter activity.
Our results support a model which proposes cross-talk between promoter and
enhancer binding factors as the basic mechanism for kappa enhancer-mediated c-
myc activation in BL cells.

PMID: 10637333 [PubMed - indexed for MEDLINE]


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18: J Immunol. 1999 Jan 1;162(1):314-22. 

A NF-kappa B/c-myc-dependent survival pathway is targeted by corticosteroids in
immature thymocytes.

Wang W, Wykrzykowska J, Johnson T, Sen R, Sen J.

Rosenstiel Research Center and Department of Biology, Brandeis University,
Waltham, MA 02254, USA.

Glucocorticoid hormones modulate T cell maturation in vivo. While low levels of
hormones are required for appropriate T cell development, high levels of
glucocorticoid hormones target immature developing thymocytes for cell death
during systemic stress. In this report, we propose a molecular mechanism for the
induction of apoptosis in CD4+CD8+ double-positive thymocytes by dexamethasone
in vivo. Dexamethasone injection induced the expression of IkappaBalpha and
IkappaBbeta in thymocytes and down-regulated NF-kappaB DNA binding activated by
intrathymic signals. Down-regulation of NF-kappaB DNA binding preceded cell
death, suggesting that NF-kappaB may be important for the survival of immature
thymocytes. In addition, ex vivo treatment of thymocyte single-cell suspension
with dexamethasone accelerated p65/RelA down-regulation and cell death.
Conversely, NF-kappaB induction diminished dexamethasone-induced death.
Expression of the c-myc proto-oncogene, a NF-kappaB target, was also reduced in
thymocytes of dexamethasone-treated animals, and ectopic transgenic expression
of c-myc in mice provided partial rescue of double-positive thymocytes from
dexamethasone mediated cell death. These observations suggest that viability of
CD4+CD8+ thymocytes may be maintained by an NF-kappaB/c-myc-dependent pathway in
vivo.

PMID: 9886401 [PubMed - indexed for MEDLINE]


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19: J Immunol. 1998 Nov 1;161(9):4572-82. 

Adenovirus-mediated expression of a dominant negative mutant of p65/RelA
inhibits proinflammatory gene expression in endothelial cells without
sensitizing to apoptosis.

Soares MP, Muniappan A, Kaczmarek E, Koziak K, Wrighton CJ, Steinhauslin F,
Ferran C, Winkler H, Bach FH, Anrather J.

Immunobiology Research Center, Beth Israel Deaconess Medical Center, Harvard
Medical School, Boston, MA 02115, USA.

We hypothesized that blocking the induction of proinflammatory genes associated
with endothelial cell (EC) activation, by inhibiting the transcription factor
nuclear factor kappaB (NF-kappaB), would prolong survival of vascularized
xenografts. Our previous studies have shown that inhibition of NF-kappaB by
adenovirus-mediated overexpression of I kappaB alpha suppresses the induction of
proinflammatory genes in EC. However, I kappaB alpha sensitizes EC to
TNF-alpha-mediated apoptosis, presumably by suppressing the induction of the
NF-kappaB-dependent anti-apoptotic genes A20, A1, manganese superoxide dismutase
(MnSOD), and cellular inhibitor of apoptosis 2. We report here that adenovirus
mediated expression of a dominant negative C-terminal truncation mutant of
p65/RelA (p65RHD) inhibits the induction of proinflammatory genes, such as
E-selectin, ICAM-1, VCAM-1, IL-8, and inducible nitric oxide synthase, in EC as
efficiently as does I kappaB alpha. However, contrary to I kappaB alpha, p65RHD
does not sensitize EC to TNF-alpha-mediated apoptosis although both inhibitors
suppressed the induction of the anti-apoptotic genes A20, A1, and MnSOD equally
well. We present evidence that this difference in sensitization of EC to
apoptosis is due to the ability of p65RHD, but not I kappaB alpha, to inhibit
the constitutive expression of c-myc, a gene involved in the regulation of
TNF-alpha-mediated apoptosis. These data demonstrate that it is possible to
block the expression of proinflammatory genes during EC activation by targeting
NF-kappaB, without sensitizing EC to apoptosis and establishes the role of c-myc
in controlling induction of apoptosis during EC activation. Finally, these data
provide the basis for a potential approach to suppress EC activation in vivo in
transgenic pigs to be used as donors for xenotransplantation.

PMID: 9794384 [PubMed - indexed for MEDLINE]


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20: Blood. 1998 Jun 1;91(11):4136-44. 

NF-kappaB transcription factors are involved in normal erythropoiesis.

Zhang MY, Sun SC, Bell L, Miller BA.

Department of Pediatrics, Pennsylvania State University College of Medicine,
Milton S. Hershey Medical Center, Hershey, PA 17033-0850, USA.

NF-kappaB/Rel designates a widely distributed family of transcription factors
involved in immune and acute phase responses. Here, the expression and function
of NF-kappaB factors in erythroid proliferation and differentiation were
explored. In an erythroleukemia cell line, TF-1, high levels of p105/p50,
p100/p52, p65, and IkappaBalpha were detected 24 hours after growth factor
deprivation. In response to granulocyte-macrophage colony-stimulating factor
(GM-CSF) stimulation, significant induction of p52 expression was observed.
GM-CSF also induced nuclear translocation of both p52 and p65. No induction of
NF-kappaB factors was observed with erythropoietin stimulation of TF-1 cells.
Overexpression of p52 and p65 in TF-1 cells by transient transfection resulted
in significant induction of a kappaB-TATA-luciferase reporter plasmid, showing
that these factors are functional in vivo in erythroid cells. To determine
whether NF-kappaB factors may play a role in normal erythropoiesis, levels of
these factors were determined in burst-forming unit-erythroid (BFU-E)-derived
cells at different stages of differentiation. The NF-kappaB factors p105/p50,
p100/p52, and p65 were highly expressed in early BFU-E-derived precursors, which
are rapidly proliferating, and declined during maturation. Furthermore, nuclear
levels of NF-kappaB factors p50, p52, and p65 were higher in less mature
precursors (day 10 BFU-E-derived cells) compared with more differentiated (day
14) erythroblasts. In nuclear extracts from day 10 BFU-E-derived cells, p50,
p52, and p65 were able to form complexes, which bound to kappaB sites in the
promoters of both the c-myb and c-myc genes, suggesting that c-myb and c-myc may
be among the kappaB-containing genes regulated by NF-kappaB factors in normal
erythroid cells. Taken together, these data show that NF-kappaB factors are
modulated by GM-CSF and suggest they function to regulate specific kappaB
containing genes involved in erythropoiesis.

PMID: 9596659 [PubMed - indexed for MEDLINE]


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21: J Biol Chem. 1998 Jan 2;273(1):592-9. 

Discrimination between RelA and RelB transcriptional regulation by a dominant
negative mutant of IkappaBalpha.

Ferreira V, Tarantino N, Korner M.

Laboratoire d'Immunologie Cellulaire, CNRS URA 625, Bat. CERVI, Hopital de la
Pitie Salpetriere, 83, Bd. de l'Hopital, 75013 Paris, France.

RelA and RelB belong to the nuclear factor-kappaB (NF-kappaB-Rel) transcription
factor family. Both proteins are structurally and functionally related, but
their intracellular and tissue distributions are different. In resting cells,
RelB is found mostly in the nucleus, whereas RelA is sequestered in the cytosol
by protein inhibitors, among which IkappaBalpha is the dominant form in
lymphocytes. Upon cellular activation IkappaBalpha is proteolyzed, allowing RelA
dimers to enter the nucleus and activate target genes. To study the selectivity
of gene regulation by RelA and RelB, we generated T cell lines stably expressing
a dominant negative mutant of IkappaBalpha. We show that selective inhibition of
RelA-NF-kappaB decreased induction of NFKB1, interleukin-2, and
interleukin-2Ralpha genes but not c-myc. Transcription driven by the
IkappaBalpha promoter was blocked by the transgenic IkappaBalpha; however, wild
type IkappaBalpha was expressed in the transgenic cell clones but with much
slower kinetics than that in control cells. Wild type IkappaBalpha expression
was concomitant with RelB up-regulation, suggesting that RelB could be involved
in transcription of IkappaBalpha through binding to an alternative site. These
results indicate that RelB and RelA have both distinct and overlapping effects
on gene expression.

PMID: 9417120 [PubMed - indexed for MEDLINE]


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22: Mol Cell Biol. 1997 Dec;17(12):7375-85. 

I kappaB alpha physically interacts with a cytoskeleton-associated protein
through its signal response domain.

Crepieux P, Kwon H, Leclerc N, Spencer W, Richard S, Lin R, Hiscott J.

Terry Fox Molecular Oncology Group, Lady Davis Institute for Medical Research,
Department of Medicine, McGill University, Montreal, Que., Canada.

The I kappaB alpha protein is a key molecular target involved in the control of
NF-kappaB/Rel transcription factors during viral infection or inflammatory
reactions. This NF-kappaB-inhibitory factor is regulated by posttranslational
phosphorylation and ubiquitination of its amino-terminal signal response domain
that targets I kappaB alpha for rapid proteolysis by the 26S proteasome. In an
attempt to identify regulators of the I kappaB alpha inhibitory activity, we
undertook a yeast two-hybrid genetic screen, using the amino-terminal end of I
kappaB alpha as bait, and identified 12 independent interacting clones. Sequence
analysis identified some of these cDNA clones as Dlc-1, a sequence encoding a
small, 9-kDa human homolog of the outer-arm dynein light-chain protein. In the
two-hybrid assay, Dlc-1 also interacted with full-length I kappaB alpha protein
but not with N-terminal-deletion-containing versions of I kappaB alpha. I kappaB
alpha interacted in vitro with a glutathione S-transferase-Dlc-1 fusion protein,
and RelA(p65) did not displace this association, demonstrating that p65 and
Dlc-1 contact different protein motifs of I kappaB alpha. Importantly, in HeLa
and 293 cells, endogenous and transfected I kappaB alpha coimmunoprecipitated
with Myc-tagged or endogenous Dlc-1. Indirect immunofluorescence analyzed by
confocal microscopy indicated that Dlc-1 and I kappaB alpha colocalized with
both nuclear and cytoplasmic distribution. Furthermore, Dlc-1 and I kappaB alpha
were found to associate with the microtubule organizing center, a perinuclear
region from which microtubules radiate. Likewise, I kappaB alpha colocalized
with alpha-tubulin filaments. Taken together, these results highlight an
intriguing interaction between the I kappaB alpha protein and the human homolog
of a member of the dynein family of motor proteins and provide a potential link
between cytoskeleton dynamics and gene regulation.

PMID: 9372968 [PubMed - indexed for MEDLINE]


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23: Eur J Immunol. 1997 Sep;27(9):2269-78. 

Proteasome regulation of Fas ligand cytotoxicity.

Matsui K, Omura S, Cui H, Schauer SL, Sonenshein GE, Ju ST.

Department of Pathology and Laboratory Medicine, Boston University School of
Medicine, MA 02118, USA.

The role of NF-kappa B in regulating FasL-mediated cytotoxicity was investigated
by using lactacystin. Lactacystin is a microbial metabolite known to inhibit
only the protease activity of the proteasome, which is required for NF-kappa B
translocation. When activated by immobilized anti-CD3 monoclonal antibody,
hybridoma T cells (5D5) degraded I kappa B beta, translocated NF-kappa B into
the nucleus, transcribed immediate-early genes and the Fas ligand (FasL) gene,
and expressed FasL-mediated cytotoxicity. Lactacystin strongly blocked I kappa B
beta degradation and the translocation of NF-kappa B (p50/RelA heterodimer), but
had little effect on the expression of the transcription factors, Oct-1 and
AP-1. Moreover, lactacystin did not inhibit the nuclear translocation of NF-ATp
whereas cyclosporin A inhibited the translocation of both NF-kappa B and NF-ATp.
The expression of c-myc and nur77, two immediate-early genes implicated in FasL
gene activation, was blocked by lactacystin. Subsequently, the expression of
FasL gene and FasL-mediated cytotoxicity was inhibited. LLnL, a well-known
peptide aldehyde which inhibits the protease activities of the proteasome and
cysteine proteases, also inhibited NF-kappa B translocation and FasL-mediated
cytotoxicity. However, these events were not inhibited by the highly specific
cysteine protease inhibitor E64. These observations provide further evidence
that FasL cytotoxicity is regulated by the proteasome. Furthermore, lactacystin
must be added early in order to efficiently inhibit the induction of FasL
cytotoxicity, indicating that the early events are critical for FasL gene
activation. Our study integrates the proteasome-dependent I kappa B degradation
and NF-kappa B translocation into a T cell activation cascade which results in
FasL gene activation and the expression of FasL-mediated cytotoxicity.

PMID: 9341769 [PubMed - indexed for MEDLINE]


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24: J Virol. 1997 Aug;71(8):5972-81. 

VBP and RelA regulate avian leukosis virus long terminal repeat-enhanced
transcription in B cells.

Curristin SM, Bird KJ, Tubbs RJ, Ruddell A.

Department of Microbiology and Immunology and Cancer Center, University of
Rochester, School of Medicine and Dentistry, New York 14642, USA.

The avian leukosis virus (ALV) long terminal repeat (LTR) contains a compact
transcription enhancer that is active in many cell types. A major feature of the
enhancer is multiple CCAAT/enhancer element motifs that could be important for
the strong transcriptional activity of this unit. The contributions of the three
CCAAT/enhancer elements to LTR function were examined in B cells, as this cell
type is targeted for ALV tumor induction following integration of LTR sequences
next to the c-myc proto-oncogene. One CCAAT/enhancer element, termed a3, was
found to be the most critical for LTR enhancement in transiently transfected B
lymphoma cells, while in chicken embryo fibroblasts all three elements
contributed equally to enhancement. Gel shift assays demonstrated that
vitellogenin gene-binding protein (VBP), a member of the PAR subfamily of C/EBP
factors, is a major component of the nuclear proteins binding to the a3
CCAAT/enhancer element. VBP activated transcription through the a3
CCAAT/enhancer element, supporting the idea that VBP is important for LTR
enhancement in B cells. A member of the Rel family of proteins was also
identified as a component of the a3 protein binding complex in B cells. Gel
shift and immunoprecipitation assays indicated that this factor is RelA. Gel
shift assays demonstrated that while RelA does not bind directly to the LTR
CCAAT/enhancer elements, it does interact with VBP to potentiate VBP DNA binding
activity. The synergistic interaction of VBP and RelA increased CCAAT/enhancer
element-mediated transcription, indicating that both factors may be important
for viral LTR regulation and also for expression of many cellular genes.

PMID: 9223487 [PubMed - indexed for MEDLINE]


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25: Oncogene. 1994 Aug;9(8):2391-8. 

Failure of the splicing variant p65 delta of the NF-kappa B subunit p65 to
transform fibroblasts.

Grimm S, Baeuerle PA.

Institute of Biochemistry, Albert-Ludwigs-University, Freiburg, Germany.

A naturally occurring splice variant of the p65 subunit of the inducible
transcription factor NK-kappa B, called p65 delta, has recently been reported to
transform rat-1 fibroblasts in transfection experiments. Criteria for
transformation included focus formation, growth in soft agar and tumor formation
in athymic mice. In the present study we provide evidence that p65 delta cannot
transform either rat-1 or rat-2 cells although p65 delta was efficiently
expressed and showed all transcriptional activities reported previously. Eleven
rat-1 fibroblasts cells lines selected for stably overexpressing p65 delta also
showed neither focus formation nor anchorage-independent growth in soft agar. No
transformation of primary rat fibroblasts was evident when p65 delta was
cotransfected with either ras or myc oncogenes, whereas a combination of the two
oncogenes caused focus formation. We submit that the transforming potential of
p65 delta is highly conditional and presumably restricted to a particular
subclone of rat-1 cells.

PMID: 8036023 [PubMed - indexed for MEDLINE]


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26: Mol Cell Biol. 1994 Aug;14(8):5349-59. 

Sequential induction of NF-kappa B/Rel family proteins during B-cell terminal
differentiation.

Liou HC, Sha WC, Scott ML, Baltimore D.

Rockfeller University, New York, New York 10021.

The NF-kappa B/Rel family of at least five transcription factor polypeptides is
thought to function both as a developmental regulator in B cells and as a rapid
response system in all cells. To examine this notion in more detail, we
determined the protein contents of both the inducible and constitutive NF-kappa
B/Rel activities in a pre-B-cell line, 70Z/3, and a mature B-cell line, WEHI
231. NF-kappa B p50/p65 is the major inducible nuclear complex after
lipopolysaccharide or phorbol myristate acetate treatment of 70Z/3 cells. The
constitutive and inducible complexes in WEHI 231 cells are mainly composed of
p50 and Rel. The constitutive or induced activities are all sensitive to I kappa
B-alpha, but this inhibitor is very short-lived in WEHI 231 cells, suggesting
that the balance between synthesis and degradation of I kappa B-alpha determines
whether a particular cell lineage has constitutive activity. A patterned
expression of the NF-kappa B/Rel activator proteins emerges from an analysis of
other B-lineage cell lines and splenic B cells: mainly p50 and p65 in pre-B (and
non-B) cells, a predominance of Rel and p50 in mature B cells, and expression of
p52 and RelB in plasmacytoma lines. This ordered pattern of regulators may
reflect the requirement for expression of different genes during terminal B-cell
differentiation because different combinations of NF-kappa B/Rel family members
preferentially activate distinct kappa B sites in reporter constructs.

PMID: 8035813 [PubMed - indexed for MEDLINE]


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