1: Oncogene. 2005 Sep 29;24(43):6574-83. 

Immortalized fibroblasts from NF-kappaB RelA knockout mice show phenotypic
heterogeneity and maintain increased sensitivity to tumor necrosis factor alpha
after transformation by v-Ras.

Gapuzan ME, Schmah O, Pollock AD, Hoffmann A, Gilmore TD.

Department of Biology, Boston University, 5 Cummington Street, Boston, MA 02215,
USA.

Activation of the NF-kappaB pathway can either promote or block apoptosis and
oncogenesis in different cell types and circumstances. In this report, we show
that independently derived immortalized mouse embryonic fibroblast cell lines
prepared from RelA knockout mice have different phenotypes, based on their
sensitivity to tumor necrosis factor alpha (TNFalpha)-induced apoptosis,
morphology, ability to form colonies in soft agar, and the presence of distinct
kappaB site-binding complexes. In addition, these RelA-deficient cell lines
appear to have distinct alterations in the p53 pathway, which correlate with the
normal vs transformed status of individual cell lines. We have also infected
mouse embryonic fibroblasts lacking RelA, c-Rel or p50 with a retrovirus for the
expression of v-Ha-Ras to determine whether individual NF-kappaB family members
are required for Ras-mediated transformation. All three NF-kappaB-deficient cell
types could be transformed by v-Ha-Ras. However, v-Ras-infected RelA-deficient
cells formed colonies in soft agar at an approximately fourfold reduced
efficiency compared to v-Ras-transformed control mouse 3T3 and p50-deficient
cells. Ras transformation did not alter the sensitivity of RelA-deficient cells
to TNFalpha-induced apoptosis, and Ras transformation did not affect the general
resistance of 3T3, c-Rel-deficient, and p50-deficient cells to TNFalpha-induced
apoptosis. However, TNFalpha specifically and dose-dependently decreased the
ability of v-Ras-transformed RelA-deficient cells to form colonies in soft agar.
These results suggest that RelA is a potential protein target for human tumors
driven by oncogenic Ras mutations, but caution that inhibition of RelA may
promote tumorigenesis in some circumstances.

PMID: 16027734 [PubMed - indexed for MEDLINE]


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2: Oncogene. 2005 Apr 14;24(16):2635-46. 

Epstein-Barr virus latent membrane protein 1 represses p53-mediated DNA repair
and transcriptional activity.

Liu MT, Chang YT, Chen SC, Chuang YC, Chen YR, Lin CS, Chen JY.

National Health Research Institutes, 3F No. 109, Section 6, Min-Chuan East Road,
Taipei 114, Taiwan.

The latent membrane protein 1 (LMP1) of Epstein-Barr virus (EBV), a viral
oncogene, is essential for transformation of resting B cells by the virus. We
previously demonstrated that LMP1 could repress DNA repair in p53-wild-type and
p53-deficient human epithelial cells. In this study, using a host cell
reactivation (HCR) assay, we demonstrated that p53-enhanced DNA repair was
repressed by LMP1 in p53-deficient cells. Moreover, we found that LMP1 was able
to repress p53-dependent transcriptional activity. Regarding the mechanisms of
p53 repression by LMP1, we found that LMP1 did not inhibit p53 function through
direct interaction, by promoting protein degradation or reducing its DNA-binding
ability. Using chimeric proteins in the reporter assay, we demonstrated that
LMP1 inhibited p53 transactivation by influencing the N-terminal transactivation
domain of p53. Subsequent experiments using various LMP1 deletion mutants
indicated that a C-terminus-activating region of LMP1, CTAR1 or CTAR2, is
responsible for the repression of p53-mediated DNA repair and p53-dependent
transcription, which is correlated with the region responsible for NF-kappaB
activation. Furthermore, blockage of NF-kappaB signalling by IkappaB-DeltaN was
shown to abolish the repression of p53 by LMP1, suggesting that LMP1 likely
repressed p53 function through the NF-kappaB pathway. Based on these results, we
propose that inhibition of p53-dependent transcriptional activity and DNA repair
by LMP1 results in the loss of p53 activity for maintaining genomic stability,
which may contribute to the oncogenesis of LMP1 in human epithelial cells.

PMID: 15829976 [PubMed - indexed for MEDLINE]


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3: Biochem Biophys Res Commun. 2005 May 20;330(4):1034-40. 

TNF-alpha promotes Doxorubicin-induced cell apoptosis and anti-cancer effect
through downregulation of p21 in p53-deficient tumor cells.

Cao W, Chi WH, Wang J, Tang JJ, Lu YJ.

Laboratory of Medical Molecular Virology, Fudan University, 138 Yixueyuan Road,
Shanghai 200032, China.

p53 is a key regulator in cell apoptosis, and cancer cells deficient in p53
expression fail to respond to chemotherapy. Here we show that effective
Doxorubicin (DOX)-induced apoptosis is p53-dependent. However, an alternative
treatment of DOX/TNF-alpha/DOX restored sensitivity of p53-deficient cells to
DOX-induced apoptosis. Treatment of cells with TNF-alpha resulted in a decrease
of p21 (waf1/cip1/sdi1) expression following second dose of DOX. In previous
work, we demonstrated that p21 suppressed DOX-induced apoptosis via its
(cyclin-dependent kinase) CDK-binding and CDK-inhibitory activity. Thus, we
propose that TNF-alpha enhances the anti-cancer effect of DOX through
suppressing the anti-apoptotic activity of p21, and that a combined treatment
TNF-alpha/Dox is an effective chemotherapeutic strategy for p53-deficient
cancers.

PMID: 15823547 [PubMed - indexed for MEDLINE]


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4: EMBO J. 2005 Mar 23;24(6):1157-69. Epub 2005 Mar 10. 

Regulation of NF-kappaB and p53 through activation of ATR and Chk1 by the ARF
tumour suppressor.

Rocha S, Garrett MD, Campbell KJ, Schumm K, Perkins ND.

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

The ARF tumour suppressor is a central component of the cellular defence against
oncogene activation. In addition to activating p53 through binding Mdm2, ARF
possesses other functions, including an ability to repress the transcriptional
activity of the antiapoptotic RelA(p65) NF-kappaB subunit. Here we demonstrate
that ARF induces the ATR- and Chk1-dependent phosphorylation of the RelA
transactivation domain at threonine 505, a site required for ARF-dependent
repression of RelA transcriptional activity. Consistent with this effect, ATR
and Chk1 are required for ARF-induced sensitivity to tumour necrosis factor
alpha-induced cell death. Significantly, ATR activity is also required for
ARF-induced p53 activity and inhibition of proliferation. ARF achieves these
effects by activating ATR and Chk1. Furthermore, ATR and its scaffold protein
BRCA1, but not Chk1, relocalise to specific nucleolar sites. These results
reveal novel functions for ARF, ATR and Chk1 together with a new pathway
regulating RelA NF-kappaB function. Moreover, this pathway provides a mechanism
through which ARF can remodel the cellular response to an oncogenic challenge
and execute its function as a tumour suppressor.

PMID: 15775976 [PubMed - indexed for MEDLINE]


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5: J Biol Chem. 2005 Apr 29;280(17):16843-50. Epub 2005 Jan 25. 

Function of polo-like kinase 3 in NF-kappaB-mediated proapoptotic response.

Li Z, Niu J, Uwagawa T, Peng B, Chiao PJ.

Department of Surgical Oncology, University of Texas M. D. Anderson Cancer
Center, Houston, Texas 77030, USA.

RelA, the p65 subunit of NF-kappaB transcription factors, plays a key role in
regulation of antiapoptotic and proapoptotic responses. However, the downstream
target genes regulated by RelA-NF-kappaB in the initiation of proapoptotic
signaling were not identified. We previously showed that RelA-NF-kappaB
functioned as a proapoptotic factor by activating the p53-signaling pathway in
response to doxycycline-induced superoxide. In the present study, we demonstrate
that the ability of doxycycline/superoxide to induce expression of polo-like
kinase 3 (Plk3) depends on NF-kappaB activity. We identified a kappaB binding
site in the promoter of Plk3, and this kappaB site is directly involved in its
induction by the RelA-NF-kappaB complex. Plk3 formed a complex with p53 and was
involved in the phosphorylation of p53 on Ser-20 in response to superoxide.
Inhibition of Plk3 expression by Plk3 small interfering RNA suppressed the
doxycycline/superoxide-mediated apoptosis. Overexpression of wild-type Plk3 in
HCT116 p53+/+ cells induced rapid apoptosis, whereas overexpression of wild-type
Plk3 in HCT116 p53-/- cells and the kinase-defective mutant Plk3(K91R) in p53+/+
cells induced delayed onset of apoptosis. Furthermore, mutagenesis of Plk3
showed that the N-terminal domain (amino acids 1-26) is essential for the
induction of delay onset of apoptosis. These data show that Plk3 is a
RelA-NF-kappaB-regulated gene that induces apoptosis in both p53-dependent and
-independent signaling pathways, suggesting a possible mechanism for
RelA-NF-kappaB-regulated proapoptotic responses.

PMID: 15671037 [PubMed - indexed for MEDLINE]


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6: J Biol Chem. 2005 Mar 18;280(11):10326-32. Epub 2004 Dec 20. 

A novel NF-kappaB pathway involving IKKbeta and p65/RelA Ser-536 phosphorylation
results in p53 Inhibition in the absence of NF-kappaB transcriptional activity.

Jeong SJ, Pise-Masison CA, Radonovich MF, Park HU, Brady JN.

Virus Tumor Biology Section, Laboratory of Cellular Oncology, Center for Cancer
Research, NCI, National Institutes of Health, Bethesda, Maryland 20892-5055,
USA.

Nuclear factor kappaB (NF-kappaB) plays an important role in regulating cellular
transformation and apoptosis. The human T-cell lymphotropic virus type I
protein, Tax, which is critical for viral transformation, modulates the
transcription of several cellular genes through activation of NF-kappaB. We have
demonstrated previously that Tax inhibits p53 activity through the p65/RelA
subunit of NF-kappaB. We now present evidence that suggests that the upstream
kinase IKKbeta plays an important role in Tax-induced p53 inhibition through
phosphorylation of p65/RelA at Ser-536. First, mouse embryo fibroblast (MEF)
IKKbeta-/-cells did not support Tax-mediated p53 inhibition, whereas MEFs
lacking IKKalpha allowed Tax inhibition of p53. Second, transfection of IKKbeta
wild type (WT), but not a kinase-dead mutant, into IKKbeta-/-cells rescued p53
inhibition by Tax. Third, the IKKbeta-specific inhibitor SC-514 decreased the
ability of Tax to inhibit p53. Fourth, we show that phosphorylation of p65/RelA
at Ser-536 is important for Tax inhibition of p53 using MEF p65/RelA-/-cells
transfected with p65/RelA WT or mutant plasmids. Moreover, Tax induced p65/RelA
Ser-536 phosphorylation in WT or IKKalpha-/- cells but failed to induce the
phosphorylation of p65/RelA Ser-536 in IKKbeta-/-cells, suggesting a link
between IKKbeta and p65/RelA phosphorylation. Consistent with this observation,
blocking IKKbeta kinase activity by SC-514 decreases the phosphorylation of
p65/RelA at Ser-536 in the presence of Tax in human T-cell lymphotropic virus
type I-transformed cells. Finally, the ability of Tax to inhibit p53 is
distinguished from the NF-kappaB transcription activation pathway. Our work,
therefore, describes a novel Tax-NF-kappaB p65/RelA pathway that functions to
inhibit p53 but does not require NF-kappaB transcription activity.

PMID: 15611068 [PubMed - indexed for MEDLINE]


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7: FEBS Lett. 2004 Jul 16;570(1-3):7-12. 

ING4 induces G2/M cell cycle arrest and enhances the chemosensitivity to
DNA-damage agents in HepG2 cells.

Zhang X, Xu LS, Wang ZQ, Wang KS, Li N, Cheng ZH, Huang SZ, Wei DZ, Han ZG.

State Key Laboratory of Bioreactor Engineering, New World Institute of
Biotechnology, East China University of Science and Technology, Shanghai 200237,
China.

The known members of inhibitor of growth (ING) gene family are considered as
candidate tumor suppressor genes. ING4, a novel member of ING family, is
recently reported to interact with tumor suppressor p53, p300 (a major component
of histone acetyl transferase complexes), and p65(RelA) subunit of NF-kappaB. In
this study, we investigated the cellular behaviors of HepG2 cells with exogenous
ING4. Interestingly, the overexpression of ING4 negatively regulated the cell
growth with significant G2/M arrest of cell cycle, and moreover, enhanced the
cell apoptosis triggered by serum starvation in HepG2 cells. Furthermore, the
exogenous ING4 could upregulate endogenous p21 and Bax in HepG2 cells, not in
p53-deficient Saos-2 cells, suggesting that G2/M arrest induced by ING4 could be
mediated by the increased p21 expression in a p53-dependent manner, although
there is no significant increase of p53 expression in HepG2 cells. Moreover,
HepG2 cells with exogenous ING4 could significantly increase cell death, as
exposed to some DNA-damage agents, such as etoposide and doxorubicin, implying
that ING4 could enhance chemosensitivity to certain DNA-damage agents in HepG2
cells.

PMID: 15251430 [PubMed - indexed for MEDLINE]


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8: Blood. 2004 Sep 1;104(5):1490-7. Epub 2004 May 20. 

HTLV-I Tax induces a novel interaction between p65/RelA and p53 that results in
inhibition of p53 transcriptional activity.

Jeong SJ, Radonovich M, Brady JN, Pise-Masison CA.

Virus Tumor Biology Section, Basic Research Laboratory, Center for Cancer
Research, National Cancer Institute/NIH, Bldg 41/B303, 41 Library Drive,
Bethesda, MD 20892, USA.

Nuclear factor kappaB (NF-kappaB) activation plays a critical role in
oncogenesis by human T-cell lymphotrophic virus type I (HTLV-I), the etiologic
agent of adult T-cell leukemia (ATL), and is indispensable for maintenance of
the malignant phenotype. In T lymphocytes, Tax-mediated p53 inhibition is
dependent on Tax activation of the NF-kappaB pathway and is linked to p53
phosphorylation. We now report that blocking NF-kappaB transcriptional
activation in HTLV-I-transformed cells restores p53 activity. Further, using
mouse embryo fibroblast (MEF) null cells and antisense oligonucleotides to
inhibit expression of NF-kappaB family members, we demonstrate that the p65
subunit of NF-kappaB is uniquely involved in p53 inhibition.
Coimmunoprecipitation assays demonstrate an interaction between p65 and p53 in
HTLV-I-transformed cells. In transient transfection assays, we demonstrate that
Tax induces the p53-p65 interaction. Phosphorylation of p53 at serines 15 and
392 is critical for complex formation. Importantly, Tax-mediated p53 inhibition
correlates with p65 and p53 interaction. By using chromatin immunoprecipitation
(ChIP) assays, we find that in HTLV-I-transformed cells p53 and p65 form a
complex on the inactive, p53-responsive murine double minute 2 (MDM2) promoter.
Consistent with reduced transcriptional activity, transcription factor IID
(TFIID) binding is not observed. These studies identify a unique mechanism for
p53 regulation by the p65/RelA subunit of NF-kappaB.

PMID: 15155458 [PubMed - indexed for MEDLINE]


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9: J Biol Chem. 2004 Jun 25;279(26):27549-59. Epub 2004 Apr 21. 

Stabilization of p53 is a novel mechanism for proapoptotic function of
NF-kappaB.

Fujioka S, Schmidt C, Sclabas GM, Li Z, Pelicano H, Peng B, Yao A, Niu J, Zhang
W, Evans DB, Abbruzzese JL, Huang P, Chiao PJ.

Department of Surgical Oncology, The University of Texas M. D. Anderson Cancer
Center, Houston, Texas 77030, USA.

Both pro- and antiapoptotic activities of NF-kappaB transcription factor have
been observed; however, less is known about the mechanism by which NF-kappaB
induces apoptosis. To elucidate how NF-kappaB regulates proapoptotic signaling,
we performed functional analyses using wild-type, ikk1(-/-), ikk2(-/-),
rela(-/-) murine fibroblasts, MDAPanc-28/Puro, MDAPanc-28/IkappaBalphaM, and
HCT116/p53(+/+) and HCT116/p53(-/-) cells with investigational anticancer agent
doxycycline as a superoxide inducer for generating apoptotic stimulus. In this
report, we show that doxycycline increased superoxide generation and
subsequently activated NF-kappaB, which in turn up-regulated p53 expression and
increased the stability and DNA binding activity of p53. Consequently,
NF-kappaB-dependent p53 activity induced the expression of p53-regulated genes
PUMA and p21(waf1) as well as apoptosis. Importantly, lack of RelA, IKK, and p53
as well as expression of a dominant negative IkappaBalpha (IkappaBalphaM)
inhibited NF-kappaB-dependent p53 activation and apoptosis. The
doxycycline-induced NF-kappaB activation was not inhibited in HCT116/p53(-/-)
cells. Our results demonstrate that NF-kappaB plays an essential role in
activation of wild-type p53 tumor suppressor to initiate proapoptotic signaling
in response to overgeneration of superoxide. Thus, these findings reveal a
mechanism of NF-kappaB-regulated proapoptotic signaling.

PMID: 15102862 [PubMed - indexed for MEDLINE]


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10: J Biol Chem. 2004 Jun 18;279(25):26115-25. Epub 2004 Apr 8. 

p53 induces NF-kappaB activation by an IkappaB kinase-independent mechanism
involving phosphorylation of p65 by ribosomal S6 kinase 1.

Bohuslav J, Chen LF, Kwon H, Mu Y, Greene WC.

Gladstone Institute of Virology and Immunology and the Department of Medicine,
University of California, San Francisco, California 94143-1234, USA.

Apoptosis induced by p53 has been proposed to involve activation of the
transcription factor NF-kappaB. Here we describe the novel molecular mechanism
through which p53 and DNA-damaging agents activate NF-kappaB. NF-kappaB
induction by p53 does not occur through classical activation of the IkappaB
kinases and degradation of IkappaBalpha. Rather, p53 expression stimulates the
serine/threonine kinase ribosomal S6 kinase 1 (RSK1), which in turn
phosphorylates the p65 subunit of NF-kappaB. The lower affinity of
RSK1-phosphorylated p65 for its negative regulator, IkappaBalpha, decreases
IkappaBalpha-mediated nuclear export of shuttling forms of NF-kappaB, thereby
promoting the binding and action of NF-kappaB on cognate kappaB enhancers. These
findings highlight a rather unusual pathway of NF-kappaB activation, which is
utilized by the p53 tumor suppressor.

PMID: 15073170 [PubMed - indexed for MEDLINE]


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11: J Neurosci. 2004 Mar 24;24(12):2963-73. 

Nuclear factor-(kappa)B modulates the p53 response in neurons exposed to DNA
damage.

Aleyasin H, Cregan SP, Iyirhiaro G, O'Hare MJ, Callaghan SM, Slack RS, Park DS.

Ottawa Health Research Institute, Neurosciences, East Division, Ottawa, Ontario,
Canada K1H 8M5.

Previous studies have shown that DNA damage-evoked death of primary cortical
neurons occurs in a p53 and cyclin-dependent kinase-dependent (CDK) manner. The
manner by which these signals modulate death is unclear. Nuclear factor-kappaB
(NF-kappaB) is a group of transcription factors that potentially interact with
these pathways. Presently, we show that NF-kappaB is activated shortly after
induction of DNA damage in a manner independent of the classic IkappaB kinase
(IKK) activation pathway, CDKs, ATM, and p53. Acute inhibition of NF-kappaB via
expression of a stable IkappaB mutant, downregulation of the p65 NF-kappaB
subunit by RNA interference (RNAi), or pharmacological NF-kappaB inhibitors
significantly protected against DNA damage-induced neuronal death. NF-kappaB
inhibition also reduced p53 transcripts and p53 activity as measured by the
p53-inducible messages, Puma and Noxa, implicating the p53 tumor suppressor in
the mechanism of NF-kappaB-mediated neuronal death. Importantly, p53 expression
still induces death in the presence of NF-kappaB inhibition, indicating that p53
acts downstream of NF-kappaB. Interestingly, neurons cultured from p65 or p50
NF-kappaB-deficient mice were not resistant to death and did not show diminished
p53 activity, suggesting compensatory processes attributable to germline
deficiencies, which allow p53 activation still to occur. In contrast to acute
NF-kappaB inhibition, prolonged NF-kappaB inhibition caused neuronal death in
the absence of DNA damage. These results uniquely define a signaling paradigm by
which NF-kappaB serves both an acute p53-dependent pro-apoptotic function in the
presence of DNA damage and an anti-apoptotic function in untreated normal
neurons.

PMID: 15044535 [PubMed - indexed for MEDLINE]


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12: J Biol Chem. 2004 Feb 6;279(6):4877-86. Epub 2003 Nov 10. 

An alternative splice form of Mdm2 induces p53-independent cell growth and
tumorigenesis.

Steinman HA, Burstein E, Lengner C, Gosselin J, Pihan G, Duckett CS, Jones SN.

Department of Pathology, University of Michigan Medical School, Ann Arbor,
Michigan 48109, USA.

The Mdm2 gene is amplified in approximately one-third of human sarcomas and
overexpressed in a variety of other human cancers. Mdm2 functions as an
oncoprotein, in part, by acting as a negative regulator of the p53 tumor
suppressor protein. Multiple spliced forms of Mdm2 transcripts have been
observed in human tumors; however, the contribution of these variant transcripts
to tumorigenesis is unknown. In this report, we isolate alternative splice forms
of Mdm2 transcripts from sarcomas that spontaneously arise in
Mdm2-overexpressing mice, including Mdm2-b, the splice form most commonly
observed in human cancers. Transduction of Mdm2-b into a variety of cell types
reveals that Mdm2-b promotes p53-independent cell growth, inhibits apoptosis,
and up-regulates the RelA subunit of NFkappaB. Furthermore, expression of Mdm2-b
induces tumor formation in transgenic mice. These results identify a
p53-independent role for Mdm2 and determine that an alternate spliced form of
Mdm2 can contribute to formation of cancer via a p53-independent mechanism.
These findings also provide a rationale for the poorer prognosis of those
patients presenting with tumors harboring multiple Mdm2 transcripts.

PMID: 14612455 [PubMed - indexed for MEDLINE]


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13: Mol Cell Biol. 2003 Nov;23(22):8070-83. 

IkappaB kinase-independent IkappaBalpha degradation pathway: functional
NF-kappaB activity and implications for cancer therapy.

Tergaonkar V, Bottero V, Ikawa M, Li Q, Verma IM.

Laboratory of Genetics, The Salk Institute for Biological Studies, 10010 North
Torrey Pines Road, La Jolla, CA 92037, USA.

Antiapoptotic activity of NF-kappaB in tumors contributes to acquisition of
resistance to chemotherapy. Degradation of IkappaB is a seminal step in
activation of NF-kappaB. The IkappaB kinases, IKK1 and IKK2, have been
implicated in both IkappaB degradation and subsequent modifications of NFkappaB.
Using mouse embryo fibroblasts (MEFs) devoid of both IKK1 and IKK2 genes
(IKK1/2(-/-)), we document a novel IkappaB degradation mechanism. We show that
this degradation induced by a chemotherapeutic agent, doxorubicin (DoxR), does
not require the classical serine 32 and 36 phosphorylation or the PEST domain of
IkappaBalpha. Degradation of IkappaBalpha is partially blocked by
phosphatidylinositol 3-kinase inhibitor LY294002 and is mediated by the
proteasome. Free NF-kappaB generated by DoxR-induced IkappaB degradation in
IKK1/2(-/-) cells is able to activate chromatin based NF-kappaB reporter gene
and expression of the endogenous target gene, IkappaBalpha. These results also
imply that modification of NF-kappaB by IKK1 or IKK2 either prior or subsequent
to its release from IkappaB is not essential for NF-kappaB-mediated gene
expression at least in response to DNA damage. In addition, DoxR-induced cell
death in IKK1/2(-/-) MEFs is enhanced by simultaneous inhibition of NF-kappaB
activation by blocking the proteasome activity. These results reveal an
additional pathway of activating NF-kappaB during the course of anticancer
therapy and provide a mechanistic basis for the observation that proteasome
inhibitors could be used as adjuvants in chemotherapy.

PMID: 14585967 [PubMed - indexed for MEDLINE]


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14: Mol Cell. 2003 Jul;12(1):15-25. 

p53- and Mdm2-independent repression of NF-kappa B transactivation by the ARF
tumor suppressor.

Rocha S, Campbell KJ, Perkins ND.

School of Life Sciences, Division of Gene Regulation and Expression, MSI/WTB
Complex, Dow Street, University of Dundee, Dundee, DD1 5EH, Scotland, United
Kingdom.

One mechanism by which a cell affords protection from the transforming effects
of oncogenes is via the action of the tumor suppressor, ARF, which activates p53
by inactivating Mdm2. Many oncogenes have also been shown to activate the
transcription factor NF-kappa B, which can contribute toward the malignant
phenotype in many ways, including an ability to antagonize p53. Here we find
that ARF inhibits NF-kappa B function and its antiapoptotic activity independent
of Mdm2 and p53. ARF represses the transcriptional activation domain of the
NF-kappa B family member RelA by inducing its association with the histone
deacetylase, HDAC1. Further, we show that the response of NF-kappa B to the
oncogene Bcr-Abl is determined by the ARF status of the cell. These results
reveal an important function of ARF that can regulate the NF-kappa B response to
oncogene activation.

PMID: 12887889 [PubMed - indexed for MEDLINE]


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15: Mol Cell Biol. 2003 Jan;23(2):721-32. 

Regulation of RelA (p65) function by the large subunit of replication factor C.

Anderson LA, Perkins ND.

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

The RelA (p65) subunit of NF-kappaB is an important regulator of inflammation,
proliferation, and apoptosis. We have discovered that the large subunit, p140,
of replication factor C (RFC) can function as a regulator of RelA. RFC is a
clamp loader, facilitating the addition and removal of proliferating-cell
nuclear antigen from DNA during replication and repair but can also interact
directly with the retinoblastoma tumor suppressor protein and the transcription
factor C/EBPalpha. We find that RFC (p140) interacts with RelA both in vitro and
in vivo and stimulates RelA transactivation. In contrast, coexpression of
fragments of RFC (p140) that mediate the interaction with RelA results in
transcriptional inhibition. The significance of this regulation was confirmed by
using short interfering RNA oligonucleotides targeted to RFC (p140). Down
regulation of endogenous RFC (p140) inhibits expression from a chromosomally
integrated reporter plasmid induced by endogenous, TNF-alpha-activated
NF-kappaB. Dominant negative fragments of RFC (p140) also cooperate with
overexpressed RelA to induce cell death. Interestingly, RFC (p140) also
interacts with the tumor suppressor p53. Taken together, these observations
suggest that, in addition to its previously described function in DNA
replication and repair, RFC (p140) has an important role as a regulator of
transcription and NF-kappaB activity.

PMID: 12509469 [PubMed - indexed for MEDLINE]


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16: Cancer Cell. 2002 Oct;2(4):335-46. 

A novel protein overexpressed in hepatoma accelerates export of NF-kappa B from
the nucleus and inhibits p53-dependent apoptosis.

Higashitsuji H, Higashitsuji H, Nagao T, Nonoguchi K, Fujii S, Itoh K, Fujita J.

Department of Clinical Molecular Biology, Faculty of Medicine, Kyoto University,
54 shogoin Kawaharacho, Sakyo-ku, 606-8507, Kyoto, Japan.

NF-kappa B is a transcription factor that can protect from or contribute to
apoptosis. Here we report identification of HSCO that binds to NF-kappa B and
inhibits apoptosis. HSCO mRNA was overexpressed in 20 of 30 hepatocellular
carcinomas analyzed. Overexpression of HSCO inhibited caspase 9 activation and
apoptosis induced by DNA damaging agents, while it augmented apoptosis induced
by TNFalpha. Like I kappa B alpha, HSCO inhibited NF-kappa B activity and
abrogated p53-induced apoptosis. However, the underlying mechanism was
different. HSCO is a nuclear-cytoplasmic shuttling protein, bound to RelA
NF-kappa B, and HSCO sequestered it in the cytoplasm by accelerating its export
from the nucleus. These results suggest that overexpression of HSCO suppresses
p53-induced apoptosis by preventing nuclear localization of NF-kappa B during
signaling and thus contributes to hepatocarcinogenesis.

PMID: 12398897 [PubMed - indexed for MEDLINE]


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17: 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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18: Mol Endocrinol. 2002 Aug;16(8):1793-809. 

Estrogen up-regulation of p53 gene expression in MCF-7 breast cancer cells is
mediated by calmodulin kinase IV-dependent activation of a nuclear factor
kappaB/CCAAT-binding transcription factor-1 complex.

Qin C, Nguyen T, Stewart J, Samudio I, Burghardt R, Safe S.

Department of Veterinary Physiology & Pharmacology, Texas A&M University,
College Station, Texas 77843-4466, USA.

This study investigates the mechanism of hormonal regulation of p53 gene
expression in MCF-7 human breast cancer cells. 17beta-Estradiol (E2) induced a
2-fold increase in p53 mRNA levels and a 2- to 3-fold increase in p53 protein.
Analysis of the p53 gene promoter has identified a minimal E2-responsive region
at -106 to -40, and mutation/deletion analysis of the promoter showed that
motifs that bind CCAAT-binding transcription factor-1 (CTF-1) and nuclear factor
kappaB (NFkappaB) proteins are required for hormone responsiveness. The p65
subunit of NFkappaB was identified in both nuclear and cytosolic fractions of
untreated MCF-7 cells; however, formation of the nuclear NFkappaB complex was E2
independent. Hormonal activation of constructs containing p53 promoter inserts
(-106 to -40) and the GAL4-p65 fusion proteins was inhibited by the
intracellular Ca2+ ion chelator EGTA-AM and Ca2+/calmodulin-dependent protein
kinase (CaMK) inhibitor KN-93. Constitutively active CaMKIV but not CaMKI
activated p65, and treatment of MCF-7 cells with E2 induced phosphorylation of
CaMKIV but not CaMKI. The results indicate that hormonal activation of p53
though nongenomic pathways was CaMKIV-dependent and involved cooperative
p65-CTF-1 interactions.

PMID: 12145335 [PubMed - indexed for MEDLINE]


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19: Blood. 2002 May 1;99(9):3367-75. 

MDM2 induces NF-kappaB/p65 expression transcriptionally through Sp1-binding
sites: a novel, p53-independent role of MDM2 in doxorubicin resistance in acute
lymphoblastic leukemia.

Gu L, Findley HW, Zhou M.

Division of Pediatric Hematology/Oncology/BMT, Emory University School of
Medicine, Atlanta, GA 30322, USA.

MDM2 protein is thought to exhibit tumorigenic activity by binding to the p53
tumor-suppressor protein and inhibiting its function. Alternatively, MDM2 may
have oncogenic roles other than those resulting from p53 interactions. Here we
report that MDM2 can induce expression of the p65 subunit of NF-kappaB, which is
an anti-apoptotic factor expressed in certain neoplastic cells in response to
chemotherapy. Initially, we noted that the overexpression of MDM2 protein in
leukemic bone marrow cells of patients with B-cell precursor acute lymphoblastic
leukemia (BCP-ALL), and an ALL cell line (EU-4) transfected with the MDM2 gene
was associated with elevated expression of p65 and in vitro resistance to
doxorubicin (Adriamycin). By cotransfection of the MDM2 gene and
p65-promoter-reporter constructs into EU-4 cells, we found that transient and
high-level MDM2 expression induced p65 promoter activity. In the presence of
wild-type (wt) p53, MDM2 increased p65 promoter activity by reversing
p53-mediated suppression of p65. In the absence of p53, MDM2 directly increased
p65 promoter activity. Deletion and mutation analysis of the p65 promoter
indicated that the region between nt -575 and -178, which contains the first and
second Sp1-binding sites, was required for activation by MDM2. Further studies
using chromatin immunoprecipitation (CHIP) and electrophoretic mobility shift
assay (EMSA) showed that MDM2 was able to directly bind to the Sp1 site of the
p65 promoter. Our findings suggest that by inducing p65 expression, MDM2 has a
p53-independent role in tumorigenesis, which may further elucidate the
association between MDM2 overexpression and resistant disease in childhood ALL.

PMID: 11964305 [PubMed - indexed for MEDLINE]


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20: Cell Death Differ. 2002 Mar;9(3):252-63. 

Role of glutathione depletion and reactive oxygen species generation in
apoptotic signaling in a human B lymphoma cell line.

Armstrong JS, Steinauer KK, Hornung B, Irish JM, Lecane P, Birrell GW, Peehl DM,
Knox SJ.

Department of Radiation Oncology, Stanford University, Stanford, California, CA
94305-5105, USA.

The primary objective of this study was to determine the sequence of biochemical
signaling events that occur after modulation of the cellular redox state in the
B cell lymphoma line, PW, with emphasis on the role of mitochondrial signaling.
L-Buthionine sulphoximine (BSO), which inhibits gamma glutamyl cysteine
synthetase (gammaGCS), was used to modulate the cellular redox status. The
sequence and role of mitochondrial events and downstream apoptotic signals and
mediators was studied. After BSO treatment, there was an early decline in
cellular glutathione (GSH), followed by an increase in reactive oxygen species
(ROS) production, which induced a variety of apoptotic signals (detectable at
different time points) in the absence of any external apoptotic stimuli. The
sequence of biochemical events accompanying apoptosis included a 95% decrease in
total GSH and a partial (25%) preservation of mitochondrial GSH, without a
significant increase in ROS production at 24h. Early activation and nuclear
translocation of the nuclear factor kappa B subunit Rel A was observed at
approximately 3h after BSO treatment. Cytochrome c release into the cytosol was
also seen after 24h of BSO treatment. p53 protein expression was unchanged after
redox modulation for up to 72 h, and p21waf1 independent loss of cellular
proliferation was observed. Surprisingly, a truncated form of p53 was expressed
in a time-dependent manner, beginning at 24h after BSO incubation. Irreversible
commitment to apoptosis occurred between 48 and 72 h after BSO treatment when
mitochondrial GSH was depleted, and there was an increase in ROS production.
Procaspase 3 protein levels showed a time-dependent reduction following
incubation with BSO, notably after 48 h, that corresponded with increasing ROS
levels. At 96 h, caspase 3 cleavage products were detectable. The pan-caspase
inhibitor zVADfmk, partially blocked the induction of apoptosis at 48 h, and was
ineffective after 72 h. PW cells could be rescued from apoptosis by removing
them from BSO after up to 48, but not 72 h incubation with BSO. Mitochondrial
transmembrane potential (DeltaPsi(m)) remained intact in most of the cells
during the 72 h observation period, indicating that DeltaPsi(m) dissipation is
not an early signal for the induction of redox dependent apoptosis in PW cells.
These data suggest that a decrease in GSH alone can act as a potent early
activator of apoptotic signaling. Increased ROS production following
mitochondrial GSH depletion, represents a crucial event, which irreversibly
commits PW cells to apoptosis.

PMID: 11859408 [PubMed - indexed for MEDLINE]


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21: Zhonghua Zhong Liu Za Zhi. 2001 May;23(3):199-201. 

[EBV latent membrane protein 1 induces p53 expression via NF-kappa B in
nasopharyngeal carcinoma]

[Article in Chinese]

Liao W, Tang M, Yin L.

Cancer Research Institute, Hunan Medical University, Changsha, Hunan 410078,
China.

OBJECTIVE: To ascertain if EBV-encoded latent membrane protein 1 (LMP1) induces
p53 expression via NF-kappa B signaling. METHODS: A nasopharyngeal carcinoma
cell line, Tet-on-LMP1 HNE2, transfected with LMP1, the expression of which was
regulated by tetracycline, was used in this study. Functional activity of
NF-kappa B was determined by luciferase reporter assay and expression of p53 and
bcl-2 was detected by Western blot. RESULTS: LMP1 induced p53 expression via
NF-kappa B signaling pathway. Induction of p53 expression could be blocked by
phosphorothiate analogs of antisense oligonucleotides to NF-kappa B p65 and
LMP1, but not by NF-kappa B p50. However, it seemed that LMP1 had no influence
on bcl-2 expression in nasopharyngeal carcinoma. CONCLUSION: Induction
expression of p53 by EBV-encoded LMP1 implies that p53 may act as a mediator in
apoptosis triggered by LMP1, which brings about a complex balance in
nasopharyngeal carcinogenesis.

PMID: 11783085 [PubMed - indexed for MEDLINE]


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22: Leukemia. 2001 May;15(5):808-13. 

NF-kappaB (p65/RelA) as a regulator of TNFalpha-mediated ML-1 cell
differentiation.

Mudipalli A, Li Z, Hromchak R, Bloch A.

Department of Molecular Pharmacology and Therapeutics, Roswell Park Cancer
Institute, Buffalo, NY 14263, USA.

ML-1 human myeloblastic leukemia cells, suspended in serum-depleted medium,
proliferate when the insulin-like growth factor-1 (IGF-1) and transferrin (Tf)
are supplied, but differentiate to monocytes when these factors are replaced by
the tumor necrosis factor-alpha (TNF-alpha). Induction of differentiation, but
not of proliferation, involved the selective activation of diverse members of
the NF-kappaB family of proteins. In differentiation-induced cells, NF-kappaB
(p65) was translocated from the cytoplasm to the nucleus, whereas NF-kappaB
(p75) remained localized to the cytoplasm. In contrast, NF-kappaB (p52) was
present in the nuclei of proliferation- as well as of differentiation-induced
ML-1 cells. The differentiation-specific translocation of NF-kappaB (p65) from
the cytoplasm to the nucleus was mediated by an increase in the level of NIK,
the NF-kappaB-inducing kinase which, through phosphorylation of IkappaB kinase
alpha (Ikappakalpha), causes a decrease in the level of IkappaBalpha, allowing
p65 to move from the cytoplasm to the nucleus. The p52/p65 heterodimer formed in
the nucleus, bound specifically to the promoter of the tumor suppressor protein
p53, effecting a 25 to 30-fold increase in the level of this protein. As we
reported previously (Li et al, Cancer Res 1998; 58: 4282-4287), that increase
led to the decreased expression of proliferating cell nuclear antigen (PCNA) and
to the loss of proliferation-associated DNA synthesis. The ensuing uncoupling of
growth from differentiation was followed by the initiation of the
monocyte-specific differentiation program.

PMID: 11368442 [PubMed - indexed for MEDLINE]


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23: Nat Cell Biol. 2001 Apr;3(4):409-16. 

Regulation of death receptor expression and TRAIL/Apo2L-induced apoptosis by
NF-kappaB.

Ravi R, Bedi GC, Engstrom LW, Zeng Q, Mookerjee B, Gelinas C, Fuchs EJ, Bedi A.

Johns Hopkins Oncology Center, The Johns Hopkins University School of Medicine,
Bunting-Blaustein Cancer Research Building, 1650 Orleans Street, Baltimore,
Maryland 21231, USA.

TRAIL (tumour-necrosis factor-related apoptosis ligand or Apo2L) triggers
apoptosis through engagement of the death receptors TRAIL-R1 (also known as DR4)
and TRAIL-R2 (DR5). Here we show that the c-Rel subunit of the transcription
factor NF-kappaB induces expression of TRAIL-R1 and TRAIL-R2; conversely, a
transdominant mutant of the inhibitory protein IkappaBalpha or a
transactivation-deficient mutant of c-Rel reduces expression of either death
receptor. Whereas NF-kappaB promotes death receptor expression,
cytokine-mediated activation of the RelA subunit of NF-kappaB also increases
expression of the apoptosis inhibitor, Bcl-xL, and protects cells from TRAIL.
Inhibition of NF-kappaB by blocking activation of the IkappaB kinase complex
reduces Bcl-x L expression and sensitizes tumour cells to TRAIL-induced
apoptosis. The ability to induce death receptors or Bcl-xL may explain the dual
roles of NF-kappaB as a mediator or inhibitor of cell death during immune and
stress responses.

PMID: 11283615 [PubMed - indexed for MEDLINE]


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24: Mol Carcinog. 2000 Nov;29(3):159-69. 

Induced expression of dominant-negative c-jun downregulates NFkappaB and AP-1
target genes and suppresses tumor phenotype in human keratinocytes.

Li JJ, Cao Y, Young MR, Colburn NH.

Gene Regulation Section, National Cancer Institute, National Institutes of
Health, Frederick, Maryland 21702-1201, USA.

Neoplastically transformed mouse and human keratinocytes elevate transactivation
of both activator protein 1 (AP-1) and nuclear factor kappaB (NFkappaB)
transcription factors. The present study addresses the question of whether
elevated NFkappaB in addition to elevated AP-1-dependent gene expression is
necessary for maintaining the tumor cell phenotype. When a
tetracycline-regulatable dominant-negative c-jun (TAM67, having a truncated
transactivation domain) was expressed in tumorigenic human keratinocytes, AP-1-
and NFkappaB- but not p53-dependent reporter activity was inhibited by 40-60%.
Tumor phenotype, as measured by anchorage-independent growth, was inhibited by
90%. Neither AP-1/NFkappaB activation nor expression of tumor phenotype was
inhibited in TAM67-harboring keratinocytes under noninducing conditions.
Electrophoretic mobility shift analysis showed that induction of TAM67
expression slightly increased AP-1- but reduced NFkappaB DNA-binding activity.
Immunoprecipitation showed that TAM67 interacted in keratinocyte nuclei with
NFkappaB p65, suggesting that inhibition of NFkappaB by TAM67 is mediated by
direct protein-protein interactions, possibly producing decreased binding to DNA
or inactivating p65. To analyze the putative effector genes that may be targeted
by TAM67, expression of genes responsive to AP-1 or NFkappaB was measured by
reverse transcriptase-polymerase chain reaction in TAM67 transfectants with or
without TAM67 induction. Induction of TAM67 inhibited or reduced the expression
of collagenase I, stromelysin I (AP-1 responsive), and interleukins 1 and 6
(NFkappaB responsive). These results indicate that genes controlled by NFkappaB
and by AP-1 may be transformation-relevant targets of TAM67 and that TAM67 may
inhibit NFkappaB activation through direct interaction with NFkappaB p65.
Moreover, the findings provide proof for the principle of using inducible TAM67
as a gene therapy to suppress tumor phenotype in human carcinoma cells.

PMID: 11108661 [PubMed - indexed for MEDLINE]


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25: J Biol Chem. 2000 Aug 18;275(33):25336-41. 

Apoptosis induced by the nuclear death domain protein p84N5 is associated with
caspase-6 and NF-kappa B activation.

Doostzadeh-Cizeron J, Yin S, Goodrich DW.

Department of Cancer Biology, University of Texas M.D. Anderson Cancer Center,
Houston 77030, USA.

Although the mechanisms involved in responses to extracellular or mitochondrial
apoptotic signals have received considerable attention, the mechanisms utilized
within the nucleus to transduce apoptotic signals are not well understood. We
have characterized apoptosis induced by the nuclear death domain-containing
protein p84N5. Adenovirus-mediated N5 gene transfer or transfection of p84N5
expression vectors induces apoptosis in tumor cell lines with nearly 100%
efficiency as indicated by cellular morphology, DNA fragmentation, and annexin V
staining. Using peptide substrates and Western blotting, we have determined that
N5-induced apoptosis is initially accompanied by activation of caspase-6.
Activation of caspases-3 and -9 does not peak until 3 days after the peak of
caspase-6 activity. Expression of p84N5 also leads to activation of NF-kappaB as
indicated by nuclear translocation of p65RelA and transcriptional activation of
a NF-kappaB-dependent reporter promoter. Changes in the relative expression
level of Bcl-2 family proteins, including Bak and Bcl-Xs, are also observed
during p84N5-induced apoptosis. Finally, we demonstrate that p84N5-induced
apoptosis does not require p53 and is not inhibited by p53 coexpression. We
propose that p84N5 is involved in an apoptotic pathway distinct from those
triggered by death domain-containing receptors or by p53.

PMID: 10840029 [PubMed - indexed for MEDLINE]


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26: Biochem Biophys Res Commun. 2000 Jun 7;272(2):375-9. 

p300/CBP-dependent and -independent transcriptional interference between
NF-kappaB RelA and p53.

Ikeda A, Sun X, Li Y, Zhang Y, Eckner R, Doi TS, Takahashi T, Obata Y, Yoshioka
K, Yamamoto K.

Department of Molecular Pathology, Cancer Research Institute, Kanazawa
University, 13-1 Takaramachi, Kanazawa, 920-0934, Japan.

p53 and NF-kappaB RelA are activated by various genotoxic agents and mutually
suppress each other's ability to activate transcription, most likely through
competition for transcriptional coactivators such as CBP or p300. However, we
found that the inhibition by RelA of p53 transcriptional activity is not
completely restored by CBP/p300 overexpression and that a p53 mutant can not
suppress RelA activity despite of its ability to bind CBP/p300. In the present
study, we further present evidence that these two transcriptional factors
directly interact both in vivo and in vitro. These results therefore indicate
that the cross transcriptional interference between p53 and RelA is partly
caused by the direct interaction between these two transcription factors which
is mediated by their dimerization/tetramerization domains and results in
inhibition of each other's transcriptional activity. Finally, cells derived from
RelA knockout mice showed enhanced p53 transcriptional activity, suggesting that
this cross transcriptional interference is physiologically important in cellular
response to genotoxic stress. Copyright 2000 Academic Press.

PMID: 10833421 [PubMed - indexed for MEDLINE]


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27: Nature. 2000 Apr 20;404(6780):892-7. 

Role of NF-kappaB in p53-mediated programmed cell death.

Ryan KM, Ernst MK, Rice NR, Vousden KH.

Regulation of Cell Growth Laboratory, NCI-FCRDC, Frederick, Maryland 21702-1201,
USA.

The tumour suppressor p53 inhibits cell growth through activation of cell-cycle
arrest and apoptosis, and most cancers have either mutation within the p53 gene
or defects in the ability to induce p53. Activation or re-introduction of p53
induces apoptosis in many tumour cells and may provide effective cancer therapy.
One of the key proteins that modulates the apoptotic response is NF-kappaB, a
transcription factor that can protect or contribute to apoptosis. Here we show
that induction of p53 causes an activation of NF-kappaB that correlates with the
ability of p53 to induce apoptosis. Inhibition or loss of NF-kappaB activity
abrogated p53-induced apoptosis, indicating that NF-kappaB is essential in
p53-mediated cell death. Activation of NF-kappaB by p53 was distinct from that
mediated by tumour-necrosis factor-alpha and involved MEK1 and the activation of
pp90rsk. Inhibition of MEK1 blocked activation of NF-kappaB by p53 and
completely abrogated p53-induced cell death. We conclude that inhibition of
NF-kappaB in tumours that retain wild-type p53 may diminish, rather than
augment, a therapeutic response.

PMID: 10786798 [PubMed - indexed for MEDLINE]


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28: Mol Cell Biol. 2000 May;20(10):3377-86. 

Inactivation of p53 by human T-cell lymphotropic virus type 1 Tax requires
activation of the NF-kappaB pathway and is dependent on p53 phosphorylation.

Pise-Masison CA, Mahieux R, Jiang H, Ashcroft M, Radonovich M, Duvall J,
Guillerm C, Brady JN.

Virus Tumor Biology Section, Laboratory of Receptor Biology and Gene Expression,
National Cancer Institute, National Institutes of Health, Bethesda, Maryland
20892, USA. masionc@dce41.nci.nih.gov

p53 plays a key role in guarding cells against DNA damage and transformation. We
previously demonstrated that the human T-cell lymphotropic virus type 1 (HTLV-1)
Tax can inactivate p53 transactivation function in lymphocytes. The present
study demonstrates that in T cells, Tax-induced p53 inactivation is dependent
upon NF-kappaB activation. Analysis of Tax mutants demonstrated that Tax
inactivation of p53 function correlates with the ability of Tax to induce
NF-kappaB but not p300 binding or CREB transactivation. The Tax-induced p53
inactivation can be overcome by overexpression of a dominant IkappaB mutant.
Tax-NF-kappaB-induced p53 inactivation is not due to p300 squelching, since
overexpression of p300 does not recover p53 activity in the presence of Tax.
Further, using wild-type and p65 knockout mouse embryo fibroblasts (MEFs), we
demonstrate that the p65 subunit of NF-kappaB is critical for Tax-induced p53
inactivation. While Tax can inactivate endogenous p53 function in wild-type
MEFs, it fails to inactivate p53 function in p65 knockout MEFs. Importantly,
Tax-induced p53 inactivation can be restored by expression of p65 in the
knockout MEFs. Finally, we present evidence that phosphorylation of serines 15
and 392 correlates with inactivation of p53 by Tax in T cells. This study
provides evidence that the divergent NF-kappaB proliferative and p53 cell cycle
arrest pathways may be cross-regulated at several levels, including
posttranslational modification of p53.

PMID: 10779327 [PubMed - indexed for MEDLINE]


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29: Mol Cell Biol. 1999 May;19(5):3485-95. 

Transcriptional cross talk between NF-kappaB and p53.

Webster GA, Perkins ND.

Department of Biochemistry, Division of Gene Regulation and Expression,
University of Dundee, Dundee DD1 5EH, Scotland, United Kingdom.

Many cellular stimuli result in the induction of both the tumor suppressor p53
and NF-kappaB. In contrast to activation of p53, which is associated with the
induction of apoptosis, stimulation of NF-kappaB has been shown to promote
resistance to programmed cell death. These observations suggest that a
regulatory mechanism must exist to integrate these opposing outcomes and
coordinate this critical cellular decision-making event. Here we show that both
p53 and NF-kappaB inhibit each other's ability to stimulate gene expression and
that this process is controlled by the relative levels of each transcription
factor. Expression of either wild-type p53 or the RelA(p65) NF-kappaB subunit
suppresses stimulation of transcription by the other factor from a reporter
plasmid in vivo. Moreover, endogenous, tumor necrosis factor alpha-activated
NF-kappaB will inhibit endogenous wild-type p53 transactivation. Following
exposure to UV light, however, the converse is observed, with p53 downregulating
NF-kappaB-mediated transcriptional activation. Both p53 and RelA(p65) interact
with the transcriptional coactivator proteins p300 and CREB-binding protein
(CBP), and we demonstrate that these results are consistent with competition for
a limiting pool of p300/CBP complexes in vivo. These observations have many
implications for regulation of the transcriptional decision-making mechanisms
that govern cellular processes such as apoptosis. Furthermore, they suggest a
previously unrealized mechanism through which dysregulated NF-kappaB can
contribute to tumorigenesis and disease.

PMID: 10207072 [PubMed - indexed for MEDLINE]


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30: J Biol Chem. 1999 Jan 22;274(4):1879-82. 

CREB-binding protein is a nuclear integrator of nuclear factor-kappaB and p53
signaling.

Wadgaonkar R, Phelps KM, Haque Z, Williams AJ, Silverman ES, Collins T.

Vascular Research Division, Department of Pathology, Brigham and Women's
Hospital and Harvard Medical School, Boston, Massachusetts 02115, USA.

Transcriptional coactivators may function as nuclear integrators by coordinating
diverse signaling events. Here we show that the p65 (RelA) component of nuclear
factor-kappaB (NF-kappaB) and p53 mutually repress each other's ability to
activate transcription. Additionally, tumor necrosis factor-activated NF-kappaB
is inhibited by UV light-induced p53. Both p65 and p53 depend upon the
coactivator CREB-binding protein (CBP) for maximal activity. Increased levels of
the coactivator relieve p53-mediated repression of NF-kappaB activity and
p65-mediated repression of p53-dependent gene expression. Nuclear competition
for limiting amounts of CBP provides a novel mechanism for altering the balance
between the expression of NF-kappaB-dependent proliferation or survival genes
and p53-dependent genes involved in cell cycle arrest and apoptosis.

PMID: 9890939 [PubMed - indexed for MEDLINE]


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31: Oncogene. 1998 Dec 3;17(22):2889-99. 

Prostate carcinoma cell death resulting from inhibition of proteasome activity
is independent of functional Bcl-2 and p53.

Herrmann JL, Briones F Jr, Brisbay S, Logothetis CJ, McDonnell TJ.

The Department of Genitourinary Medical Oncology, The University of Texas MD
Anderson Cancer Center, Houston 77030, USA.

The ATP/ubiquitin-dependent 26S proteasome is a central regulator of cell cycle
progression and stress responses. While investigating the application of peptide
aldehyde proteasome inhibitors to block signal-induced IkappaBalpha degradation
in human LNCaP prostate carcinoma cells, we observed that persistent inhibition
of proteasomal activity signals a potent cell death program. Biochemically, this
program included substantial upregulation of PAR-4 (prostate apoptosis
response-4), a putative pro-apoptotic effector protein and stabilization of
c-jun protein, a potent pro-death effector in certain cells. We also observed
modest downregulation of bcl-XL, a pro-survival effector protein. However, in
contrast to some recent reports stable, high level, expression of functional
bcl-2 protein in prostate carcinoma cells failed to signal protection against
cell death induction by proteasome inhibitors. Also in disagreement to a recent
report, no evidence was found for activation of the JNK stress kinase pathway. A
role for p53, a protein regulated by the proteasome pathway, was ruled out,
since comparable cell death induction by proteasome inhibitors occurred in PC-3
cells that do not express functional p53 protein. These data signify that the
ubiquitin/proteasome pathway represents a potential therapeutic target for
prostate cancers irrespective of bcl-2 expression or p53 mutations.

PMID: 9879995 [PubMed - indexed for MEDLINE]


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32: Cancer Res. 1998 Oct 15;58(20):4531-6. 

p53-mediated repression of nuclear factor-kappaB RelA via the transcriptional
integrator p300.

Ravi R, Mookerjee B, van Hensbergen Y, Bedi GC, Giordano A, El-Deiry WS, Fuchs
EJ, Bedi A.

Johns Hopkins Oncology Center, The Johns Hopkins University School of Medicine,
Baltimore, Maryland 21287-8967, USA.

The p53 tumor suppressor gene plays an instrumental role in transcriptional
regulation of target genes involved in cellular stress responses. p53-dependent
transactivation and transrepression require its interaction with p300/CBP, a
coactivator that also interacts with the RelA subunit of nuclear factor-kappaB.
We find that p53 inhibits RelA-dependent transactivation without altering RelA
expression or inducible kappaB-DNA binding. p53-mediated repression of RelA is
relieved by p300 overexpression and the increased RelA activity conferred by
p53-deficiency is counteracted by either transactivation domain-deficient p300
fragments that bind RelA or a transdominant mutant of IkappaB alpha. Our results
suggest that p53 can regulate diverse kappaB-dependent cellular responses.

PMID: 9788595 [PubMed - indexed for MEDLINE]


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33: Proc Natl Acad Sci U S A. 1994 Jun 21;91(13):5957-61. 

Sequence-independent induction of Sp1 transcription factor activity by
phosphorothioate oligodeoxynucleotides.

Perez JR, Li Y, Stein CA, Majumder S, van Oorschot A, Narayanan R.

Division of Oncology, Roche Research Center, Hoffmann-La Roche, Inc., Nutley, NJ
07110.

Modified analogues of antisense oligodeoxynucleotides (ODNs), particularly
phosphorothioates ([S]ODNs), have been extensively used to inhibit gene
expression. The potential sequence specificity of antisense oligomers makes them
attractive as molecular drugs for human diseases. The use of antisense [S]ODNs
to inhibit gene expression has been complicated by frequent nonspecific effects.
In this study we show in diverse cell types that [S]ODNs, independent of their
base sequence, mediated the induction of an Sp1 nuclear transcription factor.
The [S]ODN-mediated Sp1 induction was rapid and was associated with elevated
levels of Sp1 protein. This induction was dependent on NF-kappa B activity,
since inhibition of NF-kappa B activity abolished the [S]ODN-induced Sp1
activity. [S]ODN-induced Sp1 activity was seen in mouse spleen cells following
in vivo administration. Sp1 activity induced by [S]ODNs required the tyrosine
kinase pathway and did not have transactivating potential. These results may
help to explain some of the non-specific effects often seen with [S]ODNs.

PMID: 8016096 [PubMed - indexed for MEDLINE]


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