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NF-kB is a cellular transcription factor that is
essential for B cell differentiation and cell survival. B cell lines
latently infected with human gammaherpesviruses have constitutive
NF-kB activity. Inhibition of this activity results in cell death.
We hypothesized that the inhibition of NF-kB activity in S11E cells
a murine B cell line latently infected with murine gammaherpesvirus
68 (gHV68) would result in apoptotic death. In order to monitor
NF-kB activity we established a NF-kB dependent luciferase assay
system. This required the optimization of S11E cell transfection
with the Amaxa nucleofection system a new proprietary transfection
system that delivers plasmids directly to the nucleus. Using a green-fluorescent
protein (GFP)-expression construct I determined the optimal transfection
solution conditions and plasmid input amounts that would yield maximal
GFP expression with minimal impact on cell viability. Next we verified
that NF-kB activity could be modulated in S11E cells by cotransfection
of a NF-kB-luciferase reporter construct with a NF-kB activator
pMEKK and an inhibitor pIkBaM. Finally we monitored the inhibitory
drug Bay11-7082 the SN50 peptide and the pIkBaM construct for their
ability to inhibit NF-kB in the luciferase assay and induce apoptosis
by Annexin V staining. We found a correlation between NF-kB inhibition
and apoptotic induction. This indicates that NF-kB is important
for maintaining gammaherpesvirus latency. These studies provide
a strong foundation for future investigations of the protective
effect that NF-kB provides and the mechanism by which gHV68 maintains
NF-kB activity.
Multiple upstream events can lead to NF-kB activation.
Well-characterized signaling events such as membrane receptor interaction
with extracellular tumor necrosis factor (TNF) lead to the phosphorylation
and activation of the IkB kinase (IKK) complex. The IKK complex
then phosphorylates the cytoplasmic IkBa-NF-kB-complex. Phosphorylation
of IkBa leads to its ubiquitination and proteosomal degradation
allowing NF-kB to translocate to the nucleus. NF-kB can then turn
on genes that are responsible for cell survival inflammation and
cell proliferation. There are several methods for modulating NF-kB
in vitro. NF-kB can be activated by the addition of TNF to the culture
medium or by transfection with upstream signaling proteins such
as a mitogen activated protein kinase kinase (MEKK). Conversely
NF-kB activity can be inhibited by treatments with: i) the drug
Bay11-7082 that blocks IkBa phosphorylation (blue bar) ii) the cell
permeable SN50 peptide that masks the nuclear localization sequence
of NF-kB (red bar) and iii) transfection with a constitutively active
mutant form of IkBa with amino acids 32 and 36 changed from serine
to alanine preventing phosphorylation and subsequent release of
NF-kB.
90% transfection efficiency of S11E cells can be obtained with
the Amaxa nucleofection system using 10 mg plasmid DNA solution
V and program O-17. NF-kB activity can be monitored in S11E cells
by luciferase assay. NF-kB activity can be inhibited by transfection
with a constitutively active IkBa mutant the peptide SN50 and the
drug Bay 11-7082. Inhibition of NF-kB by the SN50 and Bay11-7082
is associated with increased apoptosis.
Fine-tuning the system
Optimize the amount and duration of treatment with the inhibitor.
Verify apoptotic induction by determining caspase-3 activation --How
does the virus keep NF-kB active during latency? Which gHV68 proteins
are essential for inducing NF-kB? What signalling pathway is utilized
for NF-kB activation?
Regulation of lytic reactivation
Will the inhibition of NF- kB allow the virus to enter the lytic
stage? Can the overexpression of NF-kB block lytic cycle progression?
We especially thank lab members Janice Moser, Nat Moorman, and
Dave Willer for their technical advice and reagents. KAH was supported
by a Minority Undergraduate Research Fellowship from American Society
for Microbiology and Howard Hughes Medical Institute grant No. 52003727.
For the past two months I have intensely looked at B-cell apoptosis
cell death with a cell line that is latently infected with a gammaherpesvirus.
We began by optimizing the transfection procedure for our S11E cell
line with the Amaxa nucleofector. The Amaxa nucleofector uses electroporation
to deliver the DNA to the nucleus. After optimizing the transfection
procedure we then used a NF-kB reporter construct to monitor levels
of NF-kB activity in the S11 cell line. Various other plasmids were
also transfected as controls activators and others to detect a dose
response to an inhibition of NF-kB by IkBaM. IkBaM is a protein
involved in trapping NF-kB in the cytoplasm when it is not in an
activated state. The 'M' states that it is a mutant form of IkBa.
The mutantion occurs at amino acids 32 and 36 - from serines to
alanines. The mutant form is unable to be phosphorylated and degragated
in the cytoplasm of the cell - allowing for the NF-kB-IkBa complex
to remain trapped in the cytoplasm. After determining an amount
of IkBaM that we could use to block NF-kB activity we then looked
at apoptosis by means of different drugs and peptides. Apoptosis
was determined by Annexin V-GFP staining. Annexin V binds to a structural
protein that is flipped to the outer membrane of the cell upon entering
the beginning stages of apoptosis. GFP analysis was done by fluorescence
activated cell sorting which analyzes cells bases on GFP intensity
and cell viability - depending on what view you are looking at.
GFP analysis determined that we were seeing a large increase in
cell death by blocking NF-kB activity.
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