Download NF-kB expression by HPV-16 E6 and E7 genes in human epithelial

NF-kB expression by HPV-16 E6 and E7 genes in human epithelial cells
Courtney Grayson
Dr. Craig D. Woodworth
Problem Definition/Description
Cervical cancer is a major health problem worldwide, particularly in underdeveloped
nations where women have poor access to medical care. In the United States alone, this cancer is
diagnosed in over 9,700 women and takes the lives of over 3,700 each year. The major risk
factor for cervical cancer is infection by specific strains of the human papillomavirus (HPV).
Although there are over 100 different HPV types, infection with fifteen “high risk” HPV strains
accounts for greater than 99% of all cervical cancers. Cervical cancer could be averted by either
preventing HPV infection or by proper diagnosis and treatment of existing infections. Although
an effective vaccine to protect against HPV has been developed, progress in the prevention and
treatment of this disease continues to be essential. The vaccine protects against only two
common “high risk” strains of HPV and two “low risk” strains that cause genital warts. The
vaccine is also very expensive and thus, not feasible for widespread use in women who do not
have health insurance and critically need protection. Seventy five percent of sexually active
women have contracted HPV; thus, therapies for existing infections will be necessary in the
coming decades.
Preventing infection by HPV and providing treatment for existing infections requires a
better understanding of how HPV evades detection by the immune system and hides in the
cervix. In am interested in the specific mechanisms used by HPV to evade immune recognition,
allowing the infection to persist and progress to cancer. My research with Dr. Craig Woodworth
at Clarkson University is focused on the most common “high risk” strain of the virus, HPV-16.
In particular I am interested in two viral oncoproteins, E6 and E7, which promote malignant
transformation by blocking the normal function of two tumor suppressor proteins, p53 and the
retinoblastoma protein. Once the virus blocks the function of these tumor suppressors, the
infected cells fail to correct DNA mutations and divide without control. These rapidly growing
cells can then develop mutations and rapidly progress to cancer. The E6 and E7 genes are
critical for malignant development. They are retained and expressed in nearly all cervical
cancers, and continued expression of the E6 and E7 genes is required for cancer growth. Thus,
these specific genes are fundamentally important in understanding the mechanisms by which
HPV evades detection by the immune system and promotes cancer.
Abbreviations
BCA; Bicinchoninic acid protein assay, HPV; Human Papillomavirus, HRP; Horseradish peroxidase,
KSFM; Keratinocyte Serum Free Media, NF-kB; Nuclear factor-kappa B, p53; Protein 53, PBS;
Phosphate Buffered Saline, pRb; Retinoblastoma protein, TAE; Tris-acetate-EDTA buffer, TNFα; Tumor
Neucrosis Factor α.
Studying the transcription factor NF-kB, which serves to alert the immune system to the
presence of infections agents, is an important step in the advancement of HPV treatment and
cervical cancer prevention. Activation of the transcription factor NF-kB is an important barrier
against persistent HPV infection and cervical cancer. Thus, my research focuses on this central
mediator of immune and inflammatory responses. NF-kB serves to bring the immune response to
an infection site by stimulating the expression of genes that encode growth factors, cytokines,
and adhesion molecules, which are necessary to combat infections such as HPV. NF-kB is
thought to play a crucial role in malignant development, and thus is being investigated as a
potential target for cancer therapy.
The goal of my research is to determine the role the E6 and E7 genes play in the
regulation of NF-kB activity when expressed over an extended period of time. This is a
medically important question because the genes must be expressed for many years in order for
malignant development to occur. My work is built upon previous experiments performed in Dr.
Woodworth’s lab, which have shown that the E6 and E7 genes encode proteins that directly alter
the activity and function of NF-kB. This research indicated that NF-kB activity is increased
immediately upon infection by HPV, serving as a mechanism to alert the immune system to the
present infection. While prior experiments indicated that expression of the E6 and E7 genes
contribute to this initial increase in NF-kB activity, little is known about the role of these genes
in NF-kB expression over a longer range of time.
Inhibition of NF-kB is a potentially novel mechanism for HPV to evade detection by the
immune system. If NF-kB could be “turned on” in infected cells to counteract the effects of the
E6 and E7 viral genes, the immune system would be alerted to combat the infection, halting
malignant development. My results suggest that treating HPV infections with drugs known to
increase NF-kB activity could potentially prevent persistent HPV infection and cervical cancer.
My research, in conjunction with other questions being investigated in Dr. Woodworth’s lab,
such as how NF-kB activity levels differ in each stage of cancer, will pinpoint possibilities for
NF-kB-targeted therapies. Using NF-kB as a target for treating HPV infection and cervical
cancer will offer hope to those already infected with the virus, as well as those not able to afford
the vaccine. My work could be important to a number of studies working to develop improved
vaccines to prevent HPV infection or therapeutic vaccines to treat existing cervical cancers.
Background
Mediation of cell cycle by NF-kB
NF-kB is a eukaryotic transcription factor that serves as a central mediator for immune
and inflammatory responses. NF-kB contributes to the induction of several classes of genes;
genes that have products involved in negative-feedback control of NF-kB, serve as regulators of
the immune system, promote cell proliferation, and inhibit apoptosis (programmed cell death).
Genes of each of these classes can contribute to tumorgenesis. For this reason, NF-kB is thought
to play an important role in malignant development, and is being investigated as a potential
target for cancer therapy. NF-kB has also been shown to function as a tumor suppressor under
certain conditions, and is thus said to have a paradoxical function (Perkins 2006).
In its inactive form, NF-kB is bound to the IkB complex in the cytoplasm. It is activated
to initiate gene expression by stimuli such as cytokines, free radicals, bacterial or viral antigens,
and other forms of cell stress, including cancer. Activation by these stimuli occurs through the
“classical pathway” of activation, in which a pathogen phophorolates the IkB complex, labeling
it for ubiquitin dependent degradation (Figure 1). The IkB complex is then degraded by a
protease and broken into subunits, which can then translocate into the nucleus (Perkins 2006).
The NF-kB family consists of five structurally related protein subunits: c-Rel, RelA (p65), RelB,
p50/p105, and p52/p100. These three proteins all contain amino acid sequences that are essential
for dimerization, DNA binding and nuclear transport. Once in the nucleus, the subunits can
initiate gene expression, and are further regulated by phosphorylation (Perkins 2006).
Figure 1 The classical NF-kB pathway is activated by signals such as stress, bacterial and viral antigens,
and cytokines. This activation allows the NF-kB subunits to translocate into the nucleus and code for
genes regulating NF-kB response, inflammation, cell migration and immune response.
Source:http://www.bio.davidson.edu/Courses/immunology/Students/spring2006/Magargal/NFkB.htm
The NF-kB subunits code for the production of IkBα, which inactivates subunits in the nucleus.
This serves as a negative feedback control mechanism for NF-kB, and thus its expression is short
lived. It is thought that activation of NF-kB by this classical pathway could be an important
mediator of inflammation-induced tumor growth and progression (Karin 2005). This suggests
that NF-kB could be the mechanistic link between inflammation and cancer.
Human Papillomavirus and Cervical Cancer
Infection by certain strains of Human Papillomavirus (HPV) is a major risk factor for the
development of cervical cancers. Of about one hundred strains of the virus, approximately thirty
are sexually transmitted. Of these sexually transmitted strains, only a few have the potential to
develop into cancer. HPV strains are divided into two categories; “high risk” and “low risk.”
“Low risk” strains of the virus typically cause benign changes or abnormalities in the cervix,
such as genital warts. “High risk” strains of the virus are those that have the ability to cause
cervical cancer. 99.7% of cervical cancers are directly linked to infection by a “high risk” strain
of HPV (McIntosh 2000). HPV-16 is the most common “high risk” strain of the virus.
Malignant Promotion by HPV-16 E6 and E7 oncogenes
Two specific early region HPV-16 genes, E6 and E7, act as oncogenes to promote
malignant transformation by blocking the effects of tumor suppressors p53 and Rb respectively
(Kuroda etal 2005). Once the function of these tumor suppressors is blocked by the virus, the
infected cells are allowed to reproduce without control. The rapidly growing cells can then
develop changes in the genetic structures of the cells, ultimately leading to the development of
cancer. Cancers tend to develop in a specific region of the cervix, the transformation zone. This
area is characterized by the point where the flat squamous cells of the ecto cervix meet the
glandular cells of the endo cervix. It is unclear why cancerous development is limited to
infections in this specific area of the cervix.
Literature Review
Several studies have been done to determine the role of HPV E6 and E7 genes in
carcinogenic development and NF-kB activation. Balance between tumor cell proliferation and
spontaneous cell death via apoptosis have been shown to have an important role in regulation of
tumor cell growth (Divya 2006). In vivo studies have shown that expression of the E7 oncogene
leads to increased cell proliferation, inhibition of differentiation, and finally apoptosis, which are
all mediated by pRb. With co-expression of E6 and E7 in vivo in a p53 null background,
apoptosis can be blocked. Similar in vivo studies have shown that the E6 oncogene can prevent
p53-independent apoptosis. These results suggest that the E6 and E7genes are capable of
modulating the frequency apoptosis, which could be an important part of the mechanism by
which these genes contribute to carcinogenesis (Jansen-Dürr 1996). Immediately upon infection
by an HPV, NF-kB activity is increased due to the stress placed upon the cells by the virus. This
serves as a mechanism to alert the immune system of the present infection. Experiments done in
Dr. Woodworth’s lab have shown that both the E6 and E7 genes both contribute to this initial
increase in NF-kB activity. It is possible, however, that the one or both E6 and E7 early genes
could act to inhibit NF-kB activity when expressed stably for a longer period of time. This could
serve as a survival mechanism for the HPV virus, allowing it to evade the immune system.
Future work needs to address the role of E6 and E7 genes in NF-kB activity when stably
expressed in human cervical cells, which will be addressed in this study.
In both normal cervix and cervical intraepithelial neoplasia lesion, RelA (p65) is mainly
concentrated in the cytoplasm (Branca et al 2006). In general, the over expression of NF-kB is
associated with the progression of cervical intraepithelial neoplasia lesions to cancer. NF-kB
precursors, p100 and p105 have been found in high levels in HPV-16 cervical carcionomaderived keritinocytes and keratinocytes stably transfected with HPV 16 E6 or E7 oncogenes.
The p100 and p105 proteins were found to be predominately cytoplasmic in cells expression E7,
and localized in the nucleus in cells expressing E6. This suggests that HPV 16 E6 and E7
oncogenes modulate the expression and subcellular localization of p100 and p105 NF-kB
precursors (Harvard et al 2004). Past studies have also examined the subcellular localization of
NF-kB subunits in E6 and E7 infected cells using immunohistochemistry to visualize the
subunits. This technique makes it difficult to quantify, however. Using a Western Blotting
technique will allow the concentrations of each subunit protein to be quantified in cytoplasmic
and nuclear fractions. In order to better understand the role of the E6 and E7 genes in the
subcellular localization of NF-kB subunits, the RelA (p65) subunit must also be studied in
conjunction with the p50 and p52 subunits. This project will address this question, and differs
from past work in that it will examine these three subunits, rather and the p100 and p105
precursors. This study is also important in that it will be performed using human cervical cells,
whereas many past studies on the role of the E6 and E7 genes on NF-kB activity and subcellular
localization are performed using foreskin cells or cell lines.
Materials and Methods
Overview
Reporter gene plasmid DNAs were first replicated in competent E.coli and purified.
Restriction digest and separation of fragments by agarose gel electrophoresis were then used to
confirm the identity of each DNA sample. Plasmid DNA was introduced into cultured human
cervical cells using a lipofectamine transfection to measure NF-kB activity in retrovirallyinfected cervical cells expressing the PLXSN (the empty retroviral vector), the E6 or E7 gene
alone, or E6 and E7 co-expressed. The level of NF-kB activity was mesured using a dualluciferase reporter gene assay, which measures the level of reporter gene expression based on the
level of luminesence. In separate experiments, the quantity of p50 and RelA (p65) NF-kB
subunits in cytoplasmic and nuclear fractions of retrovirally-infected cervical cells will be
determined using polyacrilamide gel electrophoresis (PAGE) and Western Blotting.
Replication and Purification of Recombinant Plasmid DNA
Plasmid reporter gene DNA was replicated in order to have sufficient quantities for future
experiments. Plasmid reporter gene DNA was replicated by transforming competent E.coli
bacteria. 20 μl of glycerol stock was inoculated into a 15 ml conical tube containing 5 ml of LB
broth with 5 μl of ampicillin and allowed to grow in a shaker incubator at 37°C. Once the broth
appeared cloudy, signifying bacterial growth, the broth was transferred into a 500 ml flask
containing 200 ml of LB broth and allowed to grow overnight. The recombinant DNA was
purified using a Maxi prep kit from Marligen Biosciences. The purified DNA was suspended in
1x TE buffer, and the concentration was determined using a BioRad spectrophotometer.
Plasmid reporter gene DNA was then cut at specific points using restriction digest. The
DNA fragments were then separated by molecular weight using agarose gel electrophoresis and
compared to a molecular weight ladder to confirm the identity of each sample. One μg of each
DNA sample was allowed to digest in an eppendorf tube for 1 hour in a 37°C water bath with 1
μl of the appropriate enzyme, 2 μl enzyme buffer, and enough deionized water to bring the total
volume to 20 μl. The reaction was then transferred to a 60°C water bath to inactivate the
enzyme. 4 μl of 6x loading buffer was added to each eppendorf tube and mixed. An agarose gel,
containing 0.8% aragose in TAE buffer and ethidium bromide at a concentration of 0.5 μg/ml,
was placed in a get box, and enough TAE buffer was added to the box to completely cover the
gel. 10 μl of each sample was then loaded into a separate well of the gel. 3 μl of a 1 kB ladder of
DNA molecular weight makers was loaded into the last well to use as a way to determine the
molecular weights of the restriction fragments for comparison to expected values. The fragments
were separated by agarose gel electrophoresis at 90 V for 1-2 minutes, then at 60-70 V for 60
minutes. The gel was then photographed under exposure to UV light, and bands were compared
to 1 kB ladder to confirm the identity of each recombinant DNA.
Culture of Human Cervical Cells
Human cervical cells were cultured using KSFM media with antibiotics. Before reaching
100% confluence, cells were split. In a 100mm dish, the cells were washed with 5 ml PBS, and
then 4 ml of working trypsin solution was placed on the cells. The cells were then incubated at
37°C for 5-15 minutes, until the cells detached from the bottom of the plate. 4 ml of KSFM with
5% FBS was added to neutralize the trypsin, and then the media containing the cells was
centrifuged in a conical tube for 5 minutes to pellet the cells. The cell pellet was then
resuspended in KSFM and plated at the desired concentration onto dishes containing additional
KSFM.
Reporter Gene Assay to Measure NF-kB Activity
A dual-luciferase reporter gene assay was used to introduce purified plasma DNA into
cultured human cervical cells and measure the level of NF-kB activity based on the level of
luminescence measured. In order to measure NF-kB activity, reporter genes were introduced
into human cervical cells using lipofectamine transfection. Once cells split into 12-well plates
reached 50-80% confluence, the media was replaced with 500 μl KSFM without antibiotics, and
antibiotics were removed from the media in all subsequent steps of the transfection procedure, in
order to minimize toxicity to cells. The desired quantity of reporter DNA (composed of 90%
NK-kB luciferase plasmid DNA and 10% renilla luciferase plasmid DNA) was placed in an
eppendorf tube with 50 μl of basal media and the desired amount of PLUS reagent, mixed, and
incubated at room temperature for 15 minutes. In a second eppendorf tube, 50 ml basal media
and desired amount of lipofectamine reagent were mixed and incubated at room temperature for
15 minutes. The contents of the two tubes were then mixed together and allowed to incubate for
an additional 15 minutes to allow for liposome formation. In liposome formation, positively
charged lipids coat negatively charged DNA, allowing cells to take up the DNA through the cell
membrane. 100 μl of the final mixture was placed into each dish and cultured for 24 hours to
allow cells to take the liposomes in through the cell membrane and express the reporter DNA.
Varied amounts of DNA and lipofectamine were repeated in triplicate to find optimum amounts
for future experiments. Amounts of DNA used for optimization were 0.1 μg, 0.25 μg and 0.75
μg. Lipofectamine values used were 0.5 μl, 1.0 μl, 1.5 μl and 2.0 μl. Subsequent transfection
experiments were performed using 0.25 μg of DNA, 2.0 μl of lipofectamine and 2 μl of PLUS
reagent, the determined optimum values.
Once the human cervical cells had been exposed to liposomes for 24 hours, growth media
was removed from cells, and then cells were rinsed with 1x PBS. 200 μl 1x lysis buffer was
added to each well, and dishes were rocked for 15 minutes to ensure complete coverage. Cells
were then scraped from each dish into an eppendorf tube, and subjected to two freeze-thaw
cycles to complete cell lysis. Cell lysates were stored at -80°C until needed for analysis.
NF-kB activity was measured from lysates using a dual-luciferase reporter gene assay kit
from Promega. Signals of the two reporter genes were measured using a TD-20/20 luminometer.
Luciferase assay agent II (LAR II) and Stop and Glo reagent were prepared according to kit
instructions. 20 μl of lysate expressing the reporter genes was added to 100 μl LAR II in an
eppendorf tube, and luminescence was measured using the luminometer. The NF-kB reporter
gene contains an NF-kB promoter cloned in front of a firefly luciferase gene, allowing NF-kB
activity to be measured based on the level of luminescence. The renilla luciferase gene is active
in all cells, and therefore used as a baseline for comparison. The first measure of luminescence
indicated the strength of the NF-kB reporter gene signal. 100 μl of Stop and Glo reagent was
then added to the tube, eliminating the firefly signal and causing the renilla luciferase to
luminesce. A second reading was taken with the luminometer, which then reported renilla
reporter gene activity, normalizing the reading. The ratio between the signal strength of the NFkB and renilla reporter genes was then acquire, indicating the actual level NF-kB activity.
PAGE and Western Blotting to Measure Steady State Levels of NF-kB Subunits
The quantity of p50 and RelA (p65) NF-kB subunits in cytoplasmic and nuclear fractions
of retrovirally-infected cervical cells will be determined using polyacrilamide gel electrophoresis
(PAGE) and Western Blotting. For Western Blot experiments measuring the concentration of
NF-kB subunits, cytoplasm and nuclear protein fractions will be separated using the protocol
outlined in Current Protocols in Molecular Biology (Abmayr SM 1990). Protein concentrations
will be determined using a BCA assay. Samples will then be vortexed and centrifuged for 2
minutes at 12000xg and diluted to 1 μg/μl in lysis buffer with 5% bromophenol blue and 2.5%
Beta-mercaptoethanol. Samples will then be vortexed for 5 seconds, and boiled with molecular
weight ladder and standards for 5 minutes, then immediately put on ice. Diluted samples will
then be vortexed for 5 seconds and centrifuged for 3 minutes at 12000xg. Running buffer will
then be prepared using 100 ml of BioRad 10x Tris/Glycine/SDS buffer and 900 ml of nanopure
water and poured into gel box. Fifteen μl of each diluted sample, the standards, and molecular
weight ladder will then be loaded into a polyacrilimide gel. The samples will be separated by
polyacrilimide gel electrophoresis at 80V for 90 minutes. Transfer buffer will be prepared using
500 ml of BioRad 10xTris/Glycine, 1000 ml methanol and 3500 ml nanopure water. The
transfer apparatus will be set up to transfer the protein on the gel to the nitrocellulose membrane,
and then run at 80 V for 90 minutes. Nitrocellulose membrane will be rinsed with TBS for 5
minutes, then incubated for 60 minutes at room temperature in 1x Western Block Reagent. The
nitrocellulose membrane will then be incubated in primary antibody in a 0.5x Western Block
Solution at 4˚C overnight with gentle shaking. The following morning, the membrane will be
rinsed with TBS+1%Tween (TBS-T) and then rinsed in 5x3 minutes in TBS-T. Membrane will
then be incubated in appropriate HRP-linked secondary antibody diluted 1:5000 in 0.5x Western
Blocking Reagent for 90 minutes at room temperature with gentle shaking. The membrane will
be rinsed with TBS+1%Tween (TBS-T) and then rinsed in 5x3 minutes in TBS-T. The
membrane will then be incubated in luminal reagent for 30 seconds and visualized in a dark
room.
Results
Quality Control for Recombinant Plasmid DNA
Agarose gel electrophoresis of plasmid DNA restriction fragments produced diagnostic
bands based on the molecular weights of the samples (Figure 2).
NF-kB
IKBα
LZRS
Renilla
p65
Hind III, uncut Eco RI uncut Eco RI uncut Bam HI, uncut Hind III, uncut
NOT I
EcoRI
Xba I
1kb ladder
3 Kb
1.6 Kb
1 Kb
Figure 2. Restriction fragments for NF-kB, IkBa, LZRS, renilla and p65 plasmid DNAs
produced separate bands, indicating where restriction enzymes cleaved the DNA. The left lane
of each sample is cut by a specific enzyme(s), while the right lane contains the undigested
plasmid DNA. The 1 Kb ladder produces bands of known molecular weights for comparison to
the restriction fragments.
Based on comparison to the 1 Kb ladder, each of the DNA samples was cut by the appropriate
restriction enzyme(s) at the expected locations, indicating that each sample is of the expected
identity. The lack of smearing of the bands indicates that there is no bacterial RNA or DNA
contamination. The results indicate that the plasmid DNAs were suitable for use in transfection
assays.
Optimization of Reporter Gene Assay
In order to optimize the reporter gene assay, normal cells were transfected with varying
amounts of DNA and lipofectamine. As amounts of DNA and lipofectamine were varied,
individual reporter gene signals varied significantly. The highest individual signals were
achieved using 0.25 μg of DNA, 2.0 μl of lipofectamine and 2 μl of PLUS reagent (Figure 3).
Figure 3. As the amount of lipofectamine reacted was increased, individual NF-kB and renilla
signals increased. Larger quantities of DNA also increased individual reporter gene signals.
The ratio of NF-kB luciferase signal to the renilla luciferase signal indicates actual NFkB activity. All ratios were under 2, indicating that a minimal amount of toxicity and stress was
placed on the cells. Minimal toxicity in a transfection is characteristic of normal cells, while
retroviral infected cells will have a higher baseline level of NF-kB activity as a result of the
stress placed on the cells by the infection. Though baseline levels would be elevated in retroviral
infected cells, our work has shown that the trends in NF-kB activity and reporter gene signal
strength with varied amounts of DNA and lipofectamine would be preserved. The lowest levels
of NF-kB activity were achieved under the following conditions: 0.25 μg DNA, 0.5 μl
lipofectamine and 2 μl of PLUS reagent; 0.25 μg DNA, 2.0 μl lipofectamine and 2 μl of PLUS
reagent; 0.75 μg DNA, 1.0 μl lipofectamine and 5 μl of PLUS reagent (Figure 4).
3
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Figure 4. The ratio of NF-kB luciferase signal to renilla luciferase signal indicates the level of
NF-kB activity under each set of conditions.
An optimum transfection provides the strongest reporter gene signals possible, while
minimizing the amount of toxicity and stress placed on the cells. Higher values for the
individual NF-kB luciferase and renilla luciferase reporter gene signals are necessary to limit
background noise and achieve accurate results. The ratio of the two signals, which is
proportional to the actual level of NF-kB activity, is an indicator of the amount of toxicity and
stress placed upon the cells. Any stress placed upon the cells will falsely elevate baseline NF-kB
activity levels, and thus it is important to minimize cell stress in order to achieve accurate results.
Based the data from this experiment, 0.25 μg of reporter DNA (0.22 μg NF-kB luciferase and
0.03 μg of renilla luciferase), 2.0 μl of lipofectamine and 2 μl of PLUS reagent are optimal, as
these values produced the highest individual signals and one of the lowest levels of toxicity.
These values were used in future transfection experiments to achieve the greatest reporter gene
signals while minimizes cell toxicity and stress.
Effect of HPV-16 E6 and E7 Genes on NF-kB Activity
Preliminary results show that the E6 viral gene increases NF-kB activity, the E7 gene
decreases NF-kB activity, and the combination of the E6 and E7 genes inhibits NF-kB as
compared to the baseline level of activation by the empty viral vector, PLXSN (Figure 5).
30
25
20
PLXSN
E6
E7
E6/E7
15
10
5
0
Normal KSFM
Ca+2 Media
TNFα
Figure 5. In normal KSFM media, calcium media, and TNFα the E6 genes tends to upregulate
NF-kB activity, E7 tends to downregulate activity, and the combination of the E6 and E7 genes
produce variable results. PLXSN represents cervical cells retroviral-infected with the empty
viral vector as a control.
The combination of the E6 and E7 genes is of particular interest, because both genes are
normally co-expressed in infected cervical cells. There is some variation in response, as the
cervical cells are derived from individuals who respond differently to the infection. Normal
KSFM media is simply the growth media that the cell culutres are grown in. Calcium (Ca+2)
media is used to promote differentiation of the cells, and TNFα is added as a known inducer of
NF-kB activity. The general upregulation of NF-kB activity by E6 and the downregulation by
E7 is somewhat consistent in normal KSFM, Ca+2 media, and in TNFα. Statistical tests will be
performed to determine whether the differneces in NF-kB activity produced by the E6 and E7
genes are significantly different from the baseline levels.
Goal/Expected Results
The next step in my research is to measure the concentrations of p50 and p65 NK-kB
subunits in nuclear and cytoplasmic cell fractions using a Western Blot technique. I expect that
the concentrations of each NF-kB subunit protein in the nuclear fraction will be the highest in
cells expressing the E6 oncogene. The subunits will likely be located predominately in the
cytoplasm in cells expressing the E7 gene and PLXSN, the empty retroviral vector. I expect the
majority of the subunit concentration to be in the cytoplasm, with a slightly higher level of
expression above the baseline in cells co-expressing the E6/E7 genes. These results would be
consistent with the observed increase in NF-kB activity in E6- infected cells and the decrease in
activity in E7- infected cells in experiments utilizing the reporter gene assay to measure NF-kB
activity. It is possible, however, that one or two subunits may be responsible for the activation of
NF-kB by translocating to the nucleus in response to infection by HPV oncogenes, while another
remains in the cytoplasm.
Timeline
I am currently working on finalizing my procedure for the Western Blotting experiments
and ordering necessary reagents. This semester, I will perform a “test” Western Blot experiment
on normal cervical cells and work out any problems with the new procedure. By the end of this
semester, I plan to begin Western Blot experiments to detect p50 and p65 NF-kB subunit
concentrations in the nucleus and cytoplasm. I also plan to complete drafts of Chapters 1-3 of
my thesis. During the fall semester of my junior year, I will continue Western Blotting Analysis
to detect the p50 and p65 subunits. I also plan to complete a draft of my thesis during the fall
semester of my senior year. The spring semester of my senior year will be devoted to finalizing
the written aspect of my thesis.
References
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Branca et al. 2006. Upregulation of NF-kB is related to the grade of cervical intraepithelial
neoplasia, but is not an independent predictor of high-risk human papillomavirus or
disease outcome in cervical cancer. Diagnostic Cytopathology. 34:8.
Divya CS. 2006. Antitumor action of curcumin in human papillomavirus associated cells
involves downregulation of viral oncogenes, prevention of NFkB and AP-1 translocation,
and modulation of apoptosis. Molecular Carcinogenesis. 45(5), 320-332.
Jansen-Dürr P. 1996. Viral oncogenesis and cell cycle control. Virus Research. 42(1-2), 187-191.
Karin M, Greten FR. 2005. NF-kB: Linking inflammation and immunity to cancer development
and progression. Nature. 5:749-759.
Kuroda M etal. 2005. The human papillomavirus E6 and E7 inducible oncogene, hWAPL,
exhibits potential as a therapeutic target. British Journal of Cancer 92, 290-293.
L. Harvard et al. 2004. High levels of p105 (NFKB1) and p100 (NFKB2) proteins in HPV16transformed keratinocytes: role of E6 and E7 oncoproteins. Virology. 331(2), 357-366.
McIntosh N. MD. 2000. JHPIEGO Corporation. Human Papillomavirus and Cervical Cancer.
United States Agency for International Development.
Perkins ND, Gilmore TD. 2006. Good cop, bad cop: the different faces of Nf-kB. Nature.
13:759-772.