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Role of TLR4 and AID in lymphomagenesis induced by obesity and hepatitis C virus

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Role of TLR4 and AID in lymphomagenesis induced by obesity and hepatitis C virus

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Content i

ROLE OF TLR4 AND AID IN LYMPHOMAGENESIS INDUCED BY
OBESITY AND HEPATITIS C VIRUS
by
Rajeshwar Nitiyanandan

A Thesis Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(MOLECULAR MICROBIOLOGY AND IMMUNOLOGY)

August 2013

Copyright 2013 Rajeshwar Nitiyanandan
ii

ACKNOWLEDGEMENTS

I would like to take this opportunity to thank my thesis advisor, Dr. Keigo
Machida, for being an excellent mentor and for being very supportive of my work
in lab during the past two years. He has always given me the liberty to come up
with my own ideas and implement them in the project but at the same time has
given excellent inputs to improve the experiments I have performed.
I want to take this opportunity to thank Ms. Chia-Lin Chen for teaching me all the
techniques I had needed to complete this project successfully and also for her
valuable insights regarding the experiments.
I am also grateful to Felix Liu for training me to handle animals and perform
surgeries on them and also to my fellow lab members Dinesh Babu and Ambika
Ramrakhiani for their help during the course of this project. I am also extremely
thankful to Mrs. Michelle MacVeigh-Aloni who helped me with Confocal
microscopy.
I am extremely thankful to Mr. Sinivasan for his financial support which helped
me pursue my Masters degree at USC.
Finally I would like to thank my parents who have been the biggest source of
moral support for me during all these years and all my friends who have helped
in one way or another especially in the past two years.

iii

TABLE OF CONTENTS

Acknowledgements ii

List of Tables

iv
List of Figures

v
Abstract

vi
1. Introduction 1
1.1 Hepatitis C Virus 1
1.2 Lymphoma 3
1.3 Toll-Like Receptor 4 5
1.4 Activation-Induced Cytidine Deaminase 5
1.5 Background and Hypothesis 6

2. Materials and Methods 11
3. Results 17
4. Discussion

26
5. Summary 28
6. Future Directions

References

29

30
iv

LIST OF TABLES
Table 1. Primer sets for qRT-PCR 15

v

LIST OF FIGURES

Figure 1. Hepatitis C Virus Complete Genome 1
Figure 2. Histological comparison of Normal and Diseased Spleen.
HCV gene expression studies 8
Figure 3. Occurrence of Myc-Igh translocations in AID proficient mice 9
Figure 4. Hypothetical Model 10
Figure 5. AID and Ig mRNA expression studies in Normal and Core
positive splenocytes 18
Figure 6. Splenocyte proliferation studies in Core positive and
Normal mice 20
Figure 7. AID and C-Myc gene expression studies 22
Figure 8. PCR study to test presence of Myc-IgH translocations in
High fat diet fed Core positive mice 23
Figure 9. Immunofluorescence studies for C-Myc expression in
Lymphoma and Tumor tissues 25

vi

ABSTRACT
Role of TLR4 and AID in Lymphomagenesis induced by Obesity and
Hepatitis C Virus
Lymphomas are cancers occurring in T and B lymphocytes which affect nearly
700,000 people in the United States. It has been suggested that Hepatitis C virus
which is known to predominantly cause liver associated diseases is also
responsible for B cell related disorders in a chronic state. Recent research has
shown that the risk of Lymphoma incidence increases with an intake of high fat
diet. AID or activation induced cytidine deaminase has been found to promote
Lymphomagenesis via induction of chromosomal translocations (Okazaki, Kotani,
& Honjo, 2007). Toll like receptor a known driver of AID expression has been
found over expressed in HCV infected cells (Machida et al., 2006). We proposed
that the HCV infection and intake of a high fat diet may have synergistic effect on
AID expression leading to chromosomal aberrations which induces
Lymphomagenesis. To test this we performed gene expression studies for AID,
Immunoglobulins and C-Myc and also detected the presence of chromosomal
translocations in infected cells through PCR and Immunofluorescence studies.

1

CHAPTER 1

INTRODUCTION

1.1 Hepatitis C Virus
Hepatitis C virus, an enveloped RNA virus is a part of the Flaviviridae
family and the genus Hepacivirus. It has a single stranded positive sense RNA
which contains a lone Open Reading Frame (ORF) consisting of 9600 base
pairs. This RNA being positive sense can be directly translated into proteins. The
genome codes for 3 structural and 7 non-structural proteins while also containing
untranslated regions (UTR) in the 3’ and 5’ ends. The 10 proteins being coded for
are the Core, E1, E2, NS1, NS2, NS3, NS4A, NS4B, NS5A and NS5B. There is
also a protein encoded by an alternate reading frame known as the F protein(De
Francesco, 1999; KATO, 2000).

Fig 1. Complete genome of Hepatitis C Virus. Adapted from (Chevaliez &
Pawlotsky, 2006).
The E1 and E2 envelope glycoproteins and Core(C) proteins act as the 3
structural proteins. The core protein is an RNA binding protein which functions
principally as a nuclear capsid (McLauchlan, 2000) and has tendencies to
interact with a few cellular proteins and play a role in the viral lifecycle and
2

activation of genes like C-Myc (Ray, Martin Lagging, Meyer, Steele, & Ray, 1995;
Shih, Lo, Miyamura, Chen, & Lee, 1993). A mutated form of the core protein is
present and is known as F protein and is the result of a -2/+1 frame shift
translation in the core encoding region . Envelope glycoproteins E1 and E2 form
the viral envelope and are critical for viral entry. They are 35 and 72kDa in size
respectively (Bartosch, Dubuisson, & Cosset, 2003). Studies with respect to E2
have shown that it initiates the infectivity process by interacting with cell surface
receptors like CD81, SCARB1, Claudin and Occludin (Hsu et al., 2003; Rosa et
al., 1996; Zeisel, Barth, Schuster, & Baumert, 2009).
The non-structural proteins are mainly required for viral transcription and
replication processes except for the p7 protein. The p7 proteins is important in
the infectivity process as any mutations in its cytoplasmic loop decreased the
infectivity of the virus (Carrère-Kremer et al., 2002). It is also a viroporin
(Gonzalez & Carrasco, 2003) and may help in the release of newly produced
virus particles.
The NS2 gene codes for a zinc dependent metalloprotease which cleaves the
NS2-NS3 region and after cleavage the proteolytic ability is lost and the NS2
protein is degraded by a proteasome in phosphorylation dependent manner
(Yamaga & Ou, 2002).
NS3-NS4A is protease which has several functions. It is a serine protease and it
helps cleave the NS3-NS4A, NS4A-NS4B, NS4B-NS5A and NS5A-NS5B
junctions (Grakoui, Wychowski, Lin, Feinstone, & Rice, 1993). The NS4A region
3

acts as a co-factor to the protease activity of the NS3 protein. The protein is also
found to block dsRNA mediated interferon regulatory pathway (IRF) and RIG-I
pathway (Foy et al., 2003). The NS3 protein is also known to have helicase
(Gwack, Kim, Han, & Choe, 1997; Tai, Chi, Chen, & Hwang, 1996) and NTP
hydrolysis activity.
NS4B is a 27kDA integral membrane protein, It is hydrophobic and contains 4
transmembrane proteins. It’s main function is changing the structure of the
endoplasmic reticulum to a structure commonly known as the membranous web
(Hügle et al., 2001) and is also a part of the RNA replication complex (Piccininni
et al., 2002).
NS5A which is a phosphorylated zinc metalloprotein plays a role in interferon
resistance by binding to PKR and also seems to associate with lipid rafts (Gao,
Aizaki, He, & Lai, 2004; Shi, Lee, Aizaki, Hwang, & Lai, 2003). NS5B is an RNA
dependent RNA polymerase(RdRp) (Ivashkina et al., 2002) and is part of a family
of proteins known as tail anchored proteins (Brass et al., 2010).
1.2 Lymphoma
Lymphomas are a type of Blood cancer which primarily affect the T and B
lymphocytes. These lymphomas may present itself in various organs like the
spleen, lymph node, bone marrow, blood etc. The lymphomas are part of larger
group of neoplastic diseases known as hematological neoplasms. The first type
of Lymphoma was described by Dr. Thomas Hodgkin.
4

The Lymphomas were first categorized into Hodgkin’s and Non-Hodgkin’s
lymphomas but due to the presence of many variations between each type of
lymphoma this classification was stopped. The most commonly used
classification system used right now is the WHO classification system (Jaffe,
2001) in which the Lymphomas are classified on the basis of their type i.e. B-cell,
T-Cell and Natural Killer cells. Lymphomas are the most common type of Blood
related malignancy and make nearly 5.5% of all types of cancers and 56% of
blood cancers (Horner et al., 2009). Patients with weakened immune systems as
is the case with people suffering from SCID, CVID and AIDS have a higher
tendency to get lymphomas.
The prognosis changes according to each lymphoma, some lymphomas are mild
and can be easily treated with chemo- or radiotherapy and patients can lead a
normal life but cannot be completely cured, other types are extremely aggressive
and are fatal within months of diagnosis.
Some of the common symptoms of Lymphomas are fever, night sweats and
substantial weight loss and also include anorexia and fatigue. Two very common
types of lymphomas present in HCV patients are Diffuse Large B cell Lymphoma
and Marginal Zone B cell lymphomas.

5

1.3 Toll Like Receptor-4
The TLR4 protein is encoded by the TLR4 gene and is part of the toll like
receptor family. This receptor can detect bacterial Lipopolysaccharide(LPS) and
induce the innate immunity.
The toll like receptors are highly conserved in species from drosophila to humans
and are highly expressed in Lymphocytes. The TLR4 mostly uses the MyD88
pathway for signal transduction and is dependent upon Rab1. Alcohols are
supposed to be potent agonists of TLR4 and prior research showed that ethanol
can activate the TLR4 in the absence of LPS (Blanco, Vallés, Pascual, & Guerri,
2005). It has also been reported that a mutation in the TLR4 gene resulted in a
reduction of alcoholism in mice.
1.4 Activation induced Cytidine Deaminase
Activation induced cytidine de-aminase or AID is coded for by the gene
AICDA. It is essentially a 24kDA enzyme whose basic function is the deamination
of Cytosine to form Uracil which in turn is recognized as a Thymine resulting in
mutations. AID also uses three mechanisms for the antibody diversification
process namely 1. Somatic Hypermutation 2. Class Switch Recombination 3.
Gene Conversion. Somatic Hypermutation is generally used to cause small
mutations which helps in enhancing affinity towards a particular antigen. Class
switch recombination is used to convert IgM to other types of Ig. The deamination
process of AID happens only in transcriptionally active regions and this might be
due to its high activity in single stranded DNA. The AID protein is generally
6

present in the germinal center where the B-Cells undergo activation, proliferation
and antibody diversification processes. It has been found that certain mutations
in or a transcriptionally activated AID leads to mutations in the B cells resulting
Lymphomas.
1.5 Background and Hypothesis
Hepatitis C Virus is known to be conducive to incidence of Lymphoma
specifically of the B cell type, Tsukiyama-Kohara and Colleagues had shown
significant incidence of lymphomas in RzCD19Cre mice which expressed the
complete genome of the HCV specifically in the B cells (Kasama et al., 2010),
They observed a 25% incidence of Diffuse Large B cell lymphomas in their case
study, Similarly a Cohort Study done by Kimberly Bertrand et al indicates a link
between a higher BMI with a increased risk of NHLs. It was shown that a women
and men with a BMI> 30 had a 20% and 63% higher risk of lymphoma incidence
respectively. HCV in general tends to have a synergistic effect along with a high
fat diet in the incidence of hepatic diseases and this might be replicated in the
case of Lymphomagenesis. It has been discovered that the expression of TLR4
was found to be much higher in the case of HCV infected cells as compared to
cells infected with inactivated virus and mock infected cells (Machida et al.,
2006). The induction of germinal center derived Lymphomagenesis was found to
be highly dependent on the presence of AID protein with the knockout mice
showing no induction of lymphomas (Pasqualucci et al., 2007) .It was also found
that these AID expressing mice with lymphomas had translocations between the
7

C-Myc and IgH regions. Shyam Unniraman and his group found that AID is
required for Myc-Ig translocations to take place in an in-vivo setting (Unniraman,
Zhou, & Schatz, 2004). Prior research work has shown that binding of the HCV
E2 protein to CD81 results in the cell escaping apoptosis mediated by the fas
ligand (Chen et al., 2011) and it was shown by (Rosa et al., 2005) that the
activation of CD81 resulted in transformation of Naïve B Lymphocytes into
Memory B lymphocytes leading to various lymphocytic disorders.
8

Fig 2 A. shows a histological comparison between normal and lymphoma
affected spleen tissues. A diffuse growth pattern is observed in the affected
tissue.
B. PCR experiments performed show the expression of HCV genome in the
RzCD19cre mice (Adapted from (Kasama et al., 2010).
9

Fig 3. Presence of Myc-IgH translocations compared between AID knockout and
Wild type mice by PCR (Adapted from (Pasqualucci et al., 2007).
Prior research suggested a link between high fat and combined with a chronic
HCV infection. In the following experiments, we attempt to decipher if the
enhanced expression of TLR due to the infection by HCV is leading to a trans-
activated AID protein. We hypothize that the trans-activated AID demethylates
the C-Myc gene thereby enhancing the translocation process between Ig and C-
Myc. Since the Ig promoter is highly transcribed, the translocation of C-Myc to
the Ig promoter region leads to its constitutive expression. The constitutive
expression of the C-Myc oncogene and Transactivation of AID leads to further
deleterious mutations in the genome leading to Lymphomagenesis. To confirm
our hypothesis, we performed qRT-PCR, immunostaining studies for the
10

expression of the AID, C-Myc genes and proliferation studies of Core expressing
splenocytes.

Fig 7. Hypothetical Model
CD81
HCV
E2
AID
TLR4
Obesity
C-Jun
NF- κB
CORE
Ig
Genomic Instability
C-Myc IgH translocations
Lymphomagenesis
11

CHAPTER 2

MATERIALS AND METHODS

2.1 Cell Culture

HEK293T, Raji and BJAB cells were used for this study. HEK 293T is a
variant type of the Human embryonic kidney 293 cells containing the large T-
antigen of the SV40 virus. This variant helps achieve episomal replication of
transfected plasmids and generally used in retroviral vectors. These cells form
the basis for a lot of retroviral packaging cell lines. Raji cells are lymphoblastoid
cells which were derived from Human Burkitt's Lymphoma and is widely used for
detection of immune complexes. HEK 293T cells were cultured in Dulbecco's
modified Eagle's medium containing 10% Fetal Bovine Serum(FBS). Raji cells
were cultured in RPMI 1640 supplemented with 20%FBS, 1% Penicillin-
Streptomycin-Glutamate(PSG) and 1% Non Essential Amino Acid. B-Jab cells
were grown in RPMI 1640 media containing 10%FBS.
2.2 Plasmids and Electroporation
The reporter plasmid(Addgene Plasmid 14886) for analysis of
Immunoglobulin Kappa Chain promoter activity was obtained from Addgene(). A
control Renila luciferase expression plasmid pRL-SV40 was obtained from
Amersham Biosciences. The Raji cells were transfected via electroporation by
mixing the cells with the plasmid DNA and were pulsed at 950µF and 220V. The
cells were then grown in RPMI 1640 media containing 20%FBS, 1%PSG and 1%
NEAA.
12

2.3 In-Vitro RNA synthesis and Transfection
In-vitro transcription of HCV(SB/JFH1 Chimaeric) RNA was performed as
using MEGAscript T7 Kit(Ambion) with small modifications in protocol (Wakita et
al., 2005). Transfection of Raji Cells was performed by electroporation by mixing
cells along with viral DNA and pulsing them at 950µF and 220V. The media was
collected 7 days post infection to get the maximum titer of the virus. The medium
was then centrifuged at 2000rpm for 10minutes and the supernatant was filtered
through a 0.45µm filter(Millipore) before use for infection.
2.4 Live Virus infection
The Raji were cultured on 6 well plates. These cells were infected with the
SB/JFH1 chimaeric strain virus and were also stimulated with Bacterial
Lipopolysaccharide(25µg/ml). The cells were either infected with a live virus or an
UV inactivated form (3 minutes inactivation) and treated with LPS for various time
points i.e. 6hr, 12hr and 24hr time points. The cells were collected at given time
points and centrifuged. The RNA was then isolated from the cell pellet and used
for qRT-PCR studies.

13

2.5 Mice
Mice which express the Hepatitis C Virus Core protein of the HCV
genotype 1b under the human elongation factor 1a promoter were generated and
further bred at the USC transgenic mouse facility. TLR4 -/- knockout mice were
obtained from The Jackson Laboratories(Bar Harbor, Maine) .The mice were fed
with Lieber-DeCarli containing 3.5% Ethanol for 12 months based on the
intragastric ethanol infusion model (Tsukamoto. H et al). Following 12months the
mice were sacrificed and spleen and tumor tissues were collected. All
procedures were performed in accordance to protocol approved by the
Institutional Animal Care and Use Committee protocol at the University of
Southern California.

2.6 C-Myc IgH translocation PCR
Translocations between C-Myc and IgH regions were studied using the
PCR conditions and primers designed by Muller et al (Müller, Jones, Janz, &
Potter, 1997) . The PCR reaction was a nester PCR which followed the following
conditions: 5 minute initial Denaturation at 95
o
C followed by 40 cycles of
Denaturation(95
o
C for 15seconds), Annealing(65
o
c for 15 seconds) and
Extension(72
o
C for 2minutes). 1ul of the reaction product from the first round of
PCR was used for the second round of the nested PCR with identical conditions.
14

The Primers that were used for this reaction are Ig1, 5'-AGC-TCA-TTC-CAG-
CTC-AGC-TCA-GCC-T-3'; Ig2,5'-AGC-TCA-GCT-CAG-CCT-ARC-CCA-GCT-C-
3′; Myc1, 5′-AGG - GAT - ACC - CGC - GGA - TCC - CAA - GTA - GGA - ATG -
TGA -GG-3′; Myc2, 5′-CCA-AGT-CAA-CGA-ATC-GGT-CAC-ATC-CCT-GTC-
CCA-AT-3′; Ig1 and Myc1 primers were used for the first round PCR reaction
followed by Ig2 and Myc2 primers for the second reaction. The expected size of
the products was around 500bp to 2.4kb.

2.7 Quantitative Real-time RT-PCR
Total RNA was isolated using Qiagen RNeasy mini kit(Qiagen, Inc., Venlo,
Netherlands) according the manufacturer's protocol, and the RNA concentration
was measured using Thermo Scientific NanoDrop
TM
Spectrophotometer. cDNA
was synthesized from the RNA templates using Random primers and 10mM
dNTPs under the following conditions- 16
o
C for 30min, 42
o
C for 30min and 85
o
C
for 5min. Real time PCR analysis was performed on ABI 7900 HT QPCR
system(Life Technologies, Carlsbad, CA) using SYBR Green QPCR Master Mix
(Stratagene) according to the manufacturers' instructions. Primer sets for Ig, AID,
C-Myc, β-Actin are listed in Table. β-Actin was used as endogenous reference
control.

15

Target Primers
IgH hFW1(F) 5'-AGGTGCAGCTGGWGSAGTSKGG-3'
hM3(R) 5'-GGAAAAGGGTTGGGGCGGAT-3'
AID (F) 5'-AAATGTCCGCTGGGCTAAGG-3'
(R) 5'-GGAGGAAGAGCAATTCCACGT-3'
C-Myc (F) 5'-CTTCTCTGAAAGGCTCTCCTTG-3'
(R) 5'-GTCGAGGTCATAGTTCCTGTTG-3'
β-Actin (F) 5'-TTCTACAATGAGCTGCGTGTG-3'
(R) 5'-GGGGTGTTGAAGGTCTCAAA-3'

Table 1. Primer Sets for Quantitative RT-PCR.

2.8 Immunofluorescence
Tissue sections were prepared from paraffin embedded cassettes of
Mouse spleen. Lymphoma tissues were deparaffinized at 55
o
C overnight and
then immersed in Xylene three times for 5 minutes followed by immersion in
varying concentrations of ethanol(100%,95% , and 50%) for 5 minutes each , and
were washed with 1XPBS for 5 minutes. Antigen retrieval was performed on the
paraffin sections by heating in 10mM sodium citrate buffer(pH6.0) for 15 minutes
at 90
o
C and then washed for 15 minutes in 1XPBST. The tissue sections were
then blocked for 1 hr in a blocking buffer(5% goat sera in PBS) and probed with
16

the required primary antibodies with a dilution of 1:200 in a dilution buffer. The
sections were incubated overnight at 4
o
C followed by a 15 minute was in 1XPBS
and then followed by conjugation with the necessary secondary antibody with a
dilution of 1:400 for 1hr at room temperature. Nuclear staining of the tissue
sections was performed using 4′,6′-diamidino-2-phenylindole
dihydrochloride(DAPI) contained in the mounting media. The various tissue
sections were then visualized using a Confocal Microscope( Zeiss LSM 510
confocal laser scanning microscope). Microscopy was carried out as per the
guidelines of the system operating software provided with the microscope and all
images were photographed in identical conditions. The microscopy services were
provided by the Center for Liver Diseases at the University of Southern
California(USC-LDRC).
2.9 Statistical Analysis
Statistical Analysis for all studies was performed using student T-Test.

17

CHAPTER 3

RESULTS

3.1 AID(activation induced cytidine deaminase) and Immunoglobulin
mRNA are over expressed in High fat diet fed HCV Core protein producing
mice presenting Splenomegaly.
To determine if there was increased expression of AID and
Immunoglobulin in Hepatitis C virus Core protein transgenic mice fed with a High
fat diet, The Core Tg and TLR4-/-, Wild Type mice were fed with a Lieber-
DeCarli high fat diet for a period of 12 months. Mice were sacrificed after
completion of the diet period of 12 months and the spleen tissues were collected.
Splenocytes were isolated from these tissues and cultured in RPMI media, after
which RNA was isolated and used for the qRT-PCR study. The splenocytes
isolated from mice which presented an enlarged spleen(Splenomegaly) showed
a significant increase in the mRNA levels of both AID and
Immunoglobulins(Figure 1), whereas mice without any enlarged spleen showed a
minor yet insignificant(Figure 1) increase of mRNA levels in both AID and
Immunoglobulins. The High fat diet tends to activate TLR4 which leads to the
activation of NF-КB which binds to its binding site in the AID promoter, leading to
the activation of the AID gene. Incidentally the HCV core protein, which
increased the expression of TLR4 as shown by K Machida et al may lead to a
constitutively activated AID gene. It has also been shown that a constitutively
18

activated AID increased the tumor incidence in B Cells (Okazaki et al., 2007).
The core protein may result in the phosphorylation of C-Jun via the JNK pathway
similar to the activation of CD81 by binding HCV E2 which leads to the
phosphorylation of C-Jun resulting in a higher expression of immunoglobulin
genes (Rosa et al., 2005).
A. B.

Figure 5. AID and Immunoglobulin mRNA are over expressed in Core transgenic
mice presenting Splenomegaly. RNA isolated from the different splenocytes was
subjected to qRT-PCR studies with the respective primers for AID and Ig. A.
Data show a significant increase in Ig mRNA levels in Core Transgenic mice
presenting splenomegaly as compared to Core negative mice. B. Core positive
mice with an enlarged spleen tend to express AID mRNA at a significantly higher
rate than Core negative mice.

19

3.2 Increased proliferation of splenocytes in Core Transgenic mice with an
enlarged spleen
AID has been shown to be a cause of germinal center derived
Lymphomagenesis (Pasqualucci et al., 2007). Hence we wondered if the
increased expression of AID could lead to enhanced proliferative rate of the
splenocytes of the core transgenic mice as compared to the Core negative mice.
For this study, splenocytes from mice presenting enlarged spleen in the core
positive group and normal sized spleens from the core negative group were
used. The splenocytes were cultured in RPMI 1640 media. As shown in Figure 2,
There was a much higher rate of proliferation in the case of splenocytes isolated
from the enlarged spleen whereas the splenocytes from the normal spleen
showed a low growth rate. This suggested that the over expressed AID might be
causing certain changes within the Core Tg splenocytes which caused it to
proliferate faster than normal splenocytes.

20

Figure 6. Enhanced Proliferation of Core Tg mice splenocytes as compared to
core negative mice.

3.3 Over expression of AID leads to induction of higher levels of C-Myc
mRNA
A time point assay was performed to determine if expression of C-Myc mRNA
correlated with the expression of AID. For this experiment Raji cells were infected
with a SB/JFH1 chimaeric strain virus along with LPS activation for 6,12 and 24
hour time points. As control the Virus was inactivated under UV for 3 minutes and
used to infect Raji cells for 6, 12 and 24 hours, Mock and LPS activated Raji cells
were also used as controls. Cells were collected at the various time points and
RNA was isolated using QIAGEN RNeasy mini kit according manufacturer's
instructions. qRT-PCR was performed to determine AID and C-Myc expression
levels at the specific time points. It was found that the maximum expression of
21

AID was at 12hrs post infection, this correlated with the C-Myc expression levels
at the same time point suggesting a relation between an increased expression of
AID and C-Myc . Both AID and C-Myc mRNA were expressed at significantly
higher levels compared to the Mock infected cells(Fig 4).

22

A.

B.
Fig 7. A. AID expression levels at different time points were studied. Maximum
expression was observed at 12hr post infection also a nearly significant increase
was seen 6hrs post infection B. C-Myc expression level at similar time points
were studied. Maximum expression was again observed at 12hr post infection
suggesting a link between the AID and C-Myc mRNA expression levels. The
increase in C-Myc mRNA was significant compared to Mock infected cells.
23

3.4 Splenocytes of Splenomegaly presenting Core Transgenic mice show
translocation between Myc-Igh regions
DNA was isolated from splenocytes of Core Tg and Wild type mice and PCR was
performed using primers designed and conditions given by Muller et al. It was
observed that 2 out of 5 samples showed translocation between C-Myc Igh
regions. No translocation was observed in Wild types DNA samples. BJAB cells
were used as a positive control for this purpose. Size of bands was expected to
be within a range <500bp up to 2.4kb.

Fig 8. Myc-IgH Translocations are indicated by arrows. Lane 2 and Lane 5 are
positive for translocations. Lane 12 is used as a positive control. Lane 7-11 are
negative controls.
Lane 2 Lane 5 Lane 12
24

3.5 AID protein over expression due to HCV leads to translocations
between C-Myc and IgH regions
It has been shown that AID expression leads production of C-Myc-Igh
translocations in Germinal center derived Lymphomagenesis (Laura Pasqualucci
et al). Hence to test if a similar mechanism of action is taking place in this
scenario we performed a Nested PCR and Immunofluorescence study. The
Nested PCR study done using primers for Ig and C-Myc designed by Muller et
al. showed the presence of translocations in 2 out of 5 Core transgenic mice fed
with a high fat diet and Wild type mice fed with a control diet did not exhibit any
translocations. The Immunofluorescence study was a comparative study
between a Lymphoma tissue, Normal Spleen Tissue and a Liver Tumor Tissue.
The Confocal images for the study showed a localization of C-Myc in both the
Cytoplasm and Nucleus(marked by arrow heads) in the Lymphoma tissue(Figure
3A) which is indicative of a translocation between the C-Myc and IgH regions in
Lymphomas as shown by prior research evidence that nuclear and cytoplasmic
localization is a hallmark of C-Myc IgH translocation whereas only cytoplasmic
localization is present with the lack of any such kind of translocation (Ruzinova,
Caron, & Rodig, 2010). The Liver tumor tissue section showed localization of C-
Myc in the cytoplasm which indicates that there is no translocation in the C-Myc
IgH regions(Figure 3B).
25

A. B.
Figure 9. Immunofluorescence studies done by Confocal Microscopy with DAPI
and FITC stains. A. Arrowheads mark the positions of C-Myc localization in
cytoplasm and nucleus indicating presence of translocation. B. C-Myc
localization present only in cytoplasm as marked by arrowheads indicates lack of
translocation.
26

CHAPTER 4

DISCUSSION

4.1 Translocations are a typical phenomenon in multiple types of
lymphomas
Many different types of lymphomas present themselves with a multitude of
chromosomal aberrations which lead to translocations between oncogenes and
highly transcribed regions. One of the most commonly translocated regions is the
Immunoglobulin promoter regions. Cancers such as Diffuse large B cell
lymphoma, Burkitt’s Lymphoma etc have been found to contain translocations
between C-Myc and Ig regions. These lymphomas alternatively might also have
translocations between BCL6 and Ig regions with Ig being a common
denominator in all these translocations. The preference for these translocations
to happen in the Ig regions is due to it being a highly transcribed region which
results in oncogenes like C-Myc and BCL6 being over expressed. Enzymes like
AID have been found to be drivers for these translocations due to their ability to
cause mutations in the genome or by demethylation thereby leaving the DNA
susceptible to these translocation processes.
4.2 Obesity increases the propensity for Lymphomagenesis
Various epidemiological studies have shown that a high fat diet increases risk of
Lymphoma. A diet consisting of red meat ups the chances of lymphomas
especially in women (Zhang et al., 1999). Research has shown the differences in
risk of NHL induction in diets consisting of red meat, fried meat and vegetables
27

and it was seen that both red meats and fried meats significantly increased
chances of NHL (Chang et al., 2005).
4.3 Activation of CD81 may play an important role in induction of
Lymphomagenesis
CD81 is one of the 4 most important receptors for HCV binding and entry.
Research work has shown that CD81 helps the B-cell avoid apoptosis via the
Fas-mediated pathway (Chen et al., 2011) and activation of CD81 by the HCV E2
protein leads to transformation of Naive B-cells into memory B-cells and this
process follows the JNK pathway resulting in accumulation of Phospo C-Jun
(Rosa et al., 2005) which lead to transactivation of the immunoglobulin promoter.
It has also been shown that E2-CD81 interaction induces hypermutations in the B
cell genome and also leads to increased AID expression, Incidentally this hyper
mutation process is lost with the silencing of the AID gene showing a direct link
between E2-CD81 interaction and AID over expression (Machida, Cheng, Pavio,
Sung, & Lai, 2005). The expression of CD81 has also been found to be really
high in B cells present in the germinal center (Luo et al., 2010) where the
expression of AID is at its peak. All these seem to suggest that CD81 may play
an important role in Lymphomagenesis induction.

28

CHAPTER 5
SUMMARY
The aim of this study was to determine the link between high fat diet and
Hepatitis C virus infection in induction of Lymphomagenesis, for this purpose we
performed gene expression studies to determine if AID, C-Myc and
Immunoglobulin genes were over expressed. We also found a correlation
between the expression of AID and the expression of C-Myc as seen in fig . To
test if this hypothesis stands true in an in-vivo setting we isolated splenocytes
from transgenic mice expressing core protein and also from wild type mice to be
used as control. It was observed that the transgenic mice presenting with an
enlarged spleen showed an increased expression of AID and Immunoglobulin
genes. We also found that the core expressing splenocytes showed a higher
tendency to proliferate as compared to the wild type splenocytes. To test the
presence of translocations we performed PCR and Immunofluorescence studies.
Translocations were found in 40% of the cases in during the PCR studies as
seen in fig. Translocation in the C-Myc region was confirmed by staining for C-
Myc protein in Lymphoma tissue section which showed both nuclear and
cytoplasmic localization of C-Myc which is a hallmark of C-Myc- IgH
translocation.

29

CHAPTER 6
FUTURE DIRECTIONS
Having found that the activation of TLR4 and CD81 is directly leading to an
increased expression of AID and C-Myc and may lead to chromosomal
aberrations and translocations between C-Myc and IgH regions as shown in
Lymphoma cases. It is now imperative to show direct mechanistic evidence of
this activation causing such translocations. For this purpose Raji cells cannot be
used as they already contain the specific translocation and hence a cell line
lacking the translocation needs to be used. A B cell lymphoma cell line known as
SU- DHL 5 which lacks the translocation has been provided by Dr. Alan Epstein.
The cell line will be infected with HCV pseudo particles along with LPS activation
for different time points and as controls the cell line will be transformed to have
silenced CD81 and AID and similar time point assay will be performed. qRT-
PCR will be performed to quantify AID, C-Myc and Immunoglobulin gene
expression. Western blotting will also be performed to test the expression of
AID, C-Myc and Immunoglobulin proteins. Ki67 staining will be used to confirm
proliferation of splenocytes in core transgenic mice.

30

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Abstract (if available)

Abstract Lymphomas are cancers occurring in T and B lymphocytes which affect nearly 700,000 people in the United States. It has been suggested that Hepatitis C virus which is known to predominantly cause liver associated diseases is also responsible for B cell related disorders in a chronic state. Recent research has shown that the risk of Lymphoma incidence increases with an intake of high fat diet. AID or activation induced cytidine deaminase has been found to promote Lymphomagenesis via induction of chromosomal translocations (Okazaki, Kotani, & Honjo, 2007). Toll like receptor a known driver of AID expression has been found over expressed in HCV infected cells (Machida et al., 2006). We proposed that the HCV infection and intake of a high fat diet may have synergistic effect on AID expression leading to chromosomal aberrations which induces Lymphomagenesis. To test this we performed gene expression studies for AID, Immunoglobulins and C-Myc and also detected the presence of chromosomal translocations in infected cells through PCR and Immunofluorescence studies.

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Asset Metadata

Creator Nitiyanandan, Rajeshwar (author)

Core Title Role of TLR4 and AID in lymphomagenesis induced by obesity and hepatitis C virus

School Keck School of Medicine

Degree Master of Science

Degree Program Molecular Microbiology and Immunology

Publication Date 08/01/2013

Defense Date 05/29/2013

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag activation induced cytidine deaminase,hepatitis c virus,lymphoma,OAI-PMH Harvest,obesity,TLR4

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Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Machida, Keigo (committee chair), Epstein, Alan L. (committee member), Ou, J.-H. James (committee member)

Creator Email nitiyana@usc.edu,nrajeshniti@gmail.com

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