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CpG poor promoter SULT1C2 regulated by DNA methylation and is induced by cigarette smoke condensate in lung cell lines

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CpG poor promoter SULT1C2 regulated by DNA methylation and is induced by cigarette smoke condensate in lung cell lines

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Content CpG Poor Promoter SULT1C2 Regulated by DNA
Methylation and is Induced by Cigarette Smoke
Condensate in Lung Cell Lines

By

Candace Jeanette-Sukey Johnson

A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA

In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(GENETICS MOLECULAR AND CELLULAR BIOLOGY)

August 2015

i

ABSTRACT

We used the Illumina Infinium HumanMethylation27 BeadChip to investigate DNA
methylation in lung cancer tissue and adjacent non tumor lung (AdjNTL) from sixty subjects
with lung adenocarcinoma. Significant hypomethylation of a SULT1C2 promoter CpG
(cg13968390) dinucleotide was noted in AdjNTL from Asians (most of whom were female non-
smokers) vs. Caucasians (most of whom were smokers). SULT1C2 is a sulfotransferase, a phase
II enzyme that transfers sulfur groups to various substrates, including xenobiotics and chemical
carcinogens, thereby improving water-solubility to facilitate clearing via urine or bile. However,
in the process of xenobiotic modification, sometimes carcinogenic compounds are created as
by products. In the sixty subjects, SULT1C2 methylation and expression in AdjNTL were
inversely correlated. SULT1C2 expression is not normally detected in the adult lung. We
hypothesized that environmental exposures might induce SULT1C2 through demethylation. We
investigated the effect of demethylation on the SULT1C2 CpG-poor promoter using 5-Aza-2’-
deoxycytidine, a drug that is incorporated into the DNA of dividing cells and inhibits DNA
methylation. Indeed, when methylation was prevented, expression of SULT1C2 increased in PC3
and H2347 lung adenocarcinoma cell lines and also in immortalized bronchial epithelial cells
(BEAS2B). We performed a luciferase assay driven by a 1.8 kb fragment including the SULT1C2
promoter and part of exon1 into a CpG-less vector and observed that methylation of the
promoter significantly decreased luciferase activity. Finally, as a model for the effect of
environmental factors such as cigarette smoke on the expression of SULT1C2, we treated the
cell lines with cigarette smoke condensate (CSC) at increasing doses for 24, 48, and 72 hours.
ii

Since the Aryl hydrocarbon receptor (AHR) plays a key role in activating phase I enzymes such
as CYP1B1, we searched the SULT1C2 promoter sequence for AHR binding sites and detected
one 1.3Kb upstream of the transcription start site. AHR expression increased concordantly with
SULT1C2 expression. Our results suggest that in non-smokers exposed to environmental
carcinogens or secondary tobacco smoke, AHR binding to the SULT1C2 promoter induced and
activated the expression of SULT1C2 possibly causing the long term epigenetic changes
reflected in lower DNA methylation levels at cg13968390.

iii

ACKNOWLEDGEMENTS

Firstly, I would like to thank Ite Laird-Offringa for pushing me to be “proactive” in my endeavor
of obtaining this degree. She was an integral part of my best getting better every single year in
this program. The late nights and weekends were all worth it.
Next I would like to thank my mother. From the beginning, she caused me to believe that if an
opportunity presents itself, be an opportunist. For all of the times I caused her to worry about
my welfare and never saying a negative word, thank you Mommy.
Most of all, I would like to acknowledge my husband Keith and my two daughters Sydney
and Simone. They are my inspiration and my foundation that keep me grounded when my head
gets too far in the clouds. They are the best thing I never knew I needed…family.
I would like to thank my laboratory daughter Diane Lee for always telling me to “Google it” and
Evelyn Tran for all the weight lifting sessions when it all became too much. I would like to thank
Crystal Marconett and Chenchen Yang for all the wonderful data and graphs provided from R
programming. I would like Po-Han Chen for always being level headed and showing me that just
because situations do not go my way it is no reason for panic.
Lastly, but not least, I would like to thank my committee members Dr. Michael Stallcup and
Dr. Gerry Coetzee for staying the long, long road of finally accomplishing what I began so long
ago. Truly the end of a thing is better than the beginning. Thank you for all the suggestions in
creating a better project.

iv

Table of Contents
CHAPTER 1: 5-aza-2-deoxycytidine Demethylates SULT1C2 and Induces Expression in Lung Cell Lines .. 1
Introduction .............................................................................................................................................. 1
Materials and Methods ............................................................................................................................ 6
Results..................................................................................................................................................... 10
Figure 1-1. SULT1C2 cg13968390 is hypomethylated in Asian never-smokers ................................... 13
Figure 1-2. LuAD PC3 PMR and Expression ......................................................................................... 14
Figure 1-3. H2347 PMR and Expression .............................................................................................. 15
Figure 1-4 ................................................................................................................................................ 16
Discussion ............................................................................................................................................... 17
Chapter 2: Lung Cell Lines Express SULT1C2 at Low Levels of Cigarette Smoke Exposure
but Not at High Levels ................................................................................................................................ 21
Introduction ............................................................................................................................................ 21
Materials and Methods .......................................................................................................................... 27
Results..................................................................................................................................................... 29
Figure 2-1 ................................................................................................................................................ 31
Figure 2-2 ................................................................................................................................................ 32
Figure 2-3 ................................................................................................................................................ 33
Discussion ............................................................................................................................................... 34
Chapter 3: Non-CpG Island Promoter of SULT1C2 is Regulated by DNA Methylation and
Bound by AHR ............................................................................................................................................. 37
Introduction ............................................................................................................................................ 37
Materials and Methods .......................................................................................................................... 42
Results..................................................................................................................................................... 46
Figure 3-1. . DNA ................................................................................................................................. 49
Figure 3-2. 1.8 kb SULT1C2 Fragment ................................................................................................. 50
Figure 3-3 ................................................................................................................................................ 51
Figure 3-4 ................................................................................................................................................ 52
Figure 3-5 ................................................................................................................................................ 53
Figure 3-6 ................................................................................................................................................ 56
Figure 3-7 ................................................................................................................................................ 57
Figure 3-8 ................................................................................................................................................ 58
Discussion ............................................................................................................................................... 59
v

Chapter 4: Conclusion ................................................................................................................................ 64
References .................................................................................................................................................. 68

1

CHAPTER 1: 5-aza-2-deoxycytidine Demethylates SULT1C2 and Induces
Expression in Lung Cell Lines

Introduction

As smoking in developing countries increases, lung cancer has become the most frequent
cancer-related death around the world
1
. Although lung cancer is not the leading diagnosis of
cancers, it is the leading cause of cancer deaths in both men and women in the United States
2
.
In North America, 90% of the men and 75% of the women who are patients with lung cancer
are current or former smokers
3
. However, in Taiwan, only 7% of the female patients with lung
adenocarcinoma are smokers
4
. This subtype of lung cancer is shared with smokers and non-
smokers alike. However when a non-smoker, former smoker, or Asian is diagnosed with lung
cancer, lung adenocarcinoma is the cancer 90% of the time
4, 5
. Several studies have carefully
investigated possible causes for this anomaly
6, 7
. Radon gas, asbestos, and environmental
pollutants have all been taken into consideration. One of the pollutants considered is
environmental tobacco smoke (ETS). Lee et al. found that children exposed to the highest levels
of ETS (>20 smoker-years) and in adult life(>40 smoker years) were 1.8 fold and 2.2 fold more
likely to get lung cancer than those non-smoking individuals that were never exposed
8
. To
further expound upon the effects of cigarette smoke, Novakovic et al. found that the
intrauterine environment can “program” the fetus and predispose the individual to disease in
adulthood
9
. They found that fetuses that were exposed to cigarette smoke via mother’s
smoking while pregnant had different DNA methylation pattern
2

than those fetuses not exposed. In cord blood mononuclear cells (CBMC), expression was
increased and DNA methylation of AHRR was decreased in mothers who smoked in comparison
to mothers who did not. Furthermore, although DNA methylation increased in 18 month old
individuals' peripheral blood mononuclear cells (PBMC) whose mothers smoked throughout
pregnancy, it did not reach the same DNA methylation levels as 18 month old individuals of
mothers who did not smoke
9
. Thus it is clear that second hand smoke can affect gene
expression and DNA methylation patterns.
The same is true for primary smoking. In adults, hypomethylation of CpGs is also the most
frequently seen DNA methylation alteration in smokers
10
. While some of these methylation
events appear to slowly revert after smoking cessation, others appear stable for many years
11
.
One well studied CpG, cg05575921, lies in the aryl hydrocarbon receptor repressor gene, a gene
involved in the control of the xenobiotic response. Hypomethylation of the CpG is associated
with increased expression of AHRR, as observed in blood, lung macrophages and lung tissue
exposed to smoke
12
. However, in most cases of smoking-related hypomethylation, the
functional consequences of the observed methylation loss are unknown.
Here we study a DNA hypomethylation event that appears to be linked to increased
expression of SULT1C2, another xenobiotic response gene, and that is detected in the histology
of normal lung tissue of female Asian non-smokers. Hypomethylation is correlated to SULT1C2
expression, suggesting it is somehow related to upregulation of this gene. The obsevation of
hypomethylation in the SULT1C2 promoter came from previous work in our laboratory focused
on identifying DNA methylation markers that could differentiate lung adenocarcinoma from the
adjacent non-tumor lung
13
. Sixty tumors and their matched adjacent non-tumor lung from
3

Canada were evaluated on the Illumina Infinium 27K platform. The goal was to investigate the
molecular differences (including DNA methylation) between smokers (n=30) and never smokers
(n=30, <100 cigarettes/lifetime).
Unfortunately, the sample collection was ethnically biased. 70% of the non-smokers were
Asian and less than 4% of the Asians were smokers. To investigate the possibility of
confounding factors based on ethnicity, our laboratory compared the tumor as well as the
adjacent non-tumor lung from the Asians (n=22) to that of the Caucasian patients (n=36). Our
laboratory found no significant DNA methylation differences in tumors. However, the adjacent
non-tumor lung from the two ethnicities varied significantly. SULT1C2 was statistically
significantly less methylated in Asian vs. Caucasian even after multiple comparisons correction
(p<3.25 E-07).
DNA methylation is an epigenetic change which is heritable and reversible and does not
alter the DNA sequence. This can have an effect on gene expression
14,15
by two possible
mechanisms. (1) DNA methylation could directly affect the binding of transcription factors
affecting the ability for the initiation complex to form and transcribe the RNA. (2) Methyl
groups are known to be bound by methyl-binding proteins which can act as a docking area for
histone modifying enzymes like histone deacetylases which can in turn lead to chromatin
compaction thus silencing the gene affected
16,17
.
In contrast to the genome, which is identical in all cells of the body, the epigenome, or the
epigenetic information layered on top of the genome, is different in each type of cell. When the
epigenome changes in response to an environmental exposure, its consequences will depend
4

on which cell type is exhibiting the change. This is because the effect on gene silencing or
expression will be influenced by the transcription factors present in different tissues
18
. Thus a
cell’s response to environmentally-induced epigenetic change, such as DNA methylation of a
particular CpG, could depend on the tissue type.
Based on the observation of hypomethylation of CpG cg13968390 in the histologically
normal lung of Asian lung cancer patients, most of whom were non-smokers, and the
concomitant increased expression of SULT1C2, I formulated the following hypothesis: An
environmental exposure, such as second hand smoke, caused activation of SULT1C2 and loss of
methylation at cg13968390, which is somehow related to the expression change. Activation of
SULT1C2 could lead to carcinogenic bioactivation of chemicals in secondary smoke of other
inhaled toxins, such as cooking fumes, and could thereby promote lung cancer in never
smokers. Here I study SULT1C2 hypomethylation to increase our understanding of the
environment of the epigenome and on associated gene regulatory events.
The accepted dogma is that gene silencing is associated with methylation of dense regions
of CpGs ( so-called CpG islands) found at or near promoters or enhancers
19
. Genes with
promoter CpG islands are usually housekeeping genes that play a significant role in most
tissues. However 45% of genes promoters that have a tissue-specific patterns are nearly devoid
of CpGs
20,21
. Some studies have reported that genes with CpG-poor promoters could still be
expressed even when they are methylated
22,23
, while other laboratories have reported that
there is an inverse correlation between DNA methylation and gene expression, similar to that
seen in CpG island promoters
24,25
. Oster et al. found cancer-specific DNA hypermethylation was
found in CpG islands 85% of the time and hypomethylation happened in non-CpG islands 88%
5

of the time both with an inverse correlation with gene expression
26
. Our examination of
SULT1C2 can shed light not only on the potential effects of second hand smoke, but also of the
regulatory effects of DNA methylation of CpG poor promoters. We found one such gene in
SULT1C2. Using 5-aza-2-deoxycytidine, we found that SULT1C2 is activated in lung cell lines
LuAD PC3, H2347, and BEAS-2B.

6

Materials and Methods

Reagents and antibodies
Culture grade DMSO, and rabbit polyclonal antibodies targeting SULT1C2 [sc13074] and AHR
[sc479416] were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Actin antibody
was purchased from Cytoskeleton Inc. (Denver, CO,[ EXTO1]). Cigarette smoke condensate
(CSC, [NC9028647]) was purchased from Murty Pharmaceuticals Inc. (Lexington, KY). Trypsin-
EDTA was obtained from USC Cell Culture Core Facility (Los Angeles, CA).
Cell Culture
Bronchial epithelial cell line BEAS-2B was obtained from American Type Culture Collection
(Manassas, VA). PC3 lung adenocarcinoma cell line was obtained from the Japanese Cancer
Research Resources Bank (Osaka, Japan). H2347 lung adenocarcinoma cell line was a kind gift
from Dr. Eric Haura. Cancer cell lines were maintained in RPMI-1640 from Mediatech
(Manassas, VA) and the BEAS-2B cell line was maintained in modified Eagle’s medium (USC Cell
Culture Core Facility). All cell line media was supplemented with 10% fetal bovine serum
(Tarzana, CA, [FB-11]) and 100U penicillin/streptomycin and grown in a humidified chamber
with 5% CO
2
at 37
o
C. The stock of CSC (40mg/mL) was diluted in DMSO and CSC was added to
media to the final concentration, then added to cells. DMSO was used as vehicle control for all
experiments.

7

5-Aza-CdR treatment
BEAS-2B, LuAD PC3, and H2347 cell lines were plated 24 hours prior to treatment. Cells were
treated with 5-Aza-CdR from Sigma Chemical Co. (St Louis, MO, [2353-33-5]) for 24 hours. After
the 24 hours, media with 10 % FBS and antibiotics was used for 72 hours post drug removal. At
the completion of 72 hours, cells were washed in cold PBS and harvested for RNA, DNA, and
protein. It is important to remove 5-aza-2-deoxycytidine (5-aza-CdR) after 24 hours due to its
cytotoxicity to allow the cells to recover. Laboratories that are an authority on 5-aza-CdR
27
,
advise the cells be allowed to recover for 3-5 days post treatment in order for it to capture the
DNA methyltransferases through covalent bonding.
qRT-PCR
Total RNA from BEAS-2B, LuAD PC3, and H2347 cells treated with CSC or 5-Aza-CdR was isolated
with Qiagen AllPrep DNA/RNA/Protein Kit (Valencia, CA) according to manufacturer’s protocol.
RNA was quantified using Implen Nanophotometer Pearl (Westlake Village, CA) and the quality
was verified by the A260/A280 ratio. Total RNA (500ng) was converted to cDNA using iScript
cDNA Synthesis Kit (Hercules, CA). The cDNA reaction product was amplified with primers for
ACTB Forward: 5’-GTTGAGAACCGTGTACCATGT-3’;
ACTB Reverse: 5’-TTCCCACAATTTGGCAAGAGC-3’;
AHR Forward: 5’-AGTTATCCTGGCCTCCGTTT-3’;
AHR Reverse: 5’-TCAGTTCTTAGGCTCAGCGTC-3’;
CYP1B1 Forward: 5’-CTGCACTCGAGTCTGCACAT-3’;
8

CYP1B1 Reverse: 5’-TATCACTGACATCTTCGGCG-3’;
SULT1C2 Forward: 5’-CAGCCTGCAACTGTGGACAA-3’;
SULT1C2 Reverse: 5’-GATGGCGGTGTTGGATGATG-3’.
PCR products were analyzed by cycle thresholds (Ct) using a fluorescence detecting BioRad
Real-Time System (Hercules, CA) measuring Sybr Green (BioRad).
MethyLight Assay
DNA from BEAS-2B, LuAD PC3, and H2347 cells treated with 5-Aza-CdR was isolated
simultaneously with RNA described above. 1 ug of DNA was bisulfite treated using the Zymogen
EZ DNA Methylation per manufacturer’s protocol. The bisulfite treated DNA was probed with
MethyLight primers and probe for
SULT1C2 Forward: 5’- GGGTATGGTGGCGTACGTT-3’;
SULT1C2 Reverse: 5’-AATCTTAACTCACTACAACCTCCG-3’;
SULT1C2 Probe: 5’-/6FAM-CTCCCGAATTCAAACGATTCTCCTATCTCA-BHQ-3/-3. ;
ALU Forward: 5’-AGGTCGAGGTCGGCGG-3’;
ALU Reverse: 5’-CCACGCCCGACTAATTTTATATCTT-3’;
ALU Probe: 5’-/6FAM-CAAACTAATCTCAAACTCCCGACCTCAAACGA-BHQ-1/-3’.
DNA from the control and treated cells was incubated with Taq Man enzyme from Applied
BioSystems (Carlsbad, CA), primers, and probe in a 30 ul reaction and analyzed by cycle
thresholds (Ct) using a fluorescence detecting BioRad Real-Time System (Hercules, CA). Alu
9

repeats were included in the analysis to normalize for input DNA. The percentage methylated
reference (PMR) compares the level of methylation in the sample to in vitro methylated control
DNA. It is calculated by dividing the GENE: reference ratio of a sample by the GENE: reference
ratio of M. SssI-treated in vitro methylated human DNA and multiplying by 100
28
.

10

Results

Our initial study of DNA methylation originated from the Illumina Infinium Human 27k DNA
Methylation Bead Chip. In this study, lung tumor and matching adjacent non-tumor lung
(AdNTL) of 60 patients with lung adenocarcinoma were acquired from the Canary Foundation
Early Detection Research Network collaboration, and originated in Canada. Unfortunately the
sample collection was ethnically biased. 70% of the non-smokers were Asian and less than 4%
of the Asians were smokers. To assess the effect of this potential confounding factor, the tumor
as well as the adjacent non tumor lung from the Asian subjects was compared to that of the
Caucasians. We found no significant difference in methylation of tumor lung. But when the non-
tumor lung tiiues were compared, the Asian non-tumor lung was statistically significantly
hypomethylated vs. Caucasians at cg13968390. The significance even survived a multiple
comparisons correction (Figure 1-1). We noted an inverse relationship between methylation
and expression.
To facilitate functional studies, we used three cell lines as a model system. LuAD PC3 is a lung
adenocarcinoma cell line derived from an Asian, non-smoker. H2347 is a lung adenocarcinoma
Caucasian non-smoker, and BEAS-2B cells are a cell line derived from SV40 large T antigen-
immotalized bronchial epithelial cell. We treated all three cell lines with methylation inhibitor
5-Azadeoxycytidine (AzadC). BEAS-2B cells were used for a non-cancer control, since
immortalized alveolar epithelium is not available. We treated cell lines LuAD PC3 and H2347
11

with half the clinical dose (0.15uM), clinical dose (0.30 uM), and twice the clinical dose (0.60
uM) of AzadC. BEAS-2B cells did not readily respond to the clinical dose. We thus treated
BEAS-2B cells with two times (0.6 uM), four times (1.2 uM), and eight times (2.4 uM) the clinical
dose of the drug. All cell lines were exposed to AzadC for 24 hours after which, the drug was
removed and the media was replaced. We then allowed the cells to recover for three days post
treatment. It is well established that in order for the drug to fully integrate and show
effectiveness, the cells must divide and the methylation inhibition can be evaluated in the
daughter cells
29
. We used MethyLight to determine if there was a change in methylation and
RT-PCR for evaluating changes in expression. We found that upon treatment with AzadC, there
was a dose-independent response in all cell lines. Methylation decreased in the SULT1C2 CpG
poor promoter by 2-fold (Figure 1-2A) and1.5-fold (Figure 1-3A) in LuAD PC3 and H2347 cells
respectively. Gene expression increased by 3.8-fold (Figure 1-2B) and 2.6-fold (Figure 1-3B) at
the clinical dose (0.3 uM) in LuAD PC3 and H2347 cells respectively. SULT1C2 protein expression
increased with 0.3uM and 0.6uM doses of AzadC in LuAD PC3 cells but not in H2347 cells (data
not shown). In both cell lines, 0.6uM caused cell death which decreased RNA levels. The
methylation PMR of H2347 (Figure 1-3B) cells was at thirty-two percent when untreated and
RNA levels showed high expression of the phase II enzyme in its native context. Protein levels of
SULT1C2 was also heartily expressed in untreated H2347 cells. AzadC treatment of H2347 cells
caused a decrease in methylation and an increase in RNA level of expression (Figure 1-3B) with
0.3uM AzadC. BEAS-2B cells, which did not readily respond to clinical doses of AzadC, showed a
decrease in methylation by 2-fold, 1.5-fold, and 1.8-fold at 0.6uM, 1.2uM, and 2.4uM
respectively (Figure 1-4A). RNA expression was increased maximally by 7.1-fold at 1.2uM AzadC
12

(Figure 1-4B). AzadC treatment of the cell lines lowered DNA methylation levels, which
coincided with, increased SULT1C2 mRNA expression. These results suggest that methylation
plays a significant role in the regulation of the expression of SULT1C2 in lung cell lines.

13

Figure 1-1. SULT1C2 is hypomethylated and overexpressed in the tumor lung of Asian subjects with lung cancer. A. Plot of DNA methylation
difference (beta value difference) between Asian and Caucasian non-tumor lung samples vs. statistical significance for the most variant
probes. SULT1C2 probe cg13968390 is in the top left corner (red arrow). B. Scatterplot showing significantly reduced methylation in Asian non-
tumor lung compared to Caucasian non-tumor lung for the SULT1C2 probe. The scatter plot includes all Asian cases, most of whom are never-
smokers, and a Caucasian cases, most of whom are smokers. A preliminary examinantion within never-smokers still indicates significant
hypomethylation in Asians, but the numbers are small. C. Negative correlation of SULT1C2 DNA methylation with expression for the two
SULT1C2 probes on the Affymatrix expression platform (p<0.0016 for both the red and gray probes).
Figure 1-1. SULT1C2 cg13968390 is hypomethylated in Asian never-smokers
14

Figure 1-2. LuAD PC3 PMR and Expression
PC3 CELL METHYLATION
0TRT
PBS
0.15
0.30
0.60
0
10
20
30
40
5 aza treated(uM)
PMR
PC3 mRNA EXPRESSION
0TRT
PBS
0.15
0.30
0.60
0
1
2
3
4
5
5 aza treated(uM)
FOLD CHANGE RELATIVE
TO ACTIN

Figure 1-2. LuAD PC3 cells treated with 5-aza-2-deoxycytine at 0.15 uM (half the clinical dose), 0.30 (clinical dose),
and 0.60 (double the clinical dose). Media with 5-aza-2-deoxycytidine was allowed to incubate for mRNA expression
levels with RT-PCR. Experiments were done in biological triplicate.

*
A. B.
15

Figure 1-3. H2347 PMR and Expression
H2347 CELL METHYLATION
0TRT
PBS
0.15
0.30
0.60
0
10
20
30
40
50
5 aza treated(uM)
PMR
H2347 mRNA EXPRESSION
0TRT
PBS
0.15
0.30
0.60
0
1
2
3
4
5 aza treated(uM)
FOLD CHANGE RELATIVE
TO ACTIN

Figure 1-3. H2347 cells treated with 5-aza-2-deoxycytine at 0.15 uM (half the clinical dose), 0.30 (clinical dose), and
0.60 (double the clinical dose). Media with 5-aza-2-deoxycytidine was allowed to incubate for mRNA expression
levels with RT-PCR. Experiments were done in biological triplicate.

*
A. B.
16

Figure 1-4. BEAS-2B PMR and Expression
BEAS2B CELL METHYLATION
0TRT
PBS
0.60
1.20
2.40
0
20
40
60
80
5 aza treated(uM)
PMR
BEAS2B mRNA EXPRESSION
0TRT
PBS
0.60
1.20
2.40
0
2
4
6
8
10
5 aza treated(uM)
FOLD CHANGE RELATIVE
TO ACTIN

Figure 1-4. BEAS-2B cells treated with 5-aza-2-deoxycytidine at 0.60 uM (double the clinical dose), 1.2 uM (four
times the clinical dose), and 2.4 uM (eight times the clinical dose). Media with 5-aza-2-deoxycytidine was allowed
to incubate for 24 hours. Afterward, the media was replaced and cells were allowed to recover for 72 hours. Cells
were evaluated for differential methylation by A) percent methylated reference (PMR) and B) mRNA expression
levels with RT-PCR. Experiments were done all performed in biological triplicate.

A. B.
*
17

Discussion

Sakakibara et al performed a dot blot of SULT1C2 enzyme and showed that fetal lung had
the most prominent expression when probing the clone of this enzyme
30
. It is possible that
while fetal lungs are filled with surfactant, the enzyme circulates freely detoxifying and clearing
the lungs of xenobiotic substances. When surfactant is replaced with oxygen in the lung,
SULT1C2 could be inactivated via DNA methylation and thereafter produced in organs that can
clear the body via urine or bile
31,32
. In the case of environmental toxins found in cigarette
smoke, SULT1C2 could possibly be reactivated in the lung. Substrates can then be metabolically
activated into electrophiles that can both be carcinogenic and mutagenic
33, 34, 35
.
In the evaluation of cell lines LuAD PC3 from an Asian female non-smoker, and H2347 from
female Caucasian non-smoker lung adenocarcinoma, clinical doses of 5-aza-2-deoxycytidine
were both necessary and sufficient to lead to passive demethylation of the SULT1C2 promoter
and coincident upregulation of SULT1C2 expression was seen. It is tempting to assume that the
increase expression is a direct effect of promoter demethylation, but an indirect effect cannot
be ruled out; 5-azaC is not specific for the SULT1C2 promoter, and the genes encoding other
factors such as enhancer binding proteins or transcription factors might also be demethylated
and upregulated. These might indirectly cause SULT1C2 expression to rise irrespective of
changes in DNA methylation of the SULT1C2 promoter. While we examined only two cell lines,
it is notable that the LuAD PC3 cells, derived from a female Asian non-smokers, showed a
significant increase in expression when the cells were exposed to DNA methylation inhibitor.
Although cell lines may have a different signature than in vivo cells because of the extensive
18

time spent in an “out-of-context” environment, studies show that they can maintain their basic
characteristics
36,37
. The H2347 cell line showed a similar trend, though the change did not reach
significance. Although their methylation levels were similarly around 35%, the effectiveness of
5-aza-CdR varied. One could argue that the variations could be due to cell line sensitivity, but
the foundation of those variations could still be explained possibly by ethnicity due to single
nucleotide polymorphisms (SNP) that have not yet been discovered.
Human bronchial epithelial cell line BEAS-2B is a non-cancerous cell line that is commonly
used as a control for cancer cell line studies
38,39
. BEAS-2B cells are immortalized and are one of
the only lung cell lines of its kind. Ideal cell lines for this study would have been immortalized
human alveolar epithelial cells which are not currently available. However, BEAS-2B cells are
useful in that they are “non-cancerous” and show a contrast from human lung adenocarcinoma
cell lines. Indeed, when the clinical dose was administered to these cells, they did not respond.
There was no demethylation of the DNA or increase in expression. Upon considerably higher
doses (double, quadruple, and 8 times the clinical dose), the cells responded to the
demethylating reagent. Methylation of SULT1C2 in this cell line was around 55% before
treatment and was reduced about 15% after 3 days with a significant increase in expression.
The fact that these cells are “non-cancerous” might affect their response to 5-aza-CdR.
Alternatively, different cell types may contain different levels of export proteins or other
proteins involved in detoxification.
DNA methylation is associated with gene silencing in tumor suppressor genes with a CpG
island present at or near the promoter. SULT1C2 does not possess a CGI in its promoter region
nor is it thought of as a tumor suppressor gene currently. In our laboratory, SULT1C2 was found
19

to be hypomethylated in the adjacent non-tumor lung (AdjNTL) in Asian never-smoking patients
with lung adenocarcinoma in comparison to Caucasian patients (data not shown). This could be
relevant because SULT1C2 has not been shown to be expressed in lung tissue. Studies show
that genes that lack a CGI in the promoter are usually tissue specific and that adverse effects
could result when genes that are specific to other parts of the body that are misexpressed. The
expression of SULT1C2 in the lung can be due to many environmental situations like pollution
or second hand smoke which has been shown to result in demethylation of CpGs
40
. The DNA
expression of this gene, meant to the detoxify xenobiotics could instead lead to carcinogenic of
activation of the substrate, and this might contribute to lung cancer development in never
smokers.
While we assume that the experimental demethylation we observe due to 5-azaC treatment
is passive, accomplished through cell methylation division in the absence of DNMT1 activity,
demethylation in the cells and/or the lung adenocarcinoma patients could have been active. It
has been recently established, that DNA methylation can occur without cell division by way of
DNA demethylases
41
. Ten Eleven Translocation (TET) are a family of 5-methylcytosine (5mC)
dioxygenases
42
that catalyze the oxidation of 5mC to 5-hydroxymethylcytosine (5hmC),
followed by deamination to 5-hydroxymethyluracil by cytidine deaminases
43
. This is believed to
be followed by base excision repair (BER) which replaces the 5hmuC with an unmethylated
cytosine. This is known as active DNA demethylation and has been documented in exposure to
hydroquinone, a derivative of benzene that is found in cigarette smoke
41
. Further studies in this
area would include administering various environmental insults to lung cell lines in the
presence or absence of inactivated TET proteins (e.g. through shRNA treatment) to evaluate
20

whether DNA demethylation is occurring actively or passively. By measuring 5hmC and using
chromatin immunoprecipitation (ChIP) with TET family antibodies, discovering the mechanism
of demethylation should be straight forward. SULT1C2 does indeed have a TET2 DNA binding
site within intron 1 and 800 nucleotides downstream from the gene’s non-coding exon 1.
Through these experiments, the mechanisms by which environmental insults to the lungs, like
cigarette smoke, demethylates sites within SULT1C2 and activates the gene could be
elucidated.

21

Chapter 2: Lung Cell Lines Express SULT1C2 at Low Levels of Cigarette
Smoke Exposure but Not at High Levels

Introduction

Knowledge about the SULT enzymes, as a superfamily, lags far behind that of other
detoxification enzymes. Most of what is known about this superfamily was discovered in the
late 1990s. There was a big confusion about the nomenclature of this superfamily (e.g.
PST,HAST, EST) until 1995, when the HUGO Gene Nomenclature Committee adopted the name
“SULT” for all cytosolic sulfotransferases
44
. There are two distinct classes of SULT enzymes
which have been identified 1) membrane-bound sulfotransferases that are found in the Golgi
apparatus and whose function is to sulfonate macromolecules like peptides, proteins, lipids,
and glycosaminoglycans. Sulfonation affects both structural and functional characteristics of
these substrates
45
and 2) cytosolic sulfotransferases that metabolize xenobiotics and also small
endogenous compounds like hormones, steroids, bile acids, and neurotransmitters
46
. This
thesis focuses on a cytosolic SULT.
The SULT superfamily was assigned family and subfamily designations based on their
amino acid sequence identity. SULTs with at least 45% amino acid sequence identity were
members of the same family (e.g. SULT1), and when they shared 60% or more of the same
sequence, they were considered a subfamily (e.g. SULT1C)
46,44
. Furthermore, when gene
sequences are more than 90% identical, they are designated a numeral to distinguish family
members from each other (e.g. SULT1C2 and SULT1C4).
22

SULTs catalyze the transfer of a sulfonate group (SO
3
-
) from its cofactor 3’-
phosphoadenosine-5’phosphosulfate (PAPS) to the bound substrate (e.g. xenobiotic)
47,32
to
create a more water-soluble product that can be excreted from the body via urine or bile. The
optimal characteristics of the metabolized product would be water solubility and lack of passive
penetration of cell membranes to avoid reabsorption
48
. The introduction of a negative charge
significantly increases the water solubility and also effectively decreases the passive diffusion
through the cell membranes. However, this can also be problematic as heterolytic cleavage of
the sulfate group can result in mesomerism/resonance effect
49
, which is a delocalized electron
structure. This “bioactivation” can cause reactive nucleophiles, and can induce adducts of DNA
and proteins.
In the SULT1 family, which comprises all SULT1s with at least a 60% amino acid sequence
identity, there is SULT1A, SULT1B, SULT1C, and SULT1E. All these SULT family members have
known endogenous substrates
33,34,50,51
except the SULT1C family. Currently, SULT1C2 and
SULT1C4 are the only members of this subfamily for which transcripts and protein have been
detected. SULT1C4 is found in the brain, stomach, colon, kidney, liver, and fetal lung
52,35
and its
known substrates are ethanol (drinking alcohol)
53
and flavonoids
54,55
found in fruits, vegetables,
medicinal herbs, teas, and wine
56,57,58,59
. SULT1C2 is expressed mainly in the kidney and
stomach but has also been detected in the spleen, colon, liver, and thyroid
54,60,61,52
. Identified
substrates for SULT1C2 are the carcinogen N-hydroxy-2-acetylaminofluorene (N-OH-AAF)
discovered by studies in rats
62
, p-nitrophenol (an intermediate of the pain reliever
acetaminophen)
62,61
in human and other mammals, 1a,25-Dihydroxyvitamin D
3
(1,25(OH)
2
D
3
)
63
,
and the food flavoring vanillin
64
in human SULT studies. SULT1C2 appears to be present in
23

organs which are in the first line of defense for the body when food and drink are ingested
(stomach and kidney) giving this phase II detoxifying enzyme the first opportunity to create a
more water-soluble product prepared for excretion. Since the lung is also the first line of
defense in the body for inhaled environmental pollutants, we wondered whether SULT1C2
could also be induced there. It is a logical question, knowing that other detoxifying genes like
phase I CYP1B1
65
are expressed there. Port et al. found that CYP1B1 was undetectable in mouse
lung under ordinary circumstances. However, when mice and human lung adenocarcinoma cell
lines were exposed to tobacco smoke condensate, mRNA and protein levels increased in a
dose-dependent manner. Indeed, when Yasuda et al.
35
exposed human hepatoma cell line
HepG2, primary human pulmonary artery endothelial cells (HPAEC), and human lung
microvascular endothelial cells (HLMVEC) to cigarette smoke extract (CSE) all cells generated
radioactive sulfated products in a dose dependent manner. These researchers also performed
an enzymatic assay of the 11 purified known human SULTs to identify the enzyme responsible
for generating the sulfated products when exposed to CSE. They found SULT1C2 displayed the
strongest activity with SULT1A1 following as a close second. Another reason SULT1C2 may not
have been detectable to date in human lung without induction is the process by which the lung
is investigated. In the study of other xenobiotic metabolizing enzymes (XME),

many reports on
lung are based on the evaluation of whole lung and even pooled lung samples from more than
one individual
66,67,68,69
. Lelerc et al.
70
conducted a well-designed study evaluating different
phase I and phase II XMEs in the two distinct compartments of the lungs. The bronchial mucosa
or airways describes the upper lungs (bronchus and the further branchings of these tubules)
and the pulmonary parenchyma refers to the final destination at the end of the terminus
24

bronchiole, the alveoli. The two regions of the lung have different functions and are composed
of different cell types and respond differentially when exposed to inducing xenobiotics like
cigarette smoke and its products. The investigators showed that SULT1C2 was more highly
expressed in the alveoli than in the airways. Thus, going forward with our experiments, we used
two lung adenocarcinoma cell lines, which are thought to be derived from alveolar epithelium,
and BEAS-2B cells, which are derived from immortalized bronchial epithelial cells.
One important thing to consider in the study of sulfotransferases is substrate inhibition
which is known to occur in drug metabolizing enzymes including the SULTs
71,72
. It is important
to consider because it could mean that a different biological response is seen to low vs. high
levels of exposure to xenobiotics. There are two general types of substrate inhibition with
sulfotransferase enzymes; complete and partial
73
. Complete substrate inhibition displays the
standard Michaelis-Menten parameters where the concentration of substrate directly increases
the product formation. However, with higher doses of substrates the product formation
approaches zero. This has been shown to occur by two separate mechanisms. In the first
mechanism, the active site of the SULT acquires more than one substrate molecule in the active
site which is believed to change the conformation causing a reduction in activity. In the second
mechanism, PAP fails to release from the enzyme which can still bind substrate but has no
sulfate group to transfer. SULT1A3, for example, obeys the Michaelis-Menten parameters
between the concentrations of 1-20 uM of phenolic substrate but product formation
dramatically decreases when the substrate increases to 20 uM and above.
SULT1A1, a well examined SULT, is subject to partial substrate inhibition. Like complete
substrate inhibition, at low doses of substrate, the product formation also follows the
25

Michaelis-Menten parameters. But as doses increase, the rate of product formation decreases
and reaches a minimum plateau. Phenolic substrate concentrations of 0.05-0.5 uM follow
Michaelis-Menten parameters perfectly. But as the substrate increases (as little as 1 uM), the
product formation significantly decreases or plateaus
74
. SULT1C2 and SULT1A1 both metabolize
p-nitrophenol at significantly different doses and vary in different organs
52, 61
. SULT1A1 exhibits
a K
m
at 0.6 uM for the sulfonation of p-nitrophenol while SULT1C2’s K
m
was above 10 mM
31
.
Considering substrate inhibition, it seems that SULT1C2 can facilitate mass amounts of the
intermediate drug product where SULT1A1 is limited in its metabolic activity. A more specific
substrate, 1,25(OH)
2
D
3
, requires as little as 0.0001 uM and as much as 0.01 uM to obey the
Michaelis-Menten equation for SULT1C2. Rondini et al.
63
demonstrated that upon induction of
the nuclear receptor, Vitamin D Receptor (VDR), SULT1C2 was upregulated more than two fold
in the colorectal adenocarcinoma cell line, LS180. At 0.1 uM of 1,25(OH)
2
D
3
, substrate inhibition
affected mRNA levels of the xenobiotic metabolizing enzyme. Aryl hydrocarbon receptor (AHR),
a ligand-binding transcription factor induced by a variety of compounds, was also evaluated
using 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) as a ligand and there was no increase in
mRNA levels.
Cigarette smoke contains compounds that are well-established ligand for the Aryl
hydrocarbon receptor (AHR)
75,76,77
.

It is unclear what part of the cigarette smoke activates the
receptor since there are more than 4000 identified chemicals and 60 are established
carcinogens
78
. AHR, when liganded, converts into a nuclear transcription factor that activates
phase I xenobiotic metabolizing enzymes like CYP1B1
65
. It is highly possible that AHR could also
activate phase II enzymes. AHR induces expression of known phase I genes like CYP1B1
79
. It is
26

well established that CYP1B1 is overexpressed in lung adenocarcinoma and this gene is
activated by aryl hydrocarbon receptor
80
.
Here we examine the possibility that CSC could regulate expression of SULT1C2 in lung cell
lines representing the bronchial and peripheral lung regions. To try to simulate different
exposures that might happen in subjects exposed to first hand and second hand smoke, we
treated the cell lines with a series of different CSC levels.

27

Materials and Methods
Cell Culture
Bronchial epithelial cell line BEAS-2B was obtained from American Type Culture Collection
(Manassas, VA). LuAD PC3 lung adenocarcinoma cell line was obtained from the Japanese
Cancer Research Resources Bank (Osaka, Japan). H2347 lung adenocarcinoma cell line was a
kind gift from Dr. Eric Haura. Cancer cell lines were maintained in RPMI-1640 from Mediatech
(Manassas, VA) and the BEAS-2B cell line was maintained in modified Eagle’s medium (USC Cell
Culture Core Facility). All cell line media was supplemented with 10% fetal bovine serum
(Tarzana, CA) and 100U penicillin/streptomycin and grown in a humidified chamber with 5%
CO
2
at 37
o
C. The stock of CSC (40mg/mL) was obtained from Murty Pharmaceuticals (Lexington,
KY, [NC9028647]) diluted in DMSO and added to media prior to application to culture plates.
DMSO was used as vehicle control for all experiments.
qRT-PCR
Total RNA was obtained from BEAS-2B, LuAD PC3, and H2347 cells treated with DMSO or, 10,
20, 40, or 80 ug/ml of cigarette smoke condensate (CSC) for 24, 48, and 72 hours. DNA was
isolated with Qiagen AllPrep DNA/RNA/Protein Kit (Valencia, CA) according to manufacturer’s
protocol. RNA was quantified using Implen Nanophotometer Pearl (Westlake Village, CA) and
the quality was verified by the A260/A280 ratio. Total RNA (500ng) was converted to cDNA
using iScript cDNA Synthesis Kit (Hercules, CA). The cDNA reaction product was amplified with
primers for
ACTB Forward: 5’-GTTGAGAACCGTGTACCATGT-3’;
28

ACTB Reverse: 5’-TTCCCACAATTTGGCAAGAGC-3’;
AHR Forward: 5’-AGTTATCCTGGCCTCCGTTT-3’;
AHR Reverse: 5’-TCAGTTCTTAGGCTCAGCGTC-3’;
CYP1B1 Forward: 5’-CTGCACTCGAGTCTGCACAT-3’;
CYP1B1 Reverse: 5’-TATCACTGACATCTTCGGCG-3’;
SULT1C2 Forward: 5’-CAGCCTGCAACTGTGGACAA-3’;
SULT1C2 Reverse: 5’-GATGGCGGTGTTGGATGATG-3’.
PCR products were analyzed by cycle thresholds (Ct) using a fluorescence detecting BioRad
Real-Time System (Hercules, CA) measuring Sybr Green (BioRad).

29

Results

To investigate the effects of CSC on the activation and expression of SULT1C2 in lung cells,
we treated LuAD PC3, H2347 and BEAS-2B cell lines with cigarette smoke condensate. We
measured the SULT1C2 transcriptional activation as well as that of AHR, and as a positive
control, CYP1B1. Untreated cells were maintained in a separate incubator so they would not be
affected by the CSC. Using RT-PCR, we evaluated the expression of AHR, SULT1C2, and CYP1B1.
Actin was used as a control. All cell lines were treated at 10, 20, 40, and 80 ug/ml of CSC for 24,
48, and 72 hours. We used 10 and 20 ug/ml to simulate second hand smoke exposure and 40
and 80 ug/ml to simulate a smoker and heavy smoker enviornment respectively.It is important
to use various doses because of the substrate inhibition of the phase I and phase II xenobiotic
metabolizing enzymes (XME)
81
. Lesser doses show a traditional Michaelis-Menten curve, but as
the enzyme is saturated with substrate the activity and is reduced or altogether eliminated
73
.
LuAD PC3 cells treated with CSC showed a significant in AHR expression at 20 ug/ml over
background in AHR at 48 and 72 hours but not at higher CSC concentrations. In contrast, the
positive control, CYP1B1 (Figure 2-1B), showed a strong and significant dose dependent
response that was maximal at 24 and decreased thereafter, suggesting substrate inhibition. In
these cells, significant induction of SULT1C2 was only seen at 20 ug/ml dose after72 hours of
treatment. Thus, we make several observations. First that the kinetics and responses of the
three genes differ. Second, that AHR and SULT1C2 do not respond to higher levels of CSC. And
thirdly, that SUT1C2 expression appeared to be concordant with the expression of the
transcription factor, AHR. The greastest fold increase in expression of both AHR and SULT1C2
30

was at 72 hours with a dosage of 20 ug/ml of CSC. AHR expression was not concordant with
CYP1B1 induction. However, transcriptional induction of AHR is not required for its function, as
it is activated by binding to ligand, which causes it to enter the nucleus and bind xenobiotic
respones elements, such as those present in CYP1B1 promoter
65
.
H2347 cells treated with CSC showed no significant increase in AHR and SULT1C2. H2347
cells did show dose-responsive CYP1B1 induction, emphasizing that different cells contain
different transcription factor complements and other proteins involved in the xenobiotic
response which may respond in distinct ways to CSC exposure. Like the LuAD PC3 cells, at the
highest CSC concentration, the CYP1B1 response diminished over time.
The immortalized lung epithelial cell line, BEAS-2B, showed yet another response. At
lower levels of CSC treatment we observed AHR induction at distinct timepoints, and these
coincided with significant transcriptional induction of SULT1C2. The cells showed a very strong
significant transcriptional response of CYP1B1 at 24 and 72 hours with 10 and 20 ug/ml CSC, but
interestingly, no induction was seen at the intervening 48 hour timepoint.

31

Figure 2-1. LuAD PC3 Substrate Inhibition
PC3 Cells Treated With CSC
0TRT
10 DMSO
10 CSC
20 DMSO
20 CSC
40 DMSO
40 CSC
80 DMSO
80 CSC
0
2
4
6
8
10
AHR(24HR)
AHR(48HR)
AHR(72HR)
DOSAGE (ug/ml )
FOLD CHANGE RELATIVE
TO ACTIN
PC3 Cells Treated With CSC
0TRT
10 DMSO
10 CSC
20 DMSO
20 CSC
40 DMSO
40 CSC
80 DMSO
80 CSC
0
20
40
60
80
100
CYP1B1(24HR)
CYP1B1(48HR)
CYP1B1(72HR)
DOSAGE (ug/ml )
FOLD CHANGE RELATIVE
TO ACTIN
PC3 Cells Treated With CSC
0 TRT
10 DMSO
10 CSC
20 DMSO
20 CSC
40 DMSO
40 CSC
80 DMSO
80 CSC
0
5
10
15
20
SULT1C2(24HR)
SULT1C2(48HR)
SULT1C2(72HR)
DOSAGE (ug/ml )
FOLD CHANGE RELATIVE
TO ACTIN

Figure 2-1. LuAD PC3 cells treated with DMSO or cigarette smoke condensate (CSC) at 10, 20, 40, and 80 ug/ml.
Cells were treated for 24, 48, 72 hours and mRNA expression levels were measured for A) ligand activated
transcription factor Aryl hydrocarbon receptor (AHR) B) positive control phase I detoxifying enzyme CYP1B1 and C)
phase II detoxifying enzyme SULT1C2. Treatments of 10 and 20 ug/ml were performed to simulate non-smokers
exposed to environmental tobacco smoke (ETS). 40 and 80 ug/ml were a simulation of the effects of mid-to-heavy
smokers respectively. Various exposures to CSC were performed to evaluate substrate inhibition of phase I and
phase II enzymes. All experiments were performed in biological triplicate.

*
*
*
*
*
*
*
*
*
*
*
*
*
A. B.
C.
32

Figure 2-2. H2347 Substrate Inhibition
H2347 CELLS TREATED WITH CSC
0TRT
10 DMSO
10 CSC
20 DMSO
20 CSC
40 DMSO
40 CSC
80 DMSO
80 CSC
0
1
2
3
4
5
AHR(24HR)
AHR(48HR)
AHR(72HR)
DOSAGE (ug/ml )
FOLD CHANGE RELATIVE
TO ACTIN
H2347 CELLS TREATED WITH CSC
0TRT
10 DMSO
10 CSC
20 DMSO
20 CSC
40 DMSO
40 CSC
80 DMSO
80 CSC
0
20
40
60
CYP1B1(24HR)
CYP1B1(48HR)
CYP1B1(72HR)
DOSAGE (ug/ml )
FOLD CHANGE RELATIVE
TO ACTIN
H2347 CELLS TREATED WITH CSC
0TRT
10 DMSO
10 CSC
20 DMSO
20 CSC
40 DMSO
40 CSC
80 DMSO
80 CSC
0
1
2
3
4
5
SULT1C2(24HR)
SULT1C2(48HR)
SULT1C2(72HR)
DOSAGE (ug/ml )
FOLD CHANGE RELATIVE
TO ACTIN

Figure 2-2. H2347 cells treated with DMSO or cigarette smoke condensate (CSC) at 10, 20, 40, and 80 ug/ml. Cells
were treated for 24, 48, 72 hours and mRNA expression levels were measured for A) ligand activated transcription
factor Aryl hydrocarbon receptor (AHR) B) positive control phase I detoxifying enzyme CYP1B1 and C) phase II
detoxifying enzyme SULT1C2. Treatments of 10 and 20 ug/ml were performed to simulate non-smokers exposed to
environmental tobacco smoke (ETS). 40 and 80 ug/ml were a simulation of the effects of mid-to-heavy smokers
respectively. Various exposures to CSC were performed to evaluate substrate inhibition of phase I and phase II
enzymes. All experiments were performed in biological triplicate.

*
* *
*
*
*
*
*
*
*
*
A. B.
C.
*
33

Figure 2-3. BEAS-2B Substrate Inhibition
BEAS2B CELLS TREATED WITH CSC
0TRT
10 DMSO
10 CSC
20 DMSO
20 CSC
40 DMSO
40 CSC
80 DMSO
80 CSC
0
5
10
15
20
AHR(24HR)
AHR(48HR)
AHR(72HR)
DOSAGE (ug/ml )
FOLD CHANGE RELATIVE
TO ACTIN
BEAS2B CELLS TREATED WITH CSC
0TRT
10 DMSO
10 CSC
20 DMSO
20 CSC
40 DMSO
40 CSC
80 DMSO
80 CSC
0
10
20
30
40
50
100
200
300
400
500
CYP1B1(24HR)
CYP1B1(48HR)
CYP1B1(72HR)
DOSAGE (ug/ml )
FOLD CHANGE RELATIVE
TO ACTIN
BEAS2B CELLS TREATED WITH CSC
0TRT
10 DMSO
10 CSC
20 DMSO
20 CSC
40 DMSO
40 CSC
80 DMSO
80 CSC
0
10
20
30
40
SULT1C2(24HR)
SULT1C2(48HR)
SULT1C2(72HR)
DOSAGE (ug/ml )
FOLD CHANGE RELATIVE
TO ACTIN

Figure 2-3. BEAS-2B cells treated with DMSO or cigarette smoke condensate (CSC) at 10, 20, 40, and 80 ug/ml. Cells
were treated for 24, 48, 72 hours and mRNA expression levels were measured for A) ligand activated transcription
factor Aryl hydrocarbon receptor (AHR) B) positive control phase I detoxifying enzyme CYP1B1 and C) phase II
detoxifying enzyme SULT1C2. Treatments of 10 and 20 ug/ml were performed to simulate non-smokers exposed to
environmental tobacco smoke (ETS). 40 and 80 ug/ml were a simulation of the effects of mid-to-heavy smokers
respectively. Various exposures to CSC were performed to evaluate substrate inhibition of phase I and phase II
enzymes. All experiments were performed in biological triplicate.

*
*
*
*
*
*
*
*
*
*
A. B.
C.
34

Discussion

In preliminary studies of the Laird-Offringa laboratory's evaluating the DNA methylation
in the hisotologically normal lung of 60 lung adenocarcinoma subjects, half of whom were
smokers, SULT1C2 gene was discovered as differentially methylated between Asian and
Caucasian subjects. Interestingly enough, most of the Asians were never smokers and SULT1C2
was hypo-methylated in comparison to Caucasian in adjNTL. There was no significant difference
in SULT1C2 gene methylation between smokers and non-smokers. Thus, the methylation
difference might have an ethnic basis. Indeed genomic differences have been shown to affect
DNA methylation profiles
82
. Alternatively, the SULT1C2 gene methylation difference could be
due to environmental exposure. The latter might also be related to ethnicity, if certain kinds of
exposures are more common in particular population groups. Some studies report that cooking
meats at very high temperatures in poorly ventilated spaces can aerosolize heterocyclic amines
that are inhaled, and this practice might be more common in Asian cultures. It could also be
related to the inhalation of pollution related to the burning of solid fuels like wood or coal
83
.
About half the world's population, particularly low-to-medium resource countries, use solid
fuels for both cooking and heating
84
. And of course secondhand smoke (SHS) should not be
forgotten as a cause for lung cancer. SHS is a combination of sidestream smoke (emanating
from burning cigarette) and mainstream smoke (actively exhaled by smoker) which is different
from the smoke of the active inhaler because the carcinogenic compounds are not fully
combusted. Smoking husbands could possibly explain why so many non-smoking women
develop lung cancer and not men
85
. Trichopoulos et al. found a statistically significant
35

difference between cancer cases of non-smoking women and other patients with when
evaluating husbands’ smoking habits
86
.
Our findings show SULT1C2 induction by lower dose exposures of LuAD PC3 cells to CSC.
This suggest that SULT1C2 can be activated by the components of tobacco smoke, and that low
dose exposures might be required. In addition, the seemingly parallel induction of AHR and
SULT1C2 suggests that AHR might be involved in SULT1C2 activation. This could be tested by
using shRNAs to target AHR transcripts (which should not affect AHR protein that is already
present). Normally AHR is activated by ligand binding, which should not require the synthesis of
new protein. If indeed new AHR synthesis is required for SULT1C2 induction, it is tempting to
speculate that perhaps differentially modified protein might be required. There is no evidence
of alternative splicing playing a role in different AHR isoforms. Although activation of SULT1C2
should facilitate secretion of xenobiotics, its activation can lead to bioactivation of carcinogenic
compounds. If expression of this gene is elevated in the lungs of never smokers, perhaps by low
exposures to smoke, this could have a significant role in lung cancer in never smokers.
The mechanism by which SULT1C2 is induced is currently unknown, although again, our
results hint at a possible role of AHR. SULT1C2 is tissue specific and is not usually found in lung
tissue. Expression of this gene in lung-derived cells upon exposure to CSC suggest that
compounds found in cigarette smoke are substrates for this enzyme just as they are for phase I
detoxifying enzymes like the P450s. Further studies to determine the mechanism(s) of SULT1C2
activation and expression would likely be treatment of cell lines or (even better) primary cells
from non-smoking individuals with CSC at different exposure levels. Chromatin
immunoprecipitated with an AHR antibody could then be used to determine if AHR binding to
36

the SULT1C2 promoter is induced. As suggested above, removal of AHR through shRNA or other
means could be used to test whether SULT1C2 induction is dependent on this receptor. Our
demonstration that LuAD PC3 cells respond to 20 ug/ml CSC with SULT1C2 induction provide a
nice model system to study the regulation of this gene.
It is interesting that all the studied cell lines show different responses to CSC. This could be
due to many reasons. First, the BEAS-2B are derived from bronchial epithelial cells, while the
two lung adenocarcinoma cell lines are likely derived from alveolar epithelium. Second, each of
these cell lines contains genomic differences. Some of these will be somatic and could be
related to ethinic differences, others could be acquired during tumorigenesis or the years these
cells have been cultured in vitro. Lastly, each of these cell lines was derived from a human
subject that had undergone specific exposures during their lifetime. While it will likely be
difficult to tease out which of these reasons most strongly factor into the differences in
response to CSC, the cell lines provide a system in which these questions can be explored. It
would be interesting to test a large collection of cell lines and to catalogue their different
responses.
Of the three cell lines we studied, the LuAD PC3 cells appear the most interesting. They
show transcriptional induction of SULT1C2 and AHR at low levels of CSC exposure, but a strong
and dose responsive induction of CYYP1B1. It would be very interesting to investigate the
mechanistic differences in cellular response between low and high CSC doses. This might be
used as a model for different levels of tobacco smoke exposure, such as from a second hand
and primary smoke.
37

Chapter 3: Non-CpG Island Promoter of SULT1C2 is Regulated by DNA
Methylation and Bound by AHR

Introduction

Epigenetics refers to heritable and reversible marks on DNA or proteins on the genetic
code that do not alter the DNA sequence, but affect the expression of gene(s)
87
. Examples of
epigenetic mechanisms are chromatin structure, histone modifications, associated protein
complexes, transcriptional activity, and DNA methylation. Pioneers, Holliday et al.
88
and Riggs
89
,
came to the same conclusion independently in 1975 that methylated cytosines in the context of
CpG dinucleotide could serve as an epigenetic indicator. DNA methylation is the covalent
addition of a methyl group (CH
3
-) to the 5 carbon position of a cytosine immediately followed
by a guanosine popularly known as CpG dinucleotide. Methylation of CpG dinucleotides
attaches methyl groups to both strands of DNA at coinciding cytosines which project into the
major groove (Figure 3-1). These methylated CpGs can have an effect of gene expression.
Human DNA methylation is scattered across the genome in a pattern referred to as global
methylation
90
. The position of the methylation plays a significant role in to gene expression.
DNA methylation in the body of a gene is generally correlated with transcription elongation
whereas if the methylation is at or near the transcription start site (TSS), it is usually associated
with gene silencing
91
. In human somatic cells, approximately 1% of cytosines are methylated
which when calculated, consists of about 70-80% of all CpG dinucleotides in the genome
92
. One
region where CpGs appear virtually unmethylated are CpG islands (CGIs). In 1992, Gardiner-
38

Garden and Frommer defined an CpG island as being a 200-bp region of DNA with a G+C
content >50% and an observed CpG/expected CpG ration >0.6
93
. With these criteria, the so-
called CpG islands were mostly Alu repeats. Takai and Jones redefined CpG islands as 500-bp
regions with a G+C content of >55% and an observed CpG/ expected CpG of > 0.65
20
. These
more stringent criteria eliminated the majority of Alus and unknown sequences, while only
slightly decreasing the number of CpG islands in the 5’ regions of genes. This perfectly
accommodated the 40% of genes that were calculated to have CpG islands
94
. Most CpG islands
found in promoters of a gene usually remain unmethylated and nucleosome depleted, allowing
access of the transcriptional machinery to the DNA. These conditions are usually found in genes
that are actively transcribed. CpG islands are also found in gene bodies
91
and serve a
completely different function as those found at or near the transcription start site (TSS). When
CGIs are methylated in the promoter regions near TSS, it usually blocks initiation of
transcription whereas if it is found in the gene body it can, in some circumstances, promote
transcription elongation and lead to varying splice patterns
95
. In normal tissues, methylated
promoter CGIs are usually found in genes with a repressed state that are stably silenced like in
imprinted genes, X chromosome inactivation, and silencing of repetitive DNA
96
.
The methyl group on the cytosines project into the major groove of the DNA, where they
can affect gene expression through a number of possible mechanisms. One possible way is by
directly interfering with the binding of methyl-sensitive transcription factors, blocking the
transcriptional complex that binds to promoters or enhancers at or near the methylated
CpG
97,98
. Another possibility is that the methylated CpG can be bound by methyl-binding
39

proteins, which act as scaffolds to attract and bind histone-modifying enzymes like histone
deacetylases, which could result in chromatin condensation and gene silencing
99
.
Much is known about CGIs and the effects of DNA methylation upon them. However, a
newer field of study in epigenetics is the regulation of non-CGI promoters (promoters that do
not contain a CpG ratio >0.48 in a 200-bp area
100
). More than half the genes that possess a
tissue specific pattern of expression have non-CGI promoters
20,21
. Of promoters with CGIs,
intermediate CpG content promoters, and non-CGIs promoters, the latter make up 42% of all
hypermethylated promoters
22
. Non-CGI promoters show a markedly higher frequency of DNA
methylation and a positive correlation pattern of sequence enrichment with a promoter and
CpG content. Indeed, Weber et al. found that 66% of CGIs, 41% of promoters with intermediate
CpG content, and 11% of non-CGI promoters were hypomethylated demonstrating the rarely
expressed tissue-specific genes. Tissue specific hypermethylated genes were suppressed in all
tissues investigated in Nagae et al.
101
. Non-CGI promoters also tend to be methylated in
embryonic tissues which suggests that these tissue specific genes are demethylated and
activated during terminal differentiation as evidenced by Pol II occupancy at the TSS when
these genes are unmethylated. A study of the enrichment of tissue specific hypomethylated
genes in 250 hypomethylated gene sets from gene ontology (GO) was performed, and they
found that tissue-specific hypomethylated genes expressed cell type specific functions
101
. The
majority of genes that are representative of explicit hypomethylation in differentiated cells
have hypermethylation in both embryonic and induced pluripotent cells. This suggests that
hypermethylation is the default state of non-CGIs and demethylation of these promoters occurs
in a cell-specific manner. This is seen when embryonic cell line KhES3 which is fully methylated
40

in non-CGI promoters and is gradually demethylated during in vitro differentiation. This lends
validation to the findings of hypomethylated non-CGIs promoters being present in some
cancers. Ectopic expression of testis specific genes is quite common in some tumors
102
a perfect
example of re-expression of inactivated genes in tissues when DNA hypomethylation occurs.
The fact that expression is up and methylation is down in regularly methylated non-CGI
promoters in bladder cancer
100
show differential methylation is equally significant in promoters
with very few CpG dinucleotides.
DNA demethylation can occur passively or actively. Passive demethylation takes place
when demethylating agents like 5-Aza-2’-deoxycytidine are introduced to the targeted cell
population with heavy methylation. As the DNA strands divide to replicate, a cytosine analogue
incorporates into the new strand trapping the DNA methyltransferase(s) (DNMTs) and
preventing methylation in dividing cells
103
. Active demethylation, does not require cell division
to accomplish removal of the methyl group. Ten-eleven translocation (TET) demethylases are 2-
oxogluterate, oxygen- and iron-dependent dioxygenases able to catalyze the oxidation of 5-
methylcytosine (5-mC) into 5-hydroxymethylcytosine (5-hmC). The TET enzymes (TET1, TET2,
TET3) are able to be further oxidized into substrates that thymidine DNA glycosylase (TDG) can
remove causing activation of base excision repair (BER)
104,42
. Kriaucionis et al. used non-
proliferating Purkinje cells in observing what was later discovered to be 5-hmC. He found that
the increase in 5-hmC was directly proportional to the decrease in 5-mC. That same year,
Tahiliani discovered that TET was the mechanism by assaying the catalytic activity on fully
methylated double-stranded DNA oligonucleotides resulting in a strong conversion of 5-mC to
5-hmC. In a natural context, TET is active in embryonic and/or pluripotent cells
105,106
.
41

Recent data from numerous laboratories associate cigarette smoking to DNA
demethylation of promoters that do not have a CGI
107,11
. Coulter et al. showed that the
environmental toxicant hydroquinone, a benzene metabolite found in cigarette smoke, causes
an increase in TET1 demethylase expression and activity
41
.
Given the interest in the regulation of non-CGI promoters, and our observation that
reduced methylation of cg13968390 in the promoter region of SULT1C2 (Figure 3-2) was
present in the histological normal lung of Asian lung adenocarcinoma patients (see previous
Chapters), here we investigated the role of DNA methylation in SULT1C2 expression, using a
luciferase reporter system. We examined the effects of DNA methylation in the absence or
presence of cigarette smoke condensate (CSC) to stimulate smoking exposure. Because the aryl
hydrocarbon receptor (AHR) s involved in response to xenobiotics, and has a CpG-containing
binding site in the SULT1C2 promoter, we also investigated the presence of AHR on the
promoter region, using chromatin immunoprecipitation.

42

Materials and Methods

SULT1C2 Gene Promoter Construction
The -1834 fragment for the SULT1C2 promoter and part of noncoding exon 1 was PCR amplified
from genomic DNA from lung cancer cells using the forward sequence 5’-
CATCCCAGTTCATCCTCCACAAA-3’ with a SpeI enzyme restriction cut site at the 5’ end and
reverse primer 5’-CTTCAAGTTCCAGCTGAAAGTCAGCA-3’ with a BspHI restriction enzyme cut
site at the 3’ end using Phusion High Fidelity DNA Polymerase from New England Biolabs
(Ipswich, MA). The amplified fragment was run on an ethidium bromide gel and excised. The gel
fragment was purified using Qiaquick Gel Extraction kit from Qiagen (Valencia, CA). The
fragment was then incubated with the above restriction enzymes to create restriction sites
compatible to the CpG-less vector cut with SpeI and NcoI. Both vector and fragment were
purified using the Qiagen PCR purification kit (Valencia, CA). The 1.8Kb fragment was cloned
into the CpG-less vector using Instant Sticky-end Ligase Master Mix from New England Biolabs
(NEB) (Ipswich, MA) . The reporter vector with the SULT1C2 promoter was propagated in
Invitrogen E. coli PIR1cells (Grand Island, NY). The pCpGL-SULT1C2 was isolated and purified
using OriGene PowerPrep Plasmid Kit (Rockville, MD). The promoter sequence was verified for
accuracy at Genewiz (La Jolla, CA).

43

M.SssI Treatment of Plasmid
pCpGL-SULT1C2 vector was incubated with SssI (2.5 U/ug) in the presence of 160uM
S-Adenosylmethionine (SAM) from New England Biolabs (Ipswich, MA) overnight. A second
addition of SAM was applied and the plasmid was again incubated overnight. Control plasmids
used were subjected to the same treatment without SAM or SssI. Treated vectors were purified
using EconoSpin columns with silica membrane from Epoch Life Science (Missouri City, TX).
Methylation of vector was confirmed by digestion of methylation sensitive restriction enzymes
HpaII and HhaI, and methylation insensitive MspI from New England Biolabs (Ipswich, MA).
Transfect ion and Luciferase Assay
LuAD PC3 cells were transfected with 800ng of methylated and unmethylated pCpGL-
SULT1C2 luciferase reporter vector and 200ng of Renilla luciferase control reporter plasmid
kindly donated by Dr. Kwang-Ho Lee using Invitrogen Lipofectamine 2000 in low-serum
Optimem media (Grand Island, NY). H2347 and BEAS-2B cells were transfected using FuGene
HD transfection reagent from Promega (Madison, WI) using the same amount of vector
methylated and unmethylated as described above. 24 hours post-transfection, the media was
removed and replaced with media containing 10% FBS, 20 ug/ml of cigarette smoke condensate
(CSC) from Murty Pharmaceuticals (Lexington, KY, [NC9028647]), or DMSO from Sigma [D2438]
in the absence of antibiotics for 24 hours. Cells were washed with PBS and lysed with Promega
passive lysis buffer. Cell lysates were freeze thawed and assayed for firefly and renilla luciferase
activity using Promega Dual-Luciferase Reporter Assay System (Madison, WI) on a Promega
Glomax Luminometer. pCpGL plasmid without a promoter insert and a luciferase vector driven
44

by the strong cytomegalo virus promoter were used for negative and positive controls
respectively. Experiments were done in triplicate.
Chromatin immunoprecipitation Assay
LuAD PC3, H2347, and BEAS-2B cells were grown to 85% confluency in 150 mm plates and
were treated with DMSO or 20 ug/ml cigarette smoke condensate (CSC). The LuAD PC3 cells
were treated for 72 hours, based on the findings in Chapter 2 and the H2347 and BEAS-2B cells
were treated for 24 hours. The cells were incubated in 1% formaldehyde for 10 minutes on an
oscillator to crosslink the proteins with the DNA followed by glycine at a final concentration of
125mM to quench the reaction of the formaldehyde. The media was then aspirated and the
cells were washed with cold PBS, harvested, and centrifuged. The cell pellets were re
suspended in 800 ul of cell lysis buffer with protease inhibitors from Sigma-Aldrich (St. Louis,
MO) and incubated on ice for 20 minutes. The cell lysates were sonicated for 15 minutes at 30
second intervals to shear DNA to lengths between 200bp-1000bp and then centrifuged for 15
minutes at 4
o
C. Aliquots of the supernatant was distributed for each treatment and for and
each antibody. 1% of the samples were saved for input. 45 ul of Protein A/G Plus agarose beads
from Santa Cruz Biotechnology were added to all samples and rotated for 1 hour at 4
o
C to
preclear samples. Samples were then centrifuged to pellet beads and the supernatant was
transferred to fresh microfuge tubes. AHR [ab2769] and HNF4a [ab1898] antibodies from
Abcam (Cambridge, MA) and IgG from Santa Cruz Biotechnology (Santa Cruz, CA, [sc-2027])
were put into tubes of designation and incubated overnight at 4
o
C on a VWR tube rotator
(Visalia, CA). The following day, 45 ul of agarose beads were added to all samples and rotated at
4
o
C for 1 hour. The beads were washed and rotated for 5 minutes successively with low salt,
45

high salt, lithium chloride, and two washes of TE buffer. The immunoprecipitation was
incubated with elution buffer for 15 minutes and centrifuged twice pooling the eluates. 20 ul of
5M NaCl was added to each eluted samples and the input and was incubated overnight at 65
o
C
to reverse crosslinking. The DNA was then purified by phenol-chloroform extraction and
quantified using Implen Nanophotometer Pearl (Westlake Village, CA) and the quality was
verified by the A260/A280 ratio.

46

Results
DNA Methylation of CpG-poor SULT1C2 Promoter Region Effects Gene Expression
More studies are supporting the role of CpG-poor promoters in tissue-specific expression
and reinforcing the idea that they are regulated by DNA methylation, similar to CpG island
promoters
100
. There are only 13 CpG dinucleotides in the 1.4 kb region upstream sequence
from the first transcription start site (TSS) and non-coding exon 1 of SULT1C2. More than 45%
of genes that have a tissue-specific pattern are nearly devoid of CpGs in their promoters
21, 20
. It
has been established that SULT1C2 is present in human stomach, kidney, fetal liver
108
, and
recently colorectal adenocarcinoma cell line LS180
63
. Smoking-associated cancers occur in all
these tissues
109,110,111
. Cigarette smoke enters the lungs before interfacing with any of the other
tissues, so that the lung can be considered the first line of defense for these environmental
toxins. Tsaprouni et al. found that there was a statistically significant differential DNA
methylation in smokers compared to never smokers in GPR15, a gene devoid of a CpG island in
its promoter
107
. The group found the gene to be hypomethylated and upregulated in its non-
CpG island promoter in smokers when compared to non- smokers. To
investigate the effects of DNA methylation of the promoter region of SULT1C2 on gene
expression, we cloned a 1.8Kb region upstream of a firefly luciferase reporter gene in a CpG-
less vector
112
. The CpG-less vector is an ingenious construct of different plasmid sequences
combined that are devoid of CpG dinucleotides. This plasmid allows the inserted DNA sequence
to be enzymatically methylated without methylating the backbone of the plasmid. The CpG-
poor promoter was methylated in vitro by using SssI methyltransferase. We transfected all
three cell lines with methylated or unmethylated SULT1C2-CpGless luciferase reporter with and
47

without treatment of CSC to enable us to gather direct information on the effects of
methylating the SULT1C2 promoter. In LuAD PC3 cells, luciferase activity was ~37.7 (p<0.01)
fold higher and ~4.7 fold higher than empty vector in unmethylated and methylated SULT1C2-
CpG-less vector respectively (Figure 3-3). This indicates that the 1.8 Kb SULT1C2 fragment was
sufficient for transcription. Methylation of the fragment significantly decreased luciferase
activity by ~33 fold (p<0.05). Our results are in agreement with Han et al showing that DNA
methylation directly silences genes CpG-poor promoters
100
. When treated with CSC, both
unmethylated and methylated SULT1C2 promoter luciferase activity significantly increased
above the untreated to ~67.8 (p<0.001) fold and ~34.3 (p<0.001) fold respectively when
compared to empty vector. The large fold increase in activity of the methylated SULT1C2
promoter plasmid was somewhat unexpected since methylation had effectively silenced the
gene in the absence of CSC. It suggests that the non-CSC basal activity is dependent on
sequences that become inaccessible by methylation, but that methylation-independent
mechanisms are responsible for the CSC-based induction.
Transfecting cell line H2347 proved to be difficult. Using three different transfection
reagents, luciferase activity for the 1.8 Kb SULT1C2 promoter region and non-coding exon 1 was
much less than LuAD PC3 or BEAS-2B cells. Indeed, the CMV-driven positive control plasmid was
over 10 times weaker than in the LuAD PC3 cells. We believe that because the cells become
confluent in a sporadic manner instead of the uniform confluency like the other two cell lines,
the reagent could not penetrate the cell-cell boundaries. There was only a ~2.5 fold and ~1.5
fold increase in luciferase activity of the unmethylated and methylated SULT1C2- CpG less
vector over background respectively (Figure 3-4). There was no significant decrease in fold
48

activity of the methylated in comparison to the unmethylated promoter in untreated cells, but
levels were already so low. Upon treatment of CSC, there was no increase in activity in the
H2347 treated cells in comparison to the untreated in the unmethylated or methylate SULT1C2
promoter. While these observation appear to mesh with the low responsiveness of H2347 cells
described in Chapter 2, they are difficult to interpret given the low transfection efficiency.
In BEAS-2B cells, the unmethylated and methylated 1.8Kb SULT1C2 fragment yielded
~134 fold (p<0.01) and ~6.8 (p<0.05) fold increase in luciferase activity respectively in relation
to empty vector in BEAS-2B non-tumor lung cell line (Figure 3-5). Methylation of the vector
dramatically decreased luciferase activity by ~75 fold (p<0.01). When treated with CSC,
unmethylated SULT1C2 promoter significantly increased its activity over untreated cells by 2-
fold (p<0.05), while the methylated fragment did not significantly increase in activity the over
the non CSC untreated.

49

Figure 3-1. . DNA methylation is a covalent addition of a methyl group (CH
3
-) to the carbon position of a cytosine
immediately followed by a guanosine popularly known as CpG dinucleotide. Methylation of CpG dinucleotides
attaches methyl groups to both strands of DNA at coinciding cytosines which project into the major groove. Red
dots on DNA are CpG dinucleotides bound in the major groove.

Methyl Group on
Cytosine in Major
Groove
50

Figure 3-2. The -1834 fragment for the SULT1C2 promoter and part of noncoding exon 1 was PCR amplified and
cloned into a CpG-less vector for luciferase analysis
Figure 3-2. 1.8 kb SULT1C2 Fragment
51

Figure 3-3. LuAD PC3 SULT1C2 Promoter Activity
PC3 SULT1C2 PROMOTER
No TRT
CSC
0
5
10
15
20
50
100
150
200
20000
40000
60000
80000
CpGL
CMV
SULT1C2-U
SULT1C2-M
LUCIFERASE ACTIVITY RELATIVE
TO RENILLA (FOLD)

Figure 3-3. LuAD PC3 cells were transfected with 800 ng with 1) empty firefly luciferase CpGless vector 2) CpGless
vector with a 1.8 kb SULT1C2 promoter fragment or 3) CpGless vector with fully methylated 1.8 kb SULT1C2
promoter fragment. All transfections also included 200 ng of renilla luciferase for an internal control measuring
luciferase activity. Green fluorescent protein (GFP) was added to empty vector transfection to compensate for DNA
in comparison to vectors with promoters. Statistics above indicate measurements of luciferase activity between no
treatment and cells treated with CSC. Statistics also measured SULT1C2 promoter methylated and unmethylated
within the no treatment and CSC groups.

*
*
*
52

Figure 3-4. H2347 SULT1C2 Promoter Activity
H2347 SULT1C2 PROMOTER
NO TRT
CSC
0
2
4
6
8
10
500
1000
1500
2000
2500
CpGL
CMV
SULT1C2-U
SULT1C2-M
LUCIFERASE ACTIVITY RELATIVE
TO RENILLA (FOLD)

Figure 3-4. H2347 cells were transfected with 800 ng with 1) empty firefly luciferase CpGless vector 2) CpGless
vector with a 1.8 kb SULT1C2 promoter fragment or 3) CpGless vector with fully methylated 1.8 kb SULT1C2
promoter fragment. All transfections also included 200 ng of renilla luciferase for an internal control measuring
luciferase activity. Green fluorescent protein (GFP) was added to empty vector transfection to compensate for DNA
in comparison to vectors with promoters. Statistics above indicate measurements of luciferase activity between no
treatment and cells treated with CSC. Statistics also measured SULT1C2 promoter methylated and unmethylated
within the no treatment and CSC groups.

*
53

Figure 3-5. BEAS-2B SULT1C2 Promoter Activity
BEAS2B SULT1C2 PROMOTER
No TRT
CSC
0
10
20
30
40
50
100
200
300
400
1000000
2000000
3000000
4000000
5000000
CpGL
CMV
SULT1C2-U
SULT1C2-M
LUCIFERASE ACTIVITY RELATIVE
TO RENILLA (FOLD)

Figure 3-5. BEAS-2B cells were transfected with 800 ng with 1) empty firefly luciferase CpGless vector 2) CpGless
vector with a 1.8 kb SULT1C2 promoter fragment or 3) CpGless vector with fully methylated 1.8 kb SULT1C2
promoter fragment. All transfections also included 200 ng of renilla luciferase for an internal control measuring
luciferase activity. Green fluorescent protein (GFP) was added to empty vector transfection to compensate for DNA
in comparison to vectors with promoters. Statistics above indicate measurements of luciferase activity between no
treatment and cells treated with CSC. Statistics also measured SULT1C2 promoter methylated and unmethylated
within the no treatment and CSC groups.

*
*
54

The Aryl Hydrocarbon Receptor Transcription Factor Binds SULT1C2 Promoter
The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor
113
. In its
inactive state, AHR forms a complex with heat-shock protein HSP90, X-associated protein-2
(XAP2), and p23
114
. AHR is induced by various chemicals, among them are polycyclic aromatic
hydrocarbons (PAH) like benzo(a)pyrene found in cigarette smoke
115
. It is well established that
aryl hydrocarbon receptor is a transcription factor to the phase I detoxifying enzymes
cytochrome p450 when induced by exogenous ligands
116
. There is very little evidence available
on how AHR affects phase II enzymes. To investigate the mechanism of induction of SULT1C2
via cigarette smoke condensate, we chromatin immunoprecipitate assayed AHR from cell lines
treated with and without CSC. Using Biobase
52
, we discovered an AHR transcription factor DNA
binding site which contained two CpG dinucleotides in the 1.8 kb promoter fragment of the
SULT1C2 promoter. We treated cells with 20 ug/ml of CSC to simulate second hand smoke
exposure since we have shown that SULT1C2 is induced at this dose of CSC. Quantitative real
time PCR data showed significant a 7.3 fold enrichment of SULT1C2 DNA sequence specific for
AHR in LuAD PC3 cells when compared to DMSO (Figure 3-6). Enrichment of SULT1C2 surpassed
that of positive control for AHR in the lung, ChIP of the published phase I detoxifying enzyme
55

CYP1B1 AHR binding site. A similar fold enrichment of ~6.5-fold (Figures 3-7 and 3-8) of AHR on
the SULT1C2 promoter was seen in the other two cell types when compared to DMSO. In all
cell lines, enrichment of SULT1C2 exceeded that of CYP1B1. It also appears that SULT1C2 is
AHR-ligand dependent. In the absence of ligand, there was no enrichment of SULT1C2 in DMSO
in comparison to no treatment.

56

Figure 3-6. LuAD PC3 ChIP Enrichment
ChIP of PC3 CELLS
DMSO
20 CSC
0.25
0.5
1
2
4
8
16
SULT1C2
CYP1B1
DOSAGE (ug/ml )
Log2 FOLD ENRICHMENT
RELATIVE to NSP

Figure 3-6. LuAD PC3 cells were exposed to 1) no treatment 2) DMSO or 3)20 ug/ml of CSC for 72 hours to induce
optimal induction of phase I and phase II detoxifying enzymes. Cells were formaldehyde treat to crosslink DNA and
protein to be sonicated. Crosslinks were immunoprecipitated with AHR antibody. Crosslinks were then reversed and
the DNA was isolated by phenol-chloroform extraction. DNA detection was analyzed by RT-PCR. Data was
normalized using 1% input of sample and log2 fold enrichment was calculated relative to non-specific primer (NSP).

*
*
57

Figure 3-7. H2347 ChIP Enrichment
ChIP of H2347 CELLS
DMSO
20 CSC
0.25
0.5
1
2
4
8
SULT1C2
CYP1B1
DOSAGE (ug/ml )
Log 2 FOLD ENRICHMENT
RELATIVE to NSP

Figure 3-7. H2347 cells were exposed to 1) no treatment 2) DMSO or 3)20 ug/ml of CSC for 24 hours to induce
optimal induction of phase I and phase II detoxifying enzymes. Cells were formaldehyde treat to crosslink DNA and
protein to be sonicated. Crosslinks were immunoprecipitated with AHR antibody. Crosslinks were then reversed and
the DNA was isolated by phenol-chloroform extraction. DNA detection was analyzed by RT-PCR. Data was
normalized using 1% input of sample and log2 fold enrichment was calculated relative to non-specific primer (NSP).

*
*
58

Figure 3-8. BEAS-2B ChIP Enrichment
ChIPof BEAS-2B CELLS
DMSO
20 CSC
0.25
0.5
1
2
4
8
SULT1C2
CYP1B1
Log 2 FOLD ENRICHMENT
RELATIVE to NSP

Figure 3-8. BEAS-2B cells were exposed to 1) no treatment 2) DMSO or 3)20 ug/ml of CSC for 24 hours to induce
optimal induction of phase I and phase II detoxifying enzymes. Cells were formaldehyde treat to crosslink DNA and
protein to be sonicated. Crosslinks were immunoprecipitated with AHR antibody. Crosslinks were then reversed and
the DNA was isolated by phenol-chloroform extraction. DNA detection was analyzed by RT-PCR. Data was
normalized using 1% input of sample and log2 fold enrichment was calculated relative to non-specific primer (NSP).

*
*
59

Discussion

CpG islands, which are usually unmethylated, are poised for transcription in most
tissues. Non-CpG islands promoters, on the other hand, are usually hypermethylated and silent
in non-specific tissue where the gene is not supposed to be expressed. Thus in the case of CpG
poor promoters, DNA methylation protects tissues from inappropriate expression of tissue-
specific genes. Much harm could occur when cells are exposed to expression of genes that are
indigenous to other tissues. This could lead to hormonal imbalances, hyperproliferation of cells
in certain parts of the body, and even cancer.
It is unclear whether SULT1C2 is regularly expressed in the lung in the absence of any
exposures. Another detoxifying enzyme, CYP1B1, is not expressed in lung cells unless it is
induced by an aerosolic environmental insult like cigarette smoke. To date, there is no
literature showing the expression of SULT1C2 in the lung in uninduced cells. There is evidence,
however of its expression in cells induced by cigarette smoke. Guida et al. studied DNA
methylation profiles of smokers, ex-smokers, and non-smokers. Their study showed that upon
cigarette smoke exposure, some genes with non-CpG island promoters became
hypomethylated. Their data also indicated that, upon smoking cessation, DNA methylation
returned to levels similar to that of non-smokers in a time-dependent manner. Other
60

investigators report that some but not all of the tobacco-induced hypomethylation events are
reversible
117
.
Using a reporter plasmid system with the isolated SULT1C2 promoter fragment, we show
that the 1.8 kb SULT1C2 promoter fragment is active in LuAD PC3 cells and BEAS-2B cells.
H2347 data cannot be interpeted due to low transfection efficiency. We observe that in both
LuAD PC3 cells and BEAS-2B cells, DNA methylation silences the SULT1C2 promoter. An
interesting difference between the two cell lines is observed when the cells are treated with
CSC. In LuAD PC3 cells, CSC can stimulate the methylated promoter, while BEAS-2B cells, it
cannot. There could be several different explanations for this phenomenon. One could be that
the transcription factor complement of the two cells is different. In studying the sequence of
the 1.8 kb promoter region, we found multiple DNA binding sites for transcription factors, some
of which could be affected by DNA methylation, and others of which might not be affected by
methylation. Thus, one possibility is that DNA methylation might interfere with transcription
factors or associated factors in the BEAS-2B cells, but not in the LuAD PC3 cells. Another
possibility is that CSC exposure leads to active demethylation in LuAD PC3 cells, allowing the
SULT1C2 promoter to be induced. It could be that, upon environmental tobacco exposure, DNA
demethylase TET enzymes are induced in PC3 cells and that they demethylate one or more
transcription factor binding sites within the promoter region of SULT1C2 and therby reactivate
the promoter. Investigating the mechanism by which LuAD PC3 cells can express SULT1C2 when
a methylated plasmid is introduced will be very interesting, and will require measuring
methylation on the transfected plasmid. This might be challenging if the activity is arising from
a small fraction of the plasmid that was actively demethylated. If demethylation is found,
61

further studies would involve performing chromatin immunoprecipitation using TET enzymes to
find which of the TETs (TET1, TET2, TET3) is responsible for the DNA methylation of AHR or
other sites within SULT1C2 promoter. TET2 is the most likely since investigation of the SULT1C2
sequence revealed a TET2 DNA binding site in intron 1 immediately following non-coding exon
1.
In order to test whether certain transcription factors and not active demethylation allows
LuAD PC3 cells to express the methylated SULT1C2 luciferase construct in the presence of CSC,
the 1.8 kb promoter fragment could be studied by deletion analyses or mutagenesis to
investigate putative transcription factor binding sites. In addition, any factors tht are implicated
could be studied by ChIP from CSC-treated cells, assuming a good quality ChIP antibody is
present. In that regard it is interesting that in all three cell types, AHR is induced to bind to the
SULT1C2 promotr by CSC treatment. Thus, the difference in response between the cell liine
cannot be explained by lack of response of the AHR protein.
The experiments with the luciferase plasmid show that this experimental setup can be
very useful to study the response to tovaccco smoke in tussue culture cells, and could provide
useful insight through mutagenesis studies. One limitation, as we have seen with the H2347
cells is that the cells of interest have to be transfectable. It would certainly be interesting to
explore the ability fo the lung adenocarcinoma cell lines to respond to CSC when using the
methylated SULT1C2 luciferase construct. This might help tease out the mechanism for CSC-
inducibility when a methylated promoter is provided. If active demethylation were shown to
play a role, this would be very exciting and it would be an important new insight into the
epigentic effects of tobacco smoke.
62

Here, we used a lung adenocarcinoma cell line derived from an Asian non-smoker from
Japan to try to determine if there was indeed any difference in response to ethnicity as our
preliminary studies predicted. One caveat is that DNA methylation patterns could be drastically
changed in cell lines due to cells being cultivated in culture dishes, which might be necessary to
adapt to its environment. Careful investigation of the literature, however, shows when studying
DNA methylation in other cell lines with Illumina Infinium 450k DNA Methylation Bead
Platform, that there is a still a noticible difference in cell lines between Caucasian and Asian
which can also be found in patients with lung adenocarcinoma (data not shown). In Asian cell
line, LuAD PC3, but not Caucasian cell line, H2347, cigarette smoke condensate appears to have
an effect on DNA methylation. In luciferase assays of the effects of differential DNA
methylation, cigarette smoke condensate seemed to have reversed the effects of methylated
DNA which gave little luciferase activity without treatment in LuAD PC3 cells. H2347 cells
showed the same amount of luciferase activity in unmethylated assay with and without
treatment. It is certain that DNA methylation affects SULT1C2 promoter as the luciferase
activity was significantly reduced in LuAD cells without treatment. It could be that, upon
environmental tobacco exposure, DNA demethylase TET enzymes demthylate some
transcription factor binding site within the promoter region of SULT1C2 and reactivates the
promoter by replacing the methylated cytosine with a unmethylated cytosine by base excision
repair of 5-hydroxymethyl cytosine.
This was found to be the case in cells lines that were not methylated. Cigarette smoke
condensate exposure at second hand smoke levels showed that SULT1C2 DNA was enriched
when immunoprecipitating with AHR. The AHR DNA binding site in the promoter of the gene
63

has two CpG dinucleotides within its binding sequence that could be demethylated upon
exposure to cigarette smoke. It has been shown that hydroquinone, a derivative of benzene
found in cigarette smoke, activates and demethylates DNA
41
. This is the first time that cigarette
smoke has been shown to activate SULT1C2 and that AHR is the mechanism.

64

Chapter 4: Conclusion

The effects of tobacco smoke on the epigenome is a very “hot” topic. Numerous
publications describe population-based studies in which DNA methylation is compared between
hundreds of smokers and non-smokers. Many reports describe DNA methylation changes
associated with tobacco smoke exposure. Most commonly, loss of methylation is seen. Usually,
the studies are carried out using DNA derived from whole blood, because that is often the only
tissue that is available from the subjects. While those observations are interesting, they suffer
from several drawbacks. First, tobacco smoke enters the lungs and thus one should ideally
study lung cells, particularly because tobacco smoke causes lung cancer. Second, blood consists
of numerous cell types, which makes it very difficult to interpret epigenetic information from
this DNA source. If the composition of blood cell types changes upon smoking exposure, that
could easily explain DNA methylation changes.
Recently, in collaboration with Dr. Maria Teresa Landi from the NCI, the Laird-Offringa
lab investigated the DNA methylation differences between smokers and non-smokers using
histologically normal lung tissue from lung cancer patients (manuscript in preparation). Just as
in studies of blood DNA, significant hypomethylation was observed in smokers. 8
hypomethylated CpGs were identified and 7 were validated using lung tissue from The Cancer
65

Genome Atlas (TCGA). To investigate the functional effects of the hypomethylated CpGs, the
data was integrated with bisulfite genomic sequencing and enhancer histone marks from
purified alveolar epithelial cells, and luciferase assays of certain genomic regions, similar to
what was done in this thesis. Altogether, the data suggest that 4 of the CpGs mark enhancer
elements that are activated by smoke. Indeed, three of the 4 regions contain AHR binding sites.
These sites can be taken out of the genomic context, inserted in a luciferase reported plasmid,
and show response to CSC treatment. These very recent unpublished data support many of the
observations made in this thesis.
For one, they support the association between environmental exposures and DNA
methylation. In this thesis, we study hypomethylation of a CpG in the SULT1C2 promoter. The
hypomethylation is specifically observed in Asian lung cancer patients. Why this is so remains
unclear. It could be related to ethnicity-based genomic differences that affect DNA methylation
patterns. A link between genetic variation and DNA methylation variation has been reported in
lung tissue
82
. Alternatively, the hypomethylated CpG could be related to a certain type of
exposure of the Asian patients, most of whom were non-smoking women. One such exposure
could be second hand cigarette smoke.
The data set giving rise to the SULT1C2 hypomethylation observation also showed an
inverse correlation between methylation of that CpG and expression of SULT1C2. To further
investigate this, we use three cell lines: two lung adenocarcinomas, LuAD PC3, derived from an
Asian female non-smoker, and H2347, derived from a Caucasian female non-smoker. In
addition, we use BEAS-2B cells, which are SV40 Large T-immortalized bronchial epithelial cells.
66

The cell lines were applied to study SULT1C2 and its response to CSC. In Chapter 1, we
treated all three lines with the demethylation agent 5-aza-dC, and observed increased
expression of SULT1C2 in LuAD PC3 and BEAS-2B cells. Increased expression in H2347 cells was
suggested but did not reach significance. While increased expression upon 5-aza-dC treatment
is suggestive, the drug is not very specific; it affects DNA methylation genomewide, in addition
to affecting other cellular processes. Thus, in Chapter 3, we take a 1.8 kb fragment including the
SULT1C2 promoter, and insert it in a CpG-less luciferase reporter so that promoter activity can
be measured in the absence and presence of DNA methylation. In the two successfully
transfected cell lines, LuAD PC3 and BEAS-2Bs, methylation greatly reduced SULT1C2 promoter
activity. This again supports the negative effects of DNA methylation on the SULT1C2 promoter.
The promoter contains few CpGs, and one could wonder how methylation might affect
gene expression. We have at least one clue. Examination of the promoter regions shows the
presence of an AHR binding site containing two CpGs. Potentially, exposure to tobacco smoke
could induce binding of AHR and stimulate expression, but perhaps only if the site is not
methylated. In support of this model, we detect induced binding of AHR to this region of the
genome by ChIP when the three cell lines are exposed to CSC. In addition, CSC exposure induces
the luciferase constructs but DNA methylation blocks this induction (in BEAS-2B cells).
Together, this suggests that methylation might block AHR binding and might therefore block
SULT1C2 induction. Surprisingly, in LuAD PC3 cells, the methylated luciferase construct can still
be induced by CSC, suggesting that these cells carry different transcription factors or that the
promoter in the plasmid has been actively demethylated. Our experiments raise tantalizing
questions about the epigenetic and transcriptional response to tobacco smoke.
67

We also examined the CSC response in more detail. LuAD PC3 cells and BEAS-2B cells
responded to low but not high doses of CSC by inducing SULT1C2 mRNA. Interestingly, AHR
mRNA was also induced, though the protein should not require synthesis to respond to
environmental insults. In contrast to SULT1C2, induction of the well-known CYP1B1 gene
occurred at both low and high doses of CSC in all three cell lines. These data might suggest that
SULT1C2 responds to low levels of xenobiotics, and that there may be mechanisms related to
substrate inhibition that shut the gene off transcriptionally at high doses, perhaps to limit the
extent of the response, which could be harmful when prolonged.
Our experiments illustrate that cell lines can be used as powerful models to study
epigenetic changes in response to tobacco smoke. We have provided some initial insights into
possible mechanisms but much work remains to be done, because our data appear to raise as
many questions as they answer. And THAT is the beauty of science!

68

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Abstract (if available)

Abstract We used the Illumina Infinium HumanMethylation27 BeadChip to investigate DNA methylation in lung cancer tissue and adjacent non tumor lung (AdjNTL) from sixty subjects with lung adenocarcinoma. Significant hypomethylation of a SULT1C2 promoter CpG (cg13968390) dinucleotide was noted in AdjNTL from Asians (most of whom were female non-smokers) vs. Caucasians (most of whom were smokers). SULT1C2 is a sulfotransferase, a phase II enzyme that transfers sulfur groups to various substrates, including xenobiotics and chemical carcinogens, thereby improving water-solubility to facilitate clearing via urine or bile. However, in the process of xenobiotic modification, sometimes carcinogenic compounds are created as by products. In the sixty subjects, SULT1C2 methylation and expression in AdjNTL were inversely correlated. SULT1C2 expression is not normally detected in the adult lung. We hypothesized that environmental exposures might induce SULT1C2 through demethylation. We investigated the effect of demethylation on the SULT1C2 CpG-poor promoter using 5-Aza-2’-deoxycytidine, a drug that is incorporated into the DNA of dividing cells and inhibits DNA methylation. Indeed, when methylation was prevented, expression of SULT1C2 increased in PC3 and H2347 lung adenocarcinoma cell lines and also in immortalized bronchial epithelial cells (BEAS2B). We performed a luciferase assay driven by a 1.8 kb fragment including the SULT1C2 promoter and part of exon1 into a CpG-less vector and observed that methylation of the promoter significantly decreased luciferase activity. Finally, as a model for the effect of environmental factors such as cigarette smoke on the expression of SULT1C2, we treated the cell lines with cigarette smoke condensate (CSC) at increasing doses for 24, 48, and 72 hours. Since the Aryl hydrocarbon receptor (AHR) plays a key role in activating phase I enzymes such as CYP1B1, we searched the SULT1C2 promoter sequence for AHR binding sites and detected one 1.3Kb upstream of the transcription start site. AHR expression increased concordantly with SULT1C2 expression. Our results suggest that in non-smokers exposed to environmental carcinogens or secondary tobacco smoke, AHR binding to the SULT1C2 promoter induced and activated the expression of SULT1C2 possibly causing the long term epigenetic changes reflected in lower DNA methylation levels at cg13968390.

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Creator Johnson, Candace Jeanette-Sukey (author)

Core Title CpG poor promoter SULT1C2 regulated by DNA methylation and is induced by cigarette smoke condensate in lung cell lines

School Keck School of Medicine

Degree Doctor of Philosophy

Degree Program Genetic, Molecular and Cellular Biology

Publication Date 07/22/2017

Defense Date 06/16/2015

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

Tag DNA methylation,non-CpG island promoter,OAI-PMH Harvest,SULT1C2

Format application/pdf (imt)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Laird-Offringa, Ite A. (committee chair), Coetzee, Gerhard (Gerry) A. (committee member), Stallcup, Michael R. (committee member)

Creator Email candacej@usc.edu,fudgecandi@yahoo.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c3-604118

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Legacy Identifier etd-JohnsonCan-3680.pdf

Dmrecord 604118

Document Type Dissertation

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Rights Johnson, Candace Jeanette-Sukey

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Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the a...

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