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The expression of human carboxylesterases in normal tissues and cancer cell-lines

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The expression of human carboxylesterases in normal tissues and cancer cell-lines

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THE EXPRESSION OF HUMAN CARBOXYLESTERASES
IN NORMAL TISSUES AND CANCER CELL-LINES

by

Sundeep Talwar
A Thesis Presented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(BIOCHEMISTRY AND MOLECULAR BIOLOGY)

August 2008

Copyright 2008 Sundeep Talwar

ii
TABLE OF CONTENTS

List of Tables iii
List of Figures iv
Abstract v
CHAPTER I: INTRODUCTION 1
(1) Carboxylesterases 1
(2) DNA Methylation 4

CHAPTER II: MATERIALS AND METHODS 8
(1) Autopsy Tissues 8
(2) Cell Lines and Culture Conditions 8
(3) Isolation of DNA and RNA from Cancer Cell 8
Line Cultures and Human Tissues
(4) Expression of Carboxylesterase by RT-PCR 9
(5) Bisulfite Treatment 10
(6) Combined Bisulfite Restriction Analysis 12
(7) Statistical Analysis 13

CHAPTER III: RESULTS AND CONCLUSION 15
(A) Results 15
(1) Carboxylesterase Expression in Normal Tissue 15
(2) Carboxylesterases Expression in Cancer 15
(3) DNA Methylation of Carboxylesterases in Normal Tissues 17
(4) DNA Methylation of Carboxylesterases in Cancer 18
(5) Correlation between CES DNA Methylation and CES Gene Expression 20
(B) Conclusion 21

CHAPTER IV: DISCUSSION 23
(1) Current Findings 23
(2) Future 26

Bibliography 30

iii
LIST OF TABLES

Table 1: Primer sequence for reverse transcriptase PCR 10
Table 2: Primer sequence for bisulfite PCR 13
Table 3: Comparison of CES gene expression and CES DNA methylation in 21
normal tissue and cancer cell-line

iv
LIST OF FIGURES

Figure 1: DNA Methyltransferase 4
Figure 2: DNA Methylation Establishment and Maintenance by DNA 5
Methyltransferases (DNMT)

Figure 3: Link Between Gene Expression & DNA Methylation 6

Figure 4: Representative Agarose Gels 10

Figure 5: Mechanism Behind Bisulfite Treatment 11

Figure 6: Carboxylesterase Gene Maps 14

Figure 7: mRNA Expression and DNA Methylation for Normal Tissue 16

Figure 8: mRNA Expression and DNA Methylation for Cancer Cell-Line 19

v
ABSTRACT
Carboxylesterases (CESs) are serine esterases and are known to hydrolyze a variety of
substances. CESs have been used to activate drugs that target a host of ailments,
ranging from cancer to influenza. Although, CESs play an important role in drug
therapy, little is known about their tissue distribution. Five different carboxylesterases
have been identified in humans and each shows tissue specific expression. It is
hypothesized that the effectiveness of chemotherapeutic prodrugs could be increased if
the levels of CESs were known in different cancer tissues. This study measures the
CES mRNA expression for all of the human CESs and compares the CES expression
levels between normal human tissues and cancer cell-lines. We found that CESs have
variable mRNA expression in human tissues and cancer cell-lines, and that CES
expression decreases in cancer. The decrease in mRNA expression levels in cancer
may explain why CESs are less effective in hydrolyzing carboxylester bonds in
chemotherapeutic agents.

1
CHAPTER 1: INTRODUCTION
(1) Carboxylesteres:
Carboxylesterases (CES) are a group of enzymes that belong to a super-family called
the α,β-hydrolase-fold family and are serine esterases, which are found in various
animal species and mammalian tissues (Cygler et al 1993; Satoh & Hosokawa 1998;
Satoh et al 2002). The gene products of the α,β-hydrolase-fold family are localized in
the endoplasmic reticulum and the cytosol of many tissues. Several researchers have
found that the carboxylesterases (CES) are retained on the luminal side of the
endoplasmic reticulum by a tetrapeptide HXEL consensus sequence on the carboxy-
terminal end that binds to the KDEL receptor on the endoplasmic reticulum
(Murakami et al 1993; Robbi & Beaufay 1991). CESs are able to achieve their
enzymatic activity via a triad of catalytic amino acids (Ser, His and Glu) and contain
four cysteine residues which are presumed to form disulfide bonds (Satoh &
Hosokawa 1998). Studies conducted by Kroetz and colleagues have shown that CESs
need to be glycosylated at one or more sites in order to be enzymatically active and
contain N-X-T/S sites for carbohydrate modification (Kroetz et al 1993).

CESs are believed to be involved in an array of functions, from hydrolyzing
endogenous and xenobiotic compounds to metabolizing prodrugs. CESs detoxify
foreign substances which contain either ester, amide or thioester bonds and they also
cleave carboxylesters into their corresponding alcohol and carboxylic acid groups
(Redinbo & Potter 2005; Satoh & Hosokawa 1998; Xie et al 2002). These enzymes
metabolize a wide variety of prodrugs from chemotherapeutic to antithrombogenic to
2
antihypertensive and even anti-influenza agents. CESs are also involved in
hydrolyzing commercial pesticides and organically active compounds, such as
cocaine, heroin and procaine (Pindel et al 1997; Wu et al 2003). Researchers believe
that CES might play a potential role in lipid metabolism as well as human cellular
cholesterol homeostasis. CESs have been shown to function like lipases by
hydrolyzing several long chain fatty acids, such as palmitoyl-coenzyme A, and
diacylglycerols (Becker et al 1994; Mentlein et al 1987; Nilsson et al 1990). Lastly,
CESs are expressed in active phagocytic cells, such as monocytes and macrophages,
and have been found to be active in the immune defense system. CESs play an
important role in immunological defense by initiating and modulating inflammatory
processes (Langmann et al 1997; Munger et al 1991).

CESs show a broad range of substrate specificity which allows certain forms of CESs
to be more efficient in hydrolyzing and detoxifying certain prodrugs, chemicals and
fatty acid esters. Five human carboxylesterases have been discovered: CES 1, CES 2,
CES 3, CES 4 and CES 7 but only CES 1 and CES 2 have been studied in depth in
regards to substrate specificity. Human CES 1 and CES 2 have significantly different
substrate specificity. Human CES 1 favors a substrate that has a small alcohol group
along with a large acyl group. On the other hand human CES 2 recognizes a substrate
that has a large alcohol group along with a small acyl group (Bosron & Hurley 2002;
Pindel et al 1997; Satoh et al 2002; Takai et al 1997). Examples of CESs substrate
specificity are as follows: human CES 1, but not human CES 2, hydrolyzes the methyl
ester of cocaine (Satoh et al 2002), human CES 2 has a higher enzymatic activity rate
3
for converting the chemotheauraptic agent irinotecan into its active form SN-38 than
human CES 1 (Ewesuedo & Ratain 1997; Saltz 1997; Stucky-Marshall 1999; Takai et
al 1997). Takai and colleagues have also shown that human CES 1 is more active in
hydrolyzing angiotensin-converting enzyme (ACE) inhibitors, such as temocapril and
delapril, than human CES 2 (Takai et al 1997). Tissue-dependent expression of
human CES also determines which human CES activates a prodrug. For example
capecitabine, a prodrug of 5-fluorouracil (5’-FU) is metabolized to 5’-deoxy-5-
fluorocytidine (5’-DFCR) in the liver by human CES 1 and not by human CES 2 in the
small intestine (Hosokawa et al 1995; Miwa et al 1998; Shimma et al 2000).

The five human carboxylesterase genes that have been discovered are all located on
chromosome 16 and appear to have tissue-specific expression. Satoh and others have
looked extensively at human CES 1 and human CES 2 mRNA expression in various
tissues and have found that human CES 1 is ubiquitously expressed, with the highest
expression in liver. They also discovered that human CES 2 not only has lower
mRNA expression in tissues but is not as widely expressed as human CES 1 (Satoh et
al 2002; Xie et al 2002). Mori and colleagues have found human CES 3 to be
uniquely expressed in the human brain and have shown it to have the highest mRNA
expression in the brain compared to the other four human CESs (Mori et al 1999).
Human CES 7 has relatively high mRNA expression in the male reproductive tract
compared to the other four human CESs (Ecroyd et al 2006; Mikhailov & Torrado
1999). Xie and researchers examined human CES 1, 2 and 3 expression in primary
colon cancer tissues from six different patients and found that CES 1 and CES 2
4
expression was lower in colon tumor tissue compared to the adjacent normal tissue,
but the inverse was true for human CES 3 (Xie et al 2002). Wu and authors observed
human CES 2 expression in various leukemia cancer cell-lines along with a colon and
a lung cancer cell-line by multiple tissue expression array and found human CES 2
expression decreased in all of the cancer cell-lines (Wu et al 2003).

(2) DNA Methylation:
We examined DNA methylation patterns of the different CES genes in both normal
tissues and cancer cell-lines, in order to explore if DNA methylation was responsible
for tissue-specific expression. DNA methylation is an epigenetic mechanism believed
to be involved in a variety of functions, from gene silencing to mammalian
development to X-chromosome inactivation (Bird 2002; Li 2002; Li et al 1992). DNA
methylation occurs when a methyl group is added to the 5 carbon of the cytosine base
when it is followed by a guanosine base.

Figure 1: DNA Methyltransferase. Cytosine is methylated to 5-methyl-cytosine by
DNA methyltranserases (DNMT 1, 3a, 3b or 3l). DNA methyltranserases transfer a
methyl group (CH
3
) from S-adenosylmethionine (SAM) to the 5-carbon on the
cytosine ring, thus forming 5-methyl-cytosine and leaving S-adenosyl homocysteine
(SAH) as a by product in the reaction.

5
Thus, DNA methylation only occurs at CpG sites, a cytosine followed by a guanosine.
DNA methylation reactions are carried out by enzymes called DNA
methyltransferases (DNMTs). DNMTs methylate cytosines by transferring a methyl
group from S-adenosylmethionine (SAM) to the 5-carbon position (Fig. 1).

Although, five different DNMT enzymes have been discovered, they are classified in
two general classes: de novo methylation enzymes, DNMT 3a and 3b, establish
methylation patterns early in development and maintenance enzymes, such as DNMT
1 (Fig. 2), preserve methylation patterns during cell division by methylating
hemimethylated CpG sites produced from semiconservative DNA (Holliday & Pugh
1975; Riggs 1975).

Figure 2: DNA Methylation Establishment and Maintenance by DNA
Methyltransferases (DNMT). DNMT 3a and 3b are responsible for de novo
methylation, by adding methyl groups (CH
3
) to previously unmethylated CpG sites in
DNA. Whereas, DNMT 1 is involved in DNA methylation maintenance, by adding
methyl groups (CH
3
) to hemimethylated CpG dinucleotides in DNA.
6
CpG islands, which are a group of CpG dinucleotides spanning over 500 DNA base
pairs and with a GC content greater than 55%, are usually unmethylated and
conserved in the human genome (Takai & Jones 2002). CpG islands are mostly found
in the promoter regions of many mammalian genes and when they become methylated
can cause gene silencing, with many other biological modifications (Fig. 3) (Bird
2002; Bird & Wolffe 1999; Boyes & Bird 1992; Laird 2003).

Figure 3: Link Between Gene Expression & DNA Methylation. In normal tissue
when a CpG island is unmethylated the gene is actively expressed. But, when the
CpG island in or near the promoter region of a gene becomes hypermethylated in
cancer the expression of the gene is turned off.

The objective of this study was to quantify CES mRNA expression for all of the
human CESs genes, except for brain specific CES 3, in different human tissues and
cancer cell-lines. Human CES 3 mRNA expression was not investigated, since we
were interested primarily in somatic tissues. We then compared the CES expression
7
between human tissues and cancer cell-lines and found that there is not only tissue-
specific expression but also cancer-specific expression. In order to investigate why
there is variability in tissue expression of the human CES genes, we hypothesized that
CES gene expression might be regulated by DNA methylation.

8
CHAPTER 2: MATERIALS AND METHODS
(1) Autopsy Tissues:
Thirteen non-cancerous autopsy tissues (bladder, brain, colon, esophagus, heart,
kidney, liver, lung, pancreas, prostate, spleen, stomach and testis) were used to
measure DNA methylation of the four carboxylesterase genes. All human tissues were
collected through protocols approved by the Institutional Review Board of the
University of Southern California.

(2) Cell Lines and Culture Conditions:
Eleven cancer cell lines from six different tumor types were used. T24 (bladder),
MCF-7 (breast), HCT-116 (colon), HEP-3B2 (liver), HEP-G2 (liver), HEP-SK (liver),
HL60 (acute myeloid leukemia), NB4 (acute promyelocytic leukemia), RAJI
(Burkitt’s lymphoma), K562 (chronic myelogenous leukemia) and PC3 (prostate)
were all purchased from American Type Culture Collection (Manassas, VA USA) and
were cultured using recommended conditions.

(3) Isolation of DNA and RNA from Cancer Cell Line Cultures and Human
Tissues: DNA was isolated from human tissues and cancer cell line cultures by
proteinase K digestion and phenol chloroform extraction. Total RNA was isolated
from cancer cell lines using a commercially available kit (RNeasy Protect minikit
Qiagen, Valencia, CA) following the manufacturer’s recommended protocol.

9
(4) Expression of Carboxylesterase by RT-PCR:
2 μg of total RNA was used to generate complementary DNA (cDNA) by random
hexamers and M-MuLV reverse transcriptase (NEB, Ipswich, MA). Multiplex PCR
using cDNA from seventeen different human tissues and eleven different cancer cell
lines were used to detect the expression of two out of the four different
carboxylesterase genes. The multiplex PCR used to amplify cDNA from different
samples was ran with glyceraldehyde-3-phosphate dehydrogenase (GAPDH), CES 1
and CES 2 primers. However, CES 4 and CES 7 genes were both amplified
independently. The amplified PCR products were then resolved on a 2 percent
agarose gel stained with ethidium bromide and photographed with a UV camera.
cDNA from sixteen different human tissues (brain, colon, heart, kidney, leukocyte,
liver, lung, ovary, pancreas, placenta, prostate, small intestine, skeletal muscle, spleen,
testis and thymus) was obtained from Clontech (a TAKARA bio company, Mountain
View, CA). The cDNA for the seventeenth tissue, whole blood, was generated from a
healthy donor’s blood. The expression level of the four carboxylesterase genes in
normal tissues and cancer cell lines were quantified by using the program Quant One
(Bio-Rad). The expression level of each gene in various tissues and cancer cell lines
was determined by dividing the density of the gel band for a particular tissue or cancer
cell line by the density of the GAPDH gel band for that same exact tissue or cancer
cell line. The analysis of each sample and gene was performed in triplicate. The
primer sequences and PCR conditions are listed in table 1. Fig 4a illustrates a
representative agarose gel of multiplex PCR with GAPDH, CES 1 and CES 2.
10

Figure 4: Representative Agarose Gels. A). A representative agarose gel of
multiplex PCR with GAPDH, CES 1 and CES 2, RT-PCR was used to determine the
expression of the two CES genes in various human tissues. B). A representative
agarose gel showing the COBRA results of human CES 1 in the various cancer cell-
lines tested. The presence of a 276 bp band and a 130 bp band confirms CES 1
methylation in the cancer cell-lines tested.

(5) Bisulfite Treatment:
Bisulfite conversion of genomic DNA was done using a commercially available kit
(EZ DNA Methylation-Gold Kit, Zymo Research) following the manufacturer’s
recommended protocol. CT-Conversion reagent was added to 1.5 µg of genomic
DNA and the reaction was denatured with heat, first at 95
o
C for 10 minutes and then at
11
64
o
C for 2.5 hours. After denaturation, M-Binding Buffer was added to the DNA and
then washed with M-Wash Buffer. The genomic DNA was then incubated with M-
Desulphonation Buffer for 20 minutes and cleaned afterwards with M-Wash Buffer.
The bisulfite treated DNA was eluted in 20ul of elution buffer and stored at -20
o
C
until further use. For the complete mechanism of bisulfite conversation please refer to
figure 5.

Figure 5: Mechanism Behind Bisulfite Treatment. During bisulfite conversion all
unmethylated cytosines are converted to uracils, which are further converted to
thymines by PCR. All methylated cytosines remain unchanged during bisulfite
conversion. The top figure illustrates the effects of bisulfite conversion followed by
PCR. Figure was reproduced from (Laird 2003).

12
(6) Combined Bisulfite Restriction Analysis:
The combined bisulfite restriction analysis (COBRA) method was used to evaluate
methylation of the four carboxylesterase genes, and was modified from the procedure
outlined by Xiong and Laird (Xiong & Laird 1997). The basic tenets of COBRA are
that genomic DNA is first denatured and then treated with sodium bisulfite to convert
unmethylated cytosines to uracils. The uracils are then converted to thymines by PCR.
The methylated cytosines found in the genomic DNA, on the other hand are retained
as cytosines even after the sodium bisulfite treatment and primers are then designed
around these methylated cytosines to measure the methylation status (Fig 5).
Methylation status is thus converted to a primary sequence differential where the
methylated cytosine is converted to an unmethylated uracil or thymine, which is then
detected by restriction enzyme digestion. Briefly, selected regions of the four
different carboxylesterase genes were amplified from bisulfite converted DNA of
different human tissues and cancer cell lines. The target sequences were amplified
either by semi-nested or nested PCR. The PCR products were then digested with a
restriction enzyme, which cleaved a specific CpG site present in the original genomic
DNA. The digested PCR products were resolved on a 2 percent agarose gel stained
with ethidium bromide and photographed with a UV camera. A sample was
considered methylated if the digested PCR product contained methylation-specific
restriction fragments. The percent methylation was quantified by using the program
Quant One (Bio-Rad). COBRA was repeated three times for each sample and gene.
Figure 4b illustrates a representative agarose gel of CES 1 restriction enzyme
digestion with Cla I, during COBRA. The primer sequences and the PCR conditions
13
for COBRA are stated in table 2. The specific restriction enzyme used to cleave each
CpG site for the individual CES gene can be found in Figure 6.

(7) Statistical Analysis:
Using StatView software, a non-parametric Spearman’s Rank Correlation Coefficient
test was used to analyze if a statistically significant correlation between CES gene
expression and CES DNA methylation existed. Probability (P) values of < 0.05 were
considered significant.

14

Figure 6: Carboxylesterase Gene Maps. The gene maps show the regions amplified
in each of the four human CES genes, in order to measure both mRNA expression as
well as the level of DNA methylation for the chosen CpG site, which are denoted by
black vertical lines. A CpG island contains many CpG sites but is not depicted with
black vertical lines; due to space restrictions. The restriction enzymes used for each of
the four CES genes are also shown.
15
CHAPTER 3: RESULTS AND CONCLUSION
(A) RESULT
(1) Carboxylesterase Expression in Normal Tissue:
mRNA expression of the four carboxylesterase genes in seventeen normal tissues was
determined by reverse transcriptase PCR and was normalized against GAPDH
expression. We found mRNA expression of CES 1 to be variable and liver had the
highest CES 1 expression of the seventeen tissues measured, which was consistent
with previous reports (Satoh et al 2002). CES 1 expression in liver was three times
more than the combined expression of the other three CES genes (Fig. 7a). CES 2
expression was the highest in the liver, as well (Fig. 7B). CES 2 also had variable
expression but had the highest mean mRNA expression in most of the tissues tested,
compared to the other CES genes. CES 4 expression was measured to be very low in
all of the tissues tested except for liver (Fig. 7C). CES 7 expression was also very low
in all the tissues tested except for liver and testis. Consistent with previous studies
(Ecroyd et al 2006; Mikhailov & Torrado 1999), CES 7 expression was the highest in
the testis compared to the other CES genes (Fig. 7D). We found that both CES 1 and
CES 2 expression is variable, but CES 2 has the highest mean expression in majority
of the tissues tested.

(2) Carboxylesterases Expression in Cancer:
After quantifying carboxylesterase expression in normal tissues we looked at CES
expression in cancer cell-lines. Of the eleven cancer cell-lines tested only three
16
showed significant CES 1 expression: HEP-G2, HEP-3B2 and NB4 (Fig. 8A). When

Figure 7: mRNA Expression and DNA methylation for Normal Tissue. mRNA
expression was measured by reverse transcriptase PCR and DNA methylation was
determined by COBRA. Expression of the four human CES genes in normal tissues is
shown in panels A through D and percent DNA methylation of the four human CES
genes in normal human autopsy tissues is shown in panels E through H.
17
comparing CES 1 expression levels between cancer cell-lines and their corresponding
normal tissues we found that CES 1 level decreased by more than three fold. On the
other hand, CES 2 expression decreased only 1.5 fold, between cancer cell-lines and
their appropriate normal tissues. But, CES 2 expression on the whole was higher in
the eleven cancer cell-lines tested, compared to the other CES genes (Fig. 8B). The
combined CES 2 expression in the eleven cancer cell-lines was three times the amount
of the combined CES 1, 4 and 7 expression in all of the cancer cell-lines tested. When
comparing just CES 1 and CES 2 expression we found CES 2 had more than two
times the CES 1 expression, in all of the cancer cell-lines tested. Generally, CES 2 has
higher mRNA expression than CES 1, in cancer cell-lines.

Our results illustrated CES 4 expression to be very comparable to CES 1 expression
for the majority of the cancer cell-lines tested (Fig. 8C). Of the four CES genes CES 7
had the lowest expression on average in each of the cancer cell-lines tested (Fig. 8D).
Our data is consistent with the previously published report that states CES 2
expression decreases from normal colon and blood to cancer cell-lines (Wu et al
2003). On the other hand, we found not only does CES 1 and CES 2 expression
decrease in cancer; expression of CES 1 decreases at a rate two times that of CES 2.
We also found that human CES 2 expression in cancer is higher than CES 1.

(3) DNA Methylation of Carboxylesterases in Normal Tissues:
In order to investigate why there was a decrease in carboxylesterase expression
between normal tissues and cancer cell-lines, we examined the DNA methylation
18
status of each of the four carboxylesterase genes. The gene maps for the
carboxylesterase genes show the specific CpG sites that were checked for DNA
methylation, by COBRA (Fig. 6). DNA methylation of the four carboxylesterase
genes was measured in fourteen normal tissues obtained from autopsies. We found
that CES 1 had variable DNA methylation in the normal tissues measured. CES 1 had
the highest DNA methylation in blood followed by kidney at 92% and 89%
respectively and had the lowest DNA methylation in liver at 6% (Fig. 7E).

CES 2 DNA methylation in normal tissues was relatively very low compared to the
other three carboxylesterase genes (Fig. 7F). CES 2 DNA methylation in normal
tissues ranged from 1%, in the esophagus, to 11%, in the stomach (Fig 7F). CES 4
DNA methylation was also variable. CES 4 had the highest amount of DNA
methylation in blood at 74% and the lowest amount of DNA methylation in prostate
tissue at 17% (Fig. 7G). CES 7 had the highest individual percent DNA methylation
of the four carboxylesterase genes in all but two of the fourteen autopsy tissues tested,
only CES 1 DNA methylation in blood and kidney were higher. CES 7 DNA
methylation ranged from 85% in prostate to 66% in colon (Fig. 7H).

(4) DNA Methylation of Carboxylesterases in Cancer:
We then looked at DNA methylation in cancer cell-lines to see if there was a
correlation between expression and whether DNA methylation patterns changed
between normal tissue and cancer cell-lines. DNA methylation in cancer cell lines
19
was also determined by COBRA. Three of the four carboxylesterase genes had
variable DNA methylation levels.

Figure 8: mRNA Expression and DNA Methylation for Cancer Cell-Line. mRNA
expression was measured by reverse transcriptase PCR and DNA methylation was
determined by COBRA. Expression of the four human CES genes in cancer cell-lines
is shown in panels A through D and percent DNA methylation of the four human CES
genes in cancer cell-lines is shown in panels E through H.
20
CES 1 had the highest levels of DNA methylation in HL60 at 89% and the lowest
amount of DNA methylation in K562 at 7% (Fig. 8E). CES 2 was the only gene that
was hypomethylated in all the cancer cell-lines (Fig. 8F). CES 4 had the highest
amount of DNA methylation in HL60 and NB4 at 90% and 93%, respectively and the
lowest in HEP-3B2 at 2% (Fig. 8G). CES 7 also had the highest DNA methylation in
HL60 at 91% and the lowest DNA methylation in T24 at 3% (Fig. 8H). When
comparing DNA methylation between normal tissues and their corresponding cancer
cell-lines, we found DNA methylation was variable. When comparing CES 1
methylation between normal blood and the corresponding cell-lines we found that
certain cell-lines, HL60, had relatively the same amount of methylation as in normal
blood. But, then there were other leukemia cell-lines, K562, which had 13 fold lower
the amount of DNA methylation then normal blood. These results confirm that there
is variable methylation between not only normal tissues and cancer cell-lines but also
between non-cancerous tissues and their corresponding cancer cell-lines.

(5) Correlation between CES DNA Methylation and CES Gene Expression:
To detect if a statistically significant correlation existed between CES gene expression
and CES DNA methylation we preformed a non-parametric Spearman’s Rank
Correlation Coefficient test on all of the four CES genes comparing mRNA expression
with DNA methylation, first in the matched normal tissues and then in the eleven
cancer cell-lines. Only for CES 4, when comparing mRNA expression with DNA
methylation in the eleven cancer cell-lines, there appeared to be a significant
correlation with a p-value < 0.05. But when adjusting for the eight different p-values
21
by using the Bonferroni’s adjustment the CES 4 cancer cell-line p-value was not
statistically significant. Table 3 illustrates the complete p-values and the Spearman’s
Rank Coefficient (Rho) for all of the CES genes. It should be noted that no significant
correlation existed when comparing the mRNA expression and DNA methylation
when analyzing all tissues, but this does not rule out the possibility of a significant
correlation being present within an individual tissue type when correlating mRNA
expression and DNA methylation. Multiple tissues of the same type were not
examined in this study.

(B) CONCLUSION:
We discovered that CES 1 and CES 2 have variable mRNA expression levels and it is
CES 2 which appears to have a higher mean of mRNA expression in human tissues,
contrary to previous reports. We also find that both CES 4 and CES 7 have low
mRNA expression levels in all of the tissues, except for CES 7 expression in testis and
liver. When examining CES expression in cancer we were able to ascertain that both
CES 1 and CES 2 expression decreases, but CES 1 proportionally has a larger
22
decrease than CES 2. Furthermore, we found that CES 2 has the highest mRNA
expression on average in cancer, compared to the other CES genes. Finally, we
discovered that DNA methylation does not seem to play an active role in regulating
CES expression in various normal and cancerous tissues. Based on our results we
believe CES 2 may be a better target for future prodrug design.

23
CHAPTER 4: DISCUSSION
(1) Current Findings:
Contrary, to previous findings we found that not only does human CES 1 have
variable expression in many different human tissues, but human CES 2 is also
variable. In fact, our results illustrate it is human CES 2 and not human CES 1 that
has higher mRNA expression in a majority of the human tissues, except liver. Of the
seventeen tissues tested, human CES 2 expression was higher in ten, CES 1 expression
was higher in five and human CES 1 and CES 2 expression was similar in two human
tissues. Our results also confirmed that human CES 1 has the highest level of mRNA
expression in the liver, compared to not only the other three CES genes, but also in
comparison with the other sixteen different human tissues tested. We also validated
CES 2 as having the highest expression in liver out of the seventeen different tissues,
followed by whole blood (Satoh et al 2002; Xie et al 2002).

Interestingly, we observed that the expression of human CES 2 was variable from
tissue to tissue. This is potentially important because the chemotherapeutic agent
irinotecan is believed to be hydrolyzed to its active form SN-38 by CES 2 in the small
intestine and colon. The low levels of CES 2 expression in the small intestine and
colon might help explain why irinotecan is not as efficiently converted to SN-38 by
human CES 2 as compared to rabbit CES 2 (Humerickhouse et al 2000). We found
that human CES 4 and CES 7 have relatively low mRNA expression in the various
tissues measured, except for liver and testis, in the case of human CES 7. Our findings
verify the previously published reports that the carboxylesterase present in the human
24
reproductive tract is more than likely CES 7 (Ecroyd et al 2006; Mikhailov & Torrado
1999).

One limitation of our study is that the expression levels obtained for the four CES
genes in both normal tissues and cancer cell-lines are based on comparison to the
expression levels of GAPDH. There are limitations when using GAPDH expression
as a solitary control, since its levels can vary from tissue to tissue as well as from
cancer to cancer, therefore the expression levels of the four CES genes may not be
comparable for all tissues. In order to overcome this problem additional control genes
such as PCNA, beta-actin, or elongation factors, should be used.

Xie and colleagues previously reported that both human CES 1 and CES 2 expression
decreases in colon carcinomas (Xie et al 2002). Wu and authors then reported that
human CES 2 expression also decreases in the various cancer cell-lines tested (Wu et
al 2003). But we found that not only does the mRNA expression for both human CES
1 and CES 2 decrease in cancer cell-lines it also does in primary bladder cancers (data
not shown). In short, we discovered that human CES 1 expression decreases more
than human CES 2 expression in the cancer cell-lines tested.

After discovering that human CES expression is variable between the different cancer
cell-lines we investigated if epigenetic factors were at play in determining tissue and
cancer-specific expression of the CES genes. Epigenetics is defined as the change in
DNA expression without a change in DNA sequence (Bird 2002). Several studies
25
have shown that when CpG sites, which are mostly found in or around promoter
region of genes, become hypermethylated the corresponding genes are usually down-
regulated (Bird 2002; Bird & Wolffe 1999; Boyes & Bird 1992). Knowing that the
human CES genes have variable expression in both normal and cancer tissues we
decided to look at the DNA methylation of CpG sites in each CES gene. Based on our
DNA methylation results we were able to determine that no correlation existed
between human CES gene expression and human CES DNA methylation in the
various normal tissues and cancer cell-lines. This study illustrates that DNA
methylation doesn’t seem to play an integral role in regulating CESs expression
pattern in both normal tissues and cancer cell-lines, but we cannot rule out other
epigenetic mechanisms as being responsible for differential human CES expression in
normal and cancer tissues.

The lack of a statistically significant correlation between CES mRNA expression and
CES DNA methylation needs to be discussed further. Since, the conclusion no
correlation between mRNA expression and DNA methylation in the CES genes is
based on the DNA methylation results obtained from COBRA. DNA methylation
analysis by COBRA can be limited since it investigates one individual CpG site at a
time. Various studies have shown that any particular CpG site in the promoter or
regions downstream of the promoter can be methylated and correlate with mRNA
expression (Futscher et al 2002). Therefore, if one CpG site doesn’t correlate with
expression it is not safe to rule out other CpG sites, without broader analysis. In order
to thoroughly determine if CES expression is regulated by CES DNA methylation one
26
needs to investigate multiple CpG sites. This can be achieved by performing various
experiments, such as bisulfite sequencing or pyrosequencing. These techniques exam
multiple CpG sites for DNA methylation, at once.

Another, approach that can be used to determine if DNA methylation regulates CES
mRNA expression is to treat the twelve cancer cell-lines with a DNA methylation
inhibitor, such as 5-azacytidine, and then measure the mRNA and DNA methylation
levels, of the four CES genes. 5-azacytidine is a pyrimidine analog of cytidine with a
nitrogen atom in the 5 position of the ring, instead of a carbon (Sorm et al 1964).
Several studies have shown that it plays an active role in reactivating tumor suppressor
genes by decreasing DNA methylation. 5-azacytidine decrease DNA methylation by
first incorporating into both RNA and DNA and then binding DNA
methyltransferases, thus preventing DNMTs from methylating the newly replicated
DNA strands (Paces et al 1968; Zadrazil et al 1965). The CES mRNA expression
levels can then be measured either by using reverse transcriptase PCR or real-time
PCR, with controls such as GAPDH, PCNA or beta-actin. Demonstrating activation
of CES by treatment with a DNA methylation inhibitor would support the hypothesis
that expression is controlled by DNA methylation.

(2) Future:
Substantial work still needs to be done in order to fully realize the potential of the CES
enzymes. The functional diversity of the CES enzymes has already been well
documented, but not enough progress has been made as to why there is differential
27
expression of not only a particular CES gene in different human tissues but also in
different mammalian species. This is a significant obstacle that needs to be overcome
since CESs are not only active in protecting the body from harmful endogenous
substances but also have the potential to play an essential role in helping the human
body fight against disease.

One approach that can be employed in order to attain a complete understanding of
differential CES expression in various human tissues is to measure mRNA expression
and DNA methylation levels from a single source. This can be achieved by first
measuring CES expression levels from adjacent normal tissues and then measuring the
CES mRNA levels in the corresponding cancer. Then measure the DNA methylation
levels in both the normal as well as in the cancer tissue, the same tissue source which
was also used to measure mRNA expression. This should be repeated multiple times
for each particular tissue and for each of the four CES genes, thus assuring that the
mRNA and DNA methylation levels are accurate and reliable. The mRNA levels can
be measured by either reverse transcriptase or Real Time PCR and the DNA
methylation levels can be quantified by either COBRA or pyrosequencing. To further
investigate if there is a statistically significant correlation between CES expression and
CES DNA methylation in a particular tissue a Spearman’s Rank Correlation
Coefficient test can be conducted.

Another approach that can be used to examine why CESs mRNA expression varies
from normal tissue to cancer cell-lines is to look at histone modification, because
28
previous studies have reported that certain forms of histone modification can lead to
gene inactivation in cancer (Lachner & Jenuwein 2002). Another aspect that needs to
be further studied is to improve the enzymatic efficiency of the CES enzymes in
hydrolyzing prodrugs. Wierdl and colleagues have done extensive work in attempting
to improve CES activity by developing not only a prodrug using rabbit CES but also
developing human CES 1 mutants based on the protein structure of the rabbit CES
(Wierdl et al 2008). Although, both of these approaches have considerable pitfalls and
are far from being used therapeutically in humans, in vitro both these approaches have
provided promising results.

Another approach to increase CES sensitivity to prodrugs, such as irinotecan, is to
introduce hypoxia-response elements in conjugation with the CES genes. Matzow and
authors have tested this approach in vitro as well as in vivo and have found that CES
hydrolytic activity increases along with decrease in drug cytotoxicity (Matzow et al
2007). The bases behind using hypoxia-response elements is that previous studies
have shown that hypoxia can induce the expression of certain genes and Matzow et al.,
believe that CES gene expression can also be regulated by hypoxia-response elements
(Semenza 2002; Wang et al 1995). The idea to use either mutant CES 1 genes or
hypoxia-response elements in conjugation with CES genes, via a vector, in trying to
increase human CES activity are novel and exciting but still not practical, since both
approaches are likely to generate an immune response if introduced in the human
body. Therefore, other avenues need to be explored in trying to understand the true
biological function and role of the human CES genes.
29
The results of this study have identified various facts that we believe will be helpful in
trying to explain why certain human CES genes are more effective and efficient in
hydrolyzing prodrugs in specific cancer tissues, over other human CES genes. We
also investigated if a particular epigenetic event was responsible for tissue specific
expression of human CES genes in normal tissues and cancer cell-lines and found that
DNA methylation does not correlate with human CES expression. Attaining a
complete understanding of how the expression of the human CES genes varies from
cancer to cancer will be paramount, as the knowledge will aid in developing an
optimal prodrug that is efficiently and effectively hydrolyzed to its active form by the
CES enzymes. The results of the study are a step towards gaining more knowledge of
the functionality of the human CES genes.

30
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Abstract (if available)

Abstract Carboxylesterases (CESs) are serine esterases and are known to hydrolyze a variety of substances. CESs have been used to activate drugs that target a host of ailments, ranging from cancer to influenza. Although, CESs play an important role in drug therapy, little is known about their tissue distribution. Five different carboxylesterases have been identified in humans and each shows tissue specific expression. It is hypothesized that the effectiveness of chemotherapeutic prodrugs could be increased if the levels of CESs were known in different cancer tissues. This study measures the CES mRNA expression for all of the human CESs and compares the CES expression levels between normal human tissues and cancer cell-lines. We found that CESs have variable mRNA expression in human tissues and cancer cell-lines, and that CES expression decreases in cancer. The decrease in mRNA expression levels in cancer may explain why CESs are less effective in hydrolyzing carboxylester bonds in chemotherapeutic agents.

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Asset Metadata

Creator Talwar, Sundeep (author)

Core Title The expression of human carboxylesterases in normal tissues and cancer cell-lines

School Keck School of Medicine

Degree Master of Science

Degree Program Biochemistry and Molecular Biology

Degree Conferral Date 2008-08

Publication Date 07/31/2008

Defense Date 06/24/2008

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

Tag CES expression,DNA methylation,OAI-PMH Harvest

Language English

Advisor Yang, Allen S. (committee chair), Laird-Offringa, Ite A. (committee member), Stellwagen, Robert H. (committee member)

Creator Email stalwar@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-m1469

Unique identifier UC1448546

Identifier etd-Talwar-20080731 (filename),usctheses-m40 (legacy collection record id),usctheses-c127-89901 (legacy record id),usctheses-m1469 (legacy record id)

Legacy Identifier etd-Talwar-20080731.pdf

Dmrecord 89901

Document Type Thesis

Rights Talwar, Sundeep

Type texts

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

Repository Name Libraries, University of Southern California

Repository Location Los Angeles, California

Repository Email cisadmin@lib.usc.edu