Genomic Instability Induced By Long-Term Exposure to Polychlorinated Biphenyls (PCBs)

1

Abstract

In highly industrialized cities, the byproducts of mechanized operations released into the

environment are often potential carcinogens, such as polychlorinated biphenyls (PCBs). Previous studies

have shown the oncogenic effects of PCB exposure. However, the mechanisms underlying PCB-induced

carcinogenesis are largely undefined. In our research, we analyzed the effects of PCBs on cancer

progression through a long-term study of the in vitro effects on cell lines treated with the chemical. For

the experiments, we utilized a 293T human embryonic kidney cell line, and a LS174T primary colon

carcinoma cell line. At the highest concentration of PCB treatment of 50 , we primarily observed

massive amounts of cell death. Gradually, resistant colonies emerged, that were able to grow without the

PCBs affecting their viability. These colonies were passaged for fifty generations with continuous

exposure to the chemical. The original colonies were passaged into two different cell lines: one with

continuous PCB exposure and the other pulse-treated or withdrawn from PCB exposure. Our study

analyzed the growth rates of the cell lines used. In comparison to the control-cells, the PCB-treated cells

displayed an accelerated net growth rate. Exposure to PCBs resulted in 293T cells displaying significantly

higher levels of aneuploidy than the parental lines. These findings showed that the continual treatment

with PCBs enhances the degree of chromosomal instability, and confers a faster growth rate. Our findings

suggest that PCBs could promote genetic instability as a potential mechanism promoting tumorigenesis.

2

Introduction

Environmental pollution significantly affects public health. This influence has often taken a toll

on the lives of many, resulting in significant morbidity and mortality. In other words, industrial operations

have detrimentally affected ecosystems by releasing unprecedented amounts of toxic waste products into

the air and water.

Polychlorinated biphenyls (PCBs), a group of aromatic hydrocarbons, are industrial byproducts

that play a role as chlorinated pollutants in both air and water [31]

. They have a molecular structure

composed of two benzene rings surrounded by carbon atoms that determine the specific PCB type

resulting in a high number of isoforms. This organic chemical has been associated with several health

effects such as thymus gland problems, immune deficiencies and an increased risk of developing cancer

[31]. They have been designated by the International Agency for Research on Cancer (IARC) as “probably

carcinogenic to humans” and consequently by the National Toxicology Program as “reasonably

anticipated to be human carcinogens” [30]

. Their low degree of biodegradability and their lipophilic nature

allows for increased storage in adipose tissue in both animals and humans [1, 25]

. PCBs have been shown to

have a lingering presence in the environment. In the 1970’s, these chemicals began being banned and

tightly regulated in their use. They are still found in ecosystems and remain a potential health hazard to

humans and animals alike.

A few population studies indicate that PCBs are associated with specific types of cancer in

humans and animals, such as liver cancer and breast cancer [2, 15]

. An underlying variable in the toxicity of

PCB exposure is its presence in natural environments. It is not simply found as an isolated chemical;

rather it is found as a conflation of other chemicals such as dichlorodiphenyltrichloroethane (DDT) [9]

.

Over the past century, many cases connected to the accidental release of PCBs from factories

have occurred. One well known incident is the General Electric case dealing with a massive leakage from

their facilities into the Hudson River taking place from 1947 through 1977. During these thirty years, 1.3

million pounds of PCBs were released into the Hudson River. Such incidences have led to massive

dredging projects to contain these carcinogenic chemicals [29]

. Although the spill occurred decades ago,

the effect of the chemical on the environment has not eroded with the years. Recently, researchers found

the presence of PCBs in soil surrounding industrial districts [18]

. Integration of PCBs into food chains has

led to its amplified presence in ecosystems [7]

. Although PCB exposure results in a multitude of health

problems, the interplay between exposure and cancer formation is of great concern.

The link developed between PCB exposure and cancer formation has been shown in numerous

studies. Previous research has exhibited how PCB exposure affected mitotic arrest, p53 activation, and

mitotic spindle arrest in NIH fibroblasts [2, 5]

. Using mammalian cell lines such as MDA breast cancer

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cells, these chemicals were shown to promote and initiate carcinogenesis through events such as DNA

damage, and caspase-dependent apoptosis [23]

.

Although existing research on PCBs directly discusses their carcinogenic potential [23]

, such

studies address a limited time frame for exposure to the chemical. In addition, most of our understanding

of PCBs is derived from either in vivo models of carcinogenity [7]

or global epidemiological studies of

cancer incidence [8]

. It is necessary to address PCB induced effects in the long-term or from prolonged

exposure. This is because long-term exposure mimics the level of environmental stress that organisms

face when exposed to such chemicals.

To answer this question, we made use of an in vitro, human cell line model. In this model we

looked for resistant colonies emerging from initial PCB exposure. We analyzed how PCBs function on

the cellular level. To confirm the build-up of resistance we conducted a dose response assay for the first

three days of treatment for both cell lines. The cell lines were treated with different concentrations of

PCBs which allowed us to assess the effects with varying concentrations. By conducting such an

expansive test, we located which PCB type provided resistant colonies that were to be analyzed further.

Our research essentially explores the mechanisms through which long-term exposure to the

chemical induces the cells to express genes that drive cellular abnormalities. The cell lines used have been

treated for eight months with continuous exposure to the chemical. This project established four cell lines

called: Treated continuously with PCBs, Pulse-treated (or withdrawn from PCB exposure), Parental (the

original cell line never exposed to PCBs), and the DMSO- treated cell line. To determine the longitudinal

effects of exposure to the pollutant, resistant colonies found after primary treatment were passaged for

fifty generations. It was necessary for us to evaluate how long-term exposure could prompt cells to

express abnormalities. Further analysis on the nature of the pulse-treated cell line served as a model to

understand how withdrawal of treatment from the PCBs could cause the cells to revert back to a normal

phenotype. It also established a stark contrast between the Treated and the Pulse-Treated cells, showing

that long-exposure to the chemical, not short-term exposure causes further phenotypic instability.

We also explored changes in cell growth levels after continual exposure to the chemical. The

reason for looking for an abnormal growth rate, and anchorage independent growth was to develop a

phenotype of carcinogenity. One of the hallmarks of cancer is the evasion of growth suppressors. The

cancer cell is able to bypass the role that tumors suppressor genes have imposed which limits cell growth

and proliferation [3]

. Our research seeks to develop the phenotype before even addressing the mechanism.

When analyzing these established cell lines, we looked for their anchorage independent growth

levels and their growth rates at higher passage levels. These experiments allowed us to assess the high-

grade tumor formation potential, an important component for the identification of carcinogenity.

Normally, cancer cells are able to grow in the absence of their neighboring cells and anchorage to the

4

extracellular matrix (ECM) [16]

. Cells that are not transformed normally experience apoptosis when not

adhered to a substratum [16]

. A cell that does not form colonies is a non-transformed cell [17]

. Furthermore,

acquiring anchorage independence represents a portion of a cell’s malignant transformation. This is

categorized as a chromosome mutation event which as the anchorage independent growth suggests,

permits cells to become malignant and proliferate at a faster rate.

The research sought to address the formation of chromosomal abnormalities induced by PCBs.

The cells were evaluated for their copy number frequencies in two chromosomes: chromosome

16 and 18. These two chromosomes have in previous karyotyping shown to have a normal copy number

in the cell lines used [12, 13]

. The 293T human embryonic kidney cell line showed a significantly higher

level of aneuploidy than the control lines analyzed. Previous research has shown that genetic and

phenotypic instabilities are two out of many components of cancer cell formation and that an abnormal

number of chromosomes results in destabilized mitotic genes [24]

. We found a significantly higher degree

of aneuploidy for the 293T human embryonic kidney cells, treated persistently with PCBs. On the

contrary, the LS174T cell line, displayed a level of aneuploidy that was barely distinguishable from the

controls evaluated. Previous data demonstrated that the higher the level of aneuploidy, the greater the

degree of genetic instability [24]

. This research has built a model for carcinogenesis in a particular cell line,

the 293T human embryonic kidney cell line, which has shown accelerated growth rate and a significantly

higher degree of genetic instability as a result of persistent PCB exposure. Although the LS174T cells did

not show a significantly higher rate of genetic instability, its accelerated growth rate and high tumor

formation potential can be attributed to other factors playing into carcinogenesis.

We sought to define the long term effects of PCB exposure on two cell lines by using data that

demonstrated the interplay between PCB exposure and cellular changes. Surprisingly, our data

demonstrates that in 293T human embryonic kidney cells, prolonged PCB exposure induces a high degree

of genetic instability, and an accelerated growth rate. While many previous studies have characterized the

effects of this pollutant in short term exposure and demonstrated the cellular changes that occur, none

have fully explored the extent to which long-term PCB exposure changes the cell’s stability. Our findings

suggest that the chemical treatment combined with the persistent environmental stress, permitted

remodeling of the cell’s genetic stability and development of carcinogenic potential.

Material/Methods

Cell Lines-

Two established cell lines were used for this investigation: the LS174T colon carcinoma cell line

and the 293T human embryonic kidney cell line. HEK-293T cells were maintained in Dulbecco’s

Modified Eagle Medium (DMEM) containing 10% FBS. The LS174T cell line was maintained in DMEM

consisting of L-glutamine, Na-pyruvate and 10% FBS. Cells were passaged by trypsinization in order to

5

detach the cells from the plate. They were washed with 1x Sterile Phosphate buffered saline (PBS) before

passaging. These cells were incubated in a humidified environment with 5% CO2 and kept at 37 degrees

Centigrade.

Polychlorinated Biphenyls-

The PCBs were ordered from Sigma-Aldrich in a bulk order which included several members of

the family of chemicals. The six isoforms were: Aroclor 1260, 1262, 1254, 1232, 1242, and 1248. PCBs

can be found in Aroclor form- a state at which it is dissolved in iso-octane solution. These Aroclors were

chosen especially, because they were found in toxicological and genetic studies [1, 2]

. Their isoform

number depended on their different molecular structures, specifically the number of chlorine atoms found

on the benzene rings and the otherwise subtle differences in the molecular structure. The PCBs came in a

high concentration kit which had 1000 g/ ml PCB dissolved in isooctane. Such a high concentration

made it ideal for use as an analytical standard.

Isolating the PCBs from the Aroclor solution-

The PCBs were received through Sigma-Aldrich in closed glass containers [Catalog # 44803].

When evaporating the iso-octane solution in the chemical hood, we took the necessary precautions to

keep the chemical from contacting skin. This was by wearing nitrile gloves, through which the chemical

would not be able to penetrate. The PCBs, dissolved in the iso-octane solution, were isolated using a

nitrogen flow tank. This apparatus was provided by the Sawyers Lab at MSKCC.

Table 1- The different concentrations of Aroclors were calculated using given molecular weights. The measurements were calculated

inM. Using these concentrations we conducted the 24-well plate

evaluation of the effects of each Aroclor on the cells. Table 2- Observations of the 24-well plate for Day 2 for HEK-293t cells - The percentages were used to estimate which Aroclors had least affected the cells. From such charts we were able to conclude which of the six Aroclors worked optimally with the cells and produced resistant clones. The portions highlighted in red signify the highest concentration of treatment at which we observed a potential for resistance to develop gradually. This is why the estimate given is not 100% cell death. This observation was taken on the second day of PCB treatment which explains why the highest concentration showed near to hundred percent cell death. The Aroclor plate which later had resistant colonies forming

for the HEK-293T cells was Arachlor 1242 in the 50M concentration.

Table 1

Table 2

6

This method deviates from previous methods of isolating PCBs. The iso-octane solution was hard

to evaporate because it is a stable compound that is capable of forming heavy air around the tube opening

to prevent further evaporation. For this reason, we devised a method using a nitrogen gas flow directed

towards the falcon tube interior containing the Aroclor solution. The PCBs were isolated as 1 mm.

residues found in the 15 ml falcon tube after evaporating the iso-octan e. The PCBs were then dissolved in

1 ml of dimethyl sulfoxide (DMSO). This allowed for adequate storage for long-term use in the lab and

storage at room temperature in the chemical hood.

In Vitro Exposure to PCBs-

In order to provide adequate exposure, the different Aroclors were inserted in the media

according to varying concentrations. This step required the calculation of their different M amounts: 10

M, 20M, 30M, 40M, and 50M. We used different concentrations in order to determine the

dosage response and identify which dosage level proved optimal for the selection process. This meant that

we were looking for the highest concentration at which the cells gained resistance and formed resistant

colonies. The cell lines were plated with specific concentrations of PCBs, in 24-well plates for selection.

Each column had a different dosage of the chemical treatment. This scheme gave us information from the

many replicates for each dosage concentration.

There was a column for the control cell line which had treatment with Media only. There was a

total of six different 24-well plates used for this investigation because each one contained one out of the

six-member Aroclor family. Each day a specific amount of the chemical was added to fresh media as seen

in Table 2. After a 24-hour incubation period, the apoptotic, floating cells were washed off with PBS.

Fresh media was provided to the cells along with the PCB concentration.

We treated the cells each day, for two weeks until resistant colonies emerged in select plates.

During the two–week selection process, the cells were observed for the percentage of viable cells for each

dosage. An estimate of the number alive was recorded for each day (Table 2). This helped distinguish

which plates had the highest amount of cell death and which ones were affected the slightest. By

recording an estimate for each day we found in particular plates, cells were emerging at high

concentrations, which were resistant to the chemical exposure.

Figure 1 - Plating Scheme of the HEK-293t Cells and the LS174T Cells for Dose Response Curve- Following the above arrangement of the different dosages ensured that we had three replicates for each dosage concentration for a particular day. The cells were seeded 20,000 cells per well for the HEK-293T evaluation and 10,500 cells per well for the LS174T evaluation. Each day a portion of the cell lines were counted by trypisinizing and removing the cells to Vi- Cell XR Cell Viability Analyzer.

7

Dose-Response Curve-

To map out what occurred to the normal cells during the first three days of PCB treatment, we

conducted a dose response curve. The cells were grown overnight in serum-free media to ensure that they

grew at the same rate. We used 96-well plates. The scheme of the plate is shown in Figure 1. Each day the

cells were counted using a Vi-Cell Counter. Using the Vi-Cell XR Cell Viability Analyzer machine and

software we counted the number of viable cells. This machine allowed for automatic execution of the

trypan-blue exclusion protocol. This protocol involved 500 L of a sample volume mixed with trypan

blue. Then the machine took 50 images to denote the cell viability and concentration. The parameters for

analysis of the cell lines used were already in the program because of the common use of the cell lines in

the lab. There were three replicates for each cell line for each day.

The two controls provided were the Media-Only control and the Media treated with DMSO

controls. The DMSO in the media was at the same concentration as the PCBs in the media. This vehicle

control was provided in order to allow for insight on whether the cell’s viability and growth trend was in

any way affected by DMSO exposure.

Passaging of Resistant Clones-

The resistant colonies which were isolated were passaged for eight months until Passage 50

(Figure 5). The original colonies diverged into two different cell lines: one was continually treated with

the PCBs. These were LS174T Treated, and 293T Treated. For the other line, we had withdrawn PCB

exposure. These were LS174T Pulse Treated, and 293T Pulse Treated. These were passaged for a couple

of months using the Aroclor solution administered at each passage. The cells were normally split at a 1:4

ratio every three to four days. The Treated cell line was provided with 32 L concentration at the time of

initial seeding, to ensure that the cells were exposed to the chemical in the media. The total amount of

media was 10 mL and we used 10 cm. plates for passaging. The concentration used was proportional to

the total amount of media used.

Soft Agarose Assay-

Soft agarose assays provided a means to evaluate the cells’ capability to grow without contact

from other cells and to form colonies. This assay was conducted using the standardized protocol provided

by the Chan Lab. The cells were seeded at varying concentrations per well into each six well plate. The

agarose contained a double layer: the top layer is 0.4% agarose and the bottom layer is 0.8% agarose.

They were grown for two weeks with the media changed on top to prevent the plates from drying up. The

program used for the soft agarose colony counting was called Gel Count. This was used to rapidly count

the number of colonies formed in the semi-solid agarose matrix. It also provided the average colony size

information.

8

The six-well plates have been used with varying concentrations in order to see the optimal

seeding density at which the cell suspensions grow. Provided with the information about the seeding

concentration at which the cells have grown the best, it was followed up with analysis on the four cell

lines and which ones formed the largest number of colonies.

Fluorescence in situ Hybridization (FISH)-

The procedure used was provided by the Molecular Cytogenetics Core Facility. We used

interphase nuclei with a specific subset of probes. These centromere probes were specific to short,

repeated areas of each centromere. Often times, mistakes such as cross-hybridizations with other

centromeres occurred resulting in faulty signals. The chromosomes selected were Chromosome 18 and 16,

for they proved to be part of studies including abnormality in cell lines. However, they were not particular

to any former PCB studies [12, 13]

.

The color filters with which each of the probes were analyzed are provided in Table 2. The probes

were synthetized by the cytogenetics core. The probe DNA is extracted from BAC clones and Nick

translated with the fluorescent dye to tag it. The general fluorescence in situ hybridization (FISH)

protocol we had to follow involved isolating and purifying the DNA probe, and labeling with Nick

translation using PCR. The labeled probe was then added to the slide containing the cell nuclei. After this,

we placed the probe on the slide and covered with the coverslip. The 75 degree Centigrade denaturing

step was followed by a 37 degree Centigrade hybridization period. Following this hybridization portion,

the slides were subject to washing with various detergents repeatedly to remove any extra stringy signals

and to clear the slides for further visualization. The wash was as follows: Removal of coverslips by

soaking manually taking off rubber cement, soaking in 2x SSC/0.1% Igepal. Then incubate in 0.4x

SSC/0.3% Igepal at 73 degree Centigrade for 2 minutes, and incubate in 2x SSC/0.1% Igepal at RT for 1

minute. Rinse in 2x SSC briefly and incubate in 0.08% DAPI at RT for 5 minutes and rinse in 2x SSC and

distilled H2O briefly. In the last step we air dry slides upright in the dark and mounting with antifade and

coverslip. Before using the slides we stored them in 4 degree environments in order to maintain the slides

and to make sure that the dye did not wear off.

Visualizing the Signal and Establishing the Signal Patterns-

We prepared separate FISH slides for each cell line. This resulted in a total of ten slides prepared

and used for analysis which were: LS174T Treated, LS174T- Pulse Treated, LS174T Parental, and

Probe Names Dye Absorbance Length (nm) Emission Wavelength (nm) Visible Color

Chromosome 16 PSE 16 Texas Red 615 615 Red

Chromosome 18 LI84 Fluorescein FITC 495 518 green (light)

Nuclei Imaging DAPI 345 455 Violet Blue

Table 2 - The Different Filters Used for Visualizing the different centromere signals. The dyes were protected by anti-fade solution. We used oil lenses with 40x and 63x objective to visualize the signals. In addition, we switched to different color filters to capture the nuclei separately with the DAPI stain, the centromeres with Texas Red and FITC. To avoid misinterpreting signals, the focus of the exposure time with each filter had to be optimized as well as the focus.

9

LS174T- DMSO, LS174T-P0, and 293T- Treated, 293T- Pulse Treated, 293T- Parental, 293T- DMSO-

Treated, and 293T-P0. These were, at first, manually evaluated using a hundred nuclei sample size. The

nuclei number and the signal patterns were recorded and conflated into graphs. The software used to

retain the images on the computer was called Isis. We also used Metasystems, a program used to analyze

chromosome signals. This program was used to compile information on the chromosome count for each

nucleus. This automated system proved to be efficient with the amount of times that it took for scanning

each nucleus. The program also allowed for unbiased information on the slides that were used due to the

creation of histograms that charted the number of signals in each nucleus scanned. The filters also change

and the images are finalized with the clear signals.

The approach to analyzing the slides primarily involved looking manually into the microscope

images using the system, Isis, a program which solely captures fields. First, we captured enough slides on

the Isis program and adjusted the program’s capability in capturing the red and green signals for the

centromeres for Chromosome 16 and 18. Then nuclei score sheets were used to evaluate the signals which

were captured by the microscope. This process involved looking at a small sample size which created an

estimate of the number of different, possible signal patterns. The 100-nuclei cell counts were done in the

beginning in order to familiarize ourselves with the different signal patterns, for example the split signals

which were often found on the chromosome. A larger sample size was necessary for statistical analysis.

This study expanded to become a thousand nuclei analysis of each slide. Chromosome 16 and 18 were

chosen for the two-color FISH interphase nuclei analysis due to previous karyotyping done on the cell

lines used and the chromosomes having normal signal counts [12,13]

. The experiments used established cell

lines that had been extensively karyotyped in previous papers. For the LS174T cells, previous

karyotyping determined that most of chromosome counts were normal except for two trisomies at

Chromosome 7 and 15 and a loss of an X or Y. The Representative SKY karyotype for the HEK-293T

cells had shown that there were no known abnormalities associated with Chromosome 16 and 18.

Figure 3- The Analysis and Locus Specificity Metaphase Spread for the Analysis for Normal Human Peripheral Blood- Before the nuclei were completely hybridized with the probe mixture, we needed to make sure that the probe mapped to the right area of the chromosome. By using metaphase spreads we were able to visualize the probed area. .Looking at locus specificity we ensured that the centromere region was located at the correct region. (a) The orange probe color represents Chromosome 3. The green represents Chromosome 18. (b) The red probe color represents Chromosome 16. These images also help us to evaluate the strength of the signals. These chromosomes were used for the two-color FISH interphase nuclei analysis.

(a) (b)

10

Results

Dose-Response Curve

In order to observe the effect of the PCBs on the growth rates of normal cell lines in the

beginning, they were assessed for their growth rates in the first three days following PCB exposure. This

assay was used to primarily evaluate the concentration at which the cells were killed. The cultures were

started at the same time and the cells were starved in serum-free media the day before starting the growth

curve. The results of this analysis are shown in Figure 4. Notably, the graph for the dose-response curves

shows that the PCBs have a deleterious effect on the cell’s growth capacity for the first three days. This

ensured that the cells were grown starting with the same phase of growth. In the two graphs for Figure 4

we see a clear difference between the primary responses to the chemical. For the HEK-293T cells in Fig.

4a, Day 1 shows a downward growth rate. For the LS174T cell line on Day 1 we see the upward growth

rate. The LS174T cell line showed that it was not affected by the PCB on the first day as much as the

other cell line was affected. The reason for this could be attributed to the fact that LS174T cell line is

carcinogenic and has a capability to evade growth regulation that a normal cell would face. This would

explain the upward growth we see on Day 1 instead of the downward course the curve takes for the HEK-

293T cell line. This comparison also suggests that the LS174T was more resistant to the PCB exposure

initially than the HEK-293T cell line. There was a point at which the cell lines display stability and grow

further. As seen in Figure 4a, it was by Day 2 that the net growth stabilized. Overall, for the LS174T cell

lines we observed more resistance to the PCBs from the first day of treatment.

Figure 4- The Effect of the PCBs in Varying Dosages During Primary Exposure – (a) This dose-response graph is for the HEK-293T cells. These dose

response curves were generated with cell counts taken from each group and we averaged the counts for each day and plotted them onto the graph. The

10 treatment shows an overall 40% decline in the growth rate between Day 1 and 3. The 30 treatment displays a 75% decrease in growth over the

course of three days. The 50 treatment shows an overall 90% decline in growth. These results indicate that the different doses show a trend in the cell growth. The highest dosage treatment has the lowest net growth rate whereas the lowest dosage has the greatest net growth. Furthermore, the 50 experiences stabilization in growth as Day 3 approaches. The rate of decline in growth is 81.5% between Day 0 and Day 1, and lowers to 30% between

Day 1 and Day 2. Finally it shows a 58.3% decline in growth between Day 2 and Day 3. (b) This dose-response curve is for the LS174T cell line. The 10

M concentration had an overall increase in growth by 103%. The 50showed only a slight 28.5% increase in growth. These results indicate that for the LS174T cells, the PCBs affected cell growth in a trend similar to the 293T cell line. The highest dosage showed the lowest net growth while the lowest dosage response had a higher growth rate. The control treatment showed a high proliferation rate over the three days which was expected. This data from the control lines shows us how the PCB is acting on the cells, and not the DMSO.

11

Primary Treatment of Cell Lines with PCBs Induces Low Net Growth

To assess the direct effects of the PCBs on the cell line used, the cells were carefully monitored

for two weeks, taking into account the different concentrations at which they were exposed which varied

from low to high M quantities. The Aroclor isomers were evaluated separately for their effects in each

cell line. Thus, the cells were seeded at the same density in each of six 24-well plates. Each cell line

responded to a distinct Aroclor with massive cell death in the beginning, but slow gain of resistance. For

the HEK-293T cells the plate treated with Aroclor 1242 quantity produced resistant colonies by Day 3 of

treatment. By observing using one-day intervals we monitored which of the other Aroclor plates did not

have any surviving cells for the highest concentration of PCB. For the LS174T cell line, resistant clones

emerged after treatment with Aroclor 1232 for four days.

Isolating the Clonally Selected Cells for Further Passaging

The main objective of the previous dose response graphs was to display the capacity of the PCBs

to induce cell death after primary exposure. By counting the number of cells for each day, over the course

of three days, we were able to notice how the first day of exposure with PCBs caused cells to die in

different degrees for each concentration tested. For instance, the 10 M concentration had a sparse

number of cells that were brownish in color and slightly detached from the plate. However, for the 40 M

and 50 M concentrations we saw the cells detached from the plate, with necrotic morphology. We

categorized this type of cell death as necrosis, not as apoptosis. This is because apoptosis is programmed

cell death, whereas necrotic cell death occurs passively without any underlying mechanisms [19]

.

Gradually, the cells gained resistance to the treatment. This buildup of resistance was critical to the

investigation. This experiment was continued by further passaging the resistant clones that were isolated

in the 24-well plates.

Assessing the Proliferation Rate for Higher Passages

Figure 5 - How the Cells Were Handled Following Isolation of Resistant Clones Following initial PCB exposure the cells experienced massive cell death. After the first few days, resistant colonies formed in certain plates. To make sure that these colonies were indeed resistant, they were treated for a longer time with the PCB in the media. These resistant cells were passaged further into two different cell lines. The Treated cell line was passaged for an equal amount of time as the Pulse-treated cell line. Both were given the same amount of time before passaging and grown using the same media concentration. The

continually treated cell line was exposed to 32 l of PCB. Following the long term passaging with and without the treatment, we looked for the difference between the two cell lines used.

Resistant Colony

12

After passaging the cells for 8 months, we tested the hypothesis that continual PCB treatment

caused emergence of carcinogenic capabilities. A cancer cell’s principle characteristic is its ability to

sustain proliferative growth [3]

. Oncogenic processes allow for the expression of genes that control the

increased proliferation rate and the evasion of checkpoints in the cell growth and division cycle [3]

. Using

a growth curve, we directly compared between the Treated and the Pulse-treated cell line we looked for a

difference between the two cell lines.

Two critical points arise from these growth curve charts. First, the cells which have been treated

with PCBs show a higher rate of growth when compared to the following cell lines: Pulse-Treated,

Parental, and DMSO-treated (Figure 6). This leads to the possibility that after continuous exposure to

PCBs, these cells have gained growth advantages conferred upon them starting from their time as

selective clones in Passage 1.

Secondly, the DMSO–treatment did not prompt accelerated growth. Our original intent in

maintaining a DMSO-treated cell line was to look for the effect it had on cells, and whether this effect

mirrored that of the PCB treatment. Instead, the DMSO-treated cell’s growth resembled the growth of the

Parental cell line for the first three days (Figure 5a). During the first two days there was no significant

difference between the experimental and control cells. Between Day 2 and 3 the Treated cell line grew 1.4

times faster than on Day 2. In addition, between Day 3 and 4 the LS174T cells grew 1.1 times faster. We

Figure 6- Relative Growth Curve for Cell Lines- (a) Relative Growth Rate for LS174T Colon Carcinoma Cell Lines- The cells were

seeded at 100,000 cells per well in six-well plates. There were six replicates used for each day. These were grown for four days in order to assess the total number of viable cells over time. The counts from the cell line Treated with PCBs suggest that between Day 3 and 4, these cells grew faster than the other three cell lines evaluated. It grows 8% faster than the Pulse- Treated cell line, 36.7% faster than the Parental cell line, and 50.4% faster than the DMSO treated cells. This indicates that the Treated cell line has a growth rate that may be due to accumulation of abnormalities that confer growth advantages. (b) Relative Growth Curve for HEK-293T Cell Lines- The cells were seeded at a rate of 600,000 cells per well. The results show that the Treated with PCBs cell line has a significantly higher proliferation rate than the other cell lines . It grows 23.8% faster than the Pulse-Treated cells, 44.4% faster than the Parental cells, and 23.8% faster than the DMSO treated cells. The results for both graphs show that the treated cell lines show an overall increase in growth rate which may be due to the cells becoming tumorigenic.

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13

can draw additional conclusions from Figure 5a. The Pulse-treated cell line on both Day 2 and 3 showed a

viable cell count similar to the count for the Treated cell line.

By contrast on Day 4, the Treated cell line grew 8.2% faster than the Pulse-treated cell line.

Meanwhile, the Treated LS174T cell line grew 36% faster than the Parental cell line for Day 4. This

allows us to conclude that for the LS174T colon carcinoma cell line, an already mutagenic cell line, the

continual PCB treatment has given it further capability to evade normal growth patterns and cellular

programs which normally regulate the proliferation rate.

This increased growth advantage was also seen in the 293T cell line. In Figure 5b, it can be

observed that between Day 2 and 3, the Treated cell line and the Parental cell line were growing at the

same rate. On Day 3, the Treated cell line had a two-fold increase in growth. Between Day 2 and Day 3,

the Pulse-treated cell line and the Parental cell line show a 29% and 7.6% increase in growth, respectively.

On the fourth day the Treated cell line experienced a slowdown in growth rate. The graph shows the

Treated cell line had only a 5% increase in growth from the day before, in contrast to the 47.7% increase

for the Pulse-Treated cell line, 30.9% increase seen for the Parental cell line, and the 42% increase for the

DMSO-treated cell line. This data comparison suggests that the Treated cell line in contrast to the other

three cell lines evaluated, displayed a slowing down of growth between Day 3 and Day 4 and yet, the

Treated 293T cell line had the highest amount of cell viability.

We analyzed the total cell viability over the course of four days to determine whether the PCB-

treated cell line differed in its growth rate from that of the pulse-treated cell line. For the HEK-293T cell

line we observed a significant difference between the growth rate of the Treated cell line when compared

to that of the Pulse-Treated cell line. Meanwhile, for the LS174T colon carcinoma cell line, there was no

statistical difference between the Treated and the Pulse-treated cell line on Day 4, but there is a notable

difference between the Treated and Parental.

Centromere Specific Interphase Nuclei Analysis of the Established Cell Lines

Aneuploidy is commonly viewed as a hallmark of cancer [14, 6]

. This phenomenon manifests itself

as an abnormal chromosome count. This can be found in cases of an extra chromosome which is trisomy,

the absence of a chromosome which is monosomy, and an extra set of chromosomes which is polyploidy.

These two chromosomes were chosen particularly because chromosome 16 and 18 showed no

variation in copy number from the baseline level in previous karyotypes conducted. The copy number of

these two chromosomes was set at the baseline level for the karyotype studies previously. Using the two

chromosomes being as a baseline level for the chromosome count, we then could establish whether any

variation had occurred over time. One abnormality connected to Chromosome 16 was Mosaicism or

Trisomy 16 [21]

. Chromosome 18 is also another chromosome which is connected to trisomy cases. In

humans, trisomy 18 leads to serious health problems towards a disease called Edward’s syndrome. In

14

human studies both chromosomes were shown to have deleterious effects when above the baseline copy

number. The charts in Figure 7 C, D were used to map out signal patterns divided separately into the two

different cell signals; red for Chromosome 16, and green for Chromosome 18. The signal counts were

analyzed using different samples. The final sample count that we used was a thousand nuclei count from

each of the four cell lines. The large sample size was needed to see a clear percentage of aneuploidy and

was needed to rule out any unclear signals that the hybridized slides had. Locus-specific probes cover

only a hundred kilobases, a small portion of the entire chromosome. When imaging the slides the nuclei

occasionally had weak signals which could not be used in the analysis. The reason for categorizing the

different chromosome counts was to identify in specific areas were the different cell lines had the highest

frequency of a certain signal.

Figure 7- (A, B) Metafer4 program imaging system- Here are two sample nuclei taken from the Metafer4 program scan of the HEK-293T cells. The green signals are for the centromere region of Chromosome 18 and the red stands for the centromere region of Chromosome 16. The arrow points out the split signal, a common signal observed. When we saw two signals close, connected with faint hint of color, we interpreted this as one signal. (C, D) Centromere Specific Signal Counts for Chromosome 16, 18- The automatic system on the microscope captured sets of images based on the field area chosen. The results depict frequencies for each signal count. (C) In this graph we are looking at the signal counts differences for the HEK-293Tcells. There is a significant difference between the Treated line and the Parental. For the 3R and the 3G signals which are baseline signals for the HEK-293T cells, we can see that the Parental, DMSO-treated and Pulse Treated cell line have a higher frequency than the Treated cell line. For the 4R and 4G signals the Treated show a signal frequency which is twice as high as the frequency for the Pulse- Treated Cells. (D) In the centromere signal counts for the LS174T cells we saw the Treated cell with a slightly higher signal count for the 1R and 1G signal counts. However this difference is not significant. After observing the clear difference between the Treated cell line and the other three cell lines for the signal counts, we have chosen to investigate further the degree of instability in the HEK-293T cell line. Meanwhile for the LS174T cell line, since it is known that it is carcinogenic, it was assumed to have higher rates of aneuploidy. However we find a significant difference in between the treated cell line counts in 1R monosomy for Chromosome 16 and for 1G monosomy for Chromosome 18. Thus, we see very few significant changes outside of these two chromosome counts for the LS174T cell line. This smaller percentage of genetic instability demonstrates that the PCB treatment did not propel this cell line to such a level of genetic instability. Therefore, the lower the degree of aneuploidy in the cell line, the lower its genetic instability.

(A) (B)

15

We saw a significant difference in the HEK-293T Treated cell line and the control cell lines as

seen in Figure 7C. This pronounced difference is found in the Chromosome 18 counts. For, the HEK-

293T cell line, the baseline chromosome copy number was 3n. This meant that a majority of chromosome

had three copies. This cell line was previously characterized as a hypotriploid cell line [12]

. In Figure 7C,

the Treated cell line shows double the number of signals for 4G than the Pulse-treated cell line. This is

significant because at this aneuploidy level, a demonstrated higher percentage difference shows a distinct

characteristic form for the Treated cell line. The Parental cell line and the DMSO- treated cell line have

very few nuclei with the 4 green signals. The P0 cell line shows a higher number of nuclei with the

diploid signal counts for both Chromosome 16 and 18. This tells us that the original resistant colonies had

a high percentage of nuclei that had the diploid count. In Figure 7C, we can see that the HEK-293T cells

treatment with the chemical show a higher degree of genetic instability.

Soft Agarose Results

Another aspect of our investigation which was important to analyze was the independent growth

capability of the cells. This is also known as the metastasizing capability of the cells. While analyzing the

soft agarose plate counts, we evaluated whether the PCB-treated cells had achieved an ability to

proliferate independently of both external and internal signals that normally restrain growth. An ability to

grow despite natural growth barriers indicates progression to a metastatic state. This cell transformation

assay shows how LS174T Treated cell line shows a 96% higher tumorigenic potential than the Not

Figure 8 - Soft Agarose Colony Formation for LS174T Cells- Each bar represents the number of colonies which have grown on the different soft agarose plates after three week incubation period. Throughout the three weeks, we repeatedly put media on top of the double layer of agarose to ensure that the plates would not dry out. The cells were seeded at a density of 50,000 cells per well with six replicates for each cell line. This left the use of four- 6 well plates. When calculating the average and standard deviation of the data generated, it was noticed that the highest amount of colony formation was found in the wells containing the Treated with PCB- LS174T cells. This indicates that the treated cell lines made more colonies than the carcinogenic parental controls. This suggests that the PCB grown cells have a selective advantage over the controls . However, a drawback to this data is that there is no statistically significant difference between the Treated cell line and the Parental colony count. Due to the small variation we can conclude that the LS174T cell line has evolved minimally from its original parental cell line, despite the continual PCB treatment. It maintains that the LS174T due to its carcinogenic nature has anchorage independent growth.

16

Treated cell line (Figure 8). The Treated cell line shows a significantly higher colony count than the

Pulse-Treated cell line. However, the difference between the Treated and the Parental cell lines is not

significant. The PCBs did not affect the LS174T colon carcinoma cell line in its metastatic potential. The

DMSO-treated cell line similarly showed no significant difference when compared to the cell line. This

suggests that the slight difference seen in Figure 8 can be attributed to the technical issues. It also

suggests that the treatment with PCBs caused very little change in the colon carcinoma cell line in

general.

Conclusion

In view of the analyses done on the cell lines tested with PCBs, we propose that initial PCB

exposure prompts a selection process. This process is initiated by low net growth after primary exposure

to the pollutant. Although the cells responded differently with distinct Day 1 growth rates (Figure 4), in

two days, the net growth was stabilized. After two weeks of continuously treating the cells with PCBs at a

high dosage concentration, we isolated resistant colonies that emerged in the 24-well plates. For the

LS174T cells we observed resistant colonies forming in the 50 M plate for Aroclor 1232, whereas for

the HEK-293T cells, a 50 M exposure to Aroclor 1242 produced resistant colonies.

Additonally, we observed that varying dosage exposure to PCBs caused cell death in different

degrees. For the HEK-293T cell line, the percentage of cell growth observed allowed us to locate where

resistant colonies were emerging. As we passaged the cells with PCBs continually for eight months, we

observed the cells undergo selective pressure prompted by PCB exposure. In the LS174T cell line, we

observed a significantly higher growth rate than the growth rate for the Pulse-Treated cell line. Similarly,

the Treated HEK-293T cell line, displayed a higher growth rate than the Parental cell line. In the soft

agarose assay, the HEK-293T cell line had no colony formation. The LS174T cell line, a carcinoma cell

Figure 9- In this study we sought to

identify the long term effects of PCB exposure on two different cell lines. The HEK-293T cell line is shown here, with the 4 different cell lines derived. This defines the distinctive phenotype displayed by the Treated cell line and links the increased degree of aneuploidy in the cells to continuous exposure with PCBs. The Pulse-Treated cell line which stands as a model for withdrawal from the chemical showed near to normal rates for chromosome counts. The Treated cell line is distinct from the Pulse-Treated and control cells because it shows a significantly higher level of aneuploidy within two chromosome count..

17

line, which was expected to form colonies, showed colony formation for the Treated cell line. This colony

formation was significantly higher than the colony formation calculated for the Pulse-Treated cell line.

However, we did not observe a significant difference between the Treated and the Parental cell line for

colony formation. We conducted thousand nuclei set analysis of centromere counts for two chromosomes

i.e. Chromosome16 and 18. Both have been linked with abnormalities in human studies [20, 21]

. The HEK-

293T cell line displayed a significantly higher aneuploidy count for both chromosomes. The LS174T cell

line, showed only a slightly higher aneuploidy count than the controls used. This allows us to conclude

that the HEK-293T cell line has mostly been affected by the continuous PCB exposure through the

development of carcinogenic capacity as is.

Primary Resistance to Exposure

The goal of this study was to determine the effects of a portion of the large Aroclor family: Aroclor

1242, 1232, 1260, 1262, 1254, and 1248, on two established cell lines. From the graphs we concluded that

the LS174T and the HEK-293T cells were affected by PCBs in the first three days of exposure. While the

HEK-293T showed massive cell death on Day 1 of treatment, the LS174T showed little change in growth

on Day 1. There was less of cell death by Day 3, as resistant cells emerged.

We originally conducted growth curves to evaluate the concentration at which the cells were able

to gain resistance. An underlying mechanism favored their growth in such stressful conditions. For this

portion of the project, we sought the highest concentration of the PCBs at which the cells were capable of

growing. In the two cell lines we found that they gained resistance to particular isoforms of the Aroclor at

the highest concentration of 50 M. It was determined that the cells were affected at varying degrees in

the other treatment concentrations. Our objective was to look for the highest concentration of treatment at

which we would isolate cells resistant to the PCB treatment. It is probable that the colonies emerged from

single cell survivors that had a genetic makeup that allowed for their survival. This process of selection

was observed in both standardized cell lines used. Based on the rise of the resistant colonies in the plates

at the end of two weeks, we established two cell lines derived from the original resistant colonies.

Dosage Response Trends in Cell Growth

The dosage response curves show trends in the different concentrations of the polychlorinated

biphenyls when exposed to the cells. The highest concentration of PCB treatment (50 M) was shown to

have prompt a lower net growth rate than cells grown in lower concentrations such as 10 M. Both the

dose response curve and the primary PCB exposure in the 24-well plates manifest the development of

distinct cell lines used for their selective growth advantage and drug resistance.

Passaging with Prolonged Exposure the Original Resistant Colonies

Once resistance was established as seen from the dose-response curves, the cell lines were

passaged into two separate cell lines: one with continual treatment with PCBs, and the other with pulse-

18

treatment. The experiments conducted following this passaging displayed an accumulation of further

abnormalities in such cell lines which will make them oncogenic and distinct from the parental and

DMSO-treated cell lines.

Comparison of the Growth Rates of the Cell Lines

After several passages, we compared the growth rates of the treated lines. For both HEK-293T

cell lines and the LS174T cell lines, the treated cell lines showed a capacity to proliferate at a faster rate

than the pulse treated cell line and the two controls (Figure 6). The cell line continuously treated with

PCBs displayed a growth rate that is significant above all the other cell lines that are not in the presence

of PCBs. This comparative study suggested that PCBs may be inducing a higher growth rate. The

mechanism that could possibly lead to this growth advantage is the cells up-regulating preexisting genes

that regulate the mitogenic signals of cells. In The Hallmarks of Cancer, Weinberg discusses the

importance of a cancer cells ability to bypass growth regulators through the formation of growth factor

ligands and the activation of signaling pathways downstream of these ligand accepting receptors [3]

. By

following up on this, a future study could focus on the genes connected to mitotic signaling.

Anchorage-Independent Growth

While characterizing cancer cells, it is important to refer to their capability in evading the effects

of growth suppressors. To develop on the issue of the progression to carcinogenity we looked at

anchorage independent growth in the cells. When a cell is detached from neighboring cells it is incapable

of growing. In metastatic cells, the cells are capable of growing without any contact with other cells. For

anchorage-independent growth, the 293T cells did not show any colony formation. This may be due to the

fact that the 293T is not a carcinogenic cell line. For the LS174T cell line, however, the data suggests that

a high number of colonies were capable of forming for the Treated cell lines and the Parental. The pulse-

treated cell line had the fewest colonies forming. The PCB-Treated cell line and the Parental cell lines

displayed the highest number of colonies formed. However when compared statistically, we found that the

slightly higher colony formation data for the Treated cell line was not significant. The two cell lines were

comparable in the treatment they were given, however without a significant difference between the treated

and the parental, we can assume that the PCBs had not affected these cells by giving them the capability

to metastasize as higher colony formation in soft agarose would otherwise suggest. The pulse treated cell

line presents a slower rate of colony formation that remained quite distinct from the Treated line. This

suggested that a withdrawal from the original resistant clones had not conferred a high level of metastatic

potential.

Continuous PCB Exposure and the Degree of Genetic Instability Induced

Initially, we observed the process of clonal selection, where certain cells acquired resistance to

the highest concentrations of PCBs. Directly correlated to this selective growth advantage is genetic

19

instability which directly drives this growth even after so many passages. In some cancers, aneuploidy is

evident. However, it is not clear as to whether aneuploidy is a consequence of or is a causative factor of

cancer. Research has shown that numeric aberrations in chromosome count, aneuploidy, is usually found

in cancers. The induction of aneuploidy has been correlated with the mutation of several mitotic genes [24]

.

By witnessing an elevated degree of aneuploidy in the cells, we can conclude that the cell has gone

through a transformation, driven by a chromosomal mutation event [14]

.

To prove that the experimental groups became unstable due to PCB exposure, we assessed copy

number of two chromosome centromeres to measure their frequencies in varying signal patterns.

Interphase nuclei were analyzed using fluorescence in situ hybridization (FISH), to specifically look for

the centromere signal counts for chromosome 16 and 18. In previous karyotyping studies, these two

chromosomes had a normal copy number [12, 13]

. We found significantly higher aneuploidy counts for the

293T cells treated persistently with PCBs. In the LS74T cell line, the level of aneuploidy was barely

discernible from the aneuploidy rates for the other three cell lines. We conclude that the lack of

concordance between the aneuploidy rate for the LS174T cell line and the level of carcinogenesis is a

result of this cell already being carcinogenic.

Previous research has proven that the higher the aneuploidy count observed in a cell line, the

more genetically unstable is its karyotype [24]

. Thus, it can be assumed through the aneuploidy rates seen

in the PCB-treated 293T cells, that persistent exposure is not only conferring “genetic instability” onto the

cells, but also “mutability,” a characteristic that renders capable of changing more often.

What lies distinct in this study is that it addresses PCB exposure to resistant clones over the

course of several passages and analyzes for changes on the cellular level. The study investigated the types

of abnormalities that aggregated in the cells. In addition, we are analyzing the effects of PCBs during

primary exposure. By characterizing the fates of the cells in such a long-term study, the PCBs were given

a different role through which they conferred a selective growth advantage. With the combined data, two

distinct cell lines have been formed: one with consistent PCB exposure, and the other withdrawn from

PCB exposure. Despite the different treatment of these cell lines, they were derived from the original

resistant colonies. We hypothesized that withdrawal of PCB exposure would cause the cells to revert back

to their normal phenotype. Our analyses of later passages demonstrated that continuous exposure led to

the development of genetic instability and accelerated growth for the HEK-293T cells. The Pulse-Treated

cell line has shown a smaller degree of accelerated growth and aneuploidy rates than the Treated cell line.

This suggests that withdrawal of PCB exposure from original resistant colonies leads to a return to near

normal characteristics for these cells. Thus, the Parental and Pulse-Treatment data are very similar. This

connection adds to the importance of continual in vitro treatment of resistant cells. The cells developed a

phenotype of instability that permitted the 293T cell’s transformation from normal to cancerous.

20

The classical theory of carcinogenesis involves three steps which are: initiation, promotion, and

progression [28]

. The constant exposure of the chemical to the different cell lines served as the promotion

step, where the resistant cells were expanded and treated simultaneously. This presents us with the

possibility that the changes within the cell line were not entirely a genotoxic contribution, where the

chemical affects the genetic material directly. In previous studies, other pollutants such as arsenic have

been shown to induce carcinogenesis without a genotoxic contribution but rather with activation of the

stress gene expression factors [20]

. This opens up the possibility that PCB exposure has not only introduced

an abnormal chromosome copy number, but also DNA damage and epigenetic changes. It is possible that

chemical induced stress that has driven the cells to acquire alternations in gene expression permit

successful growth. In addition, we cannot limit genetic instability to aneuploidy, as there may be

deleterious mutations, deletions, chromosomal breakages, and amplification of particular genes that result

in the Treated cells ability to grow faster than normal cells.

For future studies we will look at the changes in gene expression levels in the cell lines by using

gene expression arrays. Our goal would be to identify differentially expressed genes among the cell lines.

We also propose looking for pathways which drive tumorigenesis in the PCB-treated cells. The ‘Parallel

Pathways to Tumorigenesis’ figure, displays a high variability among the pathways taken to complete

tumorigenesis. [3]

A comparison among the cell lines will give us a better understanding of specific genes

connected to PCB-induced effects. Since we already established genetic instability using a two

chromosome analyses, we will use karyotyping to map out the full extent of genetic instability. The

karyotype analysis will be done using the HEK-293T cells, because this cell line showed distinct copy

number counts for the Treated cells. In order to uncover the cause for such chromosomal instability, we

will be looking for what drives such cellular abnormalities.

Previous research has extensively covered the linkage between PCB exposure and tumor

formation in animal models. This broad range of research pertains to characterizing PCBs as a tumor

promoter. However this research project differs from previous studies tracking cancer progression. In

summary, our analyses provides a framework for PCB-induced carcinogenesis after long term exposure

not only as result of epigenetic changes but also as direct DNA reactivity [32]

. The time frame for this

study opens up the possibility for the PCBs to induce further cellular changes.

We have established that PCBs are a family of environmental carcinogens which in long-term

exposure induce resistant cells to express abnormalities that permit accelerated growth. For a cell line to

evolve with aneuploidy as a marker for its transformation there may be mutations involved. Our objective

in using RNA expression differences between the cell lines will define what mutations correlate to this

abnormality. From the first experiment displaying a process of clonogenic selection through the

chromosome count analysis, our research has shown that the experimental cells have acquired or induced

21

the expression of intrinsic mutations which allowed for the Treated cell lines to develop a phenotype of

carcinogenity. To further elucidate the reason for such abnormalities, research is needed to identify the

mutations that are related to this genetic instability and the formation of carcinogenic potential.

Acknowledgements

Many thanks to the Chan Lab for providing me with a lab bench to work in and the time and materials

needed to formulate my project and make it a reality. They assisted with all the questions I had regarding

the research process and helped with establishing the way to approach my project and plan the next

logical step based on the data. The Cytogenetics Core Facility has also assisted with the FISH procedure

analysis. They taught me how to interpret signals, compile the signaling data into charts and make the data

coherent. I am grateful for the guidance provided to me by my Research class teacher, in formulating

logical conclusions regarding my data. I would like to thank my research mentor for patiently answering

every question I came up with.. Today, I am set on continuing research in the future and using my

knowledge and experiences from the past two years to make myself a better researcher.

22

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