Screening for Apoptosis in Breast Cancer Cell
Lines Using Gel Electrophoresis
by
Nicole A. Larrier
Submitted to the Department of Nuclear Engineering
in partial fulfillment of the requirements for the degree of
Master of Science in Nuclear Engineering
at the
MASSACHUSETTS INSTITUTE OF TECHNOLOGY
©
August 1995
Massachusetts Institute of Technology 1995. All rights reserved.
Author .................
..............................
Department of Nuclear Engineering
August 10, 1995
Certified by ............................
,
.........
Kathryn D. Held
Associate Professor, Harvard Medical School
Associate Radiation Biologist, Massachusetts General Hospital
SThesis Supervisor
Read by ..................
.......
,....
Guido R. Solares
Assistant Professor of Radiation Oncology,
Tufts University School of Medicine
Thesis Reader
Read by
.........
.
.
......
.
.
..
.
.........- "
.......
Bruce R. Rosen
Visiting Associate Professor of Nuclear Engineering, MIT
Thesis Reader
Accepted by.......
ffJkrey P. Freidberg
Chairman. Departmental Committee on Graduate Students
,.ASSACHUSETTS INSTi'U i
OF TECHNOLOGY
APR 22 1996
'citence
Screening for Apoptosis in Breast Cancer Cell Lines Using
Gel Electrophoresis
by
Nicole A. Larrier
Submitted to the Department of Nuclear Engineering
on August 10, 1995, in partial fulfillment of the
requirements for the degree of
Master of Science in Nuclear Engineering
Abstract
Apoptosis in breast cancer cell lines has not been comprehensively investigated. This
study screens for radiation- and tamoxifen-induced apoptosis in five human breast
cancer cell lines: BT20, BT549, Hs578T, HTB26 and MCF7. Conventional and
Pulsed Field Gel Electrophoresis were used to analyse the DNA and look for any
fragmentation that had occured. The results showed no evidence of radiation- or
tamoxifen-induced apoptosis. However, the irradiated cells did show significant detatchment from the plates at 25 Gy. In addition, the MCF7 cell line showed a growth
delay in response to 10 pM tamoxifen.
Thesis Supervisor: Kathryn D. Held
Title: Associate Professor, Harvard Medical School
Associate Radiation Biologist, Massachusetts General Hospital
Acknowledgments
I wish to thank the Lord for seeing me through this work. May everything I do
contribute to the furthering of Your work.
Thank you Dr. Held for allowing me to work in your laboratory over the last
two years. The experience has been invaluable. Karen Hopcia, Yvonne McCarey
and Craig Sylvester also contributed greatly to my understanding of the subject and
helped immensely with the large volumes of tissue culture required to do this work.
MIT people who contributed to this endeavor (and maybe didn't even know it) are
Professor Jacquelyn C. Yanch and Dr. Guido Solares, who always took my ambitions
seriously and encouraged me; the folks in the UAA office, who taught me how to deal
with the MIT Firehose and come out a better person for it; and friends such as, Ali
Alavi, Emanuela Binello, John de Souza, Anne Kohnen, Jacquin Niles and Olapeju
Popoola, who called, emailed, visited and made this a far more pleasant experience
than most people have!
Finally, there are the wonderful people from Barbados - Mom and Deidre, Brian
Pollard, John Skinner and Marcus St. John. You guys are awesome.
Even youths shall faint and be weary,
and young men shall fall exhausted;
but they who wait for the Lord
shall renew their strength,
they shall mount up with wings like eagles,
they shall run and not be weary,
they shall walk and not faint.
Isiah 40:30-31
Contents
1 Introduction
1.1
1.2
2
9
Mechanisms of Apoptosis ..........
.. ... ...... ... .
10
.. ... ..... .... .
10
1.1.1
Apoptosis versus Necrosis
1.1.2
Apoptosis in Breast Cells......
.. .... ..... ....
11
1.1.3
Role of bcl-2 and p-53 in Apoptosis . . . . . . . . . . . . . . .
11
1.1.4
Radiation-Induced Apoptosis
. . . . . . . . . . . . . . . . . .
12
1.1.5
Tamoxifen-Induced Apoptosis . . . . . . . . . . . . . . . . . .
13
.....
Endpoints of Apoptosis ...........
.. .... ...... ...
15
Gel Electrophoresis
2.1
Conventional Electrophoresis
2.2
Pulsed Field Gel Electrophoresis . . . . . . . . . . . . . .......
2.3
Description of the CHEF-DR II System . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . .
3 Materials and Methods
22
3.1
M aterials
3.2
Tissue Culture
. . .. .
22
3.3
Treatment of Cells used in Conventional Gel Electrophoresis . . . . .
23
3.3.1
Screening of Cell Lines for Radiation-Induced Apoptosis
. . .
25
3.3.2
Screening of Cell Lines for Tamoxifen-Induced Apoptosis . . .
25
3.3.3
DNA Isolation ........................
.. .
25
3.3.4
Conventional Gel Electrophoresis
. . . . . . . . . . . . . . . .
26
3.4
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. .
. . . . . ..
. . . . . . . . . . . . . . . . ..
Treatment of Cells used in Pulsed Field Gel Electrophoresis
. . . . .
22
26
3.4.1
3.4.2
Pulsed Field Gel Electrophoresis .
Experiment 1: Establishing PFGE Run Conditions Using HL60
cells
3.4.3
...............
. . . . . . . . . . . . . . . . . . . . . . .
Experiment 2: Analysis of Radiation-Induced Apoptosis in
Breast Cancer Cell Lines . . . . . . . . . . . . . . .......
4 Results
4.1
4.2
4.3
29
Screening on Conventional Gels . . . . . . . . . . . . . . . . . . . . .
29
4.1.1
Effect of Irradiation ........................
29
4.1.2
Effect of Tamoxifen ...........
Screening on Pulsed Field Gels
..............
29
.....................
33
4.2.1
Setting Conditions Using HL60 cells . . . . . . . . . . . . . . .
33
4.2.2
Screening of Irradiated Breast Cancer Cell Lines . . . . . . . .
33
Cell Counts after Irradiation and Tamoxifen Treatment . . . . . . . .
33
5 Analysis and Conclusions
5.1
5.2
41
Improvements in the Screening Technique
. . . . . . .
. . . . . .
43
5.1.1
Radiation-Induced Apoptosis
. . . . . . . . . .
. . . . . .
43
5.1.2
Tamoxifen-Induced Apoptosis . . . . . . . . . .
. . . . . .
43
.. ....
43
Future Work .....
...............
A Schematic of the CHEF-DR II System
....
45
List of Figures
1-1
Proposed pathways that lead to Radiation and Tamoxifeninduced apoptosis [54, 40, 13, 33] .
2-1
14
.................
Diagrams of some PFGE systems. (a) The original PFGE system developed by Schwartz and Cantor. (b) Field Inversion Gel Electrophoresis (FIGE). (c) Rotating Gel Electrophoresis.
Homogeneous Electric Field Electrophoresis.
4-1
[7]
(d) Clamped
20
. ..........
Conventional gel electrophoresis of DNA extracted from BT549
cells. The cells grown on 100 mm plates showed very faint apoptotic
ladders. When the same number of cells (106) cells were grown on
150 mm plates, no ladders are observed. The horizontal axis is time
m easured in days ....................
4-2
..........
30
Conventional gel electrophoresis of DNA extracted from BT20
and Hs578T cells following irradiation with 25 Gy. The hori31
zontal axis is measured in days. .....................
4-3
Conventional gel electrophoresis of DNA extracted from MCF7,
HTB26 and BT549 cells following treatment with 10 yM Tamoxifen.
4-4
The horizontal axis is time measured in days.
. ......
32
HL60 cells following 25 Gy of irradiation. The top panel shows a
conventional agarose gel, where the horizontal axis is time measured in
hours. The bottom panels show pulsed field gels run for 22 hours (left)
and 32 hours (right). The samples were take at 6 hours after treatment. 34
4-5
PFGE of HTB26 cells following 25 Gy of irradiation. The
bottom panel shows conventional electrophoresis of the plug wash. The
horizontal axis is time measured in days. . ..............
4-6
.
PFGE of MCF7 cells following 25 Gy of irradiation.
35
The
bottom panel shows conventional electrophoresis of the plug wash. The
horizontal axis is time measured in days. . ..............
.
36
4-7 Normalised Cell Counts for the BT20 and BT549 cell lines.
The Normalised Cell Count is the number of cells counted on Day 0
divided by the cell count on a particular day. Squares = Untreated
cells. Circles = 25 Gy. Triangles = 10 pM Tamoxifen.
4-8
. ........
38
Normalised Cell Counts for the HTB26 and MCF7 cell lines.
The Normalised Cell Count is the number of cells counted on Day 0
divided by the cell count on a particular day. Squares = Untreated
cells. Circles = 25 Gy. Triangles = 10 pM Tamoxifen.
4-9
. ........
39
Normalised Cell Counts for the Hs578T cell line. The Normalised Cell Count is the number of cells counted on Day 0 divided by
the cell count on a particular day. Squares = Untreated cells. Circles
= 25 Gy. Triangles = 10 pM Tamoxifen. . ................
40
A-1 A schematic showing the set-up of Biorad's CHEF-DR II
Pulsed Field Gel Electrophoresis System
[6]. . ..........
45
List of Tables
3.1
Characteristics of Cell Lines ...................
3.2
Maintenance of Cell Lines ...................
....
.....
23
24
Chapter 1
Introduction
Studies indicate that one in nine women in the United States will develop breast
cancer [26]. Twenty-seven percent of all malignancies in women are breast cancers
[26]. The major treatments are surgery and radiation therapy. Adjuvant therapy in
the form of hormonal therapy, chemotherapy or radiation therapy is used to prevent
or delay the recurrence of the cancer [52]. Understanding the processes involved in
tumor cell kill by these agents will help improve the survival of patients.
Apoptosis is one of the two defined types of cell death. It was first defined in
1972 when observations distinguished it as a morphologically distinct form of cell
death [30]. Many tissue types, both normal and neoplastic, undergo apoptosis [69].
Normal development of vertebrates depends on apoptosis for the shaping of organs and
cavities [1]. In mammals apoptosis, coupled with mitosis, serves to keep a constant
number of intestinal crypt epithelial cells [63, 29, 1]. Apoptosis also occurs in liver
epithelium, prostate and the adrenal cortex. In pathological conditions, apoptosis
has been associated with toxin-induced liver disease, neurodegenerative disorders and
aplastic anemia [57]. Spontaneous apoptosis also occurs in most tumors [69, 29].
The agents that cause apoptosis are diverse. They include radiation(X-rays [41]
and UV [37]) in thymocytes and leukemia cells, hormonal alteration in the prostate,
dithiothreitol (DTT) in leukemia cells, ethanol in leukemia and various chemotheraputic drugs such as cisplatin [1]. The pathway that each of these agents uses to induce
apoptosis, may be different. For example, it is known that ionizing radiation damage
may induce apoptosis via a pathway that includes the p53 tumor suppressor gene,
while glucocorticoids induce a p53 independent pathway to apoptosis [13, 67]. With
breast cells, estrogen may be involved in the pathway to apoptosis. These multiple
pathways probably converge at some point downstream to give the changes that are
identified as apoptosis [21].
1.1
1.1.1
Mechanisms of Apoptosis
Apoptosis versus Necrosis
Apoptosis and necrosis are morphologically and biochemically distinct modes of cell
death. They are observed in both normal and pathological conditions. Necrosis
is characterised by swelling of the cell and rupturing of the cell membranes [69].
The cellular organelles disintegrate, and the DNA is randomly degraded.
This is
accompanied by inflammation of the surrounding tissue.
The morphological changes associated with apoptosis are shrinking of cell size,
condensation of the chromatin and the appearance of membrane bound "apoptotic
bodies" [69, 29].
The first change that is observed in apoptotic cells is the con-
densation of the chromatin and a decrease in the size of the nucleus and the cell.
Subsequently, nuclear and cytoplasmic materials are packaged in membrane-bound
sacs called apoptotic bodies which appear on the surface of the cell. The apoptotic
bodies vary in size and content. They may contain intact organelles, DNA fragments
or simply contain cytoplasm.
In vivo, the apoptotic bodies are phagocytosed by
surrounding normal tissue, engulfed by macrophages, or in the case of epithelial cells
disposed of by emptying them into the suface of the organ's lumen. Since the remains
of apoptosis are quickly disposed of, there is no inflammatory response as in the case
of necrosis. In cultured cells, the apoptotic bodies may detach from the plate and
float in the media.
One of the earliest biochemical events associated with apoptosis was the double
stranded internucleosomal cleavage of the DNA [2, 68]. This results in DNA fragments
that are multiples of 180 - 200 base pairs.
(This fragmentation is postulated to
be caused by a calcium dependent endonuclease.)
However, the absence of these
DNA fragments has been noted in some cells that exhibit morphological features of
apoptosis [14, 15]. It has been shown that the DNA is first cleaved to 50 or 300 kbp
fragments before proceeding to the smaller 200 bp fragments [9, 45]. Cohen used
zinc to show that in thymocytes the morphological changes associated with apoptosis
could occur in the absence of DNA cleavage to 200 bp fragments [14]. Subsequently,
they showed that even though there were no 200 bp fragments, there was DNA
fragmentation to large molecular weights (30-50 kbp and 200-250 kbp) [9]. Thus, it
is inferred that morphological changes precede cleavage to 50 or 300 kbp fragments,
and subsequently cleavage to 200 bp pieces occurs.
1.1.2
Apoptosis in Breast Cells
Like many tissues, breast tissue is susceptible to apoptosis in both normal and pathological situations [3]. Normal breast tissue undergoes apoptosis in a cyclic fashion
that patterns the menstrual cycle [51]. In addition, the involution of the lactating
breast after weaning is caused by apoptosis [63, 62]. One of the drugs used to treat
estrogen receptor positive breast cancer, has been shown to induce apoptosis in breast
cancer cell lines [4]. In these experiments, the drug tamoxifen caused estrogen receptor positive MCF7 cells to undergo apoptosis while the estrogen receptor negative
HTB26 and BT20 cells did not. This suggests that it might be possible to exploit
this property of chemically (or radiation) induced apoptosis in the treatment of breast
cancer.
1.1.3
Role of bcl-2 and p-53 in Apoptosis
Since the number of cells in a tissue at any time is dependent on the balance between
cell proliferation and cell death, it is reasonable to suggest that one of the relevant
modes of cell death is apoptosis [21, 49]. This means that apoptosis is likely to be
affected by the presence or absence of oncogenes and tumor suppressor genes in the
cell [46]. An oncogene is a gene that produces net cell growth when it is mutated.
Tumor suppressors act as negative regulators of cell proliferation. When they are
mutated a cell will proliferate rapidly, while an unmutated gene keeps cell growth in
check.
The bcl-2 oncogene is intimately involved in the control of apoptosis [54, 11, 40].
The 26 kDa bcl-2 protein is located on the inner and outer membrane of mitochondria
and on the nuclear membrane. Expression of the bcl-2 product does not promote cell
proliferation, but instead enhances cell survival by preventing apoptosis [46]. Bcl-2
expression is present in both normal and cancerous tissue, and it is generally found in
tissues which contain cells undergoing apoptosis [46]. In particular, bcl-2 expression
has been observed in both normal breast tissue and in a breast cancer cell line [65].
It has also been noted that within the same cell line there may exist pathways to
apoptotsis that depend on and are independent of bcl-2 expression. In addition, the
overexpression of bcl-2 does not prevent apoptosis in response to hydrogen peroxide
[40]. Thus the necessity of bcl-2 to the induction of apoptosis is not clear.
Alternately, the overexpression of the p53 tumor suppressor gene can lead to cell
death by apoptosis. The p53 gene is frequently mutated in human cancers including
lung, colorectal, brain and breast [25, 64].
It is located on chromosome 17p and
produces a 53 kDa nuclear phosphoprotein that can bind to DNA [35]. It is present
at very low levels in normal cells. Its role in the apoptotic pathway is unclear. It
seems likely that it is involved in some, but not all, apoptotic pathways [13].
In
cases where p53 levels are influential, high levels are associated with apoptosis [47].
When thymocytes from p53 knock-out mice are subjected to radiation, the cells do
not undergo apoptosis [38]. However, thymocytes from mice with normal p53 genes
readily apoptose.
1.1.4
Radiation-Induced Apoptosis
Apoptosis can be induced in tissues and cell lines in response to ionizing radiation
[42, 66, 59]. In tumor cells, radiation-induced apoptosis is seen in murine mammary
and ovarian adenocarcinomas, sarcomas and squamous cell carcinomas subjected to
2.5 to 25 Gy of y-irradiation [39]. The HL60 human leukemia cell line also readily undergoes radiation-induced apoptosis 3-5 hours after treatment although doses
greater than 10 Gy were needed [37, 34]. It has also been observed that a radiosensitive murine ovarian carcinoma is susceptible to radiation-induced apoptosis, while
a radioresistant hepatocellular carcinoma is not [58]. This suggests that the tumors
natural radioresistance may affect its ability to apoptose.
The p53 status of a cell may affect its sensitivity to radiation [33, 32]. This is
believed to be a result of p53 levels being elevated in response to the DNA damage
resulting from the radiation [33].
The presence of mutant p53 is associated with
an increased resistance to i-irradiation. Lee and Bernstein showed that mouse p53
mutations increased the resistance of bone marrow and spleen cells to i-radiation
[33]. The effect of p53 on the cell cycle may also affect apoptotic pathways. It is
suggested that wild-type p53 acts a cell cycle checkpoint. This can lead to DNA
repair or possibly to apoptosis (see Figure 1-1). This would suggest that cells that
are going to apoptose would not undergo any cell divisions. However, this is not
always observed. Cell death has been observed shortly after radiation and after one
or more cell divisions [50].
1.1.5
Tamoxifen-Induced Apoptosis
Tamoxifen is a non-steroidal antiestrogen that is often used in the treatment of estrogen receptor positive breast cancer [27]. One of its main features is that it is a
competitive inhibitor of estrogen. There are some data that suggest that high exposure to estrogen increases susceptibility to breast cancer [44] and that estrogen
ablation induces apoptosis in the MCF7 breast cancer cell line [31]. There maybe an
association between the presence of estrogen receptors and bcl-2 protein levels [65].
In addition, the level of bcl-2 in beast tissue changes during the menstrual cycle [51].
Therefore it is possible that binding of tamoxifen to estrogen receptors leads to lower
levels of bcl-2 and hence, to apoptosis (see Figure 1-1). The presence of the wildtype
p53 gene is also associated with estrogen receptor positive cancers [10]. However, how
(or if) this correlates with apoptosis is not known.
Ionizing Radiation
Tamoxifen
Estro gen Receptors
I
Radiation Damage
bc 1-2
p53 k
-
G1 Cell Cycl e Arrest
DNA Repair
Cell Division
and Proliferation
APOPTOSIS
Morphologic Changes
50 & 300 kbp fragments
Internucleosomal cleavage
(multiples of 200 bp)
Figure 1-1: Proposed pathways that lead to Radiation and Tamoxifeninduced apoptosis [54, 40, 13, 33].
1.2
Endpoints of Apoptosis
Given that the apoptotic pathway involves many steps, there are several places that
can be used to define a cell that has undergone apoptosis. On a philosophical level
the pertinent question is, is there a lowest common denominator in the converging
pathways to apoptosis that can be used to identify a cell's commitment to apoptose.
From a practical standpoint, criteria for defining and identifying apoptosis depend on
the development of assays that can detect changes that occur during the process.
The earliest definition of apoptosis was based on morphological observations [63].
This method continues to be very popular as an end point. It is considered to be a
reliable "lowest common denominator". The characteristic changes such as cell chromatin condensation, cell blebbing and decreased cell volume can be seen using electron
or light microscopy. The percent of apoptosis in a population can be estimated by
counting the fraction of apoptotic cells in a sample. However, since this is only at
one time point, the total number of cells undergoing apoptosis cannot be counted. In
addition, counting the apoptotic bodies in a sample is a tedious, somewhat subjective
procedure.
The DNA fragmentation that occurs in apoptosis is another useful endpoint. The
DNA fragments can be separated and visualised by gel electrophoresis. The 200 bp
fragments and multiples thereof appear as a characteristic ladder pattern on conventional agarose gels. (Necrosis is shown as a smear due to the random degredation of
the DNA.) The larger 50 or 300 kbp pieces can be seen as bands on pulsed field gels
[45]. DNA end labelling techniques may also be used to analyse the DNA fragmentation quantitatively.
In tissue culture, the detatchment of apoptotic cells from the plate has been used as
an assay for apoptosis [36]. This method is based on the correlation between apoptotic
ladders being found in the floating cells and intact DNA being observed in the cells
that are still attatched. However, to be a satisfactory technique, this correlation
must be performed on every cell line under consideration. In addition, no attempt
has been made to identify large molecular weight DNA fragments. The presence of
these fragments in the floating or attached cells could change the conclusions. There
is also the possibility that necrotic cells are detatching and floating with the apoptotic
cells.
In this study, apoptosis is screened for by using electrophoresis techniques. The
presence of apoptosis should yield "ladders" and/or large molecular weight bands on
agarose gels. Identifying apoptosis by these methods will be an efficient, conclusive
indicator of its occurrence.
Chapter 2
Gel Electrophoresis
Electrophoresis refers to the separation of molecules, such as DNA or protein, by the
application of an electric field across the sample under investigation. In the system
used for these studies, the DNA samples are run through an agarose matrix. The
negatively charged DNA molecules migrate towards the positive electrode, called the
anode. The mobility and resolution of the DNA depends on many factors including
the characteristics of the agarose gel, the type of voltage gradient applied across the
gel, the temperature, the size of DNA to be separated and the running buffer.
In this analysis of DNA, two electrophoretic techniques are utilized: conventional
electrophoresis and pulsed field gel electrophoresis. Conventional electrophoresis is
used to separate the low molecular weight DNA ladders, and pulsed field gel electrophoresis is used for separation of the larger (>50 kb) molecular weight DNA [42].
The main difference between the two methods is the applied electric field. In conventional electrophoresis the electric field is static, that is, the voltage is fixed in one
direction. However, in pulsed field electrophoresis the electric field changes direction
at prescribed times that depend on the size of DNA to be separated.
2.1
Conventional Electrophoresis
In conventional electrophoresis, DNA molecules move in a straight line from the
cathode towards the anode. The agarose gel is made by pouring an agarose solution
into a mold and allowing it harden. As it solidifies, a series of interconnected channels
form. The density of the pores (and hence, how the DNA moves through the gel)
depends on the concentration of agarose used. (The empirically derived mathematical
relationship that describes this is: logpi = logplo - K7T; where p is the mobility of the
DNA and 7 is the agarose concentration [55] .) The DNA is loaded onto the gel, and
the current turned on.
The DNA moves through the gel by a process called reptation. The reptation
model was proposed by de Gennes in 1971 [18]. It suggests that a molecule moves
through the matrix in a head-on fashion. That is, the DNA can slither back and forth
in the channels formed by the agarose. Since the electric field is static, the molecules
only move in one direction (towards the anode). It is easier for small molecules to
move through the channels, and therefore they move the greatest distance on the gel.
Linear DNA migrates through the gel matrix at a rate inversely proportional to the
log of its molecular weight. Practically, this empirical relationship means that all
DNA fragments greater than about 50 kb move about the same distance in a gel and
cannot be resolved.
2.2
Pulsed Field Gel Electrophoresis
Pulsed field gel electrophoresis (PFGE) is used to separate the DNA that is larger
than 50 kb and up to megabase lengths. This large DNA encounters three problems
when moving through gel matrices. These are formation of kinks, chain pinning and
formation of knots [19].
Since the agarose channels are not exactly straight lines
between the top and bottom of the gel, parts of long DNA, in effect, become trapped
in different channels, and thus the forward motion of the DNA is hindered. These
formations are known as kinks. Chain pinning refers to the situation where a DNA
molecule becomes hooked over one of the fibers in the gel. Since the DNA molecule
is large, it takes a lot of energy to get it moving again. It is possible for DNA to
become knotted with itself or with other molecules. Longer molecules are more likely
to have intra- and inter-chain knots. Again, these knots can hinder the passage of
the molecules through the agarose matrix.
Pulsed field electrophoresis solves some of these problems by varying the direction
of the electric field and giving the DNA time to relax to an equilibrium state, before
moving again under the influence of an electric field again [53]. The time required
for the DNA to relax depends on the length of the molecule, with larger molecules
taking longer to relax. Usually, an electric field is applied in one direction and then
the field is switched to another direction. During electric field switching, the DNA
uses the first part of the time in that interval to relax, before beginning to move in the
new direction. This means that larger moleules spend less time moving than smaller
molecules. As the time the DNA spends under each electric field increases, the larger
molecules are more effectively separated. When running these gels, it is common to
continually increase this switch time so that a range of molecules will be separated.
If very large molecules are to be resolved, the electric field can be turned off between
the switch in the direction of the electric field.
Since its introduction, many variations of PFGE have emerged. The original system developed by Schwartz and Cantor consisted of subjecting an agarose gel to
one homogenous and one non-homogenous electic field (Figure 2-1) [53, 7]. Now,
variations include changing the angle between the two electric fields, using only homogenous or non-homogenous fields, and periodically rotating the gel while keeping
the electric field stationary (Figure 2-1). The most commonly used systems are field
inversion gel electrophoresis (FIGE) and clamped homogeneous electric field electrophoresis (CHEF). In FIGE only one electric field is switched, but the anode and
cathode are interchanged periodically. The ratio of the time spent in each direction
determines the ultimate movement of the DNA. The CHEF system contains twentyfour electrodes arranged in an hexagon. The two homogenous electric fields operate
in the northwest to southeast and the northeast to southwest directions relative to
the gel.
A-
B-
(a)PFGE
(b) FIGE
-V
(c) Rotating
Gel
(d) CHEF
Figure 2-1: Diagrams of some PFGE systems. (a) The original PFGE system
developed by Schwartz and Cantor. (b) Field Inversion Gel Electrophoresis (FIGE).
(c) Rotating Gel Electrophoresis. (d) Clamped Homogeneous Electric Field Elec-
trophoresis. [7]
2.3
Description of the CHEF-DR II System
The commercially available CHEF-DR II System (Biorad) was used for PFGE. This
system creates highly uniform electric fields which result in clearer gels. It consists of
six components. They are a pump, the electrophoresis chamber, a cooling apparatus,
the Drive Module, the Pulsewave 760 switcher and the Model 200/2.0 Constant Voltage Power Supply. The electrophoresis chamber has platinum wire electrodes fixed
at 1200. The gel is placed in the middle of the chamber and held in place by two
gel stops. The running buffer is pumped through the chamber in the direction that
the DNA moves, into the chiller and then back into the chamber. The power supply
applies a constant voltage across opposing electrodes, and the switcher is used to
determine how quickly the voltage switches between the electrodes and the length of
the run. (See Figure A-1.)
Chapter 3
Materials and Methods
3.1
Materials
The human breast cancer cell lines BT20, BT549, HTB26, Hs578T and MCF7 were
obtained from the American Tissue Culture Collection (ATCC). The HL60 cell line,
a human myeloid leukemia cell line, was also obtained from the ATCC. The breast
cancer cell lines grew attached to the petri plates, and the HL60 cells grew in suspension. The estrogen receptor and p53 gene status of the cell lines were obtained from
the ATCC and a review of current literature. A summary of this information is given
in Table 3.1.
3.2
Tissue Culture
Cells were grown in media as given in Table 3.2. The cells were kept in exponential
growth by passaging twice weekly. The cells were trypsinized and plated at a low
enough concentration to ensure that they were in exponential growth and did not
reach plateau phase. In addition, cell counts were done to determine the doubling
time of each line. With the exception of the HTB26 cell line, the cells were maintained
in an incubator at 37°C with 5% CO 2 . The HTB26 cells were grown at 37 0 C with no
()CO2
Table 3.1: Characteristics of Cell Lines
Estrogen receptor information and the status of the p53 and bcl-2 genes were obtained
from the ATCC catalogue and a review of the current literature.
II
I bcl-2 Status jReferences
Cell Line I Estrogen Receptors Ip53 Status
[17]
normal
null
yes
HL60
[16, 5]
point mutation unknown
BT20
no
[5]
point mutation unknown
no
BT549
unknown
ATCC, [20, 23]
mutant
no
Hs578T
[43, 56, 5]
HTB26
no
point mutation unknown
MCF7
yes
normal
normal
[3, 48, 23, 61]
3.3
Treatment of Cells used in Conventional Gel
Electrophoresis
The breast cancer cell lines were treated with either X-rays or the drug Tamoxifen
Citrate, and the DNA analysed on an agarose gel. Since these gels only run for three
to six hours, the presence of a DNA ladder is a quick indication of death by apoptosis.
The cells were treated when they were in exponential growth, and daily samples
were taken for six to eight days. The possibility of apoptotic bodies in the media
that did not form part of the pellet during centrifugation was examined in previous
experiments. In those experiments, the media was collected after centrifugation, and
any DNA present was isolated. In the cases where there were no DNA ladders present
in the DNA from the cell pellet, there was no evidence of ladders in the media. In the
one case where there was a DNA ladder from the cell pellet, there was a faint ladder
in the DNA isolated from the media. Thus it was inferred that analysing the media
was not a necessary procedure.
Table 3.2: Maintenance of Cell Lines
Doubling time (DT) information was determined for each cell line from regular maintenance of the cells.
Fetal calf serum, bovine insulin, sodium pyruvate, non-essential amino acids and
L-glutamine were obtained from Sigma Chemical Company, St.Louis, MO. PenicillinStreptomycin (100 U/ml penicillin, 100 ig/ml streptomycin) was obtained from
Gibco/BRL, Grand Island, NY.
Cell Line Growth Medium
D
HL60
BT20
24 hours
2 days
BT549
RPMI 1640; 15% FCS; 10 mM Hepes; P/S
MEM; 20% FCS; 10 mM Hepes;
1 mM Non-essential Amino Acids; P/S
RPMI 1640; 20% FCS; 10 mM Hepes;
1
1.5 days
P/S
Hs578T
HTB26
DMEM; 20% FCS; 10 mM Hepes; 10 pg/ml insulin; P/S
Leiboweitz-15; 20% FCS; 10 mM Hepes; L-glutamine;
2.5 days
2 days
P/S
MCF7
MEM; 20% FCS; 10 mM Hepes;
1 mM Non-essential Amino Acids;
1 mM Sodium Pyruvate; 10 Mg/ml insulin; P/S
2 days
3.3.1
Screening of Cell Lines for Radiation-Induced Apoptosis
One to two million cells were plated onto 100 or 150 mm plates and grown in standard
media conditions. The cells were treated the next day with 25 Gy (1.95 Gy/min) Xrays in air, using a Siemens Stabilipan 2 X-ray generator operated at 250 kVp, 12
mA, effective HVL 0.5 mm Cu. Daily counts were done using a hemocytometer and
confluency conditions noted. (It has been reported that confluent conditions may
induce apoptosis [29].) The cells were then collected, and the DNA examined for any
evidence of apoptotic ladders on an agarose gel.
3.3.2
Screening of Cell Lines for Tamoxifen-Induced Apoptosis
The cells were plated as in radiation experiments and allowed to attach overnight.
Tamoxifen Citrate (Sigma Chemical Company, St. Louis, MO) in EDTA was then
added to the media to give a final concentration of 10 pM and left on the cells for the
duration of the experiment. The cells were observed and counted daily. DNA was
isolated from the cells at the desired time points and visualized on an agarose gel.
3.3.3
DNA Isolation
The cells were detached from the plate by trypsin or scraping, and the cells and media
spun at 1000 - 1500 rpm for five minutes. The supernatent was discarded and the
cell pellet resuspended in 500 pl of 1X lysis buffer (10 mM Tris; 1 mM EDTA; 0.2%
TritonX -100; pH to 7.5). The cells and lysis buffer were then incubated at 37 0 C with
50 pg/ml of Ribonuclease A for one hour. Subsequently, 50 pg/ml of Proteinase K was
added, and the suspension incubated for another hour. The DNA was precipitated
by adding 500 pl of 100% isopropanol and 100 pl of 5 M NaC1, and leaving samples
in the -200 C freezer overnight. Afterwards, the mixture was spun at 13 000g for 10
minutes at 40 C, the supernatent discarded and the pellet allowed to air dry. The
DNA pellet was resuspended in 1X TE buffer (10 mM Tris: pH 7.4; 1 mM EDTA:
pH 8.0) to a concentration of 106 cells/100 pl.
3.3.4
Conventional Gel Electrophoresis
To observe the DNA, the samples were run on 1% agarose gels with a running buffer
of 1X TBE (0.0445 M Tris; 0.0445 M Boric Acid; 0.01 M EDTA, pH 8.0). A 1 kb
(Gibco/BRL) DNA ladder was used to estimate the sizes of the DNA in the samples.
Gel loading buffer was added to the DNA, and the samples heated to 650 C for ten
minutes. The samples were then loaded into the gel and run at either 130V for 34 hours (15 cm X 25 cm gel) or 160V for 3.5-6 hours (20 cm X 40 cm gel). Gels
were stained for 30 minutes in ethidium bromide and destained for 12-18 hours in
water. The DNA was observed by ultraviolet light, and photographs were taken with
a Polaroid MP-4 Land Camera with 667 Poloroid film under UV illumination.
3.4
Treatment of Cells used in Pulsed Field Gel
Electrophoresis
The cells were treated with X-rays and collected in a manner similar to that used
for conventional electrophoresis. Once a pellet of cells was obtained, the pellet was
resuspended in 1% low melt agarose (Biorad) in 1X lysis buffer. The agarose mixture
was then cast into the plug molds and placed at 4oC for 30 minutes to harden. Each
plug holds about 200 pl and can be used for two gels. The agarose solution was added
to the pellet so that 106 cells would be loaded onto each gel. The plugs were placed
in 1 ml of 1X lysis buffer with Proteinase K (50 pg/ml) and incubated at 37°C for 18
- 24 hours. At the end of this incubation period the agarose plugs were rinsed twice
in 1 ml of TE for 15 minutes. The plugs were stored in 1 ml of 1X TE until they were
needed for electrophoresis. In some instances, the plug wash was kept so that it could
be analysed by conventional electrophoresis. To isolate the DNA, the plug wash was
placed in the freezer overnight with 500 pl of 100% isopropanol and 100 p1 5M NaC1.
The isolated DNA was run on a conventional gel at the conditions described above.
In addition, in certain experiments the agarose plugs were also run on conventional
gels.
3.4.1
Pulsed Field Gel Electrophoresis
The agarose plugs were run on a 1% agarose gel (Pulsed Field Certified Agarose from
Biorad) made with 0.5X TBE and a running buffer of 0.5X TBE. The plugs were cut
to fit the size of the wells on the gel using a clean, sharp spatula. They were then
placed in the well, being careful to make sure that the plug made contact with the
front of the well and that all bubbles were eliminated from the wells. The wells were
then sealed along the back, the sides and the top with 0.8% low melt agarose. The
agarose hardened for half an hour at room temperature or 10 minutes in the cold
room. In addition, MegaBaseII (Gibco/BRL) and 1 kb (Gibco/BRL) DNA ladders
were loaded to determine the approximate size of the DNA fragments. (The ladders
were suspended in 0.8% low melt agarose plugs and loaded in the same manner as
the samples.)
The CHEF-DR II Electrophoresis System was filled with 2 liters of the 0.5X TBE,
the running buffer. The pump was turned on and the circulating buffer cooled to 140 C
using a Model 1000 Mini-Chiller, and then the buffer was further circulated at 140 C
for half an hour. The gel was then placed in the gel box, and the flow of the buffer
was adjusted so that it did not float away or trap bubbles under the gel. The running
buffer was drained out of the system until it was 3-4 mm above the level of the gel.
The run conditions were set with a Pulsewave 760 switcher and a Model 200/2.0
Constant Voltage Power Supply. When the gel was run for a long time, the running
buffer was changed every 48 hours. At the end of the run, the DNA was stained with
ethidium bromide, visualized with ultraviolet light and photographed.
3.4.2
Experiment 1: Establishing PFGE Run Conditions
Using HL60 cells
The HL60 cell line is known to undergo radiation-induced apoptosis starting at about
3 hours after irradiation. The cells are treated according to the method described
above, and the DNA separated by conventional and pulsed field gels. The conditions
that show large (50kb) fragments are 100V, 60s - 60s switching, for 0.3 hr, followed by
200V, ls-60s, for 22 hr. At this point the gel was stained, destained and photographed.
The gel was then run for another 19 hours at 200V, 50s-80s.
3.4.3
Experiment 2: Analysis of Radiation-Induced Apoptosis
in Breast Cancer Cell Lines
The five breast cancer cell lines were treated with 25 Gy as described above. For each
cell line daily samples were collected and made into plugs. The agarose plugs were
analysed by PFGE, and the plug wash was run on a conventional agarose gel. The
pulsed field run conditions were similar to those used with HL60 samples.
Chapter 4
Results
4.1
4.1.1
Screening on Conventional Gels
Effect of Irradiation
None of the five breast cancer cell lines showed any DNA fragmentation patterns that
would indicate apoptosis in response to radiation. The BT549 cell line showed very
faint DNA ladders in both the control and radiation experiments. However, this was
likely a result of the large number of cells present. (The plates were 90% confluent
by day 2 of the experiment.) When the experiment was repeated with larger plates
(150 mm), there was no ladder except in the case of day 7 on the untreated samples.
At early time points, all cell lines showed large molecular weight DNA. This DNA
was larger than the largest size marker (12 kb) (Figure 4-1). In many cases, there
was a large fraction of DNA in the area between the wells on the gel and the largest
marker (Figure 4-2). As the time increased, the smears of low molecular weight DNA
became more prominent. This type of smearing is indicative of necrosis.
4.1.2
Effect of Tamoxifen
No tamoxifen-induced apoptosis was detected by the formation of apoptotic DNA
ladders. The DNA smears obtained on the gels were similar to those obtained from
the radiation experiments (Figure 4-3).
BT549 (grown on 100 mm plates)
Tamoxifen
Untreated
01 2 3 456 7 8
0123 45
Radiation
MW
1 234 56 7
b(p)
2216
072
054
036
636
018
06,517
BT549 (grown on 150 mm plates)
Untreated
01 2 3 4 56
Radiation
0 1 23 4 56 7
MW
(bp)
12216
4072
3054
2036
1 636
1 018
506,517
Figure 4-1: Conventional gel electrophoresis of DNA extracted from BT549
cells. The cells grown on 100 mm plates showed very faint apoptotic ladders. When
the same number of cells (106) cells were grown on 150 mm plates, no ladders are
observed. The horizontal axis is time measured in days.
BT20
Tamoxifen
Control
1 3456 7
012 •4
Radiation
5
MW
(bp)
1 234567
12216
4072
3 054
2036
1 636
1 018
Hs578T
Control
0123
Radiation
4
0
1 2 34 5
MW
(bp)
2216
072
054
036
636
Figure 4-2: Conventional gel electrophoresis of DNA extracted from BT20
and Hs578T cells following irradiation with 25 Gy. The horizontal axis is
measured in days.
MCF7
Control
0 1 23 4 5 6
Tamoxifen
0
2
4 5 6
MW
(bp)
12216
S072
3054
2036
1636
HTB26
BT549
Tamoxifen
S1
Tamoxifen
[w
"34
A S 6
bp)
2216
072
054
036
636
018
~r_____~_
A~
r(-____-IL~-l-l
-- 1
-1--I-
r igure 4-3: .onvenuional get electropnoreis or u"tIN
j
extracteu from IMvIr t,
HTB26 and BT549 cells following treatment with 10 pM Tamoxifen. The
horizontal axis is time measured in days.
4.2
Screening on Pulsed Field Gels
4.2.1
Setting Conditions Using HL60 cells
Treating HL60 cells with 25 Gy of radiation produced the expected result of apoptotic
ladders on conventional electrophoresis gels. When this experiment was ananlysed on
pulsed field gels, two molecular weight DNA bands were observed (see Figure 4-4).
The run conditions were set according to the movement of the MegaBaseII and 1 kb
DNA ladders..
4.2.2
Screening of Irradiated Breast Cancer Cell Lines
Observing the DNA of the breast cancer cells on pulsed field gels generally did not
distinguish any significant bands of large molecular weight DNA. The smears of DNA
observed in conventional electrophoresis were also observed on pulsed field gels during
the first half of the run (200V; 1-60s; 22 hours).
These smears were obtained on
controls, as well as on the treated samples (see Figure 4-5). During the second half
of the run, no significant DNA staining occurred with the ethidium bromide. The
exception was with the MCF7 control experiment which showed a band at 291-485
kb, which decreased as time increased (see Figure 4-6). Otherwise, there were faint
DNA smears and no particular DNA bands could be identified.
4.3
Cell Counts after Irradiation and Tamoxifen
Treatment
The irradiated cells, generally decreased in number after treatment (Figures 4-7, 48, 4-9). The decreased cell count was accompanied by increasing numbers of cells
floating in the media. This was observed in all cell lines after day 3 and was particluarly noticible in BT549 cells which detatched in small clumps.
Cell lines that were treated with tamoxifen varied in their cell counts. The BT20,
BT549, Hs578t and HTB26 cell lines grew at a comparable rate to untereated controls.
HL60
Conventional Gel
0123456
M Ell1
bp)
1216
054
036
636
018
)6,517
Pulsed Field Gel
22 hours
MW
(Kbp)
0 25 Gv
-J
32 hours
XMWlI
0 25 Gv
(Kbp)
291485
48.5
48.5
Figure 4-4: HL60 cells following 25 Gy of irradiation. The top panel shows a
conventional agarose gel, where the horizontal axis is time measured in hours. The
bottom panels show pulsed field gels run for 22 hours (left) and 32 hours (right). The
samples were take at 6 hours after treatment.
Pulsed Field Gels
HTB26 - Untreated
HTB26- 25 G y
0 1
0123456
34
6 7
MW
(khn)
48.5
Conventional Gels of Plug Wash
HTB26 - Untreated
01234567
HTB26- 25 Gy
1234567
Figure 4-5: PFGE of HTB26 cells following 25 Gy of irradiation. The bottom
panel shows conventional electrophoresis of the plug wash. The horizontal axis is time
measured in days.
Pulsed Field Gels
MCF7 -Untreated
MW
MCF7 - 25Gy
012345
01234567
6 7
(kbp)
194.0
145.5
48. 5
Conventional Gel of Plug Wash
MCF7 - Untreated
01234567
MCF7 - 25 Gy
1234567
Figure 4-6: PFGE of MCF7 cells following 25 Gy of irradiation. The bottom
panel shows conventional electrophoresis of the plug wash. The horizontal axis is time
measured in days.
The tamoxifen treated MCF7 cells grew at a slower rate than the control cells ( 4-8.
Graph of Normalised Cell Count vs. Time for BT20 Cell Line
100.0
10.0
1.0
0.1
0
4
Time/Days
2
6
Graph of Cell Count vs. Time for BT549 Cell Line
100.00
10.00
1.00
0.10
0.01
0
2
4
Time/Days
Figure 4-7: Normalised Cell Counts for the BT20 and BT549 cell lines. The
Normalised Cell Count is the number of cells counted on Day 0 divided by the cell
count on a particular day. Squares = Untreated cells. Circles = 25 Gy. Triangles =
10 IiM Tamoxifen.
Graph of Normalised Cell Count vs. Time for HTB26 Cell Line
10.00
5.00
2.00
1.00
0.50
0.20
0.10
0.05
0
2
4
Time/Days
6
Graph of Normalised Cell Count vs. Time for MCF7 Cell Line
50.0
20.0
010.0
5.0
c 2.0
E 1.0
zo 0.5
0.2
0
2
4
Time/Days
Figure 4-8: Normalised Cell Counts for the HTB26 and MCF7 cell lines.
The Normalised Cell Count is the number of cells counted on Day 0 divided by the
cell count on a particular day. Squares = Untreated cells. Circles = 25 Gy. Triangles
= 10 pM Tamoxifen.
Graph of Normalised Cell Count vs. Time for Hs578T Cell Line
10.0
5.0
2.0
1.0
0.5
0.2
0.1
0
2
4
Time/Days
Figure 4-9: Normalised Cell Counts for the Hs578T cell line. The Normalised
Cell Count is the number of cells counted on Day 0 divided by the cell count on a
particular day. Squares = Untreated cells. Circles = 25 Gy. Triangles = 10 pM
Tamoxifen.
Chapter 5
Analysis and Conclusions
Screening the five breast cancer cell lines showed that they did not produce radiationor tamoxifen-induced apoptosis as revealed by DNA ladders on conventional electrophoresis gels or large molecular weight bands on pulsed field gels. The DNA that
was observed on both electrophoresis and conventional gels is indicitive of intact chromosomal DNA and necrosis. However, the ability of at least one of the cell lines to
apoptose was demonstrated when it appeared that highly confluent conditions induced a faint DNA ladder in BT549 cells. This appeared on day 5, and the pertri
plates had been 100% confluent since day 3.
The pulsed field gels were equally elusive in producing evidence of apoptosis.
Both the control and radiation experiments produced DNA around the 50 kb mark.
However, this appeared to be the top of the smears that show up as necrosis on
the conventional gels, as opposed to being distinct bands of DNA. In the MCF7
cell line it is clear that on the controls the intensity of the bands decreases as the
time increases. This corresponds with the analysis that the cells are becoming more
necrotic and the DNA being degraded as time progresses. In the radiation experiment,
the high molecular weight bands decrease after day 2, and definite smears are visible
on later days. This correlates with the observation that the cells are detatching (and
presumably dying) on day 3 and later. Similar results were obtained with the other
cell lines.
The lack of DNA fragmentation in response to radiation is interesting.
Four
out of the five breast cancer cell lines have p53 mutations. This might suggest an
explanation for their resistance to a p53 pathway to apoptosis. However, the MCF7
cell line has two copies of wildtype p53 genes and still does not undergo apoptosis.
This is consistent with suggestions that MCF7 cells and more generally solid tumor
cell line are resistant to p53-mediated radiation-induced apoptosis [22, 8]. In addition,
the possible involvement of the bcl-2 gene in the apoptotic pathway in breast cell lines
is unknown. The expression of the p53 and bcl-2 protein products in breast cell lines
was studied by Haldar et al. [24]. This work showed that in the BT20 and Hs578T
lines there were high levels of p53 and no bcl-2 present; in the MCF7 cell line, bcl-2
was present, but very little p53; and in the HTB26 line comparable levels of both
proteins were present. However, the status of bcl-2 gene mutations in the cell lines
was not reported. If wildtype bcl-2 is present in these cell lines, its expression after
radiation may serve as an inhibitor of apoptosis.
Any effects of tamoxifen on the breast cancer cell lines, if present, are more subtle
than with the radiation treatment. While the irradiated cells detatched from the
plates after a couple of days, the tamoxifen-treated cells continued to divide. Only in
the MCF7 cell line was there a slight growth delay. In both situations, the tamoxifen
did not appear to be contributing to cell death. These results do not agree with other
work which shows tamoxifen-induced apoptosis in MCF7 cells [4] and tamoxifeninduced growth delays in MCF7 and BT20 cells [12, 60]. In the study that found
apoptosis in MCF7 cells, the lack of apopotsis in BT20 and HTB26 cells was also
demonstrated [4]. The MCF7 cell line is the only one that has estrogen receptors and
the BT20 line has high affinity antiestrogen receptors [12]. This might explain the
ability of these cell lines to experience growth delays.
5.1
5.1.1
Improvements in the Screening Technique
Radiation-Induced Apoptosis
Since the irradiated cells showed a propensity to detatch from the plates, further
study of them versus the attatched cells would be useful. Isolating the media and
detatched cells and subjecting them to conventinal and pulsed field electrophoresis,
will help determine if it is purely necrotic cells that are detatching. In addition, if
there is any apoptosis that is being masked by the more dominant levels of necrosis,
it is more likely to be seen in this analysis.
Another change would be to perform the screening at different radiation doses.
Since apooptosis is seen in a wide range of doses (2.5 - 25 Gy), it is possible that the
high dose used here overrode the apoptosis mechanism and induced necrosis.
5.1.2
Tamoxifen-Induced Apoptosis
The effect of different concentrations of tamoxifen needs to be investigated. It may
also be useful to use hydroxy-tamoxifen which has much higher affinity (30 -100X)
for estrogen receptors than tamoxifen [28]. The effect of phenol red in the media on
the results should also be characterised. Phenol red is a weak estrogen and would
therefore compete with tamoxifen for receptor cites. However, on experiments done
with BT20 cell line, this effect was not major [12]. The concentration of fetal calf
serum used in the media could also affect the response to tamoxifen. Chouvet et al.
show that high serum concentration reduced the growth inhibitory effects of tamoxifen
[12]. One possibility is that there there is estrogen in the serum that competes with
tamoxifen for the estrogen receptor sites.
5.2
Future Work
Since gel electrophoresis techniques did not provide any evidence of apoptosis, it is
necessary to employ morphological techniques to screen for apoptosis. DNA end labelling techniques could be used to identify any fragmentation that occurs. However,
since DNA fragmentation does not show up on agarose gels, the efficacy of the technique is questionable. The other option is to observe the cells directly for changes in
chromatin and membrane structure.
Further investigations of apoptosis in breast cancer cell lines will require the use
of molecular biology techniques. Manipulating the expression of wildtype or mutant
forms of the p53 and bcl-2 genes will answer the question of whether their state affects
the cell death pathway in these cell lines. Investigating the status of other genes that
are involved with cell proliferation or death may also be useful in establishing the
ability of breast cells to apoptose.
Discovering mechanisms to induce or enhance apoptosis by the methods used in
the treatment of breast cancer will lead to the improvement of these treatments and
survival of the patient. Therefore work to establish the mechanisms and pathways
leading to apoptosis warrants further investigation.
Appendix A
Schematic of the CHEF-DR II
System
Model 200/2.0
Pulsewave 760
Switcher
Power Supply
Figure A-1: A schematic showing the set-up of Biorad's CHEF-DR II Pulsed
Field Gel Electrophoresis System [6].
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