ICC Final Paper

THE EFFECTS OF A PI3K INHIBITOR 1
The Effects of a PI3K Protein Inhibitor on BaF3 Cancer Cells
Christopher Schonbaum, Rosemary Zaragoza, and SoRi Jang
The University of Chicago: Research in the Biological Sciences 2012
Emma McAvoy, Victoria Rael, Elizabeth Sarkel, and Oğul Üner
13 July 2012

I. Abstract
The Phosphoinositide-3 Kinase pathway is one of the main pathways involved in cell
proliferation. Many proteins are involved in this pathway, including MYC and MCL-1. If the
PI3K pathway is inhibited, the levels of these proteins, which are also involved in cell
proliferation, should decrease and, consequently, cells should proliferate less. In this
investigation, the PI3K inhibitor LY294002 was used to treat leukemic BaF3 mouse cells. The
cell viability and levels of MCL-1 and MYC were examined in order to measure the effect of the
PI3K inhibitor on leukemic BaF3 mouse cells. It was concluded that the inhibitor did in fact have
an effect on the BaF3 cells. As shown by the cell viability assay and the levels of MYC observed
in differing concentrations of LY294002 through a Western Blot, the drug decreased cancer cell
proliferation and the levels of MYC protein.

II. Introduction
The phosphoinositide-3-kinase (PI3K) protein is an important enzyme that signals
various cellular activities, including cell growth, cell differentiation, cell proliferation and cell
survival. Because of these roles in the cell cycle, the PI3K pathways are directly related to cancer
(Jia, Roberts and Zhao, 2009). In the PI3K pathways, there exist two key catalytic subunits
related to tumor formation: p110α (coded for by oncogene PIK3CA) and p110β. In tumor cells,
some of the common mutations are mutations of the PIK3CA gene, which are found in the p110α
subunit, and mutations in the PTEN gene (a tumor suppressor gene), which are found in the
p110β subunit (Jia, Roberts and Zhao, 2009). These mutations promote tumor growth and tumor
development (Jia, Roberts and Zhao, 2009).
MCL and MYC are proteins located further down the PI3K pathway. MCL inhibits
apoptosis, while the MYC oncogene helps to regulate the cell cycle (Adhikary and Eilers, 2005).
In cancer cells, mutations on the MYC gene lead to the overexpression of the MYC proteins and
many other proteins (since the MYC gene is a transcription factor) ("Myc gene," 2012). Hence,
these proteins are involved in cell proliferation and thus can cause cancer (Adhikary and Eilers,
2005).
Since a characteristic property of cancer cells is rapid proliferation, it is necessary to test
the PI3K inhibitor on cancer cells. To further investigate what happens when the PI3K pathways
are inhibited, we used a PI3K enzyme protein inhibitor drug on leukemic BaF3 mouse cells in
order to suppress the pathways and subunits of PI3K, including the p110α and p110β subunits.
We used BaF3 cells because they are leukemic and proliferate rapidly. They were also readily
available for our use. We then performed a series of experiments to examine and analyze the
effects of varying concentrations of the PI3K inhibitor drug (LY294002) on the cancerous cells.

We first used a cell viability test to observe the number of cells killed by the differing
concentrations of the drug. After the cell viability test, we then used a Western Blot to measure
the MCL and MYC protein levels in the sample of cells treated by the different concentrations of
drug. Using the Western Blot, we examined the suppression of MCL and MYC using various
concentrations of the PI3K protein inhibitor.
Lastly, we fixed cells and treated them with Nocodazole to visually observe the
percentage of cells in mitosis in a BaF3 culture, with and without Nocodazole. The drug
Nocodazole can be used to observe mitotic cells because Nocodazole stops the cell cycle in
metaphase by interfering with the polymerization of microtubules. Therefore, it prevents the cells
from continuing dividing. Thus, because of the interference by the Nocodazole cells cannot
divide and continue the cell cycle, leading to apoptosis.
In conclusion, we are researching the effects of this PI3K inhibitor because it has the
potential to be used (alone or in combination) with other drugs in order to kill cancer cells in
humans. These series of experiments allowed us to examine the potential of this drug for
treatment of cancer, leukemia in particular.

III. Materials and Methods
For this experiment, leukemic mice BaF3 cells were used because they are cancerous and
the effects of the PI3K inhibitor LY294002 would be evident. LY294002 was used as the PI3K
inhibitor because this is the inhibitor that our lab had in stock.
Setting up the Cell Viability Culture
BaF3 cells were mixed with RPMI media. Trypan blue dye was added to a small sample
of the cells to determine the cell concentration using a Countess Cell Counter. We used this
count to determine the volume of how much cell solution and media needed to be mixed to
achieve a cell concentration of 2X10^5. Then the appropriate volume of cells was plated with
RPMI media and left to incubate overnight at 37°C and 5% CO2.
Measuring the effect of LY294002 on BaF3 Cell Viability
The cell viability assay was performed in order to examine the effect of the PI3K
inhibitor (LY294002) on leukemic BaF3 cells. The cells were taken from the incubator and the
appropriate concentrations (0, 0.6, 1.25 and 2.5 uM) of the drug LY294002 were added into the
appropriate wells. The concentrations were then added into a 96-well plate in which the drug
dilutions of the LY294002 and BaF3 cells were incubated at 37° C and 5% CO2 for 48 hours. To
perform the cell viability assay, 20 ul CellTiter reagent first had to be added into each well of the
96-well plate which contained the BaF3 cells and PI3K inhibitor drug solution and left to
incubate for 1-4 hours at at 37° C and 5% CO2. After the incubation, the 96-well plate was
placed in the iMark reader and absorbance was measured at 490 nm wavelength, which was used
to create a dose-response curve and to determine the IC50.
Preparing Cell Lysates

To observe the amounts of MCL and MYC proteins in our drug treated cells, we lysed
the cells that were treated with each concentration of LY294002 (0 uM, 0.625 uM, 1.25 uM, 2.5
uM, 5 uM, 10 uM, and 20 uM). After the centrifugation, we washed PBS, and after two washes,
treated the cells with lysis buffer for 30 minutes at room temperature. Cells were then
centrifuged again for 15 minutes at 14000 ref. After the centrifugation, we removed the
supernatant which contained the proteins and stored them at -80 degree C.
BCA Assay
In order to determine the protein concentration in our lysates, 100 ul of Working
Reagents A and B were plated with 5 ul of lysate in a 96 well plate. This was done three times
for each lysate tube. The cells were incubated for half an hour before reading with an iMark
microtiter plate reader to determined the protein concentration of each lysate.
PAGE (PolyAcrylamide Gel Electrophoresis) and Gel Transfer
After the protein concentrations were found and the appropriate volume for each lysate
was determined, a Polyacrylamide gel for electrophoresis was prepared. After loading the known
protein mass ladders and the lysate samples onto the lanes, the gel was run at 200 V for
approximately 45 minutes. Afterwards, the gel was ready for the transfer step. In order to
observe the proteins and stain them with the antibodies, the gel was transferred onto a
nitrocellulose membrane. The BioRad SemiDry transfer apparatus was used and it ran at 12 V
for 30 minutes.
Ponceau Staining
After the transfer was complete, it was necessary to verify that the proteins had
successfully transferred to the membrane from the gel. The nitrocellulose membrane was stained

with Ponceaus S to visualize the protein bands . This ensured that we had equal amounts of
proteins loaded in each lane of the gel and that the transfer step was successful.
Antibody Staining and Detection
The membranes were stained with antibodies in order to observe the amounts of MYC
and MCL. First, the membrane was blocked with 1% milk protein solution to ensure that the
entire membrane was coated in protein (decreasing the likelihood that the primary and secondary
antibodies would stick to the membrane and not the desired protein). After this, 1:200 dilutions
of the antibodies for P53, MCL, and MYC and a 1:100,000 dilution of the antibody for tubulin
were prepared. These dilutions were in milk protein. The different antibodies were used for
different stains. The membranes were allowed to incubate on a Rocker II for an hour. Then, the
membrane was washed three times in TTBS buffer. After that, 1:15,000 dilution anti-rabbit
secondary antibody was added and the membrane was incubated on the Rocker II for an
additional hour. After the incubation the membranes were washed again three times with TTBS.
The membranes were incubated for 5 minutes with chemiluminescence and then viewed by
exposing the film and then developing it.
DNA Staining
Many different methods of staining BaF3 cells were used in order to find an effective
method to look for condensed chromosomes that would indicate mitotic cells.
DNA Staining: Hoechst Stain
This method began by adding one ml of BaF3 cell culture to a microfuge tube. The cells
were then spun down to the bottom of the microfuge tube using a microcentrifuge set at 400 g
(ref) for one minute. The supernatant was removed and 100 ul PBS was added to the microfuge
tube with cells. The cell and PBS mixture was then added to a tube with 100 ul of 1mg/ml stain

of Hoechst stain and incubated for ten minutes. Finally, a slide was prepared and observed using
a WU filter.
DNA Staining: Hoechst Stain 2
First, 1 mg/ml of Hoechst stain was diluted to 0.1 mg/ml with PBS. 5 ul of this stain was
added to each of two separate microfuge tubes, each with 250 ul of BaF3 cell culture (the final
concentration of the stain was 2 ug/ml per tube). The stain incubated for 12 minutes, and then a
slide for each tube was prepared using 15 ul of the stained cells. The cells were observed with a
WU fluorescent filter.
DNA Staining: Nocodazole
First, cells were counted and diluted to a concentration of 5 x 10^5 and volume of 18 ml.
In a 6-well plate, 4 ml of the cell culture was distributed into each well. After incubating at 37° C
and 5% CO2 for seven hours, 2 ul of 1 ug/ul stock Nocodazole was each added to two wells. This
was then incubated for 11 hours, or overnight.
Then the cells were permeabilized using 4% paraformaldehyde and PBT detergent. Four
microfuge tubes with untreated cells and 4 microfuge tubes with Nocodazole-treated cells were
fixed. Finally, the cells were stained. 2 ul of Hoechst stain (100 ug/ul stock) was added to
Control Tube 2 and to Nocodazole Tube 2. 12 ul of the stained cells were then used to prepare
two slides, which were viewed under a WU filter.

IV. Results and Discussion
Cell Viability
A cell viability assay was performed to determine the IC50 of LY294002 and to examine
if the concentration of cells decreased as the concentration of drug increased.
Figure 1: The dose response curve of the PI3K inhibitor drug LY294002 obtained from the cell viability
assay.
The standard deviation of the cell viability assay was small, indicating that the results
were reproducible. In Figure 1, an explicit pattern of a sigmoidal curve can be seen. As
illustrated in Figure 1, cell viability is decreased as the concentration is increased. Although the
cell viability was higher (91.96%) at the concentration 1.25 uM of LY294002 than it was at .62
uM of LY294002 (88.61%), this is not believed to represent any significant error; it could mean
that there was a small amount of contamination, but nothing that threw off overall results. Most
of the points show that the percent of viability is inversely proportional to the (log treatment)^2.
The IC50, the concentration of an inhibitor or drug where 50% of a cell population dies,
was found to be between 9 uM and 10 uM. Compared to the actual IC50 of LY294002 (around

THE EFFECTS OF A PI3K INHIBITOR
10
10 uM), this experimental IC50 is very similar and accurate. Thus the results were expected. This
means that the results are repeatable; they were accurate and expected and had no substantial
errors. It was originally thought that the IC50 of the LY294002 was around 1.4 uM; however,
with further research, it was discovered 1.4 uM was only the IC50 of the enzyme and not the
whole cell. Thus the assay was repeated, but with higher concentrations of LY294002 to show us
more data pertaining to the IC50 found in the first experiment. The second trial produced results
as expected; as the concentration of the drug went up, cell viability went down. Overall, the
effects of the LY294002 drug concentrations on the cell viability of BaF3 leukemic cells were
observed with reasonable and expected results.

11
Western Blot Analyses
Western Blots were performed to examine the levels of the MYC, MCL, and P53
proteins, which are involved in the PI3K pathway. The first Western Blot had concentrations of
cell lysate that had been treated with 0 uM, 0.625 uM, 1.25 uM, and 2.5 uM of LY294002.
Another western blot was performed for concentrations around the determined IC50.The
expectation was that the levels of MYC and MCL would decrease and the levels of P53 to
increase as the drug concentration increased.
Figure 2: 2 minute exposure
Figures 2 and 3: The 2 and 16 minute exposure results of the cut nitrocellulose membranes. The
luminescence can be observed in both of the diagrams, except for the first 5 lanes where we can’t see the
membrane or the lanes.
The first stain performed with the first membrane was for the proteins MCL and MYC
(Fig. 2). Lanes 1-6 of the membrane were stained with MCL and lanes 7-12 (Fig. 3) were stained
with MYC. In this stain, the levels of MYC decreased as the concentration of the drug increased,

12
but there was nothing present in the MCL stain. Unfortunately, this was because the wrong
secondary antibody was used. MCL is a rabbit antibody, so we needed to use an anti-rabbit
secondary, not the anti-mouse secondary (which was used). Although this blot did not give us
information about the effect of MCL, we could conclude that the levels of MYC decreased
overall. MYC is about 50-70 kilodaltons and a band was seen at about that size. It increased in
concentration from 0 uM to 0.625 uM, but this could be due to a slight effect of the drug that
makes the levels of the protein go up, however this is not likely. It may also be caused by not
enough incubation time with the drug, which was 48 hours. The levels of MYC decreased from
the 0 uM/0.625 uM range to the 2.5 uM, consistent with what was expected. This means that
LY294002 was able to inhibit PI3K and block the production of MYC, as predicted.
Figure 4: 2 minute exposure of the MCL antibody stain

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Figure 5: 12 minute exposure of the MCL antibody stain
Figures 4 and 5: The 2 and 12 minute exposures of the nitrocellulose membrane that should have had
the MCL protein. The 5th figure connotes that there are some MCL bands, but there are still cross-
products that are approximately 60 and 70 kDa.
After this stain, the first membrane was stained again with the MCL stain with the correct
(anti-rabbit) secondary antibody (Fig. 4 and 5). The MCL membranes were expected to be seen
when exposed to the film developer. However, the MCL band was not present strongly when the
developed film was examined. It is possible that the secondary antibody cross-reacted with other
antibodies (there were other protein bands on the film). MCL was not present in noticeable
amounts, so it is inconclusive from this Western Blot whether or not LY294002 had an effect on
the levels of MCL.
After this stain, the membrane was stripped and re-stained with tubulin and MCL (Fig. 6,
7, and 8). This was necessary to verify that the lanes were loaded evenly. The membrane was
simply re-stained with MCL to observe if the protein would be present in noticeable amounts.

14
Figure 6: 20-30 Second Exposure Tubulin/MCL stain
Figure 7: 30 Second Exposure Tubulin/MCL stain
Figure 8: 20 Minute Exposure Tubulin/MCL stain
Figures 6, 7 and 8: The 20-30 second, 30 second and 20 minute exposure depictions of the membranes
stained with tubulin and MCL-1.

15
When the membrane was stained again with the MCL and tubulin, the MCL did not
appear. It is unclear why this happened. There may have been a problem with the antibody
(although the antibody did work last fall when it was used). The antibody may have also cross-
reacted with another protein and stained that protein instead.
The tubulin stain confirmed that there was less protein in some of the lanes than others
(Fig. 6, 7, and 8). There should have been less protein in Lane 4, due to dilution error.
Additionally, there was less protein in Lane 8 (0 uM) than Lanes 9 and 11 (0.625 uM and 2.5
uM, respectively) when the levels of MYC were observed. The same trend was seen with the
tubulin. There was visibly less tubulin in the 0 uM lane than the other lanes, which were about
the same levels. This shows that the 0.625 uM and 2.5 lanes were loaded with equal amounts of
protein. This stain is inconclusive about the effectiveness of LY294002 on MCL since MCL was
not seen.
After this stain was performed, the first membrane (Fig. 9 and 10) was stained with P53
antibody. P53 is a protein that is involved in apoptosis, so it is expected it in higher amounts in
cells that were treated with a higher concentration of drug. The membrane on the right is from
the second Western, which had lysates that were treated with 0 uM, 2.5 uM, 5 uM, and 10 uM
LY294002. This membrane was stained with MYC and MCL antibodies to look at the amounts
of these proteins. It was predicted that the same trends in the levels of MYC and MCL would be
seen in the second membrane stain as the first membrane stain.

16
Figures: The 3 and 16 minute exposures of the cut nitrocellulose membranes from both the western I
(left) and the western II (right). The dark bands on the ladders in both of the exposures indicate strong
reactivity, and there are tubulin bands that one can see as a control.
The P53 in Fig. 9 and 10 on the left could not be seen. The membrane looks as if it was
not completely stripped from the tubulin stain. This explains why there are massive bands where
tubulin should be. P53 is also about the size of tubulin, so it is difficult to distinguish between the
tubulin and the P53.
Furthermore, the MCL bands on our second western membrane (right) were not seen,
possibly due to an antibody problem (Fig. 9 and 10). The cause of this result is unknown, since

17
the correct secondary antibody was used and no human errors were made that could have caused
the membrane to completely be missing. The MYC bands were not explicitly observed in the
second membrane, although a protein that cross-reacted with the antibody showed the
relationship between the protein concentrations that were expected to be seen in MYC. The
levels of this protein showed a decrease as the drug concentration increased. That protein (about
90 kDa) is most likely is related to MYC, as it exhibits the trend of MYC levels we expected to
see in the MYC levels. In general, expected results were seen, with the exception of faint bands
that were seen in our second western MYC membrane and no bands that were seen in our second
western MCL.
Finally, the second membrane was stained with tubulin to make sure that the lanes were
evenly loaded. The lanes should have been evenly loaded since the Ponceau stain (Fig. 11)
showed that there was protein in all of the lanes.
Figure 11:

18
Figure 12: 20 second exposure
Figure 13: 90 second exposure
Figures: The 20 and 90 second exposures of the nitrocellulose membrane that was stained with tubulin.
Tubulin is about 50 kDa in size, which is consistent with the bands we observed above.

19
Expected results were obtained such that the bands seen in both exposures had the
approximate 50 kDa band (Fig. 12 and 13). The bands on the lanes 1 and 12 had faint bands, so
re-probing was attempted. However, similar results were obtained. It is unclear why this
occurred, since the Ponceau S stain (Fig. 11) clearly showed that there was protein present in
Lanes 1 and 12, even though there was less proteins in these lanes than the others. However, the
Ponceau S stain (Fig. 11) did not indicate that there would be a very small amount of tubulin in
those lanes, especially because tubulin is abundant and very easily detected. There was not an
issue with seeing the bands in the other lanes after short exposures. The levels of tubulin were
mostly loaded evenly into the gel for the second Western blot, as shown by the stain (Fig. 11).
For an unknown reason, tubulin does not appear in lane 12; it should be present (Fig. 12 and 13).
This stain confirmed that the decreasing trend by the unknown protein that is presumably related
to MYC is not due to uneven protein levels, but due to the increase in drug concentration.

20
DNA Stain Results
The “DNA Staining: Hoechst Stain” method for observing the number of cells
undergoing mitosis was not utilized for assessing the effectiveness of the LY294002 because the
BaF3 cells were too small. Chromosomes were not easily detected, and therefore it was difficult
to determine the stage of mitosis the cells were in.
Thus, the “DNA Staining: Hoechst Stain 2” method was used. However, this method also
proved unsuccessful. During the first attempt, the cell culture from which the cells that were
taken was old; therefore a lot of the cells in that culture were dying and could not be observed in
mitosis. A second attempt was made, and when viewing the cells under the microscope, the DNA
was visible. A number of apoptotic cells were observed, but we were not able to distinguish any
cells undergoing mitosis. This might have resulted because of a couple factors. First, there could
have actually not been very many cells in mitosis because the cells had been starving. Also the
cells may have been partially synchronized in their stages and development and the time during
which almost all of the cells were dividing could have been missed. The small cell size was
another factor that made it difficult to distinguish chromosomes. As a result of these factors, this
method was eliminated as a way to observe mitotic cells.
Fig: 14: Control (Non-Treated) Slide Cell Count (Prepared from Control Tube 2): 20X objective,
200X magnification, 500 um FOV
Picture
9 8 7 6 Average
Cell Count Per Field of View 13 14 12 10 12
Mitotic Cells 0 0 1 0 2.5
Percent in Mitosis 0% 0% 8.33% 0% 2.1%

21
Fig: 15 Nocodazole Slide Cell Count (Prepared from Nocodazole Tube 2): 20X objective, 200X
magnification, 500 um FOV
Picture 15 14 8 6 Average
Cell Count Per Field of View 13 10 5 4 8
Mitotic Cells 4 2 0 1 1.75
Percent in Mitosis 31% 20% 0% 25% 19%
Fig. 16: Nocodazole Figures: These cells are about 23 um on a 20X objective (200X magnification, 500
um field of view). These are the cells that were in the Nocodazole tube that were viewed on a microscope
slide under the microscope.

22
Fig. 17: Control Figures: These cells are about 18 um on a 20X objective (200X magnification, 500 um
field of view). The cells that were in the control tube that were viewed on a microscope slide under the
microscope.
The final method, “DNA Staining: Nocodazole” was then used. Nocodazole breaks down
the microtubules in the cells, stopping the cells in early mitosis. This way, cells could be
observed arrested in metaphase. Cells were treated with Nocodazole and observed and counted
under a fluorescent microscope.
When non-treated control cells and treated cells were observed, there were slightly more
cells on the control slides than the Nocodazole slides (Fig. 14 and 15). This is because cells on

23
the control slide did not have the Nocodazole inhibiting mitosis that prevented the cells from
dividing and proliferating.
This method proved successful to observe the cells in mitosis. The purpose of this
experiment was to confirm that cells in mitosis could be viewed by staining with Hoechst. A
total of four 200x view objectives were used when counting the number of cells in mitosis. The
average of cells in mitosis when viewing the control slide (Fig. 17) was ~2.1% (Fig. 14); this is
far less than the ~19% (Fig. 15) of arrested mitotic cells viewed in the Nocodazole treated slide.
These results tell us that Nocodazole does indeed block cells from continuing cell division at
metaphase and that it is possible to view mitotic cells with a Hoechst stain at 200X
magnification. In regards to the overall experiment, this does not lead us to any further data on
the effects of PI3K protein inhibitor. The goal of the experiment was to observe cells in mitosis
using Nocodazole to inhibit mitosis. The objective was obtained. Unfortunately, the cells that
were fixed and observed were not treated with the PI3K protein inhibitor, so the data cannot lead
us to any additional data on the effects of LY294002.
In conclusion, this project confirmed that the IC50 of LY294002 is approximately 10 uM.
It also allowed us to conclude that LY294002 is effective in limiting cell proliferation and
blocking the PI3K pathway. This was shown by the decrease in cell viability, demonstrated by
all of the viability assays, and the decreasing levels of MYC in the Western Blot. Therefore,
PI3K inhibitors do have the potential to kill cancer cells and thus are a promising treatments.

24

25
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Note: Materials and methods were taken from our RIBS 2012 lab manual.

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All contributed equally 13 July
2012
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