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

THE EFFECTS OF A PI3K INHIBITOR 2I. 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.

THE EFFECTS OF A PI3K INHIBITOR 3II. 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.

THE EFFECTS OF A PI3K INHIBITOR 4We 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.

THE EFFECTS OF A PI3K INHIBITOR 5III. 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

THE EFFECTS OF A PI3K INHIBITOR 6To 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

THE EFFECTS OF A PI3K INHIBITOR 7with 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

THE EFFECTS OF A PI3K INHIBITOR 8of 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.

THE EFFECTS OF A PI3K INHIBITOR 9IV. 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 1010 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.

THE EFFECTS OF A PI3K INHIBITOR 11Western 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

Figure 3: 16 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,

THE EFFECTS OF A PI3K INHIBITOR 12but 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

THE EFFECTS OF A PI3K INHIBITOR 13Figure 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.

THE EFFECTS OF A PI3K INHIBITOR 14Figure 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.

THE EFFECTS OF A PI3K INHIBITOR 15When 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.

THE EFFECTS OF A PI3K INHIBITOR 16Figure 9: 3 minute exposure

Figure 10: 16 minute exposure

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

the correct secondary antibody was used and no human errors were made that could have caused

THE EFFECTS OF A PI3K INHIBITOR 17the 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:

THE EFFECTS OF A PI3K INHIBITOR 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.

THE EFFECTS OF A PI3K INHIBITOR 19Expected 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.

THE EFFECTS OF A PI3K INHIBITOR 20DNA 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

Picture9 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%

THE EFFECTS OF A PI3K INHIBITOR 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.

THE EFFECTS OF A PI3K INHIBITOR 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

the control slide did not have the Nocodazole inhibiting mitosis that prevented the cells from

dividing and proliferating.

THE EFFECTS OF A PI3K INHIBITOR 23This 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.

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

THE EFFECTS OF A PI3K INHIBITOR 26All contributed equally 13 July 2012

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