The lab report is written in italics. Each section is repeated and in the second copy, hints and explanations are given.

Detection of p53 Protein in Normal Cells but Not Tumor Cells

Therese M. Poole

ABSTRACT

The P53 protein is involved in cell cycle regulation and apoptosis. Many tumors have been analyzed for presence of P53 and in over 50% of the cases, P53 activity has been lost. Using Western Blot analysis, we analyzed cells from four tumors and some normal cells to see if P53 was present. We saw that P53 was present in the normal cells and two of the tumors but not in the other two tumors. These findings support earlier conclusions that P53 is an important tumor suppressor gene.

INTRODUCTION

P53 activity is lost in over 50% of all tumors and is, therefore, considered an important tumor suppressor protein. P53 regulates the G1 to S checkpoint during the cell cycle by detecting DNA damage and halting cells in G1. When the damage is repaired, P53 levels decrease and the cell can proceed into S phase. If the damage is too great to be repaired P53 can initiate apoptosis (programmed cell death).

There are many ways for a cell to lose P53 activity. First, the gene encoding P53 can be mutated or lost. Second, the p53 gene can be silenced or not expressed. Third, the protein is expressed but its function is blocked. Fourth, the protein is expressed but is quickly degraded before it can perform its action.

We wanted to determine if cells from four different tumors had lost P53. We performed Western Blot Analysis to determine if the protein was present in the cells. In Western Blot Analysis, cellular proteins were separated using SDS-PolyAcrlyamide Gel Electrophoresis (SDS-PAGE) and then blotted onto a membrane. Antibodies directed against the protein of interest are used to detect the protein.

We determined that P53 protein was not present in two of the tumors but was present in the cells from the other two tumors and from normal cells. Our results lend support to the idea that P53 acts as a tumor suppressor protein.

MATERIALS and METHODS

Isolation of Proteins from Cells. Approximately 1 X 107 cells from each tumor, designated T1, T2, T3, and T4 and from normal tissue, designated N1, were lysed by mechanical grinding for 2 minutes in 2% SDS. Ammonium Sulfate was added to 50% saturation, stirred and the solution was centrifuged at 10000x g for 30 minutes. The resulting pellet was resuspended in 1 ml of distilled water and dialyzed against distilled water overnight.

SDS-PAGE and Blotting. Protein concentration was determined by taking an OD reading at l = 280 nm. 10 mg of protein in an SDS buffer was loaded into each lane of a 10% acrylamide gel and electrophoresed at 80 V for one hour. The gel was carefully removed from the plates and part of the gel containing the molecular weight standards was stained in Coomassie stain for 1 hour and destained in 20% methanol, 10% acetic acid for 6 hours. The other part of the gel, containing the protein samples from the two cell lines was loaded onto an electroblotting apparatus. The gel was blotted onto Nylon at V for 1 hour.

Western Blotting Analysis. The Nylon membrane was blocked with 5% dry milk in 1 X TBS buffer overnight. Then the membrane was washed 3 times (5 - 10 minutes each time) in wash solution (1 X TBS and 0.1% TWEEN 20) . Primary antibody was added at a 1/1000 dilution in antibody incubation buffer (1 X TBS, 1% dry milk and 0.1% TWEEN 20) for 1 hour at room temperature with shaking. The blot was then washed 3 times (5-10 minutes each) with wash solution. Goat anti-rabbit antibodies conjugated to horseradish peroxidase enzyme were added to the antibody incubation buffer at room temperature for one hour. The blot was washed 3 times (5-10 minutes each time) in wash buffer. The ECL chemiluminenscence kit was used for detection of antibodies. Solutions 1 and 2 were mixed and poured on blot for 1 minute. This resulting solution was carefully poured off and the blot was exposed to autoradiography film.

RESULTS

The concentrations of protein in the cell samples are listed in Table 1. They ranged from 1.2 mg/ml to 4 mg/ml. These concentrations were used to calculate the correct volume to load in order to get 10 mg of total protein in each lane.

Figure 1 shows the Western Blot generated when the nylon membrane was probed with antibodies to P53 protein. P53 antibodies bound to protein in the lanes containing normal cells and two of the tumor samples whereas no band was detected in lanes containing the some of the tumor cells.

Table 1. Concentrations of protein in cell lysates

FIGURE 1: SDS-PAGE followed by Western Blotting of Normal and Tumor Cells

Figure 1: Western Blot analysis was performed as described in the Materials and Methods. 10mg of total protein was added to each lane. Lane 1 contains TML1a, Lane 2 contains TML1b, Lane 3 and 4 are from normal cells and Lane 5 contains TML2a and Lane 6 contains TML2b.

DISCUSSION

Total protein was isolated from tumor cells and from normal cells. The resulting proteins were run on SDS-PAGE and blotted onto nitrocellulose for Western Blot Analysis. A band appeared in the lanes containing normal cells and in two of the tumors that matches the expected molecular weight of P53, whereas no band appeared in the other two lanes containing tumor cells. These data indicated that P53 protein is missing from half of the tumors analyzed here. This result agrees with our expectations and with published literature where it has been reported that over 50% of all tumors lose P53 activity ().

Even though P53 is detected in two of the tumors, it is possible that P53 is not active in these tumors. Our assay only determines if P53 protein is missing. Just because P53 is present does not mean that it is active. Thus, the two tumors that contained P53 may contain inactive protein. To determine if the P53 in the two tumors is active, we would have to isolate the P53 protein and test it for its ability to halt cells in G1 or to induce apoptosis of the cells. Some tumors never lose P53 activity and thus, must become tumors through another pathway that doesn't include P53. It would be interesting to look for the loss of other tumor suppressor proteins from these tumors.

Analysis of abstract:

The P53 protein is involved in cell cycle regulation and apoptosis. Many tumors have been analyzed for presence of P53 and in over 50% of the cases, P53 activity has been lost.(SUMMARY OF INTRODUCTION) Using Western Blot analysis, we analyzed cells from four tumors and some normal cells to see if P53 was present. (SUMMARY OF METHODS) We saw that P53 was present in the normal cells and two of the tumors but not in the other two tumors. (SUMMARY OF RESULTS) These findings support earlier conclusions that P53 is an important tumor suppressor gene. (SUMMARY OF DISCUSSION/CONCLUSION)

Analysis of INTRODUCTION

P53 activity is lost in over 50% of all tumors and is, therefore, considered an important tumor suppressor protein. P53 regulates the G1 to S checkpoint during the cell cycle by detecting DNA damage and halting cells in G1. When the damage is repaired, P53 levels decrease and the cell can proceed into S phase. If the damage is too great to be repaired P53 can initiate apoptosis (programmed cell death). (WHAT IS P53 AND WHAT DOES IT DO - GENERAL BACKGROUND INFORMATION TO SET THE STAGE OR FRAME THE STORY)

There are many ways for a cell to lose P53 activity. First, the gene encoding P53 can be mutated or lost. Second, the p53 gene can be silenced or not expressed. Third, the protein is expressed but its function is blocked. Fourth, the protein is expressed but is quickly degraded before it can perform its action. (HOW IS P53 RELATED TO WHAT WE ARE LOOKING AT IN THIS EXPERIMENT? NARROWING DOWN THE INFORMATION TO RELATE SPECIFICALLY TO THIS WORK)

We wanted to determine if cells from four different tumors had lost P53. We performed Western Blot Analysis to determine if the protein was present in the cells. In Western Blot Analysis, cellular proteins were separated using SDS-PolyAcrlyamide Gel Electrophoresis (SDS-PAGE) and then blotted onto a membrane. Antibodies directed against the protein of interest are used to detect the protein. (EXPLANATION OF WHAT WAS DONE AND WHY IT WAS DONE)

We determined that P53 protein was not present in two of the tumors but was present in the cells from the other two tumors and from normal cells. Our results lend support to the idea that P53 acts as a tumor suppressor protein. (BRIEFLY STATES THE RESULTS)

Analysis of MATERIALS and METHODS

IT IS NOT NECESSARY TO LIST ALL THE SUPPLIES. NOTICE USE OF PROSE AND OF PAST TENSE - YOU ALREADY DID THE EXPERIMENT. NOTICE HOW EACH PROCEDURE GETS ITS OWN SECTION AND TITLE.

Isolation of Proteins from Cells. Approximately 1 X 107 cells from each tumor, designated T1, T2, T3, and T4 and from normal tissue, designated N1, were lysed by mechanical grinding for 2 minutes in 2% SDS. Ammonium Sulfate was added to 50% saturation, stirred and the solution was centrifuged at 10000x g for 30 minutes. The resulting pellet was resuspended in 1 ml of distilled water and dialyzed against distilled water overnight.

NOTICE USE OF CONCENTRATIONS INSTEAD OF VOLUMES (CONCENTRATION IS INDEPENDENT OF VOLUME SO ITS MORE APPLICABLE) SDS-PAGE and Blotting. Protein concentration was determined by taking an OD reading at l = 280 nm. 10 mg of protein in an SDS buffer was loaded into each lane of a 10% acrylamide gel and electrophoresed at 80 V for one hour. The gel was carefully removed from the plates and part of the gel containing the molecular weight standards was stained in Coomassie stain for 1 hour and destained in 20% methanol, 10% acetic acid for 6 hours. The other part of the gel, containing the protein samples from the two cell lines was loaded onto an electroblotting apparatus. The gel was blotted onto Nylon at 90 V for 2 hours.

Western Blotting Analysis. The Nylon membrane was blocked with 5% dry milk in 1 X TBS buffer overnight. Then the membrane was washed 3 times (5 - 10 minutes each time) in wash solution (1 X TBS and 0.1% TWEEN 20) . Primary antibody was added at a 1/1000 dilution in antibody incubation buffer (1 X TBS, 1% dry milk and 0.1% TWEEN 20) for 1 hour at room temperature with shaking. The blot was then washed 3 times (5-10 minutes each) with wash solution. Goat anti-rabbit antibodies conjugated to horseradish peroxidase enzyme were added to the antibody incubation buffer at room temperature for one hour. The blot was washed 3 times (5-10 minutes each time) in wash buffer. The ECL chemiluminenscence kit was used for detection of antibodies. Solutions 1 and 2 were mixed and poured on blot for 1 minute. This resulting solution was carefully poured off and the blot was exposed to autoradiography film.

Analysis of RESULTS

The concentrations of protein in the cell samples are listed in Table 1. They ranged from 1.2 mg/ml to 4 mg/ml. These concentrations were used to calculate the correct volume to load in order to get 10 mg of total protein in each lane. (BRIEF EXPLANATION OF HOW THE RESULTS WERE OBTAINED AND HOW THE READER CAN FIND THE SPECIFIC INFORMATION QUICKLY AND EASILY)

Figure 1 shows the Western Blot generated when the nylon membrane was probed with antibodies to P53 protein. P53 antibodies bound to protein in the lanes containing normal cells and two of the tumor samples whereas no band was detected in lanes containing the some of the tumor cells. (DESCRIPTION OF WHAT IS SEEN IN EACH OF THE TABLES AND FIGURES)

EACH TABLE SHOULD HAVE A NUMBER AND A TITLE

Table 1. Concentrations of protein in cell lysates

(AS MUCH DATA AS POSSIBLE SHOULD BE PUT INTO TABLES OR GRAPHS. TRY TO COMBINE INFORMATION TO MINIMIZE SPACE AND MAKE IT EASIER FOR THE READER TO FIND INFORMATION AND TO KNOW HOW TO RELATE THE DIFFERENT PIECES OF DATA MORE EASILY.)

FIGURE 1: SDS-PAGE followed by Western Blotting of Normal and Tumor Cells

Figure 1: Western Blot analysis was performed as described in the Materials and Methods. 10mg of total protein was added to each lane. Lane 1 contains TML1a, Lane 2 contains TML1b, Lane 3 and 4 are from normal cells and Lane 5 contains TML2a and Lane 6 contains TML2b.

Analysis of DISCUSSION

Total protein was isolated from tumor cells and from normal cells. The resulting proteins were run on SDS-PAGE and blotted onto nitrocellulose for Western Blot Analysis. (RECAP OF THE METHODS - BRIEF!) A band appeared in the lanes containing normal cells and in two of the tumors that matches the expected molecular weight of P53, whereas no band appeared in the other two lanes containing tumor cells. (RECAP OF THE IMPORTANT DATA THAT IS NEEDED TO MAKE THE CONCLUSI0N(S)) These data indicated that P53 protein is missing from half of the tumors analyzed here. This result agrees with our expectations and with published literature where it has been reported that over 50% of all tumors lose P53 activity (). (CONCLUSION - BASED ON THE RESULTS GIVEN (NOT WHAT YOU EXPECTED - WHAT YOU ACTUALLY SAW!) IN THIS PAPER) (NOTICE THE LOGICAL PROGRESSION OF THOUGHT. EVEN IF YOU DID NOT THINK OF THE RESULTS THIS WAY AHEAD OF TIME, YOU MUST WRITE CLEARLY FOR THE READER TO UNDERSTAND WHAT YOU DID.)

Even though P53 is detected in two of the tumors, it is possible that P53 is not active in these tumors. Our assay only determines if P53 protein is missing. Just because P53 is present does not mean that it is active. Thus, the two tumors that contained P53 may contain inactive protein. (POSSIBLE EXPLANATIONS OF DATA - HUMAN ERROR SHOULD NOT BE INCLUDED UNLESS YOU HAVE GOOD EVIDENCE TO SUPPORT THAT ASSERTION. IN REAL LIFE, IF YOU MADE LOTS OF ERRORS YOU WOULDN'T BE PUBLISHING THE DATA : - ) To determine if the P53 in the two tumors is active, we would have to isolate the P53 protein and test it for its ability to halt cells in G1 or to induce apoptosis of the cells. (WHAT COULD BE DONE NEXT) Some tumors never lose P53 activity and thus, must become tumors through another pathway that doesn't include P53. It would be interesting to look for the loss of other tumor suppressor proteins from these tumors. (ANOTHER POSSIBILITY AND ANOTHER POSSIBLE WAY TO TEST IT)