OVERVIEW
My lab uses bioinformatics, a method that uses computer science to interpret biological data, to understand sarcoma. Sarcomas are soft tissue (like fat, nerve sheaths, connective tissue, smooth muscle, and vessels) and bone cancers that make up about 1% of all cancers. The subtype that I work with is dedifferentiated liposarcoma (DDLPS). Lipo- meaning “fat” and dedifferentiated meaning the cells revert from specialized fat-cell properties to STEM-cell properties so that they can replicate.
DDLPS naturally have an amplified expression of a protein, CDK4, which acts like a traffic light during the cell cycle when it connects with its partner, cyclin D. The cell cycle is split up into different phases so the cell has time to prepare nutrients and duplicate its cellular components (G1), duplicate DNA (S), prepare for mitosis (G2), and then divide (M). The first traffic light is between the G1/S phase and when CDK4 is amplified, the light is green, even if the cellular components are not prepared in cancerous cells. Overexpression of CDK4 then leads to uncontrollable cell division and growth.
Therefore, using a CDK4-inhibitor would be an attractive targeted therapy for patients with DDLPS. Unfortunately, while these drugs have positive outcomes, some patients develop resistance or are innately resistant to the drug. My project aims to develop palbociclib (a CDK4-inhibitor) resistant DDLPS cell lines to determine what proteins are changing expression levels to promote resistance. From here, if I know that Protein ABC increases and Protein 123 decreases in resistant cells, then I probably should not give palbociclib to patients who have high ABC and low 123 because they are more likely to develop resistance. I can also use this information to develop combination therapies. If ABC is increased in resistant cells, then maybe we should treat patients with CDK4 and ABC inhibitors as a double-attack.
HYPOTHESIS
The cell cycle is complex and relies on many protein interactions to activate/deactivate the traffic lights. There are proteins upstream (proteins that can activate/deactivate CDK4) and downstream (CDK4 activates/deactivates those proteins) that we can look at. In the schematic above, p16 is an upstream protein that deactivates CDK4 (the T-shaped arrow means inhibit) so we would expect p16 to decrease in CDK4-inhibitor-resistant cells (if we want to continue replicating, we need CDK4, so we don’t want p16 to inhibit CDK4). On the other hand, if CDK4 is inhibited, we might rely on another CDK-cyclin pair to activate the traffic light. CDK2 and Cyclin E can do that, so I expect both of those downstream proteins to increase to accommodate for inhibited CDK4.
I expect to see the loss of p16 and Rb and an increase in Cyclin E, CDK2, and CDK6.
RESULTS
I am in the process of developing novel, palbociclib-resistant, DDLPS cell lines. These cell lines were grown over 6 months while being exposed to the drug. Resistant cell lines demonstrated decreased protein expression of Rb, p16, and CDK2. I also wanted to see what the initial response was to survive drug treatment. Interestingly, the response to palbociclib treatment was to increase CDK2, CDK6, Cyclin E, and decrease pRb—almost opposite from long-term acquired resistant cells. Nonetheless, clinically, patients with DDLPS that have lower p16 or Rb may be more likely to demonstrate resistance to palbociclib. Next-generation sequencing of DNA and RNA expression in parent and resistant lines are underway.
METHODS & DAY IN THE LAB ROUTINE
- MONDAY
- 7:00 AM – Cell Culture: Since cancerous cells rapidly divide, I have to remove some of the cells from their storage dish so that there’s room for them to continue to grow. This process of cell culture is called “splitting” because we split the population of cells. During this time, if I want to collect cells for experiments, I would do that here. Typically, if I don’t need to collect cells, the ones I remove are put in the waste. The ones I collect can be stored in the freezer. I take care of 4 cell lines and have three plates of each so I have 12 plates in total. I work in a fume hood so I have a sterile working place to prevent contamination of the cells.
- 8:30 AM – Prepare drug inhibition assay: My project assesses how cell lines react to a drug called palbociclib. We can place a select amount of cells in small wells of a plate and then treat each well with increasing doses of the drug to determine the concentration that kills half of the cells. This dose is called the IC50. We would expect resistant lines to have a higher IC50 than non-resistant cell lines. At this point, I would count how many cells I have in total so I can evenly distribute them in each well. I’ll let them grow and get used to their new environment for a day, then I’ll treat them with drugs.
- 9:30 AM – Quantify protein from cells: Now I can leave the fume hood and go back to my lab bench. I collected some cells at 7 AM so I’ll sonicate my cells which is like a hard-core blender to break open the cell membrane. I can then boil my smoothie of cells to unravel the protein structure. Then I’ll use a color-changing solution that turns dark purple in the presence of protein. The darker the color, the more protein there is. I can use known concentrations of protein to compare my samples to determine how much protein I have.
- 10:30 AM – It is important to know how much protein I have because if I want to compare the concentration of a certain protein (like CDK4), I need to make sure the total protein concentration of all my samples is the same. If they aren’t, then I can dilute some of the samples so that they are all the same. We will be preparing for an experiment that tells us how much of a specific protein we have in different samples. The first thing to do is prepare a gel. It feels very similar to Jell-o and is about 1.5 mm thick. The gel can be thought of like dryer sheets–like a matrix of fibers. Later on, I will place my proteins at the top of the gel and run electricity through the gel so the proteins move. The protein I look for can range between 16-250 kDaltons and this matrix will allow for smaller proteins to move faster through the matrix but will slow down the bigger proteins at the top so this gel will separate my proteins by size.
- 11:30 AM – Clean Up and Leave: Time to clean up my station, wash any dishes that I used, and prepare stock solutions for anything that may be running out.
- TUESDAY
- 7:00 AM – SDS-PAGE: The experiment that separates proteins by size is called SDS-PAGE. I’ll take the gel that I made on Monday and the samples that I want to look at and put the samples at the top of the gel. Then I’ll run electricity through it for about 2 hours. The samples have been stained with a dye and I also placed a dye marker at the top of the gel so I can track where the proteins are in the gel. The marker will show bands of different colors to act as a guide so I can see how far along the proteins are moving.
- 9:30 AM – Transfer the gel: I’ll then take the gel and transfer all the protein onto a membrane using electricity. This takes about 45 minutes.
- 9:45 AM – While I wait, I can treat the cells from Monday with drugs.
- 10:30 AM – Identify the protein I want to look at: I can identify a specific protein on my membrane by letting my membrane soak in a solution with a primary antibody against a protein like CDK4. The antibody will only interact with the CDK4 protein. I’ll let that solution sit overnight so we are sure that the antibody is attached to the protein.
- 10:45 AM – Clean Up and Leave
- WEDNESDAY
- 7:00 AM – I’ll take out the membrane and “wash” it by soaking it in another solution. I can then soak it in another solution with a secondary antibody that will attach to the antibody we placed on the membrane on Tuesday. This secondary antibody has a fluorescent marker on it.
- 9:00 AM – Scan the membrane in a dark room: Like photo film, we can soak the membrane in a fluorescence solution and then put it in a scanner so we can get an image of the protein concentration across different samples. This process is called Western Blotting.
- 10:00 AM – I have to repeat this process with a control to make sure I loaded the sample concentration of protein in all my samples. I’ll soak the membrane in a different primary antibody overnight.
- 10:30 AM – Clean Up and Leave
- THURSDAY
- 7:00 AM – Split the cells again. I’ll also treat the experimental plate with all the cells and drug concentrations with a color-changing solution that turns orange if cells are alive. I can interpret how many cells are alive at certain drug concentrations by how orange the solution turns.
- 9:00 AM – Repeat the washing and secondary antibody process and then scan
- 12:00 PM – Clean Up and Leave
- FRIDAY
- 12:00 PM – Attend lab meeting with my Principal Investigator (PI/mentor) and other students to share my results and listen to other students’ results.