By Amanda Cordle, August 2021
Dr. Jason Rawlings, a Furman Biology professor that specializes in Immunology, and his group of research students—Peyton Wortz, Eme de Graaf, Chase Hudson, Ansley Roberts, Kelsey Saunders, and Jonathan Davis — are studying the fundamentals of cell proliferation in hopes that in the future, doctors will be able to manipulate cell proliferation to treat certain health concerns.
Cells multiply through the process of mitosis so that our bodies can grow and continue normal function. There are times when the rate of proliferation may naturally increase or decrease to keep us healthy. For instance, if a pathogen enters the human body, immune system cells begin to multiply very quickly to fight off the illness. However, there are health risks associated with any cells proliferating too slowly or too rapidly. For instance, tumors, many of which are cancerous, are marked by an extremely high rate of cell division.
One way we can treat these issues is to change the rate of cell proliferation through medication, but researchers must learn more about the mechanisms that control cell division before pharmaceutical research can begin.
The goal in Dr. Rawlings’ lab this summer is just that—to learn about the mechanisms that control cell division. There are two broad areas of focus within this summer research—analyzing the role of intracellular calcium on chromatin decondensation and studying the effectiveness of synthesized molecules to inhibit cell division.
Roll of Intracellular Calcium on Chromatin Decondensation
Furman biology majors Peyton, Eme, Chase, and Ansley are analyzing how intracellular calcium in immune system cells influences chromatin reconfiguration—the first step of cell division.
“In Dr. Rawlings’ past research, he found that alpha-beta T cells rely on intracellular calcium to decondense their chromatin,” Eme explains.
Because alpha-beta T cells and B cells are similar in that they both proliferate rapidly during an immune response to attack pathogens, Eme and Peyton are researching whether intracellular calcium has the same effect on B cells as it does on alpha-beta T Cells.
“We have a single cell suspension taken from the spleen of mice. We place the cells in different groups and test if we take away calcium in some groups and have calcium in other groups, which one inhibits chromatin decondensation,” Peyton shares.
So far, there is preliminary evidence to suggest that B cells do require intracellular calcium for their chromatin to decondense.
Chase and Ansley are also studying the effect of intracellular calcium on cell proliferation, but instead of B Cells, they are researching gamma-delta T Cells. In addition to analyzing the role of calcium in these cells, Chase and Ansley are also hoping to learn more about these cells in general.
“The Bio World doesn’t really know much about gamma-delta T Cells and the mechanisms that control them, so…we’re studying if they are similar to the alpha-beta T cells that Dr. Rawlings has studied in past experiments,” Ansley explains.
Biologists don’t know a lot about gamma-delta T Cells simply because they are harder to study.
“The reason that we don’t have a lot of data on gamma-delta T Cells is because they’re a very small subset of the T cell population [in the spleen which is where samples are typically taken from], and so, we have to use a lot more cells in our experiments to analyze the gamma-delta T Cells compared to the alpha-beta T Cells,” rising senior Chase explains.
However, there are important things we do know about gamma-delta T Cells. For instance, they are common in mucosal tissue, such as the gut, and there are two different subtypes of them.
“One of the features of gamma-deltas that makes them interesting is that a subset of them seems to have an activated phenotype already, meaning that even though you aren’t sick, they’re already in an activated state,” Dr. Rawlings shares. “Our data shows that they have a baseline chromatin status that is more decondensed than an alpha-beta T Cell…meaning that they should be able to respond to infection faster.”
This data may have far-reaching effects within the medical world. For instance, better understanding gamma-delta T Cells could lead to better treatment for autoimmune disorders which cause immune cells to attack healthy cells.
“Because of their location, gamma-deltas are also implicated in certain autoimmune diseases, for example, Crohn’s,” Dr. Rawlings explains. “Crohn’s disease is an autoimmune disease that affects the gut, and so…Let’s target those gamma-deltas and keep them from proliferating; that might be a great treatment for Crohn’s.”
In addition to learning that some gamma-deltas have automatically decondensed chromatin, Chase and Ansley have also discovered that the gamma-delta T cells that are not of this subtype behave similarly to alpha-beta T cells. Furthermore, their evidence suggests that intracellular calcium is necessary for gamma-deltas to proliferate.
Hibiscone C and its Effects on The Cell Cycle
Rising seniors Kelsey and Jonathan are also researching ways to inhibit cell proliferation, but their focus is on the PI3 Kinase inhibitor, Hibiscone C, which Chemistry professor Dr. Goess synthesized and introduced to Dr. Rawlings about a decade ago.
At the time, Dr. Rawlings had experience with a molecule called Wortmannin, which is also a PI3 Kinase inhibitor, although less stable than Hibiscone C.
“PI3 Kinase is an enzyme that is found in almost every cell of the body,” Dr. Rawlings explains. “It regulates cellular metabolism through another protein called AKT. So, if a cell wants to ramp up metabolic activity, it needs to turn on AKT. AKT gets turned on by a phosphorylation event—when it’s phosphorylated it is on.”
Understanding the PI3 Kinase and AKT relationship is important because if scientists could decrease AKT phosphorylation, it would create an opportunity for the development of new cancer treatments. Cancer cells have a very high rate of metabolism to sustain their rampant rate of proliferation. This high metabolic rate is caused by an increase in AKT due to elevated expression of PI3 Kinase, which is the same mechanism that causes T Cells to divide quickly during an immune response.
Due to Dr. Rawlings’ work with T cells, as well as his prior experience with Wortmannin, he and Dr. Goess began a collaborative research project on Hibiscone C about ten years ago.
Over the past decade, Dr. Rawlings’ students have used a Western Blot Assay to study Hibiscone C. Through this research, they were able to draw important conclusions about the biologic activity of Hibiscone C.
“We published a paper a few years back…showing that Hibiscone C does have biological activity. It does inhibit the phosphorylation of AKT, and it does have impacts on cell viability and so on,” Dr. Rawlings shares.
While this published information is promising, there are weaknesses associated with the Western Blot Assay, such as the fact that it is not a quantitative measurement tool, which is why Kelsey and Jonathan are continuing research on Hibiscone C this summer.
“If we want to be able to quantitate the differences between Hibiscone C and Wortmannin or make modifications to Hibiscone C…in order to increase its potency or increase its selectivity, we need to have a way to quantify whether or not we’re making changes,” Dr. Rawlings explains. “And so, the way the project works historically between our two labs, is that the Goess lab will synthesize a derivative of Hibiscone C by altering part of the molecule, and then we test the biological activity and let them know yeah this increased the potency or this decreased the potency, and that helps guide the synthesis of new molecules.”
Kelsey and Jonathan’s goal this summer is to create a flow cytometric assay for phosphorylated AKT so that future research students can quantitatively study the effects of Hibiscone C molecules on PI3 Kinase in a variety of conditions.
“It’s been a lot of trial and error … but we have made some progress. We’re just trying to create the best assay possible to analyze our data,” Kelsey explains.
While this may sound straightforward, the project is very complex.
“Our days consist of culturing cells…We always begin with dissecting mice, taking the spleen, and activating them in culture. We keep the lymphocytes alive for…five-ish days, which is when the lymphocytes are in their fully activated state and proliferating like crazy….and that’s the point in which we will treat them with drugs,” Jonathan shares.
In addition to going through this whole process to simply have testable cells, the actual act of treating them is incredibly difficult.
“With regard to Kelsey and Jonathan, they are trying to use the flow cytometer to detect an intracellular protein AKT …The way we do this is we use an antibody to detect that target protein, but antibodies can’t penetrate the surface of a cell…and so we have to actually open that cell up and allow that antibody in,” Dr. Rawlings explains. “If you open the cells up…it causes them to fall apart, so we have to fix the cells first.”
However, if the cells are overly preserved, then the antibody cannot get in. Creating the right conditions so that the fixation allows holes to be poked in the cell, but that it also keeps the cell from falling apart, has been a trial-and-error process for Kelsey and Jonathan.
Jonathan and Kelsey’s main goal this summer is to create an assay that can consistently quantify the results of different Hibiscone C molecules. Since their focus is on the assay itself and not on analyzing the results of the molecule on PI3 Kinase, Jonathan and Kelsey have not drawn any conclusions about which Hibiscone C derivative is strongest, or other ramifications it may have on cells besides inhibiting PI3 Kinase. However, they did show that AKT phosphorylation behaves differently in activated and inactivated T Cells.
While the students in Dr. Rawlings’ lab are working on different research projects, the overall focus of Dr. Rawlings’ lab is to gain a better understanding of the mechanisms of cell proliferation. This research could have far-reaching effects in the medical world, such as the creation of new cancer treatments that stop cancer’s rampant proliferation and spread, or a new way to inhibit lymphocyte proliferation in autoimmune diseases.