Jessica Hess, PhD student
University of Texas at El Paso, TX, USA
Jessica is a 2nd year PhD student at The University of Texas at El Paso. Jessica's research involves high-throughput screening of novel compounds for anticancer activity. She is currently focused on identifying the protein target of a potent compound that she found after screening more than 2,000 unique molecules. Most of Jessica's time in the lab is spent culturing various cancer and normal cell lines for use in cytotoxicity and mechanistic assays. The next step in her research is to analyze the transcriptome of cells treated with her compound to identify differentially expressed genes. Throughout the academic year, she also serves as a Teaching Assistant for undergraduate Biology lab courses. Outside of the lab, you can find Jessica working on science-themed stickers and stationery with the goal of encouraging science-driven communication through art. She hopes that her colorful designs provide scientists an opportunity to engage in meaningful conversation about their research with non-experts.
Learn about Jessica’s research
Title: Investigating how Tpz-1 may be used as a novel therapeutic agent in the treatment of human hematologic cancers
- Anti-cancer drug discovery
- High-throughput drug screening
Thienopyrazole derivatives have recently emerged as effective antitumoral agents with kinase inhibitory activity. In this study, a novel thienopyrazole derivative with potent and selective in vitro cytotoxicity was identified in a high-throughput chemical library screening to identify novel anti-cancer drugs. After 48 hours of exposure, compound Tpz-1 demonstrated potent and consistent cytotoxicity against a panel of human hematologic cells at nanomolar to low micromolar concentrations; an effect which was greatly diminished against the HS-27 non-cancerous foreskin fibroblast cell line. Mechanistic analyses subsequently conducted against the CEM T cell leukemia cell line revealed that Tpz-1 induces cell death via apoptosis and interferes with cell cycle progression in a dose-dependent manner. These findings merit the further development of Tpz-1 as a novel therapeutic agent in the treatment of human hematologic cancers.
Watch the webinar
Moderator: Hello everyone, and welcome to today’s webinar, Investigating how Tpz-1 May Be Used as a Novel Therapeutic Agent in the Treatment of Human Hematologic Cancers, presented by Jessica Hess, Ph.D. student, the University of Texas at El Paso. My name is Christy Jewel and I’ll be your moderator for today’s event. Today’s educational web seminar is presented by LabRoots and brought to you by Thermo Fisher Scientific. (00:34) For more information on our sponsor, please visit thermofisher.com/cellcultureheroes.
Now before we get started, I would like to remind everyone that this event is interactive. We encourage you to participate by submitting as many questions as you want, at any time you want during the presentation. To do so, simply type them into the Ask a Question box and click Send. We will answer as many questions as we have time for at the end of the presentation. (1:00)
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I now present today’s speaker, Jessica Hess, Ph.D. student, the University of Texas at El Paso. (1:29) For a complete biography on Jessica, please visit the Biography tab at the top of your screen. Jessica, you may now begin your presentation.
Jessica Hess: Hello everyone, my name is Jessica Hess, I’m a Ph.D. student in the Department of Biological Sciences at the University of Texas at El Paso. Today I’ll be discussing my experimental compound, Tpz-1, and its development as a potential therapeutic agent in the treatment of blood cancers.
In this webinar, I’ll start off by sharing a bit about myself, some cancer statistics, discuss current challenges of chemotherapy, then walk you through the initial process of identifying novel cancer killing compounds via high surface screening and cell-based assays. I’ll be using Tpz-1 as an example throughout. My work on Tpz-1 is very much ongoing, so I’ll conclude with some future directions for this project and leave those of you new to cell culture with some useful tips that have helped me along the way. (2:30) I hope you enjoy this presentation on my research and I look forward to answering some of your questions at the end.
So hello, and thank you for your time. That’s me, Jessica, and I’m just starting my second year in Dr. Renato Aguilera’s lab, where I’m working to find novel cancer killing compounds. Prior to starting my Ph. D., I earned my Bachelor’s degree in chemistry from Virginia Commonwealth University and worked as a nursing assistant on a pediatric medical/surgical unit. I actually had no formal lab experience when I started my Ph. D. journey. My motivation to pursue cancer research was largely because I have a strong family history of cancer diagnoses. (3:09)
In a random twist of fate, my family is originally from El Paso, so I feel extremely fortunate to have the opportunity to study cancer here. I’ve really grown to love what I do, and I couldn’t be more excited to talk about my project today. When I’m not in the lab, you’ll find me designing science-themed stickers and stationary for my business, Cell Kulture Co., which I started back in January of this year, after creating a fun logo for our lab. If you’re interested in seeing more of my art, you can find the complete catalogue of my creations at CellKultureCo.com. (3:41)
In addition to my art, I enjoy sharing my Ph. D. experience and a little bit of science through social media, primarily through Instagram and Twitter. I’m very, very excited to be this month’s cell culture hero, because as you may have guessed, cell culture is truly one of my favorite parts of my job.
Moving on to cancer statistics, this year in the United States, the American Cancer Society expects approximately 1.7 million new cancer cases. That’s the equivalent of over 4800 cases each day. They also predict around 600,000 cancer deaths. All these new cases and deaths—lymphoma, myeloma, and leukemia—will be responsible for about 10 percent. This is why the development of novel treatments for these cancers is important, and why these blood cancers will be the stars of this presentation.
The specific cancer treatment a patient will receive depends on the type of cancer they have and if it has spread and where. But most often, patients receive chemotherapy in conjunction with other treatment. Unfortunately, many chemotherapies have highly toxic side effects that lead to things like hair loss, nerve damage and GI issues. Some effects can even persist for years or be permanent. Depending on a person’s tolerance for these side effects, they can really limit clinicians from using effective drug doses to treat their cancer. (5:06) In addition, cancer cells are constantly acquiring new mutations and finding a way to evade death, so drug resistance is a recurring issue. There’s a continuous need for novel cancer therapies which address these challenges and are more targeted towards cancer cells. Knowing this, the lab I work in conducts high-throughput compound screening to identify novel compounds, which are effective in killing cancer cells and will hopefully kill them via protein target that is expressed primarily in cancer.
High-throughput screening is a method used to analyze large compound libraries. In an initial compound screening, I can test up to 80 individual compounds in one 96-well plate. At this stage, I only use one concentration, typically five micromolars. The purpose of the initial screen is just to identify compounds with the ability to kill 50 percent or more of the cells. (6:01) After a compound has been identified in an initial screening, I test it again in a secondary screening to confirm its activity. At this stage, I treat cells with a gradient of the compound to calculate CC50 or the concentration of the drug which kills 50 percent of the cells. Drugs with CC50s in the nanomolar range are typically what I look for. So if a drug has a low CC50, I’ll start working my way through cell-based assays to try and clarify its mechanism of action.
The differential nuclear staining assay is a live-cell imaging technique that I use to determine cytotoxicity. It requires seeding my cells in a 96-well plate, exposing those cells to a drug, which in my case is Tpz-1, and leaving them to incubate usually for 24 to 72 hours. Then I come back and add two nuclear dyes, called Hoechst and propidium iodide, and then acquire cell images. (6:57) I use the GE IN Cell Analyzer 2000. Finally, after I take those images, I analyze them to quantify the living and dead cell population. This assay works by distinguishing these populations based on membrane integrity. Hoechst is a dye capable of permeating the membrane of all cells in my sample, whereas propidium iodide will only permeate those cells with compromised membrane. Therefore, colocalization of the two dyes is representative of the dead cell population. (7:30) I use this assay in primary high-throughput screenings, as well as when I’m retesting the compounds to find their CC50.
Now, I’d like to introduce you to Tpz-1. Unfortunately, I can’t show you the full structure at this time, but I can tell you that it’s a thienopyrazole derivative. This compound is special because thienopyrazoles are a largely under-explored class of compounds which generates less than ten results when I search the term on PubMed. However, the publications associated with these compounds identify them as effective antioxidants, antitumor agents and kinase inhibitors. (8:03) I found this compound while conducting a high-throughput screening project of over 2000 compounds and have been working with it ever since. All the data I’m about to show you has not yet been published but merits the continued study of Tpz-1 and its potential as an anticancer agent.
In the table shown, you’ll find the CC50s I calculated by testing Tpz-1 against a panel of human blood cancers, as well as one, non-tumorigenic cell line. Tpz-1 demonstrated potent cytotoxicity in the nanomolar to low micromolar range against all the cancer cell lines I used. Interestingly enough, the cytotoxic effect was greatly diminished upon testing Tpz-1 against the non-cancerous cell line (8:53) Because of its low CC50 and my familiarity with the stem cell line, this cell line was selected for use in subsequent assays to determine if Tpz-1 kills cells via apoptosis. Apoptosis is a form of programmed cell death which involves the controlled dismantling of intracellular components, avoiding inflammation and damage to surrounding cells. All of the assays I will discuss from this point on were analyzed by flow cytometry.
The first apoptosis experiment I conducted was an Annexin V-FITC/PI assay, to determine if phosphatidylserine was being externalized. Phosphatidylserine, which is normally present on the inner leaflet of the plasma membrane, is flipped to the cell surface during the early stages of apoptosis. In the body, this serves as an eat-me signal, prompting phagocytes to engulf the cell. Annexin V is a cellular protein with high affinity for phosphatidylserine. (Efflorescent 50 conjugate) was used to selectively label cells undergoing apoptosis. (9:59) In contrast, propidium iodide was used to detect necrosis because it will only permeate the cells which have lost their membrane integrity. Total apoptosis was calculated by summing early apoptotic and later apoptotic cell populations, which are both Annexin V-FITC positive and can be either PI positive or negative. The Annexin V-FITC negative but PI positive population is representative of necrosis. (10:28) The results of this assay demonstrate that Tpz-1 treatment preferentially induces cell death by apoptosis.
Next, I went on to a caspase-3 assay. Caspases are proteases which play an essential role in apoptosis. Initiator caspases, as their name implies, initiate apoptosis. They go on to activate executioner caspases, such as caspase-3, which then go on to demolish key structural proteins leading to the classic apoptotic morphological features, such as DNA fragmentation and membrane (inaudible). (11:03) Tpz-1 treatment elicited dose-dependent caspase-3 activation at eight hours, further implying that cell death occurs by apoptosis.
Next, I evaluated the accumulation of reactive oxygen species in cells treated with Tpz-1. Reactive oxygen species are indicative of oxidative stress and could mean that mitochondria are being affected. Tpz-1 treatment behaves similarly to my positive control, hydrogen peroxide, and induced significant ROS accumulation in cells after 18 hours of exposure. The next step after conducting this experiment is typically a JC-1 assay, which detects changes in mitochondrial membrane potential. (11:45) I performed the JC-1 assay, but my results were inconclusive, so it’s still not clear to me if my cells are dying via the intrinsic apoptotic pathway.
Finally, I examined the effect of Tpz-1 on the cell cycle. This assay worked by using DNA-binding dyes that bind in proportion to the amount of DNA present in the cell. Therefore cells in the S phase will take up more dye than cells in G-1, but less than cells in the G-2 phase. When compared with the profile of untreated and DMSO-treated cells, Tpz-1 treatment caused significant dose-dependent disruption. It’s important to note that accumulation of cells in the sub-G0/G1 phase was observed, and fragmentation due to apoptosis.
Currently I’m working on my first manuscript for Tpz-1, and I’m in the process of sorting through transcriptome data. The transcriptome provides me with the gene expression profile of cancer cells after treating them with Tpz-1 and will help me in piecing together Tpz-1’s function and deciding which assays I should perform next. Also in the future, I’ll be looking into some structural analogues of Tpz-1 to compare their cytotoxic activity, and also assessing Tpz-1 against more cancer types and non-tumorigenic cell lines, to determine if the compound is at all selective towards one type versus another. (13:17)
For those of you just starting cell culture, don’t worry, there’s lots of resources out there to get you started. For any general cell line information you may need and culturing methods, I recommend checking out the American Type Culture Collection website shown here. For all cell culture basics, Gibco has you covered with virtual labs, tutorials and handbooks, which are really useful if you’re a hands-on learner like me. (14:49) My personal advice I’ll leave you with is this.
One, seek the advice of colleagues with experience culturing the cell lines you’ll be using. They’ll be able to really tell you if they used an alternative media from that listed in ATPC, or if they had any struggles in maintaining viability of their culture.
Two, be prepared to spend lots of time getting to know your cells. Each cell line is unique. Some prefer to be in closer proximity, while others like to be more spread out. (14:16) Some cells do well with passaging every two or three days, while others I’ve had to passage almost daily.
Three, consider how the cells in your culture are communicating with each other when you’re passaging them. Viability can be really ard to recover if you accidentally over-dilute them, so it’s better to err on the side of caution.
And lastly, always handle your cells carefully and while using your best aseptic technique. I always make sure to visually inspect my cells before and after handling them, this way I can spot any contaminates, weird morphology changes and see if I’ve diluted my cells to a suitable density.
With that, I conclude my presentation, and I’d like to extend a special thank you to everyone that has helped me thus far, especially those mentioned here. I’d now like to open it up to questions, so please feel free to ask away.
Moderator: Thank you Jessica, for your informative presentation. Now we will start the live Q&A portion of our webinar. If you have a question you’d like to ask, please do so now. Just click on that Ask a Question box located on the far left of your screen, and we will answer as many of your questions that we have time for. Okay, let’s get started, and wow, we’ve got some great questions coming in. All right, Jessica, let’s start with this question here. How does the differential nuclear staining assay differ from more common cytotoxicity assays, such as MTS and why does your lab use it?
Jessica Hess: Thank you, Christy, that’s a great question. MTS uses enzymatic activity to measure viability, where DMS measures viability based on membrane integrity. My lab uses it because it only has a few steps, most of which are simple. We don’t have to wash cells or manipulate them really at all after they’ve been seeded. So first I plate the cells and then I apply my treatment and then I just pop them in the incubator until I’m ready to add dye. (16:32) Then once I add the dyes, I wait for two hours, image them and I’m able to segment those images using a software that gives me an Excel sheet that will tell me percentages of living and dead cells.
One of the limitations would be the high cost of the equipment. We’re really fortunate to have a high content analyzer, like the GE IN Cell, which automates the imaging process for us. (17:01) It uses a 10x objective to capture a 2x2 montage on two different fluorescent channels at once. So we’re able to visualize thousands of cells, versus the few hundred that would be possible with manual counting.
Moderator: Thank you Jessica. Can you share with us, what are you plans for when you graduate? Are you still hoping to pursue cell culture?
Jessica Hess: Ideally yes, I really find it enjoyable. I like the challenge of getting to know all my different cell lines. As I mentioned in my (inaudible @ 0:17:49 - interference), they’re all kind of different. So when I first sort some cells out, I check on them every day, really, and I really kind of try and get to know how fast they divide, how confluent they like to be and that can sometimes be a real challenge. (18:07) So I hope to continue doing cell based assays. I would like to continue in cancer research, as well.
Moderator: Can you please explain why you selected DMSO as a control?
Jessica Hess: So DMSO serves as my vehicle control, it’s what I dissolve Tpz-1 in. Some drugs don’t dissolve in PBS or saline, and mine is a prime example, so I have to use DMSO.
Moderator: I have another question for you here. Why can you not reveal the structure of Tpz-1?
Jessica Hess: Well, everything I presented today is unpublished. I’m in the process of piecing together the first draft of my manuscript, but it’s been kind of a slow process for me, because it’s my first time. Also, I’m not sure if the structure has been patented or not, so that may be looked into in the future as well.
Moderator: Thank you Jessica. Can you please elaborate on how the reactive oxygen species assay detects ROS?
Jessica Hess: Okay, so the ROS assay uses (inaudible @ 0:19:29) permeable dye, that generates green florescence when it interacts with reactive oxygen species. I usually treat my cells with my 24-hour CC50 that I’ve calculated and twice that concentration, and then I incubate for 18 hours before assessing for the reactive oxygen species.
Moderator: Here’s a great question. Jessica, can you share with us, do you have any advice for a new Ph. D. student entering their program with no experience in the field?
Jessica Hess: Try not to get caught up in feeling like you’re behind because you maybe started off with less knowledge than your peers. I believe that I was accepted to my program for a reason, and that’s true for everyone that’s been accepted to a Ph. D. program, they really saw some potential in you. So just be patient with yourself and give yourself plenty of time to review and get used to grad school, in general. (20:37) It’s a huge change from being an undergrad or just having a job, in general. It kind of helps, but it’s a lot more enjoyable to study and learn, because you’re finally being taught all the things that you probably wanted to learn about your whole life. So that’s (inaudible).
Moderator: Thank you for that. Let’s go back to Tpz-1. Does Tpz-1 share any similarities with other lead compounds your group has identified?
Jessica Hess: As far as I know, my compound has a pretty unique structure. It also looks different than the other active compounds I found within my own high-throughput screening. I did find a couple others, which just happened to be analogues of some other compounds we’re working on. But I haven’t really seen a similar structure. (21:38) If you look for recent publications with Renato J. Aguilera as an author, you can see some of the compounds that we found recently, and also learn a little bit more about the assays that I discussed today, because we all follow a similar initial screening process.
Moderator: Thank you Jessica. Do you have any final comments for our audience?
Jessica Hess: Yes, I just wanted to thank Gibco and LabRoots so much for this opportunity, especially you, Christy and Chelsea, for your guidance, and to everyone in the audience for taking the time to watch my first ever presentation on my research. If you have any questions or you’d like to collaborate, please feel free to contact me via social media, my handle is cellkultureco. You can message me through my website, cellkultureco.com or email me at email@example.com. (22:44)
I would suggest that you all check my website after this presentation for a little celebration sale. Finally, I just wanted to go back to these cell culture tips. I really, really recommend copying down these websites and using them for all things cell culture. We actually use the virtual labs in our facility to kind of help train the orientees. Gladys was kind enough to show me exactly the ones that she sent yesterday, and I was kind of surprised to see it’s on this exact same page as the cell culture heroes. So keep an eye out for that.
Moderator: Thank you Jessica. I want to thank our audience for joining us today and for their interesting questions. We had some great questions coming in. The questions that we did not have time for today and those that continue to come in during the on-demand period, will be addressed by Jessica via the contact information you provided at the time of registration. Jessica, we’d like to thank you today for your time and for your important research.
I would also like to thank LabRoots and I’d like to thank our sponsor, Thermo Fisher Scientific, for underwriting today’s educational webcast. This webcast can be viewed on demand, and LabRoots will alert you via email when it’s available for replay. (24:17) We encourage you to share that email with your colleagues who may have missed today’s live event. That’s all for now, we hope to see you again soon. Have a great day.
End Presentation (24:27)
Get to know Jessica
Is outreach/STEM important to you? Why?
STEM outreach is important to me because public funding is critical to the progression of scientific research. Outreach through social media and community events allow scientists an opportunity to connect with individuals that would otherwise not understand how their tax dollars and donations are spent. Outreach initiatives have the potential to empower more individuals to vote or donate in support of research initiatives.
Why did you become a scientist?
Cancer treatments, because of their off-target effects, can be as fear-inducing as the disease itself. Some side effects can persist for years, long after treatments have stopped. I became a scientist because there is a critical need for more targeted treatment options for all cancers.
“I am among those who think that science has great beauty.” —Marie Curie
Which scientist current or past would you most like to meet and why?
I would really like to meet James P. Allison because I admire his work on CTLA-4. Who would have thought the immune system could be harnessed to fight cancer?! Incredible.
If you could choose any other career what would it be? Why?
I have always wanted to be a scientist in some capacity, but I also really enjoy direct patient care and children. In another life, I could see myself being a pediatric oncologist.
Who is the person you are closest with?
I’m closest with my mom. She has shown me what it means to be a strong, independent Latina that believes in her potential.
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