Glucose is an energy metabolite necessary for cellular survival and growth whether or not the cell is part of a tumor. Not only do cancer cells switch from oxidative phosphorylation to aerobic glycolysis (the Warburg effect) to gain more glucose, a hallmark of cancer, but they also increase the amount of glucose taken up from the surrounding extracellular space. However, the lack of glucose can have a negative effect on cells, causing them to become apoptotic in the absence of this metabolite. Cancer cells have methods to get around the requirement for glucose, including upregulating glucose transporters to improve access to the energy metabolite. In this Redox Biology article, researchers describe how activating androgen receptor in response to a lack of glucose affects the amount of GLUT1 expressed on prostate cancer cells, making the cells resistant to glucose deprivation.
To set the stage, two prostate cancer cell lines, LNCaP, an androgen-sensitive cell line, and LNCaP-R, an androgen-insensitive cell line, were deprived of glucose. Both cell lines showed signs of cell death, but LNCaP-R cells died in greater numbers. To probe how LNCaP cells died, several inhibitors (a pan-caspase inhibitor, two necroptosis inhibitors and a ferroptosis inhibitor) were added but did not change the way the cells died. However, an autophagy inhibitor enhanced cell death, suggesting the cells were necrotic not apoptotic. Teasing apart if the necrosis of LNCaP cells was due to glucose availability or merely disrupted glycolysis, the glucose analog 2DG was added to the medium with glucose. The cells survived when treated with 2DG, suggesting it was the absence of glucose that induced necrosis. When LNCaP cells were cultivated in medium that replaced glucose with mannose or fructose, the cells survived, another point in favor of sugar depletion causing cell death. Continue reading “How Prostate Cancer Cells Survive Glucose Deprivation”
Glycobiology is the study of glycans, the carbohydrate molecules that cover the surface of most human cells. Glycans attach to cell surface proteins and lipids, in a process called glycosylation. These cell surface structures are responsible for processes as varied at protein folding, cell signaling and cell-cell recognition, including sperm-egg recognition and immune cell interactions. Glycans play important roles in the red blood cell antigens that distinguish blood types O, A and B.
Opportunities in Glycomics Research
As more is learned about the role of glycans in cell communication, they are becoming important disease research targets, particularly the role of glycans in cancer and inflammatory diseases (2).
When someone is admitted to a hospital for an illness, the hope is that medical care and treatment will help them them feel better. However, nosocomial infections—infections acquired in a health-care setting—are becoming more prevalent and are associated with an increased mortality rate worldwide. This is largely due to the misuse of antibiotics, allowing some bacteria to become resistant. Furthermore, when an antibiotic wipes out the “good” bacteria that comprise the human microbiome, it leaves a patient vulnerable to opportunistic infections that take advantage of disruptions to the gut microbiota.
One such bacteria, Clostridium difficile, is of growing concern world-wide since it is resistant to many different antibiotics. When a patient is treated with an antibiotic, C. difficile can thrive in the intestinal tract without other bacteria populating the gut. C. difficile infection is the leading cause of antibiotic-associated diarrhea. While symptoms can be mild, aggressive infection can lead to pseudomembranous colitis—a severe inflammation of the colon which can be life-threatening.
Over a hundred years ago William B Coley, the “Father of Immunotherapy”, discovered that injection of bacteria or bacterial toxins into tumors could cause those tumors to shrink. The introduction of bacteria had the side-effect of stimulating the immune system to attack the tumor. The field of cancer immunotherapy research—which today includes many different approaches for generating anti-tumor immune responses—originated with these early experiments.
Use of bacteria is one way to stimulate the immune system to attack cancer cells, others include use of cytokines, immune checkpoint blockades and vaccines. This Nature animation provides a simple overview of these methods.
The keynote speaker for this year’s International Symposium on Human Identification (ISHI), Andrew Hessle, describes himself as a catalyst for big projects and ideas (1). In biology, catalysts are enzymes that alter the microenvironment and lower the energy of activation so that a chemical reaction that would proceed anyway happens at a much faster rate—making a reaction actually useful to the biological system in which it occurs.
In practical terms, Andrew Hessel is the person who helps us over our inertia. Instead of waiting for someone else, he sees a problem, gathers an interested group of people with diverse skills and perspectives, creates a microenvironment for these people to interact, and runs with them straight toward the problem. Boom. Reaction started.
Transfection can sometimes seem more like an art than a science—the perfect transfection experiment being dependent on optimization of conditions, including cell density, transfection reagent and DNA:reagent ratio. No one reagent is perfect for every cell type, so there is the added challenge of optimizing performance in your cell line of choice—which may fall into the well-populated “difficult-to-transfect” category that includes many primary cells.
Among transfection reagents, Lipofectamine® (Thermofisher), and FuGENE® (Promega) are popular and widely used choices. Viafect™ Transfection Reagent is newer and less well-known, but gaining popularity as a high-performance, low-toxicity reagent that performs well across a wide range of cell lines. In head-to-head comparisons with FuGENE and Lipofectamine, Viafect outperformed or equaled the others for expression of transfected reporter genes and resulting cell viability (see the data in this article).
The story of ViaFect begins with Promega Custom Assay Services (CAS), a group that uses Promega technologies to construct made-to-order assays, typically in a cell line. Many projects from the CAS group involve transfecting cells with expression vectors and reporter vectors. In some instances, customers contact CAS to have an assay constructed in a difficult cell line, after attempting and failing, or experiencing difficulty building the assay themselves.
CAS projects start with a proof-of-concept experiment using transient transfection before moving on to production of a clonal, stable cell line. For difficult cell lines, the CAS group previously turned to electroporation after exhausting lipid-based transfection options. Electroporation often worked, but success came with a price—cytotoxicity. The CAS group challenged R&D to find a better solution—better transfection with low toxicity for difficult-to-use cells. The result of that challenge is the ViaFect™ Transfection Reagent. Continue reading “ViaFect™ Reagent: Building Assays in Difficult Cells”
Real-time, up-to-the-minute access to information provides new opportunities for scientists to monitor cellular events in ever more meaningful ways. Real-time cytotoxicity and cell viability assay reagents now allow constant monitoring of cell health status without the need to lyse or remove aliquots from plates for measurement. With a real-time approach, data can be collected from cell cultures or microtissues at multiple time points after addition of a drug compound or other event, and the response to treatment continually observed.
The CellTox™ Green assay is a real-time assay that monitors cytotoxicity using a fluorescent DNA binding dye, which binds DNA released from cells upon loss of membrane integrity. The dye cannot enter intact, live cells and so fluorescence only occurs upon cell death, correlating with cytotoxicity. Here’s a quick overview showing how the assay works:
More Data Using Fewer Samples and Reagents
The ability to continually monitor cytotoxicity in this way makes it easy to conduct more than one type of analysis on a single sample. Assays can be combined to determine not only the timing of cytotoxicity, but to also understand related events happening in the same cell population. As long as the readouts can be distinguished from one another multiple assays can be performed in the same well, providing more informative data while using less cells, plates and reagents.
Combining assays in this way can reveal critical information regarding mechanism of cell death. For example, assay combinations can be used to determine whether cells are dying from apoptosis or necrosis, or to distinguish nonproliferation from cell death. Combining CellTox Green with an endpoint luminescent caspase assay or a real-time apoptosis assay allows you to determine whether observed cytotoxic effects are due to apoptosis. Cytotoxic and anti-proliferative effects can be distinguished by combining the cytotoxicity assay with a luminescent or fluorescent cell viability assay. Continue reading “A Better Way to Understand How and Why Cells Die”
Cell viability assays are a bread-and-butter method for many researchers using cultured cells —everyday lab tools that are a part of many newsworthy papers, but rarely make news themselves.
Over time, cell viability assays have become easier to use and more “plug ‘n play”. Among modern assays, luminescent plate-reader based systems have been a favorite for several years because of their superior sensitivity, robustness, simple protocols and uncomplicated equipment requirements (all you need is a plate-reading luminometer). These qualities combine to allow easy scalability and adaptability from bench research to high throughput applications.
CellTiter-Glo® Luminescent Cell Viability Assay is an accepted go-to viability assay for many researchers. The assay measures ATP as an indicator of metabolically active cells. A quick search on Google Scholar returns 3,990 CellTiter-Glo results for 2017 and over 500 so far in January and February of 2018. A sampling of these recent publications gives a snapshot of some of the ways the CellTiter-Glo assay is used to support key areas of research today.
Does a treatment kill cells?
The obvious application of a cell viability assay is to understand whether cells are alive. In cancer research, the CellTiter-Glo assay is often used to confirm killing of tumor cells and to verify that normal cells survive. Therefore, these assays are a key part of the evaluation and screening of drug candidates and other therapies for cancer. Many papers reporting use of CellTiter-Glo are developing and evaluating the effectiveness of novel anti-cancer treatments. Continue reading “A Cell Viability Assay for Today”
Recently, I had the opportunity to attend a fascinating symposium held at Promega featuring conservationist Steward Brand, where he described some of the projects developed by his foundation, Revive & Restore.
The organization’s mission is to apply emerging biotechnology techniques to endangered and extinct species with the intent to increase genetic diversity, provide disease resistance and facilitate adaptation to changing climates. Although the overall message of enhancing biodiversity through the application of new genetic technology was inspiring, the project that resonated most for me was related to the plight of horseshoe crabs.
Horseshoe crabs, often referred to as living fossils, include four extant species with origins dating back about 450 million years. Although they look like crabs, they belong to their own subphylum and are more closely related to spiders. When horseshoe crabs spawn, they leave their usual habitat on the ocean floor and migrate to shore in large numbers. As a result, they have been exploited for bait and fertilizer for decades.
Enter endotoxins, an indicator for bacterial contamination in biologicals, drugs and medical devices. U.S. Food & Drug Administration regulations dictate that finished products be tested for the presence of endotoxins. These pyrogenic compounds, found in the cell wall of Gram-negative bacteria, can cause fever and affect a wide range of biological activity, possibly leading to aseptic shock and death. The most common method for testing is the gel clot and Limulus Amebocyte Lysate (LAL) Test.
I first learned about the LAL test during graduate school, where it was presented as a ubiquitous and standard requirement for testing bacterial contamination in injectable drugs. I remember being fascinated that horseshoe crabs (Limulus sp.), contain a substance that could be used to detect endotoxins. Although the instructors mentioned the need to collect blood from horseshoe crabs in order to produce the test, the method or scale of this harvest wasn’t discussed, nor were the true costs of using this method of endotoxin testing.
The LAL test has served as a faster, more inexpensive replacement for the rabbit pyrogens test for the past 35 years. Every year during mating season horseshoe crabs move to shallow water, where they are removed in huge numbers. (To get an idea of scale for the harvest and read a much more comprehensive investigation of the issue, check out this article in The Atlantic, which features an archive photo of Delaware Bay horseshoe crab harvest from 1928—for fertilizer, not pharmaceutical testing.)
After collection, the crabs end up in a lab where up to 30% of their blood is drained from a needle stuck in tissue around their heart. The LAL is extracted from the blood and can yield a product worth up to $15,000/quart. In order to avoid recollection, the crabs are returned to the ocean far from the shore where they were collected a few days before. Although it’s estimated that only 10-30% of these crabs die as a result of the process, there are indications that the horseshoe crab population and their ecosystems are impacted in other ways.
Researchers at the University of New Hampshire and Plymouth State University used accelerometers attached to recently bled female horseshoe crabs to test the hypothesis that harvesting for LAL was affecting their ability to spawn. While the research supported previous estimates with a death rate of 18%, females were found to be less likely to mate after being bled.
During his talk, Brand shared results from a study still in review that confirm the effect of over-harvesting Limulus on the survival of long distance migratory shorebirds. These birds synchronize their migration with horseshoe crab spawning, which provides a needed feast of eggs before the homestretch of their journey. Along with other ecosystem threats from climate change, the accelerated decline in the horseshoe crab population and dependency of migratory birds will likely to lead to a devastating ecological domino effect.
Fortunately, a synthetic alternative to LAL, recombinant factor C (rFC), has been available for nearly 20 years. Alas, there has been no significant shift by pharmaceutical companies away from the test based on horseshoe crab blood. rFC was patented and licensed to one company, Lonza, which Brand posited as one reason for the reluctance of drug companies to adopt its use.
Obviously, relying on one source for an essential testing reagent with no competition to temper cost is quite unattractive. But that argument has less bearing now that the patent is scheduled to expire in a few months, opening the door for additional manufacturers and creating an economic incentive for switching to the synthetic test.
Another reason may be that implementing a new test would require additional resources to validate the synthetic test for products that are already being tested with the LAL. Since the LAL has been specified in FDA guidance documents on endotoxin testing for decades, quality standards for existing products are based on the LAL, limiting momentum to change.
If both tests offered the same benefits, these arguments would make sense; however, research by one of the discoverers of rFC, Jeak Ling Ding of the National University of Singapore, shows that in many respects rFC is more efficacious than LAL. Since the raw material for the LAL test depends on an organism, there is seasonal variation in the components of the processed blood that must be taken into account. The reaction of the LAL also depends on a cascade of multiple compounds that can be affected by temperature, pH and proteins—leaving the test vulnerable to false positive results.
Although Eli Lilly is the only pharmaceutical company to date to use rFC in place of LAL, It seems the tide may be turning. According to Brand, others are interested in making the transition. It seems foolish not to, given the source for LAL shows signs of dwindling due to overexploitation. Perhaps pharmaceutical companies are beginning to see the value of a “slower/better” philosophy (the cornerstone of the Long Now Foundation, another brainchild of Brand’s), rather than “faster/cheaper.” I have certainly gained a new perspective on endotoxin testing and a deep appreciation for the work of Brand and his foundation.
Does your organization use the LAL test? What is preventing you from switching to the synthetic alternative? Let us know!
Ubiquitin modification of a protein directs events such as targeting for proteasomal degradation. Targeting a protein for degradation through ubiquitin modification is one way to regulate the amount of time a signaling protein, such as a kinase or other enzyme, is available to participate in cell signaling events. Deubiquitinases (DUBs) are enzymes that cleave the ubiquitin tags from proteins, and they have been implicated in several diseases, including cancer.
With their roles in the stabilization of proteins involved in cell cycle progression and other critical processes, DUBs are promising targets for small molecule inhibitors, particularly since they may provide a “back door” for targeting otherwise intractable, undruggable proteins by modulating their half lives. However, finding small molecule inhibitors of the ubiquitin proteases to date has not been trivial. Here we highlight two papers describing the identification and characterization of small molecule inhibitors against the DUB USP7. Continue reading “Deubiquitinases: A Backdoor into Undruggable Targets?”