At the time of writing this post, no scientist had yet discovered the secret to immortality. In our world, we’ve come to accept that living things are born, grow old and die—the circle of life.
And yet, for many years, life scientists believed that the circle of life did not apply to our constituent cells when cultured in a laboratory. That is, cultured normal human cells were immortal, and they would continue to grow and proliferate forever, as long as they were provided with the necessary nutrients.
Pioneering work published in 1961 by Leonard Hayflick and Paul Moorhead challenged that theory (reviewed in 1). Their research showed that normal cells in culture have a finite capacity to replicate. After they reach a certain number of replicative cycles, cells stop dividing. Hayflick and Moorhead made the important distinction between normal human cells and cultured cancer cells, which are truly immortal. In later years, the limit to the number of replicative cycles normal human cells can undergo became known as the Hayflick limit. Although some scientists still express skepticism about these findings, the Hayflick limit is widely recognized as a fundamental principle of cell biology.
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”
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.
In today’s post, guest blogger, Martha O’Brien, PhD, provides a preview of her upcoming AAI poster and block symposium talk on the inflammasome, caspase-1 activity and pyroptosis.
Responding rapidly to microbial pathogens and damage-associated molecular markers is critical to our innate immune system. Caspase-1 is pivotal in this process leading to processing and release of essential cytokines and an immunogenic form of cell death, termed pyroptosis. Upon sensing pathogen-associated and damage-associated molecular patterns (PAMPs and DAMPs), innate immune cells form inflammasome protein complexes that recruit and activate caspase-1 (canonical inflammasomes). In addition, other inflammatory caspases, 4 and 5 in humans and 11 in mice, directly bind bacterial lipopolysaccharides (LPS), triggering pyroptosis (non-canonical inflammasome). LPS-triggered non-canonical inflammasomes in mice and humans ultimately lead to canonical inflammasome engagement and caspase-1 activation (1–3). Caspase-1 was originally termed interleukin converting enzyme (ICE) for its well-established role in processing IL-1ß and IL-18, two important inflammation cytokines. How caspase-1 mediates pyroptosis is less well understood, but is beginning to be delineated. Recently, a substrate of the inflammatory caspases, gasdermin D, was identified and its processed fragment, gasdermin-N domain, was shown to be required for pyroptosis in non-canonical inflammasome circumstances (4, 5). The precise role of gasdermin D in canonical inflammasome-triggered pyroptosis is still under investigation. Linking inflammatory caspases directly to pyroptosis is a notable step in understanding the mechanism of this important form of cell death.
Pyroptosis is clearly one means of releasing processed IL-1ß and IL-18 from the cell. However depending on the cell type and stimulus, there is evidence for inflammasome engagement, caspase-1 activation, and release of IL-1ß in the absence of cell death (6, 7). On the flip-side there is also evidence for caspase-1 mediated pyroptosis that helps clear bacteria, independent of IL-1ß and IL-18 involvement (8). To enable further studies on the inflammasome and in particular, assessing the connections between caspase-1 activation, pyroptosis, and cytokine release, Promega developed a new tool to conveniently monitor caspase-1 activation, the Caspase-Glo® 1 Inflammasome Assay. This bioluminescent, plate-based assay is used to measure caspase-1 activity directly in cell cultures or to monitor released caspase-1 activity in culture medium from treated cells. This flexibility allows easy multiplexing to monitor all three outcomes of inflammasome stimulation; caspase-1 activity, pyroptosis, and release of IL-1ß and IL-18. Caspase-1 activation typically is monitored indirectly with western blots of processed caspase-1. Now the activity of the enzyme can be monitored directly, providing accurate information on temporal aspects of the inflammasome. The assay can be readily combined with real-time measures of cell death (e.g., CellTox™ Green Cytotoxicity Assay) and some of the culture medium can be removed for IL-1ß/IL-18 assessment, leaving the cells and remaining culture medium for caspase-1 activity measurements. At the upcoming meeting of the American Association of Immunologists (AAI) in Seattle, May 13th-17th, oral and poster presentations will highlight use of the Caspase-Glo® 1 Inflammasome Assay and its value for exploring the relationship between inflammasomes and pyroptosis.
The concept of cell death as a normal cell fate was articulated only three years after Schleiden and Schwann introduced the Cell Theory when, in 1874, Vogt described natural cell death as an integral part of toad development (as cited in Cotter and Curtin, 2003). Since these early observations, natural cell death has been described “anew” several times. In 1885 Flemming provided the first morphological description of a natural cell death process, which we now label “apoptosis”, a term coined by Kerr and colleagues to describe the unique morphology associated with a cell death that differs from necrosis (as cited in Kerr et al. 1972).
In the 1970s and 1980s, studies revealed that apoptosis not only had specific morphological characteristics but that it was also a tightly regulated process with specific biochemical characteristics. Studies of cell lineage in the nematode, Caenorhabditis elegans, showed that apoptosis was a normal feature of the nematode’s invariant developmental program (Hengartner, 1997). At the biochemical level, Wyllie showed that DNA degradation by a specific endonuclease during apoptosis resulted in a DNA ladder composed of mono- and oligonucleosomal-sized fragments (Wyllie, 1980).
These and many other studies have proven that apoptosis is a critical component of development, and when it doesn’t happen appropriately, it can be pathological, leading to cancers or other diseases. Therefore, understanding how and when apoptosis occurs and the many signals that can trigger this process is a focus of many laboratory experiments.
Determining the exact cause/effect relationship between a treatment and a cellular outcome is not a simple matter, but is critical for really understanding how therapeutic treatments affect target cells or exercise any off-target effects.
Four key factors are critical for determining whether or not a particular treatment or compound is toxic.
On Saturday I ran 12 miles. “Well, at least I have staved off apoptosis in my peripheral blood mononuclear cells” I thought as I hobbled down the stairs on Sunday morning. Normally I don’t think about mononuclear cells on Sunday mornings, only of coffee. However, a paper published last week in BMC Physiology changed that for me, at least temporarily.
The paper, by Marfe et al, investigated whether the physiological stress associated with strenuous exercise may cause apoptosis and contribute to loss of lymphocytes. This paper investigated whether apoptosis is increased in cells of the immune system after running a marathon. The authors studied the expression of various stress-related proteins in peripheral blood lymphocytes in 10 male amateur runners, examining the expression of various antioxidants, stress proteins and apoptotic markers before and after (2-hours post-race) running a marathon. They found that expression of the apoptotic marker bax was decreased significantly after the marathon, while levels of antiapoptotic bcl-2 RNA increased. The amount of propcaspase 9 did not change pre and post race, indicating that there was no change in levels of apoptosis before and after the race.
Life is complicated. So is death. And when the cells in your multiwell plate die after compound treatment, it’s not enough to know that they died. You need to know how they died: apoptosis or necrosis? Or, have you really just reduced viability, rather than induced death? Is the cytotoxicity you see dose-dependent? If you look earlier during drug treatment of your cells, do you see markers of apoptosis? If you wait longer, do you observe necrosis? If you reduce the dosage of your test compound, is it still cytotoxic? Continue reading “Describing Life and Death in the Cell”
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