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Posts Tagged ‘HypOxygen’

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Poldip2 governs cellular metabolism under hypoxic conditions

Until recently, it was understood that the addition of the prosthetic group lipoate is essential to the activity of critical mitochondrial catabolic enzymes; however, its regulation was unknown. In their paper “Poldip2 is an oxygen-sensitive protein that controls PDH and αKGDH lipoylation and activation to support metabolic adaptation in hypoxia and cancer“, Paredes et al. prove that the Poldip2-controled lipoylation of αKDH and PDH is regulated under hypoxia and in cancer cells, most notably in triple negative breast cancer cells (TNBC).

Using a Don Whitley Scientific  H35 Hypoxystation to establish accurate hypoxic environments, the authors were able to manipulate Poldip2 expression, allowing them to make substantial contributions to the understanding of Poldip2’s essential role in metabolic function during both normoxic and hypoxic conditions. Additionally, they postulate that Poldip2 deficiency may specifically impact the fate and availability of Ac-CoA due to impaired TCA cycle function, potentially providing cancer cells with an advantage for cell proliferation or hypertrophy. Further research will require investigating the interplay between Poldip2 and HIF-1α as well as the regulation of Poldip2 expression at different oxygen concentrations – a task easily achievable thanks to the Hypoxystation’s intuitive interface for effortlessly adjusting the gas composition.

The Whitley  Hypoxystation provides the user with incredible flexibility as it can control oxygen down to 0.1% while providing a temperature and humidity controlled environment with ample working space. The atmosphere is constantly monitored and adjusted by the real-time feedback system to ensure accuracy.

 

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Culturing Cells in Ambient Air is Far From Physiological

Based on the premise that the physiological range of oxygen in tissues is between 1- 8%, and pathologies from cancer to diabetes are characterised by much lower oxygen levels, researchers worldwide are cultivating their cell cultures in the Hypoxystation by Don Whitley Scientific. The Hypoxystation provides physiologically relevant conditions for cell culture and manipulation to ensure authentic behaviour of cells. User-defined parameters for temperature, CO2, O2 and humidity, plus the workstation format, where cells reside throughout the entire duration of the assays, minimise the extra-physiologic oxygen shock that is known to negatively impact cell metabolism and growth.

Numerous recent publications by our Hypoxystation users demonstrate that cell culture conditions which mimic physioxia, are essential in avoiding the significantly impaired growth rates, reduced lifespan, and altered molecular behaviour encountered in cells cultivated at ambient conditions. Oxygen levels in tissues are in constant flux; they change in response to functional status and blood delivery in the organs, and this too can be re-created in the Hypoxystation through programmed oxygen profiling.

Recent research using the Hypoxystation to investigate hypoxia inducible factors and the array of signalling pathways that regulate angiogenesis, metabolism, redox homeostasis, inflammation, and cell death, and the many other processes which enable the cellular and organismal response to hypoxia, “highlights the importance of oxygen as a cell culture parameter when making physiological inferences” (Timpano and Uniacke, 2016).

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From: Burr et al. (2016) “Mitochondrial Protein Lipoylation and the 2-Oxoglutarate Dehydrogenase Complex Controls HIF1a Stability in Aerobic Conditions” Cell Metabolism 25, 740–752


To find out more about Hypoxystations or other DWS products, please call +44 (0) 1274 595728 or email sales@dwscientific.co.uk


 

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Hypoxia and the Hallmarks of Cancer: Angiogenesis and Metastasis

The following was provided by HypOxygen, our distributor of Hypoxic Workstations in the US – Hanahan and Weinberg’s “Hallmarks of Cancer” are at the root of the multi-step progression of cancer, and they are all influenced by hypoxia in the tumor microenvironment. In this mini-review series, HypOxygen has been taking a closer look at the way Hypoxystation users worldwide are delineating the effects of hypoxia on the Hallmarks of Cancer: so far, we’ve showcased Avoiding Immune Destruction and Tumour Promoting Inflammation and Genome Instability and Mutation and Enabling Replicative Immortality.

In the Hypoxystation, researchers working with cells in culture can mimic the physiological conditions that produce those characteristic Hallmarks. The Hypoxystation enables glove-less access to cultivate and manipulate cells under physiological conditions, in a HEPA-clean environment. Oxygen levels in the Hypoxystation can be reliably and accurately adjusted to below 1%, reflecting the high metabolism, low perfusion tumor microenvironment.

 

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Hallmarks Of Cancer
1. Inducing Angiogenesis

Angiogenesis and tumor-associated neo-vascularization are central to the progression of cancer, and hypoxia in the fast-growing, poorly perfused tumor setting is one of the main factors driving the formation of new vessels. Hypoxia in the tumor activates the hypoxia stress response, which is mediated at the cellular level by HIF, VEGF and many other cytokines, growth factors and guidance molecules. As a consequence, endothelial cells and pericytes proliferate and form new blood vessels, which are, however, disorderly and leaky, in turn exacerbating hypoxia in the tumor. Cancer treatment strategies striving to normalize tumor vessels for the purpose of improved drug delivery and alleviation of hypoxia in the tumor are showing great promise.

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2. Activating Invasion and Metastasis

As with the other Hallmarks of Cancer, metastasis and cancer progression are correlated with low oxygen levels in the tumor. HIF’s activate the expression of more than 1000 genes, numerous of which play a role in inducing genes involved in the EMT, through direct interactions with HRE’s at promotor sites and other mechanisms such as epigenetic alterations, like methylation/demethylation. Hypoxia promotes migration and invasion by facilitating the endothelial-mesenchymal transition, altering cell-cell contacts, and reducing adhesion to the extra-cellular matrix. Cancer cells and neighboring cells such as fibroblasts are all influenced by hypoxia, and all contribute to the restructuring of the tumor microenvironment. The effects of the Hallmarks of Cancer continually perturb and promote each other, as when hypoxia-driven metabolic reprogramming causes acidification of the extracellular microenvironment through increased production and secretion of lactate, in turn augmenting ECM remodeling and immune evasion. Similarly, formation of novel blood vessels enables extravasation and migration of cancer cells to form new tumors.

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HypOxygen at Tumour Microenvironment Workshop in Miami

This article was written by Burga Kalz Fuller, join her and HypOxygen at the 15th International Tumour Microenvironment Workshop in Miami

In most cancers, the hypoxic microenvironment affects the development and progression of tumours, driving alterations in gene expression, metabolism and cell signalling, and significantly influencing the Hallmarks of Cancer. So what about in vitro cancer research, do culture parameters matter? Definitely! Numerous studies have shown that even very brief exposure to ambient oxygen levels and temperature significantly impacts cell culture, behaviour and function of cells in vitro.

HypOxygen will be exhibiting our Hypoxystation at the 15th International Tumour Microenvironment Workshop in Miami from 27th – 29th April. The special focus there is on “Hypoxia, Angiogenesis and Vasculature”, reflecting the critical importance of hypoxia in the context of cancer. With the Hypoxystation, cancer researchers have their finger on the pulse of physiological cell culture.

 

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The Hypoxystation mimics the hypoxic conditions present in cancer, providing a closed workstation format with contiguous, stable low oxygen down to 0.1%. Precise oxygen, carbon dioxide, and humidity control within a temperature-controlled environment, as well as ample space for cellular manipulation, assays and microscopic observation, allow researchers to recreate physiological conditions. HEPA filtration, sterile steam humidification, and remote parameter monitoring are some of the features that make the Hypoxystation so unique.

Cancer research labs, who use a Hypoxystation to re-create hypoxic conditions in the tumour microenvironment, are publishing brilliant papers which demonstrate the influence of hypoxia on the Hallmarks of Cancer. Metabolic adaptation, sustained growth, resisting cell death, and angiogenesis are just some of the Hallmarks which are affected by hypoxia. Here are some recent highlights:

Hypoxystation users are showing that “culturing cells in ambient air, or ‘normoxia’ is far from physiological.

Visit HypOxygen at the 15th International Tumor Microenvironment Workshop in Miami

Ji Zhang presenting his poster at the Keystone Symposia

Hypoxia and Tumour Metabolism in Whistler with HypOxygen

This article was written by Burga Kalz Fuller of HypOxygen, giving an account of her and HypOxygen’s recent involvement at the Keystone meeting in Whistler, Canada. 

Really, a day in Whistler doesn’t get any better: talks on the newest results on hypoxia and tumour metabolism from morning till night, and outside the snow falls all day, every day. The joint Keystone Symposia on “Adaptations to Hypoxia in Physiology and Disease” and “Tumour Metabolism: Mechanisms and Targets” in Whistler, British Columbia last week featured both skiing and science, and HypOxygen was honoured to be a part of it all.

Joint sessions every day highlighted the many ways in which hypoxia controls gene expression, influences metabolic pathways, and regulates immunological and inflammatory processes, with new data showing how hypoxia affects the Hallmarks of Cancer. North American Hypoxystation users Navdeep Chandel, Nick Denko and Brad Wouters gave talks on respiration, mitochondrial function, and hypoxic regulation of autophagy. European Hypoxystation users Almut Schulze, Janine Erler and Ester Hammond spoke about glucose/lipid metabolism, ECM remodeling and DNA replication in hypoxia. Together, a global community of cancer researchers are targeting hypoxia as a key factor underlying tumour genesis and cancer progression.

Some of our own Hypoxystation users gave poster presentations: Ji Zhang (pictured top left) from Brad Wouters’ lab at Princess Margaret Cancer Center had a poster on “Characterizing oxygen metabolism and hypoxia tolerance in pancreatic ductal adenocarcinoma“, and Sara Timpano from Dr. Jim Uniacke’s lab at University of Guelph presented “Investigating cellular metabolism, DNA damage, and oxidative stress response under physiological oxygen conditions“. Hypoxystation users Navdeep Chandel, Nick Denko and Brad Wouters gave talks on respiration, mitochondrial function, and hypoxic regulation of autophagy, to name just a few.

 

Sarah Timpano presenting her poster at the Keystone Symposia

Sarah Timpano presenting her poster at the Keystone Symposia

We spoke to many of the Keystone attendees about our Whitley i2 Instrument Workstation and the Whitley H35 HEPA Hypoxystation by Don Whitley Scientific. The closed workstation format of the Hypoxystation provides reliable hypoxia down to 0.1% for cells accustomed to the very low oxygen customary in any body tissue, and especially in the tumour microenvironment. Precise oxygen, carbon dioxide, and humidity control within a temperature-controlled environment as well as ample space for cellular manipulation, assays and microscopic observation allow researchers to mimic and monitor physiological conditions. HEPA filtration, sterile steam humidification, and remote parameter monitoring are some of the features that make the Hypoxystation so unique.

As Jim Uniacke states in this video tutorial on creating physiological oxygen, “It is important to keep cells in the hypoxia workstation up until the point of lysis, as oxygen can rapidly alter the biochemical properties of these translation factors.” His lab has been producing exciting results on translation control at hypoxia with the Hypoxystation for several years, earning him the honorary title of “cancer cells’ worst nightmare.” Dr. Uniacke and all the other researchers at the Keystone symposia are working on conquering the nightmare of cancer, Hypoxygen and Don Whitley Scientific want to assist you in that endeavour where possible.

 

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Hypoxia and Tumor Metabolism in Whistler

Information on an upcoming exhibition in Whistler for HypOxygen. Words by Dr Burga Kalz Fuller.

At 2140 feet in Whistler, BC, the air will be getting thinner at the Keystone Symposia on “Adaptations to Hypoxia in Physiology and Disease” joint with the meeting on “Tumour Metabolism: Mechanisms and Targets”. But after all, hypoxia is what we do at HypOxygen, so we are very excited to be spending time at altitude with old friends and new ones on March 5-9.

At the Keystone Symposia in Whistler, HypOxygen will be exhibiting Whitley Hypoxystations for low oxygen cell culture under in vivo conditions. Conceived as an incubator workstation, but allowing gloveless access “to avoid spikes of normoxia” for cancer cells accustomed to very low oxygen, the Hypoxystation enables researchers to culture and manipulate cells growing at consistent oxygen, CO2, humidity and temperature. Another member of the Hypoxystation family, the i2 Instrument Workstation, was developed specifically to house instrumentation such as the Agilent Seahorse XF Analyzer for metabolism assays at hypoxia.

Since seeing is believing, we are greatly looking forward to talks and posters by a number of researchers who use Hypoxystations for their hypoxic cell culture. The broad range of these researchers’ presentations clearly illustrates how closely oxygen availability is linked to cancer cell behavior and metabolism, as the Hallmarks of Cancer are influenced and even determined by hypoxia in the tumour environment. These Hypoxystation users will be presenting data in Whistler:

  • Nicholas Denko, Ohio State University, USA
    Hypoxic Regulation of Mitochondrial Function
  • Almut Schulze, University of Würzburg/Theodor-Boveri Institute, Germany
    Targeting Glucose and Lipid Metabolism in Cancer
  • Janine T. Erler, University of Copenhagen, Denmark
    Hypoxia-Driven ECM Remodelling during Cancer Progression
  • Navdeep S. Chandel, Northwestern University, USA
    Why Mammalian Cells Respire?
  • Sara M. Timpano, University of Guelph, Canada
    Human Cells Cultured Under Physiological Oxygen Utilize a Different Mode of Translation Initiation, Have Higher Proliferation Rates, Less Oxidized DNA and More Tubular Mitochondria
  • Karen H. Vousden, Beatson Institute for Cancer Research, UK
    A Role for p53 in the Adaptation to Metabolic Stress
  • Cormac Taylor, University College Dublin, Ireland
    The Role of Hypoxia in Immunity and Inflammation
  • Eyal Gottlieb, Technion Integrated Cancer Center, Israel
    Metabolic Dependencies of Leukemic Stem Cells
  • Bradly G. Wouters, University Health Network, Canada
    ULK1 Regulates Oxygen Metabolism, Hypoxia Tolerance and Is a Therapeutic Target in Pancreatic Cancer
  • Ester M. Hammond, University of Oxford, UK
    Ribonucleotide Reductase Favors the RRM2B Subunit to Maintain DNA Replication in Hypoxia

 

Please stop by our exhibit at the Whistler Conference Center to learn more about the ways the Hypoxystation can recreate the tumour environment for your cancer research. We also have a “heart-warming” gift for you!

 

Hallmarks of Cancer

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Hypoxia and the Hallmarks of Cancer

Therapeutic Targeting of Hypoxia and HIFs in Cancer. Dr Burga Kalz Fuller from US distributor HypOxygen has summarised this study that outlines the Hallmarks of Cancer.

“Tumour hypoxia and HIFs affect most of the cancer hallmarks… and contribute to chemo- and radiotherapy resistance.” In their review from 2016, Wigerup, Pahlman and Bexell of Lund University in Sweden discuss how hypoxia inducible factors HIFs regulate the hypoxic microenvironment in cancer, and the therapeutic strategies that are being developed to improve patients’ prognosis. Dr. Sven Pahlman’s lab has been using the H35 Hypoxystation for more than 5 years, to research SCLC and neuroblastoma, and their data is contributing to the understanding of the role of oxygen levels in the progression of cancer.

Hypoxia and HIF-1α and 2α expression in cancer usually signify a worse prognosis, but most hypoxia-induced transcriptional, translational, and epigenetic changes are cell-type specific. Many effects engendered by hypoxia are mediated directly or indirectly via HIF pathways, and most are causative of the iconic “Hallmarks of Cancer” that Hanahan and Weinberg introduced in 2000 and expanded in 2011. Hypoxia induces increased autophagy, apoptosis, and aberrant cell proliferation; neoangiogenesis mediated by VEGF and PDGF-β; proliferation of cancer stem cells; metabolic reprogramming to satisfy energy and synthetic requirements in proliferating cells; modulation of inflammation and immune responses; genomic instability through increased mutagenesis and diminished DNA repair; and metastasis as hypoxia induces epithelial-to-mesenchymal transition and degradation of the extracellular matrix. Assaying the relationship between hypoxia and the Hallmarks of Cancer benefits significantly from the physiological atmosphere mimicked in the Hypoxystation, a closed-culture hypoxia workstation controlling gasses, temperature and humidity.


Visit Don Whitley Scientific and HypOxygen at

Keystone Adaptations to Hypoxia and Tumour Metabolism

Location: Whistler, BC  Date: 5th – 9th March

Sven Pahlman


In their review, Wigerup and Pahlman describe the role tumour hypoxia plays for cancer therapy and treatment resistance, as oxygen levels, production of reactive oxygen species ROS, and HIF activity are intertwined actors in the cancer battle. Any and all effects of hypoxia are cell-type specific; however, numerous studies indicate that HIF’s mediate chemoresistance, suggesting that HIF-1 and 2 inhibitors can effectively support cancer therapy. The authors state that “since hypoxia is a hallmark of solid tumours and mediates aggressive, metastatic, and resistant disease, it is arguably one of the most attractive therapeutic targets in cancer.” Strategies selectively targeting hypoxia for cancer therapy include hypoxia-activated prodrugs; inhibitors of HIF mRNA and protein expression; and inhibitors of downstream HIF signalling pathways such as VEGF. Effective drug research relies on authentic replication of the hypoxic environment for cell culture: the Hypoxystation used in the Pahlman lab is able to accommodate long-term assays with sterile steam humidification and HEPA clean air. The Hypoxystation concept “Choose your Atmosphere – Define your Environment” is the best way to ensure cell culture reflects physiology in cancer research and therapy.

Hypoxia is at the heart of the Hallmarks of Cancer, and results such as these from the Pahlman lab make the cancer research community hopeful that “HIF inhibition is likely to be a powerful therapeutic approach” to eradicate cancer.

 

Hallmarks of Cancer

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Visit HypOxygen at Society for Redox Biology and Medicine

 

From November 16-19, HypOxygen will be exhibiting the Hypoxystation at the annual meeting of the SFRBM in San Francisco, please stop by our booth #4

Please visit HypOxygen to find out how ROS (reactive oxygen species) research is benefitting from the physiological conditions cells experience in the Hypoxystation. The story below, from HypOxygen, looks at research into this line of work.

Increased production of ROS, which is both a symptom and a driver of cancer Hallmarks, can push cancer cells over the cliff of oxygen homeostasis. Compounds adding oxidative pressure can thus be utilized for selective tumor therapy. In their paper “Triggering apoptosis in cancer cells with an analogue of cribrostatin 6 that elevates intracellular ROS” , Hypoxystation users Asby et al. describe their approach to chemically modifying a natural compound from marine sponges, cribrostatin 6, to enhance its cytotoxic potential. They synthesized a modified molecule 8-phenylcribrostatin 6 (8PC6) that was both more potent and more selective for breast cancer cells. Co-author Ali Tavassoli from the University of Southampton says, “We study HIF-1, so working in a hypoxic environment is critical. Besides culturing our cells in the H35, we also harvest proteins and collect RNA inside the chamber. The H35 is very easy to use; the touch screen controls are straightforward and intuitive. We have used the workstation to incubate cells in hypoxia for ~5 days, and the atmosphere remains stable over time.”

Annexin V/7-AAD staining indicated that 8PC6 induces apoptosis in cancer cells. Treatment of MCF7 cancer cells with ROS-sensing dyes and siRNA to knock down ROS-protective TIGAR demonstrated that 8PC6 increases intracellular reactive oxygen species, upsetting the delicate redox balance in highly susceptible cancer cells and leading to cell death. Hypothesizing that reduction of the cribrostatin analogue yields a semi-quinone that reacts with molecular oxygen to generate superoxide, Asby’s group decided to withdraw oxygen from the equation by incubating the MCF7 cells in the Hypoxystation at 1% O2. Pre-incubation and subsequent incubation with increasing doses of 8PC6 in normoxia versus hypoxia showed that, indeed, the IC50 was increased up to 46-fold in hypoxia due to lack of oxygen. The Hypoxystation’s closed workstation format and rigorous control of oxygen, CO2, temperature and humidity facilitates authentic cell behavior as in vivo conditions are replicated. Thus, hypoxia in the workstation equated to significant reduction in the intracellular availability of oxygen for the generation of ROS. For research being conducted on highly hypoxic tumors, the workstation atmosphere represents a close approximation of the actual conditions cells encounter.

Read more on this story

 

 

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Hypoxystation In The Lab – Sequestration In Speckles

The HIF (hypoxia-inducible factor) family of oxygen-sensing proteins are a crucial element of cells’ responses to alterations in their immediate environment, kicking off a signaling cascade involving more than 1000 genes. Hypoxystation users Taylor and See at the University of Liverpool describe novel insights into the subcellular localization of some of the HIF proteins and why the “where” determines the “how”.

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HIF-2α and HIF-1α both form heterodimers with HIF-1β, and while similarities abound between the isoforms, the two subunits are differentially expressed and regulated and have distinctly separate target genes. Taylor and See triggered HIF activation using microscope stage incubators and the Hypoxystation by Don Whitley Scientific to incubate HeLa cells in hypoxia (1%). They found that while HIF-1α distributes homogenously in the nucleus, HIF-2α diffuses freely through the nucleus but is concentrated in speckles that are tethered to nuclear structures close to active RNA polymerases. This distribution is not significantly altered by low oxygen levels.

The Hypoxystation provides physiologically relevant, in vivo conditions for cell culture and manipulation to ensure authentic behavior of cells. User-defined parameters for temperature, CO2, O2 and humidity, plus the workstation format, where cells reside throughout the entire duration of the assays, minimize the extra-physiologic shock that is known to negatively impact metabolism and growth. As the degree and duration of hypoxia are among the factors controlling HIF activity, the customizable oxygen atmosphere inside the Hypoxystation can contribute to deciphering the functionality of oxygen-sensitive signaling pathways.

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Professor Patrick Frost from UCLA speaks to HypOxygen

Patrick Frost, a professor at UCLA recently spoke to our US distributor, HypOxygen, about his work and how it is benefitted by the use of a Whitley H35 Hypoxystation. Professor Frost is investigating how the adaptive hypoxic response in mutiple myeloma cells affects tumor progression, hoping to provide insight into the pathology and chemotherapy resistance of this incurable disease.

Patrick Frost has been using the Whitley H35 Hypoxystation for almost 3 years now, and he does not miss bygone days in the lab: “We started out with just a Plexiglas box, with some valves in it, with a front cover just held on magnetically, placed inside an incubator. We would burn through a 50 L tank of nitrogen in 48 hours. I was never convinced that we had the correct level of oxygen in there”.

Now, despite two groups using the H35 intensely at 0.2 % oxygen, that gas bottle can easily last a month. Dr. Frost says, “If we shut the workstation down, the atmosphere stays stable, we don’t lose the hypoxia. It’s been a year since we replaced the CO2 bottle, and the compressed air.” Don Whitley Scientific has conducted gas consumption studies that show the Whitley H35 Hypoxystation “at rest” uses only 3.5 L of nitrogen gas/24 hour period, 47 and 28 times less than other hypoxia workstations. Part of the reason for the stability of that atmosphere is the annealed, thick acrylic build of the Hypoxystation. Dr. Frost states, “It’s pretty robust, it’s survived a move and a forklift and it never sprung a leak throughout. “

Read more about the Patrick Frost’s investigation here.


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