Molecular Targeting of Hypoxia in Radiotherapy

Most solid tumors exhibit areas of both chronic and acute hypoxia, all of them evolving dynamically as a function of cellular growth, vascularization, oxygen consuming metabolism and therapy response. Tumor hypoxia, generally far below 1% oxygen, correlates with increased recurrence rates and decreased survival rates in most cancers, so the recent review by HypOxystation users Rey et al. describing “Molecular Targeting of Hypoxia in Radiotherapy”  gives a valuable overview of the mechanisms cancer cells have developed to respond to hypoxia.

Dr. Rey of the Princess Margaret Cancer Centre in Toronto, Canada, and his co-authors Luana Schito, Marianne Koritzinsky and Brad Wouters have contributed vastly to our knowledge about the cellular response to hypoxia in the context of tumor behavior. Since 2009, they have acquired 4 HypOxystations for their lab, in order to culture cells under conditions which authentically mimic the physiological environment of cancer. The HypOxystation provides a closed workstation format for rigorous control of oxygen, CO2, temperature and humidity, facilitating accurate regulation of cell culture conditions as the in vivo tumor situation is simulated. An extensive list of publications from our HypOxystation users spans the whole breadth of research into metabolic, epigenetic and therapeutic implications of hypoxia.

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Adaptation to Stressors by Systemic Amyloidogenesis

Cells facing environmental threats have developed numerous coping mechanisms, and HypOxystation users Tim Audas and Stephen Lee have uncovered a fascinating new cellular strategy to remain viable under stress and restore homeostasis when the crisis ends. In their recent paper “Adaptation to Stressors by Systemic Protein Amyloidogenesis”, they describe a physiological process of amyloidogenesis which cells activate under stress conditions, such as hypoxia and acidosis, to remove copious amounts of heterogeneous proteins from circulation, enabling cells to survive in a dormant state. This discovery expands our current view of amyloids as a rare and pathological phenomenon associated with neuropathies such as Alzheimer’s and Parkinson’s diseases, and exposes a novel post-translational, regulatory form of protein organization.

Using a combination of Congo red staining, proteinase K digestion, and OC antibody detection on cells exposed to a variety of stimuli, Audas et al. were able to identify nuclear foci consisting of immobilized, insoluble protein in a crossed β-sheet conformation which they named A-Bodies. In amyloidogenic proteins such as VHL and RNF8, an ACM (amyloid-converting motif) containing arginine and histidine was identified as essential for capture specifically in the A-bodies; a similar motif was also identified in the pathological β-amyloid associated with Alzheimer’s disease. Upon environmental insult, the ACM interacts with ribosomal intergenic spacer RNA (rIGSRNA) to concentrate the proteins and trigger their polymerization in the A-bodies allowing the cells to enter a dormant state.

Read more: Adaptation to Stressors by Systemic Amyloidogenesis

TC-3 and TEB Bioreactors provide physiological conditions for cell culture

The TC-3 and TEB bioreactors are being used in numerous labs to provide physiological conditions for the culture of blood vessels, cardiac muscle, bone, cartilage, ligaments, tendons and skin. Here, we would like to introduce some of the research being published by these labs. 

I. A recent paper published by TC-3 users Hillary et al. (“Developing Repair Materials for Stress Urinary Incontinence to Withstand Dynamic Distension” 2016; PLoS ONE 11(3):e0149971. doi:10.1371/journal.pone.0149971) examines options for culturing adipose derived stem cells (ADSC) for use in surgical repair meshes.  Using the TC-3 to generate cyclic uniaxial distension, Hillary’s lab compared the current standard scaffold material polypropylene with Poly-L-lactic acid ((PLA) and polyurethanes (PU), focusing specifically on their supportive properties and biocompatibility, as measured by cell attachment, proliferation, and matrix production. They found that prolonged mechanical distension in vitro caused polypropylene to fail, while a combination of PLA with PU greatly improved dynamic distension and cell interaction properties of the mesh. The authors conclude that “the key finding of this study is that subjecting materials in vitro to dynamic strain reveals significant changes in their mechanical properties after only 7 days. … We suggest that this dynamic assessment is crucial in the development of materials for use in the pelvic floor.”

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Natural amyloid aggregation is induced by cellular stress

Cellular ability to react to stressors such as extreme temperatures, hypoxia, determines the sustainability of the entire organism. Tim Audas at Simon Fraser University has uncovered the mechanism by which proteins are reversibly folded and immobilized in amyloid bodies in response to stress. In a presentation he gave at the Keystone Symposium on “Cellular Stress Responses and Infectious Agents” in Santa Fe, Tim Audas describes this process of amyloidogenesis as a means of ensuring survival and homeostasis under adverse conditions. Amyloidogenesis, which is well-known in the context of neurological disorders such as Alzheimer’s and Parkinson’s disease, helps the cell to enter a state of protective dormancy by sequestering diverse proteins in sub-nuclear foci, termed A-bodies. To mimic physiological stress, Dr. Audas cultured cells at 1% oxygen in the HypOxystation, also exposing them to acidosis for additional stress. The HypOxystation’s closed workstation format ensures controlled oxygen, CO2, temperature and humidity conditions throughout the duration of culture and manipulation. Dr. Audas states that, “The workstation is critical because it allows us to manipulate the cells within a low oxygen environment and, unlike an incubator, maintains the O2 levels when cells are taken in and out, avoiding spikes of normoxia“.

Read more: Natural amyloid aggregation is induced by cellular stress

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”.

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.

Read more: Sequestration in Speckles