Examining the Role of Autophagy in Hypoxic Tumours

Examining the Role of Autophagy

Hypoxystation users Tan et al. at the University of Toronto published a paper in June examining the significance of autophagy in cancer development (“Role of Autophagy as a Survival Mechanism for Hypoxic Cells in Tumors“, Neoplasia (2016) 18, 347-355). Autophagy as a means of recycling cell components is induced under stress conditions such as hypoxia, and Tan et al. investigated the correlation of hypoxia and autophagy in solid tumours in the context of resistance to cancer therapeutics.

 

Cells were cultured in the H35 Hypoxystation for up to 48 hours at hypoxia (0.2 %) and compared to cells grown at ambient oxygen level. Gene silencing of autophagy proteins ATG7 and BECLIN1 with shRNA resulted in decreased cell survival under hypoxia, and inhibition of autophagy with pantoprazole exacerbated the loss of viability in the knock-down cells under hypoxia, demonstrating the cyto-protective effects of these autophagy proteins. Using the Seahorse XFe Analyzer to assess oxygen consumption in wild-type and silenced cells, Dr. Tan’s lab found reduced respiration when autophagy is disrupted, possibly due to accumulation of dysfunctional mitochondria in these mutant cells. The H35 Hypoxystation  Dr. Tan’s lab used for these studies creates a closed environment with controlled temperature, humidity, CO2 and oxygen, in which cells are cultured and manipulated over the course of days and weeks without the need to transfer into ambient conditions. The combination of an Hypoxystation and an i2 Instrument Workstation is designed to accommodate the specific requirements of the Seahorse XFe Analyzer for the duration of the metabolism assays investigating oxygen consumption and extracellular acidification.

 

Please visit the Meintrup DWS Laborgeräte website for more published papers featuring the Whitley Workstation range or contact us to discuss your hypoxia needs.

Whitley H35 Hypoxystation used in tumour study

KONICA MINOLTA DIGITAL CAMERA

Researchers from Oxford and Stanford universities have been using an Whitley H35 Hypoxystation to look at how the hypoxic conditions of tumours repress DNA repair pathways that protect against genomic instability. Hypoxystation users Leszczynska et al describe the interaction of DNA damage kinase ATM with the ATM interactor ATMIN within hypoxia as well as the downstream consequences for DNA repair. An abstract from the article is posted below, as well as a link to the full article.

 

ATM activation is induced at severe hypoxia as a result of replication stress, and was thought to be dependent on the ATM interactor ATMIN, especially in the absence of DNA damage. Using ATMIN siRNA, ATM inhibitors, and knock-out cell lines, the authors were able to establish that ATMIN is not required for the activation of ATM in response to hypoxia-induced replication stress, and that ATMIN is repressed at hypoxia, an effect mediated by both p53 and HIF-1. The cells were exposed to varying degrees of hypoxia, from mild (2%) down to extreme (0.1%) in an Whitley H35 Hypoxystation by Don Whitley Scientific. The closed cell culture environment created in the Hypoxystation mimics physiological conditions with regard to oxygen, CO2, temperature, and humidity and enables cancer researchers to obtain a clearer picture of in vivo processes. qPCR analysis of cells in response to hypoxia and exposure to inhibitors of proteasomal degradation indicate that the repressive effect of hypoxia is due to inhibition of translation as opposed to transcription or altered stability of ATMIN.

 

Using ATMIN siRNA at various levels of hypoxia, the authors found that loss of ATMIN impairs base excision repair BER and increases sensitivity to DMA damaging agents such as methyl methanesulfonate MMS. Decreased ATMIN levels also decrease the expression of dynein light chain LC8-type 1 DYNLL1, again in a p53-dependent manner. Thus, the authors have found a new link between tumor hypoxia and ATMIN-regulated DYNLL1 expression. Loss of DYNLL1 in hypoxic tumors affects ciliogenesis, mitosis, cellular localization of proteins, to name a few, and justifies further research into the roles of ATMIN and DYNLL1 in cancer.

 

The full paper can be read here – Mechanisms and consequences of ATMIN repression in hypoxic conditions: roles for p53 and HIF-1

Whitley H35 Hypoxystation used in heart regeneration project

H35 Australia 1

Dr. Vaibhao Janbandhu is a Postdoctoral Research Scientist at the Victor Chang Cardiac Research Institute (VCCR) in Sydney. He has been in contact with Don Whitley Scientific to explain how his lab’s work has benefited from the use of a Whitley H35 Hypoxystation. Vaibhao uses the Hypoxystation to isolate, culture and characterise adult cardiac stem cells (CSCs).

 

Dr. Janbandhu had already been using a H35 that was set up at the institute for almost three years before he got his own unit installed last year. Specifically, his project is to find new ways to stimulate heart regeneration during ageing and after heart attack. For this he needs a way to isolate, culture and characterise adult CSCs. In Vaibhao’s words the H35 Hypoxystation seems well suited for this application: “the DWS Hypoxystation provides a highly stabilised HEPA-filtered environment in which levels of oxygen, carbon dioxide, temperature and humidity are precisely controlled and it will be an integral part of the project to advance the project aims”.

 

Vaibhao explains that mammalian stem cells reside in a specialised cellular microenvironment. This niche and the stem cell niche is characterised by a low partial oxygen pressure. This hypoxic niche protects stem cells from deleterious effects of O2 on proteins and DNA. These precise conditions are essential for Dr. Janbandhu’s work as they accurately replicate the in vivo environment.

 

His work also see benefits when comparing the use of a Hypoxystation as opposed to using a CO2 incubator. Typically, cell culture work involves methods which include isolating cells under their usual physiologically relevant conditions and then working with them in “bench-top conditions” where cells are exposed to non-physiological oxygen. This can then lead to altered hypoxic response, metabolism, reactive oxygen species and DNA damage response. This metabolic stress introduces unknown outcomes and may lead to results inconsistent with physiological processes. Therefore, the precise control of oxygen levels in cell culture has been shown to be vital for reproducible and physiologically relevant results, transforming the working environment in Vaibhao’s lab.

 

As well as the precise controlling of conditions, Vaibhao likes the remote access feature on his H35 Hypoxystation. The remote access allows Vaibhao to log into his Hypoxystation’s touchscreen control whilst away from the unit, offering increased flexibility in his working methods. Additionally, he likes how he can view operating conditions, set parameters and change access levels remotely.

 

Dr. Janbandhu opted to purchase a Hypoxystation from Don Whitley Scientific for the level of service and specification we were able to provide in Australia. Other companies either couldn’t fulfil configuration requirements and didn’t provide sales and or service in Australia. Don Whitley Scientific’s office in Australia, provides both fantastic sales and service nationwide. Vaibhao also states that from “discussion with other research groups across the world we felt confident to go for a DWS Hypoxystation”.

 

Dr. Vaibhao Janbandhu has this to say about Don Whitley Scientific Pty Ltd: “I would like to thank your company personnel at the customer services division in Australia for their excellent support. Your Sales & Service Manager in Australia, Grant Shallcross, took care of all my queries in a jiffy!”. Vaibhao added that the funding for the purchase came from the James N Kirby Foundation and the Victor Chang Cardiac Research Institute.

 

H35 Australia 2

Working with Whitley Workstations

Inside-Workstation_Petridishes

Don Whitley Scientific has always strived to be the most innovative manufacturer of scientific equipment. Our leading range of workstations provides some of the most sophisticated options to the microbiology and cell culture industries.

One of the more specific markets Don Whitley Scientific provides for is those wanting to carry out experiments under hypoxic conditions. Researchers may only have the use of an incubator to keep specimens in hypoxic conditions, however this can cause problems when the conditions of the incubator become interrupted, usually when the incubator door is opened to add or remove samples and equipment.

When incubators just won’t do

In a testimonial for Don Whitley Scientific, Brad Wouters from Toronto, Canada shared this issue: “An incubator just isn’t suitable for that because whenever the door is opened, the oxygen concentration changes dramatically and the cells will go through waves of deoxygenation and oxygenation.” An issue like this could then lead to inaccurate results and potentially jeopardise experiments. This is why the Whitley Hypoxystation is ideal for these situations.

Working in a Whitley Workstation

Professor Mann states in his testimonial, “The design of this workstation has also allowed us to use other essential equipment inside the chamber, such as oxygen meters and micromanipulators”. As opposed to an incubator where samples are placed and perhaps left for days without interaction, Whitley Workstations are designed specifically for scientists wanting to carry out tasks inside the workstation.  Optional features on Whitley Workstations allow them to be tailored perfectly to those who want to conduct experiments in their workstation using additional specialised equipment, for example, the removable front option.

Australian testimonial

Maintaining an outstanding international reputation, Don Whitley Scientific exports its range of products to all corners of the globe. In Australia, at the Victor Chang Cardiac Research Institute, the aim is “to reduce the incidence, severity and impact of heart diseases” and for Dr Dunn and her team, precise control of oxygen, carbon dioxide, temperature and humidity is vital. She mentions in her testimonial that with a H35 Hypoxystation she achieves “very accurate and reproducible results”. As well as providing fantastic equipment, Don Whitley Scientific ensures that the advice and service we provide to customers is of a high level, which is also noticed by Dr Dunn, who states, “Don Whitley Scientific made great efforts to ensure our purchase, installation and ongoing use of the machine has been hassle free.”

 

Dr Dunn stood next to her H35 Hypoxystation
Dr Dunn with her H35 Hypoxystation

 

Testimonials wanted

If you are working with any equipment from Don Whitley Scientific and would like to let us know about your work and your overall experience with our products, please don’t hesitate to get in touch via our contact form, Facebook or Twitter. We now feature a variety of customer testimonials on our product pages and these can be accessed through the “testimonials” tab (if we have a testimonial for that product).

Click here for Brad Wouters’ testimonial in full

Click here for Professor Mann’s testimonial in full

Click here for Dr Dunn’s testimonial in full

Don Whitley Scientific exhibit at TCES 2015

Don Whitley scientific exhibited at TCES 2015, this annual Tissue and Cell Engineering Society event is held at the University Of Southampton between the 19th-21st of July. TCES 2015 aims to bring together outstanding national and global speakers from stem cell, tissue engineering and regenerative medicine industries, exploring how our understanding of tissue development and the resulting generation of specifiable tissues can be enhanced through stem cell science and regenerative medicine.

With special focus on “Harnessing and translating interdisciplinary research for cell and tissue engineering” the meeting features a programme of workshops and speakers in an effort to justify the excitement expressed from the public and research communities towards the affect ageing and or trauma can have on disease and the loss of tissue. Representatives from Don Whitley Scientific are attending the meeting, pictured is Daniel Secker exhibiting a Whitley H35 Hypoxystation.

Cancer Conference Targets Metastatic Disease

Don Whitley Scientific sponsored the 2015 Beatson International Cancer Conference, which was held yesterday at the Beatson Institute in Glasgow. The meeting, entitled Control of Cell Polarity and Movement in Cancer, highlighted recent exciting research into the molecular and cellular events that contribute to loss of epithelial polarity during carcinogenesis, and how cancer cells acquire different types of polarity that enable them to migrate and invade. There was also a focus on the avenues for development of agents to target cells with aberrant polarity and a potential route to treatment of metastatic disease.

Attending from Don Whitley Scientific were Danny Secker (pictured) and Steve Robertson, who demonstrated a Whitley H35 Hypoxystation to delegates.

Stem Cell Research in Stockholm

 

Stem cell researchers from all over the world are currently convening in Stockholm for the annual meeting of the International Society for Stem Cell Research (ISSCR). Topics covered in the event range from pluripotency and differentiation through regeneration to disease modelling and tissue engineering. All aspects of reprogramming and stem cell maintenance on the one hand, and differentiation on the other hand, are intricately influenced by the cellular microenvironment. Oxygenation is a crucial parameter throughout all these processes, both in the natural stem cell niche and during culture in the lab. Low oxygen promotes efficient expansion of stem cells in culture while supporting maintenance of the stem cell phenotype. Hypoxia can increase the proliferation rate and inhibit senescence of stem cells, and promote healing directly and through paracrine effects. Therapies utilizing hypoxic cells exhibit improved homing and engraftment to the target tissues as compared to normoxia. Hypoxia is a major determinant of many diverse aspects of stem cell biology.
Don Whitley Scientific will be at the event exhibiting their largest cell culture workstation to date – the Whitley H135 Hypoxystation.

Hypoxia and stem cells

Later this month, the international stem cell research community will be travelling to Stockholm for the upcoming International Society for Stem Cell Research (ISSCR) meeting covering diverse topics from reprogramming and pluripotency of stem cells through tissue engineering and organ regeneration to therapy with stem cells.

Whitley H135 Hypoxystation
Whitley H135 Hypoxystation

Hypoxia is a crucial parameter determining the fate and development of stem cells, which leads Don Whitley Scientific to exhibit the Hypoxystation controlled environment workstation for low oxygen cell culture (see us on stand no. B15:33). Dr. Burga Kalz Fuller, Product Manager at our American distributor, HypOxygen, has summarized five recent papers delineating the role of hypoxia in stem cell research:

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The hypoxic secretome induces pre-metastatic bone lesions through lysyl oxidase

Whitley H35 Hypoxystation

 

A new paper has been published in Nature magazine, outlining how hypoxic cancer secretomes induce pre-metastatic bone lesions through lysyl oxidase (LOX).

The study shows how hypoxia is specifically associated with bone relapse in patients with oestrogen-receptor negative breast cancer, and identifies a novel mechanism of regulation of bone homeostasis and metastasis, opening up opportunities for novel therapeutic intervention with important clinical implications.

The researchers, who used a Whitley H35 Hypoxystation (pictured left), came to the following conclusion:

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Interrogating gynaecological cancer cell metabolism at different oxygen tensions reveals simvastatin as metabolic regulator.

Earlier this month at the Keystone Symposia, Hypoxia: From Basic Mechanisms to Therapeutics, Dr Ayse Latif (pictured below) from the University of Manchester presented a poster entitled “Interrogating gynaecological cancer cell metabolism at different oxygen tensions reveals simvastatin as metabolic regulator”.

The poster describes the background of the study
as follows:Ayse Latif Keystone

Around 200,000 new cases of gynaecological cancers are diagnosed in Europe every year. Potentially 75% of these cancers could benefit from improved treatment regimes. Gynaecological cancer cells have an increased glycolysis rate and lactate concentration which have been suggested to predict increased likelihood of metastasis, resistance to therapy and reduced survival in patients. Lactate transport in cancer cells is carried out by members of the monocarboxylate transporter (MCT) family, notably MCT1/4. Thus, we hypothesized that pharmacologic inhibition of MCTs could improve treatment outcome by reducing glycolytic potential of these tumour cells… (To continue reading, click here).

Researchers at the University of Manchester used a Whitley i2 Instrument Workstation, housing a Seahorse XF Analyzer, connected to a Whitley H35 HEPA Hypoxystation. This allowed for the preparation of cell lines under hypoxic conditions and their subsequent transfer to the i2 for analysis in a CO2 free and controlled temperature environment without exposure to ambient conditions.