Interaction of DNA damage kinase ATM with the ATM interactor ATMIN at hypoxia

DNA repair pathways protecting against genomic instability are significantly repressed under the hypoxic conditions typical of many solid tumors. HypOxystation users Leszczynska et al. describe the interaction of DNA damage kinase ATM with the ATM interactor ATMIN at hypoxia as well as the downstream consequences for DNA repair (“Mechanisms and consequences of ATMIN  repression in hypoxic conditions: roles for p53 and HIF-1” , Scientific Reports 6:21698; 2016).

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 H35 HypOxystation by Don Whitley Scientific. The closed cell culture environment created in the Don Whitley Scientific 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.

Read more: Interaction of DNA damage kinase ATM with the ATM interactor ATMIN at hypoxia

Enhancing radiosensitivity to treat lung carcinoma at hypoxia and normoxia

Treatment of non-small cell lung cancer NSCLC, the leading cause of cancer-related death, relies heavily on radiation therapy, which however is frequently compromised by development of radioresistance. HypOxystation users Jiang et al. of the University of Oxford describe their study of olaparib, an inhibitor of Poly(ADP-ribose) polymerase 1 (PARP-1) which serves to enhance radiosensitivity, to treat lung carcinoma at hypoxia and normoxia ("Hypoxia Potentiates the Radiation-Sensitizing Effect of Olaparib in Human Non-Small Cell Lung Cancer Xenografts by Contextual Synthetic Lethality"; Int J Radiation Oncol Biol Phys, Vol. 95, No. 2, pp. 772e781, 2016). The lab uses a Don Whitley H35 HypOxystation to create stable, contiguous hypoxic conditions for their cell culture.

A combination of olaparib and radiation significantly increased DNA double strand breaks, and at hypoxia (1% oxygen), the radiation-sensitizing effect of olaparib was increased relative to normoxia (21% oxygen), as evidenced by a decrease in clonogenic survival rates. In vivo effects of oxygen availability were examined in subcutaneous Calu-6 and Calu-3 xenograft assays, where Calu-6 tumors comprise extensive hypoxic areas, and assays focusing on RAD51 protein expression showed that homologous recombination was considerably impacted by olaparib, specifically in hypoxic regions. CC3 staining of Calu-6 tumors as a measure of apoptosis also showed that hypoxic areas are more severely impacted by the combination of olaparib and radiation than normoxic areas. CA9 immunostaining served as a marker for hypoxia.

Read more: Enhancing radiosensitivity to treat lung carcinoma at hypoxia and normoxia

Brad Wouters: TRACER mimics oxygen and nutrient gradients in tumors

Interview with Brad Wouters, Princess Margaret Cancer Center, Toronto.  March 4, 2016

HypOxygen wanted to find out more about Brad Wouters' recent research, especially the interesting paper newly published describing his three-dimensional tumor TRACER project, published as “A three-dimensional engineered tumour for spatial snapshot analysis of cell metabolism and phenotype in hypoxic gradients“ (Rodenhizer et al., Nature Materials 15, 227–234, 2016).

Dr. Wouters' lab uses three H35 HypOxystations and one H45 HypOxystation.

The paper describes a new device that enables us to create naturally occurring oxygen gradients, such as the ones found in tumors. The TRACER (engineered tumour roll for analysis of cellular environment and response) developed by Alison McGuigan of the University of Toronto is basically an engineered tumour that is assembled by rolling a single-component biocomposite sheet. Respiration-induced oxygen and metabolite gradients are established inside the device, so it’s an alternative to other 3D models such as spheroids. The benefit is that the TRACER rolls can be rapidly disassembled, enabling you to interrogate the biological phenotypes or properties, characteristics, and metabolites of the cells in those defined locations within the gradient, in the six layers of the tracer. The cells can be removed in a variety of ways; you can remove the cells in a viable way and process them as you would any cells, to do flow cytometry, metabolomics, cell survival, proliferation.

Read more: Brad Wouters: TRACER mimics oxygen and nutrient gradients in tumors

Targeting the adaptive hypoxic response in multiple myeloma

4/15/2016

Patrick Frost is at the Department of Hematology-Oncology at the VA Hospital in Los Angeles. He has been using an H35 HypOxystation for almost 3 years, and just in March published his most recent findings on multiple myeloma cells cultured in the HypOxystation. At lower than 1% oxygen, the atmosphere inside the chamber mimics the hypoxic niche in the bone marrow microenvironment. Patrick Frost’s group investigated the effects of a polyamide capable of binding to the hypoxia response element HRE of genes such as VEGF that are up-regulated under low oxygen conditions. They found a significant cytotoxic effect of the polyamide against MM tumor cells which they traced to an inhibition of HIF-mediated gene transcription and decreased resistance to hypoxia-induced apoptosis. These results are described in “A DNA-binding Molecule Targeting the Adaptive Hypoxic Response in Multiple Myeloma Has Potent Antitumor Activity” (Mysore et al., 2016, Mol Cancer Res; 14(3); 253-66.)

The focus of our lab’s research is multiple myeloma, specifically with the aim of targeting the adaptive hypoxic response in myeloma cells. We have a number of interesting compounds, and we are studying HIF regulation to understand the mechanisms of what happens in these cells treated with the compounds. We have a polyamide, a synthetic molecule that recognizes the hypoxia response element HRE and binds to the minor groove of the DNA. We have to use relatively high concentrations of this compound but it is a good model to block the hypoxic response. In order to characterize the effect of the molecule, we used it in some reporter assays, in reporter cells, then we tested it to see whether it could overcome their resistance to hypoxia-mediated killing. There is some thought that hypoxia confers resistance to drugs, which we did not find in our particular experiments. We found that the drugs and the hypoxia actually synergize. I think there is a need to differentiate more between resistance and cell cycle arrest in this regard, as opposed to killing the cells. The HypOxystation is very good for placing the cells at hypoxic conditions, at different levels, and sampling at different time points. The time points especially are very useful with this chamber, because with other systems such as boxes, once the cells are inside and you start doing anything with them, you’ve re-set the system; it doesn’t take very long for those media to get re-oxygenated. So we can set up to do a 24 hour assay, using the interlock to get in and out, and take a sample of the growing cells every 24 hours. That really helps to get a nice time course. Once we have the cells inside the chamber, we can assay apoptosis, gene expression, protein expression, anything. The in vitro models are really very useful, we can cover a lot of ground quickly.

Read more: Targeting the adaptive hypoxic response in multiple myeloma

UT Southwestern explores the role of genetic and epigenetic mechanisms in the regulation of fetal lung development

2/18/2015

Dr. Houda Benlhabib is in the Department of Biochemistry at UT Southwestern in Dallas, working on the role of genetic and epigenetic mechanisms in the regulation of fetal lung development as well as in reproductive and perinatal biology.

  1. “Estrogen-Related Receptor γ (ERRγ) Regulates Oxygen-Dependent Expression of Voltage-gated Potassium (K+) Channels and Tissue Kallikrein during Human Trophoblast Differentiation” (2013) Yanmin Luo, Premlata Kumar, and Carole R. Mendelson; Mol Endocrinol, Jun; 27(6): 940–952.

    Placental development during pregnancy is accompanied by increasing tissue oxygen levels, while persisting hypoxia contributes to the pathogenesis of pre-eclampsia and IUGR. HypOxystation user Carole Mendelson describes studies examining the expression patterns of genes for potassium channels and tissue kallikrein under hypoxic (2% O2) conditions. She found a novel role for estrogen-related receptor ϒ (ERRϒ) as an oxygen-responsive transcription factor during trophoblast differentiation.

  2. The c-Myc-Regulated MicroRNA-17∼92 (miR-17∼92) and miR-106a∼363 Clusters Target hCYP19A1 and hGCM1 To Inhibit Human Trophoblast Differentiation” (2013) Premlata Kumar, Yanmin Luo, Carmen Tudela, James M. Alexander, and Carole R. Mendelson; Mol Cell Biol. 2013 May; 33(9): 1782–1796.

    Mendelson’s group investigated the role of miRNA’s in regulating expression of genes involved in trophoblast differentiation. Oxygen tension and vascularization play a significant role in pre-eclampsia, one of the leading causes of fetal and maternal death. C-Myc expression was elevated at hypoxia, and translation of miR-17∼92 and miR-106a∼363 clusters was found to be increased, also. Together, these factors seem to promote stemness in the proliferating cytotrophoblasts and inhibit differentiation. Interesting commonalities with tumor cells with regard to proliferation and miRNA expression are discussed in this paper.

The lab has been using an HypOxystation for about 4 years now. HypOxygen asked how the lab uses the workstation for hypoxic cell culture.

Read more: UT Southwestern explores the role of genetic and epigenetic mechanisms in the regulation of fetal...