Jeffrey Burkeen

Circadian rhythms of extracellular ATP accumulation in suprachiasmatic nucleus cells and cultured astrocytes

The master circadian pacemaker located within the suprachiasmatic nucleus (SCN) of the mammalian brain controls system‐level rhythms in animal physiology. Specific SCN outputs synchronize circadian physiological rhythms in other brain regions. Within the SCN, communication among neural cells provides for the coordination of autonomous cellular oscillations into ensemble rhythms. ATP is a neural transmitter involved in local communication among astrocytes and between astrocytes and neurons. Using a luciferin–luciferase chemiluminescence assay, we have demonstrated that ATP levels fluctuate rhythmically within both SCN2.2 cell cultures and the rat SCN in vivo. SCN2.2 cells generated circadian oscillations in both the production and extracellular accumulation of ATP. Circadian fluctuations in ATP accumulation persisted with an average period (τ) of 23.7 h in untreated as well as vehicle‐treated and forskolin‐treated SCN2.2 cells, indicating that treatment with an inductive stimulus is not necessary to propagate these rhythms. ATP levels in the rat SCN in vivo were marked by rhythmic variation during exposure to 12 h of light and 12 h of dark or constant darkness, with peak accumulation occurring during the latter half of the dark phase or subjective night. Primary cultures of cortical astrocytes similarly expressed circadian oscillations in extracellular ATP accumulation that persisted for multiple cycles with periods of about 23 h. These results suggest that circadian oscillations in extracellular ATP levels represent a physiological output of the mammalian cellular clock, common to the SCN pacemaker and astrocytes from at least some brain regions, and thus may provide a mechanism for clock control of gliotransmission between astrocytes and to neurons.

Mitochondrial Calcium Signaling Mediates Rhythmic Extracellular ATP Accumulation in Suprachiasmatic Nucleus Astrocytes

The master circadian pacemaker located within the suprachiasmatic nuclei (SCN) controls neural and neuroendocrine rhythms in the mammalian brain. Astrocytes are abundant in the SCN, and this cell type displays circadian rhythms in clock gene expression and extracellular accumulation of ATP. Still, the intracellular signaling pathways that link the SCN clockworks to circadian rhythms in extracellular ATP accumulation remain unclear. Because ATP release from astrocytes is a calcium-dependent process, we investigated the relationship between intracellular Ca2+ and ATP accumulation and have demonstrated that intracellular Ca2+ levels fluctuate in an antiphase relationship with rhythmic ATP accumulation in rat SCN2.2 cell cultures. Furthermore, mitochondrial Ca2+ levels were rhythmic and maximal in precise antiphase with the peak in cytosolic Ca2+. In contrast, our finding that peak mitochondrial Ca2+ occurred during maximal extracellular ATP accumulation suggests a link between these cellular rhythms. Inhibition of the mitochondrial Ca2+ uniporter disrupted the rhythmic production and extracellular accumulation of ATP. ATP, calcium, and the biological clock affect cell division and have been implicated in cell death processes. Nonetheless, rhythmic extracellular ATP accumulation was not disrupted by cell cycle arrest and was not correlated with caspase activity in SCN2.2 cell cultures. Together, these results demonstrate that mitochondrial Ca2+ mediates SCN2.2 rhythms in extracellular ATP accumulation and suggest a role for circadian gliotransmission in SCN clock function.

Mesenchymal stem cells inhibit cutaneous radiation‐induced fibrosis by suppressing chronic inflammation

Exposure to ionizing radiation (IR) can result in the development of cutaneous fibrosis, for which few therapeutic options exist. We tested the hypothesis that bone marrow‐derived mesenchymal stem cells (BMSC) would favorably alter the progression of IR‐induced fibrosis. We found that a systemic infusion of BMSC from syngeneic or allogeneic donors reduced skin contracture, thickening, and collagen deposition in a murine model. Transcriptional profiling with a fibrosis‐targeted assay demonstrated increased expression of interleukin‐10 (IL‐10) and decreased expression of IL‐1β in the irradiated skin of mice 14 days after receiving BMSC. Similarly, immunoassay studies demonstrated durable alteration of these and several additional inflammatory mediators. Immunohistochemical studies revealed a reduction in infiltration of proinflammatory classically activated CD80+ macrophages and increased numbers of anti‐inflammatory regulatory CD163+ macrophages in irradiated skin of BMSC‐treated mice. In vitro coculture experiments confirmed that BMSC induce expression of IL‐10 by activated macrophages, suggesting polarization toward a regulatory phenotype. Furthermore, we demonstrated that tumor necrosis factor‐receptor 2 (TNF‐R2) mediates IL‐10 production and transition toward a regulatory phenotype during coculture with BMSC. Taken together, these data demonstrate that systemic infusion of BMSC can durably alter the progression of radiation‐induced fibrosis by altering macrophage phenotype and suppressing local inflammation in a TNF‐R2‐dependent fashion. Stem Cells 2013;31:2231–2241

MEK1/2 inhibition enhances the radiosensitivity of cancer cells by downregulating survival and growth signals mediated by EGFR ligands

The inhibition of the Ras/mitogen-activated protein kinase (Ras/MAPK) pathway through the suppression of mutated Ras or MAPK/extracellular signal-regulated kinase 1/2 (MEK1/2) has been shown to sensitize tumor cells to ionizing radiation (IR). The molecular mechanisms of this sensitization however, are not yet fully understood. In this study, we investigated the role of transforming growth factor-α (TGF-α) in the radiosensitizing effects of selumetinib, a selective inhibitor of MEK1/2. The expression of epidermal growth factor receptor (EGFR) ligands was assessed by ELISA in both Ras wild-type and Ras mutant cells that were exposed to radiation with or without selumetinib. The effects of selumetinib on the TGF-α/EGFR signaling cascade in response to radiation were examined by western blot analysis, clonogenic assay and by determing the yield of mitotic catastrophe. The treatment of cells with selumetinib reduced the basal and IR-induced secretion of TGF-α in both Ras wild-type and Ras mutant cell lines in vitro and in vivo. The reduction of TGF-α secretion was accompanied with a reduction in phosphorylated tumor necrosis factor-α converting enzyme (TACE) in the cells treated with selumetinib with or without IR. The treatment of cells with selumetinib with or without IR inhibited the phosphorylation of EGFR and check-point kinase 2 (Chk2), and reduced the expression of survivin. Supplementation with exogenous TGF-α partially rescued the selumetinib-treated cells from IR-induced cell death, restored EGFR and Chk2 phosphorylation and increased survivin expression. These data suggest that the inhibition of MEK1/2 with selumetinib may provide a mechanism to sensitize tumor cells to IR in a fashion that prevents the activation of the TGF-α autocrine loop following IR.

The Medical Student Perspective on Global Health Care in Radiation Oncology: Opportunities, Barriers to Sustainability, and Future Directions

The Medical Student Perspective on Global Health Care in Radiation Oncology: Opportunities, Barriers to Sustainability, and Future Directions Jeffrey Burkeen, MS,*,y,1 C. Norman Coleman, MD,z,y,1 Maithili Daphtary, PhD,z and Bhadrasain Vikram, MDz *Texas A&M University, School of Medicine, Bryan, Texas; zRadiation Research Program, Division of Cancer Treatment and Diagnosis, National Cancer Institute, Bethesda, Maryland; yRadiation Oncology Branch, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland

Patient-reported health-related quality of life outcomes in supportive-care interventions for adults with brain tumors: A systematic review

The objectives of this systematic review were to (a) identify supportive‐care (psychosocial/behavioral, pharmacological, complementary, or alternative) interventions that have been evaluated via randomized controlled trials (RCTs) to improve patient‐reported health‐related quality of life (HRQoL) among adults with brain tumors, (b) evaluate the quality of the intervention studies, and (c) evaluate if developed interventions have been efficacious at improving HRQoL, as compared with control conditions in RCTs.

Bullet fragment fiducials in stereotactic body radiotherapy as a bridge to transplant for hepatocellular carcinoma

Jamie S. K. Takayesu, Kathryn R. Tringale, Deborah C. Marshall, Jeffrey Burkeen, Mark A. Valasek, Alan Hemming, Todd Atwood, Daniel Simpson and Jona Hattangadi-Gluth John A. Burns School of Medicine, University of Hawaii at Manoa, Honolulu, HI, USA; Department of Radiation Medicine and Applied Sciences, University of California San Diego, La Jolla, CA, USA; Department of Pathology, University of California San Diego, La Jolla, CA, USA; Department of Surgery, University of California San Diego, La Jolla, CA, USA