\u3ci\u3e Expression of HCMV IE1 in the U87MG Cell Line Augments Resistance to Temozolomide \u3c/i\u3e

INTRODUCTION: Human cytomegalovirus (HCMV) DNA and protein are found in gliomas but not in normal brain or other primary brain tumors. The role of HCMV infection in glioma biology is unclear. While it is unlikely that HCMV infection causes glioma, viral proteins might impart a proliferative and antiapoptotic phenotype that confers a survival advantage. Does this oncomodulation translate into a clinically relevant effect in glioma cells? To answer this question, we compared the response of the U87IE1 and U87MG malignant glioma cell lines to temozolomide. U87IE1 cells are U87MG cells that have been genetically engineered to produce HCMV IE1 protein. (The U87IE1 cell line is a generous gift from Charles Cobbs.) METHODS: Approximately 5,000 U87IE1 and U87MG cells in normal culture media were placed into wells of a 96-well plate. After 24 hours, the media was replaced with culture media containing temozolomide in increasing concentration. After 48 hours, cell viability was assessed using a luminescent assay. A dose-response curve for each cell line was generated using statistical software. The concentration of temozolomide resulting in 50% of cell death (the EC50 value) for each cell line was determined. Results: The EC50 for temozolomide in the U87MG cell line is 565.6 micromolar, while in the U87IE1 cell line it is 1319 micromolar. This difference is statistically significant (p \u3c 0.0001) and indicates that the U87IE1 cells are more resistant to temozlomide than are the U87MG cells. CONCLUSION: HCMV IE1 expression by U87MG cells enhances their proliferation and survival. In this study, we show that this oncomodulatory effect is clinically relevant: the U87IE1 cell line is more resistant than the U87MG cell line to temozolomide. This finding suggests that HCMV is a viable treatment target for patients with glioma

\u3ci\u3e Leptin Promotes Glioblastoma \u3c/i\u3e

The hormone leptin has a variety of functions. Originally known for its role in satiety and weight loss, leptin more recently has been shown to augment tumor growth in a variety of cancers. Within gliomas, there is a correlation between tumor grade and tumor expression of leptin and its receptor. This suggests that autocrine signaling within the tumor microenvironment may promote the growth of high-grade gliomas. Leptin does this through stimulation of cellular pathways that are also advantageous for tumor growth and recurrence: antiapoptosis, proliferation, angiogenesis, and migration. Conversely, a loss of leptin expression attenuates tumor growth. In animal models of colon cancer and melanoma, a decline in the expression and secretion of leptin resulted in a reduction of tumor growth. In these models, positive mental stimulation through environmental enrichment decreased leptin secretion and improved tumor outcome. This review explores the link between leptin and glioblastom

\u3ci\u3e β3-adrenergic agonists mimic eustress response and reduce leptin-mediated proliferation in a GBM cell line \u3c/i\u3e

A great deal of mental stress, depression, and anxiety often overwhelm cancer patients; those diagnosed with glioblastoma multiforme (GBM) are no exception. Different types of stress invariably impact what has been termed “the brain-adipocyte BDNF/leptin axis” (Dr. Cao and colleagues of the Comprehensive Cancer Center at The Ohio State University). For example, eustress (good stress) and distress (bad stress) both lead to increased sympathetic activity and adrenal gland stimulation, yet eustress reduces leptin levels and attenuates tumor growth while distress increases leptin levels and augments tumor growth. Complicating matters in GBM is that leptin and its receptor are expressed at much higher levels than in normal glial cells and provides a potential autocrine signaling pathway. In this study, we confirm that 200 ng/mL of leptin-conditioned media increases cell proliferation of the established GBM cell line T98G. We hypothesized that elevated sympathoadrenal activity would increase cell proliferation and be additive to leptin\u27s effects. To the contrary, adding 300 pg/mL of epinephrine to leptin-conditioned media blocked leptin-mediated proliferation. Because beta3-adrenergic receptor stimulation suppresses leptin gene expression and release in adipocytes, we hypothesized that a beta3-adrenergic agonist would counteract leptin\u27s effects on T98G cell proliferation. Use of the beta3-adrenergic agonist BRL 37344 did not only counteract leptin\u27s effects but also significantly reduced T98G cell proliferation in unconditioned media. This has immediate translational value in that treating GBM with a beta3-adrenergic agonist may reduce tumor proliferation through receptor activation and by blocking the leptin-leptin receptor autocrine loop. Moreover, recent reports indicate that beta3-adrenergic agonists capable of crossing the blood-brain barrier (like SR 58611A) may be beneficial for anxiety and depression, further improving the quality of life for brain tumor patients

\u3ci\u3e Glioblastoma Derived Exosomes Induce Apoptosis in Cytotoxic T Cells Through a Fas Ligand Mediated Mechanism \u3c/i\u3e

INTRODUCTION: Glioblastoma multiforme deploy s a number of weapons to thwart the immune system. Within the tumor microenvironment, cytotoxic T cells fall victim to Fas ligand (FasL) induced apoptosis. In prostate and colorectal cancer, exosomes can mediate this FasL induced T cell apoptosis. Exosomes are tiny, membrane bound vesicles that are released from a cell. They contain functional mRNA and protein and have cell surface molecules representative of their parent cell. It is not known if GBM derived exosomes can also mediate FasL triggered apoptosis. In this study, the role of tumor derived exosomes as the delivery vehicle for FasL is explored. METHODS: Exosomes are isolated from the T98 cell line using differential ultracentrifugation. FasL expression in the cell line and derived exosomes is determined using reverse transcription polymerase chain reaction (RT-PCR) and Western blotting. GBM derived exosomes, recombinant FasL, and exosomes treated with an anti-FasL antibody are co-cultured with Jurkat A3 T cells. Apoptosis is measured using a caspase-8 luminescent assay. RESULTS: FasL is expressed by the T98 cell line and is present on the surface of the cells and their exosomes (Figure 1). Caspase-8 activation is seen in T cells treated with GBM derived exosomes and recombinant FasL, but not with exosomes treated with anti-FasL antibody or exosome free supernatant (Figure 2). CONCLUSION: GBM derived exosomes induce T cell apoptosis through a FasL mediated mechanism. This method of immune suppression has not previously been described. This research opens new avenues to antagonize GBM related immune system malfunction

Relative photometry of HAT-P-1b occultations

We present HST STIS observations of two occultations of the transiting exoplanet HAT-P-1b. By measuring the planet to star flux ratio near opposition, we constrain the geometric albedo of the planet, which is strongly linked to its atmospheric temperature gradient. An advantage of HAT-P-1 as a target is its binary companion ADS 16402 A, which provides an excellent photometric reference, simplifying the usual steps in removing instrumental artifacts from HST time-series photometry. We find that without this reference star, we would need to detrend the lightcurve with the time of the exposures as well as the first three powers of HST orbital phase, and this would introduce a strong bias in the results for the albedo. However, with this reference star, we only need to detrend the data with the time of the exposures to achieve the same per-point scatter, therefore we can avoid most of the bias associated with detrending. Our final result is a 2 sigma upper limit of 0.64 for the geometric albedo of HAT-P-1b between 577 and 947 nm.Comment: 8 pages, 2 figures, 3 table

\u3ci\u3e Phenytoin reduces 5-ala mediated fluorescence in glioblastoma cells \u3c/i\u3e

Glioblastoma multiforme (GBM) is a devastating form of cancer, and essentially all GBM tumors recur causing fatality. A new surgical technique, fluorescence-guided resection of GBM using 5-aminolevulinic acid (5-ala), improves the extent of resection and positively impacts the length and quality of patient survival. The fluorescence achieved in neoplastic tissue depends directly on the accumulation of porphyrins derived from the metabolism of the 5-ala prodrug within the cancer cell. However, 5-ala induced fluorescence has been reported to be inconsistent. In an effort to determine the cause of the inconsistent fluorescence, the authors investigated the effect of medications commonly prescribed to brain tumor patients on 5-ala induced fluorescence. A model was developed to quantify intracellular porphyrin accumulation using a U87MG GBM cell line constitutively expressing yellow fluorescent protein (YFP-U87). 5-ala mediated fluorescence within the cells was standardized to cell number via the fluorescence emission spectra ratio of porphyrin (405 nm) to YFP (525 nm). 5-ala induced accumulation of porphyrins was measured after treating YFP-U87 cells with phenytoin, dexamethasone, or desipramine for 3 days. After a 6 hour incubation with 5-ala, no significant difference in porphyrin accumulation was observed in cells treated with dexamethasone or desipramine. Phenytoin, however, significantly reduced the accumulation of fluorescent porphyrins within the YFP-U87 cell line by nearly 30% compared to the control. To optimize fluorescence during surgery and improve patient survival these results suggest that further investigations are warranted to determine the effects of commonly administered medications on 5-ala fluorescence-guided resection of GBM

The Transit Ingress and the Tilted Orbit of the Extraordinarily Eccentric Exoplanet HD 80606b

We present the results of a transcontinental campaign to observe the 2009 June 5 transit of the exoplanet HD 80606b. We report the first detection of the transit ingress, revealing the transit duration to be 11.64 +/- 0.25 hr and allowing more robust determinations of the system parameters. Keck spectra obtained at midtransit exhibit an anomalous blueshift, giving definitive evidence that the stellar spin axis and planetary orbital axis are misaligned. The Keck data show that the projected spin-orbit angle is between 32-87 deg with 68.3% confidence and between 14-142 deg with 99.73% confidence. Thus the orbit of this planet is not only highly eccentric (e=0.93), but is also tilted away from the equatorial plane of its parent star. A large tilt had been predicted, based on the idea that the planet's eccentric orbit was caused by the Kozai mechanism. Independently of the theory, it is noteworthy that all 3 exoplanetary systems with known spin-orbit misalignments have massive planets on eccentric orbits, suggesting that those systems migrate differently than lower-mass planets on circular orbits.Comment: ApJ, in press [13 pg

\u3ci\u3e Glioblastoma derived exosomes contribute to tumor immune evasion \u3c/i\u3e

Glioblastoma multiforme (GBM) is the most frequent and lethal primary brain tumor in adults. Despite intense biomedical research, the median survival after diagnosis is 15 months. One factor contributing to this poor prognosis is the immune protection afforded by the tumor microenvironment. Tumors have a diverse repertoire of immune-evasive techniques. One method of evasion not well explored is the release of tumor-derived exosomes. Exosomes are tiny membrane-bound vesicles of endocytic origin that contain viable mRNA and functional proteins that can affect the physiology of recipient cells. Exosome release has been reported for numerous cancer types, including GBM. Exosomes from colon cancer have been shown to carry Fas ligand (FasL) and to induce apoptosis of activated T cells. The aim of this study was to elucidate whether the same immune-evasive technique is used in GBM. GBM exosomes were isolated from the serum-free culture medium of U87 MG and U138 MG cells by using differential ultracentrifugation and were then resuspended in phosphate-buffered saline. The protein concentration of the resulting exosome pellet was determined, and subsequent exosome treatments were based on protein concentration. A3T T cells were plated at a concentration of 10,000 cells per well in 96-well plates and were treated with quantified exosome fractions or with recombinant FasL, and T cell proliferation was determined. Our data demonstrated that tumor-derived exosomes significantly inhibited the proliferation of T cells and that the cellular inhibition resulting from the exosomes was comparable to that seen with the recombinant FasL. These results suggest that targeting FasL in GBM could greatly decrease the amount of immune suppression that occurs at the tumor site