ABC TRANSPORTERS IN GLIOBLASTOMA: ANTICANCER DRUG TRANSPORT AND TRANSPORTER REGULATION AT THE BLOOD-BRAIN BARRIER

Glioblastoma is one of the deadliest cancers, with a median survival of only one year. Even after aggressive treatment consisting of surgical resection, radiation, and chemotherapy, most glioblastoma patients suffer from tumor recurrence within 6-9 months. One reason for treatment failure of anticancer drugs is the blood-brain barrier that protects the brain by impeding xenobiotic uptake from the blood. To this end, efflux transporters at the human blood-brain barrier, such as P-glycoprotein (ABCB1) and Breast Cancer Resistance Protein (ABCG2), prevent many compounds, including anticancer drugs, from entering the brain. Thus far, approaches to deliver anticancer drugs across the blood-brain barrier have been unsuccessful in clinical trials. Therefore, novel therapeutic strategies are needed to overcome the blood-brain barrier for improved glioblastoma treatment. Here, I address this need in 3 independent aims: Elucidate the involvement and cooperation of ABC transporters in anticancer drug transport at the blood-brain barrier Establish and characterize human glioblastoma models Evaluate the impact of dual PI3K/Akt inhibition on brain uptake of anticancer drugs Aim 1: While Abcb1/Abcg2 inhibition improved survival in mouse glioblastoma models, clinical trials had to be terminated due to a lack of efficacy, sparking a discussion that other ABC transporters might be involved in this process. To discern how multiple ABC transporters cooperate in restricting anticancer drug uptake at the blood-brain barrier, I evaluated the effect of several efflux transporters at the blood-brain barrier on the brain level of anticancer drugs using transporter inhibitors or knockout mice. The results from this study suggest that Abcc4 works in concert with Abcb1/Abcg2 in restricting brain access of the tested anticancer drugs in mice. Further experiments are necessary to confirm this cooperation at the human blood-brain barrier. In part, these findings might provide one possible explanation why therapeutic strategies that solely focus on ABCB1/ABCG2 failed to improve treatment outcomes for glioblastoma patients. Aim 2: Successful treatment of glioblastoma requires reliable preclinical animal models to evaluate novel approaches and assess their potential therapeutic benefit. While many different glioblastoma models exist, most are not well characterized and only recapitulate a subset of glioblastoma characteristics. Here, I describe and compare two human glioblastoma models, U87-luc2 and U251-FLuc. While both models behave similarly in vitro, they have different in vivo tumor characteristics, such as invasiveness and blood-brain barrier disruption. Together, the two glioblastoma models recapitulate the tumor characteristics of a majority of patients. Aim 3: Direct transporter inhibition is unsuccessful in improving glioblastoma patient survival due to the low efficacy of inhibitors and adverse effects associated with combination treatment. However, efflux transporter regulation could open a “window-in-time” to allow anticancer drug uptake into the brain. Here, I tested a novel molecular switch approach to overcome Abcb1/Abcg2-mediated efflux at the blood-brain barrier. My data indicate that PI3K/Akt could serve as a molecular switch to transiently turn off Abcb1/Abcg2 at the blood-brain barrier and increase brain levels of anticancer drugs

Chandra X-ray spectroscopy of the focused wind in the Cygnus X-1 system. I. The non-dip spectrum in the low/hard state

We present analyses of a 50 ks observation of the supergiant X-ray binary system Cygnus X-1/HDE 226868 taken with the Chandra High Energy Transmission Grating Spectrometer (HETGS). Cyg X-1 was in its spectrally hard state and the observation was performed during superior conjunction of the black hole, allowing for the spectroscopic analysis of the accreted stellar wind along the line of sight. A significant part of the observation covers X-ray dips as commonly observed for Cyg X-1 at this orbital phase, however, here we only analyze the high count rate non-dip spectrum. The full 0.5-10 keV continuum can be described by a single model consisting of a disk, a narrow and a relativistically broadened Fe Kalpha line, and a power law component, which is consistent with simultaneous RXTE broad band data. We detect absorption edges from overabundant neutral O, Ne and Fe, and absorption line series from highly ionized ions and infer column densities and Doppler shifts. With emission lines of He-like Mg XI, we detect two plasma components with velocities and densities consistent with the base of the spherical wind and a focused wind. A simple simulation of the photoionization zone suggests that large parts of the spherical wind outside of the focused stream are completely ionized, which is consistent with the low velocities (<200 km/s) observed in the absorption lines, as the position of absorbers in a spherical wind at low projected velocity is well constrained. Our observations provide input for models that couple the wind activity of HDE 226868 to the properties of the accretion flow onto the black hole.Comment: 16 pages, 15 figures, uses emulateapj, published as ApJ 690:330-346, 2009 January

Multi-Satellite Observations of Cygnus X-1 to Study the Focused Wind and Absorption Dips

High-mass X-ray binary systems are powered by the stellar wind of their donor stars. The X-ray state of Cygnus X-1 is correlated with the properties of the wind which defines the environment of mass accretion. Chandra-HETGS observations close to orbital phase 0 allow for an analysis of the photoionzed stellar wind at high resolution, but because of the strong variability due to soft X-ray absorption dips, simultaneous multi-satellite observations are required to track and understand the continuum, too. Besides an earlier joint Chandra and RXTE observation, we present first results from a recent campaign which represents the best broad-band spectrum of Cyg X-1 ever achieved: On 2008 April 18/19 we observed this source with XMM-Newton, Chandra, Suzaku, RXTE, INTEGRAL, Swift, and AGILE in X- and gamma-rays, as well as with VLA in the radio. After superior conjunction of the black hole, we detect soft X-ray absorption dips likely due to clumps in the focused wind covering >95 % of the X-ray source, with column densities likely to be of several 10^23 cm^-2, which also affect photon energies above 20 keV via Compton scattering.Comment: 10 pages, contributed talk at the 7th Microquasar Workshop, Foca, Turkey, Sept. 1-5, 200

The Ionized Stellar Wind in Vela X-1 During Eclipse

We present a first analysis of a high resolution X-ray spectrum of the ionized stellar wind of Vela X-1 during eclipse. The data were obtained with the High Energy Transmission Grating Spectrometer onboard the Chandra X-ray Observatory. The spectrum is resolved into emission lines with fluxes between 0.02 and 1.04x10^4 ph/cm^2/s. We identify lines from a variety of charge states, including fluorescence lines from cold material, a warm photoionized wind. We can exclude signatures from collisionally ionized plasmas. For the first time we identify fluorescence lines from L-shell ions from lower Z elements. We also detect radiative recombination continua from a kT = 10 eV (1.2 x 10^5 K) photoionized optically thin gas. The fluorescence line fluxes infer the existence of optically thick and clumped matter within or outside the warm photoionized plasma.Comment: 4 pages, 2 figures, accepted by ApJ letter

Characterization and Comparison of Human Glioblastoma Models

AbstractGlioblastoma (GBM) is one of the deadliest cancers. Treatment options are limited, and median patient survival is only several months. Translation of new therapies is hindered by a lack of GBM models that fully recapitulate disease heterogeneity. Here, we characterize two human GBM models (U87-luc2, U251-RedFLuc). In vitro, both cell lines express similar levels of luciferase and show comparable sensitivity to temozolomide and lapatinib exposure. In vivo, however, the two GBM models recapitulate diferent aspects of the disease. U87-luc2 cells quickly grow into large, well-demarcated tumors; U251-RedFLuc cells form small, highly invasive tumors. Using a new method to assess GBM invasiveness based on detecting tumor-specifc anti-luciferase staining in brain slices, we found that U251-RedFLuc cells are more invasive than U87-luc2 cells. Lastly, we determined expression levels of ABC transporters in both models. Our fndings indicate that U87-luc2 and U251-RedFLuc GBM models recapitulate diferent aspects of GBM heterogeneity that need to be considered in preclinical research

High resolution Chandra HETG and RXTE observations of GRS 1915+105 : A hot disk atmosphere & cold gas enriched in Iron and Silicon

The time-averaged 30 ks Chandra HETGS observation of the micro-quasar GRS 1915+105 in the low hard state reveals for the first time in this source neutral K absorption edges from Fe, Si, Mg, & S. Ionized resonance absorption from H-, and He-like Fe (XXV, XXVI), Ca XX and possibly emission from neutral Fe Kalpha and ionized Fe XXV (forbidden, or the resonance emission component of a P-Cygni profile) are also seen. We report the tentative detection of the first astrophysical signature of XAFS in the photoelectric edge of Si (and possibly Fe and Mg), attributed to material in grains. The large column densities measured from the neutral edges reveal anomalous Si and Fe abundances. Scenarios for which the anomalous abundances can be attributed to surrounding cold material associated with GRS 1915+105 and/or that the enrichment may signify either a highly unusual supernova/hypernova, or external supernova activity local to the binary are discussed. We attribute the ionized features to a hot disk, disk-wind, or corona environment. These features allow for constraints on the ionization parameter (log xi > 4.15), temperature (T > 2.4 x 10^6 K), and hydrogen equivalent number density (n > 10^{12} cm^{-3}) for this region. Variability studies with simultaneous RXTE data show that the light curve count rate tracks changes in the disk blackbody and the power-law flux. Spectral changes in the Chandra data also track the behavior of the light curve, and may point to changes in both the ionizing flux and density of the absorber. A 3.69 Hz QPO and weak first harmonic is seen in the RXTE data.Comment: Accepted for publication in ApJ., 9 pages, 5 figure

Double-Peaked X-Ray Lines from the Oxygen/Neon-Rich Accretion Disk in 4U1626-67

We report on a 39 ks observation of the 7.7-s low-mass X-ray binary pulsar 4U1626-67 with the High Energy Transmission Grating Spectrometer (HETGS) on the Chandra X-Ray Observatory. This ultracompact system consists of a disk-accreting magnetic neutron star and a very low mass, hydrogen-depleted companion in a 42-min binary. We have resolved the previously reported Ne/O emission line complex near 1 keV into Doppler pairs of broadened (2500 km/s FWHM) lines from highly ionized Ne and O. In most cases, the blue and red line components are of comparable strength, with blueshifts of 1550-2610 km/s and redshifts of 770-1900 km/s. The lines appear to originate in hot (10^6 K), dense material just below the X-ray-heated skin of the outer Keplerian accretion disk, or else possibly in a disk wind driven from the pulsar's magnetopause. The observed photoelectric absorption edges of Ne and O appear nearly an order of magnitude stronger than expected from interstellar material and are likely formed in cool, metal-rich material local to the source. Based on the inferred local abundance ratios, we argue that the mass donor in this binary is probably the 0.02 M_sun chemically fractionated core of a C-O-Ne or O-Ne-Mg white dwarf which has previously crystallized.Comment: 9 pages. Accepted for publication in ApJ. Table 2 correcte

Correction:Prevalence and Cardiovascular Associations of Diabetic Retinopathy and Maculopathy: Results from the Gutenberg Health Study

Diabetic retinopathy (DR) is the leading cause of blindness in people of working age. The purpose of this paper is to report the prevalence and cardiovascular associations of diabetic retinopathy and maculopathy (DMac) in Germany.The Gutenberg Health Study (GHS) is a population-based study with 15,010 participants aged between 35 at 74 years from the city of Mainz and the district of Mainz-Bingen. We determined the weighted prevalence of DR and DMac by assessing fundus photographs of persons with diabetes from the GHS data base. Diabetes was defined as HbA1c ≥ 6.5%, known diagnosis diabetes mellitus or known diabetes medication. Furthermore, we analysed the association between DR and cardiovascular risk factors and diseases.Overall, 7.5% (1,124/15,010) of the GHS cohort had diabetes. Of these, 27.7% were unaware of their disease and thus were newly diagnosed by their participation in the GHS. The prevalence of DR and DMac was 21.7% and 2.3%, respectively among patients with diabetes. Vision-threatening disease was present in 5% of the diabetic cohort. In the multivariable analysis DR (all types) was associated with age (Odds Ratio [95% confidence interval]: 0.97 [0.955-0.992]; p = 0.006) arterial hypertension (1.90 [1.190-3.044]; p = 0.0072) and vision-threatening DR with obesity (3.29 [1.504-7.206]; p = 0.0029). DR (all stages) and vision-threatening DR were associated with duration of diabetes (1.09 [1.068-1.114]; p<0.0001 and 1.18 [1.137-1.222]; p<0.0001, respectively).Our calculations suggest that approximately 142 000 persons aged between 35 and 74 years have vision threatening diabetic retinal disease in Germany [corrected].Prevalence of DR was lower in the GHS compared to East-Asian studies. Associations were found with age, arterial hypertension, obesity, and duration of diabetes mellitus

Availability of phosphate for phytoplankton and bacteria and of labile organic carbon for bacteria at different pCO2 levels in a mesocosm study

Availability of phosphate for phytoplankton and bacteria and of glucose for bacteria at different pCO2 levels were studied in a mesocosm experiment (PeECE III). Using nutrient-depleted SW Norwegian fjord waters, three different levels of pCO2 (350 μatm: 1×CO2; 700 μatm: 2×CO2; 1050 μatm: 3×CO2) were set up, and nitrate and phosphate were added at the start of the experiment in order to induce a phytoplankton bloom. Despite similar responses of total particulate P concentration and phosphate turnover time at the three different pCO2 levels, the size distribution of particulate P and 33PO4 uptake suggested that phosphate transferred to the >10 μm fraction was greater in the 3×CO2 mesocosm during the first 6–10 days when phosphate concentration was high. During the period of phosphate depletion (after Day 12), specific phosphate affinity and specific alkaline phosphatase activity (APA) suggested a P-deficiency (i.e. suboptimal phosphate supply) rather than a P-limitation for the phytoplankton and bacterial community at the three different pCO2 levels. Specific phosphate affinity and specific APA tended to be higher in the 3×CO2 than in the 2×CO2 and 1×CO2 mesocosms during the phosphate depletion period, although no statistical differences were found. Glucose turnover time was correlated significantly and negatively with bacterial abundance and production but not with the bulk DOC concentration. This suggests that even though constituting a small fraction of the bulk DOC, glucose was an important component of labile DOC for bacteria. Specific glucose affinity of bacteria behaved similarly at the three different pCO2 levels with measured specific glucose affinities being consistently much lower than the theoretical maximum predicted from the diffusion-limited model. This suggests that bacterial growth was not severely limited by the glucose availability. Hence, it seems that the lower availability of inorganic nutrients after the phytoplankton bloom reduced the bacterial capacity to consume labile DOC in the upper mixed layer of the stratified mesocosms

Revealing the Dusty Warm Absorber in MCG--6-30-15 with the Chandra HETG

We present detailed evidence for a warm absorber in the Seyfert 1 galaxy MCG--6-30-15 and dispute earlier claims for relativistic O line emission. The HETG spectra show numerous narrow, unresolved (FWHM < 200 km/s) absorption lines from a wide range of ionization states of N, O, Mg, Ne, Si, S, Ar, and Fe. The O VII edge and 1s^2--1snp resonance line series to n=9 are clearly detected at rest in the AGN frame. We attribute previous reports of an apparently highly redshifted O VII edge to the 1s^2--1snp (n > 5) O VII resonance lines, and a neutral Fe L absorption complex. The shape of the Fe L feature is nearly identical to that seen in the spectra of several X-ray binaries, and in laboratory data. The implied dust column density agrees with that obtained from reddening studies, and gives the first direct X-ray evidence for dust embedded in a warm absorber. The O VIII resonance lines and weak edge are also detected, and the spectral rollover below 2 keV is explained by the superposition of numerous absorption lines and edges. We identify, for the first time, a KLL resonance in the O VI photoabsorption cross section, giving a measure of the O VI column density. The O VII (f) emission detected at the systemic velocity implies a covering fraction of ~5% (depending on the observed vs. time-averaged ionizing flux). Our observations show that a dusty warm absorber model is not only adequate to explain all the spectral features > 0.48 keV (< 26 \AA) the data REQUIRE it. This contradicts the interpretation of Branduardi-Raymont et al. (2001) that this spectral region is dominated by highly relativistic line emission from the vicinity of the black hole.Comment: 4.5 pages, 1 color figure, accepted (April 2001) for publication in ApJL, not many changes from the initial submission - updated/added some measuements for the O VII resonance series, and added a discussion about FeO2 grain