Evolutionary optimisation of antibiotic dosing regimens for bacteria with different levels of resistance.

Antimicrobial resistance is one of the biggest threats to global health, food security, and development. Antibiotic overuse and misuse are the main drivers for the emergence of resistance. It is crucial to optimise the use of existing antibiotics in order to improve medical outcomes, decrease toxicity and reduce the emergence of resistance. We formulate the design of antibiotic dosing regimens as an optimisation problem, and use an evolutionary algorithm suited to continuous optimisation (differential evolution) to solve it. Regimens are represented as vectors of real numbers encoding daily doses, which can vary across the treatment duration. A stochastic mathematical model of bacterial infections with tuneable resistance levels is used to evaluate the effectiveness of evolved regimens. The objective is to minimise the treatment failure rate, subject to a constraint on the maximum total antibiotic used. We consider simulations with different levels of bacterial resistance, two ways of administering the drug (orally and intravenously), as well as coinfections with two strains of bacteria. Our approach produced effective dosing regimens, with an average improvement in lowering the failure rate 30%, when compared with standard fixed-daily-dose regimens with the same total amount of antibiotic.

Stromal Fibroblasts Counteract the Caveolin-1-Dependent Radiation Response of LNCaP Prostate Carcinoma Cells.

In prostate cancer (PCa), a characteristic stromal-epithelial redistribution of the membrane protein caveolin 1 (CAV1) occurs upon tumor progression, where a gain of CAV1 in the malignant epithelial cells is accompanied by a loss of CAV1 in the tumor stroma, both facts that were correlated with higher Gleason scores, poor prognosis, and pronounced resistance to therapy particularly to radiotherapy (RT). However, it needs to be clarified whether inhibiting the CAV1 gain in the malignant prostate epithelium or limiting the loss of stromal CAV1 would be the better choice for improving PCa therapy, particularly for improving the response to RT; or whether ideally both processes need to be targeted. Concerning the first assumption, we investigated the RT response of LNCaP PCa cells following overexpression of different CAV1 mutants. While CAV1 overexpression generally caused an increased epithelial-to-mesenchymal phenotype in respective LNCaP cells, effects that were accompanied by increasing levels of the 5'-AMP-activated protein kinase (AMPK), a master regulator of cellular homeostasis, only wildtype CAV1 was able to increase the three-dimensional growth of LNCaP spheroids, particularly following RT. Both effects could be limited by an additional treatment with the SRC inhibitor dasatinib, finally resulting in radiosensitization. Using co-cultured (CAV1-expressing) fibroblasts as an approximation to the in vivo situation of early PCa it could be revealed that RT itself caused an activated, more tumor-promoting phenotype of stromal fibroblats with an increased an increased metabolic potential, that could not be limited by combined dasatinib treatment. Thus, targeting fibroblasts and/or limiting fibroblast activation, potentially by limiting the loss of stromal CAV1 seems to be absolute for inhibiting the resistance-promoting CAV1-dependent signals of the tumor stroma.

Donor-Specific Antibodies Against Donor Human Leukocyte Antigen are Associated with Graft Inflammation but Not with Fibrosis Long-Term After Liver Transplantation: An Analysis of Protocol Biopsies.

BACKGROUND: Donor-specific antibodies (DSA) against donor human leukocyte antigen after liver transplantation, which are associated with histological changes, have been widely studied with respect to their sustained impact on transplant function. However, their long-term impact after liver transplantation remains unclear. METHODS: We performed a cross-sectional analysis from June 2016 to July 2017 that included all patients who presented themselves for scheduled follow-up after receiving a liver transplantation between September 1989 and December 2016. In addition to a liver protocol biopsy, patients were screened for human leukocyte antigen antibodies (HLAab) and donor-specific antibodies. Subsequently, the association between human leukocyte antigen antibodies, donor-specific antibodies, histologic and clinical features, and immunosuppression was analyzed. RESULTS: Analysis for human leukocyte antigen antibodies and donor-specific antibodies against donor human leukocyte antigen was performed for 291 and 271 patients. A significant association between higher inflammation grades and the presence of human leukocyte antigen antibodies and donor-specific antibodies was detected, while fibrosis stages remained unaffected. These results were confirmed by multivariate logistic regression for inflammation showing a significant increase for presence of human leukocyte antigen antibodies and donor-specific antibodies (OR: 4.43; 95% CI: 1.67-12.6; p=0.0035). Furthermore, the use of everolimus in combination with tacrolimus was significantly associated with the status of negative human leukocyte antigen antibodies and donor-specific antibodies. Viral etiology for liver disease, hepatocellular carcinoma (HCC) and higher steatosis grades of the graft were significantly associated with a lower rate of human leukocyte antigen antibodies. The impact of human leukocyte antigen antibodies and donor-specific antibodies against donor human leukocyte antigen was associated with higher levels of laboratory parameters, such as transaminases and bilirubin. CONCLUSION: Donor-specific antibodies against donor human leukocyte antigen are associated with histological and biochemical graft inflammation after liver transplantation, while fibrosis seems to be unaffected. Future studies should validate these findings for longer observation periods and specific subgroups.

Caveolin-1 regulates the ASMase/ceramide-mediated radiation response of endothelial cells in the context of tumor-stroma interactions.

The integral membrane protein caveolin-1 (CAV1) plays a central role in radioresistance-mediating tumor-stroma interactions of advanced prostate cancer (PCa). Among the tumor-stroma, endothelial cells (EC) evolved as critical determinants of the radiation response. CAV1 deficiency in angiogenic EC was already shown to account for increased apoptosis rates of irradiated EC. This study explores the potential impact of differential CAV1 levels in EC on the acid sphingomyelinase (ASMase)/ceramide pathway as a key player in the regulation of EC apoptosis upon irradiation and cancer cell radioresistance. Enhanced apoptosis sensitivity of CAV1-deficient EC was associated with increased ASMase activity, ceramide generation, formation of large lipid platforms, and finally an altered p38 mitogen-activated protein kinase (MAPK)/heat-shock protein 27 (HSP27)/AKT (protein kinase B, PKB) signaling. CAV1-deficient EC increased the growth delay of LNCaP and PC3 PCa cells upon radiation treatment in direct 3D spheroid co-cultures. Exogenous C6 and C16 ceramide treatment in parallel increased the growth delay of PCa spheroids and induced PCa cell apoptosis. Analysis of the respective ceramide species in PCa cells with increased CAV1 levels like those typically found in radio-resistant advanced prostate tumors further revealed an upregulation of unsaturated C24:1 ceramide that might scavenge the effects of EC-derived apoptosis-inducing C16 ceramide. Higher ASMase as well as ceramide levels could be confirmed by immunohistochemistry in human advanced prostate cancer specimen bearing characteristic CAV1 tumor-stroma alterations. Conclusively, CAV1 critically regulates the generation of ceramide-dependent (re-)organization of the plasma membrane that in turn affects the radiation response of EC and adjacent PCa cells. Understanding the CAV1-dependent crosstalk between tumor cells and the host-derived tumor microvasculature and its impact on radiosensitivity may allow to define a rational strategy for overcoming tumor radiation resistance improving clinical outcomes by targeting CAV1.

Progression-Related Loss of Stromal Caveolin 1 Levels Mediates Radiation Resistance in Prostate Carcinoma via the Apoptosis Inhibitor TRIAP1.

Tumour resistance to chemo- and radiotherapy, as well as molecularly targeted therapies, limits the effectiveness of current cancer treatments. We previously reported that the radiation response of human prostate tumours is critically regulated by CAV1 expression in stromal fibroblasts and that loss of stromal CAV1 expression in advanced tumour stages may contribute to tumour radiotherapy resistance. Here we investigated whether fibroblast secreted anti-apoptotic proteins could induce radiation resistance of prostate cancer cells in a CAV1-dependent manner and identified TRIAP1 (TP53 Regulated Inhibitor of Apoptosis 1) as a resistance-promoting CAV1-dependent factor. TRIAP1 expression and secretion was significantly higher in CAV1-deficient fibroblasts and secreted TRIAP1 was able to induce radiation resistance of PC3 and LNCaP prostate cancer cells in vitro, as well as of PC3 prostate xenografts derived from co-implantation of PC3 cells with TRIAP1-expressing fibroblasts in vivo. Immunohistochemical analyses of irradiated PC3 xenograft tumours, as well as of human prostate tissue specimen, confirmed that the characteristic alterations in stromal-epithelial CAV1 expression were accompanied by increased TRIAP1 levels after radiation in xenograft tumours and within advanced prostate cancer tissues, potentially mediating resistance to radiation treatment. In conclusion, we have determined the role of CAV1 alterations potentially induced by the CAV1-deficient, and more reactive, stroma in radio sensitivity of prostate carcinoma at a molecular level. We suggest that blocking TRIAP1 activity and thus avoiding drug resistance may offer a promising drug development strategy for inhibiting resistance-promoting CAV1-dependent signals.

Connecting Artificial Brains to Robots in a Comprehensive Simulation Framework: The Neurorobotics Platform.

Combined efforts in the fields of neuroscience, computer science, and biology allowed to design biologically realistic models of the brain based on spiking neural networks. For a proper validation of these models, an embodiment in a dynamic and rich sensory environment, where the model is exposed to a realistic sensory-motor task, is needed. Due to the complexity of these brain models that, at the current stage, cannot deal with real-time constraints, it is not possible to embed them into a real-world task. Rather, the embodiment has to be simulated as well. While adequate tools exist to simulate either complex neural networks or robots and their environments, there is so far no tool that allows to easily establish a communication between brain and body models. The Neurorobotics Platform is a new web-based environment that aims to fill this gap by offering scientists and technology developers a software infrastructure allowing them to connect brain models to detailed simulations of robot bodies and environments and to use the resulting neurorobotic systems for in silico experimentation. In order to simplify the workflow and reduce the level of the required programming skills, the platform provides editors for the specification of experimental sequences and conditions, environments, robots, and brain-body connectors. In addition to that, a variety of existing robots and environments are provided. This work presents the architecture of the first release of the Neurorobotics Platform developed in subproject 10 "Neurorobotics" of the Human Brain Project (HBP). At the current state, the Neurorobotics Platform allows researchers to design and run basic experiments in neurorobotics using simulated robots and simulated environments linked to simplified versions of brain models. We illustrate the capabilities of the platform with three example experiments: a Braitenberg task implemented on a mobile robot, a sensory-motor learning task based on a robotic controller, and a visual tracking embedding a retina model on the iCub humanoid robot. These use-cases allow to assess the applicability of the Neurorobotics Platform for robotic tasks as well as in neuroscientific experiments.

Progression-related loss of stromal Caveolin 1 levels fosters the growth of human PC3 xenografts and mediates radiation resistance.

Despite good treatment results in localized prostate tumors, advanced disease stages usually have a pronounced resistance to chemotherapy and radiotherapy. The membrane protein caveolin-1 (Cav1) functions here as an important oncogene. Therefore we examined the impact of stromal Cav1 expression for tumor growth and sensitivity to ionizing radiation (IR). Silencing of Cav1 expression in PC3 cells resulted in increased tumor growth and a reduced growth delay after IR when compared to tumors generated by Cav1-expressing PC3 cells. The increased radiation resistance was associated with increasing amounts of reactive tumor stroma and a Cav1 re-expression in the malignant epithelial cells. Mimicking the human situation these results were confirmed using co-implantation of Cav1-silenced PC3 cells with Cav1-silenced or Cav1-expressing fibroblasts. Immunohistochemically analysis of irradiated tumors as well as human prostate tissue specimen confirmed that alterations in stromal-epithelial Cav1 expressions were accompanied by a more reactive Cav1-reduced tumor stroma after radiation and within advanced prostate cancer tissues which potentially mediates the resistance to radiation treatment. Conclusively, the radiation response of human prostate tumors is critically regulated by Cav1 expression in stromal fibroblasts. Loss of stromal Cav1 expression in advanced tumor stages may thus contribute to resistance of these tumors to radiotherapy.

The HIV-derived protein Vpr52-96 has anti-glioma activity in vitro and in vivo.

Patients with actively replicating human immunodeficiency virus (HIV) exhibit adverse reactions even to low irradiation doses. High levels of the virus-encoded viral protein R (Vpr) are believed to be one of the major underlying causes for increased radiosensitivity. As Vpr efficiently crosses the blood-brain barrier and accumulates in astrocytes, we examined its efficacy as a drug for treatment of glioblastoma multiforme (GBM).In vitro, four glioblastoma-derived cell lines with and without methylguanine-DNA methyltransferase (MGMT) overexpression (U251, U87, U251-MGMT, U87-MGMT) were exposed to Vpr, temozolomide (TMZ), conventional photon irradiation (2 to 6 Gy) or to combinations thereof. Vpr showed high rates of acute toxicities with median effective doses of 4.0±1.1 µM and 15.7±7.5 µM for U251 and U87 cells, respectively. Caspase assays revealed Vpr-induced apoptosis in U251, but not in U87 cells. Vpr also efficiently inhibited clonogenic survival in both U251 and U87 cells and acted additively with irradiation. In contrast to TMZ, Vpr acted independently of MGMT expression.Dose escalation in mice (n=12) was feasible and resulted in no evident renal or liver toxicity. Both, irradiation with 3x5 Gy (n=8) and treatment with Vpr (n=5) delayed intracerebral tumor growth and prolonged overall survival compared to untreated animals (n=5; p3x5 Gy<0.001 and pVpr=0.04; log-rank test).Our data show that the HIV-encoded peptide Vpr exhibits all properties of an effective chemotherapeutic drug and may be a useful agent in the treatment of GBM.

Cellular Pathways in Response to Ionizing Radiation and Their Targetability for Tumor Radiosensitization.

During the last few decades, improvements in the planning and application of radiotherapy in combination with surgery and chemotherapy resulted in increased survival rates of tumor patients. However, the success of radiotherapy is impaired by two reasons: firstly, the radioresistance of tumor cells and, secondly, the radiation-induced damage of normal tissue cells located in the field of ionizing radiation. These limitations demand the development of drugs for either radiosensitization of tumor cells or radioprotection of normal tissue cells. In order to identify potential targets, a detailed understanding of the cellular pathways involved in radiation response is an absolute requirement. This review describes the most important pathways of radioresponse and several key target proteins for radiosensitization.

Inhibition of 13-cis retinoic acid-induced gene expression of reactive-resistance genes by thalidomide in glioblastoma tumours in vivo.

The cell differentiation potential of 13-cis retinoic acid (RA) has not succeeded in the clinical treatment of glioblastoma (GBM) so far. However, RA may also induce the expression of resistance genes such as HOXB7 which can be suppressed by Thalidomide (THAL). Therefore, we tested if combined treatment with RA+THAL may inhibit growth of glioblastoma in vivo. Treatment with RA+THAL but not RA or THAL alone significantly inhibited tumour growth. The synergistic effect of RA and THAL was corroborated by the effect on proliferation of glioblastoma cell lines in vitro. HOXB7 was not upregulated but microarray analysis validated by real-time PCR identified four potential resistance genes (IL-8, HILDPA, IGFBPA, and ANGPTL4) whose upregulation by RA was suppressed by THAL. Furthermore, genes coding for small nucleolar RNAs (snoRNA) were identified as a target for RA for the first time, and their upregulation was maintained after combined treatment. Pathway analysis showed upregulation of the Ribosome pathway and downregulation of pathways associated with proliferation and inflammation. In conclusion, combined treatment with RA + THAL delayed growth of GBM xenografts and suppressed putative resistance genes associated with hypoxia and angiogenesis. This encourages further pre-clinical and clinical studies of this drug combination in GBM.

Characterization of Dynamic Behaviour of MCF7 and MCF10A Cells in Ultrasonic Field Using Modal and Harmonic Analyses.

Treatment options specifically targeting tumour cells are urgently needed in order to reduce the side effects accompanied by chemo- or radiotherapy. Differences in subcellular structure between tumour and normal cells determine their specific elasticity. These structural differences can be utilised by low-frequency ultrasound in order to specifically induce cytotoxicity of tumour cells. For further evaluation, we combined in silico FEM (finite element method) analyses and in vitro assays to bolster the significance of low-frequency ultrasound for tumour treatment. FEM simulations were able to calculate the first resonance frequency of MCF7 breast tumour cells at 21 kHz in contrast to 34 kHz for the MCF10A normal breast cells, which was due to the higher elasticity and larger size of MCF7 cells. For experimental validation of the in silico-determined resonance frequencies, equipment for ultrasonic irradiation with distinct frequencies was constructed. Differences for both cell lines in their response to low-frequent ultrasonic treatment were corroborated in 2D and in 3D cell culture assays. Treatment with ~ 24.5 kHz induced the death of MCF7 cells and MDA-MB-231 metastases cells possessing a similar elasticity; frequencies of > 29 kHz resulted in cytotoxicity of MCF10A. Fractionated treatments by ultrasonic irradiation of suspension myeloid HL60 cells resulted in a significant decrease of viable cells, mostly significant after threefold irradiation in intervals of 3 h. Most importantly in regard to a clinical application, combined ultrasonic treatment and chemotherapy with paclitaxel showed a significantly increased killing of MCF7 cells compared to both monotherapies. In summary, we were able to determine for the first time for different tumour cell lines a specific frequency of low-intensity ultrasound for induction of cell ablation. The cytotoxic effect of ultrasonic irradiation could be increased by either fractionated treatment or in combination with chemotherapy. Thus, our results will open new perspectives in tumour treatment.

In vivo Quantification of the Effects of Radiation and Presence of Hair Follicle Pores on the Proliferation of Fibroblasts in an Acellular Human Dermis in a Dorsal Skinfold Chamber: Relevance for Tissue Reconstruction following Neoadjuvant Therapy.

INTRODUCTION: In neoadjuvant therapy, irradiation has a deleterious effect on neoangiogenesis. The aim of this study was to examine the post-implantation effects of neoadjuvant irradiation on the survival and proliferation of autologous cells seeded onto an acellular human dermis (hAD; Epiflex). Additionally, we examined the influence of dermal hair follicle pores on viability and proliferation. We used dorsal skinfold chambers implanted in rats and in-situ microscopy to quantify cell numbers over 9 days. METHODS: 24 rats received a skinfold chamber and were divided into 2 main groups; irradiated and unirradiated. In the irradiated groups 20Gy were applied epicutaneously at the dorsum. Epiflex pieces were cut to size 5x5mm such that each piece had either one or more visible hair follicle pores, or no such visible pores. Fibroblasts were transduced lentiviral with a fluorescent protein for cell tracking. Matrices were seeded statically with 2.5x104 fluorescent fibroblasts and implanted into the chambers. In each of the two main groups, half of the rats received Epiflex with hair follicle pores and half received Epiflex without pores. Scaffolds were examined in-situ at 0, 3, 6 and 9 days after transplantation. Visible cells on the surface were quantified using ImageJ. RESULTS: In all groups cell numbers were decreased on day 3. A treatment-dependent increase in cell numbers was observed at subsequent time points. Irradiation had an adverse effect on cell survival and proliferation. The number of cells detected in both irradiated and non-irradiated subjects was increased in those subjects that received transplants with hair follicle pores. DISCUSSION: This in-vivo study confirms that radiation negatively affects the survival and proliferation of fibroblasts seeded onto a human dermis transplant. The presence of hair follicle pores in the dermis transplants is shown to have a positive effect on cell survival and proliferation even in irradiated subjects.

The influence of retinoic Acid and thalidomide on the radiosensitivity of u343 glioblastoma cells.

BACKGROUND/AIM: 13-cis-retinoic acid (RA) and thalidomide have shown a synergistic anti-proliferative effect on U343 glioblastoma (GBM) cells. In the present study, we test if their combined treatment might enhance the radiosensitivity of these cells. MATERIALS AND METHODS: The radiosensitivity of U343 GBM cells was determined by the colony formation assay. Fibroblast growth factor-2 (FGF2) gene expression was determined by a quantitative polymerase chain reaction (qPCR). RESULTS: RA up-regulated FGF2 gene expression, which was abrogated by thalidomide. No radiosensitisation by RA was observed under standard culture conditions with 10% serum, but enhanced radiosensitivity was observed under 1% serum during irradiation. However, a synergistic effect with thalidomide was not observed. CONCLUSION: Growth factors in the culture medium may mask radiosensitization by RA while autocrine expression of FGF2 did not seem to be protective. Importantly, the anti-proliferative effect of RA in combination with thalidomide would not compromise the radiosensitivity of these GBM cells.

Radioprotection of normal tissue cells.

Improvements of radiotherapy in combination with surgery and systemic therapy have resulted in increased survival rates of tumor patients. However, radiation-induced normal tissue toxicity is still dose limiting. Several strategies have been pursued with the goal to develop substances which may prevent or reduce damage to normal tissue. Drugs applied before radiotherapy are called radioprotectors; those given after radiotherapy to reduce long-term effects are radiomitigators. Despite more than 50 years of research, until now only two substances, amifostine and palifermin, have overcome all obstacles of clinical approval and are applied during radiotherapy of head and neck cancer or total body irradiation, respectively. However, better understanding of the cellular pathways involved in radiation response has allowed the development of several highly promising drugs functioning as scavengers of reactive oxygen species or targeting specific molecules involved in regulation of cell death pathways or cell cycle arrest. The present review describes the major targets for radioprotectors or radiomitigators currently tested in clinical trials.

Ischemia-like oxygen and glucose deprivation mediates down-regulation of cell surface γ-aminobutyric acidB receptors via the endoplasmic reticulum (ER) stress-induced transcription factor CCAAT/enhancer-binding protein (C/EBP)-homologous protein (CHOP).

Cerebral ischemia frequently leads to long-term disability and death. Excitotoxicity is believed to be the main cause for ischemia-induced neuronal death. Although a role of glutamate receptors in this process has been firmly established, the contribution of metabotropic GABAB receptors, which control excitatory neurotransmission, is less clear. A prominent characteristic of ischemic insults is endoplasmic reticulum (ER) stress associated with the up-regulation of the transcription factor CCAAT/enhancer-binding protein-homologous protein (CHOP). After inducing ER stress in cultured cortical neurons by sustained Ca(2+) release from intracellular stores or by a brief episode of oxygen and glucose deprivation (in vitro model of cerebral ischemia), we observed an increased expression of CHOP accompanied by a strong reduction of cell surface GABAB receptors. Our results indicate that down-regulation of cell surface GABAB receptors is caused by the interaction of the receptors with CHOP in the ER. Binding of CHOP prevented heterodimerization of the receptor subunits GABAB1 and GABAB2 and subsequent forward trafficking of the receptors to the cell surface. The reduced level of cell surface receptors diminished GABAB receptor signaling and, thus, neuronal inhibition. These findings indicate that ischemia-mediated up-regulation of CHOP down-regulates cell surface GABAB receptors by preventing their trafficking from the ER to the plasma membrane. This mechanism leads to diminished neuronal inhibition and may contribute to excitotoxicity in cerebral ischemia.

The disparate twins: a comparative study of CXCR4 and CXCR7 in SDF-1α-induced gene expression, invasion and chemosensitivity of colon cancer.

PURPOSE: In colorectal cancer, increased expression of the CXC chemokine receptor 4 (CXCR4) has been shown to provoke metastatic disease due to the interaction with its ligand stromal cell-derived factor-1 (SDF-1). Recently, a second SDF-1 receptor, CXCR7, was found to enhance tumor growth in solid tumors. Albeit signaling cascades via SDF-1/CXCR4 have been intensively studied, the significance of the SDF-1/CXCR7-induced intracellular communication triggering malignancy is still only marginally understood. EXPERIMENTAL DESIGN: In tumor tissue of 52 patients with colorectal cancer, we observed that expression of CXCR7 and CXCR4 increased with tumor stage and tumor size. Asking whether activation of CXCR4 or CXCR7 might result in a similar expression pattern, we performed microarray expression analyses using lentivirally CXCR4- and/or CXCR7-overexpressing SW480 colon cancer cell lines with and without stimulation by SDF-1α. RESULTS: Gene regulation via SDF-1α/CXCR4 and SDF-1α/CXCR7 was completely different and partly antidromic. Differentially regulated genes were assigned by gene ontology to migration, proliferation, and lipid metabolic processes. Expressions of AKR1C3, AXL, C5, IGFBP7, IL24, RRAS, and TNNC1 were confirmed by quantitative real-time PCR. Using the in silico gene set enrichment analysis, we showed that expressions of miR-217 and miR-218 were increased in CXCR4 and reduced in CXCR7 cells after stimulation with SDF-1α. Functionally, exposure to SDF-1α increased invasiveness of CXCR4 and CXCR7 cells, AXL knockdown hampered invasion. Compared with controls, CXCR4 cells showed increased sensitivity against 5-FU, whereas CXCR7 cells were more chemoresistant. CONCLUSIONS: These opposing results for CXCR4- or CXCR7-overexpressing colon carcinoma cells demand an unexpected attention in the clinical application of chemokine receptor antagonists such as plerixafor.

Mitogenic signalling in the absence of epidermal growth factor receptor activation in a human glioblastoma cell line.

Epidermal growth factor receptor (EGFR) gene amplification and overexpression are commonly present in glioblastoma, and confer advantages of growth, invasiveness and radio/chemotherapy-resistance for tumour cells. Here, we assessed the role of EGFR activation for downstream mitogenic signalling in the commonly used glioblastoma cell line U251. Despite the high expression level, activation of EGFR under standard culture conditions was low. Intact EGFR function was verified by the rapid phosphorylation of EGFR and downstream mitogen-activated protein(MAP) kinase ERK1/2 upon addition of exogenous EGF to serum-starved cells. By contrast, addition of fetal bovine serum (FBS) activated downstream ERK1/2 via the MAP kinase kinase without phosphorylating EGFR. A phosphoreceptor tyrosine kinase array showed FBS-induced activation of insulin-like growth factor-1 receptor (IGF-1R),and the IGF-1R inhibitor AG1024 inhibited FBS-induced phosphorylation of ERK1/2, implying IGF-1R as the major driver of FBS-associated mitogenic signalling in the absence of exogenous EGF. These findings have important implications for in vitro drug testing in glioblastoma. Moreover, activation of ERK1/2 was also strongly influenced by growth state and cell density of U251 cultures. Re-seeding exponentially growing cultures at high cell density induced p27/CDKN1B expression and suppressed P-ERK1/2 indicating a certain regulation of proliferation by contact inhibition. Strikingly, highly activated ERK1/2 signalling and cell cycle progression occurred when cells were released from plateau phase regardless of high seeding density. This phenomenon might implicate a proliferation response in the early recurrence observed after clinical therapy in glioblastoma patients. However, whether it will recapitulate in vivo remains to be demonstrated.

CXCR4 Expression and Treatment with SDF-1α or Plerixafor Modulate Proliferation and Chemosensitivity of Colon Cancer Cells.

BACKGROUND: Signaling through stromal cell-derived factor-1α (SDF-1α), strongly secreted by bone marrow stromal cells and the CXC chemokine receptor 4 (CXCR4) exposed on tumor cells has pivotal roles in proliferation, metastasis, and tumor cell "dormancy." Dormancy is associated with cytostatic drug resistance and is probably a property of tumor stem cells and minimal residual disease. Thus, hampering the SDF-1α/CXCR4 cross talk by a CXCR4 antagonist like Plerixafor (AMD3100) should overcome tumor cell dormancy bymobilization of tumor cells from "sanctuary" niches. Our aim was to elucidate the direct effects exerted by SDF-1α and Plerixafor on proliferation, chemosensitivity, and apoptosis of CXCR4-expressing tumor cells. METHODS: The ability of SDF-1α and Plerixafor to regulate intracellular signaling, proliferation, and invasion was investigated using two colon cancer cell lines (HT-29 and SW480) with either high endogenous or lentiviral expression of CXCR4 compared to their respective low CXCR4-expressing counterparts as a model system. Efficacy of Plerixafor on sensitivity of these cell lines against 5-fluorouracil, irinotecan, or oxaliplatin was determined in a cell viability assay as well as stroma-dependent cytotoxicity and apoptosis assays. RESULTS: SDF-1α increased proliferation, invasion, and ERK signaling of endogenously and lentivirally CXCR4-expressing cells. Exposure to Plerixafor reduced proliferation, invasion, and extracellular signal-regulated kinase 1/2 (ERK1/2) signaling. Combination of chemotherapy with Plerixafor showed an additive effect on chemosensitivity and apoptosis in CXCR4-overexpressing cells. An SDF-1-secreting feeder layer provideda"protective niche" for CXCR4-overexpressing cells resulting in decreased chemosensitivity. CONCLUSION: CXCR4-antagonistic therapy mobilizes and additionally sensitizes tumor cells toward cytoreductive chemotherapy.

Localization microscopy (SPDM) reveals clustered formations of P-glycoprotein in a human blood-brain barrier model.

P-glycoprotein (Pgp; also known as MDR1, ABCB1) is the most important and best studied efflux transporter at the blood-brain barrier (BBB); however, the organization of Pgp is unknown. The aim of this study was to employ the recently developed super-resolution fluorescence microscopy method spectral precision distance microscopy/spectral position determination microscopy (SPDM) to investigate the spatial distribution of Pgp in the luminal plasma membrane of brain capillary endothelial cells. Potential disturbing effects of cell membrane curvatures on the distribution analysis are addressed with computer simulations. Immortalized human cerebral microvascular endothelial cells (hCMEC/D3) served as a model of human BBB. hCMEC/D3 cells were transduced with a Pgp-green fluorescent protein (GFP) fusion protein incorporated in a lentivirus-derived vector. The expression and localization of the Pgp-GFP fusion protein was visualized by SPDM. The limited resolution of SPDM in the z-direction leads to a projection during the imaging process affecting the appeared spatial distribution of fluorescence molecules in the super-resolution images. Therefore, simulations of molecule distributions on differently curved cell membranes were performed and their projected spatial distribution was investigated. Function of the fusion protein was confirmed by FACS analysis after incubation of cells with the fluorescent probe eFluxx-ID Gold in absence and presence of verapamil. More than 112,000 single Pgp-GFP molecules (corresponding to approximately 5,600 Pgp-GFP molecules per cell) were detected by SPDM with an averaged spatial resolution of approximately 40 nm in hCMEC/D3 cells. We found that Pgp-GFP is distributed in clustered formations in hCMEC/D3 cells while the influence of present random cell membrane curvatures can be excluded based on the simulation results. Individual formations are distributed randomly over the cell membrane.

Modulation of cell surface GABA(B) receptors by desensitization, trafficking and regulated degradation.

Inhibitory neurotransmission ensures normal brain function by counteracting and integrating excitatory activity. γ-Aminobutyric acid (GABA) is the main inhibitory neurotransmitter in the mammalian central nervous system, and mediates its effects via two classes of receptors: the GABA(A) and GABA(B) receptors. GABA(A) receptors are heteropentameric GABA-gated chloride channels and responsible for fast inhibitory neurotransmission. GABA(B) receptors are heterodimeric G protein coupled receptors (GPCR) that mediate slow and prolonged inhibitory transmission. The extent of inhibitory neurotransmission is determined by a variety of factors, such as the degree of transmitter release and changes in receptor activity by posttranslational modifications (e.g., phosphorylation), as well as by the number of receptors present in the plasma membrane available for signal transduction. The level of GABA(B) receptors at the cell surface critically depends on the residence time at the cell surface and finally the rates of endocytosis and degradation. In this review we focus primarily on recent advances in the understanding of trafficking mechanisms that determine the expression level of GABA(B) receptors in the plasma membrane, and thereby signaling strength.