Inhibition of cholesterol metabolism underlies synergy between mTOR pathway inhibition and chloroquine in bladder cancer cells.

Mutations to fibroblast growth factor receptor 3 (FGFR3) and phosphatase and tensin homologue (PTEN) signalling pathway components (for example, PTEN loss, PIK3CA, AKT1, TSC1/2) are common in bladder cancer, yet small-molecule inhibitors of these nodes (FGFR/PTENi) show only modest activity in preclinical models. As activation of autophagy is proposed to promote survival under FGFR/PTENi, we have investigated this relationship in a panel of 18 genetically diverse bladder cell lines. We found that autophagy inhibition does not sensitise bladder cell lines to FGFR/PTENi, but newly identify an autophagy-independent cell death synergy in FGFR3-mutant cell lines between mTOR (mammalian target of rapamycin) pathway inhibitors and chloroquine (CQ)-an anti-malarial drug used as a cancer therapy adjuvant in over 30 clinical trials. The mechanism of synergy is consistent with lysosomal cell death (LCD), including cathepsin-driven caspase activation, and correlates with suppression of cSREBP1 and cholesterol biosynthesis in sensitive cell lines. Remarkably, loss of viability can be rescued by saturating cellular membranes with cholesterol or recapitulated by statin-mediated inhibition, or small interfering RNA knockdown, of enzymes regulating cholesterol metabolism. Modulation of CQ-induced cell death by atorvastatin and cholesterol is reproduced across numerous cell lines, confirming a novel and fundamental role for cholesterol biosynthesis in regulating LCD. Thus, we have catalogued the molecular events underlying cell death induced by CQ in combination with an anticancer therapeutic. Moreover, by revealing a hitherto unknown aspect of lysosomal biology under stress, we propose that suppression of cholesterol metabolism in cancer cells should elicit synergy with CQ and define a novel approach to future cancer treatments.

Human peripheral blood leucocyte non-obese diabetic-severe combined immunodeficiency interleukin-2 receptor gamma chain gene mouse model of xenogeneic graft-versus-host-like disease and the role of host major histocompatibility complex.

Immunodeficient non-obese diabetic (NOD)-severe combined immune-deficient (scid) mice bearing a targeted mutation in the gene encoding the interleukin (IL)-2 receptor gamma chain gene (IL2rgamma(null)) engraft readily with human peripheral blood mononuclear cells (PBMC). Here, we report a robust model of xenogeneic graft-versus-host-like disease (GVHD) based on intravenous injection of human PBMC into 2 Gy conditioned NOD-scid IL2rgamma(null) mice. These mice develop xenogeneic GVHD consistently (100%) following injection of as few as 5 x 10(6) PBMC, regardless of the PBMC donor used. As in human disease, the development of xenogeneic GVHD is highly dependent on expression of host major histocompatibility complex class I and class II molecules and is associated with severely depressed haematopoiesis. Interrupting the tumour necrosis factor-alpha signalling cascade with etanercept, a therapeutic drug in clinical trials for the treatment of human GVHD, delays the onset and progression of disease. This model now provides the opportunity to investigate in vivo mechanisms of xenogeneic GVHD as well as to assess the efficacy of therapeutic agents rapidly.

alpha7 Nicotinic receptor gene delivery into mouse hippocampal neurons leads to functional receptor expression, improved spatial memory-related performance, and tau hyperphosphorylation.

Brain alpha7 nicotinic receptors have become therapeutic targets for Alzheimer's disease (AD) based on their memory-enhancing and neuroprotective actions. This study investigated the feasibility of increasing neuronal alpha7 receptor functions using a gene delivery approach based on neuron-selective recombinant adeno-associated virus (rAAV)-derived vectors. In order to determine whether alpha7 receptor-mediated cytotoxicity was dependent on receptor density, rat alpha7 nicotinic receptors were expressed at high concentrations in GH4C1 cells as measured with nicotine-displaceable [3H]methyllycaconitine (MLA) binding. The potency of GTS-21 (an alpha7 receptor agonist) to induce cell loss was similar in these cells to that seen in pheochromocytoma (PC12) cells expressing nine-times-lower receptor levels, suggesting that cytotoxicity was more dependent on agonist concentration than receptor density. Hippocampal transduction with rat alpha7 nicotinic receptors increased [3H]MLA binding in this region in wild type and alpha7 receptor-knockout (KO) mice without apparent cytotoxicity. No difference was observed in Kd values for MLA binding between endogenous and transgenic receptors. Single cell recordings demonstrated that dentate granule cells that normally have no alpha7 receptor response did so following alpha7 receptor gene delivery in wild type mice. Recovery of alpha7 function was also observed in stratum oriens and stratum radiatum neurons of KO mice following gene delivery. Wild type mice exhibited improved acquisition performance in the Morris water task 1 month after bilateral hippocampal transductions with the rat alpha7 receptor gene compared with green fluorescent protein-transduced controls. However, both groups reached similar training levels and there was no difference in subsequent probe performance. Finally, this gene delivery approach was used to test whether alpha7 receptors affect tau-phosphorylation. Chronic (i.e. 2 month but not 2 week) expression of high levels of alpha7 receptors in hippocampus increased AT8 staining characteristic of hyperphosphorylated tau in that region, indicating that endogenous agonist-mediated receptor activation may be able to modulate this process.

Impact of respiratory motion on the detection of small pulmonary nodules in SPECT imaging.

The objective of this investigation is to determine the impact of respiratory motion on the detection of small solitary pulmonary nodules (SPN) in single photon emission computed tomographic (SPECT) imaging. We have previously modeled the respiratory motion of SPN based on the change of location of anatomic structures within the lungs identified on breath-held CT images of volunteers acquired at two different stages of respiration. This information on respiratory motion within the lungs was combined with the end-expiration and time-averaged NCAT phantoms to allow the creation of source and attenuation maps for the normal background distribution of Tc-99m NeoTect. With the source and attenuation distribution thus defined, the SIMIND Monte Carlo program was used to produce SPECT projection data for the normal background and separately for each of 150 end-expiration and time-averaged simulated 1.0 cm tumors. Normal and tumor SPECT projection sets each containing one lesion were combined with a clinically realistic noise level and counts. These were reconstructed with RBI-EM using 1) no correction (NC), 2) attenuation correction (AC), 3) detector response correction (RC), and 4) attenuation correction, detector response correction, and scatter correction (AC_RC_SC). The post-reconstruction parameters of number of iterations and 3-D Gaussian filtering were optimized by human-observer studies. Comparison of lesion detection by human-observer LROC studies reveals that respiratory motion degrades tumor detection for all four reconstruction strategies, and that the magnitude of this effect is greatest for NC and RC, and least for AC_RC_SC. Additionally, the AC_RC_SC strategy results in the best detection of lesions.

Evaluation of Multiclass Model Observers in PET LROC Studies.

A localization ROC (LROC) study was conducted to evaluate nonprewhitening matched-filter (NPW) and channelized NPW (CNPW) versions of a multiclass model observer as predictors of human tumor-detection performance with PET images. Target localization is explicitly performed by these model observers. Tumors were placed in the liver, lungs, and background soft tissue of a mathematical phantom, and the data simulation modeled a full-3D acquisition mode. Reconstructions were performed with the FORE+AWOSEM algorithm. The LROC study measured observer performance with 2D images consisting of either coronal, sagittal, or transverse views of the same set of cases. Versions of the CNPW observer based on two previously published difference-of-Gaussian channel models demonstrated good quantitative agreement with human observers. One interpretation of these results treats the CNPW observer as a channelized Hotelling observer with implicit internal noise.

Impact of Mismatched Detector-Blur Models on Ga SPECT Tumor Detection.

The quality of SPECT images suffers from the effects of photon attenuation and scatter, and from distance-dependent collimator blur, and many researchers have shown the benefit of compensating for these degradations in the inverse problem. For this work, we examined how using an incorrect collimator-blur model affects the detection and localization of (67)Ga-avid lymphomas in simulated chest scans. In particular, we considered whether blur-overcompensation can enhance reconstructed images for purposes of localizing tumors. Variations in the correct linear model for medium-energy, parallel-hole collimators were compared by means of LROC studies with human and localizing model observers. Imaging data consisted of Simind projections of the MCAT phantom, and RBI reconstructions were performed. Our results indicate that tumor-detection performance is not improved by using a mismatched RC model. Reconstruction with increased RC requires more iterations, which leads to longer noise correlations. Our results also suggest a substantial observer insensitivity to the accuracy of the RC model.

A Multiclass Model Observer for Multislice-Multiview Images.

A human-model observer for tumor detection-localization studies featuring multislice-multiview (or volumetric) image displays has been introduced. This volumetric observer, an extension of multiclass linear observers previously tested with single-slice and multislice displays, produces rating and localization data by integrating perception measurements from the different image views. A channelized NPW (CNPW) version of the observer was evaluated against humans for a background-known-exactly (BKE) detection task involving localization of Tc-99m Neotect lesions in simulated SPECT lung images. An LROC study evaluated two RBI reconstruction strategies that used different combinations of corrections for attenuation, scatter, and distance-dependent system resolution, and coronal, sagittal, and transverse slices were presented to the observers. Model-observer ranking of these strategies did not match that of the humans. Follow-up studies exploring several possible remedies for the model observer, including strategy-specific search regions and an internal-noise mechanism, showed little change. Future work will examine variations from the BKE assumption as a means of reconciling the rankings.

4-thiophenoxy-N-(3,4,5-trialkoxyphenyl)pyrimidine-2-amines as potent and selective inhibitors of the T-cell tyrosine kinase p56lck.

We have developed a series of 4-thiophenoxy-N-(3,4,5-trialkoxyphenyl) pyrimidine-2-amines as potent and selective inhibitors of p56lck tyrosine kinase activity. In particular, the most potent inhibitor shows cellular activity in T-cell receptor (TCR) stimulated models of cytokine release, which suggests an immunomodulatory role for this class of inhibitor.

Induction of uncoupling protein 1 by central interleukin-6 gene delivery is dependent on sympathetic innervation of brown adipose tissue and underlies one mechanism of body weight reduction in rats.

Interleukin-6 (IL-6) is a multifunctional cytokine that may have a role in energy regulation. Using a recombinant adeno-associated viral vector expressing murine interleukin-6 (rAAV-IL-6), we examined the chronic effects of centrally expressed IL-6 on food intake, body weight and adiposity in male Sprague-Dawley rats, and investigated the underlying mechanisms. Direct delivery of rAAV-IL-6 into rat hypothalamus suppressed weight gain and visceral adiposity without affecting food intake over a 5-week period. rAAV-IL-6 enhanced uncoupling protein 1 (UCP1) protein levels in interscapular brown adipose tissue (BAT). To investigate if the induction of UCP1 and the reduction in body weight are dependent on sympathetic innervation of BAT, we administered rAAV-IL-6 or a control vector into the hypothalamus of rats in which the interscapular BAT was unilaterally denervated. Over 21 days, there was no difference in food consumption or body weight between rAAV-IL-6- and control vector-treated rats. rAAV-IL-6 delivery increased UCP1 mRNA and protein levels in innervated BAT pads but not denervated BAT pads. Hypothalamic IL-6 signal transduction, indicated by phosphorylated signal transducer and activator of transcription 3 (P-STAT3) levels, was elevated by 2.6-fold at day 21, but returned to control levels by day 35. However, the suppressor of cytokine signaling-3 mRNA level was significantly elevated both at day 21 and day 35. These data demonstrate that chronic elevation of IL-6 in the CNS reduces body weight gain and visceral adiposity without affecting food intake. The mechanism involves sympathetic induction of UCP1 in BAT and, presumably, enhanced thermogenesis in BAT. Furthermore, chronic central IL-6 stimulation desensitizes IL-6 signal transduction characterized by reversal of elevated P-STAT3 levels.

The effects of chronic ethanol consumption on neurotrophins and their receptors in the rat hippocampus and basal forebrain.

Damage to the basal forebrain frequently results in deficits in learning and memory. Mnenonic dysfunction also occurs following prolonged ethanol consumption in humans and in animal models of chronic ethanol intake, accompanied by specific abnormalities in synaptic transmission between the basal forebrain and hippocampus. The integrity of at least some of the reciprocal neuronal connections between these brain regions is influenced by target-derived neurotrophic factors. We used a semiquantitative reverse transcription polymerase chain reaction technique to measure the messenger RNA for neurotrophins BDNF and NGF, and for their receptors trkB, trkA, and the low affinity receptor, p75(NTR) in the hippocampus and basal forebrain of rats after 28 weeks of alcohol consumption without malnutrition. This chronic ethanol treatment (CET) resulted in a marked and selective reduction in basal forebrain trkA mRNA. Western blotting revealed a similar reduction of basal forebrain trkA protein. CET effects on basal forebrain trkA may reflect impaired NGF signaling that could compromise septohippocampal synaptic connections, cholinergic differentiation, and emergent functional abilities dependent on these properties.

Transgene delivery with a cationic lipid in the presence of amyloid beta (betaAP) peptide.

The ability of a cationic lipid to deliver plasmid DNA (pDNA) in presence of the neurotoxic fragment of amyloid beta-peptide was evaluated. Pre-treatment of cells with betaAP (25-35) peptide resulted in a modest increase in transgene expression. When betaAP (25-35) peptide was mixed with the pDNA/liposome complex and used, the complexes lost their ability to transfect. However, the reverse sequenced betaAP (35-25) peptide demonstrated no significant differences in transgene expression in pre-treated cells, and in cells where betaAP (35-25) peptide was mixed with pDNA/liposome complexes and transfected. The amount of pDNA delivered to the cells was decreased in presence of betaAP (25-35) as measured with flow cytometry using fluorescently labeled liposomes. The decreased endocytosis may be due to their rod-like structure formation as demonstrated by electron microscopy and atomic force microscopy (AFM). These results demonstrate that betaAP (25-35) peptide may interfere with gene delivery with cationic systems.

Estrogens decrease reperfusion-associated cortical ischemic damage: an MRI analysis in a transient focal ischemia model.

BACKGROUND AND PURPOSE: Early identification of irreversible cerebral ischemia is critical in defining strategies that influence neuronal survival after stroke. We used MRI to investigate the effects of 17beta-estradiol (E2) on the temporal evolution of focal ischemia. METHODS: Female rats were ovariectomized and divided into 1 of 2 groups: ovariectomy alone (OVX; n=4) or ovariectomy with estrogen replacement (OVX+E2; n=3). Both groups were then subjected to 1-hour middle cerebral artery occlusion (MCAO), with the use of a standardized endovascular monofilament model, followed by reperfusion. Sequential diffusion-weighted (DWI) and T2-weighted (T2WI) MRI were obtained during and after the MCAO. In separate groups of animals (n=5 for OVX and OVX+E2), cerebral blood flow (CBF) was measured by laser-Doppler methods before, during, and after occlusion. RESULTS: DWI detected similar lesion characteristics during MCAO in both groups. In the OVX group, lesion size did not change during reperfusion, but the signal intensity ratio increased early and stabilized during the latter stages. In contrast, DWI lesion size decreased during reperfusion in OVX+E2 rats by 50% to 60% (P<0.05), a size reduction almost exclusively limited to cortical regions. During MCAO, the signal intensity ratio in OVX+E2 rats was reduced compared with OVX rats. Reperfusion further attenuated the signal intensity ratio in cortical but not subcortical regions (P<0.05 versus OVX). T2WI revealed no lesions in either group during MCAO, but it detected lesion sizes similar to that of DWI during reperfusion. Furthermore, similar patterns and magnitudes of estrogen treatment-related decrease in lesion size were noted after reperfusion. T2WI demonstrated less intense signal intensity ratio changes in both groups compared with DWI. There were no differences in CBF between groups either during occlusion, early reperfusion, or 1 day after reperfusion. CONCLUSIONS: This study strongly suggests that estrogens selectively protect cortical tissue from ischemic damage during MCAO and that this protection is exerted during both the occlusion and reperfusion phases of ischemia and does not involve an estrogen-related change in CBF.

Peroxide mediates ethanol-induced cytotoxicity in PC12 cells.

Pheochromocytoma (PC12) cell cultures exhibited a loss of cells and increase in intracellular oxidative stress when exposed to ethanol (EtOH) for 24 h. Catalase, an enzyme that hydrolyzes hydrogen peroxide (H(2)O(2)) to O(2) and H(2)O can attenuate EtOH-induced cell loss and oxidative stress in PC12 cells. This study provides the first clear evidence that oxidative stress in the form of elevated intracellular H(2)O(2) is a primary mechanism of EtOH neurotoxicity in PC12 cells.

Matching prescription claims with medication data for nursing home residents: implications for prescriber feedback, drug utilisation studies and selection of prescription claims database.

Medication data retrieved from Australian Repatriation Pharmaceutical Benefits Scheme (RPBS) claims for 44 veterans residing in nursing homes and Pharmaceutical Benefits Scheme (PBS) claims for 898 nursing home residents were compared with medication data from nursing home records to determine the optimal time interval for retrieving claims data and its validity. Optimal matching was achieved using 12 weeks of RPBS claims data, with 60% of medications in the RPBS claims located in nursing home administration records, and 78% of medications administered to nursing home residents identified in RPBS claims. In comparison, 48% of medications administered to nursing home residents could be found in 12 weeks of PBS data, and 56% of medications present in PBS claims could be matched with nursing home administration records. RPBS claims data was superior to PBS, due to the larger number of scheduled items available to veterans and the veteran's file number, which acts as a unique identifier. These findings should be taken into account when using prescription claims data for medication histories, prescriber feedback, drug utilisation, intervention or epidemiological studies.

LROC analysis of detector-response compensation in SPECT.

Localization ROC (LROC) observer studies examined whether detector response compensation (DRC) in ordered-subset, expectation-maximization (OSEM) reconstructions helps in the detection and localization of hot tumors. Simulated gallium (Ga-67) images of the thoracic region were used in the study. The projection data modeled the acquisition of attenuated 93- and 185-keV photons with a medium-energy parallel-hole collimator, but scatter was not modeled. Images were reconstructed with five strategies: 1) OSEM with no DRC; 2) OSEM preceded by restoration filtering; 3) OSEM with iterative DRC; 4) OSEM with an ideal DRC; and 5) filtered backprojection (FBP) with no DRC. All strategies included attenuation correction. There were four LROC studies conducted. In a study using a single tumor activity, the ideal DRC offered the best performance, followed by iterative DRC, restoration filtering, OSEM with no DRC, and FBP. Statistical significance at the 5% level was found between all pairs of strategies except for restoration filtering and OSEM with no DRC. A similar ranking was found for a more realistic study using multiple tumor activities. Additional studies considered the effects of OSEM iteration number and tumor activity on the detection improvement that iterative DRC offered with respect to OSEM with no DRC.

Insulin-like growth factor-binding protein-3 modulates expression of Bax and Bcl-2 and potentiates p53-independent radiation-induced apoptosis in human breast cancer cells.

We report that transfection of insulin-like growth factor-binding protein-3 (IGFBP-3) cDNA in human breast cancer cell lines expressing either mutant p53 (T47D) or wild-type p53 (MCF-7) induces apoptosis. IGFBP-3 also increases the ratio of pro-apoptotic to anti-apoptotic members of the Bcl-2 family. In MCF-7, an increase in Bad and Bax protein expression and a decrease in Bcl-x(L) protein and Bcl-2 protein and mRNA were observed. In T47D, Bax and Bad proteins were up-regulated; Bcl-2 protein is undetectable in these cells. As T47D expresses mutant p53 protein, these modulations of pro-apoptotic proteins and induction of apoptosis are independent of p53. The effect of IGFBP-3 on the response of T47D to ionizing radiation (IR) was examined. These cells do not G(1) arrest in response to IR and are relatively radioresistant. Transfection of IGFBP-3 increased the radiosensitivity of T47D and increased IR-induced apoptosis but did not effect a rapid G(1) arrest. IR also caused a much greater increase in Bax protein in IGFBP-3 transfectants compared with vector controls. Thus, IGFBP-3 increases the expression of pro-apoptotic proteins and apoptosis both basally and in response to IR, suggesting it may be a p53-independent effector of apoptosis in breast cancer cells via its modulation of the Bax:Bcl-2 protein ratio.

Comparing filtered backprojection and ordered-subsets expectation maximization for small-lesion detection and localization in 67Ga SPECT.

UNLABELLED: Iterative reconstruction of SPECT images has recently become clinically available as an alternative to filtered backprojection (FBP). However, there is conflicting evidence on whether iterative reconstruction, such as with the ordered-subsets expectation maximization (OSEM) algorithm, improves diagnostic performance over FBP. The study objective was to determine if the detection and localization of small lesions in simulated thoracic gallium SPECT images are better with OSEM reconstruction than with FBP, both with and without attenuation correction (AC). METHODS: Images were simulated using an analytic projector acting on the mathematic cardiac torso computer phantom. Perfect scatter rejection was assumed. Lesion detection accuracy was assessed using localization receiver operating characteristic methodology. The images were read by 5 nuclear medicine physicians. For each reconstruction strategy and for each observer, data were collected in 2 viewing sessions of 100 images. Two-way ANOVA and, when indicated, the Scheffé multiple comparisons test were applied to check for significant differences. RESULTS: Little difference in the accuracy of detection or localization was seen between FBP with and without AC. OSEM with AC extended the contrast range for accurate lesion detection and localization over that of the other methods investigated. Without AC, no significant difference between OSEM and FBP reconstruction was detected. CONCLUSION: OSEM with AC may improve the detection and localization of thoracic gallium-labeled lesions over FBP reconstruction.

Adeno-associated virus mediated gene transfer into primary rat brain neuronal and glial cultures: enhancement with the pH-sensitive surfactant dodecyl 2-(1'-imidazolyl) propionate.

This study evaluated the effects of a novel, pH-sensitive surfactant, dodecyl 2-(1'-imidazolyl) propionate (DIP), on cationic lipid mediated transfection in primary rat brain neuronal and glial cultures. The cationic lipid complex DOTAP/DOPE (1, 2-dioleoyl-3-trimethylammonium propionate and dioleoyl phosphatidylethanolamine, respectively) was added over a range of concentrations (0-120 microg/ml) with DNA concentration kept constant (1.6 microg/ml). The neuron-specific enolase (NSE) and cytomegalovirus (CMV) promoters were found to drive green fluorescent protein (GFP) expression in neuron-enriched and glial cultures, respectively, using adeno-associated virus (AAV) derived constructs. NSE-driven GFP expression was not observed in glial cultures. Addition of DOTAP/DOPE increased transfection efficiency over a wide range of lipid concentrations (5-50 microg/ml) keeping DNA concentration constant (1.6 microg/ml). Addition of DIP to the lipid/DNA complex increased maximum transfection efficiencies in glial and neuronal cultures 2-3-fold. Transfection efficiencies were at their maximum with a similar total lipid concentration (50 microg/ml) in both cell-types in the presence of DIP. Neuronal cultures were more sensitive than glia to the toxic actions of DOTAP/DOPE, with or without DIP. These results indicate that AAV-mediated gene-transfer to neurons and glia can be facilitated by addition of a pH-sensitive surfactant to cationic liposome/DNA complexes and that endosomal escape could be a limiting factor in transgene expression.

alpha7 nicotinic receptor-mediated protection against ethanol-induced oxidative stress and cytotoxicity in PC12 cells.

Pheochromocytoma (PC12) cell cultures exhibited a concentration-dependent loss of cells and increase in intracellular oxidative stress when exposed to ethanol for 24 h. Selective activation of alpha7 nicotinic receptors with the agonist DMXB (3 microM) attenuated both of these actions of ethanol in a manner that was in turn blocked with the nicotinic antagonist methyllyconitine (1 microM). These results suggest that the cytoprotection conferred by alpha7 nicotinic receptor agonists may be mediated at least in part by reducing the formation or accumulation of reactive oxygen species.