Solid-state MAS NMR measurements of intact articular bovine cartilage

Articular cartilage (AC), an avascular connective tissue lining articulating surfaces of the long bones, comprises extracellular biopolymers. In functionally compromised states such as osteoarthritis, thinned or lost AC causes reduced mobility and increased health-care costs. Understanding of the characteristics responsible for the load bearing efficiency of AC and the factors leading to its degradation are incomplete. DTI shows the structural alignment of collagen in AC [1] and T2 relaxation measurements suggest that the average director of reorientational motion of water molecules depends on the degree of alignment of collagen in AC [2]. Information on the nature of the chemical interactions involved in functional AC is lacking. The need for AC structural integrity makes solid state NMR an ideal tool to study this tissue. We examined the contribution of water in different functional ‘compartments’ using 1H-MAS, 13C-MAS and 13C-CPMAS NMR of bovine patellar cartilage incubated in D2O. 1H-MAS spectra signal intensity was reduced due to H/D exchange without a measureable redistribution of relative signal intensity. Chemical shift anisotropy was estimated by lineshape analysis of multiple peaks in the 1H-MAS spinning sidebands. These asymmetrical sidebands suggested the presence of multiple water species in AC. Therefore, water was added in small aliquots to D2O saturated AC and the influence of H2O and D2O on organic components was studied with 13C-MAS-NMR and 13C-CPMAS-NMR. Signal intensity in 13C-MAS spectra showed no change in relative signal intensity throughout the spectrum. In 13C-CPMAS spectra, displacement of water by D2O resulted in a loss of signal in the aliphatic region due to a reduction in proton availability for cross-polarization. These results complement dehydration studies of cartilage using osmotic manipulation [3] and demonstrate components of cartilage that are in contact with mobile water.

Unswitch-ABL drugs overcome resistance in chronic myeloid leukemia

ABL inhibitors have revolutionized the clinical management of chronic myeloid leukemia, but the BCR-ABLT315I mutation confers resistance to currently approved drugs. Chan et al. show, in this issue of Cancer Cell, that " switch-control" inhibitors block BCR-ABLT315I activity by preventing ABL from switching from the inactive to active conformation.

Oncogenic BRAF induces melanoma cell invasion by downregulating the cGMP-specific phosphodiesterase PDE5A

We show that in melanoma cells oncogenic BRAF, acting through MEK and the transcription factor BRN2, downregulates the cGMP-specific phosphodiesterase PDE5A. Although PDE5A downregulation causes a small decrease in proliferation, its major impact is to stimulate a dramatic increase in melanoma cell invasion. This is because PDE5A downregulation leads to an increase in cGMP, which induces an increase in cytosolic Ca2+, stimulating increased contractility and inducing invasion. PDE5A downregulation also this leads to an increase in short-term and long-term colonization of the lungs by melanoma cells. We do not observe this pathway in NRAS mutant melanoma or BRAF mutant colorectal cells. Thus, we show that in melanoma cells oncogenic BRAF induces invasion through downregulation of PDE5A.

Activation of forkhead box O transcription factors by oncogenic BRAF promotes p21cip1-dependent senescence

Oncogene-induced senescence (OIS) is a potent tumor-suppressive mechanism that is thought to come at the cost of aging. The Forkhead box O (FOXO) transcription factors are regulators of life span and tumor suppression. However, whether and how FOXOs function in OIS have been unclear. Here, we show a role for FOXO4 in mediating senescence by the human BRAFV600E oncogene, which arises commonly in melanoma. BRAFV600E signaling through mitogen-activated protein kinase/extracellular signal-regulated kinase kinase resulted in increased reactive oxygen species levels and c-Jun NH 2 terminal kinase-mediated activation of FOXO4 via its phosphorylation on Thr223, Ser226, Thr447, and Thr451. BRAFV600E-induced FOXO4 phosphorylation resulted in p21cip1-mediated cell senescence independent of p16 ink4a or p27kip1. Importantly, melanocyte-specific activation of BRAFV600E in vivo resulted in the formation of skin nevi expressing Thr223/Ser226-phosphorylated FOXO4 and elevated p21cip1. Together, these findings support a model in which FOXOs mediate a trade-off between cancer and aging.

Identification of direct transcriptional targets of V600EBRAF/MEK signalling in melanoma

Oncogenic mutations in BRAF are common in melanoma and drive constitutive activation of the MEK/ERK pathway. To elucidate the transcriptional events downstream of V600EBRAF/MEK signalling we performed gene expression profiling of A375 melanoma cells treated with potent and selective inhibitors of V600EBRAF and MEK (PLX4720 and PD184352 respectively). Using a stringent Bayesian approach, we identified 69 transcripts that appear to be direct transcriptional targets of this pathway and whose expression changed after 6 h of pathway inhibition. We also identified several additional genes whose expression changed after 24 h of pathway inhibition and which are likely to be indirect transcriptional targets of the pathway. Several of these were confirmed by demonstrating their expression to be similarly regulated when BRAF was depleted using RNA interference, and by using qRT-PCR in other BRAF mutated melanoma lines. Many of these genes are transcription factors and feedback inhibitors of the ERK pathway and are also regulated by MEK signalling in NRAS mutant cells. This study provides a basis for understanding the molecular processes that are regulated by V600EBRAF/MEK signalling in melanoma cells.

New players for advanced prostate cancer and the rationalisation of insulin-sensitising medication

Obesity and type 2 diabetes are recognised risk factors for the development of some cancers and, increasingly, predict more aggressive disease, treatment failure, and cancer-specific mortality. Many factors may contribute to this clinical observation. Hyperinsulinaemia, dyslipidaemia, hypoxia, ER stress, and inflammation associated with expanded adipose tissue are thought to be among the main culprits driving malignant growth and cancer advancement. This observation has led to the proposal of the potential utility of “old players” for the treatment of type 2 diabetes and metabolic syndrome as new cancer adjuvant therapeutics. Androgen-regulated pathways drive proliferation, differentiation, and survival of benign and malignant prostate tissue. Androgen deprivation therapy (ADT) exploits this dependence to systemically treat advanced prostate cancer resulting in anticancer response and improvement of cancer symptoms. However, the initial therapeutic response from ADT eventually progresses to castrate resistant prostate cancer (CRPC) which is currently incurable. ADT rapidly induces hyperinsulinaemia which is associated with more rapid treatment failure. We discuss current observations of cancer in the context of obesity, diabetes, and insulin-lowering medication. We provide an update on current treatments for advanced prostate cancer and discuss whether metabolic dysfunction, developed during ADT, provides a unique therapeutic window for rapid translation of insulin-sensitising medication as combination therapy with antiandrogen targeting agents for the management of advanced prostate cancer.

The use of metabolomics to monitor simultaneous changes in metabolic variables following supramaximal low volume high intensity exercise

High Intensity Exercise (HIE) stimulates greater physiological remodeling when compared to workload matched low-moderate intensity exercise. This study utilized an untargeted metabolomics approach to examine the metabolic perturbations that occur following two workload matched supramaximal low volume HIE trials. In a randomized order, 7 untrained males completed two exercise protocols separated by one week; 1) HIE150%: 30 x 20s cycling at 150% VO2peak, 40s passive rest; 2) HIE300%: 30 x 10s cycling at 300% VO2peak, 50 s passive rest. Total exercise duration was 30 minutes for both trials. Blood samples were taken at rest, during and immediately following exercise and at 60 minutes post exercise. Gas chromatography-mass spectrometry (GC-MS) analysis of plasma identified 43 known metabolites of which 3 demonstrated significant fold changes (HIE300% compared to the HIE150% value) during exercise, 14 post exercise and 23 at the end of the recovery period. Significant changes in plasma metabolites relating to lipid metabolism [fatty acids: dodecanoate (p=0.042), hexadecanoate (p=0.001), octadecanoate (p=0.001)], total cholesterol (p=0.001), and glycolysis [lactate (p=0.018)] were observed following exercise and during the recovery period. The HIE300% protocol elicited greater metabolic changes relating to lipid metabolism and glycolysis when compared to HIE150% protocol. These changes were more pronounced throughout the recovery period rather than during the exercise bout itself. Data from the current study demonstrate the use of metabolomics to monitor intensity-dependent changes in multiple metabolic pathways following exercise. The small sample size indicates a need for further studies in a larger sample cohort to validate these findings.

Genes and gene expression in the brains of human alcoholics

Chronic alcohol misuse by human subjects leads to neuronal loss in regions such as the superior frontal cortex (SFC). Propensity to alcoholism is associated with several genes. γ-Aminobutyric acid (GABA)A receptor expression differs between alcoholics and controls, whereas glutamate receptor differences are muted. We determined whether genotype differentiated the regional presentation of GABAA and glutamate-NMDA (N-methyl-d-aspartate) receptors in SFC. Autopsy tissue was obtained from alcoholics without comorbid disease, alcoholics with liver cirrhosis, and matched controls. ADH1C, DRD2B, EAAT2, and APOE genotypes modulated GABAA-β subunit protein expression in SFC toward a less-effective form of the receptor. Most genotypes did not divide alcoholics and controls on glutamate-NMDA receptor pharmacology, although gender and cirrhosis did. Genotype may affect amino acid transmission locally to influence neuronal vulnerability.

Comorbidity and genotype modify receptor expression in human alcoholics

Chronic alcohol misuse leads to both widespread and localized damage in human cerebral cortex. The latter, as neuronal loss, is marked in superior frontal cortex (SFC) but milder in primary motor cortex (PMC) and elsewhere. Quantitative morphometry by Harper et al showed that neuronal loss is greater in alcoholics with comorbidity (Wernicke Korsakoff syndrome, liver cirrhosis). Previous work revealed a paradox: the marked differences in GABAA receptor density, pharmacology, and expression between alcoholics without cormorbidity and controls are muted or absent in cirrhotic alcoholics. This concurs with work by the Butterworth group on hepatic encephalopathy cases — most of whom had an alcoholic ætiology — who show only minor differences from controls. Glutamate receptor differences are muted in many autopsy studies, though we have evidence that NMDA site pharmacology may vary in cirrhotic alcoholics. Here we used Real-Time PCR normalized to GAPDH deltaCT to quantify NMDA NR1, NR2A and NR2B subunit expression in SFC and PMC samples obtained at autopsy from alcoholics with and without comorbid cirrhosis and matched controls. Overall subunit transcript expression was signifi cantly lower in alcoholic cirrhotics than in either of the other groups (F2,42 = 12.942, P < 0.001). The effect was most marked for the NR1 subunit; males differed from females, particularly in SFC. The data suggest that if excitotoxicity mediates neuronal loss in SFC, it may be implemented differently: passively in uncomplicated alcoholics, by altered GABAergic transmission; actively in cirrhotic alcoholics, by altered glutamatergic transmission. We also subdivided cases on a panel of genetic markers. Different genotypes interacted with NMDA and GABAA pharmacology and expression. Cirrhotic and uncomplicated alcoholics may differ pathogenically because of inherent characteristics in addition to possible neurotoxic sequelæ to the liver damage.

Microarray and proteomic analysis of the human alcoholic brain

The superior frontal cortex (SFC) is selectively damaged in chronic alcohol abuse, with localized neuronal loss and tissue atrophy. Regions such as motor cortex show little neuronal loss except in severe co-morbidity (liver cirrhosis or WKS). Altered gene expression was found in microarray comparisons of alcoholic and control SFC samples [1]. We used Western blots and proteomic analysis to identify the proteins that also show differential expression. Tissue was obtained at autopsy under informed, written consent from uncomplicated alcoholics and age- and sex-matched controls. Alcoholics had consumed 80 g ethanol/day chronically (often, 200 g/day for 20 y). Controls either abstained or were social drinkers ( 20 g/day). All subjects had pathological confirmation of liver and brain diagnosis; none had been polydrug abusers. Samples were homogenized in water and clarified by brief centrifugation (1000g, 3 min) before storage at –80°C. For proteomics the thawed suspensions were centrifuged (15000g, 50 min) to prepare soluble fractions. Aliquots were pooled from SFC samples from the 5 chronic alcoholics and 5 matched controls used in the previous microarray study [1]. 2-Dimensional electrophoresis was performed in triplicate using 18 cm format pH 4–7 and pH 6–11 immobilized pH gradients for firstdimension isoelectric focusing. Following second-dimension SDS-PAGE the proteins were fluorescently stained and the images collected by densitometry. 182 proteins differed by 2-fold between cases and controls. 141 showed lower expression in alcoholics, 33 higher, and 8 were new or had disappeared. To date 63 proteins have been identified using MALDI-MS and MS-MS. Western blots were performed on uncentrifuged individual samples from 76 subjects (controls, uncomplicated alcoholics and cirrhotic alcoholics). A common standard was run on every gel. After transfer, immunolabeling, and densitometry, the intensities of the unknown bands were compared to those of the standards. We focused on proteins from transcripts that showed clear differences in a series of microarray studies, classified into common sets including Regulators of G-protein Signaling and Myelin-associated proteins. The preponderantly lower level of differentially expressed proteins in alcoholics parallels the microarray mRNA analysis in the same samples. We found that mRNA and protein expression do not frequently correspond; this may help identify pathogenic processes acting at the level of transcription, translation, or post-translationally.

Genes and gene expression in human alcoholic brain

Chronic alcoholism leads to localized brain damage, which is prominent in superior frontal cortex but mild in motor cortex. The likelihood of developing alcohol dependence is associated with genetic markers. GABA-A receptor expression differs between alcoholics and controls, whereas glutamate receptor differences are muted. We determined whether genotype differentiated the localized expression of glutamate N-methyl-D-aspartate (NMDA) and GABA-A receptors to influence the severity of alcohol-induced brain damage. Cerebral cortex tissue was obtained at autopsy from alcoholics without disease comorbid with alcoholics, alcoholics with cirrhosis, and matched controls. DRD2A, DRD2B, GABRB2, SLC1A2, and 5HTT genotypes did not divide alcoholic cases and controls on NMDA receptor parameters. In contrast, a specific alcohol dehydrogenase (ADHIC) genotype interacted significantly with NMDA efficacy and affinity in a region-specific manner SLC1A2 (glutamate transporter-2) genotype interacted significantly with local GABAA receptor b subunit mRNA expression, and ADHIC, DRD2B, SLC1A2, and APOE genotypes with b subunit isoform protein expression. In the latter instance, possession of the alcoholism- associated allele altered b isoform protein expression patterns toward a less-efficacious form of the GABA-A receptor in the pathologically vulnerable region. GABRB2 and GRIN2B (NMDA receptor 2B subunit} Genotypes were associated with significant regional difference in the pattern of b subunit protein isoform expression, but this was not influenced by alcoholism status. Genotype may modulate amino acid transmission locally so as to mediate neuronal vulnerability. This has implications for the effectiveness of pharmacological interventions aimed at ameliorating brain damage and, possibly, dependence.

Genomic and proteomic analysis of synaptic protein expression in the brains of human alcoholics

Alcoholism results in changes in the human brain which reinforce the cycle of craving and dependency, and these changes are manifest in the pattern of expression of mRNA and proteins in key cells and brain areas. Long-term alcohol abuse also results in damage to selected regions of the cortex. We have used cDNA microarrays to show that less than 1% of mRNA transcripts differ signifi cantly between cases and controls in the susceptible area and that the expression profi le of a subset of these transcripts is suffi cient to distinguish alcohol abusers from controls. In addition, we have utilized a 2D gel proteomics based approach to determine the identity of proteins in the superior frontal cortex (SFC) of the human brain that show differential expression in controls and long term alcohol abusers. Overall, 182 proteins differed by the criterion of > 2-fold between case and control samples. Of these, 139 showed signifi cantly lower expression in alcoholics, 35 showed signifi cantly higher expression, and 8 were new or had disappeared. To date 63 proteins have been identifi ed. The expression of one family of proteins, the synucleins, has been further characterized using Real Time PCR and Western Blotting. The expression of alpha-synuclein mRNA was signifi cantly lower in the SFC of alcoholics compared with the same area in controls (P = 0.01) whereas no such difference in expression was found in the motor cortex. The expression of beta- and gamma- synuclein were not signifi cantly different between alcoholics and controls. In contrast, the pattern of alphasynuclein protein expression differs from that of the corresponding RNA transcript. Because of the key role of synaptic proteins in the pathogenesis of alcoholism, we are developing 2-D DIGE based techniques to quantify expression changes in synaptosomes prepared from the SFC of controls and alcoholics.

Interactions between serotonergic genotype and amino acid transmitter receptor expression in human alcoholics

Serotonin can modulate the activity of neural reward pathways that are strongly implicated in mediating the effects of chronic alcohol misuse, and its treatment, in human subjects. In previous work and as discussed elsewhere at this meeting, we and others have found consistent differences in the parameters of GABA and glutamate receptors, and the expression of their component subunit transcripts and proteins, in areas of the alcoholic brain that are altered by alcoholism. We did not fi nd clear changes in GABA and glutamate transport function in such samples, but a series of microarray analyses showed consistent upregulation of the presynaptic GABA/betaine transporter SLC6A12. Microarray studies showed no signifi cant differences in the expression of transcripts associated with 5HT transmission; however, only a small number of such elements were present on the arrays. Here we partitioned GABAA and NMDA pharmacology, and subunit mRNA and protein expression, measured in samples of frontal and motor cortex obtained at autopsy from alcoholics without comorbid disease, alcoholics with liver cirrhosis, and controls, according to 5HTTLPR (SLC6A4) and 5HT1B (HTR1B) polymorphisms. We found no effect of these genotypes on the expression of GABAA receptor gene products, but there was a signifi cant mRNA Transcript X Area X Group X 5HTTLPR Interaction with NMDA subunit isoform expression measured by Real Time PCR with GAPDH normalization. Further analysis showed the effect to be selective for alcoholics with cirrhosis, to be most marked in the pathologically vulnerable frontal cortex, and to vary with subunit transcript (F2,76 = 6.545, P = 0.002). NR1 expression was most affected, followed by NR2A, with NR2B expression least altered. Pilot data suggest 5HT1B genotype may also modulate NMDA subunit expression. Interactions between amino acid and serotonin transmission may infl uence susceptibility to alcohol dependence or pathogenesis