Background:
Alpha terpineol is a bioactive component of Salvia libanotica essential oil extract and has shown antitumour activity.
Materials and Methods:
The cytotoxicity of alpha terpineol towards ...different tumour cell lines was evaluated in vitro. Mechanistic characterization was performed using analysis of drug activity in a cell line panel and drug-induced gene expression perturbation using the connectivity map approach.
Results:
The small cell lung carcinoma was the cell line most sensitive to alpha terpineol. The results proposed alpha terpineol as an NF-kappa B inhibitor, which was confirmed by the observed dose-dependent inhibition of NF-kappa B translocation and activity using two NF-kappa B assays, and by the down-regulation of the expression of several NE-kappa B-related genes such as IL-1 beta and IL1R1.
Conclusion:
The results suggest that alpha terpineol inhibits the growth of tumour cells through a mechanism that involves inhibition of the NF-kappa B pathway.
Global hypomethylation and regional hypermethylation are well-known epigenetic features of cancer; however, in chronic lymphocytic leukemia (CLL), studies on genome-wide epigenetic modifications are ...limited. Here, we analyzed the global methylation profiles in CLL, by applying high-resolution methylation microarrays (27 578 CpG sites) to 23 CLL samples, belonging to the immunoglobulin heavy-chain variable (IGHV) mutated (favorable) and IGHV unmutated/IGHV3-21 (poor-prognostic) subsets. Overall, results demonstrated significant differences in methylation patterns between these subgroups. Specifically, in IGHV unmutated CLL, we identified methylation of 7 known or candidate tumor suppressor genes (eg, VHL, ABI3, and IGSF4) as well as 8 unmethylated genes involved in cell proliferation and tumor progression (eg, ADORA3 and PRF1 enhancing the nuclear factor-κB and mitogen-activated protein kinase pathways, respectively). In contrast, these latter genes were silenced by methylation in IGHV mutated patients. The array data were validated for selected genes using methylation-specific polymerase chain reaction, quantitative reverse transcriptase–polymerase chain reaction, and bisulfite sequencing. Finally, the significance of DNA methylation in regulating gene promoters was shown by reinducing 4 methylated tumor suppressor genes (eg, VHL and ABI3) in IGHV unmutated samples using the methyl-inhibitor 5-aza-2′-deoxycytidine. Taken together, our data for the first time reveal differences in global methylation profiles between prognostic subsets of CLL, which may unfold epigenetic silencing mechanisms involved in CLL pathogenesis.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
The complex interaction between cancer cells and the microenvironment plays an essential role in all stages of tumourigenesis. Despite the significance of this interplay, alterations in protein ...composition underlying tumour–stroma interactions are largely unknown. The aim of this study was to identify stromal proteins with clinical relevance in non‐small cell lung cancer (NSCLC). A list encompassing 203 stromal candidate genes was compiled based on gene expression array data and available literature. The protein expression of these genes in human NSCLC was screened using the Human Protein Atlas. Twelve proteins were selected that showed a differential stromal staining pattern (BGN, CD99, DCN, EMILIN1, FBN1, PDGFRB, PDLIM5, POSTN, SPARC, TAGLN, TNC and VCAN). The corresponding antibodies were applied on tissue microarrays, including 190 NSCLC samples, and stromal staining was correlated with clinical parameters. Higher stromal expression of CD99 was associated with better prognosis in the univariate (p = 0.037) and multivariate (p = 0.039) analysis. The association was independent from the proportion of tumour stroma, the fraction of inflammatory cells and clinical and pathological parameters like stage, performance status and tumour histology. The prognostic impact of stromal CD99 protein expression was confirmed in an independent cohort of 240 NSCLC patients (p = 0.008). Furthermore, double‐staining confocal fluorescence microscopy showed that CD99 was expressed in stromal lymphocytes as well as in cancer‐associated fibroblasts. Based on a comprehensive screening strategy the membrane protein CD99 was identified as a novel stromal factor with clinical relevance. The results support the concept that stromal properties have an important impact on tumour progression.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
While carcinoma-associated fibroblasts (CAFs) support tumorigenesis, normal tissue fibroblasts suppress tumor progression. Mechanisms behind conversion of fibroblasts into a CAF phenotype are largely ...unrevealed.
Transwell co-cultures with fibroblasts in collagen gels and squamous-cell carcinoma (SCC) cells or normal oral keratinocytes (NOKs) in inserts. Differences in fibroblast global gene expression were analyzed using Affymetrix arrays and subsequent functional annotation and cluster analysis, as well as gene set enrichment analysis were performed.
There were 52 up-regulated and 30 down-regulated transcript IDs (>2-fold, p<0.05) in fibroblasts co-cultured with SCC compared to NOKs. Functional analysis demonstrated an enrichment of collagen-related genes. There were similarities with gene sets reflecting a non-specific, innate-type response with activation of both interferon pathways and connective tissue turnover.
There were distinct differences in fibroblast gene expression between the co-culture types. Many were in genes related to an innate-type of response and to connective tissue turnover.
Primary glioblastoma cell (GC) cultures have emerged as a key model in brain tumor research, with the potential to uncover patient-specific differences in therapy response. However, there is limited ...quantitative information about the stability of such cells during the initial 20-30 passages of culture.
We interrogated 3 patient-derived GC cultures at dense time intervals during the first 30 passages of culture. Combining state-of-the-art signal processing methods with a mathematical model of growth, we estimated clonal composition, rates of change, affected pathways, and correlations between altered gene dosage and transcription.
We demonstrate that GC cultures undergo sequential clonal takeovers, observed through variable proportions of specific subchromosomal lesions, variations in aneuploid cell content, and variations in subpopulation cell cycling times. The GC cultures also show significant transcriptional drift in several metabolic and signaling pathways, including ribosomal synthesis, telomere packaging and signaling via the mammalian target of rapamycin, Wnt, and interferon pathways, to a high degree explained by changes in gene dosage. In addition to these adaptations, the cultured GCs showed signs of shifting transcriptional subtype. Compared with chromosomal aberrations and gene expression, DNA methylations remained comparatively stable during passaging, and may be favorable as a biomarker.
Taken together, GC cultures undergo significant genomic and transcriptional changes that need to be considered in functional experiments and biomarker studies that involve primary glioblastoma cells.
Acute quadriplegic myopathy (AQM) is a common debilitating acquired disorder in critically ill intensive care unit (ICU) patients that is characterized by tetraplegia/generalized weakness of limb and ...trunk muscles. Masticatory muscles, on the other hand, are typically spared or less affected, yet the mechanisms underlying this striking muscle-specific difference remain unknown. This study aims to evaluate physiological parameters and the gene expression profiles of masticatory and limb muscles exposed to factors suggested to trigger AQM, such as mechanical ventilation, immobilization, neuromuscular blocking agents, corticosteroids (CS), and sepsis for 5 days by using a unique porcine model mimicking the ICU conditions. Single muscle fiber cross-sectional area and force-generating capacity, i.e., maximum force normalized to fiber cross-sectional area (specific force), revealed maintained masseter single muscle fiber cross-sectional area and specific-force after 5 days' exposure to all triggering factors. This is in sharp contrast to observations in limb and trunk muscles, showing a dramatic decline in specific force in response to 5 days' exposure to the triggering factors. Significant differences in gene expression were observed between craniofacial and limb muscles, indicating a highly complex and muscle-specific response involving transcription and growth factors, heat shock proteins, matrix metalloproteinase inhibitor, oxidative stress responsive elements, and sarcomeric proteins underlying the relative sparing of cranial vs. spinal nerve innervated muscles during exposure to the ICU intervention.
Abstract 1837
AKN-028 (formerly BVT-II, abstract presented at EHA 2008, Eriksson A et al) is a tyrosine kinase inhibitor originally identified in a screen targeting FLT-3. In vitro testing by ...fluorometric microculture cytotoxicity assay (FMCA, Lindhagen E. et al, Nat Protoc 2008; 3:1364-9) on primary tumor cells from 29 patients with different haematological malignancies showed that the cytotoxic activity was most pronounced in acute myeloic leukaemia (AML). In vivo, efficacy was demonstrated in a hollow fiber mouse model using MV4-11 cells and primary tumor cells from AML patients.
Kinase inhibitors interacting with the ATP-binding pocket usually targets more than one kinase. PKC-412, presently in phase III trials in AML, is a staurosporine analogue targeting many kinases. In this study we show the kinase inhibition profile of AKN-028 when characterised on a broad panel of 320 different kinases. AKN-028 is relatively specific compared to the staurosporine analogues. To further analyse the difference in mechanism of action between the multi-targeting inhibitor PKC-412 and the more selective AKN-028, we have performed global gene expression analysis by Affymetrix arrays using two different leukaemia cell-lines, HL-60 and MV4-11 (expressing FLT3-ITD), and tumour cells from a patient with FLT3-ITDpos AML. The cells were treated with 10 μM of either compound or vehicle control for 6 h. Principal component (PC) analysis was used to visualise the global gene expression pattern, see fig 1. Searching for differential expression of mRNA levels showed that treatment with AKN-028 resulted in significantly altered gene expression in all three cell types tested, compared to vehicle control treated cells. 430 mRNAs were down-regulated, and 280 were up-regulated. By contrast, treatment with PKC-412 caused very few significant alterations of mRNA levels when compared to vehicle treated cells. Further analysis of gene expression patterns to elucidate the mechanism of action of AKN-028 is ongoing. In conclusion, even though AKN-028 is a relatively selective kinase inhibitor targeting FLT-3, it has more profound effects altering gene expression both in cultured AML cell-lines and a primary AML tumor sample than the multikinase inhibitor PKC-412.
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Parrow:Akinion Pharmaceuticals: Employment, Equity Ownership. Lehmann:Akinion Pharmaceuticals: Consultancy. Larsson:Akinion Pharmaceuticals: Consultancy.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
1 Department of Clinical Neurophysiology, Uppsala University Hospital, Uppsala, Sweden;
2 Research Center for Genetic Medicine, Children National Medical Center;
3 Department of Pediatrics, The ...George Washington University Medical Center, Washington, District of Columbia;
4 Department of Medical Sciences, Uppsala University Hospital, Uppsala, Sweden;
5 Department of Physiology, University of Wisconsin, Madison, Wisconsin; and
6 Department of Anesthesiology, Karolinska Institute, Stockholm, Sweden;
7 Department of Biobehavioral Health, Pennsylvania State University, University Park, Pennsylvania
Skeletal muscle wasting and impaired muscle function in response to mechanical ventilation and immobilization in intensive care unit (ICU) patients are clinically challenging partly due to 1 ) the poorly understood intricate cellular and molecular networks and 2 ) the unavailability of an animal model mimicking this condition. By employing a unique porcine model mimicking the conditions in the ICU with long-term mechanical ventilation and immobilization, we have analyzed the expression profile of skeletal muscle biopsies taken at three time points during a 5-day period. Among the differentially regulated transcripts, extracellular matrix, energy metabolism, sarcomeric and LIM protein mRNA levels were downregulated, while ubiquitin proteasome system, cathepsins, oxidative stress responsive genes and heat shock proteins (HSP) mRNAs were upregulated. Despite 5 days of immobilization and mechanical ventilation single muscle fiber cross-sectional areas as well as the maximum force generating capacity at the single muscle fiber level were preserved. It is proposed that HSP induction in skeletal muscle is an inherent, primary, but temporary protective mechanism against protein degradation. To our knowledge, this is the first study that isolates the effect of immobilization and mechanical ventilation in an ICU condition from various other cofactors.
mechanical ventilation; immobilization; muscle function; gene expression; ubiquitin proteasome system; heat shock proteins; Lim proteins; intensive care unit
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