Untangling the intracellular signalling network in cancer--a strategy for data integration in acute myeloid leukaemia

Protein and gene networks centred on the regulatory tumour suppressor proteins may be of crucial importance both in carcinogenesis and in the response to chemotherapy. Tumour suppressor protein p53 integrates intracellular data in stress responses, receiving signals and translating these into differential gene expression. Interpretation of the data integrated on p53 may therefore reveal the response to therapy in cancer. Proteomics offers more specific data - closer to "the real action" - than the hitherto more frequently used gene expression profiling. Integrated data analysis may reveal pathways disrupted at several regulatory levels. Ultimately, integrated data analysis may also contribute to finding key underlying cancer genes. We here proposes a Partial Least Squares Regression (PLSR)-based data integration strategy, which allows simultaneous analysis of proteomic data, gene expression data and classical clinical parameters. PLSR collapses multidimensional data into fewer relevant dimensions for data interpretation. PLSR can also aid identification of functionally important modules by also performing comparison to databases on known biological interactions. Further, PLSR allows meaningful visualization of complex datasets, aiding interpretation of the underlying biology. Extracting the true biological causal mechanisms from heterogeneous patient populations is the key to discovery of new therapeutic options in cancer.     

Microfluidic perfusion for regulating diffusible signaling in stem cells

Background

Autocrine & paracrine signaling are widespread both in vivo and in vitro, and are particularly important in embryonic stem cell (ESC) pluripotency and lineage commitment. Although autocrine signaling via fibroblast growth factor-4 (FGF4) is known to be required in mouse ESC (mESC) neuroectodermal specification, the question of whether FGF4 autocrine signaling is sufficient, or whether other soluble ligands are also involved in fate specification, is unknown. The spatially confined and closed-loop nature of diffusible signaling makes its experimental control challenging; current experimental approaches typically require prior knowledge of the factor/receptor in order to modulate the loop. A new approach explored in this work is to leverage transport phenomena at cellular resolution to downregulate overall diffusible signaling through the physical removal of cell-secreted ligands.

Methodology/Principal Findings

We develop a multiplex microfluidic platform to continuously remove cell-secreted (autocrine\paracrine) factors to downregulate diffusible signaling. By comparing cell growth and differentiation in side-by-side chambers with or without added cell-secreted factors, we isolate the effects of diffusible signaling from artifacts such as shear, nutrient depletion, and microsystem effects, and find that cell-secreted growth factor(s) are required during neuroectodermal specification. Then we induce FGF4 signaling in minimal chemically defined medium (N2B27) and inhibit FGF signaling in fully supplemented differentiation medium with cell-secreted factors to determine that the non-FGF cell-secreted factors are required to promote growth of differentiating mESCs.

Conclusions/Significance

Our results demonstrate for the first time that flow can downregulate autocrine\paracrine signaling and examine sufficiency of extracellular factors. We show that autocrine\paracrine signaling drives neuroectodermal commitment of mESCs through both FGF4-dependent and -independent pathways. Overall, by uncovering autocrine\paracrine processes previously hidden in conventional culture systems, our results establish microfluidic perfusion as a technique to study and manipulate diffusible signaling in cell systems.     

Osteopenia due to enhanced cathepsin K release by BK channel ablation in osteoclasts

Background

The process of bone resorption by osteoclasts is regulated by Cathepsin K, the lysosomal collagenase responsible for the degradation of the organic bone matrix during bone remodeling. Recently, Cathepsin K was regarded as a potential target for therapeutic intervention of osteoporosis. However, mechanisms leading to osteopenia, which is much more common in young female population and often appears to be the clinical pre-stage of idiopathic osteoporosis, still remain to be elucidated, and molecular targets need to be identified.

Methodology/Principal Findings

We found, that in juvenile bone the large conductance, voltage and Ca²⁺-activated (BK) K⁺ channel, which links membrane depolarization and local increases in cytosolic calcium to hyperpolarizing K⁺ outward currents, is exclusively expressed in osteoclasts. In juvenile BK-deficient (BK^−/−) female mice, plasma Cathepsin K levels were elevated two-fold when compared to wild-type littermates. This increase was linked to an osteopenic phenotype with reduced bone mineral density in long bones and enhanced porosity of trabecular meshwork in BK^−/− vertebrae as demonstrated by high-resolution flat-panel volume computed tomography and micro-CT. However, plasma levels of sRANKL, osteoprotegerin, estrogene, Ca²⁺ and triiodthyronine as well as osteoclastogenesis were not altered in BK^−/− females.

Conclusion/Significance

Our findings suggest that the BK channel controls resorptive osteoclast activity by regulating Cathepsin K release. Targeted deletion of BK channel in mice resulted in an osteoclast-autonomous osteopenia, becoming apparent in juvenile females. Thus, the BK^−/− mouse-line represents a new model for juvenile osteopenia, and revealed the BK channel as putative new target for therapeutic controlling of osteoclast activity.     

Comprehensive genetic evaluation of common E-cadherin sequence variants and prostate cancer risk: strong confirmation of functional promoter SNP

The E-cadherin gene (CDH1) has been proposed as a prostate cancer (PC) susceptibility gene in several studies. Aberrant protein expression has been related to prognosis and progression in PC. In addition, a functional promoter SNP (rs16260) has been found to associate with PC risk. We performed a comprehensive genetic analysis of CDH1 by using the method of haplotype tagged SNPs in a large Swedish population-based case-control study consisting of 801 controls and 1,636 cases. In addition, Swedish PC families comprising a total of 157 cases sampled for DNA were analyzed for selected SNPs. Seven SNPs, including the promoter SNP rs16260, that captured over 96% of CDH1 haplotype variation were selected as haplotype tagging SNPs and analyzed for associated PC risk. We observed significant confirmation of rs16260 (P=0.003) for cases with a positive family history of PC (FH+) both in an independent case-control population and in PC families. In addition, a common haplotype (HapB, 25%) including the variant allele of rs16260 was associated (P=0.004) with PC risk among FH+ cases. The promoter SNP rs16260 as well as HapB were significantly transmitted to affected offspring in PC families. We report strong confirmation of the association between PC risk in FH+ cases and a functional CDH1 promoter SNP in an independent population. In conjunction with the biological importance of CDH1 our findings encourage further evaluation of genetic variation in CDH1 in relation to PC etiology. Due to the difficulties in replication of genetic association studies, this finding is unusual and novel.     

Genotype, enzyme activity, glutathione level, and clinical phenotype in patients with glutathione synthetase deficiency

Glutathione synthetase (GS) deficiency is a rare autosomal recessive disorder. The clinical phenotype varies widely, and nearly 30 different mutations in the GSS gene have been identified. In the present study, genotype, enzyme activity, metabolite levels and clinical phenotype were evaluated in 41 patients from 33 families. From some of the patients, data on glutathione (GSH) levels and -glutamylcysteine levels in cultured fibroblasts were also available. Twenty-seven different mutations were found: 14 missense, 9 splice, 2 deletions, 1 insertion and 1 nonsense mutation. Twenty-three patients were homozygous and 18 were compound heterozygous. The moderate and severe clinical phenotypes could not be distinguished based on enzyme activity, GSH or -glutamylcysteine levels in cultured fibroblasts. However, in fibroblasts, the residual GS activity was correlated with the GSH level. All mutations causing frameshifts, premature stop codons or aberrant splicing were associated with moderate or severe clinical phenotypes including haemolytic anaemia, 5-oxoprolinuria, and (in several forms) neurodevelopmental signs. The data indicate that additional genetic or environmental factors modify at least the moderate and severe phenotypes and that the clinical classification given to the patients may be influenced by variation in follow-up. The type of mutation involved can, to some extent, predict a mild versus a more severe phenotype.Electronic Supplementary Material Suplementary material is available for this article at .Electronic database information: Online Mendelian Inheritance in Man, (MIM 266130 for GS deficiency); GenBank, ( nos. AL133324.13 for genomic DNA and NM_000178.2 for mRNA were used for sequence referencing)     

Synthesis and anti-aromatase activity of some new steroidal D-lactones

Starting from D-seco derivatives of 5-androstene 1-3, the D-homo lactones, 4 and 5, were synthesized. By the Oppenauer oxidation and/or by dehydration of 4 and 5 with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) or 2,3,5,6-tetrachloro-1,4-benzoquinone (chloranil), the corresponding D-lactones 6-12 were obtained. The structures of 6 and 10 were unambiguously proved by the appropriate X-ray structural analysis. Anti-aromatase assay showed that tested compounds possess inhibition potency, however, two to four times smaller (IC50 from 0.2 to 0.7 microM, respectively) in comparison to aminoglutethimide (AG).     

Normal human osteoclasts formed from peripheral blood monocytes express PTH type 1 receptors and are stimulated by PTH in the absence of osteoblasts

The prevailing view for many years has been that osteoclasts do not express parathyroid hormone (PTH) receptors and that PTH's effects on osteoclasts are mediated indirectly via osteoblasts. However, several recent reports suggest that osteoclasts express PTH receptors. In this study, we tested the hypothesis that human osteoclasts formed in vitro express functional PTH type 1 receptors (PTH1R). Peripheral blood monocytes (PBMC) were cultured on bone slices or plastic culture dishes with human recombinant RANK ligand (RANKL) and recombinant human macrophage colony-stimulating factor (M-CSF) for 16-21 days. This resulted in a mixed population of mono- and multi-nucleated cells, all of which stained positively for the human calcitonin receptor. The cells actively resorbed bone, as assessed by release of C-terminal telopeptide of type I collagen and the formation of abundant resorption pits. We obtained evidence for the presence of PTH1R in these cells by four independent techniques. First, using immunocytochemistry, positive staining for PTH1R was observed in both mono- and multi-nucleated cells intimately associated with resorption cavities. Second, PTH1R protein expression was demonstrated by Western blot analysis. Third, the cells expressed PTH1R mRNA at 21 days and treatment with 10(-7) M hPTH (1-34) reduced PTH1R mRNA expression by 35%. Finally, bone resorption was reproducibly increased by two to threefold when PTH (1-34) was added to the cultures. These findings provide strong support for a direct stimulatory action of PTH on human osteoclasts mediated by PTH1R. This suggests a dual regulatory mechanism, whereby PTH acts both directly on osteoclasts and also, indirectly, via osteoblasts.     

Propylphenols are metabolites in the anaerobic biodegradation of propylbenzene under iron-reducing conditions

The metabolism of monoaromatic hydrocarbons by an iron-reducing bacterial enrichment culture originating from diesel-contaminated groundwater was examined using d₇-propylbenzene as a model hydrocarbon. Sequence analysis of the 16S rDNA gene showed that the dominant part (10 of 10 clones) of the enrichment culture consisted of a bacterium closely related to clones found in benzene-contaminated groundwater and to the iron-reducing -proteobacterium, Rhodoferax ferrireducens (similarity values were 99.5% and 98.3%, respectively). In degradation studies conducted over 18 weeks, d₇-propylphenols were detected by gas chromatography–mass spectrometry (GC/MS) as intra-cellular metabolites concomitant with cell growth in the cultures. The amount of propylphenols increased during the exponential growth phase, and by the end of this phase 4 × 10^–14 moles of ferric iron were reduced and 3 × 10^–15 moles propylphenol produced for every cell formed. During the stationary growth phase the cell density was approximately 10⁷ ml^–1, with significantly correlated amounts of propylphenols. Succinate derivates of propylbenzene or phenylpropanol previously shown to be the initial metabolites in the anaerobic degradation of alkylbenzenes could not be identified. This study is the first to report that oxidation of propylbenzene to propylphenols can initiate anaerobic propylbenzene degradation and that iron-reducing bacteria are responsible for this process. In addition, the study shows the importance of taking account of the metabolites adhering to solid phases when determining the extent of biodegradation, so as not to underestimate the extent of the process.     

Contrasting ‘Top-Down’ Effects of Crustacean Zooplankton Grazing on Bacteria and Phytoflagellates

The combined effects of grazing and nutrient regeneration by Daphnia and Eudiaptomus on the growth of Rhodomonas and heterotrophic bacteria was assessed experimentally. The responses of Rhodomonas and bacteria to the grazers were measured as net specific growth rate over the entire experimental periods, as well as production and specific production at the end of the experiments. Both zooplankton species had a negative effect on Rhodomonas net specific growth rate due to grazing and a positive effect on specific primary production due to nutrient regeneration. Daphnia had no effect on bacterial net specific growth rate, bacterial production or specific bacterial production in one of two experiments. In the other experiment, however, both bacterial growth rate and production decreased as a result of grazing. Furthermore, Daphnia had a negative effect on specific bacterial production, but Eudiaptomus had a positive effect on all bacterial parameters due to nutrient regeneration, probably of phosphorus. Positive effects of copepods on bacterial growth has previously been attributed to trophic cascades via protozoa. However, the present experiments show that regeneration of nutrients, especially phosphorus, may account for a large part of the stimulation of bacterial growth.