Ethacrynic Acid Exhibits Selective Toxicity to Chronic Lymphocytic Leukemia Cells by Inhibition of the Wnt/β-Catenin Pathway

Background

Aberrant activation of Wnt/β-catenin signaling promotes the development of several cancers. It has been demonstrated that the Wnt signaling pathway is activated in chronic lymphocytic leukemia (CLL) cells, and that uncontrolled Wnt/β-catenin signaling may contribute to the defect in apoptosis that characterizes this malignancy. Thus, the Wnt signaling pathway is an attractive candidate for developing targeted therapies for CLL.

Methodology/Principal Findings

The diuretic agent ethacrynic acid (EA) was identified as a Wnt inhibitor using a cell-based Wnt reporter assay. In vitro assays further confirmed the inhibitory effect of EA on Wnt/β-catenin signaling. Cell viability assays showed that EA selectively induced cell death in primary CLL cells. Exposure of CLL cells to EA decreased the expression of Wnt/β-catenin target genes, including LEF-1, cyclin D1 and fibronectin. Immune co-precipitation experiments demonstrated that EA could directly bind to LEF-1 protein and destabilize the LEF-1/β-catenin complex. N-acetyl-L-cysteine (NAC), which can react with the α, β-unsaturated ketone in EA, but not other anti-oxidants, prevented the drug''s inhibition of Wnt/β-catenin activation and its ability to induce apoptosis in CLL cells.

Conclusions/Significance

Our studies indicate that EA selectively suppresses CLL survival due to inhibition of Wnt/β-catenin signaling. Antagonizing Wnt signaling in CLL with EA or related drugs may represent an effective treatment of this disease.     

Distributions of beta sheets in proteins with application to structure prediction

We recently developed the Rosetta algorithm for ab initio protein structure prediction, which generates protein structures from fragment libraries using simulated annealing. The scoring function in this algorithm favors the assembly of strands into sheets. However, it does not discriminate between different sheet motifs. After generating many structures using Rosetta, we found that the folding algorithm predominantly generates very local structures. We surveyed the distribution of beta-sheet motifs with two edge strands (open sheets) in a large set of non-homologous proteins. We investigated how much of that distribution can be accounted for by rules previously published in the literature, and developed a filter and a scoring method that enables us to improve protein structure prediction for beta-sheet proteins. Proteins 2002;48:85-97.     

A unique voltage sensor sensitizes the potassium channel AKT2 to phosphoregulation

Among all voltage-gated K+ channels from the model plant Arabidopsis thaliana, the weakly rectifying K+ channel (K(weak) channel) AKT2 displays unique gating properties. AKT2 is exceptionally regulated by phosphorylation: when nonphosphorylated AKT2 behaves as an inward-rectifying potassium channel; phosphorylation of AKT2 abolishes inward rectification by shifting its activation threshold far positive (>200 mV) so that it closes only at voltages positive of +100 mV. In its phosphorylated form, AKT2 is thus locked in the open state in the entire physiological voltage range. To understand the molecular grounds of this unique gating behavior, we generated chimeras between AKT2 and the conventional inward-rectifying channel KAT1. The transfer of the pore from KAT1 to AKT2 altered the permeation properties of the channel. However, the gating properties were unaffected, suggesting that the pore region of AKT2 is not responsible for the unique K(weak) gating. Instead, a lysine residue in S4, highly conserved among all K(weak) channels but absent from other plant K+ channels, was pinpointed in a site-directed mutagenesis approach. Substitution of the lysine by serine or aspartate abolished the "open-lock" characteristic and converted AKT2 into an inward-rectifying channel. Interestingly, phosphoregulation of the mutant AKT2-K197S appeared to be similar to that of the K(in) channel KAT1: as suggested by mimicking the phosphorylated and dephosphorylated states, phosphorylation induced a shift of the activation threshold of AKT2-K197S by about +50 mV. We conclude that the lysine residue K197 sensitizes AKT2 to phosphoregulation. The phosphorylation-induced reduction of the activation energy in AKT2 is approximately 6 kT larger than in the K197S mutant. It is discussed that this hypersensitive response of AKT2 to phosphorylation equips a cell with the versatility to establish a potassium gradient and to make efficient use of it.     

Alternative metabolic fates of phosphatidylinositol produced by phosphatidylinositol synthase isoforms in Arabidopsis thaliana

PtdIns is an important precursor for inositol-containing lipids, including polyphosphoinositides, which have multiple essential functions in eukaryotic cells. It was previously proposed that different regulatory functions of inositol-containing lipids may be performed by independent lipid pools; however, it remains unclear how such subcellular pools are established and maintained. In the present paper, a previously uncharacterized Arabidopsis gene product with similarity to the known Arabidopsis PIS (PtdIns synthase), PIS1, is shown to be an active enzyme, PIS2, capable of producing PtdIns in vitro. PIS1 and PIS2 diverged slightly in substrate preferences for CDP-DAG [cytidinediphospho-DAG (diacylglycerol)] species differing in fatty acid composition, PIS2 preferring unsaturated substrates in vitro. Transient expression of fluorescently tagged PIS1 or PIS2 in onion epidermal cells indicates localization of both enzymes in the ER (endoplasmic reticulum) and, possibly, Golgi, as was reported previously for fungal and mammalian homologues. Constitutive ectopic overexpression of PIS1 or PIS2 in Arabidopsis plants resulted in elevated levels of PtdIns in leaves. PIS2-overexpressors additionally exhibited significantly elevated levels of PtdIns(4)P and PtdIns(4,5)P(2), whereas polyphosphoinositides were not elevated in plants overexpressing PIS1. In contrast, PIS1-overexpressors contained significantly elevated levels of DAG and PtdEtn (phosphatidylethanolamine), an effect not observed in plants overexpressing PIS2. Biochemical analysis of transgenic plants with regards to fatty acids associated with relevant lipids indicates that lipids increasing with PIS1 overexpression were enriched in saturated or monounsaturated fatty acids, whereas lipids increasing with PIS2 overexpression, including polyphosphoinositides, contained more unsaturated fatty acids. The results indicate that PtdIns populations originating from different PIS isoforms may enter alternative routes of metabolic conversion, possibly based on specificity and immediate metabolic context of the biosynthetic enzymes.     

Historical and philosophical perspectives on the study of developmental bias

Throughout the recent history of research at the intersection of evolution and development, notions such as developmental constraint, evolutionary novelty, and evolvability have been prominent, but the term “developmental bias” has scarcely been used. And one may even doubt whether a unique and principled definition of bias is possible. I argue that the concept of developmental bias can still play a vital scientific role by means of setting an explanatory agenda that motivates investigation and guides the formulation of integrative explanatory frameworks. Less crucial is a definition that would classify patterns of phenotypic variation and unify variational patterns involving different traits and taxa as all being “bias.” Instead, what we should want is a concept that generates intellectual identity across various researchers, and that unites the diverse fields and approaches relevant to the study of developmental bias, from paleontology to behavioral biology. I point to some advantages of conducting research specifically under the label of “developmental bias,” compared with employing other, more common terms such as “evolvability.”     

The DEK protein--an abundant and ubiquitous constituent of mammalian chromatin

The protein DEK is an abundant and ubiquitous chromatin protein in multicellular organisms (not in yeast). It is expressed in more than a million copies/nucleus of rapidly proliferating mammalian cells. DEK has two DNA binding modules of which one includes a SAP box, a sequence motif that DEK shares with a number of other chromatin proteins. DEK has no apparent affinity to specific DNA sequences, but preferentially binds to superhelical and cruciform DNA, and induces positive supercoils into closed circular DNA. The available evidence strongly suggests that DEK could function as an architectural protein in chromatin comparable to the better known classic architectural chromatin proteins, the high-mobility group or HMG proteins.     

Adaptive Growth of Gymnosperm Branches-Ultrastructural and Micromechanical Examinations

The top, the lateral and the underside of basal branch segments of two gymnosperm species, spruce (Picea abies [L.] Karst.) and yew (Taxus baccata L.), were studied with respect to possible adaptation in structural and mechanical properties. Microtensile tests were performed on thin wet foils, which were removed from the periphery of the branches. Structural parameters such as density and the microfibril angle in the S2-layer were examined to investigate the structure-function relationships of the branch wood. The top, the lateral and the underside of both branches showed significant differences in their structural and mechanical properties. However, no significant variations were observed as a function of age and size development. The findings were discussed in view of adaptive growth strategies of trees, including biomechanical constraints of a horizontally growing branch.