Aplidin induces JNK-dependent apoptosis in human breast cancer cells via alteration of glutathione homeostasis, Rac1 GTPase activation, and MKP-1 phosphatase downregulation

Aplidin is an antitumor agent in phase II clinical trials that induces apoptosis through the sustained activation of Jun N-terminal kinase (JNK). We report that Aplidin alters glutathione homeostasis increasing the ratio of oxidized to reduced forms (GSSG/GSH). Aplidin generates reactive oxygen species and disrupts the mitochondrial membrane potential. Exogenous GSH inhibits these effects and also JNK activation and cell death. We found two mechanisms by which Aplidin activates JNK: rapid activation of Rac1 small GTPase and downregulation of MKP-1 phosphatase. Rac1 activation was diminished by GSH and enhanced by L-buthionine (SR)-sulfoximine, which inhibits GSH synthesis. Downregulation of Rac1 by transfection of small interfering RNA (siRNA) duplexes or the use of a specific Rac1 inhibitor decreased Aplidin-induced JNK activation and cytotoxicity. Our results show that Aplidin induces apoptosis by increasing the GSSG/GSH ratio, a necessary step for induction of oxidative stress and sustained JNK activation through Rac1 activation and MKP-1 downregulation.     

Inhibition of THP-1 cell-mediated low-density lipoprotein oxidation by the macrophage-synthesised pterin, 7,8-dihydroneopterin

Human macrophages release the pterin, 7,8-dihydroneopterin when exposed to the immune stimulant gamma-interferon (IFN-gamma). Previous in vitro studies have shown 7,8-dihydroneopterin is a potent antioxidant, which inhibits copper- and peroxyl-radical mediated low-density lipoprotein (LDL) oxidation. Using THP-1 cells, a human derived monocyte-like cell line, we have found that low micromolar concentrations of 7,8-dihydroneopterin inhibit cell mediated oxidation of LDL, as measured by electrophoretic mobility, alpha-tocopherol loss, and lipid oxidation. Stimulation of the THP-1 cells with IFN-gamma caused a significant reduction in the cells' ability to oxidise LDL. The extracellular pterin concentration increased from 0 to 16 nM with IFN-gamma stimulation, while the intracellular concentration increased from 0.21 to 1.69 nmol/mg cell protein.     

Inhibition of p38 MAPK by glucocorticoids via induction of MAPK phosphatase-1 enhances nontypeable Haemophilus influenzae-induced expression of toll-like receptor 2

Despite the importance of glucocorticoids in suppressing immune and inflammatory responses, their role in enhancing host immune and defense response against invading bacteria is poorly understood. We have demonstrated recently that glucocorticoids synergistically enhance nontypeable Haemophilus influenzae (NTHi)-induced expression of Toll-like receptor 2 (TLR2), an important TLR family member that has been shown to play a critical role in host immune and defense response. However, the molecular mechanisms underlying the glucocorticoid-mediated enhancement of TLR2 induction still remain unknown. Here we show that glucocorticoids synergistically enhance NTHi-induced TLR2 expression via specific up-regulation of the MAPK phosphatase-1 (MKP-1) that, in turn, leads to dephosphorylation and inactivation of p38 MAPK, the negative regulator for TLR2 expression. Moreover, increased expression of TLR2 in epithelial cells greatly enhances the NTHi-induced expression of several key cytokines, including tumor necrosis factor-alpha and interleukins 1beta and 8, thereby contributing significantly to host immune and defense response. These studies may bring new insights into the novel role of glucocorticoids in orchestrating and optimizing host immune and defense responses during bacterial infections and enhance our understanding of the signaling mechanisms underlying the glucocorticoid-mediated attenuation of MAPKs.     

Genome modification in two lines of baby hamster kidney fibroblasts (BHK-21).

Reasons for the different levels of 5-methyl cytosine encountered in the DNA of two baby hamsters kidney fibroblast lines, BHK-21/C13 and BHK-21/PyY have been investigated. From enzymic studies it does not seem that there are large numbers of potentially methylatable cytosine residues in the C13 line DNA which contains a lower level of 5-methyl cytosine. Rather it is possible that the difference may be due to the reiteration in the PyY strain of certain sequences containing 5-methyl cytosine which simply occur less frequently in the other line.     

Multiple signalling pathways establish cell fate and cell number in Drosophila malpighian tubules

A unique cell, the tip mother cell, arises in the primordium of each Drosophila Malpighian tubule by lateral inhibition within a cluster of achaete-expressing cells. This cell maintains achaete expression and divides to produce daughters of equivalent potential, of which only one, the tip cell, adopts the primary fate and continues to express achaete, while in the other, the sibling cell, achaete expression is lost (M. Hoch et al., 1994, Development 120, 3439-3450). In this paper we chart the mechanisms by which achaete expression is differentially maintained in the tip cell lineage to stabilise cell fate. First, wingless is required to maintain the expression of achaete in the tubule primordium so that wingless mutants lack tip cells. Conversely, increasing wingless expression results in the persistence of achaete expression in the cell cluster. Second, Notch signalling is restricted by the asymmetric segregation of Numb, as the tip mother cell divides, so that achaete expression is maintained only in the tip cell. In embryos mutant for Notch tip cells segregate at the expense of sibling cells, whereas in numb neither daughter cell adopts the tip cell fate resulting in tubules with two sibling cells. Conversely, when numb is overexpressed two tip cells segregate and tubules have no sibling cells. Analysis of cell proliferation in the developing tubules of embryos lacking Wingless after the critical period for tip cell allocation reveals an additional requirement for wingless for the promotion of cell division. In contrast, alteration in the expression of numb has no effect on the final tubule cell number.     

Hsp70-RAP46 interaction in downregulation of DNA binding by glucocorticoid receptor

Receptor-associating protein 46 (RAP46) is a cochaperone that regulates the transactivation function of several steroid receptors. It is transported into the nucleus by a liganded glucocorticoid receptor where it downregulates DNA binding and transactivation by this receptor. The N- and C-termini of RAP46 are both implicated in its negative regulatory function. In metabolic labelling experiments, we have shown that the N-terminus of RAP46 is modified by phosphorylation, but this does not contribute to the downregulation of glucocorticoid receptor activity. However, deletion of a sequence that binds 70 kDa heat shock protein (Hsp70) and the constitutive isoform of Hsp70 (Hsc70) at the C-terminus of RAP46 abrogated its negative regulatory action. Surface plasmon resonance studies showed that RAP46 binds the glucocorticoid receptor only when it has interacted with Hsp70/Hsc70, and confocal immunofluorescence analyses revealed a nuclear transport of Hsp70/Hsc70 by the liganded receptor. Together these findings demonstrate an important contribution of Hsp70/Hsc70 in the binding of RAP46 to the glucocorticoid receptor and suggest a role for this molecular chaperone in the RAP46-mediated downregulation of glucocorticoid receptor activity.     

Identification of non-amidine inhibitors of acid-sensing ion channel-3 (ASIC3)

A series of benzothiophene methyl amines were examined in an effort to identify non-amidine chemotypes with reduced polypharmacology from existing leads with the goal of finding potent ASIC3 channel blockers to advance the therapeutic evaluation of ASIC3 inhibition.