Fas-induced B cell apoptosis requires an increase in free cytosolic magnesium as an early event

Ligation of the Fas molecule expressed on the surface of a cell initiates multiple signaling pathways that result in the apoptotic death of that cell. We have examined Mg2+ mobilization as well as Ca2+ mobilization in B cells undergoing Fas-initiated apoptosis. Our results indicate that cytosolic levels of free (non-complexed) Mg2+ ([Mg2+]i) and Ca2+ ([Ca2+]i) increase in cells undergoing apoptosis. Furthermore, the percentages of cells mobilizing Mg2+, fragmenting DNA, or externalizing phosphatidylserine (PS) increase in parallel as the concentration of anti-Fas monoclonal antibody is raised. Kinetic analysis suggests that Mg2+ mobilization is an early event in apoptosis, clearly preceding DNA fragmentation and probably occurring prior to externalization of PS as well. The source of Mg2+ that produces the increases in [Mg2+]i is intracellular and most likely is the mitochondria. Extended pretreatment of B cells with carbonyl cyanide m-chlorophenylhydrazone, an inhibitor of mitochondrial oxidative phosphorylation, produces proportional decreases in the percentage of cells mobilizing Mg2+, fragmenting DNA, and externalizing PS in response to anti-Fas monoclonal antibody treatment. These observations are consistent with the hypothesis that elevated [Mg2+]i is required for apoptosis. Furthermore, we propose that the increases in [Mg2+]i function not only as cofactors for Mg2+-dependent endonucleases, but also to facilitate the release of cytochrome c from the mitochondria, which drives many of the post-mitochondrial, caspase-mediated events in apoptotic cells.     

Microsomal membrane structure and function subsequent to calcium activation of an endogenous phospholipase

An endogenous system in the membranes of rat liver endoplasmic reticulum is capable upon Ca²⁺ activation of considerable disruption of normal structure and function. Phosphatidylethanolamine (PE) and to a lesser extent phosphatidylcholine (PC) are degraded to hydrophilic products. This lipid loss is greater at an alkaline pH, preferentially utilizes millimolar Ca²⁺ rather than Mg²⁺ ions, and is inhibited by KCl. Diethyl ether has no effect on the rate of loss of PE or PC, and the Ca²⁺ ionophore A23187 does not lower the Ca²⁺ requirement. Phospholipids are most likely lost from the membranes in a two-step process. Lysophospholipids generated in the first, Ca²⁺-dependent step are removed by an endogenous lysophospholipase demonstrated by the hydrolysis of either added lyso PE or lysophospholipids generated from endogenous substrates by Naja naja phospholipase A₂. The depletion of microsomal membrane phospholipid is accompanied by a loss of glucose 6-phosphatase and of cytochrome P-450. The latter is not associated with any change in total heme content. Polyacrylamide gel electrophoresis showed no difference between the pattern or relative amounts of solubilized membrane proteins before or after depletion of membrane phospholipid. It is concluded that activation of an endogenous phospholipase by Ca²⁺ can result in significant depletion of PE and PC that is accompanied by considerable disruption of membrane function. The significance of this system with respect to the maintenance of cell integrity and its possible role in cell injury are discussed.     

Cobalt-cytochrome c. II. Magnetic resonance spectra and conformational transitions.

Between pH approximately 4 and 10 cobaltocytochrome c (Cocyt-c) gives an electron paramagnetic resonance (EPR) spectrum with g parallel = 2.035, g the perpendicular = 2.223, CoA PARALLEL = 61.4 G, CoA the perpendicular = 49.8 G, NA parallel = 15.3 G, and NA THE PERPENDICULAR = 12.5 G. Comparisons with the EPR spectra of deoxycobaltomyoglobin, deoxycobaltohemoglobin, and model compounds and together with other evidence showed cobaltocytochrome c to have Met-80 and His-18 as its axial ligands. The protons of these ligands are seen as resonances shifted by the ring-current field of the porphyrin in the 300-MHZ 1H nuclear magnetic resonance (NMR) spectra of cobalticytochrome c (Cocyt-c+). The methyl and gamma-methylene protons of Met-80 in this molecule occupy positions with respect to heme c which are somewhat different from those in ferrocytochrome c. The 1H NMR spectra also showed that the methyl groups of Leu-32, Ile-75, Thr-63, thioether bridges, and the porphyrin ring in the cobalt protein are in the same state as in native enzyme; the same is also true for Tyr-59, His-26, and His-33 and also possibly Tyr-67, Tyr-74, and Phe-82. Above pH 11, Cocyt-c is converted to a five-coordinated form having g parallel = 2.026, g the perpendicular = 2.325, CoA parallel = 80 G, CoA the perpendicular approximately 10 G, NA parallel = 17.5 G, and NA the perpendicular not resolved. Below pH 1.0 the EPR spectrum of Cocyt-c is also five-coordinated with g parallel = 2.014, g the perpendicular = 2.359, CoA parallel = 93.8 G, and CoA the perpendicular = 38.8 G. The axial ligands in the alkaline and the acidic forms of Cocyt-c are His-18 and Met-80, respectively. New prominent proton resonance peaks are observed in cobalt-cytochrome c which are either absent or weak in native cytochrome c. These are situated at 3.0, 1.7, and 1.44 ppm, attributable, respectively, to the epsilon-CH2, DELTA-CH2 + beta-CH2, and gamma-CH2 of lysyl residues in random-coil-peptides. From the areas of these peaks, it is estimated that one-two lysyl residues in Cocyt-c have been modified; four-five lysyl residues in Cocyt-c+ have been modified. These alterations of surface charged groups are probably responsible for the lowered reactivity of Cocyt-c with cytochrome oxidase and the lack of reactivity of Cocyt-c+ with several cytochrome reductase systems.     

Induction of apoptosis in human leukemia K562 cells by cardiotoxin III

Cardiotoxin III (CTX III), a basic polypeptide with 60 amino acid residues isolated from Naja naja atra venom, has been reported to have anticancer activity. CTX III was found to inhibit the growth of K562 cells in a time-and dose-dependent manner with IC50 value of 1.7 microg/ml, and it displayed several features of apoptosis including apoptotic body formation, increase of sub G1 population, DNA fragmentation and poly (ADP-ribose) polymerase (PARP) cleavage. Investigation of the mechanism of CTXIII--induced apoptosis revealed that the treatment of K562 cells with CTX III resulted in the activation of caspase-9, caspase-3 and subsequent cleavage of its substrate PARP and that CTXIII was also associated with an early release of cytochrome c from the mitochondria. These results suggest that CTX III may induce apoptosis through a mitochondria- and caspase-dependent mechanism.     

Targeted BRAF Inhibition Impacts Survival in Melanoma Patients with High Levels of Wnt/β-Catenin Signaling

Unprecedented clinical responses have been reported in advanced stage metastatic melanoma patients treated with targeted inhibitors of constitutively activated mutant BRAF, which is present in approximately half of all melanomas. We and others have previously observed an association of elevated nuclear β-catenin with improved survival in molecularly-unselected melanoma patients. This study sought to determine whether levels of Wnt/β-catenin signaling in melanoma tumors prior to treatment might predict patient responses to BRAF inhibitors (BRAFi). We performed automated quantification of β-catenin immunohistochemical expression in pretreatment BRAF-mutant tumors from 32 BRAFi-treated melanoma patients. Unexpectedly, patients with higher nuclear β-catenin in their tumors did not exhibit the survival advantage previously observed in molecularly-unselected melanoma patients who did not receive BRAFi. In cultured melanoma cells treated with long-term BRAFi, activation of Wnt/β-catenin signaling is markedly inhibited, coinciding with a loss of the enhancement of BRAFi-induced apoptosis by WNT3A observed in BRAFi-naïve cells. Together, these observations suggest that long-term treatment with BRAFi can impact the interaction between BRAF/MAPK and Wnt/β-catenin signaling to affect patient outcomes. Studies with larger patient cohorts are required to determine whether nuclear β-catenin expression correlates with clinical responses to BRAFi and to specific mechanisms of acquired resistance to BRAFi. Understanding these pathway interactions will be necessary to facilitate efforts to individualize therapies for melanoma patients.     

Genetic Variants of CD209 Associated with Kawasaki Disease Susceptibility

Background

Kawasaki disease (KD) is a systemic vasculitis with unknown etiology mainly affecting children in Asian countries. Dendritic cell-specific intercellular adhesion molecule-3 grabbing non-integrin (DC-SIGN, CD209) in humans was showed to trigger an anti-inflammatory cascade and associated with KD susceptibility. This study was conducted to investigate the association between genetic polymorphisms of CD209 and the risk KD.

Methods

A total of 948 subjects (381 KD and 567 controls) were recruited. Nine tagging SNPs (rs8112310, rs4804800, rs11465421, rs1544766, rs4804801, rs2287886, rs735239, rs735240, rs4804804) were selected for TaqMan allelic discrimination assay. Clinical phenotypes, coronary artery lesions (CAL) and intravenous immunoglobulin (IVIG) treatment outcomes were collected for analysis.

Results

Significant associations were found between CD209 polymorphisms (rs4804800, rs2287886, rs735240) and the risk of KD. Haplotype analysis for CD209 polymorphisms showed that A/A/G haplotype (P = 0.0002, OR = 1.61) and G/A/G haplotype (P = 0.0365, OR = 1.52) had higher risk of KD as compared with G/G/A haplotype in rs2287886/rs735239/rs735240 pairwise allele analysis. There were no significant association in KD with regards to CAL formation and IVIG treatment responses.

Conclusion

CD209 polymorphisms were responsible for the susceptibility of KD, but not CAL formation and IVIG treatment responsiveness.