Reciprocal Relationship Between Cytosolic NADH and ENOX2 Inhibition Triggers Sphingolipid‐Induced Apoptosis in HeLa Cells

ENOX2 (tNOX), a tumor‐associated cell surface ubiquinol (NADH) oxidase, functions as an alternative terminal oxidase for plasma membrane electron transport. Ubiquitous in all cancer cell lines studied thus far, ENOX2 expression correlates with the abnormal growth and division associated with the malignant phenotype. ENOX2 has been proposed as the cellular target for various quinone site inhibitors that demonstrate anticancer activity such as the green tea constituent epigallocatechin‐3‐gallate (EGCg) and the isoflavene phenoxodiol (PXD). Here we present a possible mechanism that explains how these substances result in apoptosis in cancer cells by ENOX2‐mediated alterations of cytosolic amounts of NAD⁺ and NADH. When ENOX2 is inhibited, plasma membrane electron transport is diminished, and cytosolic NADH accumulates. We show in HeLa cells that NADH levels modulate the activities of two pivotal enzymes of sphingolipid metabolism: sphingosine kinase 1 (SK1) and neutral sphingomyelinase (nSMase). Their respective products sphingosine 1‐phosphate (S1P) and ceramide (Cer) are key determinants of cell fate. S1P promotes cell survival and Cer promotes apoptosis. Using plasma membranes isolated from cervical adenocarcinoma (HeLa) cells as well as purified proteins of both bacterial and human origin, we demonstrate that NADH inhibits SK1 and stimulates nSMase, while NAD⁺ inhibits nSMase and has no effect on SK1. Additionally, intact HeLa cells treated with ENOX2 inhibitors exhibit an increase in Cer and a decrease in S1P. Treatments that stimulate cytosolic NADH production potentiate the antiproliferative effects of ENOX2 inhibitors while those that attenuate NADH production or stimulate plasma membrane electron transport confer a survival advantage. J. Cell. Biochem. 110: 1504–1511, 2010. © 2010 Wiley‐Liss, Inc.     

Changing nuclear landscape and unique PML structures during early epigenetic transitions of human embryonic stem cells

The complex nuclear structure of somatic cells is important to epigenomic regulation, yet little is known about nuclear organization of human embryonic stem cells (hESC). Here we surveyed several nuclear structures in pluripotent and transitioning hESC. Observations of centromeres, telomeres, SC35 speckles, Cajal Bodies, lamin A/C and emerin, nuclear shape and size demonstrate a very different “nuclear landscape” in hESC. This landscape is remodeled during a brief transitional window, concomitant with or just prior to differentiation onset. Notably, hESC initially contain abundant signal for spliceosome assembly factor, SC35, but lack discrete SC35 domains; these form as cells begin to specialize, likely reflecting cell‐type specific genomic organization. Concomitantly, nuclear size increases and shape changes as lamin A/C and emerin incorporate into the lamina. During this brief window, hESC exhibit dramatically different PML‐defined structures, which in somatic cells are linked to gene regulation and cancer. Unlike the numerous, spherical somatic PML bodies, hES cells often display ～1–3 large PML structures of two morphological types: long linear “rods” or elaborate “rosettes”, which lack substantial SUMO‐1, Daxx, and Sp100. These occur primarily between Day 0–2 of differentiation and become rare thereafter. PML rods may be “taut” between other structures, such as centromeres, but clearly show some relationship with the lamina, where PML often abuts or fills a “gap” in early lamin A/C staining. Findings demonstrate that pluripotent hES cells have a markedly different overall nuclear architecture, remodeling of which is linked to early epigenomic programming and involves formation of unique PML‐defined structures. J. Cell. Biochem. 107: 609–621, 2009. © 2009 Wiley‐Liss, Inc.     

Bidirectional effect of met-enkephalin on macrophage effector functions

Summary Met-enkephalin (ME) exerts a bimodal effect on functional activities of rat peritoneal macrophages (PM); in a range of low concentration (10^-9-10^-7 M) antibody dependent cellular cytotoxicity (ADCC)was markedly stimulated with a simultaneous decrease of Fc receptor (FcR) mediated phagocytosis while the opposite was observed at 10^-6-10^-5 M concentrations.Studying the possible underlying mechanism(s) the followings were recorded: (1) ME in all applied concentrations induced an early Na⁺ influx which was followed by a Ca²⁺ efflux in the range of low concentrations. In the range of high concentrations Na⁺ influx was accompanied by a Ca²⁺ influx. (2) ME at 10^-8 M concentration induced a rise in cGMP level with a plateau in the 60–120th min of incubation. This effect was prevented by 10^-5 M of naloxone. At 10^-6 M concentration a transient rise of cAMP level was recorded which was not affected by naloxone. (3) Verapamil in 10^-6 M abolished both the Ca²⁺ influx and the rise in cAMP level induced by 10^-6-10^-5 M ME but not the rise in cGMP level induced by lower ME concentrations. (4) cAMP elevation by high ME concentrations was abolished by enkephalinase inhibitory puromycin. (5) PM-enkephalinase as assessed by the cleavage of fluorogenic substrate L-alanine beta naphthylamide (ABNA), was inhibited by 10^-6-10^-5 M of ME. This inhibition was abolished by verapamil, but not affected by naloxone. In the range of low concentrations ME appears to act on specific delta opioid receptors and its action is positively coupled to guanylate cyclase. In relatively higher concentrations ME-action is not mediated by specific delta opioid receptors and it appears to involve Ca²⁺ influx, adenylate cyclase activation as well as the processing of hormone by PM-enkephalinase.     

Gax suppresses chemerin/CMKLR1‐induced preadipocyte biofunctions through the inhibition of Akt/mTOR and ERK signaling pathways

Adipose tissue is closely associated with angiogenesis and vascular remodeling. Chemerin is involved in inflammatory reaction and vascular dysfunction. However, the mechanisms of chemerin participating in vascular remodeling and whether Growth arrest‐specific homeobox (Gax) can effectively intervene it remain obscured. Here, 3T3‐F442A preadipocytes were cultured, injected into athymic mice to model fat pads, and treated respectively with Ad‐chemerin, Ad‐Gax, or specific inhibitors in vitro and in vivo. MTT, flow cytometry, Western blotting, and imunohisto(cyto)‐chemistry analyses showed that chemerin enhanced the expression of FABP4 and VEGF, activated Akt/mTOR and ERK pathways, increased the cell percent of S phase, decreased the percent of G0‐G1 phase and apoptotic cells, and augmented neovascular density in fat pads. Inversely, Gax suppressed the expression of these adipogenic and vasifactive markers and these signaling proteins, decreased the percent of S phase cells, and increased those of G0‐G1 phase and apoptotic cells, and reduced the neovascular density. Our results indicate that chemerin‐CMKLR1 activates Akt/mTOR and ERK pathways and facilitates preadipocyte proliferation, adipogenesis, and angiogenesis. Contrarily, Gax weakens the effect of chemerin on preadipocyte biofunctions.     

Retroviral transfer of antisense sequences results in reduction of c-Abl and induction of apoptosis in hemopoietic cells

The c-abl proto-oncogene is ubiquitously expressed during mammalian development. Activated forms of c-Abl proteins are oncogenic and have been shown to suppress apoptosis. The biological role of normal c-Abl protein is unknown. In this study, we have introduced c-abl antisense sequences into various hemopoietic cells by retroviral gene transfer. Introduction and expression of the antisense sequence effectively reduced the amount of c-Abl protein in a number of transduced hemopoietic cells, that consequently underwent apoptosis. When factor-dependent cell lines were examined, we observed that the addition of sufficient amounts of growth factors could suppress apoptosis in myeloid but not in lymphoid lines. The ability of myeloid cells to be rescued by growth factors correlated with upregulation of mRNA level of IL-3 receptor subunits. Our data suggest that c-Abl provides an anti-apoptotic signal during mammalian cell growth, and that myeloid and lymphoid cells are different in their resistance to apoptosis.     

Metabolic turnover of myelin glycerophospholipids

The apparent half life for metabolic turnover of glycerophospholipids in the myelin sheath, as determined by measuring the rate of loss of label in a myelin glycerophospholipid following radioactive precursor injection, varies with the radioactive precursor used, age of animal, and time after injection during which metabolic turnover is studied. Experimental strategies for resolving apparent inconsistencies consequent to these variables are discussed. Illustrative data concerning turnover of phosphatidylcholine (PC) in myelin of rat brain are presented. PC of the myelin membrane exhibits heterogeneity with respect to metabolic turnover rates. There are at least two metabolic pools of PC in myelin, one with a half life of the order of days, and another with a half life of the order of weeks. To a significant extent biphasic turnover is due to differential turnover of individual molecular species (which differ in acyl chain composition). The two predominant molecular species of myelin PC turnover at very different rates (16:0, 18:1 PC turning over several times more rapidly than 18:0, 18:1 PC). Therefore, within the same membrane, individual molecular species of a phospholipid class are metabolized at different rates. Possible mechanisms for differential turnover of molecular species are discussed, as are other factors that may contribute to a multiphasic turnover of glycerophospholipids.Special issue dedicated to Dr. Marjorie Lees.