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Exposure of phagocytic cells to bacterial endotoxin (lipopolysaccharide; LPS) or inflammatory cytokines confers antiapoptotic survival signals; however, in the absence of the appropriate stimulus, monocytes are programmed to undergo apoptosis. Macrophage survival may thus influence inflammatory and immune responses and susceptibility to microbial pathogens. Herein, we demonstrate that LPS and the proinflammatory cytokine, tumor necrosis factor-alpha (TNF-alpha), enhance monocytic cell survival through the induction of the antiapoptotic c-IAP2 gene in a human promonocytic THP-1 cell line. We also investigated the role of upstream signaling molecules including the mitogen-activated protein kinases, phosphatidylinositol 3-kinase, and the calcium signaling pathways in the regulation of c-IAP2 expression and eventual survival of monocytic cells. Our results suggest that LPS and TNF-alpha-induced c-IAP2 expression was regulated by calmodulin (CaM) through the activation of calmodulin-dependent protein kinase-II (CaMKII). In addition, CaM and CaMKII regulated c-IAP2 expression in LPSand TNF-alpha-stimulated cells through NF-kappaB activation. Moreover, the CaM/CaMKII pathway also regulated LPS- and TNF-alpha-mediated inhibition of apoptosis in these cells. Taken together, these results suggest that LPS- and TNF-alpha-induced c-IAP2 expression and its associated antiapoptotic survival signals in THP-1 cells are regulated selectively by CaM/CaMKII through NF-kappaB activation.

Stable expression of short-hairpin RNAs (shRNAs) directed against the X-linked inhibitor of apoptosis (XIAP) resulted in the generation of three MDA-MB-231 cell lines (XIAP shRNA cells) with reductions in XIAP mRNA and protein levels > 85% relative to MDA-MB-231 cells stably transfected with the U6 RNA polymerase III promoter alone (U6 cells). This RNA interference (RNAi) approach dramatically sensitized these cells to killing by the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Importantly, loss of XIAP also sensitized the cells to killing by taxanes but had no additional effects on killing by carboplatin and doxorubicin. The increased sensitivity of the XIAP shRNA cells to killing by TRAIL and taxanes correlated with enhanced caspase cleavage and activation, including caspase-8, and robust processing of poly(ADP-ribose) polymerase and BID compared to U6 cells. Additionally, increasing XIAP levels by adenovirus-mediated expression protected both XIAP shRNA and U6 cells from TRAIL killing in a dose-dependent manner. The effects observed by stable RNAi with respect to TRAIL sensitization were also achieved following downregulation of XIAP in Panc-1 cells treated with a second-generation, mixed-backbone antisense oligonucleotide, AEG 35156/GEM640. These data indicate that reducing XIAP protein expression by either RNAi or antisense approaches increases cancer cell susceptibility to functionally diverse chemotherapeutic agents and supports the notion that downregulation of XIAP in vivo may synergize with disease-relevant chemotherapeutic regimes, including TRAIL and taxanes, to increase the effectiveness of antineoplastic agents.

The pathways of hepatic intra- and peri-cellular lipidation of apolipoprotein A-I (apoA-I) were studied by infecting primary mouse hepatocytes from either apoA-I-deficient or ABCA1-deficient mice with a recombinant adenovirus expressing the human apoA-I (hapoA-I) cDNA (endo apoA-I) or incubating the hepatocytes with exogenously added hapoA-I (exo apoA-I) and examining the hapoA-I-containing lipoproteins formed. The cells, maintained in serum-free medium, were labeled with [(3)H]choline, and the cell medium was separated by fast protein liquid chromatography or immunoprecipitated to quantify labeled choline phospholipids specifically associated with hapoA-I. With the apoA-I-deficient hepatocytes, the high density lipoprotein fraction formed with endo apoA-I contained proportionally more phospholipids than that formed with exo apoA-I. However, the lipoprotein size and electrophoretic mobility and phospholipid profiles were similar for exo apoA-I and endo apoA-I. Taken together, these data demonstrate that a significant proportion of hapoA-I is secreted from hepatocytes in a phospholipidated state but that hapoA-I is also phospholipidated peri-cellularly. With primary hepatocytes from ABCA1-deficient mice, the expression and net secretion of adenoviral-generated endogenous apoA-I was unchanged compared with control mice, but (3)H-phospholipids associated with endo apoA-I and exo apoA-I decreased by 63 and 25%, respectively. The lipoprotein size and electrophoretic migration and their phospholipid profiles remained unchanged. In conclusion, we demonstrated that intracellular and peri-cellular lipidation of apoA-I represent distinct and additive pathways that may be regulated independently. Hepatocyte expression of ABCA1 is central to the lipidation of newly synthesized apoA-I but also contributes to the lipidation of exogenous apoA-I. However, a significant basal level of phospholipidation occurs in the absence of ABCA1.

To investigate the role of the N terminus of apolipoprotein A-I (apoA-I) in the maturation of high density lipoproteins (HDL), two N-terminal mutants with deletions of residues 1-43 and 1-65 (referred to as Delta 1-43 and Delta 1-65 apoA-I) were studied. In vitro, these deletions had little effect on cellular cholesterol efflux from macrophages but LCAT activation was reduced by 50 and 70% for the Delta 1-43 and Delta 1-65 apoA-I mutants, respectively, relative to wild-type (Wt) apoA-I. To further define the role of the N terminus of apoA-I in HDL maturation, we constructed recombinant adenoviruses containing Wt apoA-I and two similar mutants with deletions of residues 7-43 and 7-65 (referred to as Delta 7-43 and Delta 7-65 apoA-I, respectively). Residues 1-6 were not removed in these mutants to allow proper cleavage of the pro-sequence in vivo. Following injection of these adenoviruses into apoA-I-deficient mice, plasma concentrations of both Delta 7-43 and Delta 7-65 apoA-I were reduced 4-fold relative to Wt apoA-I. The N-terminal deletion mutants, in particular Delta 7-65 apoA-I, were associated with greater proportions of pre beta-HDL and accumulated fewer HDL cholesteryl esters relative to Wt apoA-I. Wt and Delta 7-43 apoA-I formed predominantly alpha-migrating and spherical HDL, whereas Delta 7-65 apoA-I formed only pre beta-HDL of discoidal morphology. This demonstrates that deletion of the first class A amphipathic alpha-helix has a profound additive effect in vivo over the deletion of the globular domain alone (amino acids 1-43) indicating its important role in the production of mature alpha-migrating HDL. In summary, the combined in vitro and in vivo studies demonstrate a role for the N terminus of apoA-I in lecithin:cholesterol acyltransferase activation and the requirement of the first class A amphipathic alpha-helix for the maturation of HDL in vivo.

We have devised a combined in vivo, ex vivo, and in vitro approach to elucidate the mechanism(s) responsible for the hypoalphalipoproteinemia in heterozygous carriers of a naturally occurring apolipoprotein A-I (apoA-I) variant (Leu(159) to Arg) known as apoA-I Finland (apoA-I(FIN)). Adenovirus-mediated expression of apoA-I(FIN) decreased apoA-I and high density lipoprotein cholesterol concentrations in both wild-type C57BL/6J mice and in apoA-I-deficient mice expressing native human apoA-I (hapoA-I). Interestingly, apoA-I(FIN) was degraded in the plasma, and the extent of proteolysis correlated with the most significant reductions in murine apoA-I concentrations. ApoA-I(FIN) had impaired activation of lecithin:cholesterol acyltransferase in vitro compared with hapoA-I, but in a mixed lipoprotein preparation consisting of both hapoA-I and apoA-I(FIN) there was only a moderate reduction in the activation of this enzyme. Importantly, secretion of apoA-I was also decreased from primary apoA-I-deficient hepatocytes when hapoA-I was co-expressed with apoA-I(FIN) following infection with recombinant adenoviruses, a condition that mimics secretion in heterozygotes. Thus, this is the first demonstration of an apoA-I point mutation that decreases LCAT activation, impairs hepatocyte secretion of apoA-I, and makes apoA-I susceptible to proteolysis leading to dominantly inherited hypoalphalipoproteinemia.

Cortical human brain tissue was obtained from 11 craniotomies for intractable epilepsy or tumor resection. Neuregen transport medium preserved viability at 4 degrees C during transfer to the culture laboratory. Cells were isolated and cultured by methods previously developed for adult rat neurons (Brewer GJ. Isolation and culture of adult rat hippocampal neurons. J. Neurosci. Meth. 1997:71:143-55). In about 40% of the cases, cultures regenerated with a majority of neuron-like cells that stained for neurofilament and not GFAP. After 3 weeks of culture from a 70 year old meningioma case, synapse-like structures were revealed by electron microscopy. Trophic support from basic human recombinant fibroblast growth factor was synergistically improved with the steroid hormone dehydroepiandrosterone 3-sulfate. Another 40% of the cases resulted in cultures that were predominantly GFAP positive astroglia. The remaining 20% of the cases did not regenerate cells with neuron-like or glial processes. Three postmortem cases did not regenerate neurites. These methods may aid development of human culture models of epilepsy as well as human pharmacology, toxicology and development of improved methods for brain grafts.

Recombinant adenoviruses with cDNAs for human apolipoprotein A-I (wild type (wt) apoA-I) and three mutants, referred to as Delta4-5A-I, Delta5-6A-I, and Delta6-7A-I, that have deletions removing regions coding for amino acids 100-143, 122-165, and 144-186, respectively, were created to study structure/function relationships of apoA-I in vivo. All mutants were expressed at lower concentrations than wt apoA-I in plasma of fasting apoA-I-deficient mice. The Delta5-6A-I mutant was found primarily in the lipid-poor high density lipoprotein (HDL) pool and at lower concentrations than Delta4-5A-I and Delta6-7A-I that formed more buoyant HDL(2/3) particles. At an elevated adenovirus dose and earlier blood sampling from fed mice, both Delta5-6A-I and Delta6-7A-I increased HDL-free cholesterol and phospholipid but not cholesteryl ester. In contrast, wt apoA-I and Delta4-5A-I produced significant increases in HDL cholesteryl ester. Further analysis showed that Delta6-7A-I and native apoA-I could bind similar amounts of phospholipid and cholesterol that were reduced slightly for Delta5-6A-I and greatly for Delta4-5A-I. We conclude from these findings that amino acids (aa) 100-143, specifically helix 4 (aa 100-121), contributes to the maturation of HDL through a role in lipid binding and that the downstream sequence (aa 144-186) centered around helix 6 (aa 144-165) is responsible for the activation of lecithin-cholesterol acyltransferase.

The contribution of the amphipathic alpha-helices of apoA-I toward lipid efflux from human skin fibroblasts and macrophage was examined. Four apoA-I mutants were designed, each by deletion of a pair of predicted adjacent helices. Three mutants lacked two consecutive central alpha-helices [Delta(100-143), Delta(122-165), and Delta(144-186)], whereas the final mutant lacked the C-terminal domain [Delta(187-243)]. When compared to recombinant wild-type apoA-I and mutants with central domain deletions, Delta(187-243) exhibited a marked reduction in its ability to promote either cholesterol or phospholipid efflux from THP-1 macrophages. This mutant also demonstrated a decreased ability to bind lipids and to form lipoprotein complexes. In contrast, the four mutants and apoA-I equally supported cholesterol efflux from fibroblasts, albeit with a reduced capacity when compared to macrophages. Delta(187-243) bound poorly to the macrophage cell surface when compared to apoA-I, and competitive binding studies with the central domain and C-terminal deletions mutants showed that only Delta(187-243) did not compete effectively with [(125)I]apoA-I. Omission of PMA during cholesterol loading enhanced cholesterol efflux to both apoA-I (1.5-fold) and the C-terminal deletion mutant (2.5-fold). Inclusion of the Sandoz ACAT inhibitor (58-035) during loading and, in the absence of PMA, increased and equalized cholesterol efflux to apoA-I and Delta(187-243). Surprisingly, omission of PMA during cholesterol loading had minimal effects on the binding of apoA-I or Delta(187-243) to the THP-1 cell surface. Overall, these results show that cholesterol efflux from cells such as fibroblasts does not require any specific sequence between residues 100 and 243 of apoA-I. In contrast, optimal cholesterol efflux in macrophages requires binding of the C-terminal domain of apoA-I to a cell surface-binding site and the subsequent translocation of intracellular cholesterol to an efflux-competent pool.

To assess the role of superoxide dismutase in protecting Escherichia coli from killing by human serum and neutrophils, we constructed isogenic, smooth-lipopolysaccharide K-12 strains, either sod wild-type, delta sodA, or delta sodA delta sodB. The delta sodA delta sodB strain was killed by serum much more readily than either the wild-type or delta sodA strain. After allowing for this serum sensitivity difference, the delta sodA delta sodB strain also showed increased susceptibility to phagocytic killing by human neutrophils. These results indicate that superoxide dismutase protects E. coli from killing by serum (complement system) and by human neutrophils, possibly by a role in maintaining bacterial membrane structure.

Redox-cycling agents catalyze the flow of reducing equivalents to oxygen; this process generates superoxide ion and other reduced oxygen species. Measurements of redox-cycling activity have been performed previously by studying cyanide-resistant oxygen consumption (respiration) of Escherichia coli cells. E. coli strain GK100, lacking both terminal oxidases, has almost no measurable respiration. We show that the use of this strain eliminates the requirement for cyanide in measurements of redox-cycling activity. The addition of either menadione sodium bisulfite or plumbagin, well-known redox-cycling agents, to GK100 cells resulted in high levels of oxygen consumption. The rate of menadione bisulfite-induced oxygen consumption in this respiration-deficient strain, in the absence of cyanide, was comparable to the cyanide-resistant respiration of isogenic respiration-proficient E. coli strains. In GK100 cells, cyanide increased menadione bisulfite-induced oxygen consumption but had no effect on plumbagin-induced oxygen consumption.