Analysis of the promoters and 5'-UTR of mouse Cd59 genes, and of their functional activity in erythrocytes.

The complement regulatory protein CD59 inhibits formation of the membrane attack complex (MAC), the terminal effector of the complement system. There are two mouse Cd59 genes in mice but only one in humans. In the work reported here we (a) mapped the promoter regions of both mCd59a and mCd59b genes, (b) identified two different promoters for each mCd59 gene, (c) defined a previously unrecognized additional exon 1 in each mCd59 gene, (d) identified that each mCd59 gene expresses two different tissue-specific transcripts that differ in their 5'-UTR, and (e) confirmed the presence of mCd59b mRNA in multiple tissues. At the functional level, comparison of the sensitivity of mCd59ab(-/-) and mCd59a(-/-) red blood cells to MAC-mediated lysis revealed that mCd59b protects RBC from MAC-mediated lysis, at least in the setting of mCd59a deficiency. Together these findings indicate that the mCd59 genes may have complex and perhaps different regulatory mechanisms in different tissues.

Anticancer effects of thiazolidinediones are independent of peroxisome proliferator-activated receptor gamma and mediated by inhibition of translation initiation.

The thiazolidinedione (TZD) class of peroxisome proliferator-activated receptor (PPAR) gamma ligands, known for their ability to induce adipocyte differentiation and increase insulin sensitivity, also exhibits anticancer properties. Currently, TZDs are being tested in clinical trials for treatment of human cancers expressing high levels of PPARgamma because it is assumed that activation of PPARgamma mediates their anticancer activity. Using PPARgamma(-/-) and PPARgamma(+/+) mouse embryonic stem cells, we report here that inhibition of cell proliferation and tumor growth by TZDs is independent of PPARgamma. Our studies demonstrate that these compounds block G(1)-S transition by inhibiting translation initiation. Inhibition of translation initiation is the consequence of partial depletion of intracellular calcium stores and the resulting activation of protein kinase R that phosphorylates the alpha subunit of eukaryotic initiation factor 2 (eIF2), thus rendering eIF2 inactive. PPARgamma-independent inhibition of translation initiation most likely accounts for the anticancer properties of thiazolidinediones.

Genomic structure, functional comparison, and tissue distribution of mouse Cd59a and Cd59b.

CD59 is a crucial complement regulatory protein that inhibits the terminal step of the complement activation cascade by interfering with the binding of C9 to C5b-8, thus preventing the formation of the membrane attack complex (MAC). We recently reported that the mouse genome contains two Cd59 genes, while the human and rat genomes each contain only one Cd59 gene (Qian et al. 2000). Here, we describe the genomic structure, comparative activity, and tissue distribution of these two mouse genes, designated Cd59a and Cd59b. The mouse Cd59 genes encompass a total of 45.6 kb with each gene having four exons. Cd59a spans 19 kb, and Cd59b spans 15 kb, with approximately 11.6 kb of genomic DNA separating the two genes. The overall sequence similarity between Cd59a and Cd59b is approximately 60%. The sequence similarity between exon 2, exon 3, and exon 4 and the respective flanking regions between the two genes is over 85%, but exon 1 and its flanking regions are totally different. Comparative studies of the activity of both genes as inhibitors of MAC formation revealed that Cd59b has a specific activity that is six times higher than that of Cd59a. Using polyclonal antibodies specific to either Cd59a or Cd59b, we showed that Cd59a and Cd59b are both widely expressed in the kidneys, brain, lungs, spleen, and testis, as well as in the blood vessels of most mouse tissues. Interestingly, testicular Cd59a appeared to be expressed exclusively in spermatids, whereas Cd59b was expressed in more mature sperm cells. These results suggest that even though Cd59a and Cd59b are expressed in multiple tissues, they may play some different roles, particularly in reproduction.

Identification and functional characterization of a new gene encoding the mouse terminal complement inhibitor CD59.

CD59 is a 18- to 20-kDa, GPI-anchored membrane protein that functions as a key regulator of the terminal step of the complement activation cascade. It restricts binding of C9 to the C5b-8 complex, thereby preventing the formation of the membrane attack complex (C5b-9 of complement). A single human CD59 gene has been identified, and corresponding genetic homologues from rat, mouse, and pig have been characterized in previous studies. In this study, we report the discovery and functional characterization of a separate cd59 gene in the mouse (referred to as cd59b, the previously characterized mouse cd59 gene as cd59a). Mouse cd59b is 85% and 63% identical to cd59a at the nucleotide and amino acid level, respectively. In cDNA transfection experiments with Chinese hamster ovary cells, peptide-tagged cd59b was detected on the cell surface by flow cytometry and was shown to be susceptible to phosphatidylinositol-specific phospholipase C cleavage. Chinese hamster ovary cells expressing cd59b were significantly more resistant than control cells to human and mouse complement-mediated lysis. These results suggest that cd59b encodes a GPI-anchored protein that is functionally active as a membrane attack complex inhibitor. Northern blot analysis revealed that cd59b is expressed selectively in the mouse testis. In contrast, the major transcript of cd59a was shown to be expressed at high levels in the heart, kidney, liver, and lung, but only minimally in the testis. These results revealed the existence of two distinct cd59 genes in the mouse that are differentially regulated and that may have nonoverlapping physiological functions in vivo.

Inhibition of translation initiation mediates the anticancer effect of the n-3 polyunsaturated fatty acid eicosapentaenoic acid.

Eicosapentaenoic acid (EPA), an n-3 polyunsaturated fatty acid that is abundant in the fish-based diets of populations that exhibit a remarkably low incidence of cancer, exerts anticancer activity in vitro and in animal models of experimental cancer. Here we define the molecular basis for the anticancer effects of EPA. EPA inhibits cell division by inhibiting translation initiation. This is a consequence of the ability of EPA to release Ca2+ from intracellular stores while inhibiting their refilling via capacitative Ca2+ influx that results in partial emptying of intracellular Ca2+ stores and thereby activation of protein kinase R. Protein kinase R phosphorylates and inhibits eukaryotic initiation factor 2alpha, resulting in inhibition of protein synthesis at the level of translation initiation, preferentially reducing the synthesis and expression of growth-regulatory proteins, including G1 cyclins, and causes cell cycle arrest in G1. In a KLN-205 squamous cell carcinoma mouse model, daily oral administration of EPA resulted in a significant reduction of tumor size and expression of cyclin D1 in the tumor tissues. Furthermore, EPA-treated tumors showed a significant increase in the proportion of diploid cells, indicative of cell cycle arrest in G0-G1, and a significant reduction of malignant hypertetraploid cells. These results characterize EPA as a member of an emerging new class of anticancer compounds that inhibit translation initiaton.

Setting health standards for the 21st century.

OBJECTIVES: To describe the nature, substance, and impact of health standards and to speculate on how they may change during the early years of the 21st century. SUMMARY: The health care system has experienced dramatic changes over the past three decades. Health standards now cover the spectrum from statutes to regulations to guidelines, as well as less compulsory forms, and address every aspect of health, including the availability, quality, and cost of services; the safety, efficacy, and quality of products; the adequacy of processes and analytic methods; and the value and cost-effectiveness of health technologies and services. Standards cover promotion, protection, and gatekeeping functions. They are established in response to specific needs and are driven by social and political agendas; social policy will drive standards development to a greater extent in the 21st than in the 20th century. Technology advances are stimulating new forms of standards, primarily gatekeeping standards. Standards raise the question of compliance. The trend is increasingly toward less compulsory, more discretionary standards, with a greater percentage being established by quasi-official and voluntary bodies. CONCLUSION: 21st century health standards must balance the rights of access with protection from unsafe, ineffective, and poor quality products and services during a period when decisions must be made faster. They must be more flexible, enabling consumers to choose their level of protection. Such standards will pose the same concerns about adequacy, equity, and freedom of choice as health standards pose now. Whether we will use 21st century standards to improve and advance our health as well as systems, products, and services that contribute to health more effectively than we have in the 20th century is a question to ponder.

Depletion of intracellular Ca2+ stores, phosphorylation of eIF2alpha, and sustained inhibition of translation initiation mediate the anticancer effects of clotrimazole.

Regulation of translation initiation plays a critical role in the control of cell growth and division in eukaryotic cells. Translation of many growth regulatory proteins including cyclins depends critically on translation initiation factors because their mRNAs are translated inefficiently. We report that clotrimazole, a potent antiproliferative agent both in vitro and in vivo, inhibits cell growth by interfering with translation initiation. In particular, clotrimazole causes a sustained depletion of intracellular Ca2+ stores, which results in activation of PKR, phosphorylation of eIF2alpha, and thereby in inhibition of protein synthesis at the level of translation initiation. Consequently, clotrimazole preferentially decreases the expression of the growth promoting proteins cyclin A, E and D1, resulting in inhibition of cyclin-dependent kinase activity and blockage of cell cycle in G1.

Characterization of neuronal cell death induced by complement activation.

The complement system plays an important role in human immune defense mechanism. Its activation via either the classical or the alternative pathway can lead to the formation of membrane attack complex (MAC) and subsequently kills target cells. Activation of the classical pathway can be initiated with binding of C1q which is first factor of complement cascade to the Fc (fragment crystalline) region of immunoglobulin. This triggers a cascade of proteolytic events resulting in the activation of C5 convertase which cleaves C5 into C5b and C5a. The C5b then binds C6, C7, C8 to form a C5b-8 complex. Binding of C9 molecules to C5b-8 forms C5b-9, the MAC, which pore size increases as the number of C9 in the complex increases. If this membrane lesion persists and results in uncontrolled ion fluxes, the cells swell and eventually lyse. To restrict the activity of the complement system, endogenous complement inhibitors are available to regulate complement-mediated cytolysis. This enables the complement system to distinguish "self" from "foreign" and protect the host from inadvertent complement attack. Activation of the classical complement cascade has been reported in Alzheimer's disease and other neurodegenerative disorders. Recently, we demonstrated that complement activation causes neuronal cell death in vitro, and this neurodegenerative process is regulated by homologous restriction. In this article, we describe the use of two cell lines as in vitro models to evaluate cell injury/cell death induced by complement activation.

The transient pore formed by homologous terminal complement complexes functions as a bidirectional route for the transport of autocrine and paracrine signals across human cell membranes.

BACKGROUND: We have previously shown that the membrane attack complex (MAC) of complement stimulates cell proliferation and that insertion of homologous MAC into the membranes of endothelial cells results in the release of potent mitogens, including basic fibroblast growth factor (bFGF). The mechanism of secretion of bFGF and other polypeptides devoid of signal peptides, such as interleukin 1 (IL-1) is still an open problem in cell biology. We have hypothesized that the homologous MAC pore itself could constitute a transient route for the diffusion of biologically active macromolecules in and out of the target cells. MATERIALS AND METHODS: Human red blood cell ghosts and artificial lipid vesicles were loaded with labeled growth factors, cytokines and IgG, and exposed to homologous MAC. The release of the 125I-macromolecules was followed as a function of time. The incorporation of labeled polypeptides and fluorescent dextran (MW: 10,000) was measured in MAC-impacted human red blood cells and human umbilical endothelial cells (HUVEC), respectively. RESULTS: Homologous MAC insertion into HUVEC resulted in the massive uptake of 10-kD dextran and induced the release of bFGF, in the absence of any measurable lysis. Red blood cell ghosts preloaded with bFGF, IL-1 beta, and the alpha-chain of interferon-gamma (IFN-gamma) released the polypeptides upon MAC insertion, but they did not release preloaded IgG. MAC-impacted ghosts took up radioactive IFN-gamma from the extracellular medium. Vesicles loaded with IL-I released the polypeptide when exposed to MAC. CONCLUSIONS: The homologous MAC pore in its nonlytic form allows for the export of cytosolic proteins devoid of signal peptides that are not secreted through the classical endoplasmic reticulum/Golgi exocytotic pathways. Our results suggest that the release, and perhaps the uptake, of biologically active macromolecules through the homologous MAC pore is a novel biological function of the complement system in mammals.

Clotrimazole inhibits cell proliferation in vitro and in vivo.

Cell proliferation is critically dependent on the regulated movement of ions across various cellular compartments. The antimycotic drug clotrimazole (CLT) has been shown to inhibit movement of Ca2+ and K+ across the plasma membrane. Our results show that CLT inhibits the rate of cell proliferation of normal and cancer cell lines in a reversible and dose-dependent manner in vitro. Moreover, CLT depletes the intracellular Ca2+ stores and prevents the rise in cytosolic Ca2+ that normally follows mitogenic stimulation. In mice with severe combined immunodeficiency disease (SCID) and inoculated intravenously with MM-RU human melanoma cells, daily subcutaneous injections of CLT induced a significant reduction in the number of lung metastases. Modulation of early ionic mitogenic signals and potent inhibition of cell proliferation both in vitro and in vivo are new and potentially useful clinical effects of CLT.

Complement-mediated neurotoxicity is regulated by homologous restriction.

The ability of beta-amyloid peptides to activate the classical complement cascade and the presence of various complement proteins including the membrane attack complex (C5b-9) on dystrophic neurites in Alzheimer's disease brains, raises the possibility that the complement system may contribute to this neurodegenerative disorder. To address this issue, we have studied the effect of complement activation on nerve growth factor (NGF)-differentiated rat pheochromocytoma PC12 cells, and on retinoic acid (RA)-differentiated human neuroblastoma SH-SY5Y cells. Although incubation of both cell types with human serum resulted in activation of complement, as indicated by iC3b formation, only PC12 but not SH-SY5Y cells were killed by human serum treatment. In contrast, heat-inactivated serum (56 degrees C, 45 min) was not neurotoxic. On SH-SY5Y cells, both PCR amplification and immunocytochemistry demonstrated the presence of CD59, a glycosylphosphatidylinositol-anchored protein that restricts homologous complement activation by inhibiting the formation of the membrane attack complex. The presence of CD59 probably accounts for the inability of human complement to lyse the human cell lines. Indeed, removal of glycosylphosphatidylinositol (GPI)-anchored proteins with phosphatidylinositol-specific phospholipase C (PI-PLC) rendered SH-SY5Y cells vulnerable to complement attack and eventually led to serum-medicated cell death. Reconstituted C5b-9 was also toxic to both PC12 and PI-PLC-pretreated SH-SY5Y cells. These observations suggest that complement activation can cause neuronal cell death and that this process is regulated by homologous restriction.

Terminal complement proteins C5b-9 release basic fibroblast growth factor and platelet-derived growth factor from endothelial cells.

Interactions between endothelium and vascular smooth muscle cells play a major role in the biology of the blood vessel wall. Growth factors released from endothelial cells control in part the normal and pathological proliferation of vascular smooth muscle cells. Endothelial deposits of C5b-9 proteins, the membrane attack complex of complement (MAC), have been found in a variety of pathological tissues in which cell proliferation is an early characteristic abnormality, including atherosclerosis. We have explored a possible bridging role for terminal complement C5b-9 proteins in eliciting focal signals for cell proliferation by releasing growth factors from endothelial cells. We found that both bovine aortic and human umbilical vein cells respond to the MAC by releasing basic fibroblast growth factor and platelet-derived growth factor. These mitogens stimulate DNA synthesis in Swiss 3T3, vascular smooth muscle, and glomerular mesangial cells. Based on these findings, we propose that complement-induced release of mitogens from endothelial cells is a novel pathogenic mechanism for proliferative disorders.

Activated complement directly modifies the performance of isolated heart muscle cells from guinea pig and rat.

The complement system has been implicated in the pathogenesis of cardiovascular disorders including ischemia and atherosclerosis. Selective deposition of C5b-9, the membrane attack complex of complement, has been histochemically documented in human myocardium early after reperfusion of ischemic areas and in infarcted zones. However, functional sequelae of the C5b-9 complex binding to myocardial cells have not been identified. Insertion of C5b-9 complexes into the membrane of other cell types can generate transient changes in membrane permeability in the absence of cell lysis. We demonstrate in beating isolated adult guinea pig and rat cardiac myocytes that human derived C5b-9 can transiently augment in a dose-dependent manner both basal cytosolic calcium concentration and calcium transients, resulting in a temporary increase in contractility. If similar changes occur in human heart cells in vivo, they could significantly affect myocardial performance and contribute to functional abnormalities seen in ischemia and other pathological conditions associated with complement activation.

Terminal complement complex C5b-9 stimulates mitogenesis in 3T3 cells.

The membrane attack complex of complement (MAC) can induce reversible changes in cell membrane permeability resulting in significant but transient intracellular ionic changes in the absence of cell lysis. Because ion fluxes and cytosolic ionic changes are integral steps in the signaling cascade initiated when growth factors bind to their receptors, we hypothesized that the MAC-induced reversible changes in membrane permeability could stimulate cell proliferation. Using purified terminal complement components we have documented a mitogenic effect of the MAC for quiescent murine 3T3 cells. The MAC enhances the mitogenic effects of serum and PDGF, and also stimulates cell proliferation in the absence of other exogenous growth factors. MAC-induced mitogenesis represents a novel effect of the terminal complement complex that could contribute to focal tissue repair or pathological cell proliferation locally at sites of complement activation.

Membrane signaling by complement C5b-9, the membrane attack complex.

The terminal complement complexes C5b-7, C5b-8 and C5b-9 are able to generate nonlethal cell signals. One universal consequence of a cell being targeted by C5b-8 or C5b-9 is an influx of Ca2+. In addition, other second messengers, including cAMP, inositol phosphate intermediates and arachidonate metabolites, are generated by the terminal complement complexes in specific cell types. In vivo, terminal complement complexes have been found in a wide variety of inflammatory processes in humans and in experimental animal models. Some of these models of inflammation putatively induced by terminal complement complexes have been tested in complement-deficient animals, and indeed no inflammation results, which supports the critical role of the terminal complement complexes in the pathogenesis of the lesion.

Transient changes in erythrocyte membrane permeability are induced by sublytic amounts of the complement membrane attack complex (C5b-9).

We have previously shown that sublytic heterologous complement induces large but transient increases in erythrocyte membrane permeability. We now report that when erythrocytes are bystanders in zymosan-activated autologous serum, they increase their Na+ permeability 10-fold, indicating that autologous complement can also induce transient membrane lesions. When we isolated the effect of the C5b-9 membrane attack complex of complement by using human C5b-9 assembled from purified components, we found there was minimal lysis but efficient Na+ uptake. Suspension of the sublytically damaged erythrocytes in K+ medium caused the cells to lyse, which is consistent with the cells recruiting a compensatory K+ efflux similar to that observed when human erythrocytes were exposed to heterologous complement. Sublytic C5b-9 exposure also became lytic when extracellular Ca2+ was limited and when the cells were exposed to charybdotoxin, an inhibitor of the Ca(2+)-activated K+ channel. This indicates that Ca2+ is required for the functional termination of the C5b-9 lesion. We also show that the membrane hyperpolarization resulting from activation of the Ca(2+)-dependent K+ efflux does not influence the termination of the C5b-9 lesion. Thus, the influx of Ca2+ through the complement lesion initiates at least two apparently independent adaptive responses: (1) a process that terminates the leak; and (2) a K+ efflux that has a volume regulatory function. Our data support the potential of the sublytic C5b-9 lesion to act as a physiologic mediator for autologous erythrocytes.