Characterization of inhibitory and stimulatory forms of the murine natural killer cell receptor 2B4

The receptor 2B4 belongs to the Ig superfamily and is found on the surface of all murine natural killer (NK) cells as well as T cells displaying non-MHC-restricted cytotoxicity. Previous studies have suggested that 2B4 is an activating molecule because cross-linking of this receptor results in increased cytotoxicity and γ-interferon secretion as well as granule exocytosis. However, it was recently shown that the gene for 2B4 encodes two different products that arise by alternative splicing. These gene products differ solely in their cytoplasmic domains. One form has a cytoplasmic tail of 150 amino acids (2B4L) and the other has a tail of 93 amino acids (2B4S). To determine the function of each receptor, cDNAs for 2B4S and 2B4L were transfected into the rat NK cell line RNK-16. Interestingly, the two forms of 2B4 had opposing functions. 2B4S was able to mediate redirected lysis of P815 tumor targets, suggesting that this form represents an activating receptor. However, 2B4L expression led to an inhibition of redirected lysis of P815 targets when the mAb 3.2.3 (specific for rat NKRP1) was used. In addition, 2B4L constitutively inhibits lysis of YAC-1 tumor targets. 2B4L is a tyrosine phosphoprotein, and removal of domains containing these residues abrogates its inhibitory function. Like other inhibitory receptors, 2B4L associates with the tyrosine phosphatase SHP-2. Thus, 2B4L is an inhibitory receptor belonging to the Ig superfamily.

Human PEX1 cloned by functional complementation on a CHO cell mutant is responsible for peroxisome-deficient Zellweger syndrome of complementation group I

The peroxisome biogenesis disorders (PBDs), including Zellweger syndrome (ZS) and neonatal adrenoleukodystrophy (NALD), are autosomal recessive diseases caused by defects in peroxisome assembly, for which at least 10 complementation groups have been reported. We have isolated a human PEX1 cDNA (HsPEX1) by functional complementation of peroxisome deficiency of a mutant Chinese hamster ovary (CHO) cell line, ZP107, transformed with peroxisome targeting signal type 1-tagged “enhanced” green fluorescent protein. This cDNA encodes a hydrophilic protein (Pex1p) comprising 1,283 amino acids, with high homology to the AAA-type ATPase family. A stable transformant of ZP107 with HsPEX1 was morphologically and biochemically restored for peroxisome biogenesis. HsPEX1 expression restored peroxisomal protein import in fibroblasts from three patients with ZS and NALD of complementation group I (CG-I), which is the highest-incidence PBD. A CG-I ZS patient (PBDE-04) possessed compound heterozygous, inactivating mutations: a missense point mutation resulting in Leu-664 → Pro and a deletion of the sequence from Gly-634 to His-690 presumably caused by missplicing (splice site mutation). Both PBDE-04 PEX1 cDNAs were defective in peroxisome-restoring activity when expressed in the patient fibroblasts as well as in ZP107 cells. These results demonstrate that PEX1 is the causative gene for CG-I peroxisomal disorders.

Inhibition of hypoxia-inducible factor 1 activity by nitric oxide donors in hypoxia

Nitric oxide (NO) is known to have various biologic and pathophysiologic effects on organisms. The molecular mechanisms by which NO exerts harmful effects are unknown, although various O2 radicals and ions that result from reactivity of NO are presumed to be involved. Here we report that adaptive cellular response controlled by the transcription factor hypoxia-inducible factor 1 (HIF-1) in hypoxia is suppressed by NO. Induction of erythropoietin and glycolytic aldolase A mRNAs in hypoxically cultured Hep3B cells, a human hepatoma cell line, was completely and partially inhibited, respectively, by the addition of sodium nitroprusside (SNP), which spontaneously releases NO. A reporter plasmid carrying four hypoxia-response element sequences connected to the luciferase structural gene was constructed and transfected into Hep3B cells. Inducibly expressed luciferase activity in hypoxia was inhibited by the addition of SNP and two other structurally different NO donors, S-nitroso-l-glutathione and 3-morpholinosydnonimine, giving IC50 values of 7.8, 211, and 490 μM, respectively. Inhibition by SNP was also observed in Neuro 2A and HeLa cells, indicating that the inhibition was not cell-type-specific. The vascular endothelial growth factor promoter activity that is controlled by HIF-1 was also inhibited by SNP (IC50 = 6.6 μM). Induction generated by the addition of cobalt ion (this treatment mimics hypoxia) was also inhibited by SNP (IC50 = 2.5 μM). Increased luciferase activity expressed by cotransfection of effector plasmids for HIF-1α or HIF-1α-like factor in hypoxia was also inhibited by the NO donor. We also showed that the inhibition was performed by blocking an activation step of HIF-1α to a DNA-binding form.

Role of p38 mitogen-activated protein kinase in HIV type 1 production in vitro

The proinflammatory cytokines interleukin (IL)-1 and tumor necrosis factor (TNF) promote HIV type 1 viral replication in vitro. In the present studies, HIV production was increased in the macrophagic U1 cell line expressing the HIV genome after exposure to IL-1β, osmotic stress, or surface adhesion, suggesting a confluence of signaling pathways for proinflammatory cytokines and cell stressors. The p38 mitogen-activated protein kinase (MAPK) mediates both cytokine and stress responses; thus the role of this kinase in HIV production was investigated. HIV production as measured by p24 antigen correlated with changes in the expression of a specific (non-alpha) isoform of p38 MAPK. In the presence of a specific p38 MAPK inhibitor (p38 inh), IL-1β-induced HIV production was suppressed by more than 90% and IL-1β-induced IL-8 production was suppressed completely, both with IC50 of 0.01 μM. p38 inhibition blocked cell-associated p24 antigen and secreted virus to a similar extent. The p38 inh also decreased constitutive HIV production in freshly infected peripheral blood mononuclear cells by up to 50% (P < 0.05). Interruption of p38 MAPK activity represents a viable target for inhibition of HIV.

Cloning and characterization of human inducible nitric oxide synthase splice variants: A domain, encoded by exons 8 and 9, is critical for dimerization

The inducible nitric oxide synthase (iNOS) contains an amino-terminal oxygenase domain, a carboxy-terminal reductase domain, and an intervening calmodulin-binding region. For the synthesis of nitric oxide (NO), iNOS is active as a homodimer. The human iNOS mRNA is subject to alternative splicing, including deletion of exons 8 and 9 that encode amino acids 242–335 of the oxygenase domain. In this study, iNOS8−9− and full-length iNOS (iNOSFL) were cloned from bronchial epithelial cells. Expression of iNOS8−9− in 293 cell line resulted in generation of iNOS8−9− mRNA and protein but did not lead to NO production. In contrast to iNOSFL, iNOS8−9− did not form dimers. Similar to iNOSFL, iNOS8−9− exhibited NADPH-diaphorase activity and contained tightly bound calmodulin, indicating that the reductase and calmodulin-binding domains were functional. To identify sequences in exons 8 and 9 that are critical for dimerization, iNOSFL was used to construct 12 mutants, each with deletion of eight residues in the region encoded by exons 8 and 9. In addition, two “control” iNOS deletion mutants were synthesized, lacking either residues 45–52 of the oxygenase domain or residues 1131–1138 of the reductase domain. Whereas both control deletion mutants generated NO and formed dimers, none of the 12 other mutants formed dimers or generated NO. The region encoded by exons 8 and 9 is critical for iNOS dimer formation and NO production but not for reductase activity. This region could be a potential target for therapeutic interventions aimed at inhibiting iNOS dimerization and hence NO synthesis.

Cyclic AMP regulates potassium channel expression in C6 glioma by destabilizing Kv1.1 mRNA

The tissue distributions and physiological properties of a variety of cloned voltage-gated potassium channel genes have been characterized extensively, yet relatively little is known about the mechanisms controlling expression of these genes. Here, we report studies on the regulation of Kv1.1 expressed endogenously in the C6 glioma cell line. We demonstrate that elevation of intracellular cAMP leads to the accelerated degradation of Kv1.1 RNA. The cAMP-induced decrease in Kv1.1 RNA is followed by a decrease in Kv1.1 protein and a decrease in the whole cell sustained K+ current amplitude. Dendrotoxin-I, a relatively specific blocker of Kv1.1, blocks 96% of the sustained K+ current in glioma cells, causing a shift in the resting membrane potential from −40 mV to −7 mV. These data suggest that expression of Kv1.1 contributes to setting the resting membrane potential in undifferentiated glioma cells. We therefore suggest that receptor-mediated elevation of cAMP reduces outward K+ current density by acting at the translational level to destabilize Kv1.1 RNA, an additional mechanism for regulating potassium channel gene expression.

Antibodies to several conformation-dependent epitopes of gp120/gp41 inhibit CCR-5-dependent cell-to-cell fusion mediated by the native envelope glycoprotein of a primary macrophage-tropic HIV-1 isolate

The β-chemokine receptor CCR-5 is essential for the efficient entry of primary macrophage-tropic HIV-1 isolates into CD4+ target cells. To study CCR-5-dependent cell-to-cell fusion, we have developed an assay system based on the infection of CD4+ CCR-5+ HeLa cells with a Semliki Forest virus recombinant expressing the gp120/gp41 envelope (Env) from a primary clade B HIV-1 isolate (BX08), or from a laboratory T cell line-adapted strain (LAI). In this system, gp120/gp41 of the “nonsyncytium-inducing,” primary, macrophage-tropic HIV-1BX08 isolate, was at least as fusogenic as that of the “syncytium-inducing” HIV-1LAI strain. BX08 Env-mediated fusion was inhibited by the β-chemokines RANTES (regulated upon activation, normal T cell expressed and secreted) and macrophage inflammatory proteins 1β (MIP-1β) and by antibodies to CD4, whereas LAI Env-mediated fusion was insensitive to these β-chemokines. In contrast soluble CD4 significantly reduced LAI, but not BX08 Env-mediated fusion, suggesting that the primary isolate Env glycoprotein has a reduced affinity for CD4. The domains in gp120/gp41 involved in the interaction with the CD4 and CCR-5 molecules were probed using monoclonal antibodies. For the antibodies tested here, the greatest inhibition of fusion was observed with those directed to conformation-dependent, rather than linear epitopes. Efficient inhibition of fusion was not restricted to epitopes in any one domain of gp120/gp41. The assay was sufficiently sensitive to distinguish between antibody- and β-chemokine-mediated fusion inhibition using serum samples from patient BX08, suggesting that the system may be useful for screening human sera for the presence of biologically significant antibodies.

The CXC chemokine stromal cell-derived factor 1 is not responsible for CD8+ T cell suppression of syncytia-inducing strains of HIV-1

Primary CD8+ T cells from HIV+ asymptomatics can suppress virus production from CD4+ T cells acutely infected with either non-syncytia-inducing (NSI) or syncytia-inducing (SI) HIV-1 isolates. NSI strains of HIV-1 predominantly use the CCR5 chemokine receptor as a fusion cofactor, whereas fusion of T cell line-adapted SI isolates is mediated by another chemokine receptor, CXCR4. The CCR5 ligands RANTES (regulated on activation, normal T cell expressed and secreted), macrophage inflammatory protein 1α (MIP-1α), and MIP-1β are HIV-1 suppressive factors secreted by CD8+ cells that inhibit NSI viruses. Recently, the CXC chemokine stromal cell-derived factor 1 (SDF-1) was identified as a ligand for CXCR4 and shown to inhibit SI strains. We speculated that SDF-1 might be an effector molecule for CD8+ suppression of SI isolates and assessed several SDF-1 preparations for inhibition of HIV-1LAI-mediated cell–cell fusion, and examined levels of SDF-1 transcripts in CD8+ T cells. SDF-1 fusion inhibitory activity correlated with the N terminus, and the α and β forms of SDF-1 exhibited equivalent fusion blocking activity. SDF-1 preparations having the N terminus described by Bleul et al. (Bleul, C.C., Fuhlbrigge, R.C., Casasnovas, J.M., Aiuti, A. & Springer, T.A. (1996) J. Exp. Med. 184, 1101–1109) readily blocked HIV-1LAI-mediated fusion, whereas forms containing two or three additional N-terminal amino acids lacked this activity despite their ability to bind and/or signal through CXCR4. Though SDF-1 is constitutively expressed in most tissues, CD8 T cells contained extremely low levels of SDF-1 mRNA transcripts (<1 transcript/5,000 cells), and these levels did not correlate with virus suppressive activity. We conclude that suppression of SI strains of HIV-1 by CD8+ T cells is unlikely to involve SDF-1.

Yersinia signals macrophages to undergo apoptosis and YopJ is necessary for this cell death

Pathogenic Yersinia spp. carry a large common plasmid that encodes a number of essential virulence determinants. Included in these factors are the Yersinia-secreted proteins called Yops. We analyzed the consequences of wild-type and mutant strains of Yersinia pseudotuberculosis interactions with the macrophage cell line RAW264.7 and murine bone marrow-derived macrophages. Wild-type Y. pseudotuberculosis kills ≈70% of infected RAW264.7 macrophages and marrow-derived macrophages after an 8-h infection. We show that the cell death mediated by Y. pseudotuberculosis is apoptosis. Mutant Y. pseudotuberculosis that do not make any Yop proteins no longer cause host cell death. Attachment to host cells via invasin or YadA is necessary for the cell death phenotype. Several Yop mutant strains that fail to express one or more Yop proteins were engineered and then characterized for their ability to cause host cell death. A mutant with a polar insertion in YpkA Ser/Thr kinase that does not express YpkA or YopJ is no longer able to cause apoptosis. In contrast, a mutant no longer making YopE or YopH (a tyrosine phosphatase) induces apoptosis in macrophages similar to wild type. When yopJ is added in trans to the ypkAyopJ mutant, the ability of this strain to signal programmed cell death in macrophages is restored. Thus, YopJ is necessary for inducing apoptosis. The ability of Y. pseudotuberculosis to promote apoptosis of macrophages in cell culture suggests that this process is important for the establishment of infection in the host and for evasion of the host immune response.

The role of polyamine catabolism in polyamine analogue-induced programmed cell death

N1-ethyl-N11-[(cyclopropyl)methyl]-4,8,-diazaundecane (CPENSpm) is a polyamine analogue that represents a new class of antitumor agents that demonstrate phenotype-specific cytotoxic activity. However, the precise mechanism of its selective cytotoxic activity is not known. CPENSpm treatment results in the superinduction of the polyamine catabolic enzyme spermidine/spermine N1-acetyltransferase (SSAT) in sensitive cell types and has been demonstrated to induce programmed cell death (PCD). The catalysis of polyamines by the SSAT/polyamine oxidase (PAO) pathway produces H2O2 as one product, suggesting that PCD produced by CPENSpm may be, in part, due to oxidative stress as a result of H2O2 production. In the sensitive human nonsmall cell line H157, the coaddition of catalase significantly reduces high molecular weight (HMW) DNA (≥50 kb) and nuclear fragmentation. Important to note, specific inhibition of PAO by N,N′-bis(2,3-butadienyl)-1,4-butane-diamine results in a significant reduction of the formation of HMW DNA and nuclear fragmentation. In contrast, the coaddition of catalase or PAO inhibitor has no effect on reducing HMW DNA fragmentation induced by N1-ethyl-N11-[(cycloheptyl)methyl]-4,8,-diazaundecane, which does not induce SSAT and does not deplete intracellular polyamines. These results strongly suggest that H2O2 production by PAO has a role in CPENSpm cytotoxicity in sensitive cells via PCD and demonstrate a potential basis for differential sensitivity to this promising new class of antineoplastic agents. Furthermore, the data suggest a general mechanism by which, under certain stimuli, cells can commit suicide through catabolism of the ubiquitous intracellular polyamines.

Human deoxycytidine kinase is located in the cell nucleus

Human deoxyribonucleoside kinases are required for the pharmacological activity of several clinically important anticancer and antiviral nucleoside analogs. Human deoxycytidine kinase and thymidine kinase 1 are described as cytosolic enzymes in the literature, whereas human deoxyguanosine kinase and thymidine kinase 2 are believed to be located in the mitochondria. We expressed the four human deoxyribonucleoside kinases as fusion proteins with the green fluorescent protein to study their intracellular locations in vivo. Our data showed that the human deoxycytidine kinase is located in the cell nucleus and the human deoxyguanosine kinase is located in the mitochondria. The fusion proteins between green fluorescent protein and thymidine kinases 1 and 2 were both predominantly located in the cytosol. Site-directed mutagenesis of a putative nuclear targeting signal, identified in the primary structure of deoxycytidine kinase, completely abolished nuclear import of the protein. Reconstitution of a deoxycytidine kinase-deficient cell line with the wild-type nuclear or the mutant cytosolic enzymes both restored sensitivity toward anticancer nucleoside analogs. This paper reports that a deoxyribonucleoside kinase is located in the cell nucleus and we discuss the implications for deoxyribonucleotide synthesis and phosphorylation of nucleoside analogs.

Transcriptional regulation of the rat Müllerian inhibiting substance type II receptor in rodent Leydig cells

Müllerian inhibiting substance (MIS) causes regression of the fetal Müllerian duct on binding a heteromeric complex of types I and II cell-surface receptors in the fetal urogenital ridge. The MIS type II receptor (MISRII), which provides specificity for MIS, is also expressed in the adult testis, ovary, and uterus. The rat MISRII promoter was cloned to study the molecular mechanisms underlying its temporal and cell-specific expression. The 1.6-kilobase (kb) promoter contained no recognizable TATA or CAAT box, but there was a consensus Sp1 site upstream of the transcription initiation site. Two binding sites for the orphan nuclear receptor steroidogenic factor-1 (SF-1) are occupied in vitro by using nuclear extracts from R2C cells, an MIS-responsive rat Leydig cell line that expresses endogenous MISRII, with differing affinities, indicating that the distal SF-1 site is bound more avidly than is the proximal SF-1 site. R2C cells transfected with MISRII promoter/luciferase reporter constructs show a 12-fold induction with the 1.6-kb fragment and deletion of sequences upstream of −282-bp lowered luciferase expression to one-third. Mutation of both SF-1 sites greatly inhibited luciferase expression, whereas mutation of either site alone resulted in continuing activation by endogenous SF-1, indicating redundancy. In vitro binding and transcriptional analyses suggest that a proximal potential Smad-responsive element and an uncharacterized element also contribute to activation of the MISRII gene. R2C cells and MISRII promoter regulation can now be used to uncover endogenous transcription factors responsible for receptor expression or repression.

Novel Ras antagonist blocks human melanoma growth

During past decades, knowledge of melanoma biology has increased considerably. Numerous therapeutic modalities based on this knowledge are currently under investigation. Advanced melanoma, nevertheless, remains a prime example of poor treatment response that may, in part, be the consequence of activated N-Ras oncoproteins. Besides oncogenic Ras, wild-type Ras gene products also play a key role in receptor tyrosine kinase growth factor signaling, known to be of importance in oncogenesis and tumor progression of a variety of human neoplasms, including malignant melanoma; therefore, it is reasonable to speculate that a pharmacological approach that curtails Ras activity may represent a sensible approach to inhibit melanoma growth. To test this concept, the antitumor activity of S-trans, trans-farnesylthiosalicylic acid (FTS), a recently discovered Ras antagonist that dislodges Ras from its membrane-anchoring sites, was evaluated. The antitumor activity of FTS was assessed both in vitro and in vivo in two independent SCID mouse xenotransplantation models of human melanoma expressing either wild-type Ras (cell line 518A2) or activated Ras (cell line 607B). We show that FTS (5–50 μM) reduces the amounts of activated N-Ras and wild-type Ras isoforms both in human melanoma cells and Rat-1 fibroblasts, interrupts the Ras-dependent extracellular signal-regulated kinase in melanoma cells, inhibits the growth of N-Ras-transformed fibroblasts and human melanoma cells in vitro and reverses their transformed phenotype. FTS also causes a profound and statistically significant inhibition of 518A2 (82%) and 607B (90%) human melanoma growth in SCID mice without evidence of drug-related toxicity. Our findings stress the notion that FTS may qualify as a novel and rational treatment approach for human melanoma and possibly other tumors that either carry activated ras genes or rely on Ras signal transduction more heavily than nonmalignant cells.

DAD1, the defender against apoptotic cell death, is a subunit of the mammalian oligosaccharyltransferase

DAD1, the defender against apoptotic cell death, was initially identified as a negative regulator of programmed cell death in the BHK21-derived tsBN7 cell line. Of interest, the 12.5-kDa DAD1 protein is 40% identical in sequence to Ost2p, the 16-kDa subunit of the yeast oligosaccharyltransferase (OST). Although the latter observation suggests that DAD1 may be a mammalian OST subunit, biochemical evidence to support this hypothesis has not been reported. Previously, we showed that canine OST activity is associated with an oligomeric complex of ribophorin I, ribophorin II, and OST48. Here, we demonstrate that DAD1 is a tightly associated subunit of the OST both in the intact membrane and in the purified enzyme. Sedimentation velocity analyses of detergent-solubilized WI38 cells and canine rough microsomes show that DAD1 cosediments precisely with OST activity and with the ribophorins and OST48. Radioiodination of the purified OST reveals that DAD1 is present in roughly equimolar amounts relative to the other subunits. DAD1 can be crosslinked to OST48 in intact microsomes with dithiobis(succinimidylpropionate). Crosslinked ribophorin II–OST48 heterodimers, DAD1–ribophorin II–OST48 heterotrimers and DAD1–ribophorin I–ribophorin II–OST48 heterotetramers also were detected. The demonstration that DAD1 is a subunit of the OST suggests that induction of a cell death pathway upon loss of DAD1 in the tsBN7 cell line reflects the essential nature of N-linked glycosylation in eukaryotes.

The α1 domain of HLA-G1 and HLA-G2 inhibits cytotoxicity induced by natural killer cells: Is HLA-G the public ligand for natural killer cell inhibitory receptors?

We have investigated the protective role of the membrane-bound HLA-G1 and HLA-G2 isoforms against natural killer (NK) cell cytotoxicity. For this purpose, HLA-G1 and HLA-G2 cDNAs were transfected into the HLA class I-negative human K562 cell line, a known reference target for NK lysis. The HLA-G1 protein, encoded by a full-length mRNA, presents a structure similar to that of classical HLA class I antigens. The HLA-G2 protein, deduced from an alternatively spliced transcript, consists of the α1 domain linked to the α3 domain. In this study we demonstrate that (i) HLA-G2 is present at the cell surface as a truncated class I molecule associated with β2-microglobulin; (ii) NK cytolysis, observed in peripheral blood mononuclear cells and in polyclonal CD3− CD16+ CD56+ NK cells obtained from 20 donors, is inhibited by both HLA-G1 and HLA-G2; this HLA-G-mediated inhibition is reversed by blocking HLA-G with a specific mAb; this led us to the conjecture that HLA-G is the public ligand for NK inhibitory receptors (NKIR) present in all individuals; (iii) the α1 domain common to HLA-G1 and HLA-G2 could mediate this protection from NK lysis; and (iv) when transfected into the K562 cell line, both HLA-G1 and HLA-G2 abolish lysis by the T cell leukemia NK-like YT2C2 clone due to interaction between the HLA-G isoform on the target cell surface and a membrane receptor on YT2C2. Because NKIR1 and NKIR2, known to interact with HLA-G, were undetectable on YT2C2, we conclude that a yet-unknown specific receptor for HLA-G1 and HLA-G2 is present on these cells.