Overexpression of rho effector rhotekin confers increased survival in gastric adenocarcinoma

Like many epithelial-derived cancers, gastric cancer (GC) results from a multistep tumorigenic process. However, the detailed mechanisms involved in GC formation are poorly characterized. Using an ordered differential display method, we have identifiedrhotekin (RTKN), the gene coding for the Rho effector, RTKN, as one of the genes differentially expressed in human GC. Northern analysis using human multiple tissue blots showed thatRTKN is predominantly expressed in the kidney and spinal cord, and, to a lesser degree, in the thyroid, tongue, liver, brain, prostate, trachea, and stomach. RT-PCR analysis confirmed thatRTKN was overexpressed in most (5/7; 71%) GC examined. By analyzing the Stanford Microarray Database for the expression profiles of gastric tissues, we also found a progressional increase inRTKN expression in nonneoplastic mucosa, GC, and then lymph node metastases (p<0.005 by Jonckheere-Terpstra test), suggesting thatRTKN expression correlates with GC progression. The role of RTKN in the pathogenic development of GC was investigated by transfection and expression ofRTKN in AGS gastric cells, which express endogenous RTKN at a low basal level. Flow-cytometric analysis showed thatRTKN-transfected AGS cells were significantly more resistant than vector-transfected cells to apoptosis upon treatment with sodium butyrate. To explore the mechanisms underlying RTKN-mediated cell survival, a reporter assay was performed. Since the NF-B activation is known to promote cell survival and Rho GTPase may lead to NF-B activation, we transfected AGS cells with the RTKN expression vector along with a pNF-B-Luc reporter plasmid. Our results showed that overexpression of RTKN induced robust activation of NF-B, and RTKN-mediated NF-B activation was suppressed significantly by C3 transferase, an inhibitor of the small GTPase Rho. We conclude that Rho/RTKN-mediated NF-B activation leading to cell survival may play a key role in gastric tumorigenesis. This study provides original documentation for the overrepresentation of the Rho GTPase effector rhotekin in human cancer and its links to cancer formation.     

Matrix metalloproteinase-2: Mechanism and regulation of NF-κB-mediated activation and its role in cell motility and ECM-invasion

Matrix metalloproteinases belong to a family of enzymes that degrade the extracellular matrix (ECM) components and play an important role in tissue repair, tumor invasion, and metastasis. ECM proteins, cytokines, and certain other factors regulate MMP activity. OPN, an ECM protein, has been found to be overexpressed in various cancers, and it has been shown to correlate with the metastatic potential. Although such reports indicate that OPN plays an important role in the ability of tumor cells to survive and metastasize to secondary sites, the mechanism by which OPN regulates these processes is yet to be understood. In this study we report that native purified human OPN can induce cell migration and ECM invasion. Increased invasiveness and migration correlates with enhanced expression and activation of MMP-2. Our study provides evidence showing that OPN increases gelatinolytic activity by inducing MT1-MMP expression via activation of the NF-B pathway. Suppression of MMP-2 by ASMMP-2 reduces the OPN-induced cell migration and ECM invasion. Curcumin blocks OPN-induced MT1-MMP expression and pro-MMP-2 activation. Curcumin, a known anti-inflammatory and anticarcinogenic compound, suppresses OPN-induced cell migration, invasion and induces apoptotic morphology in OPN-treated cells. The mechanism by which curcumin suppresses the OPN-induced effects has also been delineated. Curcumin inhibits MT1-MMP gene expression by blocking signals leading to IKK activation. This in turn inhibits IB phosphorylation and NF-B activation. Published in 2004.     

Phosphoinositide 3-kinase activity leads to silica-induced NF-κB activation through interacting with tyrosine-phosphorylated IκB-α and contributing to tyrosine phosphorylation of p65 NF-κB

The role of the subunits of phosphoinositide (PI) 3-kinase in NF-B activation in silica-stimulated RAW 264.7 cells was investigated. Results indicate that PI3-kinase activity was increased in response to silica. The p85 subunit of PI3-kinase interacted with tyrosine-phosphorylated IB- in silica-stimulated cells. PI3-kinase specific inhibitors, such as wortmannin and LY294003, substantially blocked both silica-induced PI3-kinase and NF-B activation. The inhibition of NF-B activation by PI3-kinase inhibitors was also observed in pervanadate-stimulated but not in LPS-stimulated cells. Furthermore, tyrosine phosphorylation of NF-B p65 was enhanced in cells stimulated with silica, pervanadate or LPS, and wortmannin substantially inhibited the phosphorylation event induced by the first two stimulants but not LPS. Antioxidants, such as superoxide dismutase (SOD), N-acetylcysteine (NAC) and pyrrolidine dithiocarbamate (PDTC), blocked silica-induced PI3-kinase activation, suggesting that reactive oxygen species may be important regulatory molecules in NF-B activation by mediating PI3-kinase activation. Our data suggest that p85 and p110 subunits of PI3-kinase play a role in NF-B activation through interaction with tyrosine-phosphorylated IB- and contributing to tyrosine phosphorylation of p65 NF-B.     

Quercetin suppresses proinflammatory cytokines production through MAP kinases and NF-κB pathway in lipopolysaccharide-stimulated macrophage

Quercetin is a flavonoid molecule ubiquitous in nature and functions as an anti-oxidant and anti-inflammatory agent with little toxicity in vivo and in vitro. Dose- and time-dependent effect of quercetin has been investigated on proinflammatory cytokine expression and NO production, focusing on its effects on the MAP kinases and the NF-B signal transduction pathways in LPS-stimulated RAW 264.7 cells by using RT-PCR and immunoblotting. Quercetin strongly reduced activation of phosphorylated ERK kinase and p38 MAP kinase but not JNK MAP kinase by LPS treatment. In addition, quercetin treatment inhibited NF-B activation through stabilization of the NF-B/IB complex and IB degradation and proinflammatory cytokines and NO/iNOS expression. Quercetin may exert its anti-inflammatory and immunomodulatory properties in the effect molecules such as proinflammatory cytokines and NO/iNOS by suppressing the activation of ERK and p38 MAP kinase, and NF-B/IB signal transduction pathways.     

IκB/NF-κB mediated cisplatin resistance in HeLa cells after low-dose γ-irradiation is associated with altered SODD expression

Fractionated -irradiation (15 × 2 Gy in 3 weeks) induces a cellular resistance in HeLa cells against cisplatin exposure but not against irradiation. The mechanisms underlying this cellular resistance are associated with major changes in the TNFR1-dependent transduction pathway. The resistant HeLa/B cells exhibit increased levels of NF-B with temporally independent regulation of the subunits NF-B50 and NF-B65. Blocking IB degradation by the proteasome inhibitor PSI, which abolishes the release of the active NF-B protein, induces cell death much more effectively in the parental than in the resistant HeLa/B cells. The translocation of NF-B does not seem to be affected in a similar manner since masking of the translocation sequence by NF-B SN50 enhances cisplatin toxicity to the same degree in both cell lines and overcomes drug resistance. Changes in upstream signaling are suggested by increased sensitivity of the parental HeLa cells to cisplatin in the presence of neutralizing anti-TNFR1. In HeLa/B cells, reduced expression of the 50 kDa silencer of death domain, SODD, is accompanied by constitutive overexpression of a 40–42 kDa SODD-like protein. A possible involvement of SODD in cisplatin resistance is discussed, which may shift the balance between life and death in the TNF receptor pathway to increased NF-B activation.     

Anti-apoptotic action of API2-MALT1 fusion protein involved in t(11;18)(q21;q21) MALT lymphoma

At least three distinct chromosomal translocations, t(11;18)(q21;q21), t(1;14)(p22;q32) and t(14;18)(q32;q21) involving the API2 (also known as c-IAP2)-MALT1 fusion protein, BCL10, and MALT1, respectively, have been implicated in the molecular pathogenesis of mucosa associated lymphoid tissue (MALT) lymphoma. Our findings showed that several variants of the API2-MALT1 fusion protein can occur in patients with t(11;18)(q21;q21), and that API2-MALT1 can potently enfance activation of nuclear factor (NF)-B signaling, which may be relevant to the pathogenesis of MALT lymphomas. We also found that MALT1 is rapidly degraded via the ubiquitin-proteasome pathway, as is the case with API2, but upon the synthesis of fusion, API2-MALT1 becomes stable against this pathway. This stability of API2-MALT1 may thus result in inappropriate nuclear factor (NF)-B activation, thereby contributing to the pathogenesis of MALT lymphoma. Recent biochemical and genetic studies have clearly shown that BCL10 and MALT1 form a physical and functional complex and are both required for NF-B activation by antigen receptor stimulation in T and B lymphocytes. It has also been shown that CARMA1, a newly discovered member of the membrane-associated guanylate kinase (MAGUK) families, is critical for antigen receptor-stimulated NF-B activation. It can be assumed that API2-MALT1 can bypass this normal BCL10/MALT1 cellular signaling pathway linked to NF-B activation, thereby inducing antigen receptor-independent proliferation of lymphocytes. Furthermore, BCL10/MALT1- and API2-MALT1-induced NF-B activation may contribute to anti-apoptotic action probably through NF-B-mediated upregulation of apoptotic inhibitor genes. We recently provided direct evidence that API2-MALT1 indeed exerts anti-apoptotic action, in part, through its direct interaction with apoptotic regulators including Smac. Taken together, these findings prompt us to hypothesize that the anti-apoptotic action of API2-MALT1 may be mediated partly by the direct interaction with apoptotic regulators as well as partly by upregulation of apoptotic inhibitor genes. Further studies can be expected to stimulate research into the development of therapeutic drugs that specifically inhibit the antigen receptor signaling-stimulated NF-B activation pathway: such molecule targeting drugs should be useful for interfering with inappropriate proliferation of lymphocytes associated with inflammatory and neoplastic disorders.     

Evolution of parallel β/α-barrel enzyme family lightened by structural data on starch-processing enzymes

The parallel /-barrel domain consisting of eight parallel -sheets surrounded by eight -helices has been currently identified in crystal structures of more than 20 enzymes. This type of protein folding motif makes it possible to catalyze various biochemical reactions on a variety of substrates (i.e., it seems to be robust enough so that different enzymatic functionalities could be designed on it). In spite of many efforts aimed at elucidation of evolutionary history of the present-day /-barrels, a challenging question remains unanswered: How has the parallel /-barrel fold arisen? Although the complete sequence comparison of all /-barrel amino acid sequences is not yet available, several sequence similarities have been revealed by using the highly conserved regions of -amylase as structural templates. Since many starch-processing enzymes adopt the parallel /-barrel structure these enzymes might be useful in the search for evolutionary relationships of the whole parallel eight-folded /-barrel enzyme family.     

Curcumin inhibits ultraviolet light induced human immunodeficiency virus gene expression

Recently, we reported that the herbal drug St. John's Wort is a potent inhibitor of UV-induced HIV-LTR activation in stably transfected HIVcat/HeLa cells [35]. Our previous studies have demonstrated that the activation of p38 MAP kinase (stress-activated protein kinase-2) and NF-B are both required for a full UV-induced HIV gene expression response. In this study we have investigated the mechanism by which curcumin inhibits UV-activated HIV-LTR gene expression. We found that treatment of HIVcat/HeLa cells with micromolar concentrations of curcumin completely abolished UV activation of HIV gene expression. Curcumin treatment at similar doses as those used to inhibit HIV gene expression also effectively blocked UV activation of NF-B, as demonstrated by electrophoretic mobility shift assay. In contrast, curcumin did not inhibit UV-induced phosphorylation of p38 MAP kinase. This observation was also supported by findings that curcumin did not inhibit UV-induced phosphorylation of CREB/ATF-1 and ATF-2. Although curcumin was ineffective in preventing UV-induced p44/42 MAP kinase phosphorylation, the JNK (1 and 2) and AP-1 activation were efficiently blocked by curcumin in HeLa cells. We conclude that the mechanism by which curcumin modulates UV activation of HIV-LTR gene expression mainly involves the inhibition of NF-B activation.     

Isolation of full-length cDNA and chromosomal localization of human NF-κB modulator NEMO to Xq28

NEMO is an essential component of the IB kinase complex. Others have shown that expression of mouse NEMO can complement the lack of responsiveness to NF-B stimuli in two NEMO-deficient cell lines. Here we report the isolation of a full-length human NEMO cDNA. Virtual translation of human NEMO cDNA predicts a 48-kD coiled-coil protein which shares 87.9% identity and 90.5% similarity with the mouse homolog. By sequence alignment, we mapped the human NEMO gene to chromosome Xq28. We note that the NEMO and the G6PD (glucose-6-phosphate dehydrogenase) loci are arranged in a head-to-head orientation separated by no more than 800 bp. This map location is further supported by the sequence of an alternatively spliced variant of human NEMO mRNA. Thus, human NEMO is an X-linked gene closely adjacent to the G6PD locus.     

Interactions between NFκB and Its Inhibitor iκB: Biophysical Characterization of a NFκB/iκB-α Complex

The N-terminal domain (1–318 amino acids) of mouse NFB (p65) has been purified to homogeneity from the soluble fraction of Escherichia coli cells expressing this protein. Its complex with a full-length iB- (MAD3, 1–317 amino acids) molecule was generated by binding the E. coli-derived iB- to the purified NFB and purifying the complex by sequential chromatography. The stoichiometry of NFB to iB in the complex was determined to be 2 to 1 by light scattering and SDS–polyacrylamide gel electrophoresis. The secondary structure of the NFB (p65) determined by Fourier-transform infrared (FTIR) spectroscopy is in good agreement with that of the p50 in the crystal structure of the p50/DNA complex, indicating that no significant structural change in NFB occurs upon binding of DNA. The FTIR spectrum of the NFB/iB complex indicates that its secondary structure is composed of 17% -helix, 39% -strand, 18% irregular structures, and 26% -turns and loops. By comparing these data to the FTIR data for NFB alone, it is concluded that the iB (MAD3) in the complex contains 35% -helix, 27% -strand, 22% irregular structures, and 16% -turns and loops. Circular dichroism (CD) analysis of a shorter form of iB (pp40) indicates that it contains at least 20% -helix and that the iB subunit accounts for nearly all of the -helix present in the NFB/iB complex, consistent with the FTIR results. The stabilities of NFB, iB, and their complex against heat-induced denaturation were investigated by following changes in CD signal. The results indicate that the thermal stability of iB is enhanced upon the formation of the NFB/iB complex.     

Receptor activator of NF-κB ligand induces the fusion of mononuclear preosteoclasts into multinucleated osteoclasts

The osteoclasts are bone-resorbing multinucleatedcells formed by the fusion of mononuclearpreosteoclasts (pOCs) of hematopoietic origin.Although receptor activator of NF-Bligand (RANKL) has been shown to regulate osteoclastdifferentiation and function, its effect on the fusionof pOCs into multinucleated osteoclast-like cells(OCLs) has not been known. Using our fusion assaysystem, that is not contaminated with multinucleatedcells (MNCs) and osteoblastic cells, we determined theeffect of RANKL on the fusion of pOCs into MNCs. WhenpOCs were cultured on the plates, most of pOCs diedand disappeared from the plates within 24 h in theabsence of additives, but pOCs were fused to MNCswithin 6 h in the presence of RANKL. RANKL-inducedMNCs showed typical properties of OCL such astartrate-resistant acid phosphatase (TRAP) activity,actin ring formation, and bone-resorbing activity. Thefusion of pOCs into OCLs induced by osteoblastic cellsor RANKL was inhibited by OPG/OCIF, but that inducedby IL-1 was not. Both RANKL- andIL-1-induced OCL formation from pOCs wasinhibited by ZLLL-H, a peptide inhibitor ofproteasome. These findings indicate that RANKLsupports the survival of pOCs and induces the fusionof pOCs into OCLs and suggest that NF-Bactivation is involved in these processes induced byRANKL and IL-1.     

A robust and cost-effective method for the production of Val, Leu, Ile (δ1) methyl-protonated 15N-, 13C-, 2H-labeled proteins

A selective protonation strategy is described that uses [3-2H] 13C -ketoisovalerate to introduce (1H- methyl)-leucine and (1H- methyl)-valine into 15N-, 13C-, 2H-labeled proteins. A minimum level of 90% incorporation of label into both leucine and valine methyl groups is obtained by inclusion of 100 mg/L -ketoisovalerate in the bacterial growth medium. Addition of [3,3-2H2] -ketobutyrate to the expression media (D2O solvent) results in the production of proteins with (1H-1 methyl)-isoleucine (>90% incorporation). 1H-13C HSQC correlation spectroscopy establishes that CH2D and CHD2 isotopomers are not produced with this method. This approach offers enhanced labeling of Leu methyl groups over previous methods that utilize Val as the labeling agent and is more cost effective.     

δ and κ Opiate receptors in primary astroglial cultures from rat cerebral cortex

The effects of , , and receptor-agonists on forskolin stimulated cyclic adenosine-3, 5-monophosphate (cAMP) formation were examined in astroglial enriched primary cultures from the cerebral cortex of newborn rats. Intracellular cAMP accumulation was quantified by radioimmunoassay. Morphine was used as a -receptor agonist, D-Ala-D-Leu-Enkephalin (DADLE) as a -receptor agonist and dynorphine 1–13 (Dyn) as a -receptor agonist. Basal cAMP levels were unaffected by either the opiate agonists or the antagonists used. In the presence of the cAMP stimulator forskolin, morphine had no significant effect on the cytoplasmic cAMP levels. DADLE caused a dose related inhibition of the forskolin stimulated cAMP accumulation. The effects of this receptor stimulation was blocked with the selective antagonist ICI 174.864. In the presence of Dyn, the forskolin stimulated cAMP accumulation was inhibited in a dose related manner. This receptor stimulation was blocked with the selective antagonist MR 2266. Co-administration of DADLE and Dyn resulted in a non additive inhibition of the forskolin stimulated accumulation of cAMP. These findings indicate that astroglial enriched cultures from the cerebral cortex of rats express and -receptors co-localized ont he same population of cells, and that these receptors are inhibitory coupled to adenylate cyclase.     

Neuronal Apoptosis Induced by Endoplasmic Reticulum Stress

Apoptosis is a conserved active cellular mechanism occurring under a range of physiological and pathological conditions. In the nervous system, apoptosis plays crucial roles in normal development and neuronal degenerating diseases. Various deleterious conditions, including accumulation of the mutant proteins in the endoplasmic reticulum (ER) and inhibition of ER to Golgi transport of proteins, may result in apoptosis. In this study, we examined the downstream events of apoptosis in differentiated PC 12 cells under ER stress induced by brefeldin A, an inhibitor of ER to Golgi protein transport. Activation of NF-B and degradation of I-B were observed within 2 hours, followed by up-regulation of GRP78 protein level in treated cells. Caspase-12 only appeared around 24 hours after brefeldin A treatment, coincident with cell nuclei fragmentation. These results suggest that neuronal apoptosis may be induced by ER stress through a NF-B and caspase related pathway.     

Linkage mapping of ‘25-kDa globulin’ genes on homoeologous group-1 chromosomes of bread and durum wheat

Acid polyacrylamide-gel electrophoresis (A-PAGE) of ethanol-soluble proteins from the endosperm of bread and durum wheats reveals some bands encoded by genes on the homoeologous group-1 chromosomes with higher mobility than the -gliadins. The isolation of these proteins showed that they were the previously described 25-kDa globulins encoded by genes at the Glo-A1, Glo-B1, and Glo-D1 loci. The variability found among a collection of 51 bread and 81 durum wheats was very low: two allelic variants at Glo-A1 and no variants at Glo-B1 in each of the two species, and two allelic variants at Glo-D1 in bread wheat. Inheritance studies of 25-kDa globulin genes on group-1 chromosomes of bread and durum wheat were carried out on the F₂ progeny from four crosses, two in bread wheat and two in durum wheat. The linkage mapping of the 1A 25-kDa globulin genes of bread wheat was done based on four prolamin loci: Glu-A1, Glu-A3, Gli-A1 and Gli -A3. The percentages of recombination and the distances found allowed a re-evaluation of the linkage map of endosperm protein loci on this chromosome. The Glo-A1 locus was found to be located at the distal end of the short arm of 1A chromosome, at a distance of 5.23±1.99 cM from Gli-A1, 6.85±2.22 cM from Glu-A3, 22.64±3.62 cM from Gli-A1, and at a recombination percentage of 49.30±4.40 from Glu-A1. A similar distance between Gli-A1 and Glo-A1 (4.82±1.75 and 6.66±2.26 cM) was found in durum wheat. The distance between Gli-D1 and Glo-D1 on chromosome 1D was 2.86±1.39 cM.     

Endogenous interferon α/β produced by Kupffer cells inhibits interleukin-1, tumor necrosis factor α production and interleukin-2-induced activation of nonparenchymal liver cells

Summary We have previously reported liver-specific interferon (IFN) / production by murine Kupffer cells that was not observed with other tissue macrophages incubated in the absence of stimulators such as IFN or lipopolysaccharide (LPS). Consequently, while interleukin-2 (IL-2) alone induced pronounced lymphokine-activated killer (LAK) activity from splenocytes, combination of anti-IFN/ antibody with IL-2 was required to generate significant LAK activity from nonparenchymal liver cells. This endogenous IFN/ production by Kupffer cells was not induced by LPS because (a) addition of polymyxin B did not abolish the positive effects of anti-IFN/ antibody on nonparenchymal liver cells, and (b) similar results were obtained when comparing the responses of LPS-responsive C3HeB/FeJ and LPS-hyporesponsive C3H/HeJ mice. The possibility of hepatotropic infection was also ruled out in that anti-IFN/ antibody enhanced hepatic but not splenic LAK cell induction in vitro in both conventional and germfree C3H/HeN mice. IFN/ played an autoregulatory role by down-regulating the production of IL-1 and tumor necrosis factor by Kupffer cells. However, the augmenting effect of anti-IFN/ antibody on LAK induction from non-parenchymal liver cells was not mediated through an increase in the level of either IL-1 or TNF, as specific antisera against either cytokine did not abrogate this positive effect. Finally, flow-cytometry analysis showed that IFN/ significantly diminished the expression of IL-2 receptor chain, indicating an inhibition of LAK cell generation at a relatively early stage of induction.This work is supported by NIH grant RO1-28 835 and by Medical Research Funds from the Veterans Administration     

Immunogenicity and immunosensitivity of ex vivo human carcinomas: interferon γ and tumour necrosis factor α treatment of tumour cells potentiates their interaction with autologous blood lymphocytes

Human carcinoma cells vary appreciably in the expression of MHC class I, class II, ICAM-1 (CD54) and B7 (CD80) molecules. Short-term in vitro exposure of ex vivo carcinoma cells to interferon and tumour necrosis factor elevated/induced the surface expression of MHC class I, class II and ICAM-1, but only rarely of B7. We found that cytokine treatment elevated the cytotoxic susceptibility and the stimulatory potential of ex vivo tumour cells. This was demonstrated (a) by the increased frequency and elevated level of auto-tumour lysis and (b) by induction of DNA synthesis and generation of cytotoxic lymphocytes in autologous mixed lymphocyte/tumour cell culture (MLTC). The MHC class I and ICAM-1 molecules on the tumour cells were required for interaction with the lymphocytes as indicated by the inhibitory effect of specific mAb both in the stimulation and in the cytotoxic tests. While the cytokine-induced increases in MHC and ICAM-1 on the low-expression tumours were probably important for the modification of functional interaction with the autologous lymphocytes, it is likely that alterations in other properties of tumour cells were also induced which contributed to the phenomenon. This was indicated by the results obtained with several tumours, which expressed indigenously high levels of these molecules but activated the autologous lymphocytes only after cytokine treatment. In several experiments the untreated targets that did not activate the lymphocytes were sensitive to the cytotoxicity of the effectors activated in MLTC. The results show that the afferent and efferent arms of the immune response have different requirements for functional interactions between lymphocytes and tumour cells.