NUPR1, a new target in liver cancer: implication in controlling cell growth, migration, invasion and sorafenib resistance.

Sorafenib, an oral multikinase inhibitor, is the only approved agent for the treatment of advanced hepatocellular carcinoma (HCC). However, its benefits are modest, and as its mechanisms of action remain elusive, a better understanding of its anticancer effects is needed. Based on our previous study results, we investigated here the implication of the nuclear protein 1 (NUPR1) in HCC and its role in sorafenib treatment. NUPR1 is a stress-inducible protein that is overexpressed in various malignancies, but its role in HCC is not yet fully understood. We found that NUPR1 expression was significantly higher in primary human HCC samples than in the normal liver. Knockdown of NUPR1 significantly increased cell sensitivity to sorafenib and inhibited the cell growth, migration and invasion of HCC cells, both in vitro and in vivo. Moreover, NUPR1 silencing influenced the expression of RELB and IER3 genes. Unsurprisingly, RELB and IER3 knockdown also inhibited HCC cell viability, growth and migration. Using gene expression profiling of HCC cells following stable NUPR1 knockdown, we found that genes functionally involved in cell death and survival, cellular response to therapies, lipid metabolism, cell growth and proliferation, molecular transport and cellular movement were mostly suppressed. Network analysis of dynamic gene expression identified NF-κB and ERK as downregulated gene nodes, and several HCC-related oncogenes were also suppressed. We identified Runt-related transcription factor 2 (RUNX2) gene as a NUPR1-regulated gene and demonstrated that RUNX2 gene silencing inhibits HCC cell viability, growth, migration and increased cell sensitivity to sorafenib. We propose that the NUPR1/RELB/IER3/RUNX2 pathway has a pivotal role in hepatocarcinogenesis. The identification of the NUPR1/RELB/IER3/RUNX2 pathway as a potential therapeutic target may contribute to the development of new treatment strategies for HCC management.

Structure of human type II 5 alpha-reductase gene.

The best known activity of steroid 5 alpha-reductase is the transformation of testosterone into dihydrotestosterone, the most potent androgen. Two types of human steroid 5 alpha-reductase cDNAs and the type I gene have previously been isolated and characterized. This report describes the isolation and characterization of the human type II 5 alpha-reductase gene, the gene most likely responsible for male pseudohermaphroditism due to 5 alpha-reductase deficiency as well as the one presumed to be involved in a major androgen-related diseases such as prostate cancer and benign prostatic hyperplasia. The type II 5 alpha-reductase gene contains five exons of 352, 164, 102, 151 and 1695 bp, respectively, which share 43.8% to 64.1% homology with exons of the corresponding type I gene. These exons are separated by four introns of greater than 29, and approximately 2.3, 2.0 and 3.0 kb. Analysis of primer extension products by polyacrylamide gel electrophoresis as well as by subcloning and sequencing reveals a start site located 71 nucleotides upstream the ATG initiating codon.

Antagonist-induced intracellular sequestration of rabbit bradykinin B(2) receptor.

In a contractility assay based on the rabbit jugular vein, the structurally related drugs NPC 17731 or icatibant (1 to 3 nmol/L) were insurmountable antagonists of bradykinin (BK) B(2) receptors (B(2)Rs). After ample washing (3 hours), the antagonism exerted by these peptides was not reversible. By contrast, the antagonist LF 16. 0687 (30 to 100 nmol/L) was competitive and reversible. A rabbit B(2)R-green fluorescent protein (B(2)R-GFP) conjugate was expressed in mammalian cells. In COS-1 cells, it exhibited an affinity for [3H]BK (K(D)=1.61 nmol/L) similar to that of the wild-type rabbit B(2)R. The stably expressed construction in HEK-293 cells was functionally active (phospholipase A(2) assay), and the antagonists mentioned above retained their respective surmountable or insurmountable behavior. Competition of [(3)H]BK binding to B(2)R-GFP by the antagonists or BK was largely reversible after a 3-hour washout period at 0 degrees C; at 37 degrees C, icatibant or NPC 17731 effects were not reversible. B(2)R-GFP was visualized in the plasma membranes of HEK-293 cells and rapidly internalized in response to BK. NPC 17731 or icatibant slowly translocated B(2)R-GFP into cells over 24 hours, whereas LF 16.0687 had no effect on the subcellular distribution of B(2)R-GFP. Cell extract immunoblotting with anti-GFP antibodies revealed a 101- to 105-kDa protein that was not significantly degraded on 24 hours of cell treatment with any of the ligands but was translocated in part to the 15 000-g pellet of the extract on treatment with BK or the noncompetitive antagonists. NPC 17731 and icatibant are noncompetitive, nonequilibrium antagonists that promote the cellular sequestration of rabbit B(2)R expressed in an heterologous system.

Heterogeneity in the Xenopus ribosomal transcription factor xUBF has a molecular basis distinct from that in mammals.

The Xenopus polymerase I transcription factor xUBF is an HMG-box protein which has been purified as two polypeptides of approximately 82 and 85 kDa. Recently a cDNA sequence predicted an xUBF protein (xUBF1) of 677 amino acids (79 kDa) containing five tandem HMG-boxes. Here a second and distinct xUBF cDNA has been isolated and characterised. This cDNA codes an xUBF protein (xUBF2) of 701 amino acids (82 kDa), having 93% homology with xUBF1 but containing an insertion of 22 amino acids between HMG-boxes 3 and 4. In vitro translation of synthetic mRNAs derived from the xUBF1 and 2 cDNAs was used to show that the electrophoretic mobility of the gene products accounted for the major xUBF molecular weight heterogeneity noted in vivo. It is also shown that the Xenopus laevis genome contains 3 or 4 distinct loci which hybridise with xUBF coding sequences, leaving open the possibility of yet further unrecognised heterogeneity in xUBF.

Regulation of kinin-induced contraction and DNA synthesis by inflammatory cytokines in the smooth muscle of the rabbit aorta.

1. In rabbit aortic rings, the contractile response to kinins is mediated by the B1 receptors for kinins; the response is upregulated from an initial null level in a time- and protein synthesis-dependent manner. Incubation (3 h) with human recombinant interleukin-1 beta (IL-1 beta) selectively amplified the contractile response to the B1 receptor agonist Sar-[D-Phe8]des-Arg9-BK, while it did not affect the contractile effect of other agents (angiotensin II, endothelin-1, phenylephrine). 2. Oncostatin M (OSM), but not macrophage migration inhibitory factor (MIF), increased the contractile response to the B1 receptor agonist, des-Arg9-bradykinin (des-Arg9-BK). 3. Cultured smooth muscle cells derived from the rabbit aorta exhibit a significant des-Arg9-BK-induced increase in [3H]-thymidine incorporation if pretreated with a cyclo-oxygenase inhibitor (diclofenac) and concomitantly treated with the cytokines IL-1 or OSM. Angiotensin II, endothelin-1 or phenylephrine, alone or in the presence of IL-1 beta, exerted little effect on DNA synthesis in these cells. 4. The pharmacological characterization of the mitogenic response to kinins using a set of agonist and antagonist analogues is consistent with mediation by B1 receptors. Des-Arg9-BK-induced DNA synthesis is suppressed by prostaglandin E2 by a prostacyclin mimetic (iloprost), by the Ser/Thr protein kinase inhibitor, H-7, and by a tyrosine kinase inhibitor (i.e. an erbstatin analogue). 5. B1 receptor-mediated responses and their capacity to be regulated by cytokines, are retained in rabbit aortic smooth muscle cells. Such responses could be relevant to tissue repair mechanisms and hypertrophic medial responses to injury in arteries.

Absence of ligand-induced regulation of kinin receptor expression in the rabbit.

The induction of B(1) receptors (B(1)Rs) and desensitization or down-regulation of B(2) receptors (B(2)Rs) as a consequence of the production of endogenous kinins has been termed the autoregulation hypothesis. The latter was investigated using two models based on the rabbit: kinin stimulation of cultured vascular smooth muscle cells (SMCs) and in vivo contact system activation (dextran sulphate intravenous injection, 2 mg kg(-1), 5 h). Rabbit aortic SMCs express a baseline population of B(1)Rs that was up-regulated upon interleukin-1beta treatment ([(3)H]-Lys-des-Arg(9)-BK binding or mRNA concentration evaluated by RT - PCR; 4 or 3 h, respectively). Treatment with B(1)R or B(2)R agonists failed to alter B(1)R expression under the same conditions. Despite consuming endogenous kininogen (assessed using the kinetics of immunoreactive kinin formation in the plasma exposed to glass beads ex vivo) and producing hypotension mediated by B(2)Rs in anaesthetized rabbits, dextran sulphate treatment failed to induce B(1)Rs in conscious animals (RT - PCR in several organs, aortic contractility). By contrast, lipopolysaccharide (LPS, 50 microg kg(-1), 5 h) was an effective B(1)R inducer (kidney, duodenum, aorta) but did not reduce kininogen reserve. We tested the alternate hypothesis that endogenous kinin participate in LPS induction of B(1)Rs. Kinin receptor antagonists (icatibant combined to B-9858, 50 microg kg(-1) of each) failed to prevent or reduce the effect of LPS on B(1)R expression. Dextran sulphate or LPS treatments did not persistently down-regulate vascular B(2)Rs (jugular vein contractility assessed ex vivo). The kinin receptor autoregulation hypothesis is not applicable to primary cell cultures derived from a tissue known to express B(1)Rs in a regulated manner (aorta). The activation of the endogenous kallikrein-kinin system is ineffective to induce B(1)Rs in vivo in an experimental time frame sufficient for B(1)R induction by LPS.

Non-competitive pharmacological antagonism at the rabbit B(1) receptor.

The B(1) receptor for kinins, stimulated by kinin metabolites without the C-terminal Arg residue (e.g., des-Arg(9)-bradykinin (BK) and Lys-des-Arg(9)-BK), is an increasingly recognized molecular target for the development of analgesic and anti-inflammatory drugs. Recently developed antagonists of this receptor were compared to a conventional antagonist, Ac-Lys-[Leu(8)]-des-Arg(9)-BK, in pharmacological assays based on the rabbit B(1) receptor. B-9858 (Lys-Lys-[Hyp(3), Igl(5), D-Igl(7), Oic(8)]des-Arg(9)-BK) and three other analogues possessing the alpha-2-indanylglycine(5) (Igl(5)) residue (order of potency B-9858 approximately B-10146>B-10148>B-10050) were partially insurmountable antagonists of des-Arg(9)-BK in the contractility assay based on rabbit aortic rings. B-9858-induced depression of the maximal effect was more pronounced in tissues treated with the protein synthesis inhibitor cycloheximide to block the spontaneous increase of response attributed to the post-isolation formation of B(1) receptors, and only partly reversible on washing. By comparison, Ac-Lys-[Leu(8)]des-Arg(9)-BK was a surmountable antagonist (pA(2) 7. 5), even in cycloheximide-treated tissues. B-9958 (Lys-[Hyp(3), CpG(5), D-Tic(7), CpG(8)]des-Arg(9)-BK) was also surmountable (pA(2) 8.5). The binding of [(3)H]-Lys-des-Arg(9)-BK to recombinant rabbit B(1) receptors expressed in COS-1 cells was influenced by two of the antagonists: while Ac-Lys-[Leu(8)]des-Arg(9)-BK competed for the radioligand binding without affecting the B(max), B-9858 decreased the B(max) in a time-dependent and washout-resistant manner. B-9858 and analogues possessing Igl(5) are the first reported non-competitive, non-equilibrium antagonists of the kinin B(1) receptor.

Cloning and preliminary pharmacological characterization of the anaphylatoxin C5a receptor in the rabbit.

1 The rabbit receptor for C5a was cloned from a genomic library and found to be 79.5% identical to the human homologue, the highest degree of similarity found so far in nonprimate laboratory animals. 2 The rabbit C5a receptor stably expressed in RBL cells binds human 125I-C5a (2 nM). Unlabelled C5a and the C-terminal analogue N-acetyl-Tyr-Ser-Phe-Lys-Pro-Met-Pro-Leu-D-Ala-Arg (Ac-YSFKPMPLaR) were found to be competitors of that binding, the peptide analogue retaining approximately 0.1% of the affinity of human C5a. 3 The order of potency human C5a>Ac-YSFKPMPLaR was conserved in bioassays based on rabbits (relaxation of the isolated portal vein and pulmonary artery; acute in vivo neutropenia), but with a decreasing potency gap between the two compounds, a likely consequence of the resistance to peptidases of the analogue. 4 The molecular definition of the rabbit C5a receptor evidenced a high preservation degree of sequence and pharmacologic properties relative to the human ortholog receptor, thus defining a set of molecular tools for the investigation of the role of C5a in physiologic and pathologic models based on the rabbit (e.g. atherosclerosis, inflammation).

Altered frequency of a promoter polymorphism of the kinin B2 receptor gene in hypertensive African-Americans.

Components of the kallikrein kinin system have been associated with the pathophysiology of hypertension in animal and human studies. In this study, we examined the distribution of four different polymorphisms of the kinin B1 and B2 receptor genes in a population of 120 normotensive and 77 hypertensive African-Americans. Allelic frequencies for three of the four polymorphisms were significantly different from those previously reported in Caucasian populations. Among the polymorphisms analyzed, a potentially functionally significant polymorphism in the core promoter of the kinin B2 receptor (C-58-->T transition) displayed an increased prevalence of the C-58 allele in the hypertensive patients as compared with the controls (0.75 v. 0.62, P = .009). Thus, this B2 receptor promoter polymorphism may represent a susceptibility marker for essential hypertension in African-Americans.

Mapping of a sequence essential for the nuclear transport of the Xenopus ribosomal transcription factor xUBF using a simple coupled translation-transport and acid extraction approach.

The amino acid sequences directing the nuclear transport of the RNA polymerase I transcription factor xUBF have been studied by a novel combination of in oocyte-coupled translation-nuclear transport and selective HCl extraction. Synthetic mRNA was used to direct the translation of labeled xUBF and its mutants in microinjected oocytes. After manual dissection of nuclei and cytoplasm, labeled xUBF and mutants were isolated essentially pure by HCl extraction. Using deletion mutations, a sequence essential, but not necessarily sufficient, for nuclear transport was mapped to a 29-amino-acid segment lying between the most carboxy-terminal putative HMG-box DNA-binding domain, HMG-box 5, and the highly acidic carboxy-terminal domain. It was shown that deletion of only 5 amino acids from this segment eliminated xUBF transport, and it could be deduced that at least 11 of the 29 amino acids were essential for nuclear transport. The segment of xUBF necessary for nuclear transport contains a sequence conforming to the bipartite nuclear transport motif consensus, but this sequence in itself was insufficient for nuclear transport.

Kinin receptors.

Rapid developments are expected in the molecular pharmacology of both B1, and B2 types of kinin receptors, since the underlying genetic structures are now known and widely studied. The consequences of kinin receptor duality and physiopathological regulation have not yet been fully appreciated. Medicinal chemistry is also an active front of research in kinin pharmacology, as more effective drugs targeted at kinin receptors are regularly reported. Various complementary molecular approaches (the receptor binding, cloning, immunoreacting, mutagenesis, inactivation, the study of regulation, allelic polymorphisms, and so forth) are expanding our knowledge of the role of kinins in allergy, inflammation, and singularly, renal medicine.

Construction of a Co1E1 plasmid bearing inducible high-copy-number phenotype.

In order to construct plasmids bearing inducible high-copy-number phenotype, the cloning plasmid pBR322 was modified as follows: a DNA fragment containing a strong synthetic promoter (P1), synthetic lac operator (O1), DNA sequence corresponding to the RNAI/RNAII region of the Co1E1 replicon and the CAT gene transcription terminator was substituted for the 29 bp EcoRI/HindIII DNA fragment. Two types of plasmids were constructed in this way, differing in the orientation of the RNAI/RNAII fragment. Depending on the orientation these plasmids coded for RNA molecules representing either RNAI or RNAII domains. It was found that when RNAII molecules were overproduced the plasmid copy number was about 4 times higher than that of pBR322 and only negligible change in the plasmid copy-number value was observed upon overproduction of RNAI molecules.

Detection of human papillomavirus DNA in gynaecological swabs by filter in situ hybridization.

Gynaecological smears from the endo- and ectocervix of women with and without cytological and colposcopic abnormalities of the epithelium were investigated for human papillomavirus (HPV) types 6, 11, 16, and 18 by filter in situ hybridization (FISH). The data were compared with cytological, colposcopic, and histological findings. Of the 266 gynaecological smears, HPV DNA was detected in 84 (32%); of 101 cytologically and colposcopically HPV negative cases, HPV DNA was found in 10%. Of 56 women, cytologically and colposcopically positive for HPV infection, HPV DNA was detected in 68%. The sensitivity of the method was controlled by comparing the results of FISH with those of Southern-blot analysis of five cervical tumour biopsies. The data presented demonstrate the necessity of FISH for identification of the HPV type that might be of prognostic value in cervical pathology. Cytological and colposcopic positivity is a reliable sign in about 70% of the cases where HPV infection was proved by FISH.

Gene expression profiling in the remnant kidney model of wild type and kinin B1 and B2 receptor knockout mice.

Angiotensin-converting enzyme inhibitors are the most efficient pharmacologic agents to delay the development of end-stage renal disease (ESRD). This is a multipharmacologic approach that inhibits angiotensin II formation while increasing kinin concentrations. Considerable attention has been focused on the role of decreased angiotensin II levels; however, the role of increased kinin levels is gaining in interest. Kinins affect cellular physiology by interacting with one of two receptors being the more inducible B1 and the more constitutive B2 receptors. This study utilizes the mouse remnant kidney of 20 weeks duration as a model of ESRD. Whole mouse genome microarrays were used to evaluate gene expression in the remnant kidneys of wild type, B1 and B2 receptor knockout animals. The microarray data indicate that gene families involved in vascular damage, inflammation, fibrosis, and proteinuria were upregulated, whereas gene families involved in cell growth, metabolism, lipid, and protein biosynthesis were downregulated in the remnant kidneys. Interestingly, the microarray analyses coupled to histological evaluations are suggestive of a possible protective role of kinins operating through the B2 receptor subtype in this model of renal disease. The results highlight the potential of microarray technology for unraveling complex mechanisms contributing to chronic renal failure.

The RNA polymerase I transcription factor xUBF contains 5 tandemly repeated HMG homology boxes.

The RNA polymerase I transcription factor UBF has been identified in human, mouse, rat and Xenopus and the primary structure of the human protein has been determined. Human UBF was shown to contain four tandem homologies to the folding domains of the HMG1 and 2 proteins and hence to belong to a previously unrecognised family of 'HMG-box' transcription factors. Here, cDNA clones encoding the Xenopus laevis UBF (xUBF) have been isolated and sequenced. Northern and Southern blots revealed that in tissue culture cells, xUBF is coded on a single major mRNA size species by a small number of genes. The deduced primary structure of xUBF is highly homologous with the human protein except for a central deletion which removes most of one HMG-box. This explains the major size difference between the X. laevis and human proteins and may well explain their different transcriptional specificities. It is shown that xUBF contains 5 tandemly repeated HMG-boxes and that by analogy the human protein contains 6.

Determination of plasmid copy number by the "boiling" method.

A fast and reliable approach for determination of plasmid copy number in Escherichia coli is proposed, based on the "boiling" method (5) for separation of plasmid and chromosomal DNA. The method includes in vivo uniform labeling of total bacterial DNA, separation of DNA into plasmid and chromosomal DNA fractions, and quantitation of DNA in the two fractions by radioactivity measurement. No isolation and purification of native DNA are necessary.

Large scale purification of plasmid DNA.

A simple and rapid procedure for large scale purification of plasmid DNA is described. The procedure consists of two main steps: 1. Alkaline extraction of plasmid DNA (by a slight modification of the method of Birnboim and Doly (1)) and 2. Purification of the crude extract by hydroxyapatite chromatography. The plasmids obtained are biologically active and can be used in gene manipulation experiments.

Does the concentration of DNA (Co) and the time of incubation (t) as parameters of Cot influence the thermal stability of the DNA duplexes?

It has been shown in a previous paper (8) that the prime product of reassociation of related DNA sequences under open experimental conditions are mismatched duplexes which undergo 'maturation' upon further incubation. Due to this feature, the Tm value of the duplexes of a large number of DNAs is strongly dependent on the Cot value. Here we present data showing that the Tm of the duplexes of such type of DNAs depends also on the concentration of DNA in the range of one and the same Cot value. The significance of this finding in studying the taxonomic relationship by DNA-DNA hybridisation is discussed.

Sequence heterogeneity of the human alphoid satellite DNA and thermal stability of mismatched alphoid DNA duplexes.

1. 340 bp (dimer) and 680 bp (tetramer) fractions of the human alphoid satellite DNA (h alpha RI DNA) were isolated after complete cleavage of total human DNA with EcoR I and cloned in pBR 32.5. 2. Ten clones containing 340 bp inserts and one clone containing 680 bp insert were sequenced in order to investigate the sequence heterogeneity of this satellite DNA and the sequence data were compared with the consensus h alpha RI DNA sequence of Wu and Manuelidis (1980). 3. It was shown that in all clones studied the mutations are nonrandomly distributed along the human alphoid monomers forming distinct conservative and variable regions. 4. This mutation distribution pattern was compared with the nucleotide variations between the consensus sequences of different primate alphoid DNAs and it was found that the interspecies nucleotide divergency of this satellite DNA is quite similar to the intragenomic one. 5. The sequenced h alpha RI DNA clones were used for preparation of DNA-DNA hybrids with a known percentage of base pair mismatching. 6. These hybrids were melted on hydroxyapatite (HAP) and the results obtained were used to determine the relationship between the thermal stability (Tm) and the extent of base pair mismatching for naturally diverged DNA sequences. 7. A value of 0.7 degrees C decrease in Tm per 1% base pair mismatching was found.

Characterization of a polymorphism in the coding region of the human C5a anaphylatoxin receptor.

A polymorphism was identified in the coding region of the human C5a anaphylatoxin receptor gene leading to C to T transition at nucleotide position 450 (a silent substitution in the Ala150 codon, GCC to GCT). Its distribution was studied in a population of healthy volunteers from the Québec city region (prevalence of 2.8%) and among patients with end-stage renal failure who had previously undergone renal graft (prevalence 1.4%, not significantly different from that of the control group). This new marker provides a valuable tool to assess the risk for putative C5a-associated disorders with genetic determinism.