High levels of p19/nm23 protein in neuroblastoma are associated with advanced stage disease and with N-myc gene amplification.

The gene encoding a novel protein designated nm23-H1, which was recently identified as identical to the A subunit of nucleotide diphosphate kinase from human erythrocytes, has been proposed to play a role in tumor metastasis suppression. We report that untreated neuroblastoma tumors contain a cellular polypeptide (Mr = 19,000) designated p19, identified in two-dimensional electrophoretic gels, which occurs at significantly higher levels (P = 0.0001) in primary tumors containing amplified N-myc gene. The partial amino acid sequence obtained for p19 is identical to the sequence of the human nm23-H1 protein. An antibody to the A subunit of erythrocyte nucleotide diphosphate kinase reacted exclusively with p19. In this study, significantly higher levels of p19/nm23 occurred in primary neuroblastoma tumors from patients with advanced stages (III and IV) relative to tumors from patients with limited stages (I and II) of the disease. Even among patients with a single copy of the N-myc gene, tumors from patients with stages III and IV had statistically significantly higher levels of p19/nm23 than tumors from patients with stages I and II. Our findings indicate that, in contrast to a proposed role for nm23-H1 as a tumor metastasis suppressor, increased p19/nm23 protein in neuroblastoma is correlated with features of the disease that are associated with aggressive tumors. Therefore, nm23-H1 may have distinct if not opposite roles in different tumors.

Transcription vectors that facilitate the identification and mapping of RNA splice sites in genomic DNA.

Two transcription vectors were constructed that can identify the splice sites at exon-intron boundaries of inserted DNA fragments possessing the complementary splice site. One vector contains the 5' splice donor site and flanking exon-intron sequences from the 3' end of the adenovirus first late leader. The other vector contains the 3' splice acceptor site and the branch acceptor site, plus the flanking exon-intron sequences from the 5' end of the adenovirus second late leader. Both vectors contain a multiple cloning site for insertion of DNA fragments. DNA fragments supplying the complementary splice site, including the adjacent exon and intron sequences, were inserted into the vectors. The vectors were used as templates for the synthesis of chimeric RNA transcripts that were spliced in in vitro splicing extracts. Chimeric transcripts from the vectors containing complementary splice site boundary regions from the human growth hormone gene were accurately spliced in vitro. A splice site from a human growth hormone intron that is not normally spliced in vitro was spliced when paired with an adenovirus splice site. These vectors can be used to identify splice sites and to determine the lengths of exons and their attached introns within a DNA fragment of unknown coding content.

Mechanism of interaction of Salmonella and Schistosoma species.

In endemic areas where Salmonella and Schistosoma species co-occur, several lines of evidence suggest a synergistic bacteria-parasite interaction that results in a protracted course for the salmonella infection that has proven difficult to diagnose and therapeutically remedy. In an in vitro system using a pilus-negative and a pilus-producing transductant strain of Salmonella typhimurium we show that pili are the ligands for bacterial adherence to the schistosome surface tegument. Antipili antibodies produced in rabbits against purified pili, purified and digested to monovalent (Fab) fragments, blocked the association of Salmonella sp. to the surface tegument of Schistosoma sp., further demonstrating that pili are the appendages necessary for bacteria-parasite surface interaction. The use of carbohydrates, lectins, and enzymes demonstrated that the bacteria-parasite surface interaction was specific, mediated by pili that specifically recognize and bind to mannose-like receptors, probably glycolipids, on the surface of the worms. We suggest that prolonged salmonellosis in schistosome-infected patients is due to an association of Salmonella sp. with the schistosome worms themselves and further that the schistosome worms provide a multiplication focus for these bacteria in the portal mesenteric system, with a persisting bacteremia following.

Characterization of the gene for a proliferation-related phosphoprotein (oncoprotein 18) expressed in high amounts in acute leukemia.

The oncoprotein 18 (Op18) gene encodes a proliferation-related cytosolic phosphoprotein, which is induced in normal lymphocytes following mitogenic stimulation. Studies of the Op18 gene are of particular interest because of the proposed role of Op18 protein in signal transduction and because of its occurrence in markedly increased amounts in acute leukemia cells. We have recently reported the cloning and sequencing of two cDNA clones for Op18 (1 and 1.5 kilobases). Both clones code for the same 149-amino acid polypeptide; however, they differ in their 3'-region as a result of alternative polyadenylation. We report here the sequencing of the Op18 gene and describe its expression in leukemia. The Op18 gene, which is 6.3 kilobases in length, is comprised of five exons and four introns and exhibits features that are common to other genes involved in cellular growth and proliferation. The increase in Op18 polypeptide in leukemia is associated with increased RNA transcription without gene amplification or rearrangement. Treatment of K562 leukemia cell line with hemin that induces terminal differentiation resulted in decreased expression of Op18. Our findings suggest that the high amount of Op18 protein in acute leukemia results from increased expression of a structurally unaltered gene.

Involvement of OP18 in cell proliferation.

Op18 is a highly conserved major cytosolic phosphoprotein that is expressed at high levels in acute leukemia and in neuroblastoma. In this study we present evidence pointing to a role for Op18 in cellular proliferation. Blocking of Op18 mRNA translation using antisense oligonucleotides delayed entrance of mitotically stimulated normal peripheral blood lymphocytes into the S phase. Moreover treatment of HL-60 promyelocytic leukemia cells with DMSO or PMA which induced terminal differentiation resulted in a decrease in the level of Op18 RNA and protein. Inhibition of lymphoid proliferation with cyclosporin also resulted in reduced Op18 levels.

Antibodies specific for branched ribonucleic acids.

A chemically synthesized branched tetranucleotide, G3'p5'A [2'p5'G]3'p5'C corresponding to the consensus sequence at the branch point in introns undergoing RNA splicing, was used as a hapten to elicit antibranch antibodies. Binding assays with 32P-labeled hapten and unlabeled structurally related haptens indicated that the antibodies are highly specific for the branch structure and have some specificity for the A2'p5'G sequence at the branch point, but have essentially none for a variety of other 2'p5' or 3'p5' dinucleotides or for the linear trinucleotide G3'p5'A3'p5'C. Purification of these antibodies by binding to A2'p5'G covalently linked to Sepharose followed by covalent attachment of the purified antibodies to protein A-Sepharose has provided an adsorbent that immunospecifically retains branched oligonucleotides as well as branched introns released from RNAs during in vitro splicing.

Activation of resting peripheral blood lymphocytes through the T cell receptor induces rapid phosphorylation of Op18.

Op18 is a highly conserved major cytosolic phosphoprotein that has been implicated in signal transduction in a wide variety of cell types. Freshly isolated peripheral blood lymphocytes (PBL) constitutively express low levels of mostly unphosphorylated Op18. After mitogenic stimulation of PBL, Op18 synthesis is induced at a time when cells are entering S-phase. In this study, we have examined the phosphorylation of Op18 in freshly isolated PBL after activation of the T cell receptor by OKT3. Quantitative analysis of Op18 phosphorylation was undertaken by metabolic labeling with 32Pi and PhosphorImager analysis of two-dimensional gels. After 10 or 15 min of activation by OKT3, one of the three major phosphorylated forms of Op18, designated Op18c, increased approximately 10-fold, which represented a most pronounced change among a large number of phosphoproteins analyzed. In time course experiments, increased Op18 phosphorylation to yield Op18c was observed as early as 2 min. Continued OKT3-induced activation for 20 to 72 h resulted in a further increase in phosphorylated Op18 forms, which paralleled new Op18 synthesis and occurred at a time when cells were entering S-phase, as determined by [3H]-thymidine incorporation. Inhibitors of lymphoid proliferation, cyclosporin A and RPM, had no effect on early (less than 15 min) phosphorylation. Addition of calphostin C, a specific inhibitor of protein kinase C, 1 min prior to stimulation of resting T cells with OKT3 completely inhibited further phosphorylation of Op18. Incubation of PBL with calphostin C for 75 min decreased constitutive levels of phosphorylated Op18. In contrast, inhibition of cyclic nucleotide-dependent protein kinases with HA1004 had no effect on Op18 phosphorylation. Activation of cAMP-dependent protein kinase with Forskolin or 8Br-cAMP did not increase Op18 phosphorylation. Our results suggest that Op18 phosphorylation is mediated by protein kinase C activation as an early event in T cell activation through the T cell receptor.