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 (M, = 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 pl9 is identical to the sequence ofthe 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 mI and IV had statistically significantly higher levels of p19/nm23 than tumors from patients with stages I and H. 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. (J. Clin. Invest. 1991. 88:341-345.)
High level expression of the nm23-Hl gene, which encodes for a nucleoside diphosphate kinase, has been found to correlate with diminished metastasis in some tumors but not in others. We have previously identified the protein product of the nm23-H1 gene in two-dimensional electrophoretic gels and have designated it p19/nm23. In neuroblastoma, higher levels of p19/ nm23, which are associated with amplification of the N-myc oncogene, large tumor mass, and metastasis, were observed in advanced stage tumors compared with limited stage disease. Because of the variable expression of nm23-H1 in different tumors, we have investigated the relationship between amounts of the protein and cell proliferation. The levels of p19/nm23 were compared between resting and mitotically stimulated normal human PBLs and in leukemia cells. The amount of p19/ nm23 increased in normal lymphocytes in response to mitotic stimulation and paralleled the increase in DNA synthesis. In leukemia cells obtained from patients with different subtypes of acute leukemia, p19/nm23 levels were also increased relative to resting normal lymphocytes. Treatment of mitotically stimulated lymphocytes with cyclosporin, which inhibits proliferation, blocked the increase in pl9/nm23; treatment of the leukemia cell line HL-60 with dimethylsulfoxide, which induces terminal differentiation, resulted in diminished levels of p19/nm23. Our data therefore provide evidence that nm23-Hl expression is related to cell proliferative activity. (J. Clin. Invest. 1992. 89:919-924.)
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.
Oncoprotein 18 (Op18) is a major cystolic phosphoprotein conlation, thus coupling of Op18 phosphorylation to cell proliferstituent of leukemia cells. There is cumulative evidence that ation remained intact. Materials and methods
Two-dimensional (2D) PAGE, using carrier ampholytes for the first-dimension separation, has provided a tool for the simultaneous analysis of cellular proteins. To extend the utility of 2D PAGE to the preparative level, we have investigated the use of immobilized pH gradients (IPG) for the first-dimension separation. The results we have obtained indicate that as much as 1 mg of cellular protein can be loaded onto a single IPG gel without loss of resolution. Mutant polypeptides previously detected in carrier ampholyte-based 2D gels were equally detectable in IPG-based 2D gels. With IPG gels several hundred cellular polypeptides can be isolated, from as few as 10 gels, in sufficient amount for sequencing with current sequencing technology. We therefore conclude that IPG greatly enhances the prospects for the large-scale sequencing of cellular proteins for the development of 2D gel-related protein data bases and for the identification of new polypeptide gene products, with the attendant implications for a genome sequencing effort.The structure or function of most proteins in a cell remains unknown. From the analytical point of view, two-dimensional (2D) PAGE is unmatched in its ability to simultaneously resolve hundreds of cellular polypeptides (1). However, its potential to contribute to our understanding of molecular processes involving proteins remains largely untapped, in part because ofdifficulty in establishing structural identity for polypeptides observed in 2D gels. The ability to scale up the amount of protein loaded onto the isoelectric focusing gels, to yield sufficient amount of the polypeptide of interest for sequencing, is limited by loss of resolution at higher protein loadings. As a result, various strategies have been devised for structural studies, including protein enrichment procedures, pooling of polypeptide spots from a large number ofgels, and reliance on improved techniques for sequencing.As an alternative to the use of carrier ampholytes (CA) for 2D electrophoresis, we have utilized immobilized pH gradients (IPG) for the first-dimension separation (2, 3). IPG presents certain advantages because the pH gradient is an integral part of the polyacrylamide gel matrix. Thus, the pH separation range can be defined from broad to extremely narrow. Basic polypeptides can be resolved well, and precast gels can be produced in large batches for subsequent use as individual strips, as needed, based on the number of samples in an electrophoretic run.In this report, we describe the preparative use of IPG gels for the first-dimension separation step of 2D PAGE. Preparative loads of 1 mg of protein were applied onto individual IPG strips without loss of resolution; this substantially augments the number of polypeptides that can be sequenced from a small number of 2D gels. MATERIALS AND METHODSPreparation of IPG Strips for Electrophoresis. IPG precast gels were purchased from Pharmacia LKB; they were cast based on specifications provided to the manufacturer. Gradient slab gels of 0.5 mm in thickness we...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.