Cytosolic high K-m 5 '-nucleotidase and 5 '(3 ')-deoxyribonucleotidase in substrate cycles involved in nucleotide metabolism

5'-Nucleotidases are the catabolic members of the substrate cycles postulated to be involved in the regulation of intracellular deoxyribonucleoside triphosphate pools. Here, we attempt to identify the nature of the nucleotidases. Earlier, we constructed various mammalian cell lines that can be induced to overproduce the high K(m) 5'-nucleotidase (hkm-NT) or the 5'(3')-deoxynucleotidase (dNT-1). Now we labeled control and induced human 293 cells and hamster V79 cells with radioactive hypoxanthine or uridine and during a chase measured quantitatively the metabolism of ribo- and deoxyribonucleotides, DNA replication, and excretion of nucleosides into the medium. Overproduction of hkm-NT greatly increased excretion of inosine and guanosine but did not affect adenosine or deoxyribonucleosides. dNT-1 overproduction increased excretion of deoxycytidine, thymidine, and in particular deoxyuridine but also uridine and cytidine. We conclude that the hkm-NT is not involved in the regulation of deoxyribonucleotide pools but affects IMP and GTP pools. dNT-1, instead, appears to be the catabolic arm of substrate cycles regulating pyrimidine nucleotide pools

The relative endogenous expression levels of the IFNAR2 isoforms influence the cytostatic and pro-apoptotic effect of IFNalpha on pleomorphic sarcoma cells

Based on our previous studies where we found that IFNAR2-1, the short IFNalpha/beta receptor variant, was expressed in pleomorphic sarcoma cells, we decided to determine the relative levels of expression of IFNAR2.1 versus the longer form, named IFNAR2.2, in different pleomorphic sarcoma cells in relation to their response to interferon alpha treatment. When examining a panel of PS cells isolated from surgical specimens, we found that IFNAR2.1 prevailed in 6 out 7 lines analysed and that these generally showed cell cycle arrest and low levels of apoptosis upon IFNalpha treatment. The reverse ratio, i.e. higher constitutive levels of IFNAR2.2 than IFNAR2.1, was associated with an irreversible inhibition of cell growth and pronounced apoptosis. Impairment of tumour growth by low- and high-dose IFNalpha treatment of nude mice inoculated with PS cells expressing predominantly IFNAR2.1 further asserted the effect of the cytokine also in vivo. A proteomic analysis of 120 signalling components in growth arrested, apoptotic PS cells harbouring higher levels of IFNAR2.2 revealed engagement of the canonical Jak/Stat/ISGF3-pathway, the activation of the mitochodrial apoptotic pathway and a potentially novel mechanism of cell cycle blockade unrelated to down-regulation of cyclin A/B and their interacting/regulating kinases. Our results confirm the dominant negative role of IFNAR2.1, but also suggest that the relative endogenous levels of the two IFNalpha/beta receptor isoforms may dictate the signalling pathways triggered by the ligand, such as to cause exclusively cell cycle arrest or induce programmed cell death. This parameter may be of importance for the clinical outcome of IFNalpha treatment of PS

The relative endogenous expression levels of the IFNAR2 isoforms influence the cytostatic and pro-apoptotic effect of IFNa on pleomorphic sarcoma cells

Based on our previous studies where we found that IFNAR2-1, the short IFNalpha/beta receptor variant, was expressed in pleomorphic sarcoma cells, we decided to determine the relative levels of expression of IFNAR2.1 versus the longer form, named IFNAR2.2, in different pleomorphic sarcoma cells in relation to their response to interferon alpha treatment. When examining a panel of PS cells isolated from surgical specimens, we found that IFNAR2.1 prevailed in 6 out 7 lines analysed and that these generally showed cell cycle arrest and low levels of apoptosis upon IFNalpha treatment. The reverse ratio, i.e. higher constitutive levels of IFNAR2.2 than IFNAR2.1, was associated with an irreversible inhibition of cell growth and pronounced apoptosis. Impairment of tumour growth by low- and high-dose IFNalpha treatment of nude mice inoculated with PS cells expressing predominantly IFNAR2.1 further asserted the effect of the cytokine also in vivo. A proteomic analysis of 120 signalling components in growth arrested, apoptotic PS cells harbouring higher levels of IFNAR2.2 revealed engagement of the canonical Jak/Stat/ISGF3-pathway, the activation of the mitochodrial apoptotic pathway and a potentially novel mechanism of cell cycle blockade unrelated to down-regulation of cyclin A/B and their interacting/regulating kinases. Our results confirm the dominant negative role of IFNAR2.1, but also suggest that the relative endogenous levels of the two IFNalpha/beta receptor isoforms may dictate the signalling pathways triggered by the ligand, such as to cause exclusively cell cycle arrest or induce programmed cell death. This parameter may be of importance for the clinical outcome of IFNalpha treatment of PS

Human high Km 5'-nucleotidase: effects of overexpression of the cloned cDNA in cultured human cells.

Deoxynucleoside triphosphates (dNTPs) used for mitochondrial DNA replication are mainly formed by phosphorylation of deoxynucleosides imported into mitochondria from the cytosol. We earlier obtained evidence for a mitochondrial 5'-nucleotidase (dNT2) with a pronounced specificity for dUMP and dTMP and suggested that the enzyme protects mitochondrial DNA replication from excess dTTP. In humans, accumulation of dTTP causes a mitochondrial genetic disease. We now establish that dNT2 in vivo indeed is located in mitochondria. The native enzyme shows the same substrate specificity and affinity for inhibitors as the recombinant dNT2. We constructed ponasterone-inducible cell lines overproducing dNT2 with and without the green fluorescent protein (GFP) linked to its C terminus. The fusion protein occurred in mitochondria mostly in an inactive truncated form, with only a short C-terminal fragment of dNT2 linked to GFP. No truncation occurred when dNT2 and GFP were not linked. The cell mitochondria then contained a large excess of active dNT2 with or without the mitochondrial presequence. After removal of ponasterone overproduced dNT2 disappeared only slowly from the cells, whereas dNT2-mRNA was lost rapidly. Overproduction of dNT2 did not lead to an increased excretion of pyrimidine deoxyribonucleosides, in contrast to overproduction of the corresponding cytosolic deoxynucleotidase, suggesting that the mitochondrial enzyme does not affect overall cellular deoxynucleotide turnover

Targeted discovery tools: proteomics and chromatin immunoprecipitation-on-chip

Item does not contain fulltextDespite the availability of several completely sequenced genomes, we are still, for the most part, ignorant about how genes interact and regulate each other within a given cell type to specify identity, function and cellular memory. A realistic model of cellular regulation based on current knowledge indicates that many interacting networks operate at the epigenetic, transcriptional, translational and post-translational levels, with feedback between the various levels. Protein-protein and protein-DNA interactions help to define which genes may be activated in a particular cell, and determine whether external cues cause activation or repression. New technologies, e.g. proteomics using mass spectrometry, high-density DNA or oligonucleotide microarrays (chips), and chromatin immunoprecipitation (ChIP), provide new and exciting tools for deciphering the pathways and proteins controlling gene expression. Analysis of these pathways offers new insight that aids targeted drug development