Cell proliferation and drug sensitivity of human glioblastoma cells are altered by the stable modulation of cytosolic 5'-nucleotidase II.

Cytosolic 5'-nucleotidase II (cN-II) has been reported to be involved in cell survival, nucleotide metabolism and in the cellular response to anticancer drugs. With the aim to further evaluate the role of this enzyme in cell biology, we stably modulated its expression the human glioblastoma cell ADF in which the transient inhibition of cN-II has been shown to induce cell death. Stable cell lines were obtained both with inhibition, obtained with plasmids coding cN-II-targeting short hairpin RNA, and stimulation, obtained with plasmids coding Green Fluorescence Protein (GFP)-fused wild type cN-II or a GFP-fused hyperactive mutant (GFP-cN-II-R367Q), of cN-II expression. Silenced cells displayed a decreased proliferation rate while the over expressing cell lines displayed an increased proliferation rate as evidenced by impedance measurement using the xCELLigence device. The expression of nucleotide metabolism relevant genes was only slightly different between cell lines, suggesting a compensatory mechanism in transfected cells. Cells with decreased cN-II expression were resistant to the nucleoside analog fludarabine confirming the involvement of cN-II in the metabolism of this drug. Finally, we observed sensitivity to cisplatin in cN-II silenced cells and resistance to this same drug in cN-II over-expressing cells indicating an involvement of cN-II in the mechanism of action of platinum derivatives, and most probably in DNA repair. In summary, our findings confirm some previous data on the role of cN-II in the sensitivity of cancer cells to cancer drugs, and suggest its involvement in other cellular phenomenon such as cell proliferation.

The purine analog fludarabine acts as a cytosolic 5'-nucleotidase II inhibitor.

For several years the IMP/GMP-preferring cytosolic 5'-nucleotidase II (cN-II) has been considered as a therapeutic target in oncology. Indeed, various reports have indicated associations between cN-II expression level and resistance to anticancer agents in several cancer cell lines and in patients affected with neoplasia, mainly by hematologic malignancies. In this paper we present evidence showing that, among the commonly used cytotoxic nucleoside analogs, fludarabine can act as a cN-II inhibitor. In vitro studies using the wild type recombinant cN-II demonstrated that fludarabine inhibited enzymatic activity in a mixed manner (Ki 0.5 mM and Ki' 9 mM), whereas no inhibition was observed with clofarabine and cladribine. Additional experiments with mutant recombinant proteins and an in silico molecular docking indicated that this inhibition is due to an interaction with a regulatory site of cN-II known to interact with adenylic compounds. Moreover, synergy experiments between fludarabine and 6-mercaptopurine in human follicular lymphoma (RL) and human acute promyelocytic leukemia (HL-60) cells transfected with control or cN-II-targeting shRNA-encoding plasmids, showed synergy in control cells and antagonism in cells with decreased cN-II expression. This is in line with the hypothesis that fludarabine acts as a cN-II inhibitor and supports the idea of using cN-II inhibitors in association with other drugs to increase their therapeutic effect and decrease their resistance.

On the physiological role of cytosolic 5'-nucleotidase II (cN-II): pathological and therapeutical implications.

Among the members of the 5'-nucleotidase family, there is only one membrane-bound ectosolic isoenzyme. This esterase prefers AMP as substrate but can hydrolyze a number of purine and pyrimidine phosphorylated compounds, indicating that no evolutive pressure to develop a more restricted specificity was exerted on this enzyme. On the contrary, five cytosolic isoforms have been evolved, probably by convergent evolution, showing different and restricted substrate specificity. The different isoforms have different level of expression and distribution in organs of vertebrates. The cytosolic nucleotidase specific for IMP and GMP (cN-II), is an enzyme allosterically regulated, structurally strongly conserved and expressed at a low but constant level in all organs and tissues in vertebrates. As far as we know, alteration of cN-II expression is limited to pathological conditions. In this review, we report the results of the modulation of cN-II specific activity exerted by silencing or hyperexpression in different cell types, in the attempt to better understand its role and implications in pathology and therapy.

5 '-Amino-4-imidazolecarboxamide riboside induces apoptosis in human neuroblastoma cells via the mitochondrial pathway.

5'-Amino-4-imidazolecarboxamide (AICA) riboside induces apoptosis in neuronal cell models. In order to exert its effect, AICA riboside must enter the cell and be phosphorylated to the ribotide. In the present work, we have further studied the mechanism of apoptosis induced by AICA riboside. The results demonstrate that AICA riboside activates AMP-dependent protein kinase (AMPK), induces release of cytochrome c from mitochondria and activation of caspase 9. The role of AMPK in determining cell fate is controversial. In fact, AICA riboside has been reported to be neuroprotective or to induce apoptosis depending on its concentration, cell type or apoptotic stimuli used. In order to clarify whether the activation of AMPK is related to apoptosis in our model, we have used another AMPK stimulator, metformin, and we have analysed its effects on cell viability, nuclear morphology and AMPK activity. Five mM metformin increased AMPK activity, inhibited viability, and increased the number of apoptotic nuclei. AICA riboside, which can be generated from the ribotide (an intermediate of the purine de novo synthesis) by the action of the ubiquitous cytosolic 5'-nucleotidase (cN-II), may accumulate in those individuals in which an inborn error of purine metabolism causes both a building up of intermediates and/or an increase of the rate of de novo synthesis, and/or an overexpression of cN-II. Therefore, our results suggest that the toxic effect of AICA riboside on some types of neurons may participate in the neurological manifestations of syndromes related to purine dismetabolisms.

Identification of the 5'-nucleotidase activity altered in neurological syndromes.

5'-Nucleotidases comprise a family of enzymes involved in the regulation of intracellular and extracellular nucleotide concentration. There is increasing knowledge about an involvement of these activities in the aetiology of neurological disorders. In this paper we present a protocol for the identification of the altered enzyme in fibroblasts primary culture from patients and controls.

5'-aminoimidazole-4-carboxamide riboside induces apoptosis in human neuroblastoma cells.

5'-Aminoimidazole-4-carboxamide riboside (AICA riboside) has been previously shown to be toxic to two neuronal cell models [Neuroreport 11 (2000) 1827]. In this paper we demonstrate that AICA riboside promotes apoptosis in undifferentiated human neuroblastoma cells (SH-SY5Y), inducing a raise in caspase-3 activity. In order to exert its effect on viability, AICA riboside must enter the cells and be phosphorylated to the ribotide, since both a nucleoside transport inhibitor, and an inhibitor of adenosine kinase produce an enhancement of the viability of AICA riboside-treated cells. Short-term incubations (2 h) with AICA riboside result in five-fold increase in the activity of AMP-dependent protein kinase (AMPK). However, the activity of AMPK is not significantly affected at prolonged incubations (48 h), when the apoptotic effect of AICA riboside is evident. The results demonstrate that when the cell line is induced to differentiate both toward a cholinergic phenotype (with retinoic acid) or a noradrenergic phenotype (with phorbol esters), the toxic effect is significantly reduced, and in the case of the noradrenergic phenotype differentiation, the riboside is completely ineffective in promoting apoptosis. This reduction of effect correlates with an overexpression of Bcl-2 during differentiation. AICA riboside, derived from the hydrolysis of the ribotide, an intermediate of purine de novo synthesis, is absent in normal healthy cells; however it may accumulate in those individuals in which an inborn error of purine metabolism causes an increase in the rate of de novo synthesis and/or an overexpression of cytosolic 5'-nucleotidase, that appears to be the enzyme responsible for AICA ribotide hydrolysis. In fact, 5'-nucleotidase activity has been shown to increase in patients affected by Lesch-Nyhan syndrome in which both acceleration of de novo synthesis and accumulation of AICA ribotide has been described, and also in other neurological disorders of unknown etiology. Our results raise the intriguing clue that the neurotoxic effect of AICA riboside on the developing brain might contribute to the neurological manifestations of syndromes related to purine dismetabolisms.

Catabolism of exogenous deoxyinosine in cultured epithelial amniotic cells.

Uptake and catabolism of purine nucleosides have been commonly considered as means to salvage the purine ring for nucleic acid synthesis, usually neglecting the destiny of the pentose moiety. With the aim to ascertain if deoxyribose derived from exogenous DNA can be utilised as a carbon and energy source, we studied the catabolism of exogenous deoxyinosine in a cell line derived from human amnion epithelium (WISH). Intact WISH cells catabolise deoxyinosine by conversion into hypoxanthine. The nucleoside enters the cell through a nitrobenzylthioinosine-insensitive equilibrative transport. Deoxyinosine undergoes a phosphorolytic cleavage inside the cell. The purine base diffuses back to the external medium, while the phosphorylated pentose moiety can be further catabolised to glycolysis and citric acid cycle intermediates. Our results indicate that the catabolism of the deoxynucleoside can be considered mainly as a means to meet the carbon and energy requirements of growing cells.

Bovine cytosolic 5'-nucleotidase acts through the formation of an aspartate 52-phosphoenzyme intermediate.

Cytosolic 5'-nucleotidase/phosphotransferase (cN-II), specific for purine monophosphates and their deoxyderivatives, acts through the formation of a phosphoenzyme intermediate. Phosphate may either be released leading to 5'-mononucleotide hydrolysis or be transferred to an appropriate nucleoside acceptor, giving rise to a mononucleotide interconversion. Chemical reagents specifically modifying aspartate and glutamate residues inhibit the enzyme, and this inhibition is partially prevented by cN-II substrates and physiological inhibitors. Peptide mapping experiments with the phosphoenzyme previously treated with tritiated borohydride allowed isolation of a radiolabeled peptide. Sequence analysis demonstrated that radioactivity was associated with a hydroxymethyl derivative that resulted from reduction of the Asp-52-phosphate intermediate. Site-directed mutagenesis experiments confirmed the essential role of Asp-52 in the catalytic machinery of the enzyme and suggested also that Asp-54 assists in the formation of the acyl phosphate species. From sequence alignments we conclude that cytosolic 5'-nucleotidase, along with other nucleotidases, belong to a large superfamily of hydrolases with different substrate specificities and functional roles.

By releasing ADP, Acanthamoeba castellanii causes an increase in the cytosolic free calcium concentration and apoptosis in wish cells.

The role played by soluble molecules that may participate in acanthamoebal cytopathogenicity has yet to be fully characterized. We demonstrate here that Acanthamoeba castellanii trophozoites constitutively release ADP in the medium. Cell-free supernatants prepared from A. castellanii, by interaction with specific P(2y2) purinoceptors expressed on the Wish cell membrane, caused a biphasic rise in [Ca(2+)](i), extensive cell membrane blebbing, cytoskeletal disorganization, and the breakdown of nuclei. Cell damage induced by amoebic supernatants was blocked by the P(2y2) inhibitor Suramin. The same results were found in Wish cells exposed to purified ADP. These findings suggest that pathogenic free-living A. castellanii may have a cytopathic effect on human epithelial cells through ADP release, by a process that begins with a rise of cytosolic free-calcium concentration, and culminates in apoptosis.

Local delivery of human tissue kallikrein gene accelerates spontaneous angiogenesis in mouse model of hindlimb ischemia.

BACKGROUND: Human tissue kallikrein (HK) releases kinins from kininogen. We investigated whether adenovirus-mediated HK gene delivery is angiogenic in the context of ischemia. METHODS AND RESULTS: Hindlimb ischemia, caused by femoral artery excision, increased muscular capillary density (P:<0.001) and induced the expression of kinin B(1) receptor gene (P:<0.05). Pharmacological blockade of B(1) receptors blunted ischemia-induced angiogenesis (P:<0.01), whereas kinin B(2) receptor antagonism was ineffective. Intramuscular delivery of adenovirus containing the HK gene (Ad. CMV-cHK) enhanced the increase in capillary density caused by ischemia (969+/-32 versus 541+/-18 capillaries/mm(2) for control, P:<0.001), accelerated blood flow recovery (P:<0.01), and preserved energetic charge of ischemic muscle (P:<0.01). Chronic blockade of kinin B(1) or B(2) receptors prevented HK-induced angiogenesis. CONCLUSIONS: HK gene delivery enhances the native angiogenic response to ischemia. Angiogenesis gene therapy with HK might be applicable to peripheral occlusive vascular disease.

Role of the phosphorolysis of deoxyadenosine in the cytotoxic effect of the combination of deoxyadenosine and deoxycoformycin on a human colon carcinoma cell line (LoVo).

In LoVo cells, phosphorolytic activity acting on deoxyadenosine plays a major role in the resistance to the cytotoxic effect of the combination of deoxynucleoside with deoxycoformycin. In fact, the observed dependence of toxicity on cell density appears to be related to the metabolic conversion of deoxyadenosine into adenine. The phosphorylation of the deoxynucleoside, which represents the first step towards the formation of the cytotoxic agent dATP, proceeds at a significantly lower rate as compared to the phosphorolysis of deoxyadenosine. The analysis of the levels of deoxyadenosine and its derivatives in the incubation media reveals that the rates of disappearance of deoxyadenosine and of formation of adenine increase in concert with the reduction of the effect on cell survival.

Cytosolic 5'-nucleotidase hyperactivity in erythrocytes of Lesch-Nyhan syndrome patients.

Lesch-Nyhan syndrome is a metabolic-neurological syndrome caused by the X-linked deficiency of the purine salvage enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRT). Metabolic consequences of HGPRT deficiency have been clarified, but the connection with the neurological manifestations is still unknown. Much effort has been directed to finding other alterations in purine nucleotides in different cells of Lesch-Nyhan patients. A peculiar finding was the measure of appreciable amount of Z-nucleotides in red cells. We found significantly higher IMP-GMP-specific 5'-nucleotidase activity in the erythrocytes of seven patients with Lesch-Nyhan syndrome than in healthy controls. The same alteration was found in one individual with partial HGPRT deficiency displaying a severe neurological syndrome, and in two slightly hyperuricemic patients with a psychomotor delay. Since ZMP was a good substrate of 5'-nucleotidase producing Z-riboside, we incubated murine and human cultured neuronal cells with this nucleoside and found that it is toxic for our models, promoting apoptosis. This finding suggests an involvement of the toxicity of the Z-riboside in the pathogenesis of neurological disorders in Lesch-Nyhan syndrome and possibly in other pediatric neurological syndromes of uncertain origin.

Dilated and failing cardiomyopathy in bradykinin B(2) receptor knockout mice.

BACKGROUND: The activation of B(2) receptors by kinins could exert cardioprotective effects in myocardial ischemia and heart failure. METHODS AND RESULTS: To test whether the absence of bradykinin B(2) receptors may affect cardiac structure and function, we examined the developmental changes in blood pressure (BP), heart rate, and heart morphology of bradykinin B(2) receptor gene knockout (B(2)(-/-)), heterozygous (B(2)(+/-)), and wild-type (B(2)(+/+)) mice. The BP of B(2)(-/-) mice, which was still normal at 50 days of age, gradually increased, reaching a plateau at 6 months (136+/-3 versus 109+/-1 mm Hg in B(2)(+/+), P<0.01). In B(2)(+/-) mice, BP elevation was delayed. At 40 days, the heart rate was higher (P<0.01) in B(2)(-/-) and B(2)(+/-) than in B(2)(+/+) mice, whereas the left ventricular (LV) weight and chamber volume were similar among groups. Thereafter, the LV growth rate of B(2)(-/-) and B(2)(+/-) mice was accelerated, leading at 360 days to a LV weight-to-body weight ratio that was 9% and 17% higher, respectively, than that of B(2)(+/+) mice. In B(2)(-/-) mice, hypertrophy was associated with a marked chamber dilatation (42% larger than that of B(2)(+/+) mice), an elevation in LV end-diastolic pressure (25+/-3 versus 5+/-1 mm Hg in B(2)(+/+) mice, P<0.01), and reparative fibrosis. CONCLUSIONS: The disruption of the bradykinin B(2) receptor leads to hypertension, LV remodeling, and functional impairment, implying that kinins are essential for the functional and structural preservation of the heart.

6-thioguanine resistance in a human colon carcinoma cell line with unaltered levels of hypoxanthine guanine phosphoribosyltransferase activity.

Cell populations resistant to high doses (30 microM) of 6-thioguanine (6-TG, 6-TG(r) cells) were selected from a human colon carcinoma cell line, LoVo. This cell line, which lacks hMSH2, a component of the human mismatch binding heterodimer hMutSalpha, is resistant to low doses of 6-TG. The level of activity of hypoxanthine-guanine phosphoribosyltransferase, the enzyme responsible for the phosphoribosylation of the thiopurine, was comparable to that expressed in the parental cells. No significant difference was found in the levels of enzyme activities involved in the conversion of 6-TG or its derivatives into non-toxic compounds. In contrast, a significant difference was found in the uptake kinetics of 6-TG in the 2 cell types. Net uptake of 6-TG ceased after 100-sec incubation in the 6-TG(r) cells, while it appeared to continue throughout the 10-min incubation in the wild-type cells. As a consequence, after 10-min incubation, the total amount of 6-TG taken up by the parental LoVo cells was approximately 3 times higher than that present in the 6-TG(r) cells.

Identification, separation and characterisation of two forms of cytosolic 5'-nucleotidase/nucleoside phosphotransferase in calf thymus.

Cytosolic 5'-nucleotidase, acting preferentially on IMP, GMP and their deoxyderivatives, endowed with phosphotransferase activity, is a widespread enzyme responsible for the regulation of intracellular IMP and GMP concentrations and the phosphorylation of purine nucleoside pro-drugs. The enzyme activity is stimulated by ATP, ADP and 2,3-bisphosphoglycerate (BPG), and is inhibited by phosphate. Calf thymus possesses two active proteins with a different electrophoretic mobility. In this report we show that the two forms can be separated by ADP-agarose affinity chromatography. Whereas form A binds weakly to the column, form B is tightly bound and is released by the addition of ADP into the elution buffer. The two enzyme forms differ in terms of electrophoretic, chromatographic behaviour and regulatory characteristics. Form B, as already described for the enzyme purified from the same source (Pesi et al., 1996, Biochim Biophys Acta 294, 191-194), exhibits three different sites for the three activators with a synergistic effect between ADP and BPG. Form A has a high affinity regulatory site for BPG, while ADP and ATP appear to share the same low affinity site and no synergistic effect is observed.

Deoxyadenosine metabolism in a human colon-carcinoma cell line (LoVo) in relation to its cytotoxic effect in combination with deoxycoformycin.

We have assessed the intracellular metabolism of 2'-deoxyadenosine in a human colon-carcinoma cell line (LoVo), both in the absence and in the presence of deoxycoformycin, the powerful inhibitor of adenosine deaminase. The combination of 2'-deoxyadenosine and deoxycoformycin has been reported to inhibit the growth of LoVo cells in culture. In this paper we demonstrate that the observed toxic effect is strictly dependent on cell density. In the absence of deoxycoformycin, 2'-deoxyadenosine is primarily deaminated to 2'-deoxyinosine and then converted into hypoxanthine. In the presence of the inhibitor, the deoxynucleoside, in addition to a phosphorylation process, undergoes phosphorolytic cleavage giving rise to adenine. The conversion of 2'-deoxyadenosine to adenine might represent a protective device, emerging when the activity of adenosine deaminase is reduced or inhibited. There is much evidence to indicate that the enzyme catalyzing this process may be distinct from methylthioadenosine phosphorylase and S-adenosyl homocysteine hydrolase, which are the enzymes reported to be responsible for the formation of adenine from 2'-deoxyadenosine in mammals.

Channelling of deoxyribose moiety of exogenous DNA into carbohydrate metabolism: role of deoxyriboaldolase.

In bacteria, the addition of (deoxy)nucleosides or (deoxy)ribose to the growth medium causes induction of enzymes involved in their catabolism, leading to the utilisation of the pentose moiety as carbon and energy source. In this respect, deoxyriboaldolase appears the key enzyme, allowing the utilisation of deoxyribose 5-P through glycolysis. We observed that not only deoxynucleosides, but also DNA added to the growth medium of Bacillus cereus induced deoxyriboaldolase; furthermore, the switch of the culture from aerobic to anaerobic conditions caused a further increase in enzyme activity, leading to a more efficient channelling of deoxyribose 5-P into glycolysis, probably as a response to the low energy yield of the sugar fermentation. In eukaryotes, the catabolism of (deoxy)nucleosides is well known. However, the research in this field has been mainly devoted to the salvage of the bases formed by the action of nucleoside phosphorylases, whereas the metabolic fate of the sugar moiety has been largely neglected. Our results indicate that the deoxyriboaldolase activity is present in the liver of several vertebrates and in a number of cell lines. We discuss our observations looking at the nucleic acids not only as informational molecules, but also as a not negligible source of readily usable phosphorylated sugar.

Bovine cytosolic IMP/GMP-specific 5'-nucleotidase: cloning and expression of active enzyme in Escherichia coli.

A cDNA coding for bovine cytosolic IMP/GMP-specific 5'-nucleotidase endowed with phosphotransferase activity was cloned from calf thymus RNA, by 5' and 3' rapid amplification of cDNA ends protocols (5' and 3' RACE). Two products were isolated: a 5' RACE 1.6 kb fragment and a 3' RACE 2.0 kb fragment, with an overlapping region of 505 bp, leading to a total length of approx. 2951 bp. The similarity in the coding region to that of the human 5'-nucleotidase cDNA sequence [Oka, Matsumoto, Hosokawa and Inoue (1994) Biochem. Biophys. Res. Commun. 205, 917-922], indirectly identified as a 5'-nucleotidase, was 94% and the deduced amino acid sequences were 99.5% identical. The bovine cDNA sequence included the sequences codifying for six peptides obtained from 5'-nucleotidase/phosphotransferase purified from calf thymus. Northern blots of human mRNA species from different tissues showed a 3.6 kb mRNA expressed at equal levels in most tissues. The cDNA was cloned into a pET-28c expression vector and the protein obtained after induction had a molecular mass of 61 kDa under SDS/PAGE. It exhibited both 5'-nucleotidase and phosphotransferase activity, as well as immunological and kinetic properties similar to those of the enzyme purified from calf thymus. This is the first time that a fully active recombinant 5'nucleotidase has been described.