Emerging Role of Purine Metabolizing Enzymes in Brain Function and Tumors

The growing evidence of the involvement of purine compounds in signaling, of nucleotide imbalance in tumorigenesis, the discovery of purinosome and its regulation, cast new light on purine metabolism, indicating that well known biochemical pathways may still surprise. Adenosine deaminase is important not only to preserve functionality of immune system but also to ensure a correct development and function of central nervous system, probably because its activity regulates the extracellular concentration of adenosine and therefore its function in brain. A lot of work has been done on extracellular 5'-nucleotidase and its involvement in the purinergic signaling, but also intracellular nucleotidases, which regulate the purine nucleotide homeostasis, play unexpected roles, not only in tumorigenesis but also in brain function. Hypoxanthine guanine phosphoribosyl transferase (HPRT) appears to have a role in the purinosome formation and, therefore, in the regulation of purine synthesis rate during cell cycle with implications in brain development and tumors. The final product of purine catabolism, uric acid, also plays a recently highlighted novel role. In this review, we discuss the molecular mechanisms underlying the pathological manifestations of purine dysmetabolisms, focusing on the newly described/hypothesized roles of cytosolic 5'-nucleotidase II, adenosine kinase, adenosine deaminase, HPRT, and xanthine oxidase

Cytosolic 5′-nucleotidase II silencing in a human lung carcinoma cell line opposes cancer phenotype with a concomitant increase in p53 phosphorylation

Purine homeostasis is maintained by a purine cycle in which the regulated member is a cytosolic 5′-nucleotidase II (cN-II) hydrolyzing IMP and GMP. Its expression is particularly high in proliferating cells, indeed high cN-II activity or expression in hematological malignancy has been associated to poor prognosis and chemoresistance. Therefore, a strong interest has grown in developing cN-II inhibitors, as potential drugs alone or in combination with other compounds. As a model to study the effect of cN-II inhibition we utilized a lung carcinoma cell line (A549) in which the enzyme was partially silenced and its low activity conformation was stabilized through incubation with 2-deoxyglucose. We measured nucleotide content, reduced glutathione, activities of enzymes involved in glycolysis and Krebs cycle, protein synthesis, mitochondrial function, cellular proliferation, migration and viability. Our results demonstrate that high cN-II expression is associated with a glycolytic, highly proliferating phenotype, while silencing causes a reduction of proliferation, protein synthesis and migration ability, and an increase of oxidative performances. Similar results were obtained in a human astrocytoma cell line. Moreover, we demonstrate that cN-II silencing is concomitant with p53 phosphorylation, suggesting a possible involvement of this pathway in mediating some of cN-II roles in cancer cell biology

Studio del meccanismo di apoptosi indotto dal trattramento con deossicoformicina e deossiadenosina in cellule di neuroblastoma umano SH-SY5Y

Il dismetabolismo delle purine è frequentemente associato a disfunzioni psicomotorie, ma il legame molecolare tra lo squilibrio metabolico e le manifestazioni neurologiche, nella maggior parte dei casi, è ancora poco chiaro. La deficienza di adenosina deaminasi (ADA), enzima che catalizza la deaminazione dell’adenosina e della deossiadenosina, è associata ad una grave immunodeficienza e ad alterazioni nel funzionamento di molti organi come osso, fegato, reni e sistema nervoso. I disturbi neurologici includono disordini nella coordinazione motoria, disabilità nell’apprendimento, iperattività, epilessia e deficit uditivi. Il trapianto di midollo osseo è in grado di risolvere i problemi del sistema immunitario ma non quelli neurologici. Per lo studio dei possibili meccanismi alla base delle manifestazioni neurologiche associate alla deficienza di ADA è stato utilizzato un modello di cellule neuronali, la linea SH-SY5Y, derivata da un neuroblastoma umano, trattate con un inibitore di questo enzima, la deossicoformicina (dCF), e l’aggiunta di deossiadenosina (dAdo), che si accumula in assenza della suddetta attività enzimatica. E' stato indagato l'effetto di questo trattamento sulla vitalità cellulare, sui livelli di ATP e dATP e su l'attività delle caspasi iniziatrici -8 e -9 ed effettrici (caspasi-3) e l'effetto sia di antiossidanti che di molecole che alterano i livelli di calcio intracellulare. I risultati indicano che l'inibizione di ADA e l'accumulo di dAdo diminuiscono la vitalità nel modello utilizzato. Nifedipina, rianodina, BAPTA-AM, EDTA, N-acetilcisteina, melatonina, acido nordiidroguaiaretico e baicaleina non determinano un effetto protettivo sulla vitalità nelle cellule trattate. La 5'-ammino-5'-deossiadenosina, un inbitore della dAdo chinasi, aumenta significativamente la vitalità cellulare, sebbene dopo 15 e 24 ore di trattamento non si sia osservato un aumento di dATP. Le caspasi -9 e -8 si attivano, rispettivamente, dopo 6 e 8 ore di trattamento, mentre la caspasi-3 mostra una fase precoce e una più tardiva di attivazione. Se le condizioni sperimentali rispecchiano la deficienza di ADA, è possibile ipotizzare che l'effetto nocivo sui neuroni descritto in questa tesi abbia un ruolo nel malfunzionamento di alcuni circuiti neuronali nei pazienti ADA-SCID. The purine metabolic disorders are frequently associated with psychomotor disturbances, but the molecular link between the metabolic imbalance and the neurological manifestations, in most cases, is still unclear. The deficiency of adenosine deaminase (ADA), the enzyme which catalyzes the deamination of adenosine and deoxyadenosine, is associated with a severe immunodeficiency and to abnormalities in functioning of many organs such as bone, liver, kidney and nervous system. Neurological disorders include disturbances in motor coordination, learning disabilities, hyperactivity, epilepsy and hearing impairment. The transplantation of bone marrow is able to solve the problems of the immune system but not the neurological ones. For the study of the possible mechanisms underlying the neurological manifestations associated with deficiency of ADA, a model of neuron, the SH-SY5Y line, derived from a human neuroblastoma was used. Cells were treated with an inhibitor of ADA, deoxycoformycin (dCF) and the addition of deoxyadenosine (dAdo), which is known to accumulate intracellulary in the absence of the aforementioned enzymatic activity. It has been studied the effect of this treatment on cellular viability, on ATP and dATP levels and on the activity of initiator caspases -9 and -8 and effector (caspase-3), and the effect either of antioxidants or molecules altering intracellular calcium levels. The results indicate that ADA inhibition and dAdo accumulation decrease viability in this model. Nifedipine, ryanodine, BAPTA-AM, EDTA, N-acetylcysteine, melatonin, baicalein and nordihydroguaiaretic acid do not determine a protective effect on viability of treated cells. 5'-ammino-5'-deoxyadenosine, a dAdo kinase inhibitor, significantly increases cell viability although it has not been observed an increase in dATP following 15 or 24 hours of treatment. Caspase-9 and 8 are activated after 6 h and 8 h of treatment respectively, while caspase-3 shows both an early and a late phase of activation. If these experimental conditions mirror ADA deficiency, it is possible to hypothesize a role of the noxius effect on neurons described in the present Thesis on the malfunctioning of some neuronal circuits in ADA-SCID patients

Effects of silencing of cytosolic 5’-nucleotidase II (cN-II) on cancer cell lines

Cytosolic 5’-nucleotidase II (cN-II) is an IMP/GMP preferring 5’-nucleotidase whose activity, structure and expression has drawn the attention of several research groups from various fields such as biochemistry, crystallography, molecular biology, oncology, pharmacology and genetics. Despite a good knowledge of its tissue distribution, structure, catalytic features, allosteric regulation and possible physiological roles, cN-II still remains a clue in how it could influence cellular vital functions. In this PhD project we investigated the role of this enzyme on nucleotide content, mitochondrial mass, mitochondrial reactive oxygen species (ROS) and mitochondrial membrane potential, protein synthesis and autophagy, migration and proliferative capacity, modulation of immune cell cytotoxicity. We utilized three human cancer cell lines: lung carcinoma cells A549, glioblastoma cells ADF, triple negative breast cancer cells MDA-MB-231. In order to evaluate the role of cN-II, the expression of this enzyme was stably decreased. In A549 and ADF cells the transfection with cN-II targeting shRNA decreased the expression about 50%. In MDA-MB-231 the cN-II expression was completely abolished by using CRISPR/Cas9 technique. Our results demonstrate that high cN-II expression is associated with a glycolytic, highly proliferating phenotype, while silencing causes a reduction of proliferation, protein synthesis and migration ability, and an increase of oxidative performances. Moreover, we demonstrate that cN-II silencing is concomitant with p53 phosphorylation, suggesting a possible involvement of this pathway in mediating some of cN-II roles in cancer cell biolog

Transcriptional and Metabolic Investigation in 5′-Nucleotidase Deficient Cancer Cell Lines

International audienceEnzymes of nucleoside and nucleotide metabolism regulate important cellular processes with potential impacts on nucleotide-unrelated parameters. We have used a set of CRISPR/Cas9-modified cell models expressing both, one, or none of the 5′-nucleotidases cN-II and CD73, together with RNA sequencing and targeted metabolomics, to decipher new regulatory roles of these proteins. We observed important transcriptional modifications between models as well as upon exposure to adenosine. Metabolite content varied differently between cell models in response to adenosine exposure but was rather similar in control conditions. Our original cell models allowed us to identify a new unobvious link between proteins in the nucleotide metabolism and other cellular pathways. Further analyses of our models, including additional experiments, could help us to better understand some of the roles played by these enzymes