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1.
Front Pharmacol ; 13: 970891, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36199684

RESUMO

Guanine-based purines (GBPs) exert numerous biological effects at the central nervous system through putative membrane receptors, the existence of which is still elusive. To shed light on this question, we screened orphan and poorly characterized G protein-coupled receptors (GPRs), selecting those that showed a high purinoreceptor similarity and were expressed in glioma cells, where GBPs exerted a powerful antiproliferative effect. Of the GPRs chosen, only the silencing of GPR23, also known as lysophosphatidic acid (LPA) 4 receptor, counteracted GBP-induced growth inhibition in U87 cells. Guanine (GUA) was the most potent compound behind the GPR23-mediated effect, acting as the endpoint effector of GBP antiproliferative effects. Accordingly, cells stably expressing GPR23 showed increased sensitivity to GUA. Furthermore, while GPR23 expression was low in a hypoxanthine-guanine phosphoribosyl-transferase (HGPRT)-mutated melanoma cell line showing poor sensitivity to GBPs, and in HGPRT-silenced glioma cells, GPR23-induced expression in both cell types rescued GUA-mediated cell growth inhibition. Finally, binding experiments using [3H]-GUA and U87 cell membranes revealed the existence of a selective GUA binding (KD = 29.44 ± 4.07 nM; Bmax 1.007 ± 0.035 pmol/mg prot) likely to GPR23. Overall, these data suggest GPR23 involvement in modulating responses to GUA in tumor cell lines, although further research needs to verify whether this receptor mediates other GUA effects.

2.
Front Pharmacol ; 12: 658806, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33986683

RESUMO

Neuroblastoma arises from neural crest cell precursors failing to complete the process of differentiation. Thus, agents helping tumor cells to differentiate into normal cells can represent a valid therapeutic strategy. Here, we evaluated whether guanosine (GUO), a natural purine nucleoside, which is able to induce differentiation of many cell types, may cause the differentiation of human neuroblastoma SH-SY5Y cells and the molecular mechanisms involved. We found that GUO, added to the cell culture medium, promoted neuron-like cell differentiation in a time- and concentration-dependent manner. This effect was mainly due to an extracellular GUO action since nucleoside transporter inhibitors reduced but not abolished it. Importantly, GUO-mediated neuron-like cell differentiation was independent of adenosine receptor activation as it was not altered by the blockade of these receptors. Noteworthy, the neuritogenic activity of GUO was not affected by blocking the phosphoinositide 3-kinase pathway, while it was reduced by inhibitors of protein kinase C or soluble guanylate cyclase. Furthermore, the inhibitor of the enzyme heme oxygenase-1 but not that of nitric oxide synthase reduced GUO-induced neurite outgrowth. Interestingly, we found that GUO was largely metabolized into guanine by the purine nucleoside phosphorylase (PNP) enzyme released from cells. Taken together, our results suggest that GUO, promoting neuroblastoma cell differentiation, may represent a potential therapeutic agent; however, due to its spontaneous extracellular metabolism, the role played by the GUO-PNP-guanine system needs to be further investigated.

3.
Int J Mol Sci ; 22(9)2021 Apr 27.
Artigo em Inglês | MEDLINE | ID: mdl-33925480

RESUMO

Conventional chemotherapy for acute myeloid leukemia regimens generally encompass an intensive induction phase, in order to achieve a morphological remission in terms of bone marrow blasts (<5%). The majority of cases are classified as Primary Induction Response (PIR); unfortunately, 15% of children do not achieve remission and are defined Primary Induction Failure (PIF). This study aims to characterize the gene expression profile of PIF in children with Acute Myeloid Leukemia (AML), in order to detect molecular pathways dysfunctions and identify potential biomarkers. Given that NUP98-rearrangements are enriched in PIF-AML patients, we investigated the association of NUP98-driven genes in primary chemoresistance. Therefore, 85 expression arrays, deposited on GEO database, and 358 RNAseq AML samples, from TARGET program, were analyzed for "Differentially Expressed Genes" (DEGs) between NUP98+ and NUP98-, identifying 110 highly confident NUP98/PIF-associated DEGs. We confirmed, by qRT-PCR, the overexpression of nine DEGs, selected on the bases of the diagnostic accuracy, in a local cohort of PIF patients: SPINK2, TMA7, SPCS2, CDCP1, CAPZA1, FGFR1OP2, MAN1A2, NT5C3A and SRP54. In conclusion, the integrated analysis of NUP98 mutational analysis and transcriptome profiles allowed the identification of novel putative biomarkers for the prediction of PIF in AML.


Assuntos
Biomarcadores Tumorais/genética , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Adolescente , Criança , Pré-Escolar , Estudos de Coortes , Quinase 6 Dependente de Ciclina/genética , Feminino , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Rearranjo Gênico , Histona-Lisina N-Metiltransferase/genética , Humanos , Lactente , Recém-Nascido , Masculino , Família Multigênica , Reação em Cadeia da Polimerase em Tempo Real , Reprodutibilidade dos Testes , Falha de Tratamento
4.
Int J Mol Sci ; 21(23)2020 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-33291390

RESUMO

Acute or chronic administration of guanosine (GUO) induces anxiolytic-like effects, for which the adenosine (ADO) system involvement has been postulated yet without a direct experimental evidence. Thus, we aimed to investigate whether adenosine receptors (ARs) are involved in the GUO-mediated anxiolytic-like effect, evaluated by three anxiety-related paradigms in rats. First, we confirmed that acute treatment with GUO exerts an anxiolytic-like effect. Subsequently, we investigated the effects of pretreatment with ADO or A1R (CPA, CCPA) or A2AR (CGS21680) agonists 10 min prior to GUO on a GUO-induced anxiolytic-like effect. All the combined treatments blocked the GUO anxiolytic-like effect, whereas when administered alone, each compound was ineffective as compared to the control group. Interestingly, the pretreatment with nonselective antagonist caffeine or selective A1R (DPCPX) or A2AR (ZM241385) antagonists did not modify the GUO-induced anxiolytic-like effect. Finally, binding assay performed in hippocampal membranes showed that [3H]GUO binding became saturable at 100-300 nM, suggesting the existence of a putative GUO binding site. In competition experiments, ADO showed a potency order similar to GUO in displacing [3H]GUO binding, whereas AR selective agonists, CPA and CGS21680, partially displaced [3H]GUO binding, but the sum of the two effects was able to displace [3H]GUO binding to the same extent of ADO alone. Overall, our results strengthen previous data supporting GUO-mediated anxiolytic-like effects, add new evidence that these effects are blocked by A1R and A2AR agonists and pave, although they do not elucidate the mechanism of GUO and ADO receptor interaction, for a better characterization of GUO binding sites in ARs.


Assuntos
Ansiedade/etiologia , Ansiedade/metabolismo , Guanosina/efeitos adversos , Receptor A1 de Adenosina/metabolismo , Receptor A2A de Adenosina/metabolismo , Animais , Ansiedade/psicologia , Comportamento Animal , Membrana Celular/metabolismo , Escuridão , Relação Dose-Resposta a Droga , Guanosina/metabolismo , Hipocampo/metabolismo , Luz , Ratos , Receptor A1 de Adenosina/genética , Receptor A2A de Adenosina/genética
5.
BMC Cancer ; 18(1): 1265, 2018 Dec 18.
Artigo em Inglês | MEDLINE | ID: mdl-30563495

RESUMO

BACKGROUND: Broad copy number aberrations (BCNAs) represent a common form of genome instability in colorectal cancer (CRC). CRCs show large variations in their level of aneuploidy: microsatellite-instable (MSI) tumors are known to have a near-diploid karyotype while microsatellite-stable (MSS) tumors show high level of chromosomal instability. However, MSS tumors have great heterogeneity in the number of BCNAs, with a minor percentage of samples showing an almost normal karyotype. In the present work we subdivided MSS CRCs according to a "BCNA score" and characterized their transcriptome profiles, considered as a proxy to their phenotypic features. METHODS: Microsatellite testing, genome-wide DNA copy number and whole-transcript expression analysis (HTA) were performed on 33 tumor samples and 25 normal colonic tissue samples from 32 CRC patients. 15.1% of the samples were MSI tumors (n = 5), whereas 84.9% were MSS tumors (n = 28). Gene expression data of 34 additional MSI tumors was retrieved from a public functional genomics data repository. RESULTS: Using as a threshold the first quartile of the BCNA score distribution, MSS samples were classified as low-BCNA (LB, n = 7) or high-BCNA (HB, n = 21). LB tumors were enriched for mucinous CRCs and their gene-expression profile resembled that of MSI samples for what concerns a subset of genes involved in secretory processes, mucosal protection, and extracellular matrix remodeling. HB tumors were predominantly non-mucinous adenocarcinomas and showed overexpression of a subset of genes typical of surface colonocytes and EGF signaling. A large percentage of unclassified samples according to the consensus molecular subtypes (CMS) classifier was found in the LB group (43%), whereas 76% HB tumors belonged to CMS2. CONCLUSIONS: A classification of colorectal tumors based on the number of BCNAs identifies two groups of MSS tumors which differ for histopathology and gene expression profile. Such information can be exploited for its translational relevance in different aspects of CRC clinical management.


Assuntos
Neoplasias Colorretais/genética , Variações do Número de Cópias de DNA/genética , Instabilidade Genômica/genética , Transcriptoma/genética , Idoso , Instabilidade Cromossômica/genética , Colo , Neoplasias Colorretais/patologia , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Masculino , Instabilidade de Microssatélites , Pessoa de Meia-Idade
6.
Sci Rep ; 8(1): 14826, 2018 10 04.
Artigo em Inglês | MEDLINE | ID: mdl-30287863

RESUMO

We re-examined the correlation between Broad Genomic Aberrations (BGAs) and transcriptomic profiles in Colorectal Cancer (CRC). Two types of BGAs have been examined: Broad Copy-Number Abnormal regions (BCNAs), distinguished in gain- and loss-type, and Copy-Neutral Loss of Heterozygosities (CNLOHs). Transcripts are classified as "OverT" or "UnderT" if overexpressed or underexpressed comparing CRCs bearing a specific BGA to CRCs not bearing it and as "UpT" or "DownT" if upregulated or downregulated in cancer compared to normal tissue. BGA-associated effects were evaluated by changes in the "Chromosomal Distribution Index" (CDI) of different transcript classes. Data show that UpT are more sensitive than DownT to BCNA-associated gene dosage effects. "Over-UpT" genes are upregulated in cancer and further overexpressed by gene dosage, defining the so called "positive caricature transcriptomic effect". When Over-UpT genes are ranked according to overexpression, top positions are occupied by genes implicated at the functional and therapeutic level in CRC. We show that cancer-upregulated transcripts are sensitive markers of BCNA-induced effects and suggest that analysis of positive caricature transcriptomic effects can provide clues toward the identification of BCNA-associated cancer driver genes.


Assuntos
Neoplasias Colorretais/genética , Variações do Número de Cópias de DNA/genética , Genoma Humano , Perda de Heterozigosidade/genética , Transcriptoma/genética , Cromossomos Humanos/genética , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
7.
Front Pharmacol ; 9: 110, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29515443

RESUMO

Mounting evidence suggests that the guanine-based purines stand out as key player in cell metabolism and in several models of neurodegenerative disorders, such as Parkinson's and Alzheimer's diseases. Guanosine (GUO) and guanine (GUA) are extracellular signaling molecules derived from the breakdown of the correspondent nucleotide, GTP, and their intracellular and extracellular levels are regulated by the fine-tuned activity of two major enzymes, purine nucleoside phosphorylase (PNP) and guanine deaminase (GDA). Noteworthy, GUO and GUA, seem to play opposite roles in the modulation of cognitive functions, such as learning and memory. Indeed GUO, despite exerting neuroprotective, anti-apoptotic and neurotrophic effects, causes a decay of cognitive activities, whereas GUA administration in rats results in working memory improvement (prevented by L-NAME pre-treatment). This study was designed to investigate, in a model of SH-SY5Y neuroblastoma cell line, the signal transduction pathway activated by extracellular GUA. Altogether, our results showed that: (i) in addition to an enhanced phosphorylation of ASK1, p38 and JNK, likely linked to a non-massive and transient ROS production, the PKB/NO/sGC/cGMP/PKG/ERK cascade seems to be the main signaling pathway elicited by extracellular GUA; (ii) the activation of this pathway occurs in a pertussis-toxin sensitive manner, thus suggesting the involvement of a putative G protein coupled receptor; (iii) the GUA-induced NO production, strongly reduced by cell pre-treatment with L-NAME, is negatively modulated by the EPAC-cAMP-CaMKII pathway, which causes the over-expression of GDA that, in turn, reduces the levels of GUA. These molecular mechanisms activated by GUA may be useful to support our previous observation showing that GUA improves learning and memory functions through the stimulation of NO signaling pathway, and underscore the therapeutic potential of oral administration of guanine for treating memory-related disorders.

8.
J Inorg Biochem ; 182: 92-102, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29452884

RESUMO

A novel water-soluble ionophore based on the thiocarbohydrazone moiety conjugated with glucose (GluTch) was synthesized through a simple two-step procedure. Structural elucidation was carried out in water solution by means of various spectroscopic techniques (NMR, UV-Vis, and CD), electrospray ionization mass spectrometry and density functional theory calculations. The flexible nature of the thiocarbohydrazone moiety of the new glycoderivative compound induced both different coordination motifs and stoichiometry towards copper and zinc. Cytotoxicity assays of the ligands on the human normal keratinocyte NCTC-2544, MDA-MB-231 breast cancer and PC-3 human prostate adenocarcinoma cell lines demonstrated that i) higher activity on cancer cells growth inhibition compared to a normal cell line; ii) the introduction of the glucose unit does not alter the cytotoxic activity of the underivatized ionophore ligand and iii) the presence of copper ion improves the activity of the thiocarbohydrazones.


Assuntos
Antineoplásicos/química , Antineoplásicos/farmacologia , Glucose/química , Hidrazonas/química , Hidrazonas/farmacologia , Ionóforos/química , Ionóforos/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Dicroísmo Circular , Complexos de Coordenação/química , Complexos de Coordenação/farmacologia , Cobre/química , Humanos , Espectroscopia de Ressonância Magnética , Espectrometria de Massas por Ionização por Electrospray , Zinco/química
9.
Auton Neurosci ; 210: 55-64, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29305058

RESUMO

Mutations in the HGPRT1 gene, which encodes hypoxanthine-guanine phosphoribosyltransferase (HGprt), housekeeping enzyme responsible for recycling purines, lead to Lesch-Nyhan disease (LND). Clinical expression of LND indicates that HGprt deficiency has adverse effects on gastrointestinal motility. Therefore, we aimed to evaluate intestinal motility in HGprt knockout mice (HGprt¯). Spontaneous and neurally evoked mechanical activity was recorded in vitro as changes in isometric tension in circular muscle strips of distal colon. HGprt¯ tissues showed a lower in amplitude spontaneous activity and atropine-sensitivity neural contraction compared to control mice. The responses to carbachol and to high KCl were reduced, demonstrating a widespread impairment of contractility. L-NAME was not able in the HGprt¯ tissues to restore the large amplitude contractile activity typical of control. In HGprt¯ colon, a reduced expression of dopaminergic D1 receptor was observed together with the loss of its tonic inhibitory activity present in control-mice. The analysis of inflammatory and oxidative stress in colonic tissue of HGprt¯ mice revealed a significant increase of lipid peroxidation associated with over production of oxygen free radicals. In conclusion, HGprt deficiency in mice is associated with a decrease in colon contractility, not dependent upon reduction of acetylcholine release from the myenteric plexus or hyperactivity of inhibitory signalling. By contrast the increased levels of oxidative stress could partially explain the reduced colon motility in HGprt¯ mice. Colonic dysmotility observed in HGprt¯ mice may mimic the gastrointestinal dysfunctions symptoms of human syndrome, providing a useful animal model to elucidate the pathophysiology of this problem in the LND.


Assuntos
Motilidade Gastrointestinal/genética , Regulação da Expressão Gênica/genética , Síndrome de Lesch-Nyhan/complicações , Músculo Liso/fisiopatologia , Animais , Atropina/farmacologia , Benzazepinas/farmacologia , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Carbacol/farmacologia , Citocinas/metabolismo , Modelos Animais de Doenças , Dopamina/metabolismo , Inibidores Enzimáticos/farmacologia , Potenciais Evocados/efeitos dos fármacos , Potenciais Evocados/genética , Face , Motilidade Gastrointestinal/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Hipoxantina Fosforribosiltransferase/deficiência , Hipoxantina Fosforribosiltransferase/metabolismo , Técnicas In Vitro , Síndrome de Lesch-Nyhan/genética , Síndrome de Lesch-Nyhan/patologia , Síndrome de Lesch-Nyhan/fisiopatologia , Peroxidação de Lipídeos/efeitos dos fármacos , Peroxidação de Lipídeos/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Músculo Liso/efeitos dos fármacos , NG-Nitroarginina Metil Éster/farmacologia , Neurotransmissores/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Tirosina 3-Mono-Oxigenase/genética , Tirosina 3-Mono-Oxigenase/metabolismo
10.
Pediatr Dermatol ; 34(6): e345-e346, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-29023873

RESUMO

We report the case of a 6-year-old Caucasian girl with clinical and histopathologic features of Buschke-Ollendorff syndrome. Histologic examination of skin lesions showed thick, curly, elastic fibers in the derma. Bone lesions compatible with Buschke-Ollendorff syndrome were found in the girl's mother. Mutations in LEMD3 are pathogenic for Buschke-Ollendorff syndrome. Analysis of all exons and exon-intron junctions of LEMD3 did not reveal any germline mutations.


Assuntos
Proteínas de Membrana/genética , Proteínas Nucleares/genética , Osteopecilose/genética , Dermatopatias Genéticas/genética , Pele/patologia , Criança , Proteínas de Ligação a DNA , Tecido Elástico/patologia , Feminino , Mutação em Linhagem Germinativa , Humanos , Análise de Sequência de DNA
11.
Front Pharmacol ; 7: 158, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27378923

RESUMO

Guanine-based purines (GBPs) have been recently proposed to be not only metabolic agents but also extracellular signaling molecules that regulate important functions in the central nervous system. In such way, GBPs-mediated neuroprotection, behavioral responses and neuronal plasticity have been broadly described in the literature. However, while a number of these functions (i.e., GBPs neurothophic effects) have been well-established, the molecular mechanisms behind these GBPs-dependent effects are still unknown. Furthermore, no plasma membrane receptors for GBPs have been described so far, thus GBPs are still considered orphan neuromodulators. Interestingly, an intricate and controversial functional interplay between GBPs effects and adenosine receptors activity has been recently described, thus triggering the hypothesis that GBPs mechanism of action might somehow involve adenosine receptors. Here, we review recent data describing the GBPs role in the brain. We focus on the involvement of GBPs regulating neuronal plasticity, and on the new hypothesis based on putative GBPs receptors. Overall, we expect to shed some light on the GBPs world since although these molecules might represent excellent candidates for certain neurological diseases management, the lack of putative GBPs receptors precludes any high throughput screening intent for the search of effective GBPs-based drugs.

12.
Oxid Med Cell Longev ; 2012: 498914, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22829956

RESUMO

Following previous work, we investigated in more detail the relationship between apoptosis and delayed luminescence (DL) in human leukemia Jurkat T cells under a wide variety of treatments. We used menadione and hydrogen peroxide to induce oxidative stress and two flavonoids, quercetin, and epigallocatechin gallate, applied alone or in combination with menadione or H(2)O(2). 62 MeV proton beams were used to irradiate cells under a uniform dose of 2 or 10 Gy, respectively. We assessed apoptosis, cell cycle distributions, and DL. Menadione, H(2)O(2) and quercetin were potent inducers of apoptosis and DL inhibitors. Quercetin decreased clonogenic survival and the NAD(P)H level in a dose-dependent manner. Proton irradiation with 2 Gy but not 10 Gy increased the apoptotic rate. However, both doses induced a substantial G(2)/M arrest. Quercetin reduced apoptosis and prolonged the G(2)/M arrest induced by radiation. DL spectroscopy indicated that proton irradiation disrupted the electron flow within Complex I of the mitochondrial respiratory chain, thus explaining the massive necrosis induced by 10 Gy of protons and also suggested an equivalent action of menadione and quercetin at the level of the Fe/S center N2, which may be mediated by their binding to a common site within Complex I, probably the rotenone-binding site.


Assuntos
Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Flavonoides/farmacologia , Leucemia/patologia , Luminescência , Oxidantes/toxicidade , Prótons , Catequina/análogos & derivados , Catequina/farmacologia , Ciclo Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Clonais , Humanos , Peróxido de Hidrogênio/toxicidade , Células Jurkat , Cinética , NADP/metabolismo , Teoria Quântica , Quercetina/farmacologia , Fatores de Tempo , Vitamina K 3/toxicidade
13.
Eur J Pharmacol ; 650(1): 350-5, 2011 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-20940015

RESUMO

Adenine-based purines play a pivotal role in the control of gastrointestinal motility in rodents. Recently, guanine-based purines have been also shown to exert extracellular effects in the central nervous system raising the possibility of the existence of distinct receptors for guanine-based purines. Thus, it seems likely to speculate that also guanine-based purines may play a role in the modulation of the intestinal contractility. Spontaneous and neurally-evoked mechanical activity was recorded in vitro as changes in isometric tension in circular muscle strips from mouse distal colon. Guanosine up to 3mM or guanine up to 1mM failed to affect the spontaneous mechanical activity, but reduced the amplitude of the electrical field stimulation (EFS)-induced cholinergic contractions, without affecting the early nitrergic relaxation. Both compounds failed to affect the direct contractile responses evoked by carbachol. No desensitization of the response was observed. Guanine-based purine effects were not altered by theophylline, P1 purinoceptor antagonist, by PPADS or suramin, P2 purinoceptor antagonists, by ODQ, guanilyl cyclase inhibitor, or by DDA, adenylyl cyclase inhibitor. Nucleoside uptake inhibitors, dipyridamole or 6-[(4-Nitrobenzyl)thio]-9-ß-D-ribofuranosylpurine (NBTI), antagonized the inhibitory effects induced by guanosine without interfering with guanine. On the contrary, adenine, a competitive inhibitor of nucleobase uptake, antagonized guanine-induced effects. In conclusion, our data indicate that guanosine and guanine are able to modulate negatively the excitatory cholinergic neurotransmission in the circular muscle layer of mouse colon. Guanine-based purines appear to interfere with prejunctional acethylcoline release. Their effects are dependent by their cellular uptake, and independent by adenine-based purine receptors.


Assuntos
Motilidade Gastrointestinal/efeitos dos fármacos , Guanina/farmacologia , Animais , Transporte Biológico , Colo/citologia , Colo/efeitos dos fármacos , Colo/fisiologia , Relação Dose-Resposta a Droga , Guanina/química , Guanina/metabolismo , Técnicas In Vitro , Masculino , Camundongos , Contração Muscular/efeitos dos fármacos , Fatores de Tempo
14.
Comb Chem High Throughput Screen ; 14(1): 36-46, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-20958258

RESUMO

OPLS discriminant analysis (OPLS-DA) was successfully applied for the selection of a limited number of gene transcripts necessary to discriminate PTPN11 and RAS mutated cells in acute lymphoblastic leukaemia (ALL) patients. The original set of 273 variables with VIP (1) values higher than 2.0 in the OPLS-DA model could be further reduced to 200 by elimination of less informative variables in the PCA class models adopted for SIMCA classification. The above 200 transcripts not only achieve a satisfactory discrimination accuracy between PTPN11 and RAS mutated cells but also indicate clearly that wild type samples belong to none of the mutated class models. In this list it was possible to identify candidate genes that could be involved in the molecular mechanisms discriminating PTPN11 and RAS mutations in ALL. Among them CBFA2T2, a member of the "ETO" family, is known because of its homology and association with the product of RUNX1-CBFA2T1 gene fusion generated by t(8;21) translocation, one frequent cause of acute myeloid leukemia.


Assuntos
Genes ras , Mutação , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Proteína Tirosina Fosfatase não Receptora Tipo 11/genética , RNA Mensageiro/genética , Perfilação da Expressão Gênica , Humanos , Modelos Teóricos
15.
Genes Chromosomes Cancer ; 49(11): 1014-23, 2010 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-20725993

RESUMO

We analyzed, by the latest high-resolution SNP arrays, 19 Normal Karyotype (NK)-AML patients at diagnosis (Dx) and remission (R) phases, to determine the number of tumor-associated copy number abnormalities (CNAs) and copy neutral-loss of heterozygosity (CN-LOH) regions per patient and to identify possible recurring genomic abnormalities. The number of tumor-associated CNAs was determined after comparison of matched Dx/R samples using stringent conditions able to reduce the number of false positive CNAs. With the exception of a single outlier case, a low number of CNAs per patient was detected (median value of 1 somatic loss or gain per patient). However, a high prevalence of CNAs (60-70% of the patients showed at least one tumor-associated gain or loss) and few recurring CNAs were observed, thus providing new hints towards identification of cooperating mutations. An extensive search of all tumor-associated CN-LOH regions >1 Mb revealed only three broad regions (terminal 12Mb of 22q, terminal 27Mb of 1p and the whole chromosome 21) in three patients out of 19 (16%). CN-LOH of the whole chromosome 21 was responsible for homozygosity of a missense mutation (R80C) of RUNX1/AML1. Our study suggests that a relative submicroscopic copy number stability NK-AML genomes is associated with low recurrence of specific CNAs and CN-LOH in NK-AML patient population. Sequencing of candidate genes in the identified CNAs and CN-LOH regions should be considered a priority in the search of novel driver mutations of AML.


Assuntos
Dosagem de Genes , Leucemia Mieloide Aguda/genética , Perda de Heterozigosidade , Adulto , Sequência de Bases , Cromossomos Humanos Par 21 , Cromossomos Humanos Par 22 , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Primers do DNA , Feminino , Humanos , Cariotipagem , Masculino , Pessoa de Meia-Idade , Mutação , Reação em Cadeia da Polimerase , Polimorfismo de Nucleotídeo Único
16.
Cell Biochem Biophys ; 58(3): 169-79, 2010 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-20697976

RESUMO

Menadione (MD) is an effective cytotoxic drug able to produce intracellularly large amounts of superoxide anion. Quercetin (QC), a widely distributed bioflavonoid, can exert both antioxidant and pro-oxidant effects and is known to specifically inhibit cell proliferation and induce apoptosis in different cancer cell types. We have investigated the relation between delayed luminescence (DL) induced by UV-laser excitation and the effects of MD, hydrogen peroxide, and QC on apoptosis and cell cycle in human leukemia Jurkat T-cells. Treatments with 500 µM H2O2 and 250 µM MD for 20 min produced 66.0 ± 4.9 and 46.4 ± 8.6% apoptotic cell fractions, respectively. Long-term (24 h) pre-exposure to 5 µM, but not 0.5 µM QC enhanced apoptosis induced by MD, whereas short-term (1 h) pre-incubation with 10 µM QC offered 50% protection against H2O2-induced apoptosis, but potentiated apoptosis induced by MD. Since physiological levels of QC in the blood are normally less than 10 µM, these data can provide relevant information regarding the benefits of flavonoid-combined treatments of leukemia. All the three drugs exerted significant effects on DL. Our data are consistent with (1) the involvement of Complex I of the mitochondrial respiratory chain as an important source of delayed light emission on the 10 µs-10 ms scale, (2) the ability of superoxide anions to quench DL on the 100 µs-10 ms scale, probably via inhibition of reverse electron transfer at the Fe/S centers in Complex I, and (3) the relative insensitivity of DL to intracellular OH• and H2O2 levels.


Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Peróxido de Hidrogênio/toxicidade , Quercetina/farmacologia , Vitamina K 3/farmacologia , Proteínas de Ciclo Celular/metabolismo , Citometria de Fluxo , Humanos , Células Jurkat , Cinética , Leucemia/tratamento farmacológico , Leucemia/patologia , Espectrometria de Fluorescência
17.
BMC Med Genomics ; 3: 28, 2010 Jul 06.
Artigo em Inglês | MEDLINE | ID: mdl-20602808

RESUMO

BACKGROUND: ATRX is a severe X-linked disorder characterized by mental retardation, facial dysmorphism, urogenital abnormalities and alpha-thalassemia. The disease is caused by mutations in ATRX gene, which encodes a protein belonging to the SWI/SNF DNA helicase family, a group of proteins involved in the regulation of gene transcription at the chromatin level. In order to identify specific genes involved in the pathogenesis of the disease, we compared, by cDNA microarray, the expression levels of approximately 8500 transcripts between ATRX and normal males of comparable age. METHODS: cDNA microarray was performed using total RNA from peripheral blood mononuclear cells of ATRX and normal males. Microarray results were validated by quantitative real-time polymerase chain reaction. RESULTS: cDNA microarray analysis showed that 35 genes had a lower expression (30-35% of controls) while 25 transcripts had a two-fold higher expression in comparison to controls. In the microarray results the probe for oligophrenin-1, a gene known for its involvement in mental retardation, showed a decreased hybridization signal. However, such gene was poorly expressed in blood mononuclear cells and its decrease was not confirmed in the quantitative real-time RT-PCR assay. On the other hand, the expression of an homologous gene, the GTPase regulator associated with the focal adhesion kinase 1/Oligophrenin-1-like (GRAF1/OPHN-1-L), was relatively high in blood mononuclear cells and significantly decreased in ATRX patients. The analysis of the expression pattern of the GRAF1/OPHN-1-L gene in human tissues and organs revealed the predominant brain expression of a novel splicing isoform, called variant-3. CONCLUSIONS: Our data support the hypothesis of a primary role for altered gene expression in ATRX syndrome and suggest that the GRAF1/OPHN-1-L gene might be involved in the pathogenesis of the mental retardation. Moreover a novel alternative splicing transcript of such gene, predominantly expressed in brain tissues, was identified.


Assuntos
Proteínas Ativadoras de GTPase/genética , Doenças Genéticas Ligadas ao Cromossomo X/genética , Deficiência Intelectual/genética , Talassemia alfa/genética , Adolescente , Processamento Alternativo , Criança , Proteínas Ativadoras de GTPase/metabolismo , Regulação da Expressão Gênica , Humanos , Masculino , Análise de Sequência com Séries de Oligonucleotídeos , RNA/metabolismo , Síndrome
18.
Brain Res ; 1298: 24-36, 2009 Nov 17.
Artigo em Inglês | MEDLINE | ID: mdl-19728995

RESUMO

Calcium sensing receptor (CaSR), isolated for the first time from bovine and human parathyroid, is a G-protein-coupled receptors that has been involved in diverse physiological functions. At present a complete in vivo work on the identification of CaSR mRNA-expressing cells in the adult brain lacks and this investigation was undertaken in order to acquire more information on cell type expressing CaSR mRNA in the rat brain and to analyse for the first time its expression in different experimental models of brain injury. The expression of CaSR mRNAs was found mainly in scattered cells throughout almost all the brain regions. A double labeling analysis showed a colocalization of CaSR mRNA expression in neurons and oligodendrocytes, whereas it was not found expressed both in the microglia and in astrocytes. One week after kainate-induced seizure CaSR was found in the injured CA3 region of the hippocampus and very interestingly it was found up-regulated in the neurons of CA1-CA2 and dentate gyrus. Similarly, 1 week following ibotenic acid injection in the hippocampus, CaSR mRNA expression was increased in oligodendrocytes both in the lesioned area and in the contralateral CA1-CA3 pyramidal cell layers and dentate gyrus. One week after needle-induced mechanical lesion an increase of labeled cells expressing CaSR mRNA was observed along the needle track. In conclusion, the present results contribute to extend available data on cell type-expressing CaSR in normal and injured brain and could spur to understand the role of CaSR in repairing processes of brain injury.


Assuntos
Lesões Encefálicas/metabolismo , Encéfalo/metabolismo , Receptores de Detecção de Cálcio/metabolismo , Animais , Lesões Encefálicas/induzido quimicamente , Lesões Encefálicas/genética , Contagem de Células , Ácido Ibotênico/toxicidade , Imuno-Histoquímica , Hibridização In Situ , Ácido Caínico/toxicidade , Masculino , Neurônios/metabolismo , Oligodendroglia/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Receptores de Detecção de Cálcio/genética , Convulsões/induzido quimicamente , Convulsões/metabolismo , Fatores de Tempo
19.
Invest New Drugs ; 27(3): 189-202, 2009 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-18612590

RESUMO

The novel compound N-benzoxazol-2-yl-N'-1-(isoquinolin-3-yl-ethylidene)-hydrazine (EPH136) has been shown to exhibit antitumor activity in vitro and in vivo. A COMPARE analysis showed that the patterns of cellular effects of EPH136 are not related to any of 175 standard antitumor agents with a known mechanism of action. In order to help identify the mechanism of action we employed a bioinformatics approach called partial least squares modelling in latent variables in which the expression levels of approximately 8,000 genes in each of 56 untreated NCI panel cell lines were correlated with the respective IC(50) values of each cell line following treatment with EPH136. The 60 genes found to be most important for the antiproliferative effect of EPH136 are involved in nucleoside, nucleotide, nucleic acid binding and metabolism, developmental processes, protein modification and metabolism. In addition, using a DNA microarray we measured the expression of approximately 5,000 known genes following treatment of HT-29 colon carcinoma cells with a two-fold IC(50) concentration of EPH136. The genes that were up-regulated more than two-fold compared to untreated controls belong to the same classes as found by the bioinformatic approach. Many of these proteins are regulated by oxidation/reduction and so we concluded that formation of radicals may be involved in the mechanism of action. We show here that EPH136 leads to generation of oxygen radicals, swelling of mitochondria and dissipation of the mitochondrial membrane potential. The antiproliferative activity of EPH136 was prevented by the radical scavenger N-acetylcysteine. Cells with elevated glutathione exhibited resistance to EPH136. In summary, the mechanism of the novel experimental anticancer drug EPH136 is generation of radicals and dissipation of the mitochondrial membrane potential.


Assuntos
Antineoplásicos/farmacologia , Benzoxazóis/farmacologia , Radicais Livres/metabolismo , Hidrazonas/farmacologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Antineoplásicos/química , Antioxidantes/farmacologia , Apoptose/efeitos dos fármacos , Benzoxazóis/química , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Biologia Computacional , DNA/biossíntese , Ensaios de Seleção de Medicamentos Antitumorais , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Hidrazonas/química , Análise de Sequência com Séries de Oligonucleotídeos , RNA/biossíntese , Espécies Reativas de Oxigênio/metabolismo , Ribonucleotídeo Redutases/antagonistas & inibidores
20.
Neurosci Lett ; 447(1): 20-5, 2008 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-18835325

RESUMO

Several findings have suggested the existence in the subventricular zone (SVZ) from sagittal sections of adult rat brain of a trophic mechanism, mediated by fibroblast growth factor-2 (FGF-2) and its multiple high-affinity FGF receptors (FGFRs), regulating neurogenesis mainly by controlling precursor cell proliferation. However, no clear data are available on the expression of FGF-2 and FGFRs in proliferating precursor cells of the SVZ. To address these questions we examined FGF-2 mRNA and its FGFR mRNA expression in proliferating precursor cells of the SVZ by using a double labeling procedure, combining in situ hybridization for FGF-2 and its FGFR mRNA with immunohistochemistry for bromodeoxyuridine (BrdU), a marker for proliferating cells. The results showed that FGFR1 and FGFR2 mRNAs were expressed in BrdU-labeled proliferating precursor cells, whereas FGFR3 and FGF-2 mRNAs were not, suggesting that in the SVZ the proliferating precursor cells express FGFR1 or FGFR2 and they may respond to FGF-2 released by non-proliferating cells. The FGFR4 mRNA was not found expressed in the SVZ. In the future, by identifying the cell types expressing FGFRs, it will be possible to gain insight into the functional activity of FGF2 within the SVZ.


Assuntos
Células-Tronco Adultas/metabolismo , Proliferação de Células , Ventrículos Cerebrais/citologia , Fator 2 de Crescimento de Fibroblastos/metabolismo , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/metabolismo , Animais , Encéfalo/anatomia & histologia , Bromodesoxiuridina/metabolismo , Fator 2 de Crescimento de Fibroblastos/genética , Expressão Gênica/fisiologia , Masculino , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/genética , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/genética , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/genética , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/metabolismo
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