RESUMO
Mitochondria house many metabolic pathways required for homeostasis and growth. To explore how human cells respond to mitochondrial dysfunction, we performed metabolomics in fibroblasts from patients with various mitochondrial disorders and cancer cells with electron transport chain (ETC) blockade. These analyses revealed extensive perturbations in purine metabolism, and stable isotope tracing demonstrated that ETC defects suppress de novo purine synthesis while enhancing purine salvage. In human lung cancer, tumors with markers of low oxidative mitochondrial metabolism exhibit enhanced expression of the salvage enzyme hypoxanthine phosphoribosyl transferase 1 (HPRT1) and high levels of the HPRT1 product inosine monophosphate. Mechanistically, ETC blockade activates the pentose phosphate pathway, providing phosphoribosyl diphosphate to drive purine salvage supplied by uptake of extracellular bases. Blocking HPRT1 sensitizes cancer cells to ETC inhibition. These findings demonstrate how cells remodel purine metabolism upon ETC blockade and uncover a new metabolic vulnerability in tumors with low respiration.
Assuntos
Mitocôndrias , Purinas , Humanos , Purinas/metabolismo , Purinas/farmacologia , Mitocôndrias/metabolismo , Transporte de Elétrons , Hipoxantina Fosforribosiltransferase/metabolismo , Hipoxantina Fosforribosiltransferase/genética , Via de Pentose Fosfato , Fibroblastos/metabolismo , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/tratamento farmacológico , Linhagem Celular Tumoral , Animais , Transporte BiológicoRESUMO
Lesch-Nyhan syndrome (LNS) is a congenital defect disease that results in defective purine metabolism. It is caused by pathogenic variants of the HPRT gene. Its clinical symptoms mainly include high uric acid levels, gout, and kidney stones and damage. The mechanism of LNS has not been fully elucidated, and no cure exists. Animal models have always played an important role in exploring causative mechanisms and new therapies. This study combined CRISPR/Cas9 and microinjection to knock out the HPRT gene to create an LNS rabbit model. A sgRNA targeting exon 3 of HPRT gene was designed. Subsequently, Cas9 mRNA and sgRNA were injected into rabbit zygotes, and injected embryos were transferred to the uterus. The genotype and phenotype of rabbits were analyzed after birth. Four infant rabbits (named R1, R2, R3 and R4), which showed varying levels of gene modification, were born. The gene-editing efficiency was 100%. No wild-type sequences at the target HPRT gene were detected in R4 rabbit. Next, 6-thioguanine drug testing confirmed that HPRT enzymatic activity was deficient in R4 infant rabbit. HE staining revealed kidney abnormalities in all infant rabbits. Overall, an sgRNA capable of knocking out the HPRT gene in rabbits was successfully designed, and HPRT gene-modified rabbits were successfully constructed by using CRISPR/Cas9 technology and microinjection. This study provides a new nonrodent animal model for studying LNS syndrome.
Assuntos
Sistemas CRISPR-Cas , Modelos Animais de Doenças , Hipoxantina Fosforribosiltransferase , Síndrome de Lesch-Nyhan , Animais , Coelhos , Síndrome de Lesch-Nyhan/genética , Hipoxantina Fosforribosiltransferase/genética , Hipoxantina Fosforribosiltransferase/metabolismo , Feminino , Edição de Genes , RNA Guia de Sistemas CRISPR-Cas/genética , Masculino , FenótipoRESUMO
Adenine phosphoribosyltransferase (APRT) and hypoxanthine-guanine phosphoribosyltransferase (HGPRT) are two structurally related enzymes involved in purine recycling in humans. Inherited mutations that suppress HGPRT activity are associated with Lesch-Nyhan disease (LND), a rare X-linked metabolic and neurological disorder in children, characterized by hyperuricemia, dystonia, and compulsive self-injury. To date, no treatment is available for these neurological defects and no animal model recapitulates all symptoms of LND patients. Here, we studied LND-related mechanisms in the fruit fly. By combining enzymatic assays and phylogenetic analysis, we confirm that no HGPRT activity is expressed in Drosophila melanogaster, making the APRT homolog (Aprt) the only purine-recycling enzyme in this organism. Whereas APRT deficiency does not trigger neurological defects in humans, we observed that Drosophila Aprt mutants show both metabolic and neurobehavioral disturbances, including increased uric acid levels, locomotor impairments, sleep alterations, seizure-like behavior, reduced lifespan, and reduction of adenosine signaling and content. Locomotor defects could be rescued by Aprt re-expression in neurons and reproduced by knocking down Aprt selectively in the protocerebral anterior medial (PAM) dopaminergic neurons, the mushroom bodies, or glia subsets. Ingestion of allopurinol rescued uric acid levels in Aprt-deficient mutants but not neurological defects, as is the case in LND patients, while feeding adenosine or N6-methyladenosine (m6A) during development fully rescued the epileptic behavior. Intriguingly, pan-neuronal expression of an LND-associated mutant form of human HGPRT (I42T), but not the wild-type enzyme, resulted in early locomotor defects and seizure in flies, similar to Aprt deficiency. Overall, our results suggest that Drosophila could be used in different ways to better understand LND and seek a cure for this dramatic disease.
Assuntos
Drosophila melanogaster , Síndrome de Lesch-Nyhan , Animais , Drosophila melanogaster/fisiologia , Drosophila melanogaster/genética , Síndrome de Lesch-Nyhan/genética , Síndrome de Lesch-Nyhan/metabolismo , Purinas/metabolismo , Modelos Animais de Doenças , Comportamento Animal , Hipoxantina Fosforribosiltransferase/genética , Hipoxantina Fosforribosiltransferase/metabolismo , Hipoxantina Fosforribosiltransferase/deficiência , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , LocomoçãoRESUMO
Pervasive transcriptional activity is observed across diverse species. The genomes of extant organisms have undergone billions of years of evolution, making it unclear whether these genomic activities represent effects of selection or 'noise'1-4. Characterizing default genome states could help understand whether pervasive transcriptional activity has biological meaning. Here we addressed this question by introducing a synthetic 101-kb locus into the genomes of Saccharomyces cerevisiae and Mus musculus and characterizing genomic activity. The locus was designed by reversing but not complementing human HPRT1, including its flanking regions, thus retaining basic features of the natural sequence but ablating evolved coding or regulatory information. We observed widespread activity of both reversed and native HPRT1 loci in yeast, despite the lack of evolved yeast promoters. By contrast, the reversed locus displayed no activity at all in mouse embryonic stem cells, and instead exhibited repressive chromatin signatures. The repressive signature was alleviated in a locus variant lacking CpG dinucleotides; nevertheless, this variant was also transcriptionally inactive. These results show that synthetic genomic sequences that lack coding information are active in yeast, but inactive in mouse embryonic stem cells, consistent with a major difference in 'default genomic states' between these two divergent eukaryotic cell types, with implications for understanding pervasive transcription, horizontal transfer of genetic information and the birth of new genes.
Assuntos
Genes Sintéticos , Genoma , Saccharomyces cerevisiae , Transcrição Gênica , Animais , Humanos , Camundongos , Cromatina/genética , Ilhas de CpG , Genes Sintéticos/genética , Genoma/genética , Células-Tronco Embrionárias Murinas/metabolismo , Regiões Promotoras Genéticas/genética , Saccharomyces cerevisiae/genética , Hipoxantina Fosforribosiltransferase/genética , Evolução MolecularRESUMO
Maternal hyperhomocysteinemia (HCY) induced by genetic defects in methionine cycle enzymes or vitamin imbalance is known to be a pathologic factor that can impair embryonal brain development and cause long-term consequences in the postnatal brain development as well as changes in the expression of neuronal genes. Studies of the gene expression on this model requires the selection of optimal housekeeping genes. This work aimed to analyze the expression stability of housekeeping genes in offspring brain. Pregnant female Wistar rats were treated daily with a 0.15% L-methionine solution in the period starting on the 4th day of pregnancy until delivery, to cause the increase in the homocysteine level in fetus blood and brain. Housekeeping gene expression was assessed by RT-qPCR on whole embryonic brain and selected rat brain areas at P20 and P90. The amplification curves were analyzed, and raw means Cq data were imported to the RefFinder online tool to assess the reference genes stability. Most of the analyzed genes showed high stability of mRNA expression in the fetal brain at both periods of analysis (E14 and E20). However, the most stably expressed genes at different age points differed. Actb, Ppia, Rpl13a are the most stably expressed on E14, Ywhaz, Pgk1, Hprt1 - on E20 and P20, Hprt1, Actb, and Pgk1 - on P90. Gapdh gene used as a reference in various studies demonstrates high stability only in the hippocampus and cannot be recommended as the optimal reference gene on HCY model. Hprt1 and Pgk1 genes were found to be the most stably expressed in the brain of rat subjected to HCY. These two genes showed high stability in the brain on E20 and in various areas of the brain on the P20 and P90. On E14, the preferred genes for normalization are Actb, Ppia, Rpl13a.
Assuntos
Hiper-Homocisteinemia , Feminino , Gravidez , Ratos , Animais , Hiper-Homocisteinemia/induzido quimicamente , Hiper-Homocisteinemia/genética , Ratos Wistar , Encéfalo , Metionina , Racemetionina , Hipoxantina FosforribosiltransferaseRESUMO
BACKGROUND: Reverse transcription quantitative polymerase chain reaction (RT-qPCR) can accurately detect relative gene expression levels in biological samples. However, widely used reference genes exhibit unstable expression under certain conditions. METHODS AND RESULTS: Here, we compared the expression stability of eight reference genes (RPLP0, RPS18, RPL13, EEF1A1, ß-actin, GAPDH, HPRT1, and TUBB) commonly used in liproxstatin-1 (Lip-1)-treated K562 cells using RNA-sequencing and RT-qPCR. The expression of EEF1A1, ACTB, GAPDH, HPRT1, and TUBB was considerably lower in cells treated with 20 µM Lip-1 than in the control, and GAPDH also showed significant downregulation in the 10 µM Lip-1 group. Meanwhile, when we used geNorm, NormFinder, and BestKeeper to compare expression stability, we found that GAPDH and HPRT1 were the most unstable reference genes among all those tested. Stability analysis yielded very similar results when geNorm or BestKeeper was used but not when NormFinder was used. Specifically, geNorm and BestKeeper identified RPL13 and RPLP0 as the most stable genes under 20 µM Lip-1 treatment, whereas RPL13, EEF1A1, and TUBB were the most stable under 10 µM Lip-1 treatment. TUBB and EEF1A1 were the most stable genes in both treatment groups according to the results obtained using NormFinder. An assumed most stable gene was incorporated into each software to validate the accuracy. The results suggest that NormFinder is not an appropriate algorithm for this study. CONCLUSIONS: Stable reference genes were recognized using geNorm and BestKeeper but not NormFinder. Overall, RPL13 and RPLP0 were the most stable reference genes under 20 µM Lip-1 treatment, whereas RPL13, EEF1A1, and TUBB were the most stable genes under 10 µM Lip-1 treatment.
Assuntos
Actinas , Leucemia , Humanos , Células K562 , Sequência de Bases , Análise de Sequência de RNA , Hipoxantina Fosforribosiltransferase , Proteínas de Neoplasias , Proteínas RibossômicasRESUMO
BACKGROUND: Lesch-Nyhan disease (LND) is a severe neurological disorder caused by the genetic deficiency of hypoxanthine-guanine phosphoribosyltransferase (HGprt), an enzyme involved in the salvage synthesis of purines. To compensate this deficiency, there is an acceleration of the de novo purine biosynthetic pathway. Most studies have failed to find any consistent abnormalities of purine nucleotides in cultured cells obtained from the patients. Recently, it has been shown that 5-aminoimidazole-4-carboxamide riboside 5'-monophosphate (ZMP), an intermediate of the de novo pathway, accumulates in LND fibroblasts maintained with RPMI containing physiological levels (25 nM) of folic acid (FA), which strongly differs from FA levels of regular cell culture media (2200 nM). However, RPMI and other standard media contain non-physiological levels of many nutrients, having a great impact in cell metabolism that does not precisely recapitulate the in vivo behavior of cells. METHODS: We prepared a new culture medium containing physiological levels of all nutrients, including vitamins (Plasmax-PV), to study the potential alterations of LND fibroblasts that may have been masked by the usage of non-physiological media. We quantified ZMP accumulation under different culture conditions and evaluated the activity of two known ZMP-target proteins (AMPK and ADSL), the mRNA expression of the folate carrier SLC19A1, possible mitochondrial alterations and functional consequences in LND fibroblasts. RESULTS: LND fibroblasts maintained with Plasmax-PV show metabolic adaptations such a higher glycolytic capacity, increased expression of the folate carrier SCL19A1, and functional alterations such a decreased mitochondrial potential and reduced cell migration compared to controls. These alterations can be reverted with high levels of folic acid, suggesting that folic acid supplements might be a potential treatment for LND. CONCLUSIONS: A complete physiological cell culture medium reveals new alterations in Lesch-Nyhan disease. This work emphasizes the importance of using physiological cell culture conditions when studying a metabolic disorder.
Assuntos
Síndrome de Lesch-Nyhan , Humanos , Síndrome de Lesch-Nyhan/genética , Síndrome de Lesch-Nyhan/metabolismo , Hipoxantina Fosforribosiltransferase/genética , Hipoxantina Fosforribosiltransferase/metabolismo , Células Cultivadas , Fibroblastos/metabolismo , Ácido FólicoRESUMO
The two-test in vitro battery for genotoxicity testing (Ames and micronucleus) has in the majority of cases replaced the three-test battery (as two-test plus mammalian cell gene mutation assay) for the routine testing of chemicals, pharmaceuticals, cosmetics, and agrochemical metabolites originating from food and feed as well as from water treatment. The guidance for testing agrochemical groundwater metabolites, however, still relies on the three-test battery. Data collated in this study from 18 plant protection and related materials highlights the disparity between the often negative Ames and in vitro chromosome aberration data and frequently positive in vitro mammalian cell gene mutation assays. Sixteen of the 18 collated materials with complete datasets were Ames negative, and overall had negative outcomes in in vitro chromosome damage tests (weight of evidence from multiple tests). Mammalian cell gene mutation assays (HPRT and/or mouse lymphoma assay (MLA)) were positive in at least one test for every material with this data. Where both MLA and HPRT tests were performed on the same material, the HPRT seemed to give fewer positive responses. In vivo follow-up tests included combinations of comet assays, unscheduled DNA synthesis, and transgenic rodent gene mutation assays, all gave negative outcomes. The inclusion of mammalian cell gene mutation assays in a three-test battery for groundwater metabolites is therefore not justified and leads to unnecessary in vivo follow-up testing.
Assuntos
Hipoxantina Fosforribosiltransferase , Linfoma , Camundongos , Animais , Testes de Mutagenicidade , Ensaio Cometa , Roedores , Agroquímicos , Testes para Micronúcleos , Dano ao DNARESUMO
Small cell lung cancer (SCLC) has a poor prognosis, emphasizing the necessity for developing new therapies. The de novo synthesis pathway of purine nucleotides, which is involved in the malignant growth of SCLC, has emerged as a novel therapeutic target. Purine nucleotides are supplied by two pathways: de novo and salvage. However, the role of the salvage pathway in SCLC and the differences in utilization and crosstalk between the two pathways remain largely unclear. Here, we found that deletion of the HPRT1 gene, which codes for the rate-limiting enzyme of the purine salvage pathway, significantly suppressed tumor growth in vivo in several SCLC cells. We also demonstrated that HPRT1 expression confers resistance to lemetrexol (LMX), an inhibitor of the purine de novo pathway. Interestingly, HPRT1-knockout had less effect on SCLC SBC-5 cells, which are more sensitive to LMX than other SCLC cell lines, suggesting that a preference for either the purine de novo or salvage pathway occurs in SCLC. Furthermore, metabolome analysis of HPRT1-knockout cells revealed increased intermediates in the pentose phosphate pathway and elevated metabolic flux in the purine de novo pathway, indicating compensated metabolism between the de novo and salvage pathways in purine nucleotide biosynthesis. These results suggest that HPRT1 has therapeutic implications in SCLC and provide fundamental insights into the regulation of purine nucleotide biosynthesis. IMPLICATIONS: SCLC tumors preferentially utilize either the de novo or salvage pathway in purine nucleotide biosynthesis, and HPRT1 has therapeutic implications in SCLC.
Assuntos
Neoplasias Pulmonares , Carcinoma de Pequenas Células do Pulmão , Humanos , Carcinoma de Pequenas Células do Pulmão/genética , Purinas/metabolismo , Nucleotídeos de Purina/metabolismo , Hipoxantina Fosforribosiltransferase/metabolismo , Neoplasias Pulmonares/genéticaRESUMO
Hematopoietic stem/progenitor cell (HSPC)-based anti-HIV-1 gene therapy holds great promise to eradicate HIV-1 or to provide long-term remission through a continuous supply of anti-HIV-1 gene-modified cells without ongoing antiretroviral therapy. However, achieving sufficient engraftment levels of anti-HIV gene-modified HSPC to provide therapeutic efficacy has been a major limitation. Here, we report an in vivo selection strategy for anti-HIV-1 gene-modified HSPC by introducing 6-thioguanine (6TG) chemoresistance through knocking down hypoxanthine-guanine phosphoribosyl transferase (HPRT) expression using RNA interference (RNAi). We developed a lentiviral vector capable of co-expressing short hairpin RNA (shRNA) against HPRT alongside two anti-HIV-1 genes: shRNA targeting HIV-1 co-receptor CCR5 and a membrane-anchored HIV-1 fusion inhibitor, C46, for efficient in vivo selection of anti-HIV-1 gene-modified human HSPC. 6TG-mediated preconditioning and in vivo selection significantly enhanced engraftment of HPRT-knockdown anti-HIV-1 gene-modified cells (>2-fold, p < 0.0001) in humanized bone marrow/liver/thymus (huBLT) mice. Viral load was significantly reduced (>1 log fold, p < 0.001) in 6TG-treated HIV-1-infected huBLT mice compared to 6TG-untreated mice. We demonstrated that 6TG-mediated preconditioning and in vivo selection considerably improved engraftment of HPRT-knockdown anti-HIV-1 gene-modified HSPC and repopulation of anti-HIV-1 gene-modified hematopoietic cells in huBLT mice, allowing for efficient HIV-1 inhibition.
Assuntos
HIV-1 , Transplante de Células-Tronco Hematopoéticas , Humanos , Camundongos , Animais , HIV-1/fisiologia , Hipoxantina Fosforribosiltransferase/genética , Hipoxantina Fosforribosiltransferase/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Medula Óssea/metabolismo , Tioguanina/metabolismo , Tioguanina/farmacologia , RNA Interferente Pequeno/genéticaRESUMO
Lesch-Nyhan syndrome (LNS, MIM300322) is a rare inherited disorder caused by mutations in HPRT1 gene. Here we describe the generation of induced pluripotent stem cells (iPSCs) from an infected child carrying the HPRT1 mutation c.508C > T(p.R170X) by reprogramming peripheral blood mononuclear cells (PBMCs) with episomal vectors. The obtained hiPSCs exhibited normal karyotype, expressed pluripotency markers, and possessed trilineage differentiation capacity.
Assuntos
Células-Tronco Pluripotentes Induzidas , Síndrome de Lesch-Nyhan , Criança , Humanos , Síndrome de Lesch-Nyhan/genética , Leucócitos Mononucleares , Hipoxantina Fosforribosiltransferase/genética , Mutação/genética , Diferenciação Celular , Fatores de Iniciação de Peptídeos/genéticaRESUMO
Hypoxanthine phosphoribosyl transferase 1 (HPRT1), once considered a housekeeping gene, has been identified as playing an important role in several tumors. Its role in pan-cancer, however, has not been systematically studied. This study evaluates the relationship between HPRT1 and clinical parameters, survival prognosis, and tumor immunity based on multi omics data from the Cancer Genome Atlas (TCGA) and the Gene Expression Omnibus (GEO) databases. Drug sensitivity analysis screened 16 effective drugs against HPRT1, exploring the interactions with chemicals and genes. The significance of HPRT1 in tumor immunotherapy has also been investigated. Immunohistochemistry confirmed significant differences in the expression of HPRT1 between five tumor types (colon adenocarcinoma [COAD], head-neck squamous cell carcinoma [HNSC], lung adenocarcinoma [LUAD], thyroid carcinoma [THCA], and uterine corpus endometrial carcinoma [UCEC]) and adjacent normal tissues (P < 0.05). HPRT1 competitive endogenous RNA network was constructed in HNSC. Through cytological experiments, it was verified that HPRT1 plays a carcinogenic role in HNSC and is associated with tumor cell proliferation, migration, invasion, and apoptosis. In addition, there was a significant positive correlation between HPRT1 and programmed cell death-1 (PD-1) expression in HNSC (P < 0.05). These findings suggest that HPRT1 may be a potential biomarker for predicting and treating cancer.
Assuntos
Adenocarcinoma , Neoplasias do Colo , Neoplasias Pulmonares , Neoplasias da Glândula Tireoide , Humanos , Prognóstico , Hipoxantina FosforribosiltransferaseRESUMO
Mutations in T lymphocytes (T-cells) are informative quantitative markers for environmental mutagen exposures, but risk extrapolations from rodent models to humans also require an understanding of how T-cell development and proliferation kinetics impact mutagenic outcomes. Rodent studies have shown that patterns in chemical-induced mutations in the hypoxanthine-guanine phosphoribosyltransferase (Hprt) gene of T-cells differ between lymphoid organs. The current work was performed to obtain knowledge of the relationships between maturation events during T-cell development and changes in chemical-induced mutant frequencies over time in differing immune compartments of a mouse model. A novel reverse transcriptase-polymerase chain reaction based method was developed to determine the specific T-cell receptor beta (Tcrb) gene mRNA expressed in mouse T-cell isolates, enabling sequence analysis of the PCR product that then identifies the specific hypervariable CDR3 junctional region of the expressed Tcrb gene for individual isolates. Characterization of spontaneous Hprt mutant isolates from the thymus, spleen, and lymph nodes of control mice for their Tcrb gene expression found evidence of in vivo clonal amplifications of Hprt mutants and their trafficking between tissues in the same animal. Concurrent analyses of Hprt mutations and Tcrb gene rearrangements in different lymphoid tissues of control versus N-ethyl-N-nitrosourea-exposed mice permitted elucidation of the localization and timing of mutational events in T-cells, establishing that mutagenesis occurs primarily in the pre-rearrangement replicative period in pre-thymic/thymic populations. These findings demonstrate that chemical-induced mutagenic burden is determined by the combination of mutagenesis and T-cell clonal expansion, processes with roles in immune function and in the pathogenesis of autoimmune disease and cancer.
Assuntos
Etilnitrosoureia , Linfócitos T , Camundongos , Humanos , Animais , Etilnitrosoureia/toxicidade , Mutação , Mutagênese/genética , Mutagênicos/toxicidade , Hipoxantina Fosforribosiltransferase/genéticaRESUMO
Lilial (also called lysmeral) is a fragrance ingredient presented in many everyday cosmetics and household products. The concentrations of lilial in the final products is rather low. Its maximum concentration in cosmetics was limited and recently, its use in cosmetics products was prohibited in the EU due to the classification as reproductive toxicant. Additionally, according to the European Chemicals Agency, it was under assessment as one of the potential endocrine disruptors, i.e. a substance that may alter the function of the endocrine system and, as a result, cause health problems. Its ability to act as an androgen receptor agonist and the estrogenic and androgenic activity of its metabolites, to the best of our knowledge, have not yet been tested. The aim of this work was to determine the intestinal absorption, cytotoxicity, nephrotoxicity, mutagenicity, activation of cellular stress-related signal pathways and, most importantly, to test the ability to disrupt the endocrine system of lilial and its Phase I metabolites. This was tested using set of in vitro assays including resazurin assay, the CHO/HPRT mutation assay, γH2AX biomarker-based genotoxicity assay, qPCR and in vitro reporter assays based on luminescence of luciferase for estrogen, androgen, NF-κB and NRF2 signalling pathway. It was determined that neither lilial nor its metabolites have a negative effect on cell viability in the concentration range from 1 nM to 100 µM. Using human cell lines HeLa9903 and MDA-kb2, it was verified that this substance did not have agonistic activity towards estrogen or androgen receptor, respectively. Lilial metabolites, generated by incubation with the rat liver S9 fraction, did not show the ability to bind to estrogen or androgen receptors. Neither lilial nor its metabolites showed a nephrotoxic effect on human renal tubular cells (RPTEC/TERT1 line) and at the same time they were unable to activate the NF-κB and NRF2 signalling pathway at a concentration of 50 µM (HEK 293/pGL4.32 or pGL4.37). Neither lilial nor its metabolites showed mutagenic activity in the HPRT gene mutation test in CHO-K1 cells, nor were they able to cause double-strand breaks in DNA (γH2AX biomarker) in CHO-K1 and HeLa cells. In our study, no negative effects of lilial or its in vitro metabolites were observed up to 100 µM using different in vitro tests.
Assuntos
Hipoxantina Fosforribosiltransferase , NF-kappa B , Humanos , Ratos , Animais , Células HeLa , Células HEK293 , Fator 2 Relacionado a NF-E2 , Estrogênios/toxicidade , Estrogênios/metabolismo , Androgênios , BiomarcadoresRESUMO
DESIGN: Study participants were divided equally into three groups being exclusive vapers (never smokers), current exclusive smokers and non-users. Brush biopsy samples of oral epithelial cells were collected. DNA damage quantification was assessed using LA-QPCR, and analysis interrogated a 12.2 kb region of the DNA polymerase beta gene (POLB). An additional gene, hypoxanthine phosphoribosyltransferase 1 (HPRT), was also interrogated for validity. Enzyme-linked immunosorbent assay (ELISA) was used to measure plasma cotinine levels. Breath monitoring was measured using Bedfont Micro Smokerlyzer in order to quantify exhaled CO and %COHb levels in participants. CASE SELECTION: 72 subjects, consisting of both males and females of diverse ages, races and ethnicities, were recruited. Comprehensive interviews alongside biochemical studies were used to verify smoking and vaping status. Participants classified as vapers reported a minimum use of e-cigarettes three times weekly for 6 months, with no use of cigarettes or tobacco products in their lifetime. Smokers reported cigarette consumption for a minimum of three times weekly for at least 12 months, less than five vaping sessions ever and no use of other tobacco products in the previous 6 months. Participants reporting no or less than five uses of e-cigarettes or tobacco products were classified as non-users. Former smokers, vapers and those who were dual or poly users of e-cigarettes, cigarettes or tobacco products were excluded. DATA ANALYSIS: R environment for statistical computing (RStudio), was used for data analysis. The Shapiro-Wilk test was used to evaluate the distribution of data. Student's t test allowed comparison of all variables between two groups (vapers and nonusers, smokers and nonusers, or vapers and smokers), specifically DNA damage levels. A one-way Analysis of Variance (ANOVA) followed by a post hoc Tukey HSD test allowed comparison of damage in three or more groups (heavy vapers, light vapers, and nonusers, as well as heavy smokers, light smokers, and nonusers). DNA damage was also analysed in this manner when assessing e-cigarette device type, liquid type or in non-users. Pearson correlation coefficient analysis allowed examination of relationships between different variables. RESULTS: Mean levels of DNA damage in the POLB gene was 2.6-fold higher in vapers (p = 0.005) and 2.2-fold higher in smokers (p = 0.020), when compared to non-users. On comparing POLB gene DNA damage in vapers versus smokers, the results were not statistically significant (p = 0.522). Comparing DNA damage in the HPRT gene, levels were much higher in vapers (p = 0.029) and smokers (p = 0.033) versus non-users. Similarly to the POLB gene, DNA damage levels in the HPRT gene in vapers versus smokers were not statistically significant (p = 0.578). When assessing volume of e-cigarette liquid or smoking pack years, levels of DNA damage in increased in the POLB gene in a dose-dependent manner between 'light' and 'heavy' users versus non-users (F = 4.571, p = 0.0156 | Tukey's HSD p = 0.0195 in vapers, F = 4.368, p = 0.0185 | Tukey's HSD p = 0.0135 in smokers). Vaping device type was investigated showing mean level of DNA damage in the oral cells of pod device users was 3.3-fold higher compared to non-users (F = 3.886, p = 0.0152 | Tukey's HSD p = 0.0216). This was followed by a 2.6-fold increase in oral cell DNA damage in Mod device users, and a 1.6-fold increase in multiple device users. Levels of DNA damage was higher in those who consume sweet-flavoured e-liquid (F = 3.238, p = 0.0146 | Tukey's HSD p < 0.05), followed by vapers of multiple flavours, mint or menthol and tobacco, and fruit flavours. No correlation was found between DNA damage of oral cells and cumulative nicotine consumption in vapers (r = 0.3189, p = 0.1288). Plasma cotinine levels, a validated maker of tobacco in cigarettes and e-cigarettes, were not significantly different between vapers and smokers (p = 0.607), but were significantly higher compared to non-users (p < 0.0001). Whist compared to non-users, vapers had similar levels of CO and %COHb, smokers showed significantly increased levels (p = 0.0005 and p = 0.0002, respectively). CONCLUSIONS: Based on the results of this study, there is evidence to support a dose-dependent formation of DNA damage in oral cells in those vapers who have never smoked cigarettes, and in those exclusive cigarette smokers. Additionally, e-cigarette device type and flavour, may also determine levels of DNA damage.
Assuntos
Sistemas Eletrônicos de Liberação de Nicotina , Produtos do Tabaco , Feminino , Humanos , Masculino , Cotinina , DNA , Hipoxantina Fosforribosiltransferase , FumarRESUMO
Temozolomide (TMZ) is a standard treatment for glioblastoma (GBM) patients. However, TMZ has moderate therapeutic effects due to chemoresistance of GBM cells through less clarified mechanisms. Here, we demonstrate that TMZ-derived 5-aminoimidazole-4-carboxamide (AICA) is converted to AICA ribosyl-5-phosphate (AICAR) in GBM cells. This conversion is catalyzed by hypoxanthine phosphoribosyl transferase 1 (HPRT1), which is highly expressed in human GBMs. As the bona fide activator of AMP-activated protein kinase (AMPK), TMZ-derived AICAR activates AMPK to phosphorylate threonine 52 (T52) of RRM1, the catalytic subunit of ribonucleotide reductase (RNR), leading to RNR activation and increased production of dNTPs to fuel the repairment of TMZ-induced-DNA damage. RRM1 T52A expression, genetic interruption of HPRT1-mediated AICAR production, or administration of 6-mercaptopurine (6-MP), a clinically approved inhibitor of HPRT1, blocks TMZ-induced AMPK activation and sensitizes brain tumor cells to TMZ treatment in mice. In addition, HPRT1 expression levels are positively correlated with poor prognosis in GBM patients who received TMZ treatment. These results uncover a critical bifunctional role of TMZ in GBM treatment that leads to chemoresistance. Our findings underscore the potential of combined administration of clinically available 6-MP to overcome TMZ chemoresistance and improve GBM treatment.
Assuntos
Glioblastoma , Hipoxantina Fosforribosiltransferase , Ribonucleotídeo Redutases , Animais , Humanos , Camundongos , Proteínas Quinases Ativadas por AMP , Resistencia a Medicamentos Antineoplásicos/genética , Glioblastoma/tratamento farmacológico , Glioblastoma/genética , Hipoxantinas , Mercaptopurina , Temozolomida/farmacologia , Hipoxantina Fosforribosiltransferase/genéticaRESUMO
Proteins play important roles in biology, biotechnology and pharmacology, and missense variants are a common cause of disease. Discovering functionally important sites in proteins is a central but difficult problem because of the lack of large, systematic data sets. Sequence conservation can highlight residues that are functionally important but is often convoluted with a signal for preserving structural stability. We here present a machine learning method to predict functional sites by combining statistical models for protein sequences with biophysical models of stability. We train the model using multiplexed experimental data on variant effects and validate it broadly. We show how the model can be used to discover active sites, as well as regulatory and binding sites. We illustrate the utility of the model by prospective prediction and subsequent experimental validation on the functional consequences of missense variants in HPRT1 which may cause Lesch-Nyhan syndrome, and pinpoint the molecular mechanisms by which they cause disease.
Assuntos
Hipoxantina Fosforribosiltransferase , Síndrome de Lesch-Nyhan , Humanos , Estudos Prospectivos , Hipoxantina Fosforribosiltransferase/genética , Hipoxantina Fosforribosiltransferase/metabolismo , Proteínas/genética , Mutação de Sentido IncorretoRESUMO
Lesch-Nyhan disease (LND) is a X-linked genetic disease affecting boys characterized by complex neurological and neuropsychiatric symptoms. LND is caused by loss of function mutations in the HPRT1 gene leading to decrease activity of hypoxanthine-guanine phosphoribosyl transferase enzyme (HGPRT) and altered purine salvage pathway (Lesch and Nyhan, 1964). This study describes the generation of isogenic clones with deletions in HPRT1 produced from one male human embryonic stem cell line using CRISPR/Cas9 strategy. Differentiation of these cells into different neuronal subtypes will help elucidating the neurodevelopmental events leading to LND and develop therapeutic strategies for this devastating neurodevelopmental disorder.
Assuntos
Células-Tronco Embrionárias Humanas , Síndrome de Lesch-Nyhan , Humanos , Masculino , Síndrome de Lesch-Nyhan/genética , Síndrome de Lesch-Nyhan/metabolismo , Hipoxantina Fosforribosiltransferase/genética , Hipoxantina Fosforribosiltransferase/metabolismo , Sistemas CRISPR-Cas/genética , Técnicas de Inativação de Genes , Células-Tronco Embrionárias Humanas/metabolismoRESUMO
Twelve N2'-branched acyclic nucleoside phosphonates and bisphosphonates were synthesized as potential inhibitors of Plasmodium falciparum hypoxanthine-guanine-xanthine phosphoribosyltransferase (PfHGXPRT), the key enzyme in the purine salvage pathway for production of purine nucleotides. The chemical structures were designed with the aim to study selectivity of the inhibitors for PfHGXPRT over human HGPRT. The newly prepared compounds contain 9-deazahypoxanthine connected to a phosphonate group via a five-atom-linker bearing a nitrogen atom (N2') as a branching point. All compounds, with the additional phosphonate group(s) in the second aliphatic linker attached to N2' atom, were low micromolar inhibitors of PfHGXPRT with low to modest selectivity for the parasite enzyme over human HGPRT. The effect of the addition of different chemical groups/linkers to N2' atom on the inhibition constants and selectivity is discussed.
Assuntos
Antimaláricos , Organofosfonatos , Humanos , Hipoxantina Fosforribosiltransferase/metabolismo , Hipoxantina Fosforribosiltransferase/farmacologia , Nucleosídeos/farmacologia , Nucleosídeos/química , Plasmodium falciparum , Organofosfonatos/farmacologia , Organofosfonatos/química , Antimaláricos/farmacologia , Antimaláricos/química , Pentosiltransferases , Hipoxantinas/farmacologia , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/químicaRESUMO
The highly immunosuppressive nature of head-neck squamous cell cancer (HNSCC) is not fully understood. Exosomes play crucial roles in the communication between cancer and non-cancer cells, but the clinical significance of the expression of exosome-related genes (ERGs) remains unclear in HNSCC. This study aimed to establish an HNSCC-ERGs model by using mass spectrometry (MS)-based label-free quantitative proteomics in combination with the TCGA primary HNSCC dataset. The study managed to classify the HNSCC patients into two subtypes based on the expression level of prognostic ERGs, which showed significant differences in prognosis and immune infiltration. LASSO regression algorithm was used to establish a risk prediction model based on seven risky genes (PYGL, ACTN2, TSPAN15, EXT2, PLAU, ITGA5), and the high-risk group was associated with poor survival prognosis and suppressive immune status. HPRT1 and PYGL were found to be independent prognostic factors through univariate and multivariate Cox regression analyses. Immune and ssGSEA analysis revealed that HPRT1 and PYGL were significantly related to immunosuppression, immune response, and critical signaling transduction pathways in HNSCC. Immunohistochemistry results further validated the expression level, clinical value, and immunosuppressive function of HPRT1 and PYGL in HNSCC patients. In conclusion, this study established molecular subtypes and a prediction risk model based on the ERGs. Furthermore, the findings suggested that HPRT1 and PYGL might play critical roles in reshaping the tumor microenvironment.