RESUMO
The precise characterization of oxygen-sensing pathways and the identification of pO2-regulated gene expression are both issues of critical importance. The O2-sensing system plays crucial roles in almost all the pivotal human processes, including the stem cell specification, the growth and development of tissues (such as embryogenesis), the modulation of intermediate metabolism (including the shift of the glucose metabolism from oxidative to anaerobic ATP production and vice versa), and the control of blood pressure. The solid cancer microenvironment is characterized by low oxygen levels and by the consequent activation of the hypoxia response that, in turn, allows a complex adaptive response characterized mainly by neoangiogenesis and metabolic reprogramming. Recently, incredible advances in molecular genetic methodologies allowed the genome editing with high efficiency and, above all, the precise identification of target cells/tissues. These new possibilities and the knowledge of the mechanisms of adaptation to hypoxia suggest the effective development of new therapeutic approaches based on the manipulation, targeting, and exploitation of the oxygen-sensor system molecular mechanisms.
Assuntos
Edição de Genes , Neoplasias , Humanos , Hipóxia/genética , Oxigênio/metabolismo , Hipóxia Celular/genética , Neoplasias/genética , Neoplasias/terapia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Proteína Supressora de Tumor Von Hippel-Lindau/genética , Microambiente Tumoral/genéticaRESUMO
Mutations in VHL, which encodes von Hippel-Lindau tumor suppressor (VHL), are associated with divergent diseases. We describe a patient with marked erythrocytosis and prominent mitochondrial alterations associated with a severe germline VHL deficiency due to homozygosity for a novel synonymous mutation (c.222CâA, p.V74V). The condition is characterized by early systemic onset and differs from Chuvash polycythemia (c.598CâT) in that it is associated with a strongly reduced growth rate, persistent hypoglycemia, and limited exercise capacity. We report changes in gene expression that reprogram carbohydrate and lipid metabolism, impair muscle mitochondrial respiratory function, and uncouple oxygen consumption from ATP production. Moreover, we identified unusual intermitochondrial connecting ducts. Our findings add unexpected information on the importance of the VHL-hypoxia-inducible factor (HIF) axis to human phenotypes. (Funded by Associazione Italiana Ricerca sul Cancro and others.).
Assuntos
Mutação em Linhagem Germinativa , Transtornos do Crescimento/genética , Hipoglicemia/genética , Fator 1 Induzível por Hipóxia/deficiência , Mitocôndrias/metabolismo , Proteína Supressora de Tumor Von Hippel-Lindau/genética , Expressão Gênica , Crescimento/genética , Humanos , Masculino , Metaboloma/genética , Metaboloma/fisiologia , Síndrome , Adulto JovemRESUMO
p57Kip2 protein is a member of the CIP/Kip family, mainly localized in the nucleus where it exerts its Cyclin/CDKs inhibitory function. In addition, the protein plays key roles in embryogenesis, differentiation, and carcinogenesis depending on its cellular localization and interactors. Mutations of CDKN1C, the gene encoding human p57Kip2, result in the development of different genetic diseases, including Beckwith-Wiedemann, IMAGe and Silver-Russell syndromes. We investigated a specific Beckwith-Wiedemann associated CDKN1C change (c.946 C>T) that results in the substitution of the C-terminal amino acid (arginine 316) with a tryptophan (R316W-p57Kip2). We found a clear redistribution of R316W-p57Kip2, in that while the wild-type p57Kip2 mostly occurs in the nucleus, the mutant form is also distributed in the cytoplasm. Transfection of two expression constructs encoding the p57Kip2 N- and C-terminal domain, respectively, allows the mapping of the nuclear localization signal(s) (NLSs) between residues 220-316. Moreover, by removing the basic RKRLR sequence at the protein C-terminus (from 312 to 316 residue), p57Kip2 was confined in the cytosol, implying that this sequence is absolutely required for nuclear entry. In conclusion, we identified an unreported p57Kip2 NLS and suggest that its absence or mutation might be of relevance in CDKN1C-associated human diseases determining significant changes of p57Kip2 localization/regulatory roles.
Assuntos
Síndrome de Beckwith-Wiedemann/genética , Núcleo Celular/metabolismo , Inibidor de Quinase Dependente de Ciclina p57/genética , Inibidor de Quinase Dependente de Ciclina p57/metabolismo , Mutação , Sinais de Localização Nuclear , Síndrome de Beckwith-Wiedemann/patologia , Ciclo Celular , Núcleo Celular/genética , Proliferação de Células , Células HEK293 , Células Hep G2 , HumanosRESUMO
Lithium salt is the first-line therapeutic option for bipolar disorder and has been proposed as a potential antitumoral drug. The effects of LiCl treatment were investigated in SH-SY5Y, a human neuroblastoma cell line and an in vitro model of dopaminergic neuronal differentiation. LiCl, at the dosage used in psychiatric treatment, does not affect cell proliferation, while at higher doses it delays the SH-SY5Y cell division cycle and for prolonged usage reduces cell viability. Moreover, the ion treatment affects DNA integrity as demonstrated by accumulation of p53 and γH2AX (the phosphorylated form of H2AX histone), two important markers of genome damage. p57Kip2, a CIP/Kip protein, is required for proper neuronal maturation and represents a main factor of response to stress including genotoxicity. We evaluated the effect of lithium on p57Kip2 levels. Unexpectedly, we found that lithium downregulates the level of p57Kip2 in a dose-dependent manner, mainly acting at the transcriptional level. A number of different approaches, mostly based on p57Kip2 content handling, confirmed that the CKI/Kip reduction plays a key role in the DNA damage activated by lithium and suggests the unanticipated view that p57Kip2 might be involved in DNA double-strand break responses. In conclusion, our study identified novel roles for p57Kip2 in the molecular mechanism of lithium at high concentration and, more in general, in the process of DNA repair.
Assuntos
Inibidor de Quinase Dependente de Ciclina p57/genética , Dano ao DNA , Lítio/farmacologia , Neurônios/efeitos dos fármacos , Linhagem Celular Tumoral , Inibidor de Quinase Dependente de Ciclina p57/metabolismo , Humanos , Lítio/toxicidade , Neurônios/metabolismoRESUMO
The CDKN1C gene encodes the p57Kip2 protein which has been identified as the third member of the CIP/Kip family, also including p27Kip1 and p21Cip1. In analogy with these proteins, p57Kip2 is able to bind tightly and inhibit cyclin/cyclin-dependent kinase complexes and, in turn, modulate cell division cycle progression. For a long time, the main function of p57Kip2 has been associated only to correct embryogenesis, since CDKN1C-ablated mice are not vital. Accordingly, it has been demonstrated that CDKN1C alterations cause three human hereditary syndromes, characterized by altered growth rate. Subsequently, the p57Kip2 role in several cell phenotypes has been clearly assessed as well as its down-regulation in human cancers. CDKN1C lies in a genetic locus, 11p15.5, characterized by a remarkable regional imprinting that results in the transcription of only the maternal allele. The control of CDKN1C transcription is also linked to additional mechanisms, including DNA methylation and specific histone methylation/acetylation. Finally, long non-coding RNAs and miRNAs appear to play important roles in controlling p57Kip2 levels. This review mostly represents an appraisal of the available data regarding the control of CDKN1C gene expression. In addition, the structure and function of p57Kip2 protein are briefly described and correlated to human physiology and diseases.
Assuntos
Diferenciação Celular/genética , Inibidor de Quinase Dependente de Ciclina p57/genética , Inibidor de Quinase Dependente de Ciclina p57/metabolismo , Epigenômica , Regulação da Expressão Gênica , Homeostase/genética , Animais , HumanosRESUMO
BACKGROUND: Iron overload syndromes include a wide range of diseases frequently associated with increased morbidity and mortality. Several organs are affected in patients with iron overload including liver, heart, joints, endocrine glands, and pancreas. Moreover, severe bone and hemopoietic tissue alterations are observed. Because of the role of bone marrow mesenchymal stromal cells (BM-MSCs) in bone turnover and hematopoiesis, iron effects on primary BM-MSCs cultures were evaluated. METHODS: Primary human BM-MSCs cultures were prepared and the effects of iron on their proliferation and differentiation were characterized by biochemical analyses and functional approaches. RESULTS: Addition of iron to the culture medium strongly increased BM-MSCs proliferation and induced their accelerated S phase entry. Iron enters BM-MSCs through both transferrin-dependent and transferrin-independent mechanisms, inducing the accumulation of cyclins E and A, the decrease of p27(Kip1), and the activation of MAPK pathway. Conversely, neither apoptotic signs nor up-regulation of reactive oxygen species were observed. Iron inhibited both differentiation of BM-MSCs into osteoblasts and in vitro matrix calcification. These effects result from the merging of inhibitory activities on BM-MSCs osteoblastic commitment and on the ordered matrix calcification process. CONCLUSIONS: We demonstrated that BM-MSCs are a target of iron overload. Iron accelerates BM-MSCs proliferation and affects BM-MSCs osteoblastic commitment, hampering matrix calcification. GENERAL SIGNIFICANCE: Our study reports, for the first time, that iron, at concentration found in overloaded patient sera, stimulates the growth of BM-MSCs, the BM multipotent stromal cell component. Moreover, iron modulates the physiological differentiation of these cells, affecting bone turnover and remodeling.
Assuntos
Calcificação Fisiológica , Sobrecarga de Ferro/patologia , Células-Tronco Mesenquimais/fisiologia , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Humanos , Sistema de Sinalização das MAP Quinases/fisiologia , Osteoblastos/fisiologia , Espécies Reativas de Oxigênio/metabolismoRESUMO
Congenital erythrocytosis (CE), or congenital polycythemia, represents a rare and heterogeneous clinical entity. It is caused by deregulated red blood cell production where erythrocyte overproduction results in elevated hemoglobin and hematocrit levels. Primary congenital familial erythrocytosis is associated with low erythropoietin (Epo) levels and results from mutations in the Epo receptor gene (EPOR). Secondary CE arises from conditions causing tissue hypoxia and results in increased Epo production. These include hemoglobin variants with increased affinity for oxygen (HBB, HBA mutations), decreased production of 2,3-bisphosphoglycerate due to BPGM mutations, or mutations in the genes involved in the hypoxia sensing pathway (VHL, EPAS1, and EGLN1). Depending on the affected gene, CE can be inherited either in an autosomal dominant or recessive mode, with sporadic cases arising de novo. Despite recent important discoveries in the molecular pathogenesis of CE, the molecular causes remain to be identified in about 70% of the patients. With the objective of collecting all the published and unpublished cases of CE the COST action MPN&MPNr-Euronet developed a comprehensive Internet-based database focusing on the registration of clinical history, hematological, biochemical, and molecular data (http://www.erythrocytosis.org/). In addition, unreported mutations are also curated in the corresponding Leiden Open Variation Database.
Assuntos
Bases de Dados Genéticas , Predisposição Genética para Doença , Mutação , Policitemia/congênito , Receptores da Eritropoetina/genética , Hipóxia Celular/genética , Eritropoetina/metabolismo , Humanos , Internet , Policitemia/genética , Policitemia/metabolismo , Transdução de Sinais/genéticaRESUMO
Fanconi anemia is an inherited disease characterized by congenital malformations, pancytopenia, cancer predisposition, and sensitivity to cross-linking agents. The molecular diagnosis of Fanconi anemia is relatively complex for several aspects including genetic heterogeneity with mutations in at least 16 different genes. In this paper, we report the mutations identified in 100 unrelated probands enrolled into the National Network of the Italian Association of Pediatric Hematoly and Oncology. In approximately half of these cases, mutational screening was carried out after retroviral complementation analyses or protein analysis. In the other half, the analysis was performed on the most frequently mutated genes or using a next generation sequencing approach. We identified 108 distinct variants of the FANCA, FANCG, FANCC, FANCD2, and FANCB genes in 85, 9, 3, 2, and 1 families, respectively. Despite the relatively high number of private mutations, 45 of which are novel Fanconi anemia alleles, 26% of the FANCA alleles are due to 5 distinct mutations. Most of the mutations are large genomic deletions and nonsense or frameshift mutations, although we identified a series of missense mutations, whose pathogenetic role was not always certain. The molecular diagnosis of Fanconi anemia is still a tiered procedure that requires identifying candidate genes to avoid useless sequencing. Introduction of next generation sequencing strategies will greatly improve the diagnostic process, allowing a rapid analysis of all the genes.
Assuntos
Proteínas de Grupos de Complementação da Anemia de Fanconi/genética , Anemia de Fanconi/genética , Mutação , Substituição de Aminoácidos , Linhagem Celular , Estudos de Coortes , Biologia Computacional , Bases de Dados de Ácidos Nucleicos , Efeito Fundador , Genótipo , Humanos , Itália , Mosaicismo , Polimorfismo de Nucleotídeo ÚnicoRESUMO
Plants produce a remarkable amount of low molecular mass natural products endowed with a large array of pivotal biological activities. Among these molecules, resveratrol (3,5,4'-trihydroxystilbene) has been identified as an important modulator of cell phenotype with a complex and pleiotropic mode of action. Extensive literature regarding its activity, mainly employing cellular models, suggests that this polyphenol controls cell proliferation, induces differentiation, and activates apoptosis and autophagy. The compound also modulates angiogenesis and inflammation. Similarly, studies on implanted cancers and chemical-induced tumors confirm the potential chemotherapeutical interest of the compound. Likewise, several reports clearly demonstrated, in animal models, that the compound might positively affect the development and evolution of chronic diseases including type 2 diabetes, obesity, coronary heart disease, metabolic syndrome, and neurogenerative pathologies. Finally, a number of investigations stated that the toxicity of the molecule is scarce. Despite these promising observations, few clinical trials have yet been performed to evaluate the effectiveness of the molecule both in prevention and treatment of human chronic disease. Preliminary findings therefore suggest the need for more extensive clinical investigations.
Assuntos
Antineoplásicos Fitogênicos/uso terapêutico , Pesquisa Biomédica , Senescência Celular/efeitos dos fármacos , Neoplasias/prevenção & controle , Estilbenos/uso terapêutico , Animais , Ensaios Clínicos como Assunto , Humanos , ResveratrolRESUMO
Among pro-inflammatory cytokines, Interleukin-1ß is crucially involved in several inflammatory-based diseases and even cancer. Increased Interleukin-1ß levels in oral fluids have been proposed as an early marker of periodontitis, a broadly diffused chronic inflammatory condition of periodontal-supporting tissues, leading eventually to tooth loss. We describe the development of a portable surface-plasmon-resonance-based optical fiber probe suitably coated with an anti-Interleukin-1ß antibody monolayer. A pico-nanomolar linear range of determination was obtained in both buffer solution and saliva with a rapid (3 min) incubation and high selectivity in presence of interferents. Higher Interleukin-1ß concentration in the saliva of a periodontitis patient compared to a healthy control was determined. These measurements were validated by an automated ELISA system. Our results sustain the potential applicability of the proposed SPR-POF as diagnostic point-of-care device for real-time monitoring of salivary Interleukin-1ß, that can support early detection of oral inflammatory-based pathologies and rapid and timely therapeutic decisions.
RESUMO
Hypoxia-inducible factor 2α (HIF-2α) plays a pivotal role in the balancing of oxygen requirements throughout the body. The protein is a transcription factor that modulates the expression of a wide array of genes and, in turn, controls several key processes including energy metabolism, erythropoiesis and angiogenesis. We describe here the identification of two cases of familial erythrocytosis associated with heterozygous HIF2A missense mutations, namely Ile533Val and Gly537Arg. Ile533Val is a novel mutation and represents the genetic HIF2A change nearest to Pro-531, the primary hydroxyl acceptor residue, so far identified. The Gly537Arg missense mutation has already been described in familial erythrocytosis. However, our patient is the only described case of a de novo HIF2A mutation associated with the development of congenital polycythemia. Functional in vivo studies, based on exogenous expression of hybrid HIF-2α transcription factors, indicated that these genetic alterations lead to the stabilization of HIF-2α protein. All the identified polycythemic subjects with HIF2A mutations show serum erythropoietin in the normal range, independently of the hematocrit values and phlebotomy frequency. The erythroid precursors obtained from the peripheral blood of patients showed an altered phenotype, including an increased rate of growth and a modified expression of some HIF-2α target genes. These results suggest the novel proposal that polycythemia observed in subjects with HIF2A mutations might also be due to primary changes in hematopoietic cells and not only secondary to increased erythropoietin levels.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Eritropoetina/sangue , Mutação de Sentido Incorreto/genética , Policitemia/congênito , Adolescente , Adulto , Sequência de Aminoácidos , Biomarcadores/sangue , Estudos de Coortes , Humanos , Masculino , Dados de Sequência Molecular , Policitemia/sangue , Policitemia/diagnóstico , Policitemia/genética , Valores de ReferênciaAssuntos
Anemia Hipocrômica/genética , Proteínas de Transporte de Cátions/genética , Mutação com Perda de Função , Mutação de Sentido Incorreto , Anemia Hipocrômica/terapia , Transfusão de Sangue , Éxons/genética , Feminino , Heterozigoto , Humanos , Recém-Nascido , Ferro/análise , Sobrecarga de Ferro , Fígado/química , Miocárdio/química , LinhagemRESUMO
BACKGROUND: At the end of 2019 a new respiratory syndrome emerged in China named Coronavirus disease 2019 (COVID-19) due to SARS-CoV-2 infection. Considering the severity of the disease in adult subjects with one or more chronic pathologies, it was mandatory to find simple and effective biomarkers for negative prognosis of the disease easily available at the admission to the hospital. METHODS: To identify possible parameters showing association with the outcome in COVID-19 patients with pre-existing chronic diseases, blood biochemical profiles of 511 patients, enrolled from March to June 2020, were retrospectively evaluated. The pathological conditions taken into consideration were diabetes, arterial hypertension, chronic kidney disease, cardiovascular diseases, chronic obstructive pulmonary disease, obesity, and cancer. All the data were collected upon admission to the emergency room (ER) during the indicated period. RESULTS: We observed that serum and ionized calcium were prevalently altered in our cohort. We determined that hypocalcemia was a major parameter associated with mechanical ventilation and poor prognosis, correlating also with the presence of comorbidities such as cardiovascular diseases, chronic kidney disease, and cancer. In addition, we found a positive correlation between hypocalcemia and clinical complications during hospitalizations. CONCLUSIONS: Our results strengthen the relevance of serum calcium concentration as a useful prognostic biomarker in hospitalized COVID-19 patients.
Assuntos
COVID-19 , Doenças Cardiovasculares , Hipocalcemia , Adulto , Humanos , SARS-CoV-2 , Estudos Retrospectivos , Hipocalcemia/diagnóstico , Cálcio , BiomarcadoresRESUMO
Point-of-Care tests based on biomarkers, useful to monitor acute and chronic inflammations, are required for advances in medicine. In this scope, a key role is played by pro-inflammatory cytokines, of which interleukin 6 (IL-6) is generally thought as one of the most relevant. To use IL-6 in real scenarios, detection in ultra-low concentration ranges is required. In this work, two IL-6 biosensors are obtained by exploiting the combination of the same antibody self-assembled monolayer with two different plasmonic probes. This approach has demonstrated, via experimental results, that two different IL-6 concentration ranges can be explored. More specifically, IL-6 in an atto-femto molar range can be detected via polymer-based nanoplasmonic chips. On the other hand, a pico-nano molar range is obtained by a surface plasmon resonance platform in plastic optical fibers. As a proof of concept, the detection of IL-6 at the femto molar range has been obtained in Saliva and Serum. The results show that the proposed sensing approach could be useful in developing Point-of-Care devices based on a general setup with the capability to exploit both the plasmonic biosensor chips to monitor the IL-6 in the concentration range of interest, to provide an important support for the diagnosis and monitoring of oral and systemic diseases.
Assuntos
Técnicas Biossensoriais , Interleucina-6 , Polimetil Metacrilato , Técnicas Biossensoriais/métodos , Ressonância de Plasmônio de Superfície , Testes ImediatosRESUMO
Numerous pieces of evidence demonstrate that oxidative stress impairs biological functions, speeds up aging, and has a role in a variety of human diseases, including systemic and oral inflammatory disorders, and even cancer. Therefore, technologies providing accurate measures of oxidative stress indicators or biomarkers appear essential in the identification/prevention of such diseases, and in their management. Particularly advantageous is the employement of point-of-care tests based on affordable and small biochips since they can quickly process biological samples and deliver results near the point of care for a prompt therapeutic intervention. Malondialdehyde (MDA) is a key byproduct of oxidative reaction and has been identified as an effective marker of oxidative stress. Herein, we describe the detection of MDA in buffer and in a complex matrix such as saliva, using a plasmonic optical fiber device combined with a highly selective anti-MDA antibody. The experimental results highlight the excellent performance of the proposed biosensor, as well as its ability to provide a low-cost point-of-care test (PoC-T) to be used in real life situations. We demonstrated that a single saliva dilution step and a short incubation time are required for the accurate detection of low concentrations of total MDA (free and conjugated). As a proof-of-concept of future biomedical applications, the method has been tested to determine MDA concentration in saliva of a periodontitis patient compared to that of a healthy control. The obtained findings represent the basis for developing PoC-Ts to be employed in monitoring oral diseases like periodontitis, oral cancers or systemic oxidative-stress associated pathologies. Conclusively, our study puts the ground for an oxidative stress biosensor widely-applicable to different scenarios.
Assuntos
Técnicas Biossensoriais , Periodontite , Humanos , Malondialdeído , Sistemas Automatizados de Assistência Junto ao Leito , Fibras Ópticas , Estresse Oxidativo , BiomarcadoresRESUMO
OBJECTIVES: The evaluation of salivary biomarkers has been proposed as a simple and non-invasive aid to the conventional periodontal diagnosis based on clinical-radiographic parameters. Matrix metalloproteinase-8 (MMP-8), especially in its active form, is considered one of the most reliable biomarkers of periodontitis, and point-of-care tests (POCTs) have been proposed for its clinical monitoring. In this proof-of-concept study, a novel highly sensitive POCT based on a plastic optical fiber (POF) biosensor exploiting surface plasmon resonance (SPR) to detect salivary MMP-8 is described. METHODS: A SPR-POF biosensor was functionalized with a specific antibody to develop a surface-assembled monolayer (SAM) for the detection of total MMP-8. A white light source and a spectrometer connected to the biosensor were used to quantify MMP-8 level in both buffer and real matrix (saliva) by analysing the shift of the resonance wavelength determined by the specific antigen-antibody binding upon the SAM. RESULTS: Dose-response curves by serial dilutions of human recombinant MMP-8 were realized, obtaining a limit of detection (LOD) of 40 pM (1.76 ng/ml) in buffer and 225 pM (9.9 ng/ml) in saliva and high selectivity compared to interferent analytes (MMP-2 and IL-6). CONCLUSIONS: The proposed optical fiber-based POCT was able to detect and measure total MMP-8 with high selectivity and very low LOD in both buffer and saliva. CLINICAL SIGNIFICANCE: The SPR-POF technology may be employed to create highly sensitive biosensors to monitor salivary MMP-8 levels. The possibility of specifically detecting its active, rather than total, form need to be further investigated. If confirmed and clinically validated, such a device may represent a promising tool to make an immediate, highly sensitive and reliable diagnosis of periodontitis, and to carry out a timely and targeted therapy, possibly helping to prevent the onset of local and systemic periodontitis-related complications.
Assuntos
Biomarcadores , Técnicas Biossensoriais , Metaloproteinase 8 da Matriz , Doenças Periodontais , Saliva , Humanos , Metaloproteinase 8 da Matriz/análise , Saliva/química , Técnicas Biossensoriais/métodos , Doenças Periodontais/patologia , Biomarcadores/análiseRESUMO
The analysis of salivary biomarkers as expression of periodontal health conditions has been proposed as a useful aid to conventional diagnostic approaches. In this study, we present a point-of-care test (POCT) exploiting a surface plasmon resonance (SPR)-based optical biosensor to detect salivary macrophage inflammatory protein (MIP)-1α, a promising marker of periodontitis. A plastic optical fiber (POF) was suitably modified and functionalized by an antibody self-assembled monolayer against MIP-1α for plasmonic detection. The proposed SPR-POF biosensor showed high selectivity and very low limit of detection for MIP-1α of 129 fM (1.0 pg/mL) in phosphate-buffered saline and 346 fM (2.7 pg/mL) in saliva. As a proof of concept, this POCT was also able to discriminate between a periodontitis patient and a healthy subject. The obtained results support the future application of this technology for an on-site detection and real-time monitoring of periodontal health conditions for diagnostic and therapeutic purposes.
RESUMO
It is now definitively established that a large part of the human genome is transcribed. However, only a scarce percentage of the transcriptome (about 1.2%) consists of RNAs that are translated into proteins, while the large majority of transcripts include a variety of RNA families with different dimensions and functions. Within this heterogeneous RNA world, a significant fraction consists of sequences with a length of more than 200 bases that form the so-called long non-coding RNA family. The functions of long non-coding RNAs range from the regulation of gene transcription to the changes in DNA topology and nucleosome modification and structural organization, to paraspeckle formation and cellular organelles maturation. This review is focused on the role of long non-coding RNAs as regulators of cyclin-dependent kinase inhibitors' (CDKIs) levels and activities. Cyclin-dependent kinases are enzymes necessary for the tuned progression of the cell division cycle. The control of their activity takes place at various levels. Among these, interaction with CDKIs is a vital mechanism. Through CDKI modulation, long non-coding RNAs implement control over cellular physiology and are associated with numerous pathologies. However, although there are robust data in the literature, the role of long non-coding RNAs in the modulation of CDKIs appears to still be underestimated, as well as their importance in cell proliferation control.
Assuntos
Proteínas Inibidoras de Quinase Dependente de Ciclina , RNA Longo não Codificante , Quinases Ciclina-Dependentes/metabolismo , Humanos , RNA Longo não Codificante/genéticaRESUMO
Chronic myelogenous leukemia (CML) is characterized by the expression of BCR-ABL tyrosine kinase, which results in increased cell proliferation and inhibition of apoptosis. In this study, we show that BCR-ABL-positive CML cell lines treated with imatinib (STI571) undergo G1 cell cycle arrest associated with the accumulation of p57(Kip)², a cyclin-dependent kinase inhibitor (CKI). Interestingly, p57(Kip)² increase precedes the reported STI571-dependent upregulation of p27(Kip)¹. A number of complementary approaches allow the demonstration that p57(Kip)² buildup is due to the transcriptional activation of CDKN1C, the p57(Kip)²-encoding gene, while neither p57(Kip)² half-life elongation nor its cell relocalization were observed. We also identified a heretofore undescribed pattern of p57(Kip)² phosphorylated isoforms which, however, did not change in response to STI571 cell treatment. The imatinib-dependent p57(Kip)² upregulation occurs only in STI571-responsive cells, while the CKI accumulation was not evidenced in an imatinib-resistant clone. Nilotinib and dasatinib (second-generation BCR-ABL inhibitors), at concentrations comparable to those used in therapy, increase the CKI but do not affect p27(Kip)¹ level. Finally, CD34(+) cells from CML patients display a clear imatinib-dependent p57(Kip)² upregulation, which was not observed in CD34(+) cells from control subjects. In conclusion, our study points to p57(Kip)² as a novel and precocious effector of BCR-ABL targeting drugs.
Assuntos
Inibidor de Quinase Dependente de Ciclina p57/metabolismo , Proteínas de Fusão bcr-abl/metabolismo , Leucemia Mielogênica Crônica BCR-ABL Positiva/enzimologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Tirosina Quinases/metabolismo , Antineoplásicos/farmacologia , Benzamidas , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Inibidor de Quinase Dependente de Ciclina p57/efeitos dos fármacos , Dasatinibe , Eletroforese em Gel Bidimensional , Proteínas de Fusão bcr-abl/efeitos dos fármacos , Humanos , Mesilato de Imatinib , Immunoblotting , Imunoprecipitação , Piperazinas/farmacologia , Proteínas Tirosina Quinases/efeitos dos fármacos , Pirimidinas/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tiazóis/farmacologia , Transcrição Gênica/efeitos dos fármacosRESUMO
The Cyclin-dependent kinase (CDK) regulator p27Kip1 is a gatekeeper of G1/S transition. It also regulates G2/M progression and cytokinesis completion, via CDK-dependent or -independent mechanisms. Recently, other important p27Kip1 functions have been described, including the regulation of cell motility and migration, the control of cell differentiation program and the activation of apoptosis/autophagy. Several factors modulate p27Kip1 activities, including its level, cellular localization and post-translational modifications. As a matter of fact, the protein is phosphorylated, ubiquitinated, SUMOylated, O-linked N-acetylglicosylated and acetylated on different residues. p27Kip1 belongs to the family of the intrinsically unstructured proteins and thus it is endowed with a large flexibility and numerous interactors, only partially identified. In this review, we look at p27Kip1 properties and ascribe part of its heterogeneous functions to the ability to act as an anchor or scaffold capable to participate in the construction of different platforms for modulating cell response to extracellular signals and allowing adaptation to environmental changes.