RESUMO
Purpose: In HT29 colon cancer cells, a close interplay between self-DNA-induced TLR9 signaling and autophagy response was found, with remarkable effects on cell survival and differentiation. IGF1R activation drives the development and malignant progression of colorectal cancer. IGF1R inhibition displays a controversial effect on autophagy. The interrelated roles of IGF1R inhibition and TLR9/autophagy signaling in HT29 cancer cells have not yet been clarified. In our study, we aimed to investigate the complex interplay of IGF1R inhibition and TLR9/autophagy signaling in HT29 cells. Methods: HT29 cells were incubated with tumor-originated self-DNA with or without inhibitors of IGF1R (picropodophyllin), autophagy (chloroquine), and TLR9 (ODN2088), respectively. Cell proliferation and metabolic activity measurements, direct cell counting, NanoString and Taqman gene expression analyses, immunocytochemistry, WES Simple Western blot, and transmission electron microscopy investigations were performed. Results: The concomitant use of tumor-derived self-DNA and IGF1R inhibitors displays anti-proliferative potential, which can be reversed by parallel TLR9 signaling inhibition. The distinct effects of picropodophyllin, ODN2088, and chloroquine per se or in combination on HT29 cell proliferation and autophagy suggest that either the IGF1R-associated or non-associated autophagy machinery is "Janus-faced" regarding its actions on cell proliferation. Autophagy, induced by different combinations of self-DNA and inhibitors is not sufficient to rescue HT29 cells from death but results in the survival of some CD133-positive stem-like HT29 cells. Conclusion: The creation of new types of combined IGF1R, autophagy, and/or TLR9 signaling inhibitors would play a significant role in the development of more personalized anti-tumor therapies for colorectal cancer.
Assuntos
Neoplasias do Colo , Receptor Toll-Like 9 , Autofagia , Cloroquina/farmacologia , DNA de Neoplasias , Células HT29 , Humanos , Receptor IGF Tipo 1 , Receptor Toll-Like 9/genéticaRESUMO
HGFR activation drives the malignant progression of colorectal cancer, and its inhibition displays anti-autophagic activity. The interrelated role of HGFR inhibition and TLR9/autophagy signaling in HT29 cancer cells subjected to modified self-DNA treatments has not been clarified. We analyzed this complex interplay with cell metabolism and proliferation measurements, TLR9, HGFR and autophagy inhibitory assays and WES Simple Western blot-based autophagy flux measurements, gene expression analyses, immunocytochemistry, and transmission electron microscopy. The overexpression of MyD88 and caspase-3 was associated with enhanced HT29 cell proliferation, suggesting that incubation with self-DNAs could suppress the apoptosis-induced compensatory cell proliferation. HGFR inhibition blocked the proliferation-reducing effect of genomic and hypermethylated, but not that of fragmented DNA. Lowest cell proliferation was achieved with the concomitant use of genomic DNA, HGFR inhibitor, and chloroquine, when the proliferation stimulating effect of STAT3 overexpression could be outweighed by the inhibitory effect of LC3B, indicating the putative involvement of HGFR-mTOR-ULK1 molecular cascade in HGFR inhibitor-mediated autophagy. The most intense cell proliferation was caused by the co-administration of hypermethylated DNA, TLR9 and HGFR inhibitors, when decreased expression of both canonical and non-canonical HGFR signaling pathways and autophagy-related genes was present. The observed ultrastructural changes also support the context-dependent role of HGFR inhibition and autophagy on cell survival and proliferation. Further investigation of the influence of the studied signaling pathways and cellular processes can provide a basis for novel, individualized anti-cancer therapies.
Assuntos
Neoplasias , Receptor Toll-Like 9 , Apoptose , Autofagia , Linhagem Celular Tumoral , Proliferação de Células , DNA/farmacologia , Células HT29 , Humanos , Proteínas Proto-Oncogênicas c-met , Receptor Toll-Like 9/genética , Receptor Toll-Like 9/metabolismoRESUMO
Connexin (Cx) proteins establish intercellular gap junction channels (Cx GJCs) through coupling of two apposed hexameric Cx hemichannels (Cx HCs, connexons). Pre- and post-GJ interfaces consist of extracellular EL1 and EL2 loops, each with three conserved cysteines. Previously, we reported that known peptide inhibitors, mimicking a variety of Cx43 sequences, appear non-selective when binding to homomeric Cx43 vs. Cx36 GJC homology model subtypes. In pursuit of finding potentially Cx subtype-specific inhibitors of connexon-connexon coupling, we aimed at to understand better how the GJ interface is formed. Here we report on the discovery of Cx GJC subtype-specific protein stabilization centers (SCs) featuring GJ interface architecture. First, the Cx43 GJC homology model, embedded in two opposed membrane bilayers, has been devised. Next, we endorsed the fluctuation dynamics of SCs of the interface domain of Cx43 GJC by applying standard molecular dynamics under open and closed cystine disulfide bond (CS-SC) preconditions. The simulations confirmed the major role of the unique trans-GJ SC pattern comprising conserved (55N, 56T) and non-conserved (57Q) residues of the apposed EL1 loops in the stabilization of the GJC complex. Importantly, clusters of SC patterns residing close to the GJ interface domain appear to orient the interface formation via the numerous SCs between EL1 and EL2. These include central 54CS-S198C or 61CS-S192C contacts with residues 53R, 54C, 55N, 197D, 199F or 64V, 191P, respectively. In addition, we revealed that GJC interface formation is favoured when the psi dihedral angle of the nearby 193P residue is stable around 180° and the interface SCs disappear when this angle moves to the 0° to -45° range. The potential of the association of non-conserved residues with SC motifs in connexon-connexon coupling makes the development of Cx subtype-specific inhibitors viable.
Assuntos
Conexinas , Junções Comunicantes , Conexinas/metabolismo , Junções Comunicantes/metabolismo , Canais Iônicos/metabolismo , Simulação de Dinâmica MolecularRESUMO
Part of the ventral striatal division, the nucleus accumbens (NAc) drives the circuit activity of an entire macrosystem about reward like a "flagship," signaling and leading diverse conducts. Accordingly, NAc neurons feature complex inhibitory phenotypes that assemble to process circuit inputs and generate outputs by exploiting specific arrays of opposite and/or parallel neurotransmitters, neuromodulatory peptides. The resulting complex combinations enable versatile yet specific forms of accumbal circuit plasticity, including maladaptive behaviors. Although reward signaling and behavior are elaborately linked to neuronal circuit activities, it is plausible to propose whether these neuronal ensembles and synaptic islands can be directly controlled by astrocytes, a powerful modulator of neuronal activity. Pioneering studies showed that astrocytes in the NAc sense citrate cycle metabolites and/or ATP and may induce recurrent activation. We argue that the astrocytic calcium, GABA, and Glu signaling and altered sodium and chloride dynamics fundamentally shape metaplasticity by providing active regulatory roles in the synapse- and network-level flexibility of the NAc.
Assuntos
Astrócitos/metabolismo , Plasticidade Neuronal/fisiologia , Núcleo Accumbens/citologia , Núcleo Accumbens/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Ácido Glutâmico/metabolismo , Humanos , Ácido gama-Aminobutírico/metabolismoRESUMO
The most common cystic fibrosis mutation, ΔF508 in nucleotide binding domain 1 (NBD1), impairs cystic fibrosis transmembrane conductance regulator (CFTR)-coupled domain folding, plasma membrane expression, function and stability. VX-809, a promising investigational corrector of ΔF508-CFTR misprocessing, has limited clinical benefit and an incompletely understood mechanism, hampering drug development. Given the effect of second-site suppressor mutations, robust ΔF508-CFTR correction most likely requires stabilization of NBD1 energetics and the interface between membrane-spanning domains (MSDs) and NBD1, which are both established primary conformational defects. Here we elucidate the molecular targets of available correctors: class I stabilizes the NBD1-MSD1 and NBD1-MSD2 interfaces, and class II targets NBD2. Only chemical chaperones, surrogates of class III correctors, stabilize human ΔF508-NBD1. Although VX-809 can correct missense mutations primarily destabilizing the NBD1-MSD1/2 interface, functional plasma membrane expression of ΔF508-CFTR also requires compounds that counteract the NBD1 and NBD2 stability defects in cystic fibrosis bronchial epithelial cells and intestinal organoids. Thus, the combination of structure-guided correctors represents an effective approach for cystic fibrosis therapy.
Assuntos
Aminopiridinas/farmacologia , Benzodioxóis/farmacologia , Regulador de Condutância Transmembrana em Fibrose Cística/fisiologia , Animais , Sítios de Ligação , Brônquios/citologia , Membrana Celular/metabolismo , Cricetinae , Fibrose Cística/genética , Fibrose Cística/terapia , Regulador de Condutância Transmembrana em Fibrose Cística/química , Retículo Endoplasmático/metabolismo , Células Epiteliais/metabolismo , Glicosilação , Humanos , Mutação , Nucleotídeos/química , Dobramento de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes/químicaRESUMO
Leukemic cells often express markers, which are not characteristic of their particular cell lineage. In this study, we identified the "A" subunit of coagulation factor XIII (FXIII-A) in leukemic promyelocytes in de novo AML M3 cases. The cytoplasmic presence of factor XIII-A has previously been shown only in platelets/megakaryocytes and monocytes/macrophages. Furthermore, more recently we described the presence of FXIII-A in leukemic lymphoblasts. We studied 14 patients with this rare type of acute leukemia in a period of 4 years and investigated their bone marrow samples by 3-color flow cytometry upon diagnosis, mainly focusing on FXIII-A expression of leukemic cells. We detected FXIII-A also by ELISA, Western-blot, and confocal laser scanning microscopy. This was a homogenous group of AML M3 patients with translocation t(15;17)(q22;q21) detected by fluorescence in situ hybridization (FISH). In 10 out of 14 samples, FXIII-A was detectable by flow cytometry and was coexpressed with markers characteristic for leukemic promyleocytes (CD45dim/CD13+/CD33+/CD117+/cyMPO+ and HLA-DR-/CD34-/CD14-/CD15-). Staining for the markers GPIIb and GPIX were negative, and FXIII-A was identified in the cytoplasm of the cells by confocal microscopy in a relatively high quantity, as measured by ELISA. By Western blot analysis we could identify FXIII-A in the native 82 kDa form and in cleaved forms corresponding to cleavage products observed when purified FXIII-A was treated by human neutrophil elastase. This novel expression site of FXIII-A in AML M3 can be considered as a leukemia associated immunophenotype and may have pathophysiological significance.
Assuntos
Fator XIIIa/metabolismo , Leucemia Promielocítica Aguda/metabolismo , Subunidades Proteicas/metabolismo , Adulto , Idoso , Biomarcadores/metabolismo , Western Blotting , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Células Precursoras de Granulócitos/metabolismo , Humanos , Imunofenotipagem , Leucemia Promielocítica Aguda/patologia , Masculino , Microscopia Confocal , Pessoa de Meia-IdadeRESUMO
The intracellular form of the coagulation factor XIII has previously been identified by immunomorphological techniques using polyclonal antibodies. In these studies, only the A subunit (FXIII-A) was detectable in megakaryocytes/platelets and in monocytes/macrophages. We developed several novel monoclonal antibody clones directed to both subunits (FXIII-A and FXIII-B) and investigated their appearance in normal and leukemic cells. By using 3- and 4-color flow cytometry FXIII expression was investigated in normal peripheral blood and bone marrow samples and in acute myeloblastic (AML) and lymphoblastic (ALL) leukemia cases. Samples were studied by Western blotting and confocal laser scanning microscopy. With a previously published ELISA assay applying two monoclonal antibodies directed to different epitopes in FXIII-A, we were able to measure the intracytoplasmic content of FXIII-A in normal cells and leukemic blasts. FXIII-A was detectable in normal peripheral blood monocytes and in large quantities in platelets, but both cell types were negative for FXIII-B. There was no surface staining for FXIII-A, it only appeared intracellularly. In samples derived from patients with AML M4 and M5, FXIII-A sensitively identified blast cells. Although normal lymphocytes do not express FXIII-A, 40% of ALL cases showed significant FXIII-A expression as determined by flow cytometry. FXIII-A positivity of lymphoblasts was verified by Western blotting, ELISA, and confocal laser scanning microscopy cytometry. These data provide evidence that FXIII-A is a sufficiently sensitive marker in differentiating myeloblasts and monoblasts and is suitable for identifying leukemia-associated phenotypes in ALL.
Assuntos
Biomarcadores Tumorais/metabolismo , Fator XIII/química , Fator XIII/fisiologia , Leucemia/sangue , Leucemia/diagnóstico , Doença Aguda , Animais , Plaquetas/química , Plaquetas/metabolismo , Plaquetas/patologia , Fator XIII/biossíntese , Fator XIII/genética , Citometria de Fluxo/métodos , Humanos , Leucemia/patologia , Monócitos/química , Monócitos/metabolismo , Monócitos/patologiaRESUMO
The association of coagulation factors with leukocytes have been demonstrated in several previous studies. This study was designed to study the sensitivity and specificity of factor XIII subunit A (FXIII-A) labelling in cultured myeloblastic and monoblastic cell lines and to investigate the intracytoplasmic expression of FXIII-A in de novo acute myeloid leukemia (AML) samples. Myeloblastic and a monoblastic cell lines were cultured and investigated for lineage specific maturation markers and FXIII-A expression. Furthermore, FXIII-A expression was investigated in 12 normal samples (7 bone marrow and 5 peripheral blood), 86 de novo AML samples and 6 chronic myelomonocytic leukemia (CMML) samples. In the monoblastic MonoMac6 cell line the appearance of FXIII-A preceded that of CD14 while it remained negative in the myeloblastic PLB-985 cell line throughout its maturation period. Among the AML samples the average frequency of FXIII-A positive cells in myeloblastic leukemia samples was below 10%, while in M4 and M5AML samples it was above 50% and was significantly higher than the generally used CD14 marker (p < 0.0001). In the AML M4 and M5 cases, FXIII-A proved sensitive for the identification of monoblasts. FXIII-A can be considered as a reliable intracytoplasmic marker for the monocytic and megakaryocytic series and its presence is highly predictive for mono- and megakaryocytic AML and for CMML.
Assuntos
Fator XIIIa/metabolismo , Fator XIIIa/fisiologia , Citometria de Fluxo/métodos , Leucemia Mieloide Aguda/metabolismo , Anticorpos Monoclonais/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Linhagem da Célula , Células Cultivadas , Citoplasma/metabolismo , Granulócitos/citologia , Humanos , Leucemia Mieloide Aguda/classificação , Leucócitos/metabolismo , Receptores de Lipopolissacarídeos/biossíntese , Megacariócitos/citologia , Monócitos/citologia , Monócitos/metabolismo , Células Mieloides/citologia , Sensibilidade e EspecificidadeRESUMO
Here we report the conclusions of our 20-year experience with familial occurrence of malignant hematological disorders. Between 1 January 1983 and 1 January 2003, 35 families of multigenerational hematological malignancies were identified in first or second degree relatives. We established the following conclusions in subsequent generations: (i) a significantly earlier onset (P < 0.0001); (ii) an equal or enhanced severity of the disease; and (iii) higher relative risk in offsprings for different hematological disorders. We propose to use the term double acceleration when both phenomena (earlier onset and identical or enhanced severity) are experienced simultaneously. The data emphasize the need to use detailed cytogenetic and molecular biological studies to identify the underlying defects in cases where double acceleration can be verified.
Assuntos
Neoplasias Hematológicas/epidemiologia , Linhagem , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Saúde da Família , Feminino , Neoplasias Hematológicas/diagnóstico , Humanos , Masculino , Pessoa de Meia-Idade , Fatores de TempoRESUMO
Proteins are heteropolymers with evolutionary selected native sequences of residues. These native sequences code for unique and stable 3D structures indispensable for biochemical activity and for proteolysis resistance, the latter which guarantees an appropriate lifetime for the protein in the protease rich cellular environment. Cross-links between residues close in space but far in the primary structure are required to maintain the folded structure of proteins. Some of these cross-links are covalent, most frequently disulfide bonds, but the majority of the cross-links are sets of cooperative noncovalent long-range interactions. In this paper we focus on special clusters of noncovalent long-range interactions: the Stabilization Centers (SCs). The relation between the SCs and secondary structural elements as well as the relation between SCs and functionally important regions of proteins are presented to show a detailed picture of these clusters, which are believed to be primarily responsible for major aspects of protein stability.
Assuntos
Proteínas/química , Reagentes de Ligações Cruzadas , Dissulfetos/química , Modelos Moleculares , Peptídeos/química , Conformação Proteica , Dobramento de Proteína , Estrutura Secundária de Proteína , Relação Estrutura-AtividadeRESUMO
Multidrug resistance (MDR) is a naturally occurring defense phenomenon by which cells battle against chemically foreign substances (xenobiotics), including some cytotoxic drugs. Membrane transporter hyperactivity is a major contributor to MDR and is the primary target of both diagnostic and therapeutic interventions. Multi-xenobiotic resistance can be exploited as several fluorescent indicator probes are extruded by the same drug transporters, making it possible to quantitatively measure MDR activity in cell lines and clinical samples by flow cytometry. The literature on MDR is reported in a number of different formats, making it difficult to compare data from various groups. This article will briefly review the pathomechanism, then focus upon the diagnostic approach, the interpretation of results from clinical samples and correlations with other variables. The authors believe that a standardized MDR assay, as well as a suitable monitoring test, may become a prognostic marker in several types of leukemia.
Assuntos
Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Resistência a Múltiplos Medicamentos , Resistencia a Medicamentos Antineoplásicos , Leucemia/fisiopatologia , Anticorpos/metabolismo , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Bloqueadores dos Canais de Cálcio/metabolismo , Humanos , Leucemia/tratamento farmacológico , Linfócitos/metabolismo , Prognóstico , Verapamil/metabolismoRESUMO
CD38 is expressed on the surface of leukemic cells in a significant percentage of patients with B-cell chronic lymphocytic leukaemia (CLL). From the literature it is known that CD38 expression has prognostic value in CLL, suggesting an association between CD38 expression and the mutational status of IgV genes. Peripheral blood samples from 82 patients with CLL were analyzed by flow cytometry for CD38 expression on CD19+ leukemic cells. CD38 was expressed in 30% or more of leukemic cells in 26 patients (patients with 30% or more B cells coexpressing CD19/CD38 were considered positive). Seven of the 26 patients with high CD38 expression and eight of the 56 patients with low CD38 expression had advanced-stage disease (RAI III-IV). Higher than 4 mg/l levels of beta 2-microglobulin was measured in the serum of nine of 26 CD38+ and seven of 56 CD38- patients. Our analyses showed that the high CD38 expression is associated with other risk factors, identifying an aggressive disease, which require treatment. It will be important to conduct further studies to establish the prognostic value of the high CD38 expression in early-stage disease.
Assuntos
ADP-Ribosil Ciclase/sangue , Antígenos CD/sangue , Biomarcadores Tumorais/sangue , Leucemia Linfocítica Crônica de Células B/imunologia , ADP-Ribosil Ciclase 1 , Idoso , Idoso de 80 Anos ou mais , Antígenos CD19/sangue , Apoptose/genética , Aberrações Cromossômicas , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/patologia , Masculino , Glicoproteínas de Membrana , Pessoa de Meia-Idade , Mutação , Estadiamento de Neoplasias , Valor Preditivo dos Testes , Prognóstico , Microglobulina beta-2/sangueRESUMO
Negative selection refers to the selective deletion of autoreactive thymocytes. Its molecular mechanisms have not been well defined. Previous studies in our laboratory have demonstrated that retinoic acids, physiological ligands for the nuclear retinoid receptors, selectively inhibit TCR-mediated death under in vitro conditions, and the inhibition is mediated via the retinoic acid receptor (RAR) alpha. The present studies were undertaken to investigate whether ligation of RARalpha leads to inhibition of TCR-mediated death in vivo and to identify the molecular mechanisms involved. Three models of TCR-mediated death were studied: anti-CD3-mediated death of thymocytes in wild-type mice, and Ag- and bacterial superantigen-driven thymocyte death in TCR-transgenic mice expressing a receptor specific for a fragment of pigeon cytochrome c in the context of the E(k) (class II MHC) molecule. Our data demonstrate that the molecular program of both anti-CD3- and Ag-driven, but not that of superantigen-mediated apoptosis involves up-regulation of nur77, an orphan nuclear receptor, and bim, a BH3-only member of the proapoptotic bcl-2 protein family, proteins previously implicated to participate in the negative selection. Ligation of RARalpha by the synthetic agonist CD336 inhibited apoptosis, DNA binding of nur77, and synthesis of bim induced by anti-CD3 or the specific Ag, but had no effect on the superantigen-driven cell death. Our data imply that retinoids are able to inhibit negative selection in vivo as well, and they interfere with multiple steps of the T cell selection signal pathway.