Zeolitic imidazolate frameworks (ZIFs) are widely used MOFs because of certain characteristics, but also because they can be prepared at room temperature using water as the unique solvent. However, these a priori sustainable conditions inevitably entail a huge and somehow unusable excess of linker. Here, we present the formation of ZIFs at room temperature in water, starting from mixtures with a linker/metal ratio of two, that is, coinciding with the stoichiometry found in the final MOFs, in the presence of amines. ZIF-8 can be prepared with triethylamine (TEA), giving a yield of Zn of 96.6%. Other bases, like NaOH, tetraethylammonium hydroxide or ammonium hydroxide, do not lead to ZIF-8 under the same conditions. The so-obtained ZIF-8 contains TEA inside its cavities, making it less porous than its conventionally prepared counterparts. Amine can be removed by mild thermal treatments (200-250 °C). Such thermal treatments induce the generation of g-C3N4-like species which could give added value to these materials as potential photocatalysts, increasing their affinity to CO2, as proved in this work. This methodology can be successfully extended to other amines, like N,N-dicyclohexylmethylamine, as well as to other prepared ZIFs, like Co-based ZIF-67, isostructural to ZIF-8.
Laccases are natural catalysts with remarkable catalytic activity. However, their application is limited by their lack of stability. Metal-organic frameworks (MOFs) have emerged as a promising alternative for enzyme immobilization. Enzymes can be immobilized in MOFs via two approaches: postsynthetic immobilization and in situ immobilization. In postsynthetic immobilization, an enzyme is embedded after MOF formation by covalent interactions or adsorption. In contrast, in in situ immobilization, a MOF is formed in the presence of an enzyme. Additionally, MOFs have exhibited intrinsic enzyme-like activity. These materials, known as nanozymes when they have the ability to replace enzymes in certain catalytic processes, have multiple key advantages, such as low cost, easy preparation, and large surface areas. This review presents a general overview of the most recent advances in both enzyme@MOF biocatalysts and MOF-based nanozymes in different applications, with a focus on laccase, which is one of the most widely investigated enzymes with excellent industrial potential.
Metal-Organic Frameworks , Laccase , Enzymes, Immobilized , Catalysis , Adsorption
MYC is an oncogenic transcription factor dysregulated in about half of total human tumors. While transcriptomic studies reveal more than 1000 genes regulated by MYC, a much smaller fraction of genes is directly transactivated by MYC. Virtually all Burkitt lymphoma (BL) carry chromosomal translocations involving MYC oncogene. Most endemic BL and a fraction of sporadic BL are associated with Epstein-Barr virus (EBV) infection. The currently accepted mechanism is that EBV is the BL-causing agent inducing MYC translocation. Herein we show that the EBV receptor, CR2 (also called CD21), is a direct MYC target gene. This is based on several pieces of evidence: MYC induces CR2 expression in both proliferating and arrested cells and in the absence of protein synthesis, binds the CR2 promoter and transactivates CR2 in an E-box-dependent manner. Moreover, using mice with conditional MYC ablation we show that MYC induces CR2 in primary B cells. Importantly, modulation of MYC levels directly correlates with EBV's ability of infection in BL cells. Altogether, in contrast to the widely accepted hypothesis for the correlation between EBV and BL, we propose an alternative hypothesis in which MYC dysregulation could be the first event leading to the subsequent EBV infection.
Burkitt Lymphoma , Epstein-Barr Virus Infections , Animals , Humans , Mice , B-Lymphocytes/metabolism , Burkitt Lymphoma/pathology , Epstein-Barr Virus Infections/complications , Epstein-Barr Virus Infections/genetics , Genes, myc , Herpesvirus 4, Human/genetics
Purkinje cell (PC) loss occurs at an early age in patients and animal models of Niemann-Pick Type C (NPC), a lysosomal storage disease caused by mutations in the Npc1 or Npc2 genes. Although degeneration of PCs occurs early in NPC, little is known about how NPC1 deficiency affects the postnatal development of PCs. Using the Npc1nmf164 mouse model, we found that NPC1 deficiency significantly affected the postnatal development of PC dendrites and synapses. The developing dendrites of Npc1nmf164 PCs were significantly deficient in mitochondria and lysosomes. Furthermore, anabolic (mTORC1) and catabolic (TFEB) signaling pathways were not only perturbed but simultaneously activated in NPC1-deficient PCs, suggesting a loss of metabolic balance. We also found that mice with conditional heterozygous deletion of the Phosphatase and Tensin Homolog Deleted on Chromosome 10 gene (Pten-cHet), an inhibitor of mTORC1, showed similar early dendritic alterations in PCs to those found in Npc1-deficient mice. However, in contrast to Npc1nmf164 mice, Pten-cHet mice exhibited the overactivation of the mTORC1 pathway but with a strong inhibition of TFEB signaling, along with no dendritic mitochondrial reductions by the end of their postnatal development. Our data suggest that disruption of the lysosomal-metabolic signaling in PCs causes dendritic and synaptic developmental deficits that precede and promote their early degeneration in NPC.
Niemann-Pick Disease, Type C , Purkinje Cells , Mice , Animals , Purkinje Cells/metabolism , Niemann-Pick Disease, Type C/genetics , Niemann-Pick Disease, Type C/metabolism , Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/metabolism , Disease Models, Animal , Lysosomes/metabolism
Introduction: Basal cell carcinoma and squamous cell carcinoma collectively called non-melanoma skin cancer, are the most common malignant tumors worldwide. Objective: To identify the clinical and epidemiological characteristics of patients with non-melanoma skin cancer treated with superficial radiotherapy. Methods: A descriptive, longitudinal and retrospective study was carried out at the "Conrado Benítez García" Cancer Hospital in Santiago de Cuba in the period from October 2018 to January 2021. The universe consisted of all patients (n=147). with non-melanoma skin cancer with confirmed clinical and histological diagnosis and treated with superficial radiotherapy, older than 31 years and up to 95 years of age. The data collected from the medical records were entered into a database in the statistical program SPSS version 25 for Windows, the descriptive statistics of frequency distribution and absolute numbers were used. Results: according to age groups, the most affected was 75 - 85 years, the male sex, white skin color, urban origin and the largest number of affected patients belonged to the retired sector. Most of the patients received surgical treatment prior to radiant treatment, and the most used treatment scheme was D (320 cGy/ 17 frac / 2 times a week. Conclusions: Histological type squamous cell carcinoma had the highest incidence, as well as head and neck location was the most frequent.
Introducción: El carcinoma basocelular y el carcinoma espinocelular denominados en conjunto, cáncer de piel no melanoma, son los tumores malignos más comunes a nivel mundial. Objetivo: Identificar las características clínicas y epidemiológicas de los pacientes con cáncer de piel no melanoma tratados con radioterapia superficial. Métodos: Se realizó un estudio descriptivo, longitudinal y retrospectivo en el Hospital Oncológico "Conrado Benítez García" de Santiago de Cuba en el periodo comprendido de octubre de 2018 a enero de 2021. El universo estuvo constituido por todos los pacientes (n=147) con cáncer de piel no melanoma con diagnóstico clínico e histológico confirmado y tratados con radioterapia superficial, mayores de 31 años y hasta 95 años de edad. Los datos recogidos de las historias clínicas, fueron vaciados en una base de datos en el programa estadístico SPSS versión 25 para Windows, se utilizó la estadística descriptiva distribución de frecuencias y números absolutos. Resultados: según grupos de edades el más afectado fue 75 85 años, el sexo masculino, el color de la piel blanca, la procedencia urbana y el mayor número de pacientes afectados perteneció al sector jubilado. La mayoría de los pacientes recibieron tratamiento quirúrgico previo al tratamiento radiante, siendo el esquema de tratamiento más usado fue el D (320 cGy/ 17 frac / 2 veces a la semana Conclusiones: El carcinoma epidermoide tipo histológico fue el de mayor incidencia, así como la localización de cabeza y cuello fue la más frecuente.
Introdução: O carcinoma basocelular e o carcinoma espinocelular, chamados coletivamente de câncer de pele não melanoma, são os tumores malignos mais comuns em todo o mundo. Objetivo: Identificar as características clínicas e epidemiológicas de pacientes com câncer de pele não melanoma tratados com radioterapia superficial. Métodos: Foi realizado um estudo descritivo, longitudinal e retrospectivo no Hospital de Câncer "Conrado Benítez García" de Santiago de Cuba no período de outubro de 2018 a janeiro de 2021. O universo foi constituído por todos os pacientes (n=147). câncer de pele melanoma com diagnóstico clínico e histológico confirmado e tratados com radioterapia superficial, maiores de 31 anos e até 95 anos de idade. Os dados coletados dos prontuários foram inseridos em um banco de dados no programa estatístico SPSS versão 25 para Windows, foi utilizada a estatística descritiva de distribuição de frequência e números absolutos. Resultados: segundo as faixas etárias, a mais acometida foi de 75 a 85 anos, sexo masculino, cor da pele branca, procedência urbana e o maior número de acometidos pertencia ao setor aposentado. A maioria dos pacientes recebeu tratamento cirúrgico antes do tratamento radiante, e o esquema de tratamento mais utilizado foi o D (320 cGy/ 17 frac / 2 vezes por semana). local foi o mais frequente.
Retinoic acid (RA) plays major roles during nervous system development, and during regeneration of the adult nervous system. We have previously shown that components of the RA signaling pathway are upregulated after optic nerve injury, and that exogenous application of all-trans retinoic acid (ATRA) greatly increases the survival of axotomized retinal ganglion cells (RGCs). The objective of the present study is to investigate the effects of ATRA application on the macrophages in the optic nerve after injury, and to determine whether this affects axonal regeneration. The optic nerve was crushed and treated with PBS, ATRA and/or clodronate-loaded liposomes. Nerves were examined at one and two weeks after axotomy with light microscopy, immunocytochemistry and electron microscopy. ATRA application to the optic nerve caused transient increases in the number of macrophages and microglia one week after injury. The macrophages are consistently labeled with M2-type markers, and have considerable phagocytic activity. ATRA increased ultrastructural features of ongoing phagocytic activity in macrophages at one and two weeks. ATRA treatment also significantly increased the numbers of regenerating GAP-43-labeled axons. Clodronate liposome treatment depleted macrophage numbers by 80%, completely eliminated the ATRA-mediated increase in axonal regeneration, and clodronate treatment alone decreased axonal numbers by 30%. These results suggest that the success of axon regeneration is partially dependent on the presence of debris-phagocytosing macrophages, and that the increases in regeneration caused by ATRA are in part due to their increased numbers. Further studies will examine whether macrophage depletion affects RGC survival.
Macrophages/drug effects , Nerve Regeneration/drug effects , Optic Nerve Injuries/drug therapy , Retinal Ganglion Cells/drug effects , Tretinoin/pharmacology , Animals , Liposomes , Optic Nerve Injuries/physiopathology , Rana pipiens , Retinal Ganglion Cells/physiology , Tretinoin/therapeutic use
Chronic myelogenous leukemia (CML) is a hematological malignancy that highly depends on the BCR-ABL1/STAT5 signaling pathway for cell survival. First-line treatments for CML consist of tyrosine kinase inhibitors that efficiently target BCR-ABL1 activity. However, drug resistance and intolerance are still therapeutic limitations in Ph+ cells. Therefore, the development of new anti-CML drugs that exhibit alternative mechanisms to overcome these limitations is a desirable goal. In this work, the antitumoral activity of JKST6, a naphthoquinone-pyrone hybrid, was assessed in imatinib-sensitive and imatinib-resistant human CML cells. Live-cell imaging analysis revealed JKST6 potent antiproliferative activity in 2D and 3D CML cultures. JKST6 provoked cell increase in the subG1 phase along with a reduction in the G0/G1 phase and altered the expression of key proteins involved in the control of mitosis and DNA damage. Rapid increases in Annexin V staining and activation/cleavage of caspases 8, 9 and 3 were observed after JKST6 treatment in CML cells. Of interest, JKST6 inhibited BCR-ABL1/STAT5 signaling through oncokinase downregulation that was preceded by rapid polyubiquitination. In addition, JKST6 caused a transient increase in JNK and AKT phosphorylation, whereas the phosphorylation of P38-MAPK and Src was reduced. Combinatory treatment unveiled synergistic effects between imatinib and JKST6. Notably, JKST6 maintained its antitumor efficacy in BCR-ABL1-T315I-positive cells and CML cells that overexpress BCR-ABL and even restored imatinib efficacy after a short exposure time. These findings, together with the observed low toxicity of JKST6, reveal a novel multikinase modulator that might overcome the limitations of BCR-ABL1 inhibitors in CML therapy.
Antineoplastic Combined Chemotherapy Protocols/pharmacology , Drug Resistance, Neoplasm , Fusion Proteins, bcr-abl/antagonists & inhibitors , Imatinib Mesylate/pharmacology , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/drug therapy , Naphthoquinones/pharmacology , Protein Kinase Inhibitors/pharmacology , STAT5 Transcription Factor/metabolism , Apoptosis/drug effects , Cell Cycle/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Drug Synergism , Fusion Proteins, bcr-abl/genetics , Fusion Proteins, bcr-abl/metabolism , Gene Expression Regulation, Neoplastic , Humans , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/enzymology , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/genetics , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/pathology , STAT5 Transcription Factor/genetics , Signal Transduction
The survival rate in lung cancer remains stubbornly low and there is an urgent need for the identification of new therapeutic targets. In the last decade, several members of the SWI/SNF chromatin remodeling complexes have been described altered in different tumor types. Nevertheless, the precise mechanisms of their impact on cancer progression, as well as the application of this knowledge to cancer patient management are largely unknown. In this study, we performed targeted sequencing of a cohort of lung cancer patients on genes involved in chromatin structure. In addition, we studied at the protein level the expression of these genes in cancer samples and performed functional experiments to identify the molecular mechanisms linking alterations of chromatin remodeling genes and tumor development. Remarkably, we found that 20% of lung cancer patients show ARID2 protein loss, partially explained by the presence of ARID2 mutations. In addition, we showed that ARID2 deficiency provokes profound chromatin structural changes altering cell transcriptional programs, which bolsters the proliferative and metastatic potential of the cells both in vitro and in vivo. Moreover, we demonstrated that ARID2 deficiency impairs DNA repair, enhancing the sensitivity of the cells to DNA-damaging agents. Our findings support that ARID2 is a bona fide tumor suppressor gene in lung cancer that may be exploited therapeutically.
Lung Neoplasms/drug therapy , Lung Neoplasms/metabolism , Transcription Factors/deficiency , A549 Cells , Animals , Cell Line, Tumor , Disease Progression , Female , High-Throughput Nucleotide Sequencing , Humans , Lung Neoplasms/genetics , Lung Neoplasms/pathology , Mice , Mice, Nude , Survival Rate , Transcription Factors/genetics , Transcription Factors/metabolism
The MAX network transcriptional repressor (MNT) is an MXD family transcription factor of the basic helix-loop-helix (bHLH) family. MNT dimerizes with another transcriptional regulator, MYC-associated factor X (MAX), and down-regulates genes by binding to E-boxes. MAX also dimerizes with MYC, an oncogenic bHLH transcription factor. Upon E-box binding, the MYC-MAX dimer activates gene expression. MNT also binds to the MAX dimerization protein MLX (MLX), and MNT-MLX and MNT-MAX dimers co-exist. However, all MNT functions have been attributed to MNT-MAX dimers, and no functions of the MNT-MLX dimer have been described. MNT's biological role has been linked to its function as a MYC oncogene modulator, but little is known about its regulation. We show here that MNT localizes to the nucleus of MAX-expressing cells and that MNT-MAX dimers bind and repress the MNT promoter, an effect that depends on one of the two E-boxes on this promoter. In MAX-deficient cells, MNT was overexpressed and redistributed to the cytoplasm. Interestingly, MNT was required for cell proliferation even in the absence of MAX. We show that in MAX-deficient cells, MNT binds to MLX, but also forms homodimers. RNA-sequencing experiments revealed that MNT regulates the expression of several genes even in the absence of MAX, with many of these genes being involved in cell cycle regulation and DNA repair. Of note, MNT-MNT homodimers regulated the transcription of some genes involved in cell proliferation. The tight regulation of MNT and its functionality even without MAX suggest a major role for MNT in cell proliferation.
Basic Helix-Loop-Helix Leucine Zipper Transcription Factors/genetics , Repressor Proteins/genetics , Transcription, Genetic , Amino Acid Sequence/genetics , Animals , Basic Helix-Loop-Helix Leucine Zipper Transcription Factors/chemistry , Cell Proliferation/genetics , Gene Expression Regulation/genetics , Gene Regulatory Networks/genetics , Helix-Loop-Helix Motifs/genetics , Humans , Promoter Regions, Genetic , Protein Multimerization/genetics , Proto-Oncogene Proteins c-myc/chemistry , Proto-Oncogene Proteins c-myc/genetics , Repressor Proteins/chemistry
Cell cycle stimulation is a major transforming mechanism of Myc oncoprotein. This is achieved through at least three concomitant mechanisms: upregulation of cyclins and Cdks, downregulation of the Cdk inhibitors p15 and p21 and the degradation of p27. The Myc-p27 antagonism has been shown to be relevant in human cancer. To be degraded, p27 must be phosphorylated at Thr-187 to be recognized by Skp2, a component of the ubiquitination complex. We previously described that Myc induces Skp2 expression. Here we show that not only Cdk2 but Cdk1 phosphorylates p27 at the Thr-187. Moreover, Myc induced p27 degradation in murine fibroblasts through Cdk1 activation, which was achieved by Myc-dependent cyclin A and B induction. In the absence of Cdk2, p27 phosphorylation at Thr-187 was mainly carried out by cyclin A2-Cdk1 and cyclin B1-Cdk1. We also show that Cdk1 inhibition was enough for the synthetic lethal interaction with Myc. This result is relevant because Cdk1 is the only Cdk strictly required for cell cycle and the reported synthetic lethal interaction between Cdk1 and Myc.
CDC2 Protein Kinase/metabolism , Cyclin-Dependent Kinase Inhibitor p27/metabolism , Proto-Oncogene Proteins c-myc/metabolism , Animals , CDC2 Protein Kinase/physiology , Cell Cycle , Cell Cycle Checkpoints , Cell Cycle Proteins/metabolism , Cell Division , Cell Line , Cyclin-Dependent Kinase 2 , Cyclin-Dependent Kinase 4/metabolism , Cyclin-Dependent Kinase Inhibitor p15/metabolism , Cyclin-Dependent Kinase Inhibitor p21/metabolism , Cyclin-Dependent Kinase Inhibitor p27/physiology , Cyclin-Dependent Kinases/metabolism , Cyclins/metabolism , Female , HEK293 Cells , HeLa Cells , Humans , Male , Mice , Mice, Inbred C57BL , Phosphorylation , Proto-Oncogene Proteins c-myc/physiology , Signal Transduction
Niemann Pick Type-C disease (NPC) is an inherited lysosomal storage disease (LSD) caused by pathogenic variants in the Npc1 or Npc2 genes that lead to the accumulation of cholesterol and lipids in lysosomes. NPC1 deficiency causes neurodegeneration, dementia and early death. Cerebellar Purkinje cells (PCs) are particularly hypersensitive to NPC1 deficiency and degenerate earlier than other neurons in the brain. Activation of microglia is an important contributor to PCs degeneration in NPC. However, the mechanisms by which activated microglia promote PCs degeneration in NPC are not completely understood. Here, we are demonstrating that in the Npc1nmf164 mouse cerebellum, microglia in the molecular layer (ML) are activated and contacting dendrites at early stages of NPC, when no loss of PCs is detected. During the progression of PCs degeneration in Npc1nmf164 mice, accumulation of phagosomes and autofluorescent material in microglia at the ML coincided with the degeneration of dendrites and PCs. Feeding Npc1nmf164 mice a western diet (WD) increased microglia activation and corresponded with a more extensive degeneration of dendrites but not PC somata. Together our data suggest that microglia contribute to the degeneration of PCs by interacting, engulfing and phagocytosing their dendrites while the cell somata are still present.
Dendrites/metabolism , Microglia/metabolism , Nerve Degeneration/metabolism , Niemann-Pick Disease, Type C/metabolism , Purkinje Cells/metabolism , Animals , Cerebellum/metabolism , Cerebellum/pathology , Diet, Western , Disease Models, Animal , Female , Intracellular Signaling Peptides and Proteins/genetics , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Motor Activity/genetics , Niemann-Pick C1 Protein , Phagocytosis/genetics , Phagosomes/metabolism
We have previously shown that a single application of the growth factors ciliary neurotrophic factor (CNTF) or fibroblast growth factor 2 (FGF-2) to the crushed optic nerve of the frog, Rana pipiens, increases the numbers and elongation rate of regenerating retinal ganglion cell axons. Here we investigate the effects of these factors on the numbers and types of macrophages that invade the regeneration zone. In control PBS-treated nerves, many macrophages are present 100 µm distal to the crush site at 1 week after injury; their numbers halve by 2 weeks. A single application of CNTF at the time of injury triples the numbers of macrophages at 1 week, with this increase compared to control being maintained at 2 weeks. Application of FGF-2 is equally effective at 1 week, but the macrophage numbers have fallen to control levels at 2 weeks. Immunostaining with a pan-macrophage marker, ED1, and a marker for M2-like macrophages, Arg-1, showed that the proportion of the putative M2 phenotype remained at approximately 80% with all treatments. Electron microscopy of the macrophages at 1 week shows strong phagocytic activity with all treatments, with many vacuoles containing axon fragments and membrane debris. At 2 weeks with PBS or FGF-2 treatment the remaining macrophages are less phagocytically active, containing mainly lipid inclusions. With CNTF treatment, at 2 weeks many of the more numerous macrophages are still phagocytosing axonal debris, although they also contain lipid inclusions. We conclude that the increase in macrophage influx seen after growth factor application is beneficial for the regenerating axons, probably due to more extensive removal of degenerating distal axons, but also perhaps to secretion of growth-promoting substances.
Ciliary Neurotrophic Factor/pharmacology , Ciliary Neurotrophic Factor/therapeutic use , Fibroblast Growth Factor 2/pharmacology , Fibroblast Growth Factor 2/therapeutic use , Macrophages/drug effects , Macrophages/metabolism , Optic Nerve Injuries/drug therapy , Optic Nerve Injuries/metabolism , Animals , Axons/drug effects , Axons/metabolism , Axons/ultrastructure , Immunohistochemistry , Microscopy, Electron , Rana pipiens , Vacuoles/drug effects , Vacuoles/metabolism , Vacuoles/ultrastructure
[This corrects the article DOI: 10.18632/oncotarget.11425.].
Mesoporous silica materials are promising carriers for enzyme immobilization in heterogeneous biocatalysis applications. By tailoring their pore structural framework, these materials are designable for appropriate enzyme binding capacity and internal diffusivity. To supply O2 efficiently to solid-supported immobilized enzymes represents a core problem of heterogeneously catalyzed oxidative biotransformations. In this study, therefore, we synthesized and compared three internally well-ordered and two amorphous silica materials as enzyme carriers, each of those with pore sizes of ≥10 nm, to enable the coimmobilization of d-amino-acid oxidase (79 kDa) and catalase (217 kDa). Both enzymes were fused to the silica-binding module Zbasic2 to facilitate their selective and oriented immobilization directly from crude protein mixtures on native silica materials. Analyzing the effects of varied pore architecture and internal surface area on the performance of the immobilized bienzymatic system, we showed that a uniform pore structural framework was beneficial for enzyme loading (≥70 mg protein/g carrier), immobilization yield (≥90%), surface and pore volume filling without hindered adsorption, and catalytic effectiveness (≥60%) of the coimmobilizate. Using the best carrier LP-SBA-15, we obtained a solid oxidase-catalase preparation with an activity of 2000 µmol/(min g_material) that was recyclable and stable during oxidation of d-methionine. These results affirm a strategy of optimizing immobilized O2-dependent enzymes via tunable internal structuring of the silica material used as carrier. They thus make a significant advance toward the molecular design of heterogeneous oxidation biocatalysts on mesoporous silica supports.
Silicon Dioxide/chemistry , Adsorption , Biocatalysis , Catalase , Enzymes, Immobilized , Porosity
Human Chronic Myelogenous Leukemia (CML) is a hematological stem cell disorder which is associated with activation of Bcr-Abl-Stat5 oncogenic pathway. Direct Bcr-Abl inhibitors are initially successful for the treatment of CML but over time many patients develop drug resistance. In the present study, the effects of CM363, a novel naphthoquinone (NPQ) derivative, were evaluated on human CML-derived K562 cells. CM363 revealed an effective cell growth inhibition (IC50 = 0.7 ± 0.5 µM) by inducing cancer cells to undergo cell cycle arrest and apoptosis. CM363 caused a dose- and time-dependent reduction of cells in G0/G1 and G2/M phases. This cell cycle arrest was associated with increased levels of cyclin E, pChk1 and pChk2 whereas CM363 downregulated cyclin B, cyclin D3, p27, pRB, Wee1, and BUBR1. CM363 increased the double-strand DNA break marker γH2AX. CM363 caused a time-dependent increase of annexin V-positive cells, DNA fragmentation and increased number of apoptotic nuclei. CM363 triggered the mitochondrial apoptotic pathway as reflected by a release of cytochrome C from mitochondria and induction of the cleavage of caspase-3 and -9, and PARP. CM363 showed multikinase modulatory effects through an early increased JNK phosphorylation followed by inhibition of pY-Bcrl-Abl and pY-Stat5. CM363 worked synergistically with imatinib to inhibit cell viability and maintained its activity in imatinib-resistant cells. Finally, CM363 (10 mg/Kg) suppressed the growth of K562 xenograft tumors in athymic mice. In summary, CM363 is a novel multikinase modulator that offers advantages to circumvent imanitib resistance and might be therapeutically effective in Bcrl-Abl-Stat5 related malignancies.
Antineoplastic Agents/pharmacology , Drug Resistance, Neoplasm/drug effects , Imatinib Mesylate/pharmacology , Naphthoquinones/pharmacology , Protein Kinase Inhibitors/pharmacology , Animals , Apoptosis/drug effects , Cell Cycle/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/drug effects , Disease Models, Animal , Female , Fusion Proteins, bcr-abl/genetics , Fusion Proteins, bcr-abl/metabolism , Humans , JNK Mitogen-Activated Protein Kinases/metabolism , K562 Cells , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/drug therapy , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/genetics , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/metabolism , Mice , STAT5 Transcription Factor/metabolism , Signal Transduction , Xenograft Model Antitumor Assays
MXD1 is a protein that interacts with MAX, to form a repressive transcription factor. MXD1-MAX binds E-boxes. MXD1-MAX antagonizes the transcriptional activity of the MYC oncoprotein in most models. It has been reported that MYC overexpression leads to augmented RNA synthesis and ribosome biogenesis, which is a relevant activity in MYC-mediated tumorigenesis. Here we describe that MXD1, but not MYC or MNT, localizes to the nucleolus in a wide array of cell lines derived from different tissues (carcinoma, leukemia) as well as in embryonic stem cells. MXD1 also localizes in the nucleolus of primary tissue cells as neurons and Sertoli cells. The nucleolar localization of MXD1 was confirmed by co-localization with UBF. Co-immunoprecipitation experiments showed that MXD1 interacted with UBF and proximity ligase assays revealed that this interaction takes place in the nucleolus. Furthermore, chromatin immunoprecipitation assays showed that MXD1 was bound in the transcribed rDNA chromatin, where it co-localizes with UBF, but also in the ribosomal intergenic regions. The MXD1 involvement in rRNA synthesis was also suggested by the nucleolar segregation upon rRNA synthesis inhibition by actinomycin D. Silencing of MXD1 with siRNAs resulted in increased synthesis of pre-rRNA while enforced MXD1 expression reduces it. The results suggest a new role for MXD1, which is the control of ribosome biogenesis. This new MXD1 function would be important to curb MYC activity in tumor cells.
Basic Helix-Loop-Helix Leucine Zipper Transcription Factors/metabolism , Cell Nucleolus/metabolism , Pol1 Transcription Initiation Complex Proteins/metabolism , RNA, Ribosomal/metabolism , Repressor Proteins/metabolism , Animals , Basic Helix-Loop-Helix Leucine Zipper Transcription Factors/genetics , Cells, Cultured , HEK293 Cells , HeLa Cells , Humans , Immunoprecipitation , K562 Cells , Male , Neurons/cytology , Neurons/metabolism , Protein Binding , RNA Interference , RNA, Ribosomal/genetics , Rats , Repressor Proteins/genetics , Spermatogonia/cytology , Spermatogonia/metabolism
After lesions to the mammalian optic nerve, the great majority of retinal ganglion cells (RGCs) die before their axons have even had a chance to regenerate. Frog RGCs, on the other hand, suffer only an approximately 50% cell loss, and we have previously investigated the mechanisms by which the application of growth factors can increase their survival rate. Retinoic acid (RA) is a vitamin A-derived lipophilic molecule that plays major roles during development of the nervous system. The RA signaling pathway is also present in parts of the adult nervous system, and components of it are upregulated after injury in peripheral nerves but not in the CNS. Here we investigate whether RA signaling affects long-term RGC survival at 6 weeks after axotomy. Intraocular injection of all-trans retinoic acid (ATRA), the retinoic acid receptor (RAR) type-α agonist AM80, the RARß agonist CD2314, or the RARγ agonist CD1530, returned axotomized RGC numbers to almost normal levels. On the other hand, inhibition of RA synthesis with disulfiram, or of RAR receptors with the pan-RAR antagonist Ro-41-5253, or the RARß antagonist LE135E, greatly reduced the survival of the axotomized neurons. Axotomy elicited a strong activation of the MAPK, STAT3 and AKT pathways; this activation was prevented by disulfiram or by RAR antagonists. Finally, addition of exogenous ATRA stimulated the activation of the first two of these pathways. Future experiments will investigate whether these strong survival-promoting effects of RA are mediated via the upregulation of neurotrophins.
Nerve Regeneration/drug effects , Optic Nerve Injuries/metabolism , Optic Nerve/drug effects , Tretinoin/metabolism , Animals , Anura , Benzoates/pharmacology , Chromans/pharmacology , Naphthols/pharmacology , Receptors, Retinoic Acid/agonists , Receptors, Retinoic Acid/antagonists & inhibitors , Receptors, Retinoic Acid/metabolism , Retinal Ganglion Cells/drug effects , Retinal Ganglion Cells/metabolism , Tetrahydronaphthalenes/pharmacology
Retinoic acid (RA) is important during development, in neuronal plasticity, and also in peripheral nervous system regeneration. Here we use the frog visual system as a model to investigate the changes in RA signaling that take place after axonal injury to the central nervous system. Immunocytochemistry was used to localize different components of RA signaling within sections of the retina and optic tectum, namely, the synthetic enzyme retinaldehyde dehydrogenase (RALDH), the RA binding proteins CRABPI and II, the retinoic acid receptors RARα, ß and γ, and finally the catabolic enzyme CYP26A1. The levels of these proteins were quantified in extracts of retina and tectum using Western blotting. Animals were studied at 1 week, 3 weeks and 6 weeks after optic nerve transection. At the latter time point the RGC axons were re-entering the optic tectum. All the components of RA signaling were present at low to moderate levels in retinas and tecta of control, unoperated animals. In retina, soon after optic nerve injury there was a large increase in RALDH, some increase in the CRABPs, and a large increase in RGC RARß and (expression. These increases continued as the RGC axons were regenerating, with the addition of later RARα expression at 6 weeks. At no stage did CYP26A1 expression significantly change. In the tectum the levels of RALDH increased after axotomy and during regrowth of axons (3 weeks), then decreased at 6 weeks, at which time the levels of CYP26A1 increased. Axotomy did not cause an immediate increase in tectal RAR levels but RARα and RARß increased after 3 weeks and RARγ only after 6 weeks. These results are consistent with RA signaling playing an important role in the survival and regeneration of frog RGCs.
Optic Nerve Injuries/physiopathology , Signal Transduction , Tretinoin/metabolism , Visual Pathways/physiopathology , Animals , Female , Gene Expression Regulation , Immunohistochemistry , Male , Rana pipiens , Receptors, Retinoic Acid/biosynthesis , Retina/physiopathology , Retinal Dehydrogenase/biosynthesis , Retinal Ganglion Cells/metabolism , Retinoic Acid 4-Hydroxylase/biosynthesis , Retinoic Acid 4-Hydroxylase/genetics , Retinoid X Receptors/biosynthesis , Superior Colliculi/physiopathology
A familial behavioral variant frontotemporal dementia associated with astrocyte-predominant tauopathy is described in 2 sisters born from consanguineous parents. The neuropathologic examination revealed massive accumulation of abnormally hyperphosphorylated, conformational, truncated tau at aspartic acid 421, ubiquitinated and nitrated tau at Tyr29 in cortical astrocyte (including their perivascular foot processes), and Bergmann glia. Smaller amounts of abnormal tau were observed in neurons and rarely in oligodendrocytes. There was decreased expression of glial glutamate transporter in the majority of tau-positive astrocytes. Gel electrophoresis of sarkosyl-insoluble fractions showed 2 bands of 64 and 60 kDa and a doublet of 67 to 70 kDa (which are different from those seen in Alzheimer disease and in typical 4R and 3R tauopathies) together with several bands of lower molecular weight indicative of truncated tau. Analysis of the expression of MAPT isoforms further revealed altered splicing and representation of tau isoforms involving exons 2, 3, and 10. Genetic testing revealed no known mutations in PSEN1, PSEN2, APP, MAPT, GRN, FUS, and TARDBP and no pathologic expansion in C9ORF72. However, a novel rare heterozygous sequence variant(p.Q140H) of uncertain significance was identified in FUS in both siblings.
Astrocytes/pathology , Frontotemporal Dementia/genetics , Frontotemporal Dementia/pathology , Tauopathies/genetics , Tauopathies/pathology , Female , Humans , Middle Aged
ADAM Proteins/genetics , Codon, Nonsense , Frameshift Mutation , Purpura, Thrombotic Thrombocytopenic/genetics , ADAM Proteins/biosynthesis , ADAM Proteins/chemistry , ADAM Proteins/deficiency , ADAMTS13 Protein , Diagnostic Errors , Female , Genotype , HEK293 Cells , Heterozygote , Humans , Infant , Male , Mutation, Missense , Pedigree , Protein Stability , Purpura, Thrombocytopenic, Idiopathic/diagnosis , Purpura, Thrombotic Thrombocytopenic/diagnosis , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Sequence Deletion , Transfection
