RESUMO
Several in vitro models have been developed to mimic chronic lymphocytic leukemia (CLL) proliferation in immune niches; however, they typically do not induce robust proliferation. We prepared a novel model based on mimicking T-cell signals in vitro and in patient-derived xenografts (PDXs). Six supportive cell lines were prepared by engineering HS5 stromal cells with stable expression of human CD40L, IL4, IL21, and their combinations. Co-culture with HS5 expressing CD40L and IL4 in combination led to mild CLL cell proliferation (median 7% at day 7), while the HS5 expressing CD40L, IL4, and IL21 led to unprecedented proliferation rate (median 44%). The co-cultures mimicked the gene expression fingerprint of lymph node CLL cells (MYC, NFκB, and E2F signatures) and revealed novel vulnerabilities in CLL-T-cell-induced proliferation. Drug testing in co-cultures revealed for the first time that pan-RAF inhibitors fully block CLL proliferation. The co-culture model can be downscaled to five microliter volume for large drug screening purposes or upscaled to CLL PDXs by HS5-CD40L-IL4 ± IL21 co-transplantation. Co-transplanting NSG mice with purified CLL cells and HS5-CD40L-IL4 or HS5-CD40L-IL4-IL21 cells on collagen-based scaffold led to 47% or 82% engraftment efficacy, respectively, with ~20% of PDXs being clonally related to CLL, potentially overcoming the need to co-transplant autologous T-cells in PDXs.
Assuntos
Ligante de CD40 , Proliferação de Células , Técnicas de Cocultura , Leucemia Linfocítica Crônica de Células B , Células Estromais , Humanos , Leucemia Linfocítica Crônica de Células B/patologia , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Animais , Camundongos , Células Estromais/metabolismo , Células Estromais/patologia , Ligante de CD40/metabolismo , Ligante de CD40/genética , Linfócitos T/imunologia , Linfócitos T/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , Interleucinas/genética , Interleucinas/metabolismo , Inibidores de Proteínas Quinases/farmacologiaRESUMO
The paradigm of using metal biomaterials could be viewed from two sides - treatment of wide spectrum of degenerative diseases, and debris release from materials. After implant insertion, metal nanoparticles (NPs) and ions are released not only upon the first contact with cells/tissues, but in continual manner, which is immediately recognized by immune cells. In this work, the effects of metal nanoparticles (TiO2, Ni) and ions (Ni2+, Co2+, Cr3+, Mo6+) on primary human M0 macrophages from the blood samples of osteoarthritic patients undergoing total arthroplasty were studied in order to monitor immunomodulatory effects on the cells in a real-time format. The highest NiNPs concentration of 10 µg/ml had no effect on any of macrophage parameters, while the Ni2+ ions cytotoxicity limit for the cells is 0.5 mM. The cytotoxic effects of higher Ni2+ concentration revealed mitochondrial network fragmentation leading to mitochondrial dysfunction, accompanied by increased lysosomal activity and changes in pro-apoptotic markers. The suppression of M2 cell formation ability was connected to presence of Ni2+ ions (0.5 mM) and TiO2NPs (10 µg/ml). The immunomodulatory effect of Mo6+ ions, controversially, inhibit the formation of the cells with M1 phenotype and potentiate the thread-like shape M2s with increased chaotic cell movement. To summarize, metal toxicity depends on the debris form. Both, metal ions and nanoparticles affect macrophage size, morphological and functional parameters, but the effect of ions is more complex and likely more harmful, which has potential impact on healing and determines post-implantation reactions.
Assuntos
Nanopartículas Metálicas , Metais , Humanos , Metais/farmacologia , Macrófagos , ÍonsRESUMO
OBJECTIVE: The pathophysiological processes leading to epileptogenesis and pharmacoresistance in epilepsy have been the subject of extensive preclinical and clinical research. The main impact on clinical practice is the development of new targeted therapies for epilepsy. We studied the importance of neuroinflammation in the development of epileptogenesis and pharmacoresistance in childhood epilepsy patients. METHODS: A cross-sectional study conducted at two epilepsy centers in the Czech Republic compared 22 pharmacoresistant patients and 4 pharmacodependent patients to 9 controls. We analyzed the ProcartaPlex™ 9-Plex immunoassay panel consisting of interleukin (IL)-6, IL-8, IL-10, IL-18, CXCL10/IP-10, monocyte chemoattractant protein 1 (CCL2/MCP-1), B lymphocyte chemoattractant (BLC), tumor necrosis factor-alpha (TNF-α), and chemokine (C-X3-X motif) ligand 1 (fractalkine/CXC3CL1) to determine their alterations in cerebrospinal fluid (CSF) and blood plasma, concurrently. RESULTS: The analysis of 21 paired CSF and plasma samples in pharmacoresistant patients compared to controls revealed a significant elevation of CCL2/MCP-1 in CSF (p < 0.000512) and plasma (p < 0.00.017). Higher levels of fractalkine/CXC3CL1 were revealed in the plasma of pharmacoresistant patients than in controls (p < 0.0704), and we determined an upward trend in CSF IL-8 levels (p < 0.08). No significant differences in CSF and plasma levels were detected between pharmacodependent patients and controls. CONCLUSION: Elevated CCL2/MCP-1 in CSF and plasma, elevated levels of fractalkine/CXC3CL1 in CSF, and a trend toward elevated IL-8 in the CSF of patients with pharmacoresistant epilepsy indicate these cytokines as potential biomarkers of epileptogenesis and pharmacoresistance. CCL2/MCP-1was detected in blood plasma; this assessment may be easily achieved in clinical practice without the invasiveness of a spinal tap. However, due to the complexity of neuroinflammation in epilepsy, further studies are warranted to confirm our findings.
Assuntos
Quimiocina CCL2 , Epilepsia , Humanos , Quimiocina CCL2/líquido cefalorraquidiano , Interleucina-8/líquido cefalorraquidiano , Quimiocina CX3CL1 , Doenças Neuroinflamatórias , Estudos Transversais , Epilepsia/diagnóstico , Epilepsia/tratamento farmacológico , Biomarcadores/líquido cefalorraquidianoRESUMO
BACKGROUND: Anti-CD19 chimeric antigen receptor T cells (CART-19) frequently induce remissions in hemato-oncological patients with recurred and/or refractory B-cell tumors. However, malignant cells sometimes escape the immunotherapeutic targeting by CD19 gene mutations, alternative splicing or lineage switch, commonly causing lack of CD19 expression on the surface of neoplastic cells. We assumed that, in addition to the known mechanisms, other means could act on CD19 to drive antigen-negative relapse. METHODS: Herein, we studied the mechanism of antigen loss in an in vivo CD19-negative recurrence model of chronic lymphocytic leukemia (CLL) to CART-19, established using NOD-scid IL2Rgnull mice and HG3 cell line. We validated our findings in vitro in immortalized B-cell lines and primary CLL cells. RESULTS: In our in vivo CLL recurrence model, up to 70% of CART-19-treated mice eventually recurred with CD19-negative disease weeks after initial positive response. We found that the lack of CD19 expression was caused by promoter DNA hypermethylation. Importantly, the expression loss was partially reversible by treatment with a demethylating agent. Moreover, this escape mechanism was common for 3 B-cell immortalized lines as well as primary CLL cells, as assessed by in vitro coculture experiments. CONCLUSIONS: Epigenetically driven antigen escape could represent a novel, yet at least partially reversible, means of CD19 loss to CART-19 in B-cell tumors.
Assuntos
Metilação de DNA/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Animais , Antígenos CD19/imunologia , Feminino , Humanos , Masculino , CamundongosRESUMO
There is an emerging body of evidence that patients with chronic myeloid leukaemia (CML) may carry not only breakpoint cluster region-Abelson murine leukaemia viral oncogene homologue 1 (BCR-ABL1) kinase domain mutations (BCR-ABL1 KD mutations), but also mutations in other genes. Their occurrence is highest during progression or at failure, but their impact at diagnosis is unclear. In the present study, we prospectively screened for mutations in 18 myeloid neoplasm-associated genes and BCR-ABL1 KD in the following populations: bulk leucocytes, CD34+ CD38+ progenitors and CD34+ CD38- stem cells, at diagnosis and early follow-up. In our cohort of chronic phase CML patients, nine of 49 patients harboured somatic mutations in the following genes: six ASXL1 mutations, one SETBP1, one TP53, one JAK2, but no BCR-ABL1 KD mutations. In seven of the nine patients, mutations were detected in multiple hierarchical populations including bulk leucocytes at diagnosis. The mutation dynamics reflected the BCR-ABL1 transcript decline induced by treatment in eight of the nine cases, suggesting that mutations were acquired in the Philadelphia chromosome (Ph)-positive clone. In one patient, the JAK2 V617F mutation correlated with a concomitant Ph-negative myeloproliferative neoplasm and persisted despite a 5-log reduction of the BCR-ABL1 transcript. Only two of the nine patients with mutations failed first-line therapy. No correlation was found between the mutation status and survival or response outcomes.
Assuntos
Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Seguimentos , Proteínas de Fusão bcr-abl/genética , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva/diagnóstico , Leucemia Mielogênica Crônica BCR-ABL Positiva/terapia , Mutação , Prognóstico , Estudos ProspectivosRESUMO
Recirculation of chronic lymphocytic leukemia (CLL) cells between the peripheral blood and lymphoid niches plays a critical role in disease pathophysiology, and inhibiting this process is one of the major mechanisms of action for B-cell receptor (BCR) inhibitors such as ibrutinib and idelalisib. Migration is a complex process guided by chemokine receptors and integrins. However, it remains largely unknown how CLL cells integrate multiple migratory signals while balancing survival in the peripheral blood and the decision to return to immune niches. Our study provided evidence that CXCR4/CD5 intraclonal subpopulations can be used to study the regulation of migration of CLL cells. We performed RNA profiling of CXCR4dimCD5bright vs CXCR4brightCD5dim CLL cells and identified differential expression of dozens of molecules with a putative function in cell migration. GRB2-associated binding protein 1 (GAB1) positively regulated CLL cell homing capacity of CXCR4brightCD5dim cells. Gradual GAB1 accumulation in CLL cells outside immune niches was mediated by FoxO1-induced transcriptional GAB1 activation. Upregulation of GAB1 also played an important role in maintaining basal phosphatidylinositol 3-kinase (PI3K) activity and the "tonic" AKT phosphorylation required to sustain the survival of resting CLL B cells. This finding is important during ibrutinib therapy, because CLL cells induce the FoxO1-GAB1-pAKT axis, which represents an adaptation mechanism to the inability to home to immune niches. We have demonstrated that GAB1 can be targeted therapeutically by novel GAB1 inhibitors, alone or in combination with BTK inhibition. GAB1 inhibitors induce CLL cell apoptosis, impair cell migration, inhibit tonic or BCR-induced AKT phosphorylation, and block compensatory AKT activity during ibrutinib therapy.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/biossíntese , Movimento Celular , Proteína Forkhead Box O1/metabolismo , Regulação Leucêmica da Expressão Gênica , Leucemia Linfocítica Crônica de Células B/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Regulação para Cima , Adenina/análogos & derivados , Adenina/farmacologia , Linhagem Celular Tumoral , Humanos , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Leucemia Linfocítica Crônica de Células B/patologia , Piperidinas/farmacologiaRESUMO
Histone chaperones mediate the assembly and disassembly of nucleosomes and participate in essentially all DNA-dependent cellular processes. In Arabidopsis thaliana, loss-of-function of FAS1 or FAS2 subunits of the H3-H4 histone chaperone complex CHROMATIN ASSEMBLY FACTOR 1 (CAF-1) has a dramatic effect on plant morphology, growth and overall fitness. CAF-1 dysfunction can lead to altered chromatin compaction, systematic loss of repetitive elements or increased DNA damage, clearly demonstrating its severity. How chromatin composition is maintained without functional CAF-1 remains elusive. Here we show that disruption of the H2A-H2B histone chaperone NUCLEOSOME ASSEMBLY PROTEIN 1 (NAP1) suppresses the FAS1 loss-of-function phenotype. The quadruple mutant fas1 nap1;1 nap1;2 nap1;3 shows wild-type growth, decreased sensitivity to genotoxic stress and suppression of telomere and 45S rDNA loss. Chromatin of fas1 nap1;1 nap1;2 nap1;3 plants is less accessible to micrococcal nuclease and the nuclear H3.1 and H3.3 histone pools change compared to fas1. Consistently, association between NAP1 and H3 occurs in the cytoplasm and nucleus in vivo in protoplasts. Altogether we show that NAP1 proteins play an essential role in DNA repair in fas1, which is coupled to nucleosome assembly through modulation of H3 levels in the nucleus.
Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Cromatina/genética , Cromatina/metabolismo , Adenosina Trifosfatases/genética , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Instabilidade Genômica/genética , Instabilidade Genômica/fisiologia , Chaperonas de Histonas/genética , Chaperonas de Histonas/metabolismo , Mutação/genéticaRESUMO
BACKGROUND: While achieving prolonged remissions in other B cell-derived malignancies, chimeric antigen receptor (CAR) T cells still underperform when injected into patients with chronic lymphocytic leukemia (CLL). We studied the influence of genetics on CLL response to anti-CD19 CAR T-cell therapy. METHODS: First, we studied 32 primary CLL samples composed of 26 immunoglobulin heavy-chain gene variable (IGHV)-unmutated (9 ATM-mutated, 8 TP53-mutated, and 9 without mutations in ATM, TP53, NOTCH1 or SF3B1) and 6 IGHV-mutated samples without mutations in the above-mentioned genes. Then, we mimicked the leukemic microenvironment in the primary cells by '2S stimulation' through interleukin-2 and nuclear factor kappa B. Finally, CRISPR/Cas9-generated ATM-knockout and TP53-knockout clones (four and seven, respectively) from CLL-derived cell lines MEC1 and HG3 were used. All these samples were exposed to CAR T cells. In vivo survival study in NSG mice using HG3 wild-type (WT), ATM-knockout or TP53-knockout cells was also performed. RESULTS: Primary unstimulated CLL cells were specifically eliminated after >24 hours of coculture with CAR T cells. '2S' stimulated cells showed increased survival when exposed to CAR T cells compared with unstimulated ones, confirming the positive effect of this stimulation on CLL cells' in vitro fitness. After 96 hours of coculture, there was no difference in survival among the genetic classes. Finally, CAR T cells were specifically activated in vitro in the presence of target knockout cell lines as shown by the production of interferon-γ when compared with control (CTRL) T cells (p=0.0020), but there was no difference in knockout cells' survival. In vivo, CAR T cells prolonged the survival of mice injected with WT, TP53-knockout and ATM-knockout HG3 tumor cells as compared with CTRL T cells (p=0.0485, 0.0204 and <0.0001, respectively). When compared with ATM-knockout, TP53-knockout disease was associated with an earlier time of onset (p<0.0001), higher tumor burden (p=0.0002) and inefficient T-cell engraftment (p=0.0012). CONCLUSIONS: While in vitro no differences in survival of CLL cells of various genetic backgrounds were observed, CAR T cells showed a different effectiveness at eradicating tumor cells in vivo depending on the driver mutation. Early disease onset, high-tumor burden and inefficient T-cell engraftment, associated with TP53-knockout tumors in our experimental setting, ultimately led to inferior performance of CAR T cells.
Assuntos
Antígenos CD19/uso terapêutico , Leucemia Linfocítica Crônica de Células B/genética , Receptores de Antígenos Quiméricos/imunologia , Linfócitos T/imunologia , Animais , Voluntários Saudáveis , Humanos , CamundongosRESUMO
Genetic mutations in acute myeloid leukaemia (AML) are assumed to occur in a sequential order; however, the predominant hierarchical roles of specific mutated genes have not been fully described. In this study, we aimed to determine the clonal involvement of the most frequent AML-associated mutations. Using a targeted sequencing panel for 18 genes, we traced changes and relative clonal contribution of mutations in 52 patients. We analysed 35 pairs of diagnosis and relapse samples, 27 pairs of primary samples and corresponding patient-derived xenografts, and 34 pairs of total leukocytes and corresponding isolated primitive cells or blast populations. In both relapse and xenografts, we observed conservation of main leukaemic clones and variability was limited to subclones with late-acquired mutations. AML evolution thus mainly involved modification of subclones while the clonal background remained unchanged. NPM1 mutations were identified as the most probable leukaemia-transformation lesion, remaining conserved in contrast to high variation of accompanying subclonal FLT3 and NRAS mutations. DNMT3A mutations represented the most stable mutations forming a preleukaemic background in most samples. Mutations in genes IDH1/2, TET2, RUNX1, ASXL1 and U2AF1 were detected both as preleukaemic and as subclonal lesions, suggesting a non-specific order of acquisition.
Assuntos
Genes Neoplásicos , Leucemia Mieloide Aguda/genética , Mutação , Proteínas de Neoplasias/genética , Adulto , Idoso , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Evolução Clonal , Células Clonais , Terapia Combinada , Feminino , Transplante de Células-Tronco Hematopoéticas , Xenoenxertos , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/terapia , Leucócitos , Masculino , Camundongos , Camundongos Endogâmicos NOD , Pessoa de Meia-Idade , Transplante de Neoplasias , Células-Tronco Neoplásicas , Nucleofosmina , Recidiva , Adulto JovemRESUMO
Activation-induced cytidine deaminase (AID) is a mutator enzyme essential for somatic hypermutation (SHM) and class switch recombination (CSR) during effective adaptive immune responses. Its aberrant expression and activity have been detected in lymphomas, leukemias, and solid tumors. In chronic lymphocytic leukemia (CLL) increased expression of alternatively spliced AID variants has been documented. We used real-time RT-PCR to quantify the expression of AID and its alternatively spliced transcripts (AIDΔE4a, AIDΔE4, AIDivs3, and AIDΔE3E4) in 149 CLL patients and correlated this expression to prognostic markers including recurrent chromosomal aberrations, the presence of complex karyotype, mutation status of the immunoglobulin heavy chain variable gene, and recurrent mutations. We report a previously unappreciated association between higher AID transcript levels and trisomy of chromosome 12. Functional analysis of AID splice variants revealed loss of their activity with respect to SHM, CSR, and induction of double-strand DNA breaks. In silico modeling provided insight into the molecular interactions and structural dynamics of wild-type AID and a shortened AID variant closely resembling AIDΔE4, confirming its loss-of-function phenotype.
Assuntos
Processamento Alternativo , Citidina Desaminase , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Leucemia Linfocítica Crônica de Células B , Modelos Biológicos , Proteínas de Neoplasias , Trissomia , Idoso , Animais , Cromossomos Humanos Par 12/enzimologia , Cromossomos Humanos Par 12/genética , Simulação por Computador , Citidina Desaminase/biossíntese , Citidina Desaminase/química , Citidina Desaminase/genética , Feminino , Humanos , Leucemia Linfocítica Crônica de Células B/enzimologia , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/patologia , Masculino , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , Simulação de Dinâmica Molecular , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Trissomia/genética , Trissomia/patologiaRESUMO
PURPOSE: This study aimed at analyzing the association of gene mutations and other acute myeloid leukemia (AML) characteristics with engraftment outcomes in immunodeficient mice and to select the engraftment outcomes that best reflect patient survival. METHODS: Mutations in 19 genes as well as leukemia- and patient-related characteristics were analyzed for a group of 47 de novo AML samples with respect to three engraftment outcomes: engraftment ability, engraftment intensity (percentage of hCD45+ cells) and engraftment latency. Leukemia-related characteristics were additionally analyzed in an extended group of 68 samples that included the 47 de novo samples, and additional 21 samples from refractory and relapsed cases. Engraftment outcomes were compared with overall and event-free survival of the patients. RESULTS: For the 47 de novo samples, no single mutation influenced engraftment, whereas the NPM1 mut /DNMT3A mut co-mutation was associated with higher engraftment ability. NPM1 mut /FLT3-ITD neg had lower engraftment intensity. Among leukemia-related characteristics, a complex karyotype was associated with higher engraftment intensity. Among patient-related characteristics, higher cytogenetic risk was associated with higher engraftment intensity, and failure to achieve clinical remission was associated with shorter engraftment latency. In the extended group of 68 samples, white blood count was associated with higher engraftment ability, and the presence of a complex karyotype was associated with higher engraftment intensity. Association with patient overall survival was seen only for engraftment intensity. CONCLUSIONS: The engraftment of AML was influenced by mutation-interactions and other AML characteristics, rather than by single mutated genes, and engraftment intensity best reflected clinical penetrance of AML.
Assuntos
Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Mutação , Adulto , Idoso , Animais , Xenoenxertos/patologia , Humanos , Leucemia Mieloide Aguda/sangue , Leucócitos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Pessoa de Meia-Idade , Nucleofosmina , Transplante Heterólogo , Adulto JovemRESUMO
Glioblastoma multiforme (GBM) is the most aggressive intracranial tumor characterized with infaust prognosis. Despite advances in neurosurgical and radiotherapeutic techniques and chemotherapy, the median overall survival ranges between 12-15 months from diagnosis. The main cause of treatment failure is considered the presence of tumor cells resistant to conventional therapy, mainly radiotherapy. MicroRNAs (miRNAs) are small, non-coding RNAs that function as post-transcriptional regulators of gene expression and have been repeatedly proven to play important roles in pathogenesis and biological features of many cancers, including GBM and its radioresistant phenotype. In our study, we established radioresistant cells from the commonly used human GBM cell lines T98G, U87MG and U251. Consequently, we performed global miRNA expression profiling in both radioresistant and parental cell lines and identified 113 miRNAs with significantly different expression (p<0.05) between these two groups (73 miRNAs were up-regulated, 40 miRNAs were down-regulated). Some of these miRNAs have been previously described in relation to ionizing radiation, and others were herein identified for the first time. We believe that after deeper functional investigation of identified miRNAs in relation to radioresistance, these miRNAs present potential predictive biomarkers or therapeutic targets in GBM.
Assuntos
Neoplasias Encefálicas/genética , Regulação Neoplásica da Expressão Gênica , Glioblastoma/genética , MicroRNAs/genética , Tolerância a Radiação/genética , Apoptose , Biomarcadores Tumorais/metabolismo , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Proliferação de Células , Perfilação da Expressão Gênica , Glioblastoma/patologia , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Fenótipo , PrognósticoAssuntos
Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Dano ao DNA , Regulação para Baixo , Leucemia Linfocítica Crônica de Células B/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Adulto , Idoso , Proteínas Mutadas de Ataxia Telangiectasia/genética , Western Blotting , Ciclo Celular/genética , Inibidor de Quinase Dependente de Ciclina p27/genética , Citometria de Fluxo , Humanos , Leucemia Linfocítica Crônica de Células B/genética , Mutação , Transdução de Sinais/genética , Proteína Supressora de Tumor p53/genéticaRESUMO
Fanconi anemia (FA) is a rare genetic disorder associated with bone-marrow failure, genome instability and cancer predisposition. Recently, we and others have demonstrated dysfunctional mitochondria with morphological alterations in FA cells accompanied by high reactive oxygen species (ROS) levels. Mitochondrial morphology is regulated by continuous fusion and fission events and the misbalance between these two is often accompanied by autophagy. Here, we provide evidence of impaired autophagy in FA. We demonstrate that FA cells have increased number of autophagic (presumably mitophagic) events and accumulate dysfunctional mitochondria due to an impaired ability to degrade them. Moreover, mitochondrial fission accompanied by oxidative stress (OS) is a prerequisite condition for mitophagy in FA and blocking this pathway may release autophagic machinery to clear dysfunctional mitochondria.
Assuntos
Anemia de Fanconi/fisiopatologia , Mitocôndrias/patologia , Dinâmica Mitocondrial , Mitofagia , Doenças Raras/fisiopatologia , Autofagia , Linhagem Celular , Humanos , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência , Mitocôndrias/ultraestrutura , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismoRESUMO
Pancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal malignancies. Its dismal prognosis is often attributed to the presence of cancer stem cells (CSCs) that have been identified in PDAC using various markers. However, the co-expression of all of these markers has not yet been evaluated. Furthermore, studies that compare the expression levels of CSC markers in PDAC tumor samples and in cell lines derived directly from those tumors are lacking. Here, we analyzed the expression of putative CSC markers-CD24, CD44, epithelial cell adhesion molecule (EpCAM), CD133, and nestin-by immunofluorescence, flow cytometry and quantitative PCR in 3 PDAC-derived cell lines and by immunohistochemistry in 3 corresponding tumor samples. We showed high expression of the examined CSC markers among all of the cell lines and tumor samples, with the exception of CD24 and CD44, which were enriched under in vitro conditions compared with tumor tissues. The proportions of cells positive for the remaining markers were comparable to those detected in the corresponding tumors. Co-expression analysis using flow cytometry revealed that CD24+/CD44+/EpCAM+/CD133+ cells represented a significant population of the cells (range, 43 to 72%) among the cell lines. The highest proportion of CD24+/CD44+/EpCAM+/CD133+ cells was detected in the cell line derived from the tumor of a patient with the shortest survival. Using gene expression profiling, we further identified the specific pro-tumorigenic expression profile of this cell line compared with the profiles of the other two cell lines. Together, CD24+/CD44+/EpCAM+/CD133+ cells are present in PDAC cell lines derived from primary tumors, and their increased proportion corresponds with a pro-tumorigenic gene expression profile.
Assuntos
Adenocarcinoma/metabolismo , Neoplasias Pancreáticas/metabolismo , Células-Tronco/metabolismo , Antígeno AC133/metabolismo , Adenocarcinoma/diagnóstico , Idoso , Biomarcadores Tumorais , Antígeno CD24/metabolismo , Células Cultivadas , Molécula de Adesão da Célula Epitelial/metabolismo , Feminino , Citometria de Fluxo , Regulação Neoplásica da Expressão Gênica , Humanos , Receptores de Hialuronatos/metabolismo , Masculino , Pessoa de Meia-Idade , Nestina/metabolismo , Neoplasias Pancreáticas/diagnóstico , Prognóstico , Reação em Cadeia da Polimerase em Tempo Real , TranscriptomaRESUMO
The three most frequent pediatric sarcomas, i.e., Ewing's sarcoma, osteosarcoma, and rhabdomyosarcoma, were examined in this study: three cell lines derived from three primary tumor samples were analyzed from each of these tumor types. Detailed comparative analysis of the expression of three putative cancer stem cell markers related to sarcomas-ABCG2, CD133, and nestin-was performed on both primary tumor tissues and corresponding cell lines. The obtained results showed that the frequency of ABCG2-positive and CD133-positive cells was predominantly increased in the respective cell lines but that the high levels of nestin expression were reduced in both osteosarcomas and rhabdomyosarcomas under in vitro conditions. These findings suggest the selection advantage of cells expressing ABCG2 or CD133, but the functional tests in NOD/SCID gamma mice did not confirm the tumorigenic potential of cells harboring this phenotype. Subsequent analysis of the expression of common stem cell markers revealed an evident relationship between the expression of the transcription factor Sox2 and the tumorigenicity of the cell lines in immunodeficient mice: the Sox2 levels were highest in the two cell lines that were demonstrated as tumorigenic. Furthermore, Sox2-positive cells were found in the respective primary tumors and all xenograft tumors showed apparent accumulation of these cells. All of these findings support our conclusion that regardless of the expression of ABCG2, CD133 and nestin, only cells displaying increased Sox2 expression are directly involved in tumor initiation and growth; therefore, these cells fit the definition of the cancer stem cell phenotype.
Assuntos
Biomarcadores Tumorais/metabolismo , Células-Tronco Neoplásicas/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Sarcoma/metabolismo , Sarcoma/patologia , Antígeno AC133/metabolismo , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Adolescente , Adulto , Animais , Linhagem Celular Tumoral , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Criança , Pré-Escolar , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Proteínas de Neoplasias/metabolismo , Células-Tronco Neoplásicas/patologia , Nestina/metabolismo , Osteossarcoma/metabolismo , Osteossarcoma/patologia , Rabdomiossarcoma/metabolismo , Rabdomiossarcoma/patologiaRESUMO
Abnormalities in ATM and TP53 genes represent important predictive factors in chronic lymphocytic leukemia (CLL); however, the efficacy of CD20 targeting immunotherapy is only poorly defined in the affected patients. Therefore, we tested the in vitro response to ofatumumab (OFA) and rituximab (RTX) in 75 CLL samples with clearly defined p53 or ATM inactivation. Using standard conditions allowing complement-dependent cytotoxicity, i.e., 10 µg/mL of antibodies and 20% active human serum, we observed clear differences among the tested genetic categories: ATM-mutated samples (n = 17) represented the most sensitive, wild-type samples (n = 31) intermediate, and TP53-mutated samples (n = 27) the most resistant group (ATM-mut vs. TP53-mut: P = 0.0005 for OFA and P = 0.01 for RTX). The response correlated with distinct levels of CD20 and critical complement inhibitors CD55 and CD59; CD20 level median was the highest in ATM-mutated and the lowest in TP53-mutated samples (difference between the groups P < 0.01), while the total level of complement inhibitors (CD55 plus CD59) was distributed in the opposite manner (P < 0.01). Negligible response to both OFA and RTX was noted in all cultures (n = 10) tested in the absence of active serum, which strongly indicated that complement-dependent cytotoxicity was a principal cell death mechanism. Our study shows that (1) common genetic defects in CLL cells significantly impact a primary response to anti-CD20 monoclonal antibodies and (2) ATM-mutated patients with currently poor prognosis may potentially benefit from immunotherapy targeting CD20.
Assuntos
Anticorpos Monoclonais Murinos/farmacologia , Anticorpos Monoclonais/farmacologia , Antígenos CD20/imunologia , Antígenos de Neoplasias/imunologia , Proteínas Mutadas de Ataxia Telangiectasia/genética , Genes p53 , Leucemia Linfocítica Crônica de Células B/patologia , Proteínas de Neoplasias/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Anticorpos Monoclonais Humanizados , Antígenos CD20/efeitos dos fármacos , Antígenos de Neoplasias/efeitos dos fármacos , Proteínas Mutadas de Ataxia Telangiectasia/deficiência , Proteínas Mutadas de Ataxia Telangiectasia/fisiologia , Antígenos CD55/imunologia , Antígenos CD59/imunologia , Proteínas do Sistema Complemento/imunologia , Meios de Cultura , Reparo do DNA/genética , Resistencia a Medicamentos Antineoplásicos , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Rituximab , Soro , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/deficiência , Proteína Supressora de Tumor p53/fisiologiaRESUMO
AIMS AND BACKGROUND: Tumor diseases cause 20% of deaths in Europe and they are the second most common cause of death and morbidity after cardiovascular diseases. Thus, tumor cells are target of many therapeutic strategies and tumor research is focused on searching more efficient and specific drugs as well as new therapeutic approaches. One of the areas of tumor research is an issue of external fields. In our work, we tested influence of a pulsed electromagnetic field (PEMF) and a hypothetic field of the pulsed vector magnetic potential (PVMP) on the growth of tumor cells; and further the possible growth inhibition effect of the PVMP. METHODS: Both unipolar and bipolar PEMF fields of 5 mT and PVMP fields of 0 mT at frequencies of 15 Hz, 125 Hz and 625 Hz were tested on cancer cell lines derived from various types of tumors: CEM/C2 (acute lymphoblastic leukemia), SU-DHL-4 (B-cell lymphoma), COLO-320DM (colorectal adenocarcinoma), MDA-BM-468 (breast adenocarcinoma), and ZR-75-1 (ductal carcinoma). Cell morphology was observed, proliferation activity using WST assay was measured and simultaneous proportion of live, early apoptotic and dead cells was detected using flow cytometry. RESULTS: A PEMF of 125 Hz and 625 Hz for 24 h-48 h increased proliferation activity in the 2 types of cancer cell lines used, i.e. COLO-320DM and ZR-75-1. In contrast, any of employed methods did not confirm a significant inhibitory effect of hypothetic PVMP field on tumor cells.
Assuntos
Campos Eletromagnéticos , Magnetoterapia/métodos , Campos Magnéticos , Linhagem Celular Tumoral , Proliferação de Células/efeitos da radiação , Sobrevivência Celular/efeitos da radiação , Humanos , Magnetoterapia/instrumentaçãoRESUMO
Nestin is a class VI intermediate filament protein expressed in the cytoplasm of stem and progenitor cells in the mammalian CNS during development. In adults, nestin is present only in a small subset of cells and tissues, including the subventricular zone of the adult mammalian brain, where neurogenesis occurs. Nestin expression has also been detected under such pathological conditions as ischemia, inflammation, and brain injury, as well as in various types of human solid tumors and their corresponding cell lines. Furthermore, nestin was recently found in the nuclei of glioblastoma, neuroblastoma, and angiosarcoma cells and it was proved to interact directly with the nuclear DNA in neuroblastoma cells. Here, we perform the first study of the intracellular distribution of nestin in cell lines derived from neurogenic tumors. Using immunodetection methods, we examined nestin expression in tumor-derived cell lines obtained from 11 patients with neuroblastoma, medulloblastoma, or glioblastoma multiforme. Besides its standard cytoplasmic localization, nestin was present in the nuclei of two neuroblastoma cell lines and one medulloblastoma cell line. Nestin was only present in the nuclei of cells with diffuse cytoplasmic staining for this protein, and the proportion of cells positive for nestin in nuclei, as well as the intensity of staining, varied. The presence of nestin in the nuclei was confirmed by both transmission electron microscopy and Western blotting. Our results indicate that the presence of nestin in the nuclei of tumor cells is not very rare, especially under in vitro conditions.
Assuntos
Núcleo Celular/metabolismo , Glioblastoma/metabolismo , Proteínas de Filamentos Intermediários/metabolismo , Meduloblastoma/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neuroblastoma/metabolismo , Idoso , Western Blotting , Linhagem Celular Tumoral , Núcleo Celular/química , Núcleo Celular/ultraestrutura , Criança , Pré-Escolar , Feminino , Imunofluorescência , Glioblastoma/ultraestrutura , Humanos , Imuno-Histoquímica , Lactente , Proteínas de Filamentos Intermediários/análise , Masculino , Meduloblastoma/ultraestrutura , Microscopia Eletrônica de Transmissão , Pessoa de Meia-Idade , Proteínas do Tecido Nervoso/análise , Nestina , Neuroblastoma/ultraestruturaRESUMO
PURPOSE: The aim of this study was to perform a detailed cytogenetic and molecular genetic analysis of a tumor taken from a 14.5-year-old boy with glioblastoma multiforme who showed an atypical clinical course. METHODS: Formalin-fixed, paraffin embedded tumor tissue and the corresponding HGG-02 cell line derived from this tumor were analyzed using fluorescence in situ hybridization (FISH), G-banding, multiplex ligation-dependent probe amplification (MLPA), functional analysis of separated alleles in yeast (FASAY), immunohistochemistry (IHC), and immunocytochemistry (ICC). RESULTS: Mutation of the p53 gene and hypermethylation of the MLH1 gene were detected by FASAY and MLPA, respectively. Cytogenetic analysis showed a polyploid karyotype with extensive heterogeneity in chromosome number. Using FISH, we identified a very unusual genetic change - a loss of EGFR gene copy in both the tumor tissue and the HGG-02 cell line. In accordance with the cytogenetic findings, IHC and ICC did not demonstrate overexpression of EGFR in the tumor tissue or HGG-02 cells. CONCLUSIONS: Despite his very poor prognosis, the patient experienced 34 months of event-free survival after surgery and adjuvant radiotherapy and chemotherapy. The detected loss of the EGFR gene copy may contribute to the unusual biological features of this tumor, but the forthcoming detailed expression analysis of cancer regulatory pathways is necessary to better understand this tumor phenotype.