RESUMEN
Radiation therapy is commonly used to treat glioblastoma multiforme (GBM) brain tumors. Ionizing radiation (IR) induces dose-specific variations in transcriptional programs, implicating that they are tightly regulated and critical components in the tumor response and survival. Yet, our understanding of the downstream molecular events triggered by effective vs. non-effective IR doses is limited. Herein, we report that variations in the genetic programs are positively and functionally correlated with the exposure to effective or non-effective IR doses. Genome architecture analysis revealed that gene regulation is spatially and temporally coordinated with DNA repair kinetics. The radiation-activated genes were pre-positioned in active sub-nuclear compartments and were upregulated following the DNA damage response, while the DNA repair activity shifted to the inactive heterochromatic spatial compartments. The IR dose affected the levels of DNA damage repair and transcription modulation, but not the order of the events, which was linked to their spatial nuclear positioning. Thus, the distinct coordinated temporal dynamics of DNA damage repair and transcription reprogramming in the active and inactive sub-nuclear compartments highlight the importance of high-order genome organization in synchronizing the molecular events following IR.
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Glioblastoma , Radiación Ionizante , Humanos , Reparación del ADN/genética , Radiación no Ionizante , Transporte Biológico , Glioblastoma/genética , Glioblastoma/radioterapiaRESUMEN
BACKGROUND: Animal brain-tumor models have demonstrated a synergistic interaction between radiation therapy and a ketogenic diet (KD). Metformin has in-vitro anti-cancer activity, through AMPK activation and mTOR inhibition. We hypothesized that the metabolic stress induced by a KD combined with metformin would enhance radiation's efficacy. We sought to assess the tolerability and feasibility of this approach. METHODS: A single-institution phase I clinical trial. Radiotherapy was either 60 or 35 Gy over 6 or 2 weeks, for newly diagnosed and recurrent gliomas, respectively. The dietary intervention consisted of a Modified Atkins Diet (ModAD) supplemented with medium chain triglycerides (MCT). There were three cohorts: Dietary intervention alone, and dietary intervention combined with low-dose or high-dose metformin; all patients received radiotherapy. Factors associated with blood ketone levels were investigated using a mixed-model analysis. RESULTS: A total of 13 patients were accrued, median age 61 years, of whom six had newly diagnosed and seven with recurrent disease. All completed radiation therapy; five patients stopped the metabolic intervention early. Metformin 850 mg three-times daily was poorly tolerated. There were no serious adverse events. Ketone levels were associated with dietary factors (ketogenic ratio, p < 0.001), use of metformin (p = 0. 02) and low insulin levels (p = 0.002). Median progression free survival was ten and four months for newly diagnosed and recurrent disease, respectively. CONCLUSIONS: The intervention was well tolerated. Higher serum ketone levels were associated with both dietary intake and metformin use. The recommended phase II dose is eight weeks of a ModAD combined with 850 mg metformin twice daily.
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Neoplasias Encefálicas , Glioma , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/radioterapia , Terapia Combinada , Glioma/tratamiento farmacológico , Glioma/radioterapia , Humanos , Cetonas , Metformina/uso terapéutico , Persona de Mediana Edad , Recurrencia Local de NeoplasiaRESUMEN
Malaria parasites have a complex life cycle, during which they undergo significant biological changes to adapt to different hosts and changing environments. Plasmodium falciparum, the species responsible for the deadliest form of human malaria, maintains this complex life cycle with a relatively small number of genes. Alternative splicing (AS) is an important post-transcriptional mechanisms that enables eukaryotic organisms to expand their protein repertoire out of relatively small number of genes. SR proteins are major regulators of AS in higher eukaryotes. Nevertheless, the regulation of splicing as well as the AS machinery in Plasmodium spp. are still elusive. Here, we show that PfSR1, a putative P. falciparum SR protein, can mediate RNA splicing in vitro. In addition, we show that PfSR1 functions as an AS factor in mini-gene in vivo systems similar to the mammalian SR protein SRSF1. Expression of PfSR1-myc in P. falciparum shows distinct patterns of cellular localization during intra erythrocytic development. Furthermore, we determine that the predicted RS domain of PfSR1 is essential for its localization to the nucleus. Finally, we demonstrate that proper regulation of pfsr1 is required for parasite proliferation in human RBCs and over-expression of pfsr1 influences AS activity of P. falciparum genes in vivo.
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Empalme Alternativo , Eritrocitos/parasitología , Proteínas Nucleares/metabolismo , Plasmodium falciparum/genética , Proteínas Protozoarias/metabolismo , Proteínas de Unión al ARN/metabolismo , Línea Celular , Humanos , Señales de Localización Nuclear , Proteínas Nucleares/química , Proteínas Nucleares/genética , Plasmodium falciparum/crecimiento & desarrollo , Plasmodium falciparum/metabolismo , Proteínas Protozoarias/química , Proteínas Protozoarias/genética , Precursores del ARN/metabolismo , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/genética , Factores de Empalme Serina-ArgininaRESUMEN
Alternative splicing regulators have emerged as new players in cancer development, modulating the activities of many tumor suppressors and oncogenes and regulating the signaling pathways. However, little is known about the mechanisms by which these oncogenic splicing factors lead to cellular transformation. We have shown previously that the splicing factor serine and arginine splicing factor 1 (SRSF1; SF2/ASF) is a proto-oncogene, which is amplified in breast cancer and transforms immortal cells when overexpressed. In this study, we performed a structure-function analysis of SRSF1 and found that the RNA recognition motif 1 (RRM1) domain is required for its oncogenic activity. Deletion of RRM1 eliminated the splicing activity of SRSF1 on some of its endogenous targets. Moreover, we found that SRSF1 elevates the expression of B-Raf and activates the mitogen-activated protein kinase kinase (MEK) extracellular signal-regulated kinase (ERK) pathway and that RRM1 is required for this activation as well. B-Raf-MEK-ERK activation by SRSF1 contributes to transformation as pharmacological inhibition of MEK1 inhibits SRSF1-mediated transformation. In conclusion, RRM1 of SRSF1 is both required (and when tethered to the RS domain) also sufficient to activate the Raf-MEK-ERK pathway and to promote cellular transformation.
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Transformación Celular Neoplásica/patología , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteínas Nucleares/fisiología , Empalme del ARN/genética , Proteínas de Unión al ARN/fisiología , Ribonucleótido Reductasas/fisiología , Secuencias de Aminoácidos , Animales , Western Blotting , Adhesión Celular , Proliferación Celular , Células Cultivadas , Quinasas MAP Reguladas por Señal Extracelular/genética , Células HEK293 , Humanos , Hígado/metabolismo , Hígado/patología , Ratones , Quinasas de Proteína Quinasa Activadas por Mitógenos/genética , Proteínas Quinasas Activadas por Mitógenos/genética , Mutación , Unión Proteica , Estructura Terciaria de Proteína/fisiología , Proto-Oncogenes Mas , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Ribonucleósido Difosfato Reductasa , Factores de Empalme Serina-Arginina , Transducción de Señal , Células Madre/metabolismo , Células Madre/patología , Proteína p53 Supresora de Tumor/fisiologíaRESUMEN
INTRODUCTION: Chemokine (C-X3-C Motif) Receptor 1 (CX3CR1) is present in a subset of the immune cells in the tumor microenvironment (TME) and plays an essential and diverse role in cancer progression. However, its potential function in the irradiated TME remains unknown. MATERIALS AND METHODS: A mouse lung cancer model was performed by subcutaneously inoculating Lewis Lung Carcinoma (LLC) cells expressing luciferase (Luc-2) and mCherry cells in CX3CR1GFP/GFP, CX3CR1DTR/+, and wild-type (WT) mice. Bioluminescence imaging, clonogenic assay, and flow cytometry were used to assess tumor progression, proliferation, and cell composition after radiation. RESULTS: Radiation provoked a significant influx of CX3CR1-expressing immune cells, notably monocytes and macrophages, into the TME. Co-culturing irradiated LLC cells with CX3CR1-deficient monocytes, and macrophages resulted in reduced clonogenic survival and increased apoptosis of the cancer cells. Interestingly, deficiency of CX3CR1 in macrophages led to a redistribution of the irradiated LLC cells in the S-phase, parallel to increased expression of cyclin E1, required for cell cycle G1/S transition. In addition, the deficiency of CX3CR1 expression in macrophages altered the cytokine secretion with a decrease in interleukin 6, a crucial mediator of cancer cell survival and proliferation. Next, LLC cells were injected subcutaneously into CX3CR1DTR/+ mice, sensitive to diphtheria toxin (DT), and WT mice. After injection, tumors were irradiated with 8 Gy, and mice were treated with DT, leading to conditional ablation of CX3CR1-expressing cells. After three weeks, CX3CR1-depleted mice displayed reduced tumor progression. Furthermore, combining the S-phase-specific chemotherapeutic gemcitabine with CX3CR1 cell ablation resulted in additional attenuation of tumor progression. CONCLUSIONS: CX3CR1-expressing mononuclear cells invade the TME after radiation therapy in a mouse lung cancer model. CX3CR1 cell depletion attenuates tumor progression following radiation and sensitizes the tumor to S-phase-specific chemotherapy. Thus, we propose a novel strategy to improve radiation sensitivity by targeting the CX3CR1-expressing immune cells.
RESUMEN
The role of the L-type calcium channel (Cav1.2) as a molecular switch that triggers secretion prior to Ca(2+) transport has previously been demonstrated in bovine chromaffin cells and rat pancreatic beta cells. Here, we examined the effect of specific Cav1.2 allosteric modulators, BayK 8644 (BayK) and FPL64176 (FPL), on the kinetics of catecholamine release, as monitored by amperometry in single bovine chromaffin cells. We show that 2 microm BayK or 0.5 microm FPL accelerates the rate of catecholamine secretion to a similar extent in the presence either of the permeable Ca(2+) and Ba(2+) or the impermeable charge carrier La(3+). These results suggest that structural rearrangements generated through the binding of BayK or FPL, by altering the channel activity, could affect depolarization-evoked secretion prior to cation transport. FPL also accelerated the rate of secretion mediated by a Ca(2+)-impermeable channel made by replacing the wild type alpha(1)1.2 subunit was replaced with the mutant alpha(1)1.2/L775P. Furthermore, BayK and FPL modified the kinetic parameters of the fusion pore formation, which represent the initial contact between the vesicle lumen and the extracellular medium. A direct link between the channel activity and evoked secretion lends additional support to the view that the voltage-gated Ca(2+) channels act as a signaling molecular switch, triggering secretion upstream to ion transport into the cell.
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Ácido 3-piridinacarboxílico, 1,4-dihidro-2,6-dimetil-5-nitro-4-(2-(trifluorometil)fenil)-, Éster Metílico/farmacología , Agonistas de los Canales de Calcio/farmacología , Canales de Calcio Tipo L/química , Canales de Calcio Tipo L/metabolismo , Células Cromafines/efectos de los fármacos , Conformación Proteica , Pirroles/farmacología , Animales , Bario/metabolismo , Calcio/metabolismo , Canales de Calcio Tipo L/genética , Señalización del Calcio/fisiología , Catecolaminas/metabolismo , Bovinos , Células Cultivadas , Células Cromafines/citología , Células Cromafines/metabolismo , Técnicas Electroquímicas , Lantano/metabolismo , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Mutación , Subunidades de Proteína/química , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , RatasRESUMEN
Strontium (Sr(2+)), Barium (Ba(2+)) and Lanthanum (La(3+)) can substitute for Ca(2+) in driving synaptic transmission during membrane depolarization. Ion recognition at the polyglutamate motif (EEEE), comprising the channel selectivity-filter, during voltage-driven transitions, controls the kinetics of the voltage-gated calcium channel (VGCC) and its interactions with the synaptic proteins. We tested the effect of different charge carriers on evoked-release, as a means of exploring the involvement of VGCC in the fusion pore configuration. Employing amperometry recordings in single bovine chromaffin cells we show that the size of the fusion pore, designated by the 'foot'-amplitude, was increased when Ba(2+) substituted for Ca(2+) and decreased, with La(3+). The fusion pore stability, indicated by 'foot'-width, was decreased in La(3+). Also, the mean open time of the fusion pore (tau(fp)) was significantly lower in Sr(2+) and La(3+) compared to Ba(2+) and Ca(2+). These cations when occupying the selectivity filter reduced the spike frequency in the order of Ca(2+) > Sr(2+) > Ba(2+) > La(3+), which is parallel to the reduction in total catecholamine release. The correlation between ion binding at the selectivity filter and fusion pore properties supports a model in which the Ca(2+) channel regulates secretion through a site at the selectivity filter, upstream to cation entry into the cell.