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Expansion of a single repetitive DNA sequence, termed a tandem repeat (TR), is known to cause more than 50 diseases1,2. However, repeat expansions are often not explored beyond neurological and neurodegenerative disorders. In some cancers, mutations accumulate in short tracts of TRs, a phenomenon termed microsatellite instability; however, larger repeat expansions have not been systematically analysed in cancer3-8. Here we identified TR expansions in 2,622 cancer genomes spanning 29 cancer types. In seven cancer types, we found 160 recurrent repeat expansions (rREs), most of which (155/160) were subtype specific. We found that rREs were non-uniformly distributed in the genome with enrichment near candidate cis-regulatory elements, suggesting a potential role in gene regulation. One rRE, a GAAA-repeat expansion, located near a regulatory element in the first intron of UGT2B7 was detected in 34% of renal cell carcinoma samples and was validated by long-read DNA sequencing. Moreover, in preliminary experiments, treating cells that harbour this rRE with a GAAA-targeting molecule led to a dose-dependent decrease in cell proliferation. Overall, our results suggest that rREs may be an important but unexplored source of genetic variation in human cancer, and we provide a comprehensive catalogue for further study.
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Expansión de las Repeticiones de ADN , Genoma Humano , Neoplasias , Humanos , Secuencia de Bases , Expansión de las Repeticiones de ADN/genética , Genoma Humano/genética , Neoplasias/clasificación , Neoplasias/genética , Neoplasias/patología , Análisis de Secuencia de ADN , Regulación de la Expresión Génica , Elementos Reguladores de la Transcripción/genética , Intrones/genética , Carcinoma de Células Renales/genética , Carcinoma de Células Renales/patología , Proliferación Celular/efectos de los fármacos , Reproducibilidad de los ResultadosRESUMEN
The primary cilium is thought to be disassembled prior to mitosis, freeing the centrosomes to participate in the mitotic spindle. In this issue, Paridaen et al. demonstrate that a remnant of the ciliary membrane remains attached to the mother centriole and is asymmetrically inherited in the developing neocortex.
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División Celular , Cilios/metabolismo , Células-Madre Neurales/citología , Animales , HumanosRESUMEN
BACKGROUND: Therapies for metastatic castration-resistant prostate cancer (mCRPC) include targeting the androgen receptor (AR) with androgen receptor inhibitors (ARIs) and prostate-specific membrane antigen (PSMA). Having the ability to detect AR, AR splice variant 7 (AR-V7), or PSMA in circulating tumor cells (CTCs) or circulating exosomal cell-free RNA (cfRNA) could be helpful to guide selection of the appropriate therapy for each individual patient. The Vortex Biosciences VTX-1 system is a label-free CTC isolation system that enables the detection of the expression of multiple genes in both CTCs and exosomal cfRNA from the same blood sample in patients with mCRPC. Detection of both AR-V7 and PSMA gene expression in both CTCs and cfRNA simultaneously has not yet been reported. METHODS: To characterize the combined VTX-1-AdnaDetect workflow, 22Rv1 cancer cells were spiked into blood from healthy donors and processed with the VTX-1 to mimic patient samples and assess performances (capture efficiency, purity, AR and AR-V7 expression). Then, we collected 19 blood samples from 16 patients with mCRPC and therapeutic resistance to androgen receptor inhibitors (ARIs). Plasma was separated and the plasma-depleted blood was processed further with the VTX-1 to collect CTCs. Both plasma exosomal cfRNA and CTCs were subsequently analyzed for AR, AR-V7, PSMA, and prostate-specific antigen (PSA) mRNA expression using the AdnaTest ProstateCancerPanel AR-V7 assay. RESULTS: AR-V7 expression could be detected in 22Rv1 cells spiked into blood from healthy volunteers as well as in CTCs and plasma-derived exosomal cfRNA from patients with mCRPC by processing blood with the VTX-1 CTC isolation system followed by the AdnaTest ProstateCancerPanel AR-V7 assay. 94.7% of patient blood samples (18/19) had detectable AR expression in either CTCs or exosomal cfRNA (16 in CTCs, 12 in cfRNA). 15.8% of the 19 patient blood samples (3/19) were found to have AR-V7-positive (AR-V7+) CTCs, one of which was also AR-V7+ in the exosomal cfRNA analysis. 42.1% of patient blood samples (8/19) were found to be PSMA positive (PSMA+): 26.3% (5/19) were PSMA+ in the CTC analysis and 31.6% (6/19) were PSMA+ in the exosomal cfRNA analysis. Of those 8 PSMA+ samples, 2 had detectable PSMA only in CTCs, and 3 had detectable PSMA only in exosomal cfRNA. CONCLUSION: VTX-1 enables isolation of CTCs and plasma exosomes from a single blood draw and can be used for detecting AR-V7 and PSMA mRNA in both CTCs and cfRNA in patients with mCRPC and resistance to ARIs. This technology facilitates combining RNA measurements in CTCs and exosomal cfRNA for future studies to develop potentially clinically relevant cancer biomarker detection in blood.
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Ácidos Nucleicos Libres de Células , Exosomas , Células Neoplásicas Circulantes , Neoplasias de la Próstata Resistentes a la Castración , Humanos , Masculino , Antagonistas de Receptores Androgénicos/farmacología , Antagonistas de Receptores Androgénicos/uso terapéutico , Biomarcadores de Tumor/genética , Ácidos Nucleicos Libres de Células/genética , Ácidos Nucleicos Libres de Células/metabolismo , Exosomas/genética , Exosomas/metabolismo , Células Neoplásicas Circulantes/patología , Próstata/patología , Antígeno Prostático Específico , Neoplasias de la Próstata Resistentes a la Castración/tratamiento farmacológico , Isoformas de Proteínas/genética , Receptores Androgénicos/genética , Receptores Androgénicos/metabolismo , ARN Mensajero/genéticaRESUMEN
McKittrick-Wheelock syndrome (MKWS) is an uncommon clinical manifestation of large, villous, epithelial lesions of the distal colon and rectum. Excessive secretion of electrolyte-rich mucus from these lesions leads to secretory diarrhea, electrolyte disorders and acute renal failure. Several cases of MKWS have been reported since its initial description in 1954. The definitive treatment for the great majority of MKWS cases has consisted of surgical resection of the affected part of the colorectum, usually in the form of a low anterior resection or an abdominoperineal resection with the formation of an ostomy. Recent developments in endoscopic resection techniques now offer new, minimally invasive treatment alternatives for MKWS patients. We present the first reported case in the Western world of MKWS caused by a rectal adenoma with a size of 19 × 10 cm, treated through endoscopic submucosal dissection. Through the lessons learned by this case, as well as by a thorough review of the literature, we discuss this uncommon syndrome, focusing on treatment alternatives.
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The epithelial sodium channel (ENaC) and the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel critically regulate airway surface liquid by driving fluid absorption and secretion, respectively. Their functional interplay is complex and incompletely understood. ENaC is a heteromeric channel with three well-characterized subunits (α, ß, and γ). In humans, an additional δ-ENaC subunit exists in lung and several other tissues, where it may replace the α-subunit to form δßγ-ENaC. Little is known about the physiological role of δßγ-ENaC and its possible interaction with CFTR. The aim of the present study was to investigate the effect of human CFTR on human δßγ-ENaC heterologously expressed in Xenopus laevis oocytes. In oocytes coexpressing δßγ-ENaC and CFTR the ENaC-mediated amiloride-sensitive whole cell current (ΔIami) was reduced by ~50% compared with that measured in oocytes expressing δßγ-ENaC alone. Moreover, basal level of proteolytic ENaC activation was reduced in the presence of CFTR. The inhibitory effect of CFTR on δßγ-ENaC was due to a combination of decreased average open probability (Po) and reduced channel expression at the cell surface. Interestingly, in oocytes expressing δßγ-ENaC, increasing intracellular [cAMP] by IBMX and forskolin increased ΔIami by ~50%. This stimulatory effect was not observed for human and rat αßγ-ENaC and was independent of CFTR coexpression and coactivation. Experiments with a mutant channel (δßS520Cγ-ENaC) which can be converted to a channel with a Po of nearly 1 suggested that cAMP activates δßγ-ENaC by increasing Po In conclusion, our results demonstrate that δßγ-ENaC is inhibited by CFTR but activated by cAMP.
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AMP Cíclico/farmacología , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Canales Epiteliales de Sodio/metabolismo , Oocitos/metabolismo , Subunidades de Proteína/metabolismo , Xenopus laevis/metabolismo , 1-Metil-3-Isobutilxantina/farmacología , Animales , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Quimotripsina/metabolismo , Colforsina/farmacología , Femenino , Humanos , Activación del Canal Iónico/efectos de los fármacos , Oocitos/efectos de los fármacos , Probabilidad , Proteolisis/efectos de los fármacos , RatasRESUMEN
Control of cell proliferation is a fundamental aspect of tissue formation in development and regeneration. Cells experience various spatial and mechanical constraints depending on their environmental context in the body, but we do not fully understand if and how such constraints influence cell cycle progression and thereby proliferation patterns in tissues. Here, we study the impact of mechanical manipulations on the cell cycle of individual cells within a mammalian model epithelium. By monitoring the response to experimentally applied forces, we find a checkpoint at the G1-S boundary that, in response to spatial constraints, controls cell cycle progression. This checkpoint prevents cells from entering S phase if the available space remains below a characteristic threshold because of crowding. Stretching the tissue results in fast cell cycle reactivation, whereas compression rapidly leads to cell cycle arrest. Our kinetic analysis of this response shows that cells have no memory of past constraints and allows us to formulate a biophysical model that predicts tissue growth in response to changes in spatial constraints in the environment. This characteristic biomechanical cell cycle response likely serves as a fundamental control mechanism to maintain tissue integrity and to ensure control of tissue growth during development and regeneration.
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Puntos de Control del Ciclo Celular/fisiología , Proliferación Celular , Inhibición de Contacto/fisiología , Modelos Biológicos , Animales , Fenómenos Biomecánicos , Biofisica , Perros , Cinética , Células de Riñón Canino Madin DarbyRESUMEN
Defects in the structure or function of the primary cilium, an antennae-like structure whose functional integrity has been linked to the suppression of uncontrolled kidney epithelial cell proliferation, are a common feature of genetic disorders characterized by kidney cysts. However, the mechanisms by which primary cilia are maintained remain poorly defined. von Hippel-Lindau (VHL) disease is characterized by the development of premalignant renal cysts and arises because of functional inactivation of the VHL tumour suppressor gene product, pVHL. Here, we show that pVHL and glycogen synthase kinase (GSK)3beta are key components of an interlinked signalling pathway that maintains the primary cilium. Although inactivation of either pVHL or GSK3beta alone did not affect cilia maintenance, their combined inactivation leads to loss of cilia. In VHL patients, GSK3beta is subjected to inhibitory phosphorylation in renal cysts, but not in early VHL mutant lesions, and these cysts exhibit reduced frequencies of primary cilia. We propose that pVHL and GSK3beta function together in a ciliary-maintenance signalling network, disruption of which enhances the vulnerability of cells to lose their cilia, thereby promoting cyst formation.
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Glucógeno Sintasa Quinasa 3/metabolismo , Enfermedades Renales Quísticas/metabolismo , Túbulos Renales/metabolismo , Transducción de Señal , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/metabolismo , Enfermedad de von Hippel-Lindau/metabolismo , Animales , Línea Celular , Cilios/metabolismo , Cilios/patología , Embrión de Mamíferos/citología , Células Epiteliales/metabolismo , Fibroblastos/metabolismo , Glucógeno Sintasa Quinasa 3/deficiencia , Glucógeno Sintasa Quinasa 3/genética , Glucógeno Sintasa Quinasa 3 beta , Humanos , Enfermedades Renales Quísticas/patología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Fosforilación , Interferencia de ARN , Transfección , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/genética , Enfermedad de von Hippel-Lindau/patologíaRESUMEN
Magnetic levitation-based sorting technologies have revolutionized the detection and isolation of rare cells, including circulating tumor cells (CTCs) and circulating tumor cell clusters (CTCCs). Manual counting and quantification of these cells are prone to time-consuming processes, human error, and inter-observer variability, particularly challenging when heterogeneous cell types in 3D clusters are present. To overcome these challenges, we developed "Fastcount," an in-house MATLAB-based algorithm for precise, automated quantification and phenotypic characterization of CTCs and CTCCs, in both 2D and 3D. Fastcount is 120 times faster than manual counting and produces reliable results with a ±7.3% deviation compared to a trained laboratory technician. By analyzing 400 GB of fluorescence imaging data, we showed that Fastcount outperforms manual counting and commercial software when cells are aggregated in 3D or staining artifacts are present, delivering more accurate results. We further employed Fastcount for automated analysis of 3D image stacks obtained from CTCCs isolated from colorectal adenocarcinoma and renal cell carcinoma blood samples. Interestingly, we observed a highly heterogeneous spatial cellular composition within CTCCs, even among clusters from the same patient. Overall, Fastcount can be employed for various applications with lab-chip devices, such as CTC detection, CTCC analysis in 3D and cell detection in biosensors.
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BACKGROUND: Prognostic models for patients with metastatic renal cell carcinoma (mRCC) include select laboratory values. These models have important limitations, including reliance on a limited array of laboratory tests, and use of dichotomous ("high-low") cutoffs. We applied a Laboratory-Wide Association Study (LWAS) framework to systematically evaluate common clinical laboratory results associated with survival for patients diagnosed with mRCC. METHODS: We used laboratory data for 3,385 patients diagnosed with mRCC from 2002 to 2017. We developed a LWAS framework, to examine the association with 53 common clinical laboratory tests results (641,712 measurements) and overall survival. We employed false-discovery rate to test the association of multiple laboratory tests with survival, and validated these results using 3 separate cohorts to generate a standardized hazard ratio (sHR), reported for a 1 standard deviation unit change in each laboratory test. RESULTS: The LWAS approach confirmed the association of laboratory values currently used in prognostic models with survival, including calcium (HR 1.35, 95%CI 1.24-1.48), leukocyte count (HR 1.40, 95%CI 1.30-1.51), platelet count (HR 1.36, 95%CI 1.27-1.51), and hemoglobin (HR 0.79, 95%CI 0.72-0.86). Use of these tests as continuous variables improved model performance. LWAS also identified acute phase reactants associated with survival not typically included in prognostic models, including serum albumin (HR 0.66, 95%CI 0.61-0.72), ferritin (HR 1.25, 95%CI 1.08-1.45), alkaline phosphatase (HR 1.31, 95%CI 1.23-1.40), and C-reactive protein (HR 1.70, 95%CI 1.14-2.53). CONCLUSIONS: Routinely measured laboratory tests can refine current prognostic models, facilitate comparisons across clinical trial cohorts, and match patients with specific systemic therapies.
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Carcinoma de Células Renales/sangre , Carcinoma de Células Renales/mortalidad , Neoplasias Renales/sangre , Neoplasias Renales/mortalidad , Anciano , Carcinoma de Células Renales/secundario , Estudios de Cohortes , Femenino , Pruebas Hematológicas , Humanos , Neoplasias Renales/patología , Laboratorios Clínicos , Masculino , Persona de Mediana Edad , Pronóstico , Tasa de SupervivenciaRESUMEN
Thymomas and thymic carcinomas (TC) are malignant thymic epithelial tumors (TETs) with poor outcome, if non-resectable. Metabolic signatures of TETs have not yet been studied and may offer new therapeutic options. Metabolic profiles of snap-frozen thymomas (WHO types A, AB, B1, B2, B3, n = 12) and TCs (n = 3) were determined by high resolution magic angle spinning 1H nuclear magnetic resonance (HRMAS 1H-NMR) spectroscopy. Metabolite-based prediction of active KEGG metabolic pathways was achieved with MetPA. In relation to metabolite-based metabolic pathways, gene expression signatures of TETs (n = 115) were investigated in the public "The Cancer Genome Atlas" (TCGA) dataset using gene set enrichment analysis. Overall, thirty-seven metabolites were quantified in TETs, including acetylcholine that was not previously detected in other non-endocrine cancers. Metabolite-based cluster analysis distinguished clinically indolent (A, AB, B1) and aggressive TETs (B2, B3, TCs). Using MetPA, six KEGG metabolic pathways were predicted to be activated, including proline/arginine, glycolysis and glutathione pathways. The activated pathways as predicted by metabolite-profiling were generally enriched transcriptionally in the independent TCGA dataset. Shared high lactic acid and glutamine levels, together with associated gene expression signatures suggested a strong "Warburg effect", glutaminolysis and redox homeostasis as potential vulnerabilities that need validation in a large, independent cohort of aggressive TETs. If confirmed, targeting metabolic pathways may eventually prove as adjunct therapeutic options in TETs, since the metabolic features identified here are known to confer resistance to cisplatin-based chemotherapy, kinase inhibitors and immune checkpoint blockers, i.e., currently used therapies for non-resectable TETs.
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Alterations in cellular sugar, amino acid and nucleic acid, and lipid metabolism, as well as in mitochondrial function, are a hallmark of renal cell carcinoma (RCC). The activation of oncogenes such as hypoxia-inducible factor and loss of the von Hippel-Lindau function and other tumor suppressors frequently occur early on during tumorigenesis and are the drivers for these changes, collectively known as "metabolic reprogramming," which promotes cellular growth, proliferation, and stress resilience. However, tumor cells can become addicted to reprogrammed metabolism. Here, we review the current knowledge of metabolic addictions in clear cell RCC, the most common form of RCC, and to what extent this has created therapeutic opportunities to interfere with such altered metabolic pathways to selectively target tumor cells. We highlight preclinical and emerging clinical data on novel therapeutics targeting metabolic traits in clear cell RCC to provide a comprehensive overview on current strategies to exploit metabolic reprogramming clinically.
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Carcinoma de Células Renales , Neoplasias Renales , Carcinoma de Células Renales/tratamiento farmacológico , Carcinoma de Células Renales/metabolismo , Proliferación Celular , Humanos , Neoplasias Renales/tratamiento farmacológico , Neoplasias Renales/metabolismo , Redes y Vías Metabólicas , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/metabolismoRESUMEN
To personalize treatment for renal cell carcinoma (RCC), it would be ideal to confirm the activity of druggable protein pathways within individual tumors. We have developed a high-resolution nanoimmunoassay (NIA) to measure protein activity with high precision in scant specimens (eg, fine needle aspirates [FNAs]). Here, we used NIA to determine whether protein activation varied in different regions of RCC tumors. Since most RCC therapies target angiogenesis by inhibiting the vascular endothelial growth factor (VEGF) receptor, we quantified phosphorylation of extracellular signal-regulated kinase (ERK), a downstream effector of the VEGF signaling pathway. In 90 ex vivo FNA biopsies sampled from multiple regions of 38 primary clear cell RCC tumors, ERK phosphorylation differed among patients. In contrast, within individual patients, we found limited intratumoral heterogeneity of ERK phosphorylation. Our results suggest that measuring ERK in a single FNA may be representative of ERK activity in different regions of the same tumor. As diagnostic and therapeutic protein biomarkers are being sought, NIA measurements of protein signaling may increase the clinical utility of renal mass biopsy and allow for the application of precision oncology for patients with localized and advanced RCC. PATIENT SUMMARY: In this report, we applied a new approach to measure the activity of extracellular signal-regulated kinase (ERK), a key cancer signaling protein, in different areas within kidney cancers. We found that ERK activity varied between patients, but that different regions within individual kidney tumors showed similar ERK activity. This suggests that a single biopsy of renal cell carcinoma may be sufficient to measure protein signaling activity to aid in precision oncology approaches.
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Carcinoma de Células Renales/enzimología , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Neoplasias Renales/enzimología , Carcinoma de Células Renales/química , Carcinoma de Células Renales/patología , Quinasas MAP Reguladas por Señal Extracelular/análisis , Humanos , Neoplasias Renales/química , Neoplasias Renales/patologíaRESUMEN
An important hallmark of cancer is 'metabolic reprogramming' or the rewiring of cellular metabolism to support rapid cell proliferation [1-5]. Metabolic reprogramming through oncometabolite-mediated transformation or activation of oncogenes in renal cell carcinoma (RCC) globally impacts energy production as well as glucose and glutamine utilization in RCC cells, which can promote dependence on glutamine supply to support cell growth and proliferation [6, 7]. Novel inhibitors of glutaminase, a key enzyme in glutamine metabolism, target glutamine addiction as a viable treatment strategy in metastatic RCC (mRCC). Here, we review glutamine metabolic pathways and how changes in cellular glutamine utilization enable the progression of RCC. This overview provides scientific rationale for targeting this pathway in patients with mRCC. We will summarize the current understanding of cellular and molecular mechanisms underlying anti-tumor efficacy of glutaminase inhibitors in RCC, provide an overview of clinical efforts targeting glutaminase in mRCC, and review approaches for identifying biomarkers for patient stratification and detecting therapeutic response early on in patients treated with this novel class of anti-cancer drug. Ultimately, results of ongoing clinical trials will demonstrate whether glutaminase inhibition can be a worthy addition to the current armamentarium of drugs used for patients with mRCC.
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With 15 drugs currently approved for the treatment of metastatic renal cell carcinoma (mRCC) and even more combination regimens with immunotherapy on the horizon, there remains a distinct lack of molecular biomarkers for therapeutic efficacy. Our study reports on real-world clinical outcomes of mRCC patients from a tertiary academic medical center treated with empirically selected standard-of-care therapy. We utilized the Stanford Renal Cell Carcinoma Database (RCCD) to report on various outcome measures, including overall survival (OS) and the median number of lines of targeted therapies received from the time of metastatic diagnosis. We found that most metastatic patients did not survive long enough to attempt even half of the available targeted therapies. We also noted that patients who failed to receive a clinical benefit within the first two lines of therapy could still go on to experience clinical benefit in later lines of therapy. The term, "clinical benefit" was assigned to a line of therapy if a patient remained on drug treatment for three months or longer. Moreover, patients with clinical benefit in at least one line of therapy experienced significantly longer OS compared to those who did not have clinical benefit in at least one line of therapy. Developing biomarkers that identify patients who will receive clinical benefit in individual lines of therapy is one potential strategy for achieving rational drug sequencing in mRCC.
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A long-standing hypothesis in radiotherapy is that intensity-modulated radiotherapy (IMRT) increases the risk of second cancer due to low-dose exposure of large volumes of normal tissue. Therefore, young patients are still treated with conventional techniques rather than with modern IMRT. We challenged this hypothesis in first-of-its-kind experiments using an animal model. Cancer-prone Tp53+/C273X knockout rats received mediastinal irradiation with 3 × 5 or 3 × 8 Gy using volumetric-modulated arc therapy (VMAT, an advanced IMRT) or conventional anterior-posterior/posterior-anterior (AP/PA) beams using non-irradiated rats as controls (n = 15/group, ntotal = 90). Tumors were assigned to volumes receiving 90-107%, 50-90%, 5-50%, and <5% of the target dose and characterized by histology and loss-of-heterozygosity (LOH). Irradiated rats predominantly developed lymphomas and sarcomas in areas receiving 50-107% (n = 26) rather than 5-50% (n = 7) of the target dose. Latency was significantly shortened only after 3 × 8 Gy vs. controls (p < 0.0001). The frequency (14/28 vs. 19/29; p = 0.29) and latency (218 vs. 189 days; p = 0.17) of radiation-associated tumors were similar after VMAT vs. AP/PA. LOH was strongly associated with sarcoma but not with treatment. The results do not support the hypothesis that IMRT increases the risk of second cancer. Thus the current practice of withholding dose-sparing IMRT from young patients may need to be re-evaluated.
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Modelos Animales de Enfermedad , Mediastino/efectos de la radiación , Neoplasias Inducidas por Radiación/epidemiología , Radioterapia de Intensidad Modulada/métodos , Animales , Femenino , Masculino , Órganos en Riesgo , Dosificación Radioterapéutica , Planificación de la Radioterapia Asistida por Computador/métodos , Ratas , Ratas Transgénicas , Factores de Riesgo , Proteína p53 Supresora de Tumor/genéticaRESUMEN
Centrioles are composed of long-lived microtubules arranged in nine triplets. However, the contribution of triplet microtubules to mammalian centriole formation and stability is unknown. Little is known of the mechanism of triplet microtubule formation, but experiments in unicellular eukaryotes indicate that delta-tubulin and epsilon-tubulin, two less-studied tubulin family members, are required. Here, we report that centrioles in delta-tubulin and epsilon-tubulin null mutant human cells lack triplet microtubules and fail to undergo centriole maturation. These aberrant centrioles are formed de novo each cell cycle, but are unstable and do not persist to the next cell cycle, leading to a futile cycle of centriole formation and disintegration. Disintegration can be suppressed by paclitaxel treatment. Delta-tubulin and epsilon-tubulin physically interact, indicating that these tubulins act together to maintain triplet microtubules and that these are necessary for inheritance of centrioles from one cell cycle to the next.
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Centriolos/metabolismo , Microtúbulos/metabolismo , Multimerización de Proteína , Tubulina (Proteína)/metabolismo , Eliminación de Gen , Humanos , Unión Proteica , Tubulina (Proteína)/genéticaRESUMEN
Neutrophils have a critical role in regulating the immune system. The immune system is compromised during chemotherapy, increasing infection risks and imposing a need for regular monitoring of neutrophil counts. Although commercial hematology analyzers are currently used in clinical practice for neutrophil counts, they are only available in clinics and hospitals, use large blood volumes, and are not available at the point of care (POC). Additionally, phlebotomy and blood processing require trained personnel, where patients are often admitted to hospitals when the infections are at late stage due to lack of frequent monitoring. Here, a reliable method is presented that selectively captures and quantifies white blood cells (WBCs) and neutrophils from a finger prick volume of whole blood by integrating microfluidics with high-resolution imaging algorithms. The platform is compact, portable, and easy to use. It captures and quantifies WBCs and neutrophils with high efficiency (>95%) and specificity (>95%) with an overall 4.2% bias compared to standard testing. The results from a small cohort of patients (N = 11 healthy, N = 5 lung and kidney cancer) present a unique disposable cell counter, demonstrating the ability of this tool to monitor neutrophil and WBC counts within clinical or in resource-constrained environments.
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Von Hippel-Lindau (VHL) tumor suppressor gene mutations predispose carriers to kidney cancer. The protein pVHL has been shown to interact with microtubules (MTs), which is critical to cilia maintenance and mitotic spindle orientation. However, the function for pVHL in the regulation of MT dynamics is unknown. We tracked MT growth via the plus end marker EB3 (end-binding protein 3)-GFP and inferred additional parameters of MT dynamics indirectly by spatiotemporal grouping of growth tracks from live cell imaging. Our data establish pVHL as a near-optimal MT-stabilizing protein: it attenuates tubulin turnover, both during MT growth and shrinkage, inhibits catastrophe, and enhances rescue frequencies. These functions are mediated, in part, by inhibition of tubulin guanosine triphosphatase activity in vitro and at MT plus ends and along the MT lattice in vivo. Mutants connected to the VHL cancer syndrome are differentially compromised in these activities. Thus, single cell-level analysis of pVHL MT regulatory function allows new predictions for genotype to phenotype associations that deviate from the coarser clinically defined mutant classifications.
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Imagenología Tridimensional/métodos , Microtúbulos/metabolismo , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/metabolismo , Secuencia de Aminoácidos , Análisis por Conglomerados , Proteínas Fluorescentes Verdes/metabolismo , Guanosina Trifosfato/metabolismo , Humanos , Hidrólisis/efectos de los fármacos , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/efectos de los fármacos , Datos de Secuencia Molecular , Nocodazol/farmacología , Fenotipo , Mutación Puntual/genética , Proteínas Recombinantes de Fusión/metabolismo , Tubulina (Proteína)/metabolismo , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/química , Enfermedad de von Hippel-Lindau/genéticaRESUMEN
BACKGROUND AND PURPOSE: To study whether pre-therapeutic lactate or pyruvate predict for tumour response to fractionated irradiation and to identify possible coherencies between intermediates of glycolysis and expression levels of selected proteins. MATERIALS AND METHODS: Concentrations of lactate, pyruvate, glucose and ATP were quantified via bioluminescence imaging in tumour xenografts derived from 10 human head and neck squamous cell carcinoma (HNSCC) lines. Tumours were irradiated with 30 fractions within 6 weeks. Expression levels of the selected proteins in tumours were measured at the mRNA and protein level. Tumour-infiltrating leucocytes were quantified after staining for CD45. RESULTS: Lactate but not pyruvate concentrations were significantly correlated with tumour response to fractionated irradiation. Lactate concentrations in vivo did not reflect lactate production rates in vitro. Metabolite concentrations did not correlate with GLUT1, PFK-L or LDH-A at the transcriptional or protein level. CD45-positive cell infiltration was low in the majority of tumours and did not correlate with lactate concentration. CONCLUSIONS: Our data support the hypothesis that the antioxidative capacity of lactate may contribute to radioresistance in malignant tumours. Non-invasive imaging of lactate to monitor radiation response and testing inhibitors of glycolysis to improve outcome after fractionated radiotherapy warrant further investigations.
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Adenosina Trifosfato/efectos de la radiación , Fraccionamiento de la Dosis de Radiación , Glucosa/efectos de la radiación , Ácido Láctico/efectos de la radiación , Ácido Pirúvico/efectos de la radiación , Animales , Carcinoma de Células Escamosas/radioterapia , Línea Celular Tumoral , Glucólisis , Neoplasias de Cabeza y Cuello/radioterapia , Humanos , RatonesRESUMEN
BACKGROUND: The pathogenesis and mechanisms of septic encephalopathy are not completely understood. We compared two different models of sepsis: lipopolysaccharide-induced endotoxemia and cecal ligation and puncture (CLP) bacteremia in rats with respect to changes in endothelial expression of the adhesion molecules intercellular adhesion molecule-1 (ICAM-1), platelet-endothelial cell adhesion molecule-1 (PECAM-1), and of cerebral albumin extravasation as a marker for capillary breakdown of the blood brain barrier. MATERIAL AND METHODS: Male Wistar rats were divided into control, endotoxemia, or CLP-group. Mean arterial blood pressure was measured via femoral artery catheterization. Brain tissue for immunohistochemistry was harvested at 1 h, 6 h, and 24 h after induction of sepsis. RESULTS: The CLP-group showed a decrease in mean arterial pressure after 24 h in comparison with the sham-group (P < 0.05). Cerebral ICAM-1 expression was at its maximum 24 h after induction of sepsis, with the highest expression in the CLP-group. There was no difference in PECAM-1 expression between the groups. Cerebral albumin extravasation increased early after 6 h in both septic groups with a maximum at 24 h after induction of sepsis. CONCLUSION: These results suggest that there are early changes in the integrity of the blood-brain barrier in the central nervous system in an ongoing septic progress. This provides evidence that these changes are due to inflammatory mediators, and not to the presence of live bacteria. Increased ICAM-1 expression might be an early factor involved in these pathogenic events. Although the role of PECAM-1 cannot conclusively be determined, we were able to show its expression on cerebral endothelium in all groups.