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BACKGROUND/PURPOSE: Matching phasic pressure tracings between a fluid-filled catheter and high-fidelity pressure wire has received limited attention, although each part contributes half of the information to clinical decisions. We aimed to study the impact of a novel and automated method for improving the phasic calibration of a fluid-filled catheter by accounting for its oscillatory behavior. METHODS/MATERIALS: Retrospective analysis of drift check tracings was performed using our algorithm that corrects for mean difference (offset), temporal delays (timing), differential sensitivity of the manifold transducer and pressure wire sensor (gain), and the oscillatory behavior of the fluid-filled catheter described by its resonant frequency and damping factor (how quickly oscillations disappear after a change in pressure). RESULTS: Among 2886 cases, correcting for oscillations showed a large improvement in 28 % and a medium improvement in 41 % (decrease in root mean square error >0.5 mmHg to <1 or 1-2 mmHg, respectively). 96 % of oscillators were underdamped with median damping factor 0.27 and frequency 10.6 Hz. Fractional flow reserve or baseline Pd/Pa demonstrated no clinically important bias when ignoring oscillations. However, uncorrected subcycle non-hyperemic pressure ratios (NHPR) displayed both bias and scatter. CONCLUSIONS: By automatically accounting for the oscillatory behavior of a fluid-filled catheter system, phasic matching against a high-fidelity pressure wire can be improved compared to standard equalization methods. The majority of tracings contain artifacts, mainly due to underdamped oscillations, and neglecting them leads to biased estimates of equalization parameters. No clinically important bias exists for whole-cycle metrics, in contrast to significant effects on subcycle NHPR.
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Reserva del Flujo Fraccional Miocárdico , Humanos , Artefactos , Estudios Retrospectivos , CatéteresRESUMEN
Cancer vaccines are promising treatments to prevent relapse after chemotherapy in acute myeloid leukemia (AML) patients, particularly for those who cannot tolerate intensive consolidation therapies. Here, we report the development of an AML cell membrane-coated nanoparticle (AMCNP) vaccine platform, in which immune-stimulatory adjuvant-loaded nanoparticles are coated with leukemic cell membrane material. This AMCNP vaccination strategy stimulates leukemia-specific immune responses by co-delivering membrane-associated antigens along with adjuvants to antigen-presenting cells. To demonstrate that this AMCNP vaccine enhances leukemia-specific antigen presentation and T cell responses, we modified a murine AML cell line to express membrane-bound chicken ovalbumin as a model antigen. AMCNPs were efficiently acquired by antigen-presenting cells in vitro and in vivo and stimulated antigen cross-presentation. Vaccination with AMCNPs significantly enhanced antigen-specific T cell expansion and effector function compared with control vaccines. Prophylactic vaccination with AMCNPs enhanced cellular immunity and protected against AML challenge. Moreover, in an AML post-remission vaccination model, AMCNP vaccination significantly enhanced survival in comparison to vaccination with whole leukemia cell lysates. Collectively, AMCNPs retained AML-specific antigens, elicited enhanced antigen-specific immune responses, and provided therapeutic benefit against AML challenge.
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Vacunas contra el Cáncer , Leucemia Mieloide Aguda , Nanopartículas , Animales , Presentación de Antígeno , Membrana Celular , Humanos , Inmunoterapia , Leucemia Mieloide Aguda/tratamiento farmacológico , Ratones , VacunaciónRESUMEN
Posttranscriptional regulation has emerged as a driver for leukemia development and an avenue for therapeutic targeting. Among posttranscriptional processes, alternative polyadenylation (APA) is globally dysregulated across cancer types. However, limited studies have focused on the prevalence and role of APA in myeloid leukemia. Furthermore, it is poorly understood how altered poly(A) site usage of individual genes contributes to malignancy or whether targeting global APA patterns might alter oncogenic potential. In this study, we examined global APA dysregulation in patients with acute myeloid leukemia (AML) by performing 3' region extraction and deep sequencing (3'READS) on a subset of AML patient samples along with healthy hematopoietic stem and progenitor cells (HSPCs) and by analyzing publicly available data from a broad AML patient cohort. We show that patient cells exhibit global 3' untranslated region (UTR) shortening and coding sequence lengthening due to differences in poly(A) site (PAS) usage. Among APA regulators, expression of FIP1L1, one of the core cleavage and polyadenylation factors, correlated with the degree of APA dysregulation in our 3'READS data set. Targeting global APA by FIP1L1 knockdown reversed the global trends seen in patients. Importantly, FIP1L1 knockdown induced differentiation of t(8;21) cells by promoting 3'UTR lengthening and downregulation of the fusion oncoprotein AML1-ETO. In non-t(8;21) cells, FIP1L1 knockdown also promoted differentiation by attenuating mechanistic target of rapamycin complex 1 (mTORC1) signaling and reducing MYC protein levels. Our study provides mechanistic insights into the role of APA in AML pathogenesis and indicates that targeting global APA patterns can overcome the differentiation block in patients with AML.
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Regulación Leucémica de la Expresión Génica , Leucemia Mieloide Aguda/genética , Poliadenilación , Regiones no Traducidas 3' , Células Cultivadas , Células Madre Hematopoyéticas/metabolismo , Humanos , Células Tumorales Cultivadas , Factores de Escisión y Poliadenilación de ARNm/genéticaRESUMEN
BACKGROUND: Acute myeloid leukemia (AML) with the t(8;21)(q22;q22) chromosomal translocation is among the most common subtypes of AML and produces the AML1-ETO (RUNX1-ETO, RUNX1-RUNX1T1) oncogenic fusion gene. AML1-ETO functions as an aberrant transcription factor which plays a key role in blocking normal hematopoiesis. Thus, the expression of AML1-ETO is critical to t(8;21) AML leukemogenesis and maintenance. Post-transcriptional regulation of gene expression is often mediated through interactions between trans-factors and cis-elements within transcript 3'-untranslated regions (UTR). AML1-ETO uses the 3'UTR of the ETO gene, which is not normally expressed in hematopoietic cells. Therefore, the mechanisms regulating AML1-ETO expression via the 3'UTR are attractive therapeutic targets. METHODS: We used RNA-sequencing of t(8;21) patients and cell lines to examine the 3'UTR isoforms used by AML1-ETO transcripts. Using luciferase assay approaches, we test the relative contribution of 3'UTR cis elements to AML1-ETO expression. We further use let-7b microRNA mimics and anti-let-7b sponges for functional studies of t(8;21) AML cell lines. RESULTS: In this study, we examine the regulation of AML1-ETO via the 3'UTR. We demonstrate that AML1-ETO transcripts primarily use a 3.7 kb isoform of the ETO 3'UTR in both t(8;21) patients and cell lines. We identify a negative regulatory element within the AML1-ETO 3'UTR. We further demonstrate that the let-7b microRNA directly represses AML1-ETO through this site. Finally, we find that let-7b inhibits the proliferation of t(8;21) AML cell lines, rescues expression of AML1-ETO target genes, and promotes differentiation. CONCLUSIONS: AML1-ETO is post-transcriptionally regulated by let-7b, which contributes to the leukemic phenotype of t(8;21) AML and may be important for t(8;21) leukemogenesis and maintenance.
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BACKGROUND: It would be ideal for a non-hyperaemic index to predict fractional flow reserve (FFR) more accurately, given FFR's extensive validation in a multitude of clinical settings. AIMS: The aim of this study was to derive a novel non-hyperaemic algorithm based on deep learning and to validate it in an internal validation cohort against FFR. METHODS: The ARTIST study is a post hoc analysis of three previously published studies. In a derivation cohort (random 80% sample of the total cohort) a deep neural network was trained (deep learning) with paired examples of resting coronary pressure curves and their FFR values. The resulting algorithm was validated against unseen resting pressure curves from a random 20% sample of the total cohort. The primary endpoint was diagnostic accuracy of the deep learning-derived algorithms against binary FFR ≤0.8. To reduce the variance in the precision, we used a fivefold cross-validation procedure. RESULTS: A total of 1,666 patients with 1,718 coronary lesions and 2,928 coronary pressure tracings were included. The diagnostic accuracy of our convolutional neural network (CNN) and recurrent neural networks (RNN) against binary FFR ≤0.80 was 79.6±1.9% and 77.6±2.3%, respectively. There was no statistically significant difference between the accuracy of our neural networks to predict binary FFR and the most accurate non-hyperaemic pressure ratio (NHPR). CONCLUSIONS: Compared to standard derivation of resting pressure ratios, we did not find a significant improvement in FFR prediction when resting data are analysed using artificial intelligence approaches. Our findings strongly suggest that a larger class of hidden information within resting pressure traces is not the main cause of the known disagreement between resting indices and FFR. Therefore, if clinicians want to use FFR for clinical decision making, hyperaemia induction should remain the standard practice.
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Estenosis Coronaria , Aprendizaje Profundo , Reserva del Flujo Fraccional Miocárdico , Inteligencia Artificial , Cateterismo Cardíaco , Angiografía Coronaria , Estenosis Coronaria/diagnóstico , Vasos Coronarios/diagnóstico por imagen , Humanos , Valor Predictivo de las Pruebas , Reproducibilidad de los Resultados , Índice de Severidad de la EnfermedadRESUMEN
OBJECTIVES: We sought to develop an automatic method for correcting common errors in phasic pressure tracings for physiology-guided interventions on coronary and valvular stenosis. BACKGROUND: Effective coronary and valvular interventions rely on accurate hemodynamic assessment. Phasic (subcycle) indexes remain intrinsic to valvular stenosis and are emerging for coronary stenosis. Errors, corrections, and clinical implications of fluid-filled catheter phasic pressure assessments have not been assessed in the current era of ubiquitous, high-fidelity pressure wire sensors. METHODS: We recruited patients undergoing invasive coronary physiology assessment. Phasic aortic pressure signals were recorded simultaneously using a fluid-filled guide catheter and 0.014â³ pressure wire before and after standard calibration as well as after pullback. We included additional subjects undergoing hemodynamic assessment before and after transcatheter aortic valve implantation. Using the pressure wire as reference standard, we developed an automatic algorithm to match phasic pressures. RESULTS: Removing pressure offset and temporal shift produced the largest improvements in root mean square (RMS) error between catheter and pressure wire signals. However, further optimization <1 mmHg RMS error was possible by accounting for differential gain and the oscillatory behavior of the fluid-filled guide. The impact of correction was larger for subcycle (like systole or diastole) versus whole-cycle metrics, indicating a key role for valvular stenosis and emerging coronary pressure ratios. CONCLUSIONS: When calibrating phasic aortic pressure signals using a pressure wire, correction requires these parameters: offset, timing, gain, and oscillations (frequency and damping factor). Automatically eliminating common errors may improve some clinical decisions regarding physiology-based intervention.
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Aorta/fisiopatología , Estenosis de la Válvula Aórtica/diagnóstico , Presión Arterial , Cateterismo Cardíaco/instrumentación , Catéteres Cardíacos , Estenosis Coronaria/diagnóstico , Transductores de Presión , Anciano , Algoritmos , Estenosis de la Válvula Aórtica/fisiopatología , Estenosis de la Válvula Aórtica/terapia , Automatización , Calibración , Cateterismo Cardíaco/efectos adversos , Cateterismo Cardíaco/normas , Catéteres Cardíacos/normas , Estenosis Coronaria/fisiopatología , Estenosis Coronaria/terapia , Femenino , Reserva del Flujo Fraccional Miocárdico , Humanos , Masculino , Persona de Mediana Edad , Valor Predictivo de las Pruebas , Reproducibilidad de los Resultados , Procesamiento de Señales Asistido por Computador , Transductores de Presión/normasRESUMEN
Heterozygous deletions within chromosome 20q, or del(20q), are frequent cytogenetic abnormalities detected in hematologic malignancies. To date, identification of genes in the del(20q) common deleted region that contribute to disease development have remained elusive. Through assessment of patient gene expression, we have identified STK4 (encoding Hippo kinase MST1) as a 20q gene that is downregulated below haploinsufficient amounts in myelodysplastic syndrome (MDS) and myeloproliferative neoplasm (MPN). Hematopoietic-specific gene inactivation in mice revealed Hippo kinase loss to induce splenomegaly, thrombocytopenia, megakaryocytic dysplasia, and a propensity for chronic granulocytosis; phenotypes that closely resemble those observed in patients harboring del(20q). In a JAK2-V617F model, heterozygous Hippo kinase inactivation led to accelerated development of lethal myelofibrosis, recapitulating adverse MPN disease progression and revealing a novel genetic interaction between these 2 molecular events. Quantitative serum protein profiling showed that myelofibrotic transformation in mice was associated with cooperative effects of JAK2-V617F and Hippo kinase inactivation on innate immune-associated proinflammatory cytokine production, including IL-1ß and IL-6. Mechanistically, MST1 interacted with IRAK1, and shRNA-mediated knockdown was sufficient to increase IRAK1-dependent innate immune activation of NF-κB in human myeloid cells. Consistent with this, treatment with a small molecule IRAK1/4 inhibitor rescued the aberrantly elevated IL-1ß production in the JAK2-V617F MPN model. This study identified Hippo kinase MST1 (STK4) as having a central role in the biology of del(20q)-associated hematologic malignancies and revealed a novel molecular basis of adverse MPN progression that may be therapeutically exploitable via IRAK1 inhibition.
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Neoplasias Hematológicas/genética , Síndromes Mielodisplásicos/genética , Trastornos Mieloproliferativos/genética , Proteínas Serina-Treonina Quinasas/genética , Animales , Deleción Cromosómica , Cromosomas Humanos Par 20/genética , Regulación hacia Abajo , Regulación Neoplásica de la Expresión Génica , Neoplasias Hematológicas/inmunología , Humanos , Inmunidad Innata , Péptidos y Proteínas de Señalización Intracelular , Ratones , Síndromes Mielodisplásicos/inmunología , Trastornos Mieloproliferativos/inmunología , Proteínas Serina-Treonina Quinasas/inmunologíaRESUMEN
Recent genome analysis of human prostate cancers demonstrated that both AR gene amplification and TP53 mutation are among the most frequently observed alterations in advanced prostate cancer. However, the biological role of these dual genetic alterations in prostate tumorigenesis is largely unknown. In addition, there are no biologically relevant models that can be used to assess the molecular mechanisms for these genetic abnormalities. Here, we report a novel mouse model, in which elevated transgenic AR expression and Trp53 deletion occur simultaneously in mouse prostatic epithelium to mimic human prostate cancer cells. These compound mice developed an earlier onset of high-grade prostatic intraepithelial neoplasia and accelerated prostate tumors in comparison with mice harboring only the AR transgene. Histological analysis showed prostatic sarcomatoid and basaloid carcinomas with massive squamous differentiation in the above compound mice. RNA-sequencing analyses identified a robust enrichment of the signature genes for human prostatic basal cell carcinomas in the above prostate tumors. Master regulator analysis revealed SOX2 as a transcriptional regulator in prostatic basal cell tumors. Elevated expression of SOX2 and its downstream target genes were detected in prostatic tumors of the compound mice. Chromatin immunoprecipitation analyses implicate a coregulatory role of AR and SOX2 in the expression of prostatic basal cell signature genes. Our data demonstrate a critical role of SOX2 in prostate tumorigenesis and provide mechanistic insight into prostate tumor aggressiveness and progression mediated by aberrant AR and p53 signaling pathways.
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Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , Receptores Androgénicos/fisiología , Factores de Transcripción SOXB1/fisiología , Proteína p53 Supresora de Tumor/genética , Animales , Transformación Celular Neoplásica/genética , Progresión de la Enfermedad , Eliminación de Gen , Regulación Neoplásica de la Expresión Génica , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Invasividad Neoplásica , Receptores Androgénicos/genética , Transducción de Señal/genética , TranscriptomaRESUMEN
Emerging evidence has shown that the hepatocyte growth factor (HGF) and its receptor, MET proto-oncogene, receptor tyrosine kinase (MET), promote cell proliferation, motility, morphogenesis, and angiogenesis. Whereas up-regulation of MET expression has been observed in aggressive and metastatic prostate cancer, a clear understanding of MET function in prostate tumorigenesis remains elusive. Here, we developed a conditional Met transgenic mouse strain, H11Met/+:PB-Cre4, to mimic human prostate cancer cells with increased MET expression in the prostatic luminal epithelium. We found that these mice develop prostatic intraepithelial neoplasia after HGF administration. To further assess the biological role of MET in prostate cancer progression, we bred H11Met/+/PtenLoxP/LoxP:PBCre4 compound mice, in which transgenic Met expression and deletion of the tumor suppressor gene Pten occurred simultaneously only in prostatic epithelial cells. These compound mice exhibited accelerated prostate tumor formation and invasion as well as increased metastasis compared with PtenLoxP/LoxP:PB-Cre4 mice. Moreover, prostatic sarcomatoid carcinomas and lesions resembling the epithelial-to-mesenchymal transition developed in tumor lesions of the compound mice. RNA-Seq and qRT-PCR analyses revealed a robust enrichment of known tumor progression and metastasis-promoting genes in samples isolated from H11Met/+/PtenLoxP/LoxP:PB-Cre4 compound mice compared with those from PtenLoxP/LoxP:PB-Cre4 littermate controls. HGF-induced cell proliferation and migration also increased in mouse embryonic fibroblasts (MEFs) from animals with both Met transgene expression and Pten deletion compared with Pten-null MEFs. The results from these newly developed mouse models indicate a role for MET in hastening tumorigenesis and metastasis when combined with the loss of tumor suppressors.
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Transformación Celular Neoplásica/metabolismo , Factor de Crecimiento de Hepatocito/metabolismo , Neoplasias de la Próstata/metabolismo , Proteínas Proto-Oncogénicas c-met/metabolismo , Transducción de Señal , Animales , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/patología , Factor de Crecimiento de Hepatocito/genética , Masculino , Ratones , Ratones Transgénicos , Fosfohidrolasa PTEN/genética , Fosfohidrolasa PTEN/metabolismo , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas c-met/genéticaRESUMEN
Viral gain-of-function mutations frequently evolve during laboratory experiments. Whether the specific mutations that evolve in the lab also evolve in nature and whether they have the same impact on evolution in the real world is unknown. We studied a model virus, bacteriophage λ, that repeatedly evolves to exploit a new host receptor under typical laboratory conditions. Here, we demonstrate that two residues of λ's J protein are required for the new function. In natural λ variants, these amino acid sites are highly diverse and evolve at high rates. Insertions and deletions at these locations are associated with phylogenetic patterns indicative of ecological diversification. Our results show that viral evolution in the laboratory mirrors that in nature and that laboratory experiments can be coupled with protein sequence analyses to identify the causes of viral evolution in the real world. Furthermore, our results provide evidence for widespread host-shift evolution in lambdoid viruses.
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Bacteriófago lambda/genética , Evolución Molecular , Mutación con Ganancia de Función/genética , Selección Genética , FilogeniaRESUMEN
Aims: Echocardiography and tomographic imaging have documented dynamic changes in aortic stenosis (AS) geometry and severity during both the cardiac cycle and stress-induced increases in cardiac output. However, corresponding pressure gradient vs. flow relationships have not been described. Methods and results: We recruited 16 routine transcatheter aortic valve implantations (TAVI's) for graded dobutamine infusions both before and after implantation; 0.014â³ pressure wires in the aorta and left ventricle (LV) continuously measured the transvalvular pressure gradient (ΔP) while a pulmonary artery catheter regularly assessed cardiac output by thermodilution. Before TAVI, ΔP did not display a consistent relationship with transvalvular flow (Q). Neither linear resistor (median R2 0.16) nor quadratic orifice (median R2 < 0.01) models at rest predicted stress observations; the severely stenotic valve behaved like a combination. The unitless ratio of aortic to left ventricular pressures during systolic ejection under stress conditions correlated best with post-TAVI flow improvement. After TAVI, a highly linear relationship (median R2 0.96) indicated a valid valve resistance. Conclusion: Pressure loss vs. flow curves offer a fundamental fluid dynamic synthesis for describing aortic valve pathophysiology. Severe AS does not consistently behave like an orifice (as suggested by Gorlin) or a resistor, whereas TAVI devices behave like a pure resistor. During peak dobutamine, the ratio of aortic to left ventricular pressures during systolic ejection provides a 'fractional flow reserve' of the aortic valve that closely approximates the complex, changing fluid dynamics. Because resting assessment cannot reliably predict stress haemodynamics, 'valvular fractional flow' warrants study to explain exertional symptoms in patients with only moderate AS at rest.
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Estenosis de la Válvula Aórtica/fisiopatología , Estenosis de la Válvula Aórtica/cirugía , Válvula Aórtica/fisiología , Válvula Aórtica/cirugía , Reemplazo de la Válvula Aórtica Transcatéter , Anciano de 80 o más Años , Presión Sanguínea , Femenino , Humanos , Masculino , Flujo Sanguíneo Regional , Índice de Severidad de la Enfermedad , Factores de TiempoRESUMEN
Evolutionary innovations are often achieved by repurposing existing genes to perform new functions; however, the mechanisms enabling the transition from old to new remain controversial. We identified mutations in bacteriophage λ's host-recognition gene J that confer enhanced adsorption to λ's native receptor, LamB, and the ability to access a new receptor, OmpF. The mutations destabilize λ particles and cause conformational bistability of J, which yields progeny of multiple phenotypic forms, each proficient at different receptors. This work provides an example of how nongenetic protein variation can catalyze an evolutionary innovation. We propose that cases where a single genotype can manifest as multiple phenotypes may be more common than previously expected and offer a general mechanism for evolutionary innovation.
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Bacteriófago lambda/genética , Evolución Molecular , Aptitud Genética , Variación Genética , Proteínas de la Membrana Bacteriana Externa/genética , Mutación , Porinas/genética , Receptores Virales/genética , Proteínas de la Cola de los Virus/genéticaRESUMEN
The leucine zipper tumor suppressor 2 (LZTS2) was identified as a tumor susceptibility gene within the 10q24.3 chromosomal region, and is approximately 15Mb from the PTEN locus. This region containing the both loci is frequently deleted in a variety of human malignancies, including prostate cancer. LZTS2 is a ß-catenin-binding protein and a negative regulator of Wnt signaling. Overexpression of PTEN in prostate cancer cell lines reduces ß-catenin-mediated transcriptional activity. In this study, we examined the collaborative effect of PTEN and LZTS2 using multiple in vitro and in vivo approaches. Co-expression of PTEN and LZTS2 in prostate cancer cells shows stronger repressive effect on ß-catenin mediated transcription. Using a newly generated mouse model, we further assessed the effect of simultaneous deletion of Pten and Lzts2 in the murine prostate. We observed that mice with both Lzts2 and Pten deletion have an earlier onset of prostate carcinomas as well as an accelerated tumor progression compared to mice with Pten or Lzts2 deletion alone. Immunohistochemical analyses show that atypical and tumor cells from compound mice with both Pten and Lzts2 deletion are mainly composed of prostate luminal epithelial cells and possess higher levels of cytoplasmic and nuclear ß-catenin. These cells also exhibit a higher proliferative capacity than cells isolated from single deletion mice. These data demonstrate the significance of simultaneous Pten and Lzts2 deletion in oncogenic transformation in prostate cells and implicates a new mechanism for the dysregulation of Wnt/ß-catenin signaling in prostate tumorigenesis.
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Proteínas de Ciclo Celular/genética , Transformación Celular Neoplásica/genética , Proteínas de Unión al ADN/genética , Fosfohidrolasa PTEN/genética , Neoplasias de la Próstata/patología , Proteínas Supresoras de Tumor/genética , beta Catenina/metabolismo , Animales , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Proliferación Celular/genética , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica/genética , Predisposición Genética a la Enfermedad , Humanos , Masculino , Ratones , Ratones Noqueados , Fosfohidrolasa PTEN/metabolismo , Regiones Promotoras Genéticas/genética , Interferencia de ARN , ARN Interferente Pequeño/genética , Proteínas Supresoras de Tumor/metabolismo , Vía de Señalización Wnt/genética , beta Catenina/genéticaRESUMEN
Bladder cancer represents a significant human tumor burden, accounting for about 7.7% and 2.4% of all cancer cases in males and females, respectively. While men have a higher risk of developing bladder cancer, women tend to present at a later stage of disease and with more aggressive tumors. Previous studies have suggested a promotional role of androgen signaling in enhancing bladder cancer development. To directly assess the role of androgens in bladder tumorigenesis, we have developed a novel transgenic mouse strain, R26hARLoxP/+:Upk3aGCE/+, in which the human AR transgene is conditionally expressed in bladder urothelium. Intriguingly, both male and female R26hARLoxP/+:Upk3aGCE/+ mice display a higher incidence of urothelial cell carcinoma (UCC) than the age and sex matched control littermates in response to the carcinogen, N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN). We detect expression of the human AR transgene in CK5-positive and p63-positive basal cells in bladder urothelium. Further analyses of UCC tissues from R26hARLoxP/+:Upk3aGCE/+ mice showed that the majority of tumor cells are of urothelial basal cell origin. Positive immunostaining of transgenic AR protein was observed in the majority of tumor cells of the transgenic mice, providing a link between transgenic AR expression and oncogenic transformation. We observed an increase in Ki67 positive cells within the UCC lesions of transgenic AR mice. Manipulating endogenous androgen levels by castration and androgen supplementation directly affected bladder tumor development in male and female R26hARLoxP/+:Upk3aGCE/+ mice, respectively. Taken together, our data demonstrate for the first time that conditional activation of transgenic AR expression in bladder urothelium enhances carciongen-induced bladder tumor formation in mice. This new AR transgenic mouse line mimics certain features of human bladder cancer and can be used to study bladder tumorigenesis and for drug development.
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Andrógenos , Carcinoma de Células Transicionales/etiología , Neoplasias Hormono-Dependientes/etiología , Receptores Androgénicos/fisiología , Neoplasias de la Vejiga Urinaria/etiología , Animales , Butilhidroxibutilnitrosamina , Carcinoma de Células Transicionales/inducido químicamente , Carcinoma de Células Transicionales/genética , División Celular , Transformación Celular Neoplásica , Implantes de Medicamentos , Femenino , Predisposición Genética a la Enfermedad , Humanos , Integrasas , Masculino , Ratones , Ratones Transgénicos , Neoplasias Hormono-Dependientes/inducido químicamente , Neoplasias Hormono-Dependientes/genética , Orquiectomía , Regiones Promotoras Genéticas/efectos de los fármacos , Receptores Androgénicos/genética , Proteínas Recombinantes de Fusión/metabolismo , Tamoxifeno/farmacología , Testosterona/administración & dosificación , Transgenes , Neoplasias de la Vejiga Urinaria/inducido químicamente , Neoplasias de la Vejiga Urinaria/genética , Uroplaquina III/biosíntesis , Uroplaquina III/genéticaRESUMEN
OBJECTIVES: This study classified and quantified the variation in fractional flow reserve (FFR) due to fluctuations in systemic and coronary hemodynamics during intravenous adenosine infusion. BACKGROUND: Although FFR has become a key invasive tool to guide treatment, questions remain regarding its repeatability and stability during intravenous adenosine infusion because of systemic effects that can alter driving pressure and heart rate. METHODS: We reanalyzed data from the VERIFY (VERification of Instantaneous Wave-Free Ratio and Fractional Flow Reserve for the Assessment of Coronary Artery Stenosis Severity in EverydaY Practice) study, which enrolled consecutive patients who were infused with intravenous adenosine at 140 µg/kg/min and measured FFR twice. Raw phasic pressure tracings from the aorta (Pa) and distal coronary artery (Pd) were transformed into moving averages of Pd/Pa. Visual analysis grouped Pd/Pa curves into patterns of similar response. Quantitative analysis of the Pd/Pa curves identified the "smart minimum" FFR using a novel algorithm, which was compared with human core laboratory analysis. RESULTS: A total of 190 complete pairs came from 206 patients after exclusions. Visual analysis revealed 3 Pd/Pa patterns: "classic" (sigmoid) in 57%, "humped" (sigmoid with superimposed bumps of varying height) in 39%, and "unusual" (no pattern) in 4%. The Pd/Pa pattern repeated itself in 67% of patient pairs. Despite variability of Pd/Pa during the hyperemic period, the "smart minimum" FFR demonstrated excellent repeatability (bias -0.001, SD 0.018, paired p = 0.93, r(2) = 98.2%, coefficient of variation = 2.5%). Our algorithm produced FFR values not significantly different from human core laboratory analysis (paired p = 0.43 vs. VERIFY; p = 0.34 vs. RESOLVE). CONCLUSIONS: Intravenous adenosine produced 3 general patterns of Pd/Pa response, with associated variability in aortic and coronary pressure and heart rate during the hyperemic period. Nevertheless, FFR - when chosen appropriately - proved to be a highly reproducible value. Therefore, operators can confidently select the "smart minimum" FFR for patient care. Our results suggest that this selection process can be automated, yet comparable to human core laboratory analysis.
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Cateterismo Cardíaco , Enfermedad de la Arteria Coronaria/diagnóstico , Estenosis Coronaria/diagnóstico , Vasos Coronarios/fisiopatología , Reserva del Flujo Fraccional Miocárdico , Hemodinámica , Adenosina/administración & dosificación , Algoritmos , Presión Arterial , Enfermedad de la Arteria Coronaria/fisiopatología , Estenosis Coronaria/fisiopatología , Vasos Coronarios/efectos de los fármacos , Europa (Continente) , Reserva del Flujo Fraccional Miocárdico/efectos de los fármacos , Hemodinámica/efectos de los fármacos , Humanos , Hiperemia/fisiopatología , Infusiones Intravenosas , Valor Predictivo de las Pruebas , Reproducibilidad de los Resultados , Índice de Severidad de la Enfermedad , Procesamiento de Señales Asistido por Computador , Factores de Tiempo , Estados Unidos , Vasodilatadores/administración & dosificaciónRESUMEN
The precise role of Wnt/ß-catenin signaling during prostatic development and tumorigenesis is unclear. Axin2 is a direct transcriptional target of ß-catenin. Recent studies have shown that Axin2-expressing cells have stem/progenitor cell properties in a variety of mouse tissues. Here, we genetically labeled Axin2-expressing cells at various time points and tracked their cellular behavior at different developmental and mature stages. We found that prostatic Axin2-expressing cells mainly express luminal epithelial cell markers and are able to expand luminal cell lineages during prostatic development and maturation. They can also survive androgen withdrawal and regenerate prostatic luminal epithelial cells following androgen replacement. Deletion of ß-catenin or expression of stabilized ß-catenin in these Axin2-expressing cells results in abnormal development or oncogenic transformation, respectively. Our study uncovers a critical role of Wnt/ß-catenin-responsive cells in prostatic development and regeneration, and that dysregulation of Wnt/ß-catenin signaling in these cells contributes to prostatic developmental defects and tumorigenesis.
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Proteína Axina/biosíntesis , Próstata/crecimiento & desarrollo , Próstata/metabolismo , Regeneración/fisiología , Vía de Señalización Wnt/fisiología , beta Catenina/biosíntesis , Animales , Linaje de la Célula , Células Epiteliales/metabolismo , Masculino , Ratones , Ratones Transgénicos , Organogénesis/fisiología , Próstata/citologíaRESUMEN
ZMIZ2, also named ZIMP7, is a protein inhibitor of activated STAT (PIAS)-like protein and a transcriptional coactivator. In this study, we investigated the interaction between ZMIZ2 and ß-catenin, a key regulator of the Wnt signaling pathway. We demonstrated that the expression of exogenous ZMIZ2 augments TCF (T cell factor) and ß-catenin-mediated transcription. In contrast, shRNA knockdown of ZMIZ2 expression specifically represses the enhancement of TCF/ß-catenin-mediated transcription by ZMIZ2. Using Wnt3a-conditioned medium, we demonstrated that ZMIZ2 can enhance Wnt ligand-induced TCF/ß-catenin-mediated transcription. We also showed a promotional role of ZMIZ2 in enhancing ß-catenin downstream target gene expression in human cells and in Zmiz2 null (Zmiz2(-/-)) mouse embryonic fibroblasts (MEFs). The regulatory role of Zmiz2 in Wnt-induced TCF/ß-catenin-mediated transcription can be restored in Zmiz2(-/-) MEFs that were infected with adenoviral expression vectors for Zmiz2. Moreover, enhancement of Zmiz2 on TCF/ß-catenin-mediated transcription was further demonstrated in Zmiz2 knockout and Axin2 reporter compound mice. Furthermore, the protein-protein interaction between ZMIZ2 and ß-catenin was identified by co-immunoprecipitation and in vitro protein pulldown assays. We also observed recruitment of endogenous ZMIZ2 onto the promoter region of the Axin 2 gene, a ß-catenin downstream target promoter, in a Wnt ligand-inducible manner. Finally, a promotional role of ZMIZ2 on cell growth was demonstrated in human cell lines and Zmiz2 knockout MEFs. Our findings demonstrate a novel interaction between ZMIZ2 and ß-catenin and elucidate a novel mechanism for PIAS-like proteins in regulating Wnt signaling pathways.
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular/metabolismo , Transducción de Señal , Proteínas Wnt/metabolismo , beta Catenina/metabolismo , Animales , Proteína Axina/genética , Línea Celular , Proliferación Celular , Femenino , Regulación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Receptores de Hialuranos/metabolismo , Péptidos y Proteínas de Señalización Intracelular/deficiencia , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Ratones , Regiones Promotoras Genéticas/genética , Proteínas Inhibidoras de STAT Activados , Factores de Transcripción TCF/metabolismo , Transcripción GenéticaRESUMEN
The PTEN tumor suppressor gene is frequently inactivated in human prostate cancer. Using Osr1 (odd skipped related 1)-Cre mice, we generated a novel conditional Pten knockout mouse strain, Pten(LoxP):Osr1-Cre. Conditional biallelic and monoallelic Pten knockout mice were viable. Deletion of Pten expression was detected in the prostate of Pten(LoxP/LoxP):Osr1-Cre mice as early as 2 weeks of age. Intriguingly, Pten(LoxP/LoxP):Osr1-Cre mice develop high-grade prostatic intraepithelial neoplasms (PINs) with high penetrance as early as one-month of age, and locally invasive prostatic tumors after 12-months of age. Pten(LoxP/+):Osr1-Cre mice show only mild oncogenic changes after 8-weeks of age. Castration of Pten(LoxP/LoxP):Osr1-Cre mice shows no significant regression of prostate tumors, although a shift of androgen receptor (AR) staining from the nuclei to cytoplasm is observed in Pten null tumor cells of castrated mice. Enhanced Akt activity is observed in Pten null tumor cells of castrated Pten(LoxP/LoxP):Osr1-Cre. This study provides a novel mouse model that can be used to investigate a primary role of Pten in initiating oncogenic transformation in the prostate and to examine other genetic and epigenetic changes that are required for tumor progression in the mouse prostate.
Asunto(s)
Eliminación de Gen , Ratones Transgénicos , Fosfohidrolasa PTEN/genética , Próstata/patología , Neoplasia Intraepitelial Prostática/genética , Neoplasia Intraepitelial Prostática/patología , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , Edad de Inicio , Alelos , Animales , Castración , Transformación Celular Neoplásica , Progresión de la Enfermedad , Efecto Fundador , Expresión Génica , Integrasas/genética , Integrasas/metabolismo , Masculino , Ratones , Invasividad Neoplásica , Fosfohidrolasa PTEN/deficiencia , Próstata/metabolismo , Neoplasia Intraepitelial Prostática/metabolismo , Neoplasias de la Próstata/metabolismo , Receptores Androgénicos/genética , Receptores Androgénicos/metabolismoRESUMEN
Using an Lzts2 knock-out mouse model, we characterized the biological role of Lzts2 in tumorigenesis. Both heterozygous and homozygous deletion of the Lzts2-targeted allele in mice shows an increased incidence in spontaneous tumor development, although Lzts2 homozygous knock-out mice show significantly higher incidences than heterozygous mice. Treatment of Lzts2-deficient mice with a carcinogen, N-butyl-N-(4-hydroxybutyl) nitrosamine, increases the susceptibility to N-butyl-N-(4-hydroxybutyl) nitrosamine-induced bladder carcinoma development. Examination of human prostate cancer tissue specimens shows a reduction of LZTS2 protein expression in prostate cancer cells. Further analyses of mouse embryonic fibroblasts isolated from Lzts2 knock-out embryos show that loss of Lzts2 enhances cell growth. These data provide the first line of evidence demonstrating that deletion of Lzts2 increases susceptibility to spontaneous and carcinogen-induced tumor development.
Asunto(s)
Proteínas de Ciclo Celular/biosíntesis , Proteínas de Unión al ADN/biosíntesis , Eliminación de Gen , Predisposición Genética a la Enfermedad , Neoplasias de la Próstata/metabolismo , Proteínas Supresoras de Tumor/biosíntesis , Neoplasias de la Vejiga Urinaria/metabolismo , Animales , Butilhidroxibutilnitrosamina/toxicidad , Carcinógenos/toxicidad , Proteínas de Ciclo Celular/genética , Proteínas de Unión al ADN/genética , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/genética , Células HEK293 , Heterocigoto , Humanos , Masculino , Ratones , Ratones Noqueados , Neoplasias de la Próstata/inducido químicamente , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , Proteínas Supresoras de Tumor/genética , Neoplasias de la Vejiga Urinaria/inducido químicamente , Neoplasias de la Vejiga Urinaria/genética , Neoplasias de la Vejiga Urinaria/patologíaRESUMEN
Members of the leucine zipper putative tumor suppressor (LZTS) family play crucial roles in transcription modulation and cell cycle control. We previously demonstrated that LZTS2 functions as a novel ß-catenin-interacting protein and represses ß-catenin-mediated transcription on T-cell factor/lymphoid enhancing factor. Here, we investigate the biological role of LZTS2 using newly established Lzts2 KO mice. Homozygosity for loss-of-function of the Lzts2-targeted allele resulted in severe kidney and urinary tract developmental defects, including renal/ureteral duplication, hydroureter, and hydronephrosis, which were visible prenatally. Altered ureteric bud outgrowth was identified in Lzts2 null embryos. Further analysis indicated that ß-catenin subcellular localization was altered in fibroblasts isolated from Lzts2 null embryos. In addition, Wnt growth factor-induced ß-catenin-mediated transcriptional activity was increased in Lzts2 null fibroblasts, suggesting a direct role for Lzts2 in the Wnt signaling pathway. These data demonstrate a critical role of LZTS2 in renal development and implicate LZTS2 as a critical regulator of ß-catenin-mediated nephrogenesis.