RESUMEN

With immunohistochemical (IHC) staining increasingly being used to guide clinical decisions, variability in staining quality and reproducibility are becoming essential factors in generating diagnoses using IHC tissue preparations. The current study tested a method to track and quantify the interrun, intrarun, and intersite variability of IHC staining intensity. Our hypothesis was that staining precision between laboratory sites, staining runs, and individual slides may be verified quantitatively, efficiently and effectively utilizing algorithm-based, automated image analysis. To investigate this premise, we tested the consistency of IHC staining in 40 routinely processed (formalin-fixed, paraffin-embedded) human tissues using 10 common antibiomarker antibodies on 2 Dako Omnis instruments at 2 locations (Carpinteria, CA: 30 m above sea level and Longmont, CO: 1500 m above sea level) programmed with identical, default settings and sample pretreatments. Digital images of IHC-labeled sections produced by a whole slide scanner were analyzed by a simple commercially available algorithm and compared with a board-certified veterinary pathologist's semiquantitative scoring of staining intensity. The image analysis output correlated well with pathology scores but had increased sensitivity for discriminating subtle variations and providing reproducible digital quantification across sites as well as within and among staining runs at the same site. Taken together, our data indicate that digital image analysis offers an objective and quantifiable means of verifying IHC staining parameters as a part of laboratory quality assurance systems.

Asunto(s)

Procesamiento de Imagen Asistido por Computador/métodos , Inmunohistoquímica/métodos , Algoritmos , Análisis de Varianza , Anticuerpos , Neoplasias de la Mama/inmunología , Neoplasias de la Mama/metabolismo , Neoplasias del Colon/inmunología , Neoplasias del Colon/metabolismo , Humanos , Hígado/inmunología , Hígado/metabolismo , Pulmón/inmunología , Pulmón/metabolismo , Control de Calidad , Reproducibilidad de los Resultados , Bazo/inmunología , Bazo/metabolismo , Coloración y Etiquetado , Neoplasias Gástricas/inmunología , Neoplasias Gástricas/metabolismo

RESUMEN

PURPOSE: Dopamine receptor D2 (DRD2) is a G protein-coupled receptor antagonized by ONC201, an anticancer small molecule in clinical trials for high-grade gliomas and other malignancies. DRD5 is a dopamine receptor family member that opposes DRD2 signaling. We investigated the expression of these dopamine receptors in cancer and their influence on tumor cell sensitivity to ONC201. EXPERIMENTAL DESIGN: The Cancer Genome Atlas was used to determine DRD2/DRD5 expression broadly across human cancers. Cell viability assays were performed with ONC201 in >1,000 Genomic of Drug Sensitivity in Cancer and NCI60 cell lines. IHC staining of DRD2/DRD5 was performed on tissue microarrays and archival tumor tissues of glioblastoma patients treated with ONC201. Whole exome sequencing was performed in RKO cells with and without acquired ONC201 resistance. Wild-type and mutant DRD5 constructs were generated for overexpression studies. RESULTS: DRD2 overexpression broadly occurs across tumor types and is associated with a poor prognosis. Whole exome sequencing of cancer cells with acquired resistance to ONC201 revealed a de novo Q366R mutation in the DRD5 gene. Expression of Q366R DRD5 was sufficient to induce tumor cell apoptosis, consistent with a gain-of-function. DRD5 overexpression in glioblastoma cells enhanced DRD2/DRD5 heterodimers and DRD5 expression was inversely correlated with innate tumor cell sensitivity to ONC201. Investigation of archival tumor samples from patients with recurrent glioblastoma treated with ONC201 revealed that low DRD5 expression was associated with relatively superior clinical outcomes. CONCLUSIONS: These results implicate DRD5 as a negative regulator of DRD2 signaling and tumor sensitivity to ONC201 DRD2 antagonism.

Asunto(s)

Antagonistas de los Receptores de Dopamina D2/farmacología , Neoplasias/metabolismo , Receptores de Dopamina D2/metabolismo , Receptores de Dopamina D5/metabolismo , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Biomarcadores , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Resistencia a Medicamentos/genética , Expresión Génica , Humanos , Imidazoles/farmacología , Imidazoles/uso terapéutico , Inmunohistoquímica , Imagen por Resonancia Magnética , Clasificación del Tumor , Estadificación de Neoplasias , Neoplasias/diagnóstico , Neoplasias/tratamiento farmacológico , Neoplasias/mortalidad , Pronóstico , Unión Proteica , Piridinas/farmacología , Piridinas/uso terapéutico , Pirimidinas/farmacología , Pirimidinas/uso terapéutico , Receptores de Dopamina D2/genética , Receptores de Dopamina D5/química , Receptores de Dopamina D5/genética , Transducción de Señal

RESUMEN

The supraoptic nucleus (SON) of the hypothalamus contains magnocellular neurosecretory neurons (MNC) which synthesize and release the peptide hormones vasopressin and oxytocin. Glutamate is a prominent excitatory neurotransmitter in the SON and regulates MNC excitability. NMDA receptors (NMDAR), a type of ionotropic glutamate receptor, mediate synaptic plasticity of MNCs and are necessary for characteristic burst firing patterns which serve to maximize hormone release. NMDARs are di- or tri-heteromeric complexes of NR1 and NR2 subunits. Receptor properties depend on NR2 subunit composition and variable splicing of NR1. We investigated the expression profile of NR1 and NR2 subunits in the SON at the mRNA and protein levels plus protein expression of NR1 splice variants in control and salt-loaded adult rats. There was robust mRNA expression of all subunits, with NR2D levels being the highest. At the protein level, NR1, NR2B, and NR2D were robustly expressed, while NR2A was weakly expressed. NR2C protein was not detected with either of the two antibodies tested. All four NR1 splice variant cassettes (N1, C1, C2, C2') were detected in the SON, although NR1 N1 expression was too low for accurate analysis. Three days of salt-loading did not alter mRNA, protein, or splice variant expression of NMDAR subunits in the SON. Robust NR2D protein expression has not been previously shown in MNCs and is uncommon in the adult brain. Although the functional significance of this unusual expression profile is unknown, it may contribute to important physiological characteristics of SON neurons, such as burst firing and resistance to excitotoxicity.

Asunto(s)

Perfilación de la Expresión Génica , Receptores de N-Metil-D-Aspartato/biosíntesis , Núcleo Supraóptico/metabolismo , Animales , Western Blotting , Deshidratación/metabolismo , Expresión Génica , Masculino , Isoformas de Proteínas/biosíntesis , ARN Mensajero/análisis , Ratas , Ratas Sprague-Dawley , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa

RESUMEN

Since transgenes were first cloned into recombinant adenoviruses almost 30 years ago, a variety of viral vectors have become important tools in genetic research. Viruses adeptly transport genetic material into eukaryotic cells, and replacing all or part of the viral genome with genes of interest or silencing sequences creates a method of gene expression modulation in which the timing and location of manipulations can be specific. The hypothalamo-neurohypophyseal system (HNS), consisting of the paraventricular (PVN) and supraoptic (SON) nuclei in the hypothalamus, regulates fluid balance homeostasis and is highly plastic, yet tightly regulated by extracellular fluid (ECF) osmolality and volume. Its reversible plasticity and physiological relevance make it a good system for studying interactions between gene expression and physiology. Here, four viral vectors were compared for their ability to transduce magnocellular neurosecretory neurons (MNCs) of the SON in adult rats. The vectors included an adenovirus, a lentivirus (HIV) and two serotypes of adeno-associated viruses (AAV5 and AAV2). Though adenovirus and AAV2 vectors have previously been used to transduce SON neurons, HIV and AAV5 have not. All four vectors transduced MNCs, but the AAV vectors were the most effective, transducing large numbers of MNCs, with minimal or no glial transduction. The AAV vectors were injected using a convection enhanced delivery protocol to maximize dispersal through the tissue, resulting in the transduction of neurons throughout the anterior to posterior length of the SON (∼1.5mm). AAV5, but not AAV2, showed some selectivity for SON neurons relative to those in the surrounding hypothalamus.

Asunto(s)

Vectores Genéticos/genética , Neuronas/virología , Núcleo Supraóptico/virología , Transducción Genética/métodos , Virus/genética , Animales , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Humanos , Masculino , Neuronas/metabolismo , Ratas , Ratas Sprague-Dawley , Reproducibilidad de los Resultados , Núcleo Supraóptico/metabolismo