Abstract A204: Flow cytometric assay of proteasome inhibition in whole blood.
Notice bibliographique
Résumé
Abstract Introduction: Two proteasome inhibitors are currently approved by the US FDA for the treatment of multiple myeloma: bortezomib and carfilzomib. Both drugs make a covalent bond with a Thr in the active site of the 20S catalytic β5 subunit. While the covalent bond is hydrolyzable in the case of bortezomib, its binding is characterized by a long residency time with a half-life of close to 2 hours. The current clinical surrogate assay measures the suppression of proteasome activity in pooled cell lysates from patients’ blood or tissue following the administration of inhibitor. As such, this assay depends upon the long residency time of current inhibitors. Second-generation proteasome inhibitors that are in development, such as ixazomib, have a much faster off-rate, making accurate measurement of inhibition in diluted cell lysates problematic. Our objective was to develop a surrogate proteasome assay for use in whole blood, without requiring cell lysis, thus preventing sample dilution. Methods: Whole-blood samples were pre-incubated with proteasome inhibitors for 1-2 hours at 37°C. Subsequently, samples were stimulated with LPS at 37°C, then fixed and permeabilized using PerFix-P (Beckman Coulter). Washed white blood cells were stained with IκBα-AF488 (Cell Signaling Technologies), CD45-KrO, and CD14-PC7 monoclonal antibodies (Beckman Coulter) and analyzed using a Gallios™ flow cytometer. Results and Discussion: During the classical activation of NF-κB signaling, the NF-κB inhibitor, IκBα, undergoes phosphorylation and ubiquitination, followed by rapid proteasomal degradation. Released NF-κB complexes can then translocate to the nucleus, where they initiate transcriptional activation. In this study, we analyzed the LPS-induced degradation of IκBα in whole-blood monocytes by flow cytometry. We found a 70-80% decrease in the median fluorescent intensity (MFI) of IκBα-AF488 after 10 min of LPS stimulation. The kinetic profile and magnitude of the MFI reduction were highly reproducible in whole-blood monocytes from healthy donors, as well as in a limited number of blood samples obtained from multiple-myeloma patients. The MFI reduction was inhibited in a dose-dependent manner by different proteasome inhibitors, including bortezomib, carfilzomib, and MG132. To increase the specificity of the assay, ERK phosphorylation can also be monitored simultaneously within the same cells. ERK phosphorylation in whole-blood monocytes peaks at ∼10 min after LPS stimulation, and is caused by the release of TPL2 from its complex with p105 (unprocessed NF-κB1) following the proteasomal processing of p105 into p50. Because of this, ERK phosphorylation is also inhibited by proteasome inhibitors in a dose-dependent manner, similar to IκBα. This assay can be optimized for different cell types within whole blood by using different activators, although the kinetics of IκBα degradation varies. Conclusions: We have established a fast and convenient assay to measure proteasome activity and inhibition in whole blood leukocytes. This assay can be used for investigation of the PK/PD relationships of new-generation proteasome inhibitors with fast off-rates. Disclaimer: PerFix-P and Gallios™ are for research use only. Not for use in diagnostic procedures. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A204. Citation Format: Sergei Gulnik, T. Vincent Shankey, Lidice L. Lopez, David W. Hedley, Sue Chow. Flow cytometric assay of proteasome inhibition in whole blood. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A204.
Récupéré en direct depuis OpenAlex et désinversé. Les résumés ne sont pas conservés dans cette base de données : les index inversés représentent 8,6 Go des 9,3 Go de texte de la base, et le serveur dispose de 13 Go libres.
Comment cette classification a été obtenuedéplier
Prédiction machine sur la base complète
Imitation des enseignantsNi prévalence calibrée, ni vérité terrain. Validation humaine à venir. Le volet Gemma est une étiquette directe du modèle pour chaque travail de la base, lue sur la notice réduite au titre. Le volet Codex est un classifieur appris des 10 348 étiquettes directes de Codex et calibré sur les taux pondérés de l'échantillon; les champs sans appui suffisant ne portent aucun appel Codex. Le mode candidate est l'union des deux volets; le consensus est leur intersection. Ces sorties portent le statut machine_predicted_unvalidated et ne sont pas des étiquettes humaines.
Scores du classifieur distillé par catégorie (deux têtes)
| Catégorie | Codex | Gemma |
|---|---|---|
| Métarecherche | 0,001 | 0,000 |
| Méta-épidémiologie (sens strict) | 0,001 | 0,000 |
| Méta-épidémiologie (sens large) | 0,000 | 0,000 |
| Bibliométrie | 0,002 | 0,001 |
| Études des sciences et des technologies | 0,001 | 0,000 |
| Communication savante | 0,001 | 0,001 |
| Science ouverte | 0,001 | 0,000 |
| Intégrité de la recherche | 0,001 | 0,001 |
| Charge utile insuffisante (le modèle a refusé de juger) | 0,011 | 0,004 |
Scores machine (provisoires)
Les deux têtes enseignantes du modèle étudiant, lues sur ce travail. Un score ordonne la base pour la relecture; il n'affirme jamais une catégorie, et le statut de validation accompagne chaque rangée tel quel.
Scores de référence d'un modèle non mature (critères de maturité non atteints, 7 itérations). Un score ordonne; il n'affirme jamais une catégorie.
score_only:v0-immature-baseline · tel quel depuis la passe de notation : score_only signifie que le nombre peut ordonner les travaux, et qu'aucune étiquette de catégorie n'en découleClassification
machine, non validéePrédiction automatique; un appel candidat d’une seule source (Gemma direct ou Codex distillé), pas un consensus.
Le détail, modèle par modèle et score par score, se trouve en fin de page sous « Comment cette classification a été obtenue ».