Effects of Arsenic Trioxide on Megakaryocytic Cell Lines: Apoptosis, Cell Cycle Dysfunction and Signaling Pathways.
Notice bibliographique
Résumé
Abstract Despite progress made in the elucidation of the actions of arsenic trioxide (ATO) in acute promyelocytic leukemia, the molecular mechanisms leading to apoptosis in other malignancies remain unclear. In particular, the effects of ATO on the megakaryocytic (MK) lineage have not been well characterized. In this study, we focused on two MK cell lines CHRF-288-11 (CHRF) and MEG-01, which were derived from an infant and adult acute megakaryocytic leukemia (AMKL), respectively. Our data showed that these cells underwent apoptosis within 24 – 48 h post-ATO (6 μM) treatment, as demonstrated by the Annexin V assay (Table 1). By flow cytometry, significant activation of caspase-3 was detected in the MK cells at 24 h, and was preceded by the loss of mitochondrial membrane potential (8 h) as determined by the fluorescent dye JC-1. Western blotting experiments showed that ATO induced Bax expression and down-regulated Bcl-2, which led to an increase in Bax/Bcl-2 ratio. ATO exerted immediate and significant interference on the cell cycle by delaying S-phase progression and the subsequent accumulation of cells in the G2/M phase (43.2% vs 13.6%, p < 0.01). By multivariate analysis (BrdU and 7-AAD), active caspase-3 was detected in all phases of the cell cycle. The responses of CHRF and MEG-01 cells to ATO were similar, except that the latter appeared more resistant, in terms of the dosage of ATO and the slight delayed onset of apoptosis. We screened the expression levels of 96 genes involved in apoptosis using the GEArray Q Series Human Apoptosis Gene Array at 0, 4, 8 and 16 h (each n = 2) post-ATO treatment. We identified the up-regulation of mRNA of two extrinsic components of apoptosis. Fas was progressively increased in both cell lines (up to 6.14-fold) and caspase-8 was elevated in MEG-01 (3.58-fold). The protein expressions of Fas and activated caspase-8 were demonstrated in both cell lines by flow cytometry. Increased mRNA expressions of caspase-1 (2.30-fold) and CD137 (2.33-fold) were also noted, but their significance in apoptosis of our system remained to be investigated. To demonstrate the direct effect of ATO on gene expressions in AMKL cells, a more comprehensive microarray (Human 19K Array, Ontario Cancer Institute Microarray Centre) was used. Treatment with ATO for 4 h (n = 3) prompted an elevation in the mRNA levels of stress-associated proteins, such as metallothioneins (MT1G: 6.31-fold; MT2A: 3.64-fold), Hsp72 (5.81-fold), Hsp73 (3.77-fold), Hsp90 (2.11-fold), ferritin (2.02-fold) and ubiquitin (2.76-fold). Interestingly, WT1, a cell cycle regulatory gene elevated in many types of leukemia, was induced by ATO (2.44-fold). In conclusion, our results suggested that apoptosis in AMKL cells mediated by ATO involved a switch from pro-survival in the early phase to the activation of multiple death machineries, consisting of the intrinsic (mitochondrial, Bax, Bcl-2) and the extrinsic (Fas, caspase-8) compartments. Table 1: Signals regulated by ATO in CHRF cells 0 h 24 h 48 h Mean ± SEM; * p < 0.05 compared to 0 h; # n = 2, others n = 3–5. Annexin V +/PI − (%) 4.56±0.28 8.28±0.53* 9.83±0.73* Active caspase-3 (%) 2.28±0.13 4.58±0.87* 14.7±1.16* JC-1 greenhi/redlo (%) 4.18±0.52 8.05±0.60* 20.76±8.69* Bax/Bcl-2 (Fold)# 0.63±0.08 2.65±0.68 - Fas (Fold) 1 1.73±0.17* 1.96±0.20* CD137 (Fold) 1 1.55±0.08* 1.76±0.03*
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Comment cette classification a été obtenuedéplier
Prédiction distillée sur la base complète
Imitation des enseignantsNi prévalence calibrée, ni vérité terrain. Validation humaine à venir. Apprise à partir de 10 348 étiquettes directes de Codex et de 10 348 étiquettes directes de Gemma. Le mode candidate est l'union des têtes enseignantes seuillées; le consensus est leur intersection. Ces sorties portent le statut machine_predicted_unvalidated et ne sont ni des étiquettes humaines ni des étiquettes directes de modèles de pointe.
Scores Codex et Gemma par catégorie
| Catégorie | Codex | Gemma |
|---|---|---|
| Métarecherche | 0,000 | 0,000 |
| Méta-épidémiologie (sens strict) | 0,000 | 0,000 |
| Méta-épidémiologie (sens large) | 0,000 | 0,000 |
| Bibliométrie | 0,000 | 0,000 |
| Études des sciences et des technologies | 0,000 | 0,000 |
| Communication savante | 0,000 | 0,000 |
| Science ouverte | 0,000 | 0,000 |
| Intégrité de la recherche | 0,000 | 0,000 |
| Charge utile insuffisante (le modèle a refusé de juger) | 0,000 | 0,000 |
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 tête enseignante, 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 ».