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Depleting interferon regulatory factor-1(IRF-1) with CRISPR/Cas9 attenuates inducible oxidative metabolism without affecting RA-induced differentiation in HL-60 human AML cells.

ABSTRACT: The known collaboration between all-transretinoic acid and interferon motivates this study of the dependence of RA-induced leukemic cell differentiation on interferon regulatory factor-1 (IRF-1), a transcription factor that is the main mediator of interferon effects. In the HL-60 acute myeloid leukemia (AML) model that represents a rare RA-responsive subtype of AML, IRF-1 is not expressed until RA induces its prominent expression, and ectopic IRF-1 expression enhances RA-induced differentiation, motivating interest in how IRF-1 is putatively needed for RA response. Accordingly, we created CRISPR/Cas9-mediated IRF-1 knockout HL-60 cells. Contrary to expectation, loss of IRF-1 did not diminish RA-induced cellular signaling that propels differentiation, and RA-induced cell differentiation markers, including CD38 and CD11b expression and G1/G0cell cycle arrest, were unaffected. However, elimination of IRF-1 inhibited RA-induced p47phox expression and inducible oxidative metabolism detected by reactive oxygen species (ROS), suggesting IRF-1 is essential for mature granulocytic inducible oxidative metabolism. In the case of 1,25-Dihydroxyvitamin D3-induced differentiation to monocytes, IRF-1 loss did not affect D3-induced expression of CD38, CD11b, and CD14, and G1/0 arrest; but inhibited ROS production. Our data suggest that IRF-1 is inessential for differentiation but upregulates p47phox expression for mature-cell ROS production.

SUBMITTER: Zhu K

PROVIDER: S-EPMC7325585 | biostudies-literature | 2020 Jun

REPOSITORIES: biostudies-literature

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Depleting interferon regulatory factor-1(IRF-1) with CRISPR/Cas9 attenuates inducible oxidative metabolism without affecting RA-induced differentiation in HL-60 human AML cells.

Zhu Kaiyuan K Yue Jianbo J Yen Andrew A

FASEB bioAdvances 20200522 6

The known collaboration between all-transretinoic acid and interferon motivates this study of the dependence of RA-induced leukemic cell differentiation on interferon regulatory factor-1 (IRF-1), a transcription factor that is the main mediator of interferon effects. In the HL-60 acute myeloid leukemia (AML) model that represents a rare RA-responsive subtype of AML, IRF-1 is not expressed until RA induces its prominent expression, and ectopic IRF-1 expression enhances RA-induced differentiation, ...[more]

PMID: 32617521

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Project description:BackgroundThe aryl hydrocarbon receptor (AhR) ligand 6-Formylindolo(3,2-b)carbazole (FICZ) has received increasing attention since its identification as an endogenous AhR ligand and a photoproduct of tryptophan. FICZ and its metabolites have been detected in human fluids. We recently reported that AhR promotes retinoic acid (RA)-induced granulocytic differentiation of HL-60 myeloblastic leukemia cells by restricting the nuclear abundance of the stem cell associated transcription factor Oct4. The standard clinical management of acute promyelocytic leukemia (APL) is differentiation induction therapy using RA. But RA is not effective for other myeloid leukemias, making the mechanism of RA-induced differentiation observed in a non-APL myeloid leukemia of interest. To our knowledge, this is the first study regarding the influence of FICZ on RA-induced differentiation in any type of leukemic blasts.MethodsUsing flow cytometry and Western blotting assays, we determined the effects of FICZ on RA-induced differentiation of HL-60 human leukemia cells. All experiments were performed in triplicate. The groups RA and FICZ + RA were compared using the Paired-Samples T-Test. Western blot figures present the typical blots.ResultsWe demonstrate that FICZ enhances RA-induced differentiation, assessed by the expression of the membrane differentiation marker CD11b; cell cycle arrest; and the functional differentiation marker, inducible-oxidative metabolism. FICZ causes changes in signaling events that are known to drive differentiation, and notably augments the RA-induced sustained activation of the RAF/MEK/ERK axis of the mitogen-activated protein kinase (MAPK) cascade. FICZ also augments expression of the known MAPK signaling regulatory molecules c-Cbl, VAV1, pY458 p85 PI3K, Src-family kinases (SFKs), and IRF-1, a transcription factor associated with this putative signalsome that promotes RA-induced differentiation. Moreover, FICZ in combination with RA also increases expression of AhR and even more so of both Cyp1A2 and p47phox, which are known to be transcriptionally regulated by AhR. pY1021 PDGFRβ, a marker associated with retinoic acid syndrome was also increased.ConclusionsOur data suggest that FICZ modulates intracellular signaling pathways and enhances RA-induced differentiation.

Dataset Information

Depleting interferon regulatory factor-1(IRF-1) with CRISPR/Cas9 attenuates inducible oxidative metabolism without affecting RA-induced differentiation in HL-60 human AML cells.

Publications

Depleting interferon regulatory factor-1(IRF-1) with CRISPR/Cas9 attenuates inducible oxidative metabolism without affecting RA-induced differentiation in HL-60 human AML cells.

Similar Datasets

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