Regulation of vitamin D receptor expression by retinoic acid receptor alpha in acute myeloid leukemia cells

AbstractAcute myeloid leukemia (AML) is the predominant acute leukemia among adults, characterized by an accumulation of malignant immature myeloid precursors. A very promising way to treat AML is differentiation therapy using either all-trans-retinoic acid (ATRA) or 1,25-dihydroxyvitamin D3 (1,25D), or the use of both these differentiation-inducing agents. However, the effect of combination treatment varies in different AML cell lines, and this is due to ATRA either down- or up-regulating transcription of vitamin D receptor (VDR) in the cells examined. The mechanism of transcriptional regulation of VDR in response to ATRA has not been fully elucidated. Here, we show that the retinoic acid receptor α (RARα) is responsible for regulating VDR transcription in AML cells. We have shown that a VDR transcriptional variant, originating in exon 1a, is regulated by RARα agonists in AML cells. Moreover, in cells with a high basal level of RARα protein, the VDR gene is transcriptionally repressed as long as RARα agonist is absent. In these cells down-regulation of the level of RARα leads to increased expression of VDR. We consider that our findings provide a mechanistic background to explain the different outcomes from treating AML cell lines with a combination of ATRA and 1,25D

Diverse Regulation of Vitamin D Receptor Gene Expression by 1,25-Dihydroxyvitamin D and ATRA in Murine and Human Blood Cells at Early Stages of Their Differentiation

Vitamin D receptor (VDR) is present in multiple blood cells, and the hormonal form of vitamin D, 1,25-dihydroxyvitamin D (1,25D) is essential for the proper functioning of the immune system. The role of retinoic acid receptor α (RARα) in hematopoiesis is very important, as the fusion of RARα gene with PML gene initiates acute promyelocytic leukemia where differentiation of the myeloid lineage is blocked, followed by an uncontrolled proliferation of leukemic blasts. RARα takes part in regulation of VDR transcription, and unliganded RARα acts as a transcriptional repressor to VDR gene in acute myeloid leukemia (AML) cells. This is why we decided to examine the effects of the combination of 1,25D and all-trans-retinoic acid (ATRA) on VDR gene expression in normal human and murine blood cells at various steps of their development. We tested the expression of VDR and regulation of this gene in response to 1,25D or ATRA, as well as transcriptional activities of nuclear receptors VDR and RARs in human and murine blood cells. We discovered that regulation of VDR expression in humans is different from in mice. In human blood cells at early stages of their differentiation ATRA, but not 1,25D, upregulates the expression of VDR. In contrast, in murine blood cells 1,25D, but not ATRA, upregulates the expression of VDR. VDR and RAR receptors are present and transcriptionally active in blood cells of both species, especially at early steps of blood development

Ikaros and <i>RAG-2</i>-Mediated Antisense Transcription Are Responsible for Lymphocyte-Specific Inactivation of <i>NWC</i> Promoter

<div><p>Recombination activating gene-2 (<i>RAG-2</i>) and <i>NWC</i> are strongly evolutionarily conserved overlapping genes which are convergently transcribed. In non-lymphoid cells the <i>NWC</i> promoter is active whereas in lymphocytes it is inactive due to the DNA methylation. Analysing the mechanism responsible for lymphocyte-specific methylation and inactivation of <i>NWC</i> promoter we found that Ikaros, a lymphocyte-specific transcription factor, acts as a repressor of <i>NWC</i> promoter - thus identifying a new Ikaros target - but is insufficient for inducing its methylation which depends on the antisense transcription driven by <i>RAG-2</i> promoter. Possible implications of these observations for understanding evolutionary mechanisms leading to lymphocyte specific expression of <i>RAG</i> genes are discussed.</p></div

Ikaros downregulates the activity of <i>NWC</i> promoter.

<p>(A) Real-Time RT-PCR analysis of endogenous <i>NWC</i> expression in non-lymphoid cells (HEK293T) transfected with Ikaros-expressing construct (pLVX-Ikaros-IRES-ZsGreen) or with empty vector (pLVX-IRES-ZsGreen) (100%). Transfection efficiency varied and approximated 80%. The MFI after sorting was ∼1300. Expression values were normalized to <i>GAPDH</i>. The results shown are the means of three experiments with error bars representing ±1 SD. The asterisk indicates a significant difference (p<0.05) between both sample groups. (B) Activity of <i>NWC</i> promoter fragments containing point mutations in the Ikaros/ZFP-143 binding sites tested by luciferase assay in HEK293T cells ectopically expressing Ikaros gene (white bars) or transfected with empty vector (grey bars). pNWC-NM: non-mutated binding sites, pNWC-mGC: mutation specifically affecting the Ikaros binding site (see C), pNWC-mAT: mutation affecting both Ikaros and ZFP-143 binding sites. The relative promoter activities were normalized to the activity of promoter-less vector (pGL3-Basic). Vector containing SV40 promoter was used as a control. The results shown are the means of three experiments with error bars representing ±1 SD. The asterisk indicates a significant difference (p<0.05) between the activity of the given promoter in the presence or absence of Ikaros expression. (C) EMSA experiments verifying the Ikaros-specific nature of mutations (mGC) introduced to <i>NWC</i> promoter constructs. Non-mutated probes (NM) and probes with mutations affecting both Ikaros and ZFP-143 binding (mAT) were used as controls to monitor the efficiency of Ikaros and ZFP-143 binding.</p

Accuracy of the CUETO, EORTC 2016 and EAU 2021 scoring models and risk stratification tables to predict outcomes in high-grade non-muscle-invasive urothelial bladder cancer

Purpose: Non-muscle-invasive bladder cancers (NMIBC) constitute 3-quarters of all primary diagnosed bladder tumors. For risk -adapted management of patients with NMIBC, different risk group systems and predictive models have been developed. This study aimed to externally validate EORTC2016, CUETO and novel EAU2021 risk scoring models in a multi-institutional retrospective cohort of patients with high-grade NMIBC who were treated with an adequate BCG immunotherapy.Methods: The Kaplan-Meier estimates for recurrence-free survival and progression-free survival were performed, predictive abilities were assessed using the concordance index (C-index) and area under the curve (AUC).Results: A total of 1690 patients were included and the median follow-up was 51 months. For the overall cohort, the estimates recur-rence-free survival and progression-free survival rates at 5-years were 57.1% and 82.3%, respectively. The CUETO scoring model had poor discrimination for disease recurrence (C-index/AUC for G2 and G3 grade tumors: 0.570/0.493 and 0.559/0.492) and both CUETO (C-index/AUC for G2 and G3 grade tumors: 0.634/0.521 and 0.622/0.525) EAU2021 (c-index/AUC: 0.644/0.522) had poor discrimination for disease progression.Conclusion: Both the CUETO and EAU2021 scoring systems were able to successfully stratify risks in our population, but presented poor discriminative value in predicting clinical events. Due to the lack of data, model validation was not possible for EORTC2016. The CUETO and EAU2021 systems overestimated the risk, especially in highest-risk patients. The risk of progression according to EORTC2016 was slightly lower when compared with our population analysis. (c) 2022 Published by Elsevier Inc