Elsevier

Leukemia Research

Volume 22, Issue 6, June 1998, Pages 517-525
Leukemia Research

A novel retinoic acid receptor (RAR)-selective antagonist inhibits differentiation and apoptosis of HL-60 cells: implications of RARα-mediated signals in myeloid leukemic cells

https://doi.org/10.1016/S0145-2126(98)00026-5Get rights and content

Abstract

Retinoic acid (RA) induces HL-60 cells to differentiate terminally into mature granulocytes, which subsequently die by apoptosis. The biological effects of RA are mediated by two distinct families of transcription factors: retinoic acid receptors (RARs) and retinoid X receptors (RXRs). RARs and RXRs form heterodimers and regulate retinoid-mediated gene expression. We have recently developed a novel RAR-selective antagonist (ER27191) which prevents RAR activation by retinoids. Using this RAR-selective antagonist, and RXR and RAR agonist, we demonstrate the RAR-mediated signaling pathway is important for differentiation and apoptosis of myeloid leukemic cells. Simple activation of RXRs is not sufficient to induce apoptosis of the cells. Interestingly, the combination of the RAR-selective antagonist and 9-cis RA resulted in partial differentiation and apoptosis of HL-60 and NB4 cells, whereas the RAR antagonist completely blocked all-trans RA-induced differentiation and apoptosis of the cells. Additional experiments showed that levels of BCL-2 protein decreased during differentiation of myeloid leukemic cells. Furthermore, HL-60 cells transduced with a bcl-2 expression vector showed the same differentiation response to retinoids as did parental HL-60 cells even though apoptosis was inhibited in these bcl-2-transduced cells, suggesting that differentiation and apoptosis are regulated independently in myeloid leukemic cells.

Introduction

All-trans retinoic acid (RA) and 9-cis RA induced differentiation and inhibited proliferation of human leukemic cell lines such as HL-60 cells and APL (acute promyelocytic leukemia) cells from patients 1, 2. Clinically, a high proportion of patients with APL achieve complete remission after treatment with all-trans RA 3, 4. RA-differentiated HL-60 cells subsequently can be induced to undergo apoptosis in response to various stimuli [5]. Therefore, HL-60 cells can provide a model to help study the mechanism and relationship between the induction of differentiation and apoptosis in response to retinoids. However, the effects of RA on cellular growth, differentiation and apoptosis are complicated because retinoids mediate these effects by binding and activating two distinct family of receptors: retinoic acid receptors (RARs) and retinoid X receptors (RXRs) [6]. RARs and RXRs form heterodimers (RAR/RXR) or homodimers (RXR/RXR) and stimulate gene transcription by binding to cis-acting enhancer elements, termed RA responsive elements (RAREs), in a variety of target genes [7]. The ligands for these receptors are thought to be acid derivatives of retinol. Previous studies have shown that RARs recognize both all-trans RA and 9-cis RA, whereas RXRs interact exclusively with 9-cis RA 8, 9, 10, 11. An endogenous retinol metabolic pathway was recently identified and it was shown that 4-oxoretinol can activate transcription of RARs [12]. Several studies have demonstrated that receptors, including RAR, thyroid hormone receptor (TR), and vitamin D receptor (VDR), bind to RXR as heterodimers 7, 13. Ligand-induced transcription activity for RXR has shown to be suppressed when complexed with VDR, TR, and RAR [14]. Heterodimer formation prevents RXR from binding its ligand in RAR/RXR and TR/RXR, suggesting that 9-cis RA responsiveness is not an obligatory consequence of heterodimerization with RXR 14, 15, 16. Although RXR is a silent heterodimerization partner with RAR and TR, however, it can become activated in complexes with orphan receptors, as for example NGFI-B/RXR or Nurr/RXR [17]. Thus, RXR may be shifted from an inactive to an active receptor depending its partner, and these data suggest that several potentially different pathways exist by which retinoids can induce leukemic cell differentiation and apoptosis. Recent studies suggest that the ligand activation of RARs is sufficient to induce differentiation, whereas ligand activation of RXRs is essential for the induction of apoptosis in HL-60 cells 18, 19, 20, 21. However, little is known of the exact pathway that contributes to the effects of retinoids on leukemic cells. Several studies have addressed this problem using receptor-selective synthetic analogs, which preferentially activate either RARs or RXRs 18, 19, 20, 21. Another approach to dissect this problem is to use antagonists for retinoid receptors which prevent activation of RARs by retinoids. We have recently developed a novel RAR antagonist [22], which we have applied in this study to help determine the signaling pathways in HL-60 cells by retinoids. Furthermore, we also examined how retinoid signaling effects BCL-2 in HL-60 cells.

Section snippets

Cells and cell culture

RA-sensitive HL-60 and NB4 cells (the latter a kind gift from Dr M. Lanotte, Hôpital St Louis, Paris, France) [23]were maintained in RPMI 1640 medium (GIBCO BRL, Gaithersburg, MD) supplemented with 10% fetal bovine serum (Cytosystems, Australia) and 1% penicillin and streptomycin (GIBCO). RA-resistant HL-60 cells (RS-HL-60, a gift of Dr R.E. Gallagher, Montefiore Medical Center, Bronx, NY) were maintained in serum-free culture medium (Cosmedium 001; Cosmo Bio, Tokyo, Japan) containing all-trans

ER27191 as an antagonist of RAR activation by all-trans RA

Fig. 1 shows the chemical structure of 4-[4,5,7,8,9,10-hexahydro-7,7,10,10-tetramethyl-1-(3-pyridylmethyl)anthra-[1,2-b]pyrrol-3-yl] benzoic acid (ER27191). Induction of differentiation of HL-60 cells into mature granulocytes was assesed by fluorescence-activated cell sorting (FACS) analysis to determine the percentage of CD11b positive cells. All-trans RA increased the expression of CD11b antigens in a dose-dependent manner, whereas ER27191 alone did not alter the expression of CD11b antigen (

Discussion

Using a novel RAR-selective antagonist (ER27191) that prevents transactivation of RARs (α, β, and γ) by retinoids [22], we demonstrated in this study that ligand activation of RARs was necessary to cause terminal differentiation and apoptosis of HL-60 cells, while activation solely of RXRs by an RXR-specific agonist (SR11217) did not induce either differentiation or apoptosis. These results are consistent with previous studies which using different systems 18, 19. One investigation used

Acknowledgements

This work was supported by grants from the Ministry of Education, Science and Culture in Japan, the National Grant-in-Aid for the Establishment of High-Tech Research Center in a Private University, and the Keio University Special Grants, as well as NIH grants and the Parker Hughes and Concern Foundations. H.P.K. is a member of the Jonsson Cancer Center.

References (37)

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    In regard to cells, treatment of HL60 cells with a highly selective agonist of RARα, such as AGN195183 and AM580, was also sufficient to drive differentiation towards neutrophils [17,18]. Furthermore, the RAR antagonists, ER27191 and Ro 41-5253, mostly blocked ATRA-induced differentiation in HL60 cells [19,20]. In addition, a mutation in RARα (C411T) was shown to block the ATRA-induced differentiation of HL60 cells [21,22], and the reintroduction of RARα into HL60R cells rendered these cells sensitive to ATRA [23].

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    RAR antagonists ER 27191, Ro 415253 and AGN 194301 were kindly provided by Tsukuba Research Laboratories (Ibaraki, Japan), Hoffman-La Roche Ltd (Basel, Switzerland) and Allergan Inc. (California, USA), respectively. ER 27191 is a selective antagonist of RAR α, β and γ (Ueno et al., 1998) while both Ro 415253 and AGN 194301 are selective antagonists of RAR α (Apfel et al., 1992; Kochhar et al., 1998; Teng et al., 1997). Retinol, all-trans-RA, 9-cis-RA, 13-cis-RA and retinyl palmitate were obtained from Sigma (St Louis, USA).

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