Activation of Retinoid Receptors RARa and RXRa Induces Differentiation and Apoptosis, Respectively, in HL-60 Cells’

Induction of granulocytic differentiation in HL-60 myeloid leukemia cells by retinoids is followed by their death via apoptosis. Retinoids are known to mediate their biological effects through at least two distinct types of nuclear receptors, the retinoic acid receptors and retinoid X receptors. We undertook to characterize the potential role of these receptors in inducing differentiation and apoptosis by retinoids. For this, we used a previously described variant of an HL-60 cell line (HL-60R) in which retinoid receptor function has been abrogated due to a trans-dominant negative mutation. Retroviral vector-mediated gene transfer was used to introduce the normal retinoic acid receptor (RARa) or retinoid X receptor (RXRa) into HL-60R cells. Our results suggest that ligand-Induced activation of RARa is sufficient to induce differentiation in HL-60 cells, whereas activation of RXRa can induce direct apoptosis of these cells without their prior commitment to differentiate. Introduction Normal myelopoiesis involves a series of events that leads to the differentiation of very primitive hematopoietic precursor stem cells into terminally differentiated granulocytes (1). Mature granulocytes represent the most abundant and shortest lived of all the hematopoietic cell populations. The average half-life of normal granulocytes in the circulating blood, where they function as specific effector cells in host-defense mechanisms, is about 1 2 h. After spending relatively short periods in a functional state, granulocytes are eliminated from the body through a genetically regulated mechanism called apoptosis or programmed cell death (2-4). A critical Received 9/25/95; revised 1 1/6/95; accepted 11/17/95. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to mdicate this fact. I This work is supported in part by USPHS Grant FDR000923 from the Food and Drug Administration and by a grant from Argus Pharmaceuticels, Inc. (The Woodlands, TX). 2 To whom requests for reprints should be addressed, at Department of Bioimmunotherapy, Box 60, The University of Texas M. D. Anderson Cancer Center, I 51 5 Holcombe Boulevard, Houston, TX 77030. Phone: (713) 792-2649; Fax: (713) 796-1731. balance between the life span of these cells and their regulated death is important for normal homeostasis. The human myeloblast cell line HL-60 provides a convenient model for studying the regulatory mechanisms of myeloid cell differentiation. This cell line can be induced to differentiate into a granulocytic lineage following ATRA3 treatment (5). Retinoid-differentiated HL-60 cells, like normal granulocytes, have a limited in vitro life span that could be further induced to undergo apoptosis in response to certain stimuli (6). This suggests that HL-60 cells could also serve as a model to study the mechanisms of apoptosis in terminally differentiated hematopoietic cells. However, the interpretation of retinoid effects on cell growth, differentiation, or apoptosis is difficult because retinoids mediate these effects by binding and activating two different types of nuclear receptors: the RARs and RXRs, which differ in their sequences and exhibit distinct ligand-binding properties (7-9). The RARs bind both ATRA and its 9-cis stereoisomer (9-cis RA), whereas the RXRs bind only 9-cis RA (1 0-1 2). Since most of the blood cells, including HL-60, express both types of receptors (1 3, 1 4), retinoid-induced effects in these cells may be a result of activation of either RARs, RXRs, or both types of receptors. We directly addressed this problem in a mutant subclone of the HL-6O cell line (HL-60R) in which retinoid receptor function has been abrogated as a result of a trans-dominant negative regulatory point mutation in the ligand-binding domain of the receptor (1 5). HL-60R subclones expressing specific receptors were generated by retrovirus-mediated transduction of RARaor RXRa-specific coding sequences (1 6, 17). Our results suggest that the introduction of RARa into HL-60R cells completely restored their sensitivity to ATRA-induced granulocytic differentiation. In contrast, the introduction of RXRa cDNA in HL-60R cells rendered them markedly sensitive to apoptosis in response to the RXRspecific ligand, 9-cis RA. Our observations provide direct evidence that ATRA-induced terminal differentiation of HL-60 cells is mediated by activation of RARa, whereas ligand activation of RXRa receptors triggers a signal that leads to programmed death, or apoptosis, of these cells. Results Receptor-specific Regulation of CD38 and TGase by Retinoids. Treatment of HL-60 cells with retinoids has been shown to induce the rapid and acute expression of a cell 3 The abbreviations used are: ATRA, all-trans retinoic acid; RA, retinoic acid; AAR, retinoic acid receptor; RXA, retinoid X receptor; TGase, transglutaminase; S:N ratio, signal:noise ratio; TdT, terminal deoxynucleotidyl transferase; MiT, 3-(4,5-dimethyfthiazol-2-yl)-2,5-diphenyl tetrazolium bromide.