Leukemia (1997) 11, 393–400 1997 Stockton Press All rights reserved 0887-6924/97 $12.00 Alterations of differentiation, clonal proliferation, cell cycle progression and bcl-2 expression in RARa-altered sublines of HL-60 I Grillier1, T Umiel1, E Elstner1, SJ Collins2 and HP Koeffler1 1Division of Hematology/Oncology, Cedars-Sinai Medical Center/UCLA School of Medicine, Los Angeles, CA; and 2Fred Hutchinson Cancer Research Center, Seattle, WA, USA All-trans retinoic acid (RA) induces granulocytic differentiation bind all-trans RA as well as 9-cis RA, while RXRs bind only of acute promyelocytic leukemia cells both in vivo and in vitro. 9-cis RA.19–21 Thus, RARs heterodimerize to form RAR/RXR In the HL-60 wild-type (WT) early promyelocytic leukemia cell 17,22–25 line, granulocytic differentiation appears to be directly complexes. RXRs act as a coregulator, enhancing the mediated by the nuclear receptor RARa. An HL-60 subline binding of RA, 1,25 dihydroxyvitamin D3 [1,25(OH)2D3], thy- resistant to RA (HL-60 R) contains a point mutation which roid hormone, and peroxisome-activated receptors to their results in a truncation of 52 amino acids at the COOH end of responsive elements via heterodimers.22–27 Moreover, RARs RARa. Cross-talk between differentiation, clonal inhibition of may antagonize AP-1 function by either binding (directly or growth and apoptosis was studied using HL-60 WT, HL-60 R, indirectly) to c-jun/c-fos to form an inactive complex or inter- and HL-60 R infected by a retroviral vector containing RARa (LX) as targets, which were cultured with various retinoids, vit- acting with and sequestering another nuclear accessory factor that is required for AP-1-mediated transactivation.28–30 amin D3 analogs, HMBA, or DMSO. None of these compounds induced significant differentiation of HL-60 R and HL-60 LX, but The problem of RA resistance in APL patients reflects the they did induce differentiation of HL-60 WT. In contrast, retino- limits of differentiation-inducing therapy in general. Therefore, ids inhibited the clonal proliferation of HL-60 WT, HL-60 R, and in an attempt to improve the therapeutic outcome of a single- HL-60 LX. Vitamin D3 analogs including KH1060 stimulated the agent, RA in the treatment of leukemia, we used RA and non- clonal growth of HL-60 R; but they inhibited clonal growth of HL-60 WT and LX. Levels of Bcl-2 strongly decreased in HL- retinoid agents to examine their abilities to inhibit prolifer- 60 WT and LX after treatment by retinoids, while no change in ation, and to induce differentiation and apoptosis. The RA- expression occurred in HL-60 R. Neither KH 1060 nor 9-cis RA resistant subclone of the HL-60 myeloid leukemia cell line, induced apoptosis of HL-60 R, but these agents did induce designated as HL-60 R,31–35 was used in our studies. These apoptosis in HL-60 LX WT. Taken together, we showed that HL- cells contain a mutation which results in a truncation of 52 60 R has a global defect in its ability to be induced to differen- amino acids at the COOH end of RARa resulting in resistance tiate by a variety of pathways, not merely the retinoid pathway. Furthermore, our HL-60 models showed that inhibition of pro- to RA-induced differentiation. Another model subline was HL- liferation and induction of apoptosis and differentiation can 60 LX; these cells result from infection of HL-60 R with a retro- be dissociated. Clinically, these results suggest that several viral vector containing RARa.31,32 With these sublines of HL- putative differentiation agents may have anti-cancer 60 as well as wild-type (WT) HL-60 cells as tools, we determ- (antiproliferative) activities, even though they do not induce ined that inhibition of clonal proliferation could be disas- differentiation of the cancer cells. sociated from induction of differentiation and/or apoptosis of Keywords: HL-60; resistance; differentiation; clonal growth leukemic cells, and resistance to induction of differentiation was a global defect in the HL-60 R cells. Introduction Materials and methods Retinoids exert a wide range of biological effects predomi- nantly related to cell proliferation and differentiation, includ- Cell lines ing profound effects on hematopoeitic cells.1 Retinoic acid (RA) enhances the clonal growth of normal human myeloid HL-60 WT cells were obtained from the American Type Cul- and erythroid precursors and inhibits the clonal proliferation ture Collection (Rockeville, MD, USA). The retinoic acid of leukemic cells from patients with acute myelogenous leuke- resistant subline, HL-60 R, and the HL-60 R variant infected 2–4 mia (AML) as well as myeloid leukemia cell lines in vitro. with a functional retinoic acid receptor RARa (HL-60 LX) were Moreover, treatment of acute promyelocytic leukemia (APL) generated and characterized as described previously.31,32 The with RA as a single agent leads to a high rate of complete HL-60* clone isolated by Gallagher et al,33–35 was continu- 5,6 remissions. However, these remissions are generally not ously grown in RA (10−7 m). All cells were grown in RPMI durable because RA fails to eliminate the malignant clone and ml 7–13 1640 supplemented with 10% fetal calf serum, 2 m -gluta- because clinical resistance to RA develops. The mech- mine, penicillin (100 units/ml) and streptomycin (100 mg/ml). anism for this resistance is still unclear. The cells were incubated in a humidified incubator containing The biological effects of retinoids are mediated through ° 5% CO2 at 37 C. Cell viability was determined by trypan their binding to the nuclear receptors: retinoic acid receptors blue exclusion. (RARa,b,g) and retinoid X receptors (RXR a,b,g).14–17 Both classes of receptors belong to the steroid/thyroid hormone 18 superfamily and act as inducible transcription factors. RARs Cell treatment and differentiation Differentiation of HL-60 cells was measured by reduction of Correspondence: HP Koeffler, Division of Hematology/Oncology, 36 a Cedars-Sinai Medical Center/UCLA School of Medicine, Los Angeles, nitroblue tetrazolium (NBT), nonspecific -naphthyl acetate CA 90048, USA acid esterase (NSE) (Sigma, St Louis, MO, USA)36 and mor- Received 17 June 1996; accepted 5 November 1996 phology using light microscopy of cytospin preparations Biological effects of RA I Grillier et al 394 stained with Diff-Quick Stain Set (Baxter Heathcare Corpor- Results ation, Miami, FL, USA) after 5 days cultivation of HL-60 cells in suspension with various compounds. The dimethylsulfoxide Effects of retinoids and non-retinoid agents on (DMSO) and the hexamethylbisacetamide (HMBA) were differentiation of RA-resistant HL-60 cells obtained from Sigma. Prior studies showed that HL-60 wild-type (WT) cells are induced to differentiate down the granulocytic pathway by Retinoids and vitamin D3 compounds retinoids, HMBA and DMSO; and down the monocytic path- way by 1,25(OH)2D3 and related analogs and 12-O-tetrade- 39,40 All-trans-retinoic acid (RA) (Sigma, Poole, UK); 9-cis retinoic canoylphorbol-13-acetate (TPA). In this study, we exam- acid (9-cis-RA); 4-[1-(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahy- ined the induction of differentiation of WT- and RA-resistant dro-2-naphthyl)ethenyl] benzoic acid (code name, LG1069) HL-60 cells by assessing their abilities to reduce nitroblue (kindly provided by Dr R Heymann, Ligand Pharmaceuticals, tetrazolium (NBT) (granulocytic and monocytic San Diego, CA, USA); 2-(3-4-dihydro-4,4-dimethyl-2H-1 differentiation) and to acquire nonspecific esterase (NSE) benzopyran-6-yl)-2-(4-carboxyphenyl-1,3-dithiane (code activity (monocytic differentiation) and by morphological name, SR11238) (kindly provided by Dr M Dawson, SRI Inter- differentiation. Several retinoids were examined for their abilities to induce national, Menlo Park, CA, USA; 1,25 dihydroxyvitamin D3 differentiation. The RA (10−7 m), 9-cis RA (10−6 m) and their [1,25(OH)2D3] code name, compound C); 1,25(OH)2-16ene- combination induced the differentiation of 60%, 70% and 23yne-D3 (code name, compound V) (kindly provided by Dr M Uskokovic, Hoffman-La-Roche, Nutley, NJ, USA); 75% of HL-60 WT, respectively, after 5 days of culture a a a (Table 1, Figure 1a). The induction of differentiation was time 1,25(OH)2-24 ,26 ,27 -tri-homo-22,24-diene-D3 (code name EB 1089); and 20-epi-22-oxa-24a,26a,27a-tri-homo- and concentration dependent, with up to 85% of HL-60 WT a cells becoming NBT-positive after exposure to 10−7 m RA for 1 ,25-(OH)2-D3 (code name KH 1060) (kindly provided by Dr L Binderup, Leo Pharmaceutical Products, Ballerup, Denmark) 7 days (data not shown). The RXR-selective ligand LG1069 −6 m were dissolved in ethanol. The 9-cis RA and LG1069 were (10 , 5 days) did not induce differentiation of HL-60 WT dissolved in 50% ethanol, 50% DMSO. cells (2% NBT-positive HL-60 WT cells). The 1,25(OH)2D3, another seco-steroid, and several of its analogs were also examined either alone or in combination with either 9-cis RA or RA (Table 1, Figure 1). The most potent analog was Clonogenic assay in soft agar KH 1060 (10−7 m, 5 days), which resulted in 99% NBT-posi- tive HL-60 WT; and the combination of 1,25(OH)2D3 and 9- cis RA was the most potent combination to produce differen- HL-60 (2 × 103) cells were cultured in a two-layer soft agar tiation of HL-60 WT (Table 1). In contrast, none of these com- system for 10 days according to previously described pounds, either alone or in combination with RA or 9-cis RA methods.37 induced significant differentiation (,7%) of HL-60 R down either the granulocytic or monocytic pathways (Table 1, Figure 1B).