Characterization of two novel retinoic acid-resistant cell lines derived from HL-60 cells following long-term culture with all-trans-retinoic acid.

Either all-trans-retinoic acid (RA) or vitamin D3 (VD) induces differentiation of the myeloid leukemia cell line HL-60. RA is available for the treatment of acute promyeloleukemia, although the development of resistance to the agent is a serious problem for differentiation-inducing therapy. To approach the mechanisms of resistance to RA, we developed two novel cell lines, HL-60-R2 and R9, which were subcloned after exposure to increasing concentrations of RA. The growth rate of HL-60-R2 cells was significantly increased by RA treatment, whereas the growth rate of HL-60-R9 was not affected. RA induces apoptosis in the parental HL-60 cells. The number of apoptotic cells, however, was not increased and nitroblue tetrazolium (NBT) reduction was not altered by 1 microM RA in either of the cloned cell lines. Treatment with VD induced monocytic differentiation and increased the expression of CD11b in HL-60 and HL-60-R9 cells, but not in HL-60-R2 cells. Flow cytometric and G-banding analysis demonstrated that R2 cells were near-triploid. The sequencing analysis revealed a deletion of three nucleotides in the sequence of the RAR alpha gene in HL-60-R9 cells, resulting in deletion of codon 286. No mutation was found in HL-60-R2 cells. Taken together, these data indicate that the resistance to RA is caused by the mutation in RAR alpha of HL-60-R9, but by other factor(s), which also affect the VD-response pathways, in HL-60-R2. The abnormal response to VD may be associated with the abnormal ploidy of the R2 cells.     

土槿乙酸增强9顺式维甲酸对白血病细胞生长的抑制作用

目的观察过氧化物酶体激活物活化受体γ (PPARγ)的配体土槿乙酸(PLAB)联合维甲类X受体α (RXRα)的配体9-顺式维甲酸(9-cis RA)对白血病细胞生长的影响。方法应用MTT 法检测细胞生长抑制率,流式细胞术和Hoechst33342/PI染色分析HL-60细胞凋亡及细胞周期变化,RT-PCR检测PPARγ和RXRα mRNA表达。结果HL-60细胞系上有PPARγ和RXRα的表达,经配体联合作用后能明显抑制细胞生长,呈剂量依赖关系;细胞出现明显凋亡形态学改变和亚G₁峰;PPARγ和RXRα mRNA表达明显增强。结论PLAB能显著增强9-cis RA对HL-60细胞的生长抑制作用,其机制与凋亡诱导效应有关。     

IL-6 enhanced the retinoic acid-induced differentiation of human acute promyelocytic leukemia cells

It has been shown that retinoic acid (RA) induced the expression of interleukin-6 (IL-6) in human acute promyelocytic leukemia HL-60 cells. In the present study, we examined the ability of RA to induce the expression of gp130, the signal-transducing receptor component for IL-6, in HL-60 and a RA-supersensitive cell line HL-60/S4. We found that RA induced the expression of gp130, at both the mRNA and protein levels, in HL-60 and HL-60/S4 cells. Interestingly, the induction of gp 130 expression observed in the RA-supersensitive HL-60/S4 cells was much more pronounced than that observed in HL-60 cells. Furthermore, activation of the RA-induced gp130 by exogenous IL-6 potentiated the differentiating effects of RA. The synergistic effects observed for IL-6 and RA was also much stronger in HL-60/S4 cells than in HL-60 cells. Our findings suggest that the differentiating effects of RA may partially be mediated by the up-regulation of IL-6/gp130 signaling in HL-60 and HL-60/S4 cells.     

Characterization of Two Novel Retinoic Acid-resistant Cell Lines Derived from HL-60 Cells Following Long-term Culture with all-trans-Retinoic Acid

Either all-trans-retinoic acid (RA) or vitamin D3 (VD) induces differentiation of the myeloid leukemia cell line HL-60. RA is available for the treatment of acute promyeloleukemia, although the development of resistance to the agent is a serious problem for differentiation-inducing therapy. To approach the mechanisms of resistance to RA, we developed two novel cell lines, HL-60–R2 and R9, which were subcloned after exposure to increasing concentrations of RA. The growth rate of HL-60–R2 cells was significantly increased by RA treatment, whereas the growth rate of HL-60–R9 was not affected. RA induces apoptosis in the parental HL-60 cells. The number of apoptotic cells, however, was not increased and nitroblue tetrazolium (NBT) reduction was not altered by 1 μM RA in either of the cloned cell lines. Treatment with VD induced monocytic differentiation and increased the expression of CD11b in HL-60 and HL-60–R9 cells, but not in HL-60–R2 cells. Flow cytometric and G-banding analysis demonstrated that R2 cells were near-triploid. The sequencing analysis revealed a deletion of three nucleotides in the sequence of the RARα gene in HL-60–R9 cells, resulting in deletion of codon 286. No mutation was found in HL-60–R2 cells. Taken together, these data indicate that the resistance to RA is caused by the mutation in RARα of HL-60–R9, but by other factor(s), which also affect the VD-response pathways, in HL-60–R2. The abnormal response to VD may be associated with the abnormal ploidy of the R2 cells.     

Increasing the intracellular availability of all-trans retinoic acid in neuroblastoma cells

Recent data indicate that isomerisation to all-trans retinoic acid (ATRA) is the key mechanism underlying the favourable clinical properties of 13-cis retinoic acid (13cisRA) in the treatment of neuroblastoma. Retinoic acid (RA) metabolism is thought to contribute to resistance, and strategies to modulate this may increase the clinical efficacy of 13cisRA. The aim of this study was to test the hypothesis that retinoids, such as acitretin, which bind preferentially to cellular retinoic acid binding proteins (CRABPs), or specific inhibitors of the RA hydroxylase CYP26, such as R116010, can increase the intracellular availability of ATRA. Incubation of SH-SY5Y cells with acitretin (50 microM) or R116010 (1 or 10 microM) in combination with either 10 microM ATRA or 13cisRA induced a selective increase in intracellular levels of ATRA, while 13cisRA levels were unaffected. CRABP was induced in SH-SY5Y cells in response to RA. In contrast, acitretin had no significant effect on intracellular retinoid concentrations in those neuroblastoma cell lines that showed little or no induction of CRABP after RA treatment. Both ATRA and 13cisRA dramatically induced the expression of CYP26A1 in SH-SY5Y cells, and treatment with R116010, but not acitretin, potentiated the RA-induced expression of a reporter gene and CYP26A1. The response of neuroblastoma cells to R116010 was consistent with inhibition of CYP26, indicating that inhibition of RA metabolism may further optimise retinoid treatment in neuroblastoma.     

Expression of aberrant O-glycans attached to leukosialin in differentiation-deficient HL-60 cells

Promyelocytic leukemia HL-60 cells can be induced to differentiate into granulocytic cells by various agents including retinoic acid (RA), dimethyl sulfoxide, and 6-thioguanine (6-TG). Although the induced cells are no longer capable of proliferation, a few cells continue to divide in the presence of inducers, and these cells are resistant to terminal differentiation by these inducers (R. E. Gallagher, D. A. Giangiulio, C-S. Chang, C. J. Glover, and R. L. Felsted, Blood, 68: 1402-1406, 1986). The present study examined the structures of O-glycans attached to leukosialin, a major sialoglycoprotein in HL-60 cells, and the activities of glycosyltransferases involved in O-glycan synthesis. Leukosialin from RA-resistant and 6-TG-resistant HL-60 sublines migrated much more slowly than those from wild-type HL-60 cells when applied to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The dimethyl sulfoxide-resistant HL-60 subline, on the other hand, expressed leukosialin with a molecular weight similar to wild-type HL-60 cells. RA-resistant and 6-TG-resistant HL-60 cells were found to express a significant amount of tetrasaccharides that contain no sialic acid residue, while wild-type HL-60 cells expressed mainly disialosyl hexasaccharides and contained no detectable amount of asialo-oligosaccharides. Furthermore, wild-type HL-60 cells treated with the inducers for 4 days were found to express the same saccharides present in untreated wild-type HL-60 cells, indicating that the altered O-glycans present in RA and 6-TG sublines were not caused by a direct effect of these agents but rather are intrinsically unique to these sublines. To better understand the mechanisms underlying the differences in O-glycans, the activities of four sialyltransferases were measured: Gal beta 1----3GalNAc alpha 2----3sialyltransferase, Gal beta 1----4(3) GlcNAc alpha 2----3sialyltransferase, Gal beta 1----4GlcNAc alpha 2----6sialyltransferase, and GalNAc alpha 2----6sialyltransferase. Among them, Gal beta 1----3GalNAc alpha 2----3sialyltransferase and Gal beta 1----4(3)GlcNAc alpha 2----3sialyltransferase were much lower in the RA- or 6-TG-resistant HL-60 subline than in wild-type HL-60 cells. These findings indicate that the differences in O-glycans are due to the differences in alpha 2----3sialyltransferase activities. These results strongly suggest that O-glycans associated with leukosialin may play some role in HL-60 cell differentiation.     

Variable regulation of sensitivity to retinoic acid-induced differentiation in wild-type and retinoic acid-resistant HL-60 cells

The initial cell association and metabolic conversion of retinoic acid (RA) by HL-60 cells in serum-free, transferrin/insulin-supplemented, RPMI 1640 medium was greater than or equal to 10-fold greater than in RPMI 1640 medium containing 10% fetal bovine serum (FBS). This was paralleled under the serum-free conditions by 10-fold greater sensitivity to RA-induced differentiation, which was partially reversed by the addition of purified bovine serum albumin to the same concentration present in 10% FBS. In serum-free HL-1 medium, HL-60 cell sensitivity to RA-induced differentiation was approximately 250-fold less than in serum-free RPMI 1640 medium but, in this comparison, there was little difference in RA cell association or metabolism. A greater than 200-fold RA-resistant HL-60 subline had RA cell-association and metabolism rates similar to those of wild-type cells under all culture conditions. No significant qualitative differences in the high performance liquid chromatography elution patterns of polar metabolites were observed under any circumstances. These results indicate that inherent cellular properties, not associated with gross differences in RA uptake or metabolism, primarily determined the relative sensitivity or insensitivity of HL-60 cells to RA-induced differentiation but that RA responsiveness was markedly regulated by extracellular factors, one of which, serum albumin, appeared to act by decreasing the initial cell association and metabolism of RA, whereas other, as yet unidentified exogenous factors, may have acted independently of these functions.     

PPARγ和RXRα配体联用对白血病细胞生长作用机制的研究

 目的　研究过氧化物酶体增生物活化受体（PPARγ）和维甲类X受体（RXRα）在人类白血病细胞（HL-60、K562、U937）中的表达及其对配体作用的反应。方法　采用MTT法检测细胞生长;半定量RT-PCR法检测PPARγ和RXRαmRNA的表达。结果　PPARγ和RXRα在HL-60细胞中有较强表达,在K562、U937细胞中表达较弱。PPARγ的配体土槿乙酸（PLAB）能显著抑制3株细胞的生长,PLAB与RXRα的配体9-顺式维甲酸（9-cisRA）联合作用时,与PLAB单独作用比较,对受体高表达的HL-60细胞抑制作用增强较明显（P＜0.05）,但对K562、U937的作用增强不明显（P＞0.05）。PLAB和9-cisRA分别上调HL-60细胞PPARγ和RXRα的表达,且联合作用组显著高于单独作用组（P＜0.05）。结论　在HL-60、K562、U937细胞中,HL-60细胞PPARγ和RXRα表达最强,这对配体发挥作用可能是必需的,配体发挥的抑制细胞生长的效应可能与其介导的信号途径有关。     

Granulocytic differentiation induced by retinoic acid in HL-60 cells is associated with changes in adenylate cyclase activity

The effect of all-trans Retinoic Acid (RA) on the activity of membrane bound adenylate cyclase (AC) of human malignant cell lines (HL-60 and U-937) was studied. Granulocytic terminal differentiation of the HL-60 cells was correlated to an increase of AC activity and to a potentiation of guanosine 5' triphosphate (GTP) inhibitory effects on the enzyme activity. No direct in vitro effect of RA on HL-60 membranes was found. Monocytic terminal differentiation obtained on 1 B-D arabinofuranosyl cytosine (Ara-C) treated HL-60 cells, or on RA treated U-937 cells, did not modify AC activity. The results here reported suggest relations between the modification of GTP binding protein activity and RA induced granulocytic differentiation of malignant cells.     

Granulocyte colony-stimulating factor, not granulocyte-macrophage colony-stimulating factor, co-operates with retinoic acid on the induction of functional N-formyl-methionyl-phenylalanine receptors in HL-60 cells.

The interaction of colony-stimulating factors (CSF) and retinoic acid (RA) in the proliferation and differentiation of HL-60 cells was examined. Granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulated the proliferation of HL-60 cells in a dose-dependent manner at concentrations of 0.01-100 ng/ml; however, the proliferation due to GM-CSF was suppressed by 100 nM RA. Granulocyte colony-stimulating factor (G-CSF) slightly stimulated the proliferation of HL-60 cells at concentrations above 10 ng/ml. Neither G-CSF nor GM-CSF alone induced 12-o-tetra-decanoyl-phorbol-13-acetate (TPA)- or N-formyl-methionyl-phenylalanine (FMLP)-stimulated nitro-blue tetrazolium (NBT) reduction at concentrations of 0.01-100 ng/ml. G-CSF induced TPA- and FMLP-stimulated NBT reduction in the presence of 100 nM RA, but GM-CSF induced only TPA-stimulated NBT reduction. RA in addition to G-CSF synergistically increased FMLP binding to HL-60 cells, accompanied by increased NBT reduction in response to FMLP. RA in addition to GM-CSF markedly increased FMLP binding to HL-60 cells more than that induced by RA alone, but the combined treatment with RA and GM-CSF did not increase FMLP-stimulated NBT reduction more than that induced by RA alone. The results suggest that G-CSF stimulates RA-induced morphological and functional differentiation of HL-60 cells, but the differentiation-enhancing effects of GM-CSF are limited, whereas the growth-stimulating effect of GM-CSF on HL-60 cells is greater than that of G-CSF.     

Synergistic effects of clinically achievable concentrations of 12-O-tetradecanoylphorbol-13-acetate in combination with all-trans retinoic acid, 1alpha,25-dihydroxyvitamin D3, and sodium butyrate on differentiation in HL-60 cells.

Our recent studies demonstrated that 12-O-tetradecanoylphorbol-13-acetate (TPA) has pharmacological activity for the treatment of acute myelocytic leukemia patients. In the present study, we investigated the potential synergistic effect of all-trans retinoic acid (RA), 1alpha,25-dihydroxyvitamin D3 (VD3), and sodium butyrate (NaB) on TPA-induced differentiation in HL-60 human promyelocytic leukemia cells. The cells were treated once with these agents for 48 h or treated every 24 h for 96 h. Treatment of HL-60 cells once with TPA, RA, VD3, or NaB for 48 h resulted in concentration-dependent growth inhibition and cell differentiation. At clinically achievable concentrations, TPA (0.16 nM) increased the number of adherent cells and RA (0.1-1 microM) increased the number of nitroblue tetrazolium (NBT)-positive cells. The combinations of TPA (0.16 nM) with RA (0.1-1 microM), VD3 (1 nM), or NaB (100 microM) for 48 h synergistically increased differentiation as measured by the formation of adherent cells (P < or = 0.01). Moreover, cells treated with various combinations of low concentrations of TPA, RA, VD3, and NaB every 24 h for 96 h resulted in a further decrease in cell growth and an increase in differentiation. At clinically achievable concentrations, the strongest stimulation of differentiation was achieved in cells treated with a "cocktail" that combined TPA, RA, VD3, and NaB. The synergistic effect of combinations of TPA with RA or NaB at clinically effective concentrations on HL-60 cell differentiation suggests that the combination of these agents may improve the therapeutic efficacy of TPA for the treatment of acute promyelocytic leukemia (APL) patients. A differentiation "cocktail" that combines TPA, RA, VD3, and NaB may provide an even more effective strategy for improving the therapeutic efficacy of TPA and RA.     

Inhibition of 13-cis retinoic acid-induced gene expression of homeobox B7 by thalidomide

Thalidomide and 13-cis retinoic acid (RA) show anticancer effects as sole agents or in combination with other drugs. However, induction of homeobox (HOX) gene expression by 13-cis RA may contribute to tumor progression thereby potentially limiting its efficacy. The purpose was to test if thalidomide can inhibit 13-cis RA-induced HOXB7 expression and whether thalidomide may enhance the antiproliferative effect of 13-cis RA in U343MG glioblastoma cells. Quantitative real-time PCR showed significant inhibition of 13-cis RA-induced HOXB7 expression by thalidomide with IC(50) approximately 0.1-0.2 microg/ml when given simultaneously with 13-cis RA but not when administered 18 h later (p < 0.0001). 13-cis RA alone inhibited proliferation and colony formation in a concentration-dependent manner whereas growth inhibition by thalidomide alone at 5-100 microg/ml was constant at 80-90% of controls. At 10% serum concentration, growth inhibition by a combination of the 2 drugs was additive but at 1% serum, growth inhibition was synergistic. It is concluded that thalidomide inhibits the RA-induced HOXB7 expression in glioblastoma cells and that 13-cis RA/thalidomide combinations can in principle enhance cytotoxicity. The improved cell kill induced by thalidomide is attributed to downregulation of growth stimulatory factors induced by 13-cis RA. Implications for the modus operandi of thalidomide in embryogenesis are noted.     

Bioavailability and dose-dependent anti-tumour effects of 9-cis retinoic acid on human neuroblastoma xenografts in rat.

Neuroblastoma, the most common extracranial solid tumour in children, may undergo spontaneous differentiation or regression, but the majority of metastatic neuroblastomas have poor prognosis despite intensive treatment. Retinoic acid regulates growth and differentiation of neuroblastoma cells in vitro, and has shown activity against human neuroblastomas in vivo. The retinoid 9-cis RA has been reported to induce apoptosis in vitro, and to inhibit the growth of human neuroblastoma xenografts in vivo. However, at given dosage, the treatment with 9-cis RA caused significant toxic side effects. In the present study we investigated the bioavailability of 9-cis RA in rat. In addition, we compared two different dose schedules using 9-cis RA. We found that a lower dose of 9-cis RA (2 mg day(-1)) was non-toxic, but showed no significant effect on tumour growth. The bioavailability of 9-cis RA in rat was 11% and the elimination half-life (t1/2) was 35 min. Considering the short t1/2, we divided the toxic, but tumour growth effective dose 5 mg day(-1) into 2.5 mg p.o. twice daily. This treatment regimen showed no toxicity but only limited effect on tumour growth. Our results suggest that 9-cis RA may only have limited clinical significance for treatment of children with poor prognosis neuroblastoma.     

9-顺式维甲酸诱导胃癌MGC803细胞周期阻滞和凋亡的作用机制

背景与目的:9-顺式维甲酸(9-cisretinoicacid,9-cisRA)对胃癌的抗癌活性及机制尚不清楚,本研究以MGC803为靶细胞观察9-cisRA对胃癌的作用。方法:采用RT-PCR法检测维甲类X受体α(recinoidXreceptor!,RXRα)、细胞周期蛋白CyclinD1和细胞周期蛋白依赖性激酶CDK4mRNA表达,流式细胞术检测细胞周期,MTT实验检测9-cisRA作用MGC803细胞后生长抑制情况,Hoechst33342/PI双荧光染色和琼脂糖凝胶电泳检测凋亡,免疫细胞化学检测凋亡相关基因Bcl-2蛋白表达。结果:0.1～10μmol/L9-cisRA作用MGC803细胞96h,能显著抑制细胞增殖。10μmol/L9-cisRA分别作用48、72和96h,G1期细胞随着作用时间的延长而增加,呈明显的G1期阻滞。作用72h后,细胞出现核染色质凝集、DNA片段化等凋亡特征;从作用48h开始,Bcl-2表达即显著下降。在MGC803细胞中RXRα表达较弱,经10μmol/L9-cisRA作用48h其表达水平显著增加(P<0.01);作用96h,细胞中CyclinD1和CDK4表达显著降低(P<0.01)。结论:9-cisRA能明显诱导MGC803细胞周期G1期阻滞和凋亡,该作用可能与其下调细胞周期因子CyclinD1和CDK4表达有关。     

Inhibition of cell growth and telomerase activity of breast cancer cells in vitro by retinoic acids

The effects of retinoic acid (RA) and its analogs, all-trans RA, 9-cis RA and 13-cis RA, were investigated in human breast cancer MCF-7 cells and immortalized breast epithelial cell line MCF-10A. RA inhibited the telomerase activity of MCF-7 cells in a wide range of concentrations. RA at 10 microM also inhibited the growth of MCF-7 cells in a time-dependent manner. However, no significant growth inhibition was found between untreated control and RA-treated MCF-10A cells. Moreover, a marked inhibition of telomerase activity by RA was detected early in MCF-7 cells (after 24 h of RA treatment), which was preceded by a reduction of hTERT mRNA expression (after 12 h of RA treatment). However, MCF-10A cells showed a reduction of telomerase activity and down-regulation of hTERT after 4 days of RA treatment. Simultaneous changes in hTERT mRNA expression and telomerase activity were found for MCF-10A cells. The expressions of hTR and hTEP1 telomerase component genes were not changed after RA treatment. These results indicate that the anti-breast cancer activity of RA could be mediated by its ability to down-regulate the expression of hTERT telomerase gene.     

Changes in actin and actin-binding proteins during the differentiation of HL-60 leukemia cells.

Actin and actin-binding proteins form a peripheral network on the cytosolic side of the plasma membrane. These cytoskeleton proteins are involved in functions that require cellular movement and may also have a role in modulating signal transduction during cellular proliferation and differentiation. To measure changes in F-actin and actin-binding proteins during HL-60 differentiation, cells were induced to mature along the granulocytic pathway by exposure to 1 microM retinoic acid (RA) for 5 days and were analyzed for F-actin and actin-binding proteins by flow cytometry. The amounts of F-actin and spectrin in untreated HL-60 cells and in those undergoing differentiation by treatment with the retinoid did not differ. N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)-phallacidin was used to measure F-actin content and a monoclonal antibody followed by fluorescence isothiocyanate-conjugated goat anti-mouse immunoglobulin antibody was used to measure the content of spectrin; cells were analyzed by flow cytometry. In contrast, cells exposed to RA contained larger amounts of alpha-actinin, vinculin, talin, lipocortin I, and lipocortin II, as determined with their respective antibodies followed by flow cytometric analysis as described above. An RA-supersensitive clone of HL-60, designated HL-60/S4, exhibited lower constitutive levels of alpha-actinin, vinculin, and talin but a higher constitutive level of lipocortin II than parental cells. Treatment of HL-60/S4 with RA led to increases in vinculin, talin, lipocortin I, and lipocortin II. An RA-resistant clone, designated HL-60/R3, constitutively expressed larger amounts of alpha-actinin, vinculin, lipocortin I, and lipocortin II than parental HL-60 cells. Treatment of HL-60/R3 with RA resulted in decreases in the amounts of these actin-binding proteins. Changes in actin-binding proteins that occur during the differentiation of HL-60 cells suggest that these proteins may be of importance to the expression of the mature phenotype.     

The effects of microtubule disrupting drugs on the differentiation of HL-60 leukemia cells.

We and others have previously shown that microtubules (MT) are stained more intensely and are organized differently in differentiating leukemia cells. To study the effects of the MT disrupting drugs, colchicine (Coln) and vincristine (VCR), on the maturation process, HL-60 leukemia cells were pretreated with Coln or VCR for 1 h and then exposed to either retinoic acid (RA) or dimethyl sulfoxide (DMSO). Neither Coln nor VCR induced the differentiation of HL-60 cells, but in combination with RA increased the percentage of nitroblue tetrazolium-positive cells, the expression of the mature myelocyte surface marker Mo 1, and the content of MT over the effects produced by RA alone. In contrast, pretreatment with Coln or VCR delayed the commitment to a differentiation pathway induced by DMSO. The supra-additivity exhibited between Coln and RA required the administration of Coln prior to RA; thus, Coln had no effect when given two days after the cellular exposure to RA. The findings suggest that (a) a combination of non-cytotoxic concentrations of Coln or VCR with RA may have clinical utility as inducers of leukemia cell maturation, and (b) MT may be involved in modulating signal transduction during the initiation of HL-60 cell differentiation.     

Upregulation of Bfl-1/A1 in leukemia cells undergoing differentiation by all-trans retinoic acid treatment attenuates chemotherapeutic agent-induced apoptosis.

All-trans retinoic acid (ATRA) induces differentiation of NB4 and HL-60 leukemia cells, but not R4 and HL-60/Res cells. Three agents used in cancer therapy, doxorubicin (Dox), arsenic trioxide (As(2)O(3)) and paclitaxel, induce apoptosis, but not differentiation, in all of these cell lines. The induction of apoptosis by these agents is decreased in ATRA-pretreated NB4 and HL-60 cells, but not in ATRA-pretreated R4 and HL-60/Res cells. The level of Bcl-2 protein is decreased by ATRA treatment in NB4, HL-60 and HL-60/Res cells. The level of Mcl-1 protein is increased by ATRA treatment in NB4 and R4 cells, but not in HL-60 and HL-60/Res cells. Bfl-1/A1 mRNA is not expressed in these cell lines, however, its expression is markedly induced by ATRA treatment in NB4 and HL-60 cells, but not in R4 or HL-60/Res cells, which correlates with inhibition of apoptosis. Inhibiting Bfl-1/A1 mRNA upregulation in ATRA-pretreated NB4 cells using small interfering RNA (siRNA) partly recovers cell sensitivity to Dox-induced apoptosis. These data demonstrate that ATRA induction of Bfl-1/A1 in differentiated NB4 and HL-60 cells contributes to a loss of sensitivity to chemotherapy-induced apoptosis.