Human HL-60 cells were obtained from American Type Culture Collection (Rockville, MD, USA) and cultured in RPMI-1640 medium supplemented with heat-inactivated 10% fetal bovine serum (Omega Scientific, Tarzana, CA, USA) and antibiotics at 37°C in a humidified 5% CO₂ incubator. To maintain exponential growth, cells were seeded at a concentration of 1×10⁵ cells/ml and sub-cultured every 3–4 days. ATRA, PA, SC and phorbol 12-myristate 13-acetate were from Sigma (St. Louis, MO, USA). HE and l-asparaginase (l-ASNase) were purchased from Enzo Life Sciences (Farmingdale, NY, USA) and Aviva Systems Biology (San Diego, CA, USA), respectively.

HL-60 cells at a concentration of 1.5×10⁵/ml were cultured for 72 h in the presence of ATRA and/or sesquiterpene lactones and l-ASNase. At the end of treatment, the cells were harvested by a centrifugation and incubated in PBS buffer containing 0.1% NBT (USB, Cleveland, OH, USA) and 200 ng/ml PMA for 1 h to allow the cells to form a blue-black nitroblue formazan. The differentiation-positive cells were accessed under a light microscope. At least 200 cells were counted for each culture sample, and the results were expressed as a relative percentage of NBT-positive cells to total cells.

At the end of culture, cells were collected, washed with ice-cold PBS buffer and labeled with PE-conjugated CD11b monoclonal antibody (BD Bioscience, San Jose, CA, USA) at room temperature for 15 min. Fluorescent intensity was analyzed by flow cytometric measurement using BD FACSCalibur.

HL-60 cells were treated with 50 nM ATRA alone or combination with HE, PA, or SC for 24 h. Total RNA from the cultures were isolated using TRIzol reagent (MRC, Cincinnati, OH, USA). For DNA microarray assay, fluorescence-labeled cDNA probes were obtained from 30 μg of total RNA by using SuperScript II reverse transcriptase (Gibco BRL) in a total reaction volume of 30 μl and applied to human 8.5K cDNA microarrays. The sample from untreated HL-60 cells was used as a reference for each chip assay. The experimental and analytical procedures were done as previously described (27).

The cDNA was obtained from 1 to 1.5 μg of total RNA by the RocketScript RT kit (Bioneer, Daejeon, Korea). The RT product (1 μl) was applied to each PCR reaction with the following primer sets: asparagine synthetase (ASNS; forward, 5′-acagaaggattggctgcctt-3′; reverse, 5′-cctctcactctcctcctcgg-3′), activating transcription factor 4 (ATF4); forward, 5′-aacagcaaggaggatgcctt-3′; reverse, 5′-gtgctgaggagaccccagat-3′), ATF5 (forward, 5′-ttggatactctggacttgct-3′; reverse, 5′-tccttgacgtactggatctc-3′) and β-actin (forward, 5′-agcgggaaatcgtgcgtg-3′; reverse, 5′-cagggtacatggtggtgcc-3′). The final products were analyzed on a 1.2% agarose gel with ethidium bromide staining.

The results were obtained from at least three-independent experiments. Statistical significance of the data was determined using a paired Student’s t-test. A P-value <0.05 was considered statistically significant.

To confirm the effects of STL compounds on ATRA-induced leukemia cell differentiation, HL-60 cells were treated with one of three different STLs, helenalin (HE), parthenolide (PA) or sclareolide (SC), with or without a suboptimal concentration dose (50 nM) of ATRA. As shown in Fig. 1A, HE itself induced the differentiation of HL-60 cells. Both HE and PA strongly enhanced the effect of ATRA in inducing the differentiation, but SC did not. Similarly, the surface expression of CD11b, a marker antigen of general myeloid differentiation, was increased in the cells by combination treatment of ATRA with either HE or PA (Fig. 1B).

To investigate the mechanism by which ATRA-induced HL-60 cell differentiation was enhanced by combination treatment with HE or PA, we used cDNA microarray analyses of cells treated with 50 nM ATRA alone or during co-treatment with HE, PA or SC. Microarray analyses showed that treatment with ATRA alone resulted in transcriptional changes of 111 genes, compared with the basal levels in unstimulated cells. As expected, co-treatment with ATRA and STLs resulted in transcriptional changes in greater number of genes, allowing alterations in an increased number of genes including the gene observed in ATRA-treated HL-60 cells (Fig. 2A). Furthermore, the number of genes influenced by the STLs (152 by PA, 257 by HE and 140 by SC) seems to reflect the degree by which the STL enhanced differentiation, implying that the enhancing potential of an STL in ATRA-induced HL-60 cell differentiation depends on the participation of these extra genes.

To further investigate how HE and PA, but not SC, enhance ATRA-induced granulocytic differentiation of HL-60 cells, we sought a common set of genes that were influenced in both the cells treated with HE and with PA, but not in cells treated with ATRA alone or co-treated cells with ATRA and SC. As shown in Fig. 2B, 48 genes satisfied the criteria and they are summarized in Table I. Twenty-four of these genes, including phosphogluconate dehydrogenase (PGD) and platelet factor 4 (PF4), were upregulated, whereas the remaining 20, including ASNS and ATF5, were downregulated.

Based on the genome-wide profiles from cDNA chip analysis, we chose ASNS as a target gene for further study in relation to the differentiation of HL-60 cells. We firstly validated the STL-induced change in ASNS mRNA expression using RT-PCR. As shown in Fig. 3, ASNS expression was downregulated in the cells treated with HE or PA alone, as well as in the cells co-treated with ATRA, whereas no difference was found between cells treated with SC and untreated cells. We also determined the levels of two transcription factors, ATF4 and ATF5, which are known to regulate transcription of ASNS (28,29). In our microarray analysis, like ASNS, both genes were also suppressed by HE and PA when combined with ATRA (Table I). This finding suggests that the ASNS may play a role in the differentiation of HL-60 cells.

It is well known that the protein encoded by ASNS plays an important role in asparagine synthesis. Therefore, to investigate the effect of lower ASNS expression on ATRA-induced cell differentiation, we attempted to create a similar condition by using l-asparaginase (l-ASNase). Treatment with either a suboptimal dose of ATRA (50 nM) or l-ASNase (0.1 U/ml) had little effect on the expression of CD11b on the cell surface, while a combination of both the drugs increased the expression of this antigen (Fig. 4A). The combined effect of ATRA and l-ASNase was confirmed by a NBT reduction assay (Fig. 4B). On examining nuclear morphology by Giemsa staining, we also observed a slight increase in the cytoplasm to nucleus ratio and that the nuclei were multilobed after treatment with ATRA and l-ASNase (Fig. 4C). Additionally, we examined whether depletion of asparagine could enhance differentiation in HL-60 cells treated with both ATRA and HE. To address this aim, HL-60 cells were treated with ATRA and a lower dose of HE (0.3 μM) in the presence or absence of l-ASNase. As shown in Fig. 4D and E, the addition of l-ASNase to the combination of ATRA and HE strongly increased the number of NBT-positive cells and the levels of the CD11b expression on the cell surface.