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1 Department of Hematology and Oncology, Clinical Sciences for Pathological Organs, Graduate School of Medicine, Kyoto University, Kyoto, Japan; 2 Frontier Research Program, Institute of Chemical and Physical Research, RIKEN, Saitama, Japan; and 3 Department of Dermatology, School of Medicine, University of California and Dermatology Service and Research Unit, Department of Veterans Affairs Medical Center, San Francisco, California
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The sphingolipid ceramide is recognized as a proapoptotic lipid mediator because a diverse array of cell stresses, including anti-Fas antibody cross-linking, tumor necrosis factor 
, irradiation, heat shock and anticancer drugs, increases intracellular ceramide during the execution phase of apoptosis (3, 4, 5, 6, 7)
. Cellular ceramide levels are known to be regulated by both ceramide-generating enzymes, such as ceramide synthase and sphingomyelinase, and ceramide-metabolizing enzymes, such as glucosylceramide synthase (GCS; EC 2.4.1.80; Ref. 8
), sphingomyelin synthase (9)
, and ceramidase (10)
. Among the anticancer drugs, daunorubicin caused elevation of de novo ceramide synthesis in murine leukemia P388 cells and human monoblastic leukemia U-937 cells (11)
. Similarly, 1-ß-D-arabinofuranosylcytosine increased ceramide in human leukemia HL-60 cells, but it did so through an activation of neutral sphingomyelinase (12)
. Tumor necrosis factor was reported to elevate ceramide levels by activating sphingomyelinase through p55 tumor necrosis factor receptor-associated FADD and TRADD components in 293 cells (13)
.
In contrast to these ceramide-generating systems, activation of ceramide-metabolizing enzymes can attenuate apoptosis by decreasing intracellular ceramide content (14) . For example, the conversion of ceramide to glucosylceramide by GCS has a role in anthracycline resistance of breast cancer (MCF-7/ADR) cells (15) . Restoration of doxorubicin (DOX) susceptibility by antisense-induced reduction of GCS activity in MCF-7/ADR cells revealed a critical role for GCS in apoptosis (16 , 17) . Our prior studies demonstrated that GCS activity is higher than sphingomyelinase activity in drug-resistant HL-60/ADR cells and that the apoptotic effects of sphingomyelinase-generated ceramide are attenuated by the elevation of GCS activity in these cells (18 , 19) . In fact, we recently showed not only that steady-state GCS activity was higher in DOX-resistant HL-60/ADR cells, which are myeloid derived and p53-null, than in their DOX-sensitive (HL-60) counterparts but also that GCS overexpression induced resistance to DOX in HL-60 cells (19) . Consistent with these in vitro studies, blast cells obtained from refractory leukemia patients show significantly higher GCS activities than those from chemosensitive leukemia patients, suggesting a clinical relevance for the ceramide-to-glucosylceramide conversion (19) . Thus, the regulation of ceramide content through GCS seems to be closely associated with DOX-induced cell death in leukemia cells, both in vitro and in vivo.
Increase of ceramide, either by treatment with short-chain ceramide species or by bacterial sphingomyelinase, induced up-regulation of GCS mRNA levels in B16 murine melanoma cells and in human HaCaT keratinocytes (17 , 20 , 21) . In addition, GCS overexpression decreased apoptosis in human hepatoma Huh 7 cells (17 , 20) and facilitated the growth of HaCaT cells (21) . However, the mechanism by which cells override/suppress increases in intracellular ceramide through GCS in response to DOX is unknown.
Our recent studies also demonstrated that a GC-rich, putative Sp1 transcription factor binding element in the 5' GCS promoter region is important for regulating GCS expression in both murine B16 melanoma cells (8) and normal human keratinocytes.4 Although DNA binding activity of Sp1 transcription factor was reported to increase in DOX-resistant HL-60 cells (22) , the role of Sp1 activation in resistance to apoptotic cell death in HL-60/ADR leukemia cells is also unresolved.
Therefore, we examined here whether activation of Sp1 is involved in attenuation of ceramide levels through transcriptional changes of GCS in HL-60/ADR cells. It is reported that DOX induces Sp1 activation and subsequently increases GCS mRNA and activity levels in HL-60/ADR cells, but not in HL-60 cells. In addition, double-stranded decoy oligodeoxynucleotides (ODNs) containing a GC-rich, putative Sp1 sequence (Sp1 decoy ODNs) increase DOX-induced ceramide levels through transcriptional inhibition of GCS function and suppress DOX-dependent apoptotic cell death in HL-60/ADR cells. Thus, it is suggested that transcriptional up-regulation of GCS through Sp1 activation is one potential mechanism to inhibit DOX-induced increase of ceramide and cell death in HL-60/ADR cells.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-32P]ATP and L-[14C]serine were purchased from Amersham Pharmacia Biotech (Uppsala, Sweden). L-[3H]serine was from American Radiolabeled Chemical, Inc. (St. Louis, MO). Unless indicated otherwise, the remaining chemicals were obtained from Sigma (St. Louis, MO)
Assay for Cell Viability and Apoptosis.
Apoptotic cells were assessed both by bis-benzimide (Hoechst 33258) and by trypan blue dye exclusion. Morphological changes of nuclear chromatin in apoptotic cells were assessed by staining with the DNA-binding fluorochrome Hoechst 33258 (Sigma), as described previously (24)
. Briefly, cells were fixed with 3% paraformaldehyde in PBS for 10 minutes at room temperature, followed by washing with PBS. Cells were then incubated with Hoechst 33258 (16 µg/ml). Approximately 200 cells chosen at random were assessed for chromosomal condensation using fluorescence microscopy (AxioPlan 2; Carl Zeiss, Hallobergmoos, Germany). In addition, because cells become permeable to trypan blue during the later phases of apoptosis, we also assessed viable cell number and viability by counting the number of trypan blue-positive cells after 0.025% trypan blue dye staining. Briefly, we cultured cells in three separate dishes (n = 3) for each experiment. For each dish, five separate hemacytometer readings (1 mm2) were performed, and the average viable cell number and viability (the total cell numbers were adjusted to approximately 30–50 cells for each reading) were calculated. The average cell number (Fig. 1A and B
; Fig. 6B
) and viability (Fig. 6A)
from the three separate culture dishes are reported.
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Ceramide Measurement.
Lipids were extracted by the method of Bligh and Dyer (25)
, and ceramide mass was measured using the diacylglycerol kinase assay as described previously (26
, 27)
. The solvent system to separate ceramide 1-phosphate and phosphatidic acid was chloroform/acetone/methanol/acetic acid/H20 (10:4:3:2:1, v/v). For labeling, cells were washed with PBS, seeded at 5 x 105 cells/ml in 2% fetal calf serum (FCS)-RPMI 1640 with L-[14C]serine (0.1 µCi/ml), and incubated at 37°C in 5% CO2 for 36 hours. Labeled cells were treated with DOX as described above and incubated at 37°C in 5% CO2 for 2 hours, and lipids were extracted as described above. The extracted samples were dried under N2 gas and dissolved in 100 µl of chloroform. Twenty µl were applied on Silica Gel 60 thin-layer chromatography (TLC) plates (Merck, Darmstadt, Germany), and 40 µl were used to measure phospholipid phosphate. TLC plates were developed with solvent containing chloroform/methanol/2N NH4OH (60:35:5, v/v), and spots corresponding to [14C]ceramide were counted using the BAS system (Fuji Film, Tokyo, Japan).
For the radiolabeling of ceramide, HL-60/ADR cells were first incubated with L-[3H]serine (1 µCi/ml) for 24 hours and transfected with Sp1 decoy ODNs or mock ODNs (as described below), followed by incubation with DOX for 24 hours. Lipid extracts were prepared from cellular homogenates as described above (26) , and separation of individual lipid species was achieved by high-performance TLC (21) . Radioactive counts in each lipid fraction were determined by using scintillation spectrometry.
Glucosylceramide Synthase Assay.
The assay for GCS was performed as described previously (21)
. Briefly, cells were lysed in buffer [20 mmol/L Tris-HCl (pH 7.5), 10 mmol/L EGTA, 2 mmol/L EDTA, 1 mmol/L phenylmethylsulfonyl fluoride, and 2.5 µg/ml leupeptin] by passing them through a 27-gauge needle; complete cell lysis was confirmed by microscopy. The lysate was centrifuged at 800 x g for 5 minutes at 4°C, and the supernatant was collected and used as the enzyme source. Protein concentrations were determined using the protein assay kit (Bio-Rad, Hercules, CA). Fifty µg of protein were mixed in buffer [250 µmol/L UDP-glucose, 5 mmol/L Tris-HCl (pH 7.5), 500 µmol/L EDTA, 10 µg/ml 6-(CN-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoyl)sphingosine-ceramide, and 100 µg/ml phosphatidylcholine; total, 100 µl] and incubated at 37°C for 2 hours. The reaction was terminated by the addition of 400 µl of H2O and 1 ml of chloroform/methanol (2:1, v/v), mixed well, and centrifuged. The lower phase was collected, and the solvent evaporated. Aliquots from resuspended extracts were applied to TLC plates (Merck), and glucosylceramide was resolved using a solvent system containing chloroform/methanol/12 mM MgCl2 in H2O (65:25:4, v/v). 6-(CN-(7- nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoyl)sphingosine-lipids were quantitated by fluorometry (excitation = 475 nm, emission = 525 nm).
Western Blot Analysis.
Sample aliquots (50 µg) were denatured by boiling in Laemmli’s sample buffer for 5 minutes, subjected to SDS-PAGE using a 7.5% running gel, and electroblotted to Immobilon-P transfer membrane (Millipore, Bedford, MA) as described previously (7)
. Nonspecific binding was blocked by incubation of the membrane with PBS containing 5% skim milk and 0.1% Tween 20 for more than 1 hour. The membrane was washed twice in PBS containing 0.1% Tween 20 (PBS-T) and incubated with a 1:200 dilution of anti-GCS (28)
antibody (in PBS-T buffer for 1 hour) for detection of GCS. The membrane was then washed twice in PBS-T buffer and incubated with a 1:4,000 dilution of antimouse or antirat immunoglobulin peroxidase conjugate, as appropriate. After three additional washes (5 minutes each) in PBS-T buffer, detection was performed using enhanced chemiluminescence Western blotting detection reagents (Amersham Pharmacia Biotech, Piscataway, NJ) according to the manufacturer’s protocol (4)
.
Northern Blot Analysis.
Total cellular RNA was isolated using ISOGEN (Nippon Gene, Tokyo, Japan) according to the manufacturer’s protocol. Northern blotting was performed as described previously (29)
. GCS cDNA was prepared from pCG-2 plasmid digested with EcoRI and HindIII (Stratagene, La Jolla, CA). Briefly, GCS or ß-actin probe was mixed with 1 µl of 10x protruding end kinase buffer and 5 µl of [32P]ATP, incubated at room temperature for 2 hours, and then purified twice through Sephadex G-50 spin columns. Hybridizations were performed at 42°C for 24 hours, and the membranes were subsequently washed in 2x SSC/0.1% SDS (1x SSC, 0.15 mol/L NaCl, and 15 mmol/L sodium citrate) at room temperature for 30 minutes and at 50°C for 15 minutes. The membranes were exposed to Fuji X-ray film with intensifying screens at –25°C for 2 days. Equal loading of RNA was confirmed by ß-actin mRNA signal and methylene blue staining of the membrane.
Nuclear Extracts and Gel Mobility Shift Assays.
HL-60 cell nuclear extracts were prepared from 5 x 106 cells using CelLytic NuCLEAR Extraction Kit (Sigma) according to the manufacturer’s protocols. Nuclear extracts were quantitated by the Bradford assay (Bio-Rad) and stored at –80°C. The protein concentration of each sample was diluted to be 7 mg/ml. Gel mobility shift assay was performed using Gel Shift Assay System (Promega, Madison, WI). Briefly, Sp1 consensus and mutant oligonucleotides (5'-ATTCGATCGGGGCGGGGCGAGC-3' and 5'-ATTCGATCGGTTCGGGGCGAGC-3', respectively) were end-labeled by filling in with Klenow DNA polymerase and [-32P]CTP. Approximately 1 ng of the labeled probe was mixed with 15 µg of nuclear protein in 10 µl of the binding buffer [10 mmol/L Tris-HCl (pH 7.5), 50 mmol/L NaCl, 0.5 mmol/L EDTA, 0.5 mmol/L dithiothreitol, 1 mmol/L MgCl2, and 4% glycerol] containing 0.05 mg/ml poly(deoxyinosinic-deoxyceptidylic). After incubation for 20 minutes at room temperature, the reaction mixture was separated on a 6% nondenaturing polyacrylamide gel with 0.5x TBE buffer (40 mmol/L Tris borate, 1 mmol/L EDTA). The gel was dried and subjected to autoradiography. For competition experiments, the excess of unlabeled competitor DNA was added 10 minutes before the addition of labeled probe as specified.
Transduction with GC-Rich/Sp1 Decoy Oligodeoxynucleotides.
Phosphorothioated double-stranded ODNs (decoy ODNs) containing the sequence of the GCS putative Sp1/GC-rich binding site (5'-ATTCCGGGGGCGGGGGCATG-3') and mock ODNs (5'-CATGCCATCGCTACCGGGGC-3'; Biomolecular Resource Center, University of California San Francisco, San Francisco, CA) were transfected (final concentration, 0.5 µmol/L) into HL-60/ADR cells suspended in 2.5% FCS-RPMI 1640 using TfxTM-50 reagent (Promega) for 18 hours. After transfection, cells were incubated with 10% FCS-RPMI for 6 hours and then treated with DOX.
Luciferase Assay.
The GCS promoter region was cloned from a murine genomic DNA library and inserted upstream of a luciferase reporter gene in pGL3-Basic vector (Promega), as described previously (8)
. HL-60/ADR cells were transfected with 15 µg of GCS promoter-luciferase reporter, 3 µg of Renilla luciferase-thymidine kinase-vector (Promega), and Sp1 decoy ODNs or mock ODNs for 18 hours, as described above. Cells were then incubated with DOX for 18 hours. Promoter activity was assessed using the Dual Luciferase Assay System (Promega) and a luminometer (GEM Biomedical, Inc., Hamden, CT).
Quantitative Reverse Transcription-PCR Analysis and Enzyme Activity for Glucosylceramide Synthase after Transfection of Sp1 Mock or Decoy Oligodeoxynucleotides.
HL-60/ADR cells, transfected with Sp1 decoy ODNs or mock ODNs as described above, were incubated with DOX for 18 hours. Total RNA was prepared as described previously (21)
. cDNA was prepared using SuperScriptTM II RNaseH-Reverse Transcriptase RNA (Invitrogen, Carlsbad, CA), and then 10 ng of cDNA were mixed with sets of primer pairs (final concentration, 200 pmol/L) and SYBR Green PCR mix containing Taq DNA polymerase and SYBR Green I dye (Applied Biosystems, Foster City, CA). The primers used were as follows: human GCS, 5'-TGCTCAGTACATTGCCGAAGC-3' and 5'-AACCTCCAAACGTTACAGGT-3'; and 18S rRNA, 5'-CGGCTACCACATCCAAGGAA-3' and 5'-GCTGGAATTACCGCGGCT-3'. The thermal cycling conditions were as follows: 50°C for 2 minutes, 95°C for 10 minutes, 95°C for 15 seconds, and 60°C for 1 minute; repeated 40 times on ABI Prism 7700 (Applied Biosystems). GCS RNA levels were normalized to 18S rRNA expression. GCS activity after transfection was assessed by [3H]galactose incorporation into glucosylceramide in cultured cells as described previously (21)
. Briefly, HL-60/ADR cells transduced with Sp1 decoy ODNs or mock ODNs were cultured with DOX for 18 hours and then incubated with [3H]galactose during the final 3 hours. Lipids were extracted and analyzed as described above.
Analysis of Statistical Significance.
Results shown in this work represent the mean values ± SD of three independent experiments. Significance was determined using Student’s t test.
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RESULTS |
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. As anticipated, treatment with 0.1 µmol/L DOX inhibited the growth of HL-60 cells. After 48 hours, a 90% decrease in cell number was detected as compared with the untreated control (Fig. 1A
; P < 0.01), and the percentages of apoptotic cell death assessed by PI staining (Fig. 1C)
and annexin V binding (Fig. 1D)
increased from 4.9% and 2.4% to 19.5% and 35.1%, respectively. Conversely, the number of viable HL-60/ADR cells was inhibited by 0.1 µmol/L DOX but not diminished to less than the initial concentration of 2.5 x 105 cells/ml (Fig. 1A and B)
. In addition, minimal apoptosis was evident in HL-60/ADR cells at 48 hours after 0.1 µmol/L DOX (i.e., PI staining, from 2.8% to 4.5%; annexin V binding, from 6.5% to 11.2%; Fig. 1C and D
). Moreover, although a dose-dependent inhibition of cell growth was detected in both HL-60 cells and HL-60/ADR cells when compared with controls at the same time point, the HL-60/ADR cells showed significantly decreased sensitivity to increasing DOX concentrations (Fig. 1B)
; e.g., the viable cell number of HL-60/ADR cells 24 hours after the highest concentration of DOX (0.35 µmol/L) was equivalent to that of the untreated initial controls. In addition, the results of PI staining analysis in Fig. 1C
showed that DOX induced cell cycle arrest at G2-M phase, but not apoptosis, in HL-60/ADR cells. These results suggest that although DOX-induced inhibition of cell growth is evident in both HL-60 and HL-60/ADR cells, the HL-60/ADR cells showed a cytostatic reaction in response to DOX and were more resistant to DOX-induced apoptosis than HL-60 cells.
Increased Intracellular Ceramide in HL-60 Cells, but Not in HL-60/ADR Cells, after Doxorubicin Treatment.
Baseline ceramide content, as determined by the diacylglycerol kinase assay (see Materials and Methods), was 7.3 ± 0.4 and 5.9 ± 0.5 pmol/nmol phospholipid in HL-60 and HL-60/ADR cells, respectively (data not shown). Although ceramide levels were not increased in HL-60 cells during the first 24 hours after treatment with 0.1 µmol/L DOX, significantly increased ceramide levels were evident at both 36 and 48 hours, i.e., 1.8- and 3.1-fold increase versus HL-60/ADR cells (P < 0.01 each), respectively (Fig. 2A)
. Increased ceramide became evident by 24 hours when HL-60 cells were treated with higher concentrations of DOX (i.e., 0.35 and 0.7 µmol/L; Fig. 2B
). In contrast, a faint decrease in ceramide levels was observed at any time point in HL-60/ADR cells after equivalent DOX treatment (0.1 and 0.35 µmol/L; Fig. 2A
) and after treatment with even higher concentrations of DOX (0.05–0.7 µmol/L; Fig. 2B
). In addition, when cells were prelabeled with [14C]serine and then treated with 0.35 µmol/L DOX for 24 hours, the [14C]serine-labeled ceramide levels were increased 1.5-fold in HL-60 cells over that in HL-60/ADR cells (data not shown). These results reveal that the DOX-induced increase of ceramide in HL-60 cells is not evident in HL-60/ADR cells, suggesting that the lack of ceramide accumulation after DOX treatment may be related to the diminished apoptotic response in HL-60/ADR cells.
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. After DOX treatment (0.1 µmol/L) of HL-60/ADR cells, maximum elevation of GCS mRNA, protein, and activity levels was detected at 12 (3.5-fold), 24 (2.3-fold), and 24 hours (1.8-fold), respectively, as compared with untreated control (Fig. 3C and D)
. In contrast, in HL-60 cells, the levels of GCS mRNA, protein, and activity were not altered at any time point in response to equivalent DOX treatment (Fig. 3C and D)
. Moreover, the DOX-induced increases of GCS mRNA, protein, and activity were dose dependent in HL-60/ADR cells, whereas no increases were detected in HL-60 cells even at a higher concentration of DOX (0.35 µmol/L; Fig. 3E–H
). For example, treatment of HL-60/ADR cells with 0.35 µmol/L DOX for 24 hours increased GCS mRNA, protein, and activity levels to approximately 2.3-, 1.9-, and 1.7-fold of control levels, respectively. These results reveal that HL-60/ADR cells up-regulate the levels of GCS mRNA, protein, and activity in response to DOX, suggesting that increased GCS function may account, at least in part, for the attenuation of ceramide levels in DOX-treated HL-60/ADR cells.
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B, and AP-2 binding sites (8)
. Previous and recent promoter-deletion studies in human cells have revealed a GC-rich region containing putative Sp1 binding elements/domains to be important for both baseline and inducible GCS promoter activity (8)
.4
Given that increased DNA-Sp1 binding ability has been linked to DOX resistance (22)
, we sought to determine whether Sp1 transcription factors might be involved in the DOX-mediated changes in GCS expression and ceramide content in HL-60/ADR cells. To do so, we first confirmed that GCS promoter activity was affected by DOX. As shown in Fig. 4
, Sp1 transcription factors were increased in the nucleus after treatment with 0.35 µmol/L DOX in HL-60/ADR cells, but not in HL-60 cells, as assessed by gel shift assay. The baseline level of Sp1 was also higher in HL-60/ADR cells than in HL-60 cells (Fig. 4A)
. Specificity of Sp1 binding ability was confirmed by a dilution and replacement experiment using unlabeled Sp1 and mutant Sp1 ODNs (Fig. 4B)
. Overloading of cold Sp1 ODNs diminished Sp1 binding ability, but mutant Sp1 ODNs did not affect it.
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; P < 0.01). Note that the presence of the mock ODNs (used as a control for the decoy ODN studies) did not alter the GCS promoter response to DOX. We next confirmed whether this putative Sp1 response element plays a role in the DOX-induced activation of GCS promoter by using double-stranded decoy ODNs containing this putative Sp1 sequence ("Sp1 decoy ODNs"). Although transfection of HL-60/ADR cells with Sp1 decoy ODNs did not alter the baseline GCS promoter-luciferase activity, it completely attenuated the DOX-induced GCS promoter activation (Fig. 4C)
. These results suggest that DOX-induced activation of the GCS promoter is regulated via transcription factor(s) binding to the GC-rich, Sp1 response element/domain, whereas baseline GCS promoter activity may be not primarily Sp1 dependent.
Inhibition by Sp1 Decoy Oligodeoxynucleotides of Doxorubicin-Induced Increases in Glucosylceramide Synthase mRNA and Activity and Ceramide Levels.
We next examined whether the Sp1 decoy ODN attenuation of GCS promoter activation by DOX would lead to equivalent changes in GCS mRNA expression and activity, as well as affect ceramide content in the HL-60 drug-resistant and -sensitive cells. Consistent with the promoter activation results (Fig. 4)
, quantitative real-time PCR revealed GCS mRNA levels to be significantly increased in DOX- versus vehicle-treated HL-60/ADR cells (i.e., 130% eighteen hours after DOX treatment using mock ODN-transfected control cells; Fig. 5A
; P < 0.02). However, the DOX-induced elevation of GCS mRNA levels was significantly abrogated by transfection with Sp1 decoy ODNs (Fig. 6A
; P < 0.01). Similarly, GCS activity also was increased by DOX treatment in mock ODN-transfected cells (Fig. 6B
; P < 0.02), but this elevation was significantly attenuated in Sp1 decoy ODN-transfected cells (Fig. 5B
; P < 0.01). Conversely, ceramide levels were not altered in mock ODN-transfected HL-60/ADR cells, whereas they increased to 135% of control by DOX treatment (24 h) in Sp1 decoy ODN-transfected cells (Fig. 5C
; P < 0.01). These results suggest that activation of GCS promoter via a putative Sp1/GC-rich element may be an important mechanism by which GCS expression and activity are regulated in the presence of DOX in HL-60/ADR cells. The resultant lack of ceramide accumulation in these DOX-resistant cells suggests that GCS up-regulation represents at least one mechanism by which these cells protect against anthracycline toxicity.
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; P < 0.01), although, as shown in Fig. 1A and B
, the number of HL-60/ADR cells was not decreased to less than the initial cell number after 48 hours of treatment with 0.35 µmol/L DOX. This result suggests that the increase in DOX-induced cell death may be due to an inhibition of GCS activation by Sp1-mediated events. To determine whether DOX-induced increase of cell death by Sp1 decoy ODN is due to apoptosis, we examined whether the resultant morphological changes corresponded to apoptosis. The percentage of apoptotic cells was significantly increased to 36% in Sp1 decoy ODN-transfected cells treated with DOX as compared with 14% apoptotic cells in mock ODN-transfected cells after 46 hours of treatment (data not shown). Thus, inhibition of GCS activation at the transcriptional level by Sp1 decoy ODNs and the subsequent inhibition of GCS activity and corresponding increase of ceramide levels (compare Figs. 4
and 5
) suggest that GCS activation via Sp1 and ceramide reduction are correlated to the inhibition of DOX-induced apoptosis in HL-60/ADR cells. |
DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In the present study, treatment of drug-sensitive HL-60 cells with DOX induced both significant apoptosis and a parallel increase in ceramide content, whereas DOX-induced responses in cell death were lacking in apoptosis-resistant HL-60/ADR cells. Similar to drug-resistant MCF-7/AdrR cells (34
, 35)
, basal GCS levels also were significantly higher in HL-60/ADR cells than HL-60 cells. However, distinct from MCF-7/AdrR cells (34)
, HL-60/ADR cells up-regulate the levels of GCS mRNA, protein, and enzyme activity in response to DOX (Fig. 3)
. Because HL-60/ADR cells seemed to be arrested in G2-M phase and subsequently showed a decrease of cell growth without inducing apoptosis (Fig. 1C and D)
, inhibition of ceramide increase through up-regulation of GCS may be involved in cytostatic avoidance of cell death induced in drug-resistant cells.
Although the mouse GCS promoter was originally described by Ichikawa et al. (8)
, and it was shown that the GCS 5'-promoter region of mouse lacks classic TATA and CAAT boxes, numerous putative regulatory elements were evident, including five GC-rich Sp1 sites. Among the recognized transcription factor-binding sites, which include AhR, NF-B, AP-2, and GATA-1 motifs, the GC-rich, putative Sp1 elements appeared most important for GCS promoter activation by the results of a luciferase assay using deletion mutants of the GCS 5'-promoter region (8)
.4
The human GCS promoter has yet to be characterized. As long as we searched upstream of the GCS gene within –1 kb to know the difference between human and murine GCS promoter sequences, the key Sp1 sequence, but not other transcription factors such as AP1, NF-B, and CAP, is only conserved in both the human and murine promoter regions. Therefore, we used murine promoter region containing Sp1 sites and investigated whether Sp1 is a critical factor in DOX-mediated GCS up-regulation in human HL/60ADR cells. Our data reveal that the Sp1 decoy attenuates not only GCS promoter activation but also GCS mRNA expression, GCS activity, and cell growth in HL-60/ADR cells treated with DOX (Figs. 4
5
6)
.
Based on the present studies and our prior work, it appears evident that Sp1 protein(s) represent important transcription factors for the regulation of GCS and the subsequent attenuation of ceramide increases (8
, 21)
. However, it is clear that we cannot exclude the possibility that other factor(s) also are involved in the transcriptional regulation of GCS. In fact, as we determined that Sp1 binding ability was increased in HL-60/ADR cells but not in HL-60 cells, whereas baseline levels of Sp1 activation were similar between these cell lines (Fig. 4A and B)
, and that Sp1 decoy ODNs alone did not alter basal GCS activity in HL-60/ADR cells, we speculate that other factor(s) [e.g., AP-2 and GATA-1 motifs (8)
] may be important for regulating basal GCS expression/activity, whereas activation of Sp1 might be more specifically involved in DOX-induced up-regulation of GCS.
Because decoy Sp1 ODNs increased DOX-induced apoptotic cell death in HL-60/ADR cells, it appears that the apoptosis-resistant cells can attenuate cell death, at least in part, through Sp1-dependent transcriptional up-regulation of GCS, with subsequent attenuation of DOX- induced ceramide levels. Recent studies further support an opposing relationship between Sp1 activity and cell death. For example, immortality against DOX-induced apoptosis in acute lymphoblastic leukemia cells involves MDM2 induction of the NF-B p65 subunit through Sp1 (35)
. In addition, Sp1 is involved in 12-O-tetradecanoylphorbol-13-acetate-induced resistance to methotrexate in Chinese hamster ovary cells (36)
. Both of these studies confirm a role for Sp1 activation in the inhibition of cell death. However, the mechanism by which Sp1 activation is involved in protection against apoptotic cell death from elevated ceramide has not been investigated. Given that the Sp1 decoy ODNs inhibited both DOX-increased GCS expression and activity and increased ceramide content and subsequent apoptosis in HL-60/ADR cells, the present studies reveal that DOX-induced Sp1 activation increases GCS activity and that GCS activation and ceramide reduction are negatively correlated with DOX-induced apoptotic cell death in human leukemia HL-60/ADR cells.
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FOOTNOTES |
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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 indicate this fact.
Note: Y. Hirabayashi is currently in the Laboratory for Memory and Learning, RIKEN Brain Science Institute, Saitama, Japan.
Requests for reprints: Toshiro Okazaki, Department of Hematology and Oncology, Clinical Sciences for Pathological Organs, Graduate School of Medicine, Kyoto University, 54 Shogoin-Kawaharacho, Sakyo-ku, Kyoto, Japan. Phone/Fax: 81-75-751-3154; E-mail: toshiroo{at}kuhp.kyoto-u.ac.jp
4 S. Murata, Y. Uchida, J. D. Lee, et al., Regulation of glucosylceramide synthase expression through Sp1-mediated promoter activation, submitted for publication. 
Received 5/23/03. Revised 5/21/04. Accepted 6/21/04.
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 Y.-Y. Liu, J. Y. Yu, D. Yin, G. A. Patwardhan, V. Gupta, Y. Hirabayashi, W. M. Holleran, A. E. Giuliano, S. M. Jazwinski, V. Gouaze-Andersson, et al. A role for ceramide in driving cancer cell resistance to doxorubicin FASEB J, July 1, 2008; 22(7): 2541 - 2551. [Abstract] [Full Text] [PDF]
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A. Charruyer, S. M. Bell, M. Kawano, S. Douangpanya, T.-Y. Yen, B. A. Macher, K. Kumagai, K. Hanada, W. M. Holleran, and Y. Uchida Decreased Ceramide Transport Protein (CERT) Function Alters Sphingomyelin Production following UVB Irradiation J. Biol. Chem., June 13, 2008; 283(24): 16682 - 16692. [Abstract] [Full Text] [PDF]
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