Affiliations of authors: B. J. Maurer, C. P. Reynolds, Division of Hematology-Oncology, Childrens Hospital Los Angeles and Department of Pediatrics, University of Southern California Keck School of Medicine, Los Angeles; L. Melton, C. Billups, Division of Hematology-Oncology, Childrens Hospital Los Angeles; M. C. Cabot, The John Wayne Cancer Institute at Saint John's Health Center, Santa Monica, CA.
Correspondence to: C. Patrick Reynolds, M.D., Ph.D., Division of Hematology-Oncology, MS #57, Childrens Hospital Los Angeles, 4650 Sunset Blvd., Los Angeles, CA 90027 (e-mail: preynolds{at}chla.usc.edu).
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ABSTRACT |
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INTRODUCTION |
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Ceramide, generated in separate cellular compartments by de novo synthesis or sphingomyelin breakdown, can increase ROS, promote apoptosis or necrosis, including p53-independent apoptosis under hypoxic conditions, activate pro-death JNK/stress-activated protein kinase (JNK/SAPK) in a manner opposed by sphingosine-1-phosphate activation of extracellular signal-regulated kinases (ERK1/2), and may induce Bcl2 dephosphorylation (1215). Ceramide can be metabolized to less toxic forms by glucosylation via glucosylceramide synthase (16) and by acylation via 1-O-acylceramide synthase (17). The cytotoxicity of exogenous ceramide and sphingomyelinase is potentiated by sphingoid bases (18) and by inhibitors of ceramide glycosylation (19) and opposed by ceramide activation of protein kinase C zeta (PKC- ) (20), possibly by stimulating sphingosine kinase activity (21), by increasing NF-kappaB (12,22), or by other mechanisms (23,24). A schematic of the pathways of ceramide synthesis and relevant enzymes is shown in Fig. 1
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MATERIALS AND METHODS |
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4-HPR was provided by R. W. Johnson Pharmaceuticals, Spring House, PA. Safingol and PPMP were purchased from Matreya, Inc., Pleasant Gap, PA. Thin-layer chromatography (TLC)-grade organic solvents, -thioglycerol, Hanks' balanced salt solution (HBSS), L-cycloserine, and fumonisin B1 were from Sigma Chemical Co., St. Louis, MO. Granulocytemonocyte colony-stimulating factor (GM-CSF) (Sagromostim) was from Immunex Corp., Seattle, WA. Ecolume scintillation cocktail was from ICN Biomedicals, Costa Mesa, CA. [9,10-3H(N)]Palmitic acid (50 Ci/mmol) was from Dupont NEN® Research Products, Boston, MA. Iscove's modified Dulbecco's medium (IMDM) was from BioWhittaker, Walkersville, MD. RPMI-1640 medium, Ham's F-12K medium, fetal bovine serum (FBS), and L-glutamine were from Gemini BioProducts, Calabasas, CA. Bacto agar was from DIFCO, Becton, Dickinson and Co., Franklin Lake, NJ. ITSTM Premix culture supplement (insulin, transferrin, and selenious acid) was from Collaborative Biomedical Products, Bedford, MA. Glucosylceramide (purified from Gaucher's spleen) was from Matreya, Inc., Pleasant Gap, PA. Other lipid standards were from Avanti Polar Lipids, Inc., Alabaster, AL. Uniplate Silica Gel G 250 micron TLC plates were from Analtech, Inc., Newark, DE. Specialty gases and gas mixtures were from Praxair, North Hollywood, CA. Modular incubation chambers were from Billups-Rothenberg, Del Mar, CA. Falcon Brand plasticware was from DIFCO Becton, Dickinson and Co. 4-HPR and safingol were dissolved in 95% ethanol, and PPMP was dissolved in 50% ethanol; they were stored at -20 °C. Tamoxifen citrate was freshly prepared in saline.
Cell Culture
The human neuroblastoma cell lines CHLA-79 (31), CHLA-90 (31), and CHLA-171 (32) were established from a relapsed tumor in bone marrow after myeloablative chemoradiotherapy supported by autologous bone marrow transplant (CHLA-79 and CHLA-90) or from a relapsed tumor following treatment with buthionine sulfoximine (BSO) plus melphalan (CHLA-171) and were maintained in IMDM supplemented with 0.7 mM L-glutamate, insulin, and transferrin (5 µg/mL each), selenium (5 ng/mL), and 20% heat-inactivated FBS (complete IMDM). Cultures of normal human fibroblasts CRL-2076 and CRL-2091 were maintained in complete IMDM. Pancreatic cancer cell lines PANC-1 and Hs 766T were maintained in Dulbecco's modified Eagle medium High Glucose medium and 10% heat-inactivated FBS. The colon cancer cell line LoVo, the prostate cancer cell line PC-3, and the lung carcinoma cell line A-549 were maintained in Ham's F-12K medium with 10% heat-inactivated FBS and 2 mM glutamate. Other neuroblastoma (31,33,34) and non-neuroblastoma human cell lines were maintained in RPMI-1640 medium supplemented with 10% heat-inactivated FBS without antibiotics (complete medium). Other non-neuroblastoma cell lines were purchased from the American Type Culture Collection, Manassas, VA, except DoxR MCF7, a doxorubicin-resistant MCF-7 breast cancer cell line derivative (35), which was a gift from Drs. Kenneth Cowan and Merrill Goldsmith (National Cancer Institute, Bethesda, MD), and the MCF-7/tet cell line, which is MCF-7 transfected with a doxycycline-controlled reverse Tet repressor construct (36). Cell lines were cultured at 37 °C in a humidified incubator in an atmosphere of 95% room air plus 5% CO2. Neuroblastoma cells were detached without trypsin from culture plates with the use of a modified Puck's solution A plus EDTA (Puck's EDTA) that contains 140 mM NaCl, 5 mM KCl, 5.5 mM glucose, 4 mM NaHCO3, 0.8 mM EDTA, 13 µM Phenol Red, and 9 mM HEPES buffer (pH 7.3) (34). Non-neuroblastoma cells were detached with Puck's EDTA plus 0.25% trypsin.
For cytotoxicity assays (described below) in reduced oxygen conditions, cells were prepared and treated with drugs as described previously (11). Under these conditions, medium in microplate wells attains a pO2 below the degree of hypoxia found in bone marrow (37) and in the range of hypoxia found in tumor tissue (38).
Cytotoxicity Assay
Cytotoxicity in cell lines was determined with the use of a fluorescence-based assay employing digital imaging microscopy (DIMSCAN) (11). DIMSCAN quantitates viable cells that selectively accumulate fluorescein diacetate and is capable of measuring cytotoxicity over a 4- to 5-log dynamic range by quantifying total fluorescence per well (which is proportional to viable, clonogenic cells) after eliminating background fluorescence with the use of digital thresholding and eosin Y quenching. Cell lines were seeded into 96-well plates in 100 µL of complete medium per well as described previously (11). Cells were allowed to attach overnight prior to the addition of drugs in 50-µL volumes of complete medium to various final drug concentrations in replicates of 12 wells per concentration. The effects of fumonisin B1 on cytotoxicity were assessed by preincubation of the cells with 90 µM fumonisin B1 for 16 hours before the addition of other drugs. The effects of L-cycloserine on cytotoxicity were assessed by preincubation of the cells with 2 mM L-cycloserine for 3 hours before the addition of other drugs. Control wells received ethanol (final concentration = 0.12%0.20%) in complete medium equivalent to the maximum final ethanol concentration of drug-treated wells. Plates were assayed 47 days after the initiation of drug exposure, depending on the growth properties of each cell line, to allow for maximum cell death and outgrowth of surviving cells. To measure cytotoxicity, we added fluorescein diacetate (stock solution of 1 mg/mL in dimethyl sulfoxide) in 50 µL of complete medium per well, to a final concentration of 10 µg/mL. The plates were incubated for an additional 1530 minutes at 37 °C, and then 30 µL of eosin Y (0.5% in normal saline) was added per well. The total fluorescence of each well was then measured with the use of DIMSCAN as described previously (39). For L-cycloserine treatment in the MCF-7/tet cell line, the cytotoxic effects of 4-HPR and 4-HPR/safingol are expressed as the increase in cytotoxicity above that produced by treatment with L-cycloserine alone; i.e., cytotoxic results are normalized for the cytotoxicity found in controls treated with only L-cycloserine.
The results were analyzed and expressed as survival fractions by a comparison of the quantified fluorescence of surviving cells to that of control cells with the use of Microsoft Excel 97© software (Microsoft Corp., Redmond, WA) and were graphed with the use of SigmaPlot 5.0 (Jandell Scientific, San Rafael, CA). The limits of detection were determined by the estimated number of cells present in control wells at the time that they were treated with drug, generally targeted at 10 x 103 cells, for a detection limit for cytotoxicity of one live cell at the end of the assay per 10 000 starting cells. For many cell lines, the presence of more than 10 x 103 cells on day +0 resulted in overgrowth of the control wells by the day of assay, although for slower growing cell lines, such as SMS-LHN, a greater detection limit was possible. For simplicity, all results are graphed with a detection limit of 10-4. The stability of 4-HPR, safingol, PPMP, L-cycloserine, tamoxifen, and fumonisin B1 for 47 days in the DIMSCAN cytotoxicity assay system is not known.
The cytotoxicity of drugs to human bone marrow myeloid (granulocytes and monocytes) progenitor cells was analyzed by granulocytemonocyte colony-forming unit (GM-CFU) assay. Bone marrow was diluted 1 : 1 with HBSS, and red blood cells were removed by centrifugation over Ficoll (375g for 15 minutes at room temperature in a Damon/IEC model CRU-5000 centrifuge; International Equipment Corp., Needham Heights, MA). The mononuclear cell layer was removed and washed two times in HBSS and resuspended in IMDM plus 10% heat-inactivated FBS. Aliquots (50 µL) were removed for viable cell counts with the use of trypan blue dye and turks counting (turks = 3% acetic acid and methylene blue, to lyse residual red blood cells) at 1 : 10 dilutions. Cells (10 x 106) were resuspended in 5 mL of IMDM plus 20% FBS medium in replicate T25 flasks, drug or ethanol (controls) was added to final concentrations, and flasks were incubated at 37 °C in humidified 20% oxygen plus 5% CO2 (day 0). At 24-hour intervals, the flasks were agitated, and 1 mL of medium was removed. The cells were harvested by centrifugation at 375g for 3 minutes at room temperature and resuspended in 1 mL of IMDM with 10% human serum albumin. An aliquot (0.4 mL) of resuspended cells was mixed with 0.2 mL of 8 x 103 µg/mL of GM-CSF and 3.4 mL of the following agar mix: 1.2x Iscove's medium, 0.5% bacto agar, 20% fetal calf serum, 1.6 mM L-glutamine, 100 U/mL penicillin/streptomycin, and 0.9 µM -thioglycerol at approximately 50 °C. Aliquots (1 mL) of the cellagar mix were plated in 35 x 10-mm tissue culture dishes in triplicate and incubated for 12 days at 37 °C in humidified 20% oxygen plus 5% CO2. Colonies of 50 cells or more were scored, and the results were expressed as colonies per 2 x 105 cells of the initial cell inoculum placed in the flask on day 0.
Lipid Analysis
Methods were described previously (11). Briefly, tritium-labeled lipids were prepared by [3H]palmitic acid exposure of cultured cells. Total cellular lipids were extracted as described previously (11). Commercial lipid standards (5 µg/lane) were co-spotted onto TLC plates with the cellular 3H-labeled lipids (10-µL aliquots). Ceramide was resolved in a solvent system containing chloroform/acetic acid (90 : 10, vol/vol). Glucosylceramide was resolved in a solvent system containing chloroform/methanol/ammonium hydroxide (70 : 20 : 4, vol/vol). The lipid standards were visualized by iodine vapor, and we assayed the comigrating tritiated lipid sample by scraping the TLC plate in the area of interest, adding 0.5 mL water and 4.5 mL of Ecolume scintillation cocktail, vortexing, and measuring the counts per minute of tritium by liquid scintillation counting. This value was then corrected for the amount of the original sample previously removed for other assays. Lipid changes are expressed as the mean -fold increase or decrease in three drug-treated samples as compared with that of three matched controls. For some assays, a sphinganine N-acyltransferase (dihydroceramide synthase) inhibitor (40), fumonisin B1, was added to 90 µM at 16 hours before the addition of other drugs, or a serine palmitoyltransferase inhibitor (41), L-cycloserine, was added to 2 mM 3 hours before the addition of other drugs. Drugs did not affect [3H]palmitic acid uptake. The ceramide isolated by the above assay method has been verified by mass spectroscopy to contain only ceramide species.
Statistical Analysis
Cytotoxicity and lipid data are presented as means ± 95% confidence intervals. The 95% confidence interval was calculated as 1.96/
n, where
= the standard deviation of ungrouped data and n = the number of trials. The statistical significance of differences in means was evaluated by the unpaired, two-sided Student's t test with the use of Microsoft© Excel 97 software. All P values are two-sided.
Drug-induced cytotoxic synergy was analyzed and graphed with the use of CalcuSyn software from Biosoft, Cambridge, U.K. and was expressed as the combination index at the LC99 (i.e., concentration lethal to 99% of cells) (42). The combination index is a method for quantifying drug cytotoxic synergism, based on the Mass-Action Law approach and the Median-Effect Principle derived from enzyme kinetic models as developed by Chou and Talalay, which has been widely used to evaluate interactions of antineoplastic drugs (43). With the use of the combination index, synergism is defined as a more than expected additive effect, and antagonism is defined as a less than expected additive effect. By this method, a combination index equal to 1 indicates an additive effect, a combination index less than 1 indicates synergy, and a combination index greater than 1 indicates antagonism. With the use of CalcuSyn software, synergy is further refined as synergism (combination index = 0.30.7), strong synergism (combination index = 0.10.3), and very strong synergism (combination index <0.1) (42). The combination index (CIN) value can be calculated at different "effect levels" or "fraction affected" levels (e.g., LC50 [i.e., concentration lethal to 50% of the cells] or LC99) and may vary depending on the fractional effect level at which it is calculated. The mutually nonexclusive assumption was used in these analyses. For combination index plots, the combination index is plotted as the log10 (CIN) versus fraction affected (defined as 1 survival fraction), and the 95% confidence intervals are shown where calculable, with the use of the algebraic approximation method of the CalcuSyn program. On these plots, additivity is, therefore, defined as log10 (CIN) = 0; very strong synergy is defined as log10 (CIN) = -1; and antagonism is defined as log10 (CIN) >0. Note: While synergism and cytotoxicity may be related, a combination index indicating strong synergy does not necessarily imply a high degree of absolute cytotoxicity (i.e., a small survival fraction); conversely, a combination index indicating antagonism need not exclude a high degree of cytotoxicity.
All results were experimentally reproducible.
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RESULTS |
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Safingol (14 µM) was combined with 4-HPR (312 µM) at a 3 : 1 molar ratio (4-HPR : safingol). Safingol synergized 4-HPR cytotoxicity in six neuroblastoma cell lines (LC99, combination index [CIN] values <0.1), which resulted in 3.54 logs of cell killing, including in those cell lines highly resistant to alkylating agents and etoposide (Fig. 2, A; Table 1
). Safingol plus 4-HPR (4-HPR/safingol) synergized and/or caused multilog cell killing in four lung cancer cell lines (Fig. 2, B
; Table 1
; LC99, combination index value range = <0.10.2); in two melanoma cell lines (Fig. 3, A
; Table 1
; LC99, combination index values = <0.1 and 0.2); in two prostate cancer cell lines, including one p53 null (Fig. 3, B
; Table 1
; LC99, combination index values = <0.1 and 1.0); in two colon cancer cell lines, including one p53 mutant (Fig. 3, C
; Table 1
; LC99, combination index values = 0.1 and 0.3); and in one of two pancreatic cancer cell lines (Fig. 3, E
; Table 1
; LC99, combination index value = 0.2). Safingol also synergized 4-HPR cytotoxicity in two breast cancer cell lines or their derivatives (Fig. 3, D
; Table 1
; LC99, combination index value range = 0.20.5). 4-HPR/safingol retained multilog cytotoxicity (P<.001) compared with controls in the SMS-LHN (Fig. 4, A
) and CHLA-90 (data not shown) neuroblastoma cell lines, when assayed in 2.5% oxygen. Conditions of reduced oxygen have been shown previously to reduce 4-HPR single-agent cytotoxicity in these cell lines (11).
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4-HPR/safingol was minimally cytotoxic to CRL-2076 normal human fibroblasts (Fig. 4, B). The addition of safingol (4 µM) did not affect the cytotoxicity of 4-HPR (12 µM) (P = .32), but safingol (
6 µM) increased the cytotoxicity (P<.001) at +96 hours. Similar results were observed in CRL-2091 normal human fibroblasts (data not shown). 4-HPR/safingol (9 µM/3 µM) did not cause significant cytotoxicity to bone marrow myeloid progenitors (GM-CFU) at +72 hours (P = .89; Fig. 4, C
) compared with control cells.
Effects of 4-HPR and Inhibitors of De Novo Ceramide Synthesis on Ceramide Levels
As reported previously (11), 4-HPR (10 µM) progressively increased radiolabeled ceramide levels fourfold to fivefold at +24 hours in CHLA-90 neuroblastoma cells (Fig. 5, B [P<.001]; Fig. 5, C
[P = .004]; right panel of Fig. 6, A
[P = .002]). The increase in ceramide levels preceded morphologic evidence of cytotoxicity (11). To determine the synthetic route of the ceramide increased by 4-HPR in CHLA-90 cells, we assayed the effects of two inhibitors of de novo ceramide synthesis. L-Cycloserine (2 mM) (44) is an inhibitor of serine palmitoyltransferase, the initial and rate-limiting step of de novo ceramide synthesis (45). Fumonisin B1 (90 µM), is an inhibitor of (dihydro)ceramide synthase (40). Both inhibitors prevented (P<.001) 4-HPR-induced ceramide increases at +6 hours (Fig. 5, A
). Neither drug affected the cellular uptake of [3H]palmitic acid (data not shown). The effect of more prolonged exposure of L-cycloserine on ceramide increase could not be ascertained because it proved to be too cytotoxic at +24 hours in CHLA-90 cells (not shown). However, fumonisin B1 prevented ceramide increases at +24 hours (P = .002) and at +48 hours (P<.001) (Fig. 5, B
). Together, these results strongly suggest that the 4-HPR-mediated ceramide increase was from de novo synthesis, although these data do not exclude the possibility that 4-HPR might also increase ceramide levels by decreasing its degradation into sphingosine or its conversion into sphingomyelin or glucosylceramides. However, in CHLA-90 and SMS-LHN neuroblastoma cells, 4-HPR (10 µM) increased tritium-labeled glucosylceramide levels approximately 3.8-fold (P<.001) and approximately 2.5-fold (P = .003) and sphingomyelin levels approximately threefold (P = .06) and approximately 2.8-fold (P = .006), respectively, over controls at +24 hours, indicating that neither glucosylceramide synthase nor sphingomyelin synthesis was inhibited by 4-HPR (right panel of Fig. 6, A
, and data not shown).
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Safingol Incorporated Into a Stereochemical Variant of Ceramide (cer*)
Safingol, the L-threo-isomer of native sphinganine (D-erythro-dihydrosphingosine) (Fig. 1) has been reported to be incorporated into a stereochemically variant ceramide (here termed "cer*") via acylation by (dihydro)ceramide synthase in rat liver in vivo (46,47). To confirm this, we assayed for ceramide species after safingol exposure and determined the effects of L-cycloserine (2 mM) and fumonisin B1 (90 µM) on levels of ceramide species. No drug affected the cellular uptake of [3H]palmitic acid (data not shown). Safingol (3 µM) increased the level of a ceramide species (cer*) ninefold at +6 hours (P = .002) (Fig. 5, A
) and approximately 17-fold at +24 hours (P = .004) (Fig. 5, C
) in CHLA-90 cells; however, significantly, glucosylceramide levels remained unchanged at +24 hours (P = .84) (Fig. 5, C
). The increase in ceramide species (cer*) was unaffected by L-cycloserine at +6 hours (P = .8) but was blocked completely by fumonisin B1 (P = .002) (Fig. 5, A
); taken together, these findings strongly suggest that radiolabeling was via acylation of tritiated palmitoyl-coenzyme A by (dihydro)ceramide synthase into an abundant dihydrosphingosine species that was not derived from de novo synthesis (i.e., safingol). Thus, the ceramide species increased by safingol probably represented acylated-safingol (cer*), the L-threo-stereochemical analogue of native ceramide (D-erythro-N-acylsphingosine). The accompanying lack of an increase in glucosylceramide levels (Fig. 5, C
) suggests that cer* is not readily glucosylated by glucosylceramide synthase, unlike the ceramide induced by 4-HPR (P<.001) (Fig. 5, C
; right panel of Fig. 6, A
). Combining safingol with 4-HPR (4-HPR/safingol) produced an apparent additive increase in total ceramide species (P = .12) (Fig. 5, C
) at +24 hours compared with safingol alone, presumably representing the summed production of native ceramide induced by 4-HPR and of safingol-derived, cer* (indistinguishable by this assay method). Glucosylceramide levels increased (P = .09) (Fig 5, C
) with 4-HPR/safingol only to the same extent (P = .73) as with 4-HPR alone (Fig. 5, C
), again suggesting that cer* cannot be glycosylated by glucosylceramide synthase. L-Cycloserine only somewhat decreased the total ceramide species increase induced by 4-HPR/safingol (P = .07) (Fig. 5, A
), presumably reflecting the decrease in the smaller amount of native ceramide induced by 4-HPR, without an effect on the greater incorporation of safingol into cer*. Fumonisin B1 prevented the ceramide species increase induced by 4-HPR/safingol (P = .004) (Fig. 5, A
), consistent with the interpretation that inhibiting (dihydro)ceramide synthase prevents both the increase in native ceramide levels induced by 4-HPR via de novo synthesis and the incorporation of safingol into cer*. It is interesting that, in the 4-HPR-resistant SK-N-RA cell line (Fig. 2, A
), 4-HPR (10 µM) increased ceramide levels only approximately 3.5-fold (P = .004) and safingol (3 µM) increased cer* levels only approximately sixfold (P = .001) at +24 hours (data not shown), suggesting that decreased production or increased degradation of ceramide and cer* may occur in this cell line. Since 4-HPR-induced ceramide levels increased in a time-dependent manner (11), the cumulative effects of decreased production or increased degradation may account for the decreased 4-HPR sensitivity of this cell line compared with other neuroblastoma cell lines when cytotoxicity was assayed at +5 days (see the "Methods" section). Of the cell lines tested, 4-HPR/safingol-insensitive normal fibroblasts (Fig. 4, B
) demonstrated the least increase in 4-HPR-stimulated ceramide levels, an approximately 50% increase (P = .03) at +24 hours (Fig. 5, D
), safingol only increased the levels of cer* approximately 4.5-fold (P<.001) (Fig. 5, D
), and 4-HPR/safingol did not increase the levels of ceramide species compared with safingol alone (P = .38) (Fig. 5, D
), further suggesting a relationship between induced ceramide levels and 4-HPR and 4-HPR/safingol sensitivity.
Effects of Glucosylceramide Synthase Inhibitors
Glucosylceramide synthase converts ceramide into nontoxic glucosylceramide (Fig. 1). Overexpression of glucosylceramide synthase decreases ceramide levels and increases cellular resistance after doxorubicin exposure (36). At +24 hours, 4-HPR (10 µM) increased ceramide levels in CHLA-90 cells approximately 5.5-fold (P = .002) and glucosylceramide levels approximately 3.5-fold (P = .01) (right panel of Fig. 6, A
). The glucosylceramide synthase/1-O-acylceramide transferase inhibitor PPMP (28,48) at 6 µM reduced glucosylceramide formation (P = .009) below control levels (P = .01) and increased ceramide levels (P = .05) compared with 4-HPR alone at +24 hours in CHLA-90 cells (right panel of Fig 6, A
), whereas 2 µM tamoxifena weaker, less specific glucosylceramide synthase inhibitor (30,49)reduced glucosylceramide levels (P = .02) to a lesser extent and did not increase ceramide levels (P = .52) (right panel of Fig. 6, A
). PPMP (315 µM) synergized 4-HPR cytotoxicity (1 : 1 molar ratio) in CHLA-90 cells (LC99, combination index value = 0.1) (left panel of Fig. 6, A
), but tamoxifen did not (data not shown). In 4-HPR-resistant SK-N-RA cells, 4-HPR (10 µM) increased (P = .03) ceramide levels only approximately fourfold at +24 hours, while PPMP (10 µM)/4-HPR (10 µM) increased (P = .005) ceramide levels approximately 15-fold at +24 hours (data not shown), demonstrating that large amounts of 4-HPR-induced ceramide were shunted to nontoxic glucosylceramide and/or 1-O-acylceramide, possibly accounting for 4-HPR resistance in this cell line. Both PPMP and tamoxifen (210 µM) synergized 4-HPR cytotoxicity (1 : 1 molar ratio) in SK-N-RA cells (LC99, combination index values <0.1) (left and right panels of Fig. 6, B
). PPMP (315 µM) also synergized 4-HPR cytotoxicity (1 : 1 molar ratio) in A375 melanoma cells (LC99, combination index value <0.1) and in MCF-7 breast cancer cells (LC99, combination index value = 0.2) (data not shown). Lower doses of PPMP (14 µM) also synergized multilog 4-HPR/safingol cytotoxicity (4-HPR/safingol/PPMP at 3 : 1 : 1 molar ratios) in SK-N-RA neuroblastoma cells (LC99, combination index value = 0.4) (left panel of Fig. 6, C
), in CHLA-79 neuroblastoma cells (LC99, combination index value = 0.1), and in MDA-MB-231 breast cancer cells (LC99, combination index value = 0.1) but resulted in only additive cytotoxicity in CHLA-90 neuroblastoma cells (LC99, combination index value = 1.0) (data not shown). Lower doses of tamoxifen (14 µM) synergized multilog 4-HPR/safingol cytotoxicity (4-HPR/safingol/tamoxifen at 3 : 1 : 1 molar ratios) in some cell lines, such as SK-N-RA neuroblastoma cells (LC99, combination index value = 0.2) and HT-29 colon cells (LC99, combination index value = 0.1) (center and right panels, respectively, of Fig. 6, C
), but not in all cell lines, including CHLA-90 (data not shown). The addition of safingol/tamoxifen (1 : 1 ratio) (13 µM) did not increase (P = .82) 4-HPR cytotoxicity in CRL-2091 normal human fibroblasts at +96 hours (Fig. 6, D
); however, safingol/tamoxifen (1 : 1 ratio) (4 µM) slightly increased 4-HPR (12 µM) cytotoxicity in an additive manner at +96 hours (survival fraction = 0.78 versus 0.64; P<.001; LC99, combination index value = 1.0) (Fig. 6, D
). Similar results were observed in CRL-2076 normal human fibroblasts (data not shown).
Effects of Inhibitors of De Novo Ceramide Synthesis on Cytotoxicity
To directly demonstrate the extent to which de novo ceramide production contributed to 4-HPR and 4-HPR/safingol cytotoxicity, we attempted to determine the effects of inhibitors of de novo ceramide synthesis on cytotoxicity. However, prolonged exposures (>16 hours) to L-cycloserine or -chloro-L-alanine, inhibitors of serine palmitoyltransferase, the rate-limiting step of ceramide synthesis (41), proved to be too cytotoxic in the neuroblastoma cell lines tested to be used for this purpose (data not shown) (50). Prolonged L-cycloserine exposure (>16 hours) was also cytotoxic (P<.001) to an MCF-7 breast cancer cell line derivative compared with ethanol-only-exposed controls (Fig. 7, A
), although to a lesser extent than in neuroblastoma cell lines. Within this limitation, normalized to L-cycloserine-treated controls, L-cycloserine (2 mM) significantly reduced the increase in cytotoxicity of 4-HPR (P<.001) and of 4-HPR/safingol (P<.001 at 6 µM: 2 µM; P = .02 at 9 µM: 3 µM) observed in this cell line compared with 4-HPR and 4-HPR/safingol treatment alone (Fig. 7, A
). Paradoxically, fumonisin B1, an inhibitor of ceramide synthase, slightly increased the cytotoxicity of 4-HPR in CHLA-90 cells (P<.01 at 3 µM; P = .01 at 6 µM; P<.001 at 9 µM; P<.03 at 12 µM) (left panel of Fig. 7, B
) and SK-N-RA cells (P<.001) (right panel of Fig. 7, B
), likely because of the accumulation of the cytotoxic sphingolipids sphinganine and sphingosine, resulting from fumonisin B1 exposure (51,52). An interpretation of the effect of fumonisin B1 on 4-HPR/safingol cytotoxicity in these cell lines was less clear (left and right panels of Fig. 7, B
).
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DISCUSSION |
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We attempted to demonstrate the extent to which de novo ceramide production contributed to 4-HPR cytotoxicity, both as a single agent and in 4-HPR/safingol, by using inhibitors of de novo ceramide synthesis. L-Cycloserine, an inhibitor of serine palmitoyltransferase, the rate-limiting step of ceramide synthesis, significantly reduced the cytotoxicity of 4-HPR and 4-HPR/safingol in an MCF-7 cell line derivative. However, L-cycloserine inhibits aminotransferases (53,54), decarboxylases (55), and pyruvate metabolism (56), in addition to inhibiting serine palmitoyltransferase, and prolonged exposure to L-cycloserine itself caused significant cytotoxicity. Furthermore, L-cycloserine and -chloro-L-alanine cytotoxicity cannot be rescued completely by sphingosine in Chinese hamster ovary cells (57), further suggesting that these agents significantly perturb cytotoxic, non-sphingolipid-related pathways. Thus, the reduction of 4-HPR and 4-HPR/safingol cytotoxic effects observed with L-cycloserine pretreatment must be interpreted with caution. Paradoxically, an inhibitor of ceramide synthase, fumonisin B1, slightly increased the cytotoxicity of 4-HPR in CHLA-90 and SK-N-RA cells. Since inhibition of ceramide synthase results in the accumulation of cytotoxic sphinganine and sphingosine and the depletion of higher order glycosphingolipids, these results should also be interpreted with circumspection. Thus, it is unlikely that L-cycloserine and fumonisin B1 can be used to definitely demonstrate the contribution made by de novo synthesized ceramide to the cytotoxic effects of 4-HPR and 4-HPR/safingol. However, our preliminary results with a transfected cell line that overexpresses glucosylceramide synthase (36), which converts ceramide into nontoxic glucosylceramide, indicate that overexpression of glucosylceramide synthase significantly decreases the cytotoxicity of 4-HPR and 4-HPR/safingol (data not shown).
The direct stimulatory effects of 4-HPR on serine palmitoyltransferase and ceramide synthase enzymatic activities in CHLA-90 neuroblastoma cells have been determined and will be reported elsewhere (Cabot MC: unpublished data). Etoposide has recently been reported (58) to increase de novo ceramide levels via stimulation of serine palmitoyltransferase in Molt-4 leukemia cells. Pretreatment of Molt-4 cells with fumonisin B1 prevented the etoposide-induced ceramide increase and reduced etoposide-induced cytotoxicity (58). This result is in contrast to the slight increase in 4-HPR-induced cytotoxicity observed here with fumonisin B1 pretreatment and may reflect a cell type-specific variation in response to the increase in sphinganine caused by fumonisin B1. Taken together, our results and those reported for Molt-4 leukemia (58) support a role for the overproduction of de novo ceramide in the promotion of cell death by both 4-HPR and etoposide.
The subcellular location of the machinery of de novo ceramide synthesis may provide a clue to possible pro-death functions of de novo increases in ceramide. Serine palmitoyltransferase has been enriched from the endoplastic reticulum (41), whereas ceramide synthase activity has been found in the cytosolic aspect of the endoplastic reticulum and is enriched in the mitochondrial fraction (40). Therefore, it is possible that 4-HPR stimulation of serine palmitoyltransferase activity may increase ceramide levels directly in mitochondria, in addition to increases in ceramide levels in other subcellular compartments. Both short-chain ceramides (59,60) and 4-HPR (61) have been reported to disrupt the mitochondrial respiratory chain between complex II and complex III, resulting in generation of ROS, one reported mechanism of 4-HPR cytotoxicity (8,9). Furthermore, sustained mitochondrial electron transport chain decoupling can result in depletion of cellular adenosine triphosphate stores and cell death by necrosis (62,63). In this regard, we have demonstrated previously that 4-HPR kills neuroblastoma cells via mixed apoptosis/necrosis (11), perhaps representing death by the process variously termed "necrapoptosis" (62) or "aponecrosis" (64). In addition, ceramide or its short-chain analogues have been associated with pro-death JNK/SAPK activation (12), dephosphorylation (inactivation) of apoptosis-opposing Bcl-2 via activation of mitochondrial protein phosphatase-2A (PP2A) (15), and inhibition of the phosphatidylinositol 3-kinase (PI3K)-protein kinase B/Akt1 survival pathway (65,66), all of which could potentially contribute to a ceramide-mediated component of 4-HPR cytotoxicity.
The mechanism by which safingol synergizes 4-HPR cytotoxicity is not clear. It may act by inhibiting the regulatory subunit of PKC- (25,26,67). PKC-
is activated by ceramide (12), acts to increase NFkappaB (a pro-life transcription factor) (68), may facilitate pro-life p70 S6 kinase activation (69), and also activates sphingosine kinase (12,70). Sphingosine kinase converts sphingosine, a degradation product of ceramide, to sphingosine 1-phosphate, which activates the pro-life extracellular signal-regulated kinase (ERK1/2) cascade (70,71). ERK1/2 activation acts to oppose ceramide-stimulated, pro-death-associated JNK/SAPK activity (7072), and 4-HPR has been reported to increase JNK/SAPK activity (10). In this regard, our preliminary results indicate that nontoxic doses of safingol (3 µM) more rapidly and strongly activate JNK/SAPK in CHLA-90 neuroblastoma cells than does 4-HPR itself (data not shown). We speculate that JNK/SAPK preactivation by safingol may predispose to cell death in a cellular context of de novo ceramide stress induced by 4-HPR. Safingol has also been reported to potentiate arabinofuranosylcytosine-induced apoptosis in HL-60 leukemia cells independently of ceramide-induced JNK/SAPK activation by decreasing the pro-life ERK1/2 cascade activation (73), perhaps by direct inhibition of PKC-
activation of mitogen-activated protein kinase (MAPK) kinase (MEK) (23).
Whether safingol is synergistic with 4-HPR as the parental L-threo-dihydrosphingosine compound or as its in vivo-acylated, stereochemically variant, ceramide analogue (cer*) is under investigation. It is noteworthy that there was a large accumulation of cer* at minimally toxic doses of safingol (3 µM) but that cer* was not glucosylated to glucosylceramide. This finding indicates that the variant stereochemistry of cer* significantly altered its biologic processing compared with native (D-erythro)ceramide. It is possible, in this respect, that cer* retains some but not all of the messenger functions of native ceramide (such as, perhaps, the ability to activate JNK/SAPK). In support of this hypothesis, the activation of ceramide-activated protein phosphatase-1 (PP1) and protein phosphatase-2A (PP2A) by native D-erythro-C18-ceramide is stereochemically restricted, with significantly reduced activity induced by the D-threo and L-threo diastereoisomers and the L-erythro enantiomer (74). Such stereochemical restriction may explain the minimal cytotoxicity of cer* observed when low-dose safingol was used as a single agent.
Current phase I clinical trials of 4-HPR, given orally in 1-week courses, have demonstrated that serum levels of 4-HPR, approximating or exceeding those reported here to achieve multilog cytotoxicity with safingol, are well tolerated clinically (75,76). Safingol (at 120 mg/m2) has achieved approximately 3 µM plasma levels over 1-hour infusion times without reported toxicity (77). Our preliminary washout data in vitro indicate that, in some cell lines, safingol need only be co-present for a fraction of the total 4-HPR-exposure period to obtain much of its synergistic cytotoxicity (data not shown). Taken together, these results indicate that animal toxicology studies of continuous infusion of 4-HPR/safingol should be conducted and should be followed by phase I clinical trials as warranted. Future preclinical studies and clinical trials will likely involve combining inhibitors of glucosylceramide synthase, such as tamoxifen, which is tolerated to approximately 7 µM plasma levels with pulse oral dosing (78), and/or inhibitors of 1-O-acylceramide synthase with 4-HPR and 4-HPR/safingol.
Thus, should they be tolerated clinically, combinations of 4-HPR and modulators of ceramide metabolism may form the basis for a novel chemotherapy that is p53 and caspase independent and is active under conditions of reduced oxygen.
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Certain intellectual property rights pertaining to the contents of this article may be retained by the Childrens Hospital of Los Angeles and the John Wayne Cancer Institute.
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REFERENCES |
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---|
1 Di Vinci A, Geido E, Infusini E, Giaretti W. Neuroblastoma cell apoptosis induced by the synthetic retinoid N-(4-hydroxyphenyl)retinamide. Int J Cancer 1994;59:4226.[Medline]
2 Mariotti A, Marcora E, Bunone G, Costa A, Veronesi U, Pierotti MA, et al. N-(4-hydroxyphenyl)retinamide: a potent inducer of apoptosis in human neuroblastoma cells. J Natl Cancer Inst 1994;86:12457.[Medline]
3 Ziv Y, Gupta MK, Milsom JW, Vladisavljevic A, Brand M, Fazio VW. The effect of tamoxifen and fenretinimide on human colorectal cancer cell lines in vitro. Anticancer Res 1994;14:20059.[Medline]
4 Igawa M, Tanabe T, Chodak GW, Rukstalis DB. N-(4-Hydroxyphenyl) retinamide induces cell cycle specific growth inhibition in PC3 cells. Prostate 1994;24:299305.[Medline]
5 Kalemkerian GP, Slusher R, Ramalingam S, Gadgeel S, Mabry M. Growth inhibition and induction of apoptosis by fenretinide in small-cell lung cancer cell lines. J Natl Cancer Inst 1995;87:167480.[Abstract]
6 Delia D, Aiello A, Lombardi L, Pelicci PG, Grignani F, Grignani F, et al. N-(4-Hydroxyphenyl)retinamide induces apoptosis of malignant hemopoietic cell lines including those unresponsive to retinoic acid. Cancer Res 1993;53:603641.[Abstract]
7 Formelli F, Cleris L. Synthetic retinoid fenretinide is effective against a human ovarian carcinoma xenograft and potentiates cisplatin activity. Cancer Res 1993;53:53746.[Abstract]
8
Oridate N, Suzuki S, Higuchi M, Mitchell MF, Hong WK, Lotan R. Involvement of reactive oxygen species in N-(4-hydroxyphenyl)retinamide-induced apoptosis in cervical carcinoma cells. J Natl Cancer Inst 1997;89:11918.
9 Delia D, Aiello A, Meroni L, Nicolini M, Reed JC, Pierotti MA. Role of antioxidants and intracellular free radicals in retinamide-induced cell death. Carcinogenesis 1997;18:9438.[Abstract]
10
Chen YR, Zhou G, Tan TH. c-Jun N-terminal kinase mediates apoptotic signaling induced by N-(4-hydroxyphenyl)retinamide. Mol Pharmacol 1999;56:12719.
11
Maurer BJ, Metelitsa LS, Seeger RC, Cabot MC, Reynolds CP. Increase of ceramide and induction of mixed apoptosis/necrosis by N-(4-hydroxyphenyl)retinamide in neuroblastoma cell lines. J Natl Cancer Inst 1999;91:113846.
12 Mathias S, Pena LA, Kolesnick RN. Signal transduction of stress via ceramide. Biochem J 1998;335:46580.[Medline]
13 Hannun YA, Luberto C. Ceramide in the eukaryotic stress response. Trends Cell Biol 2000;10:7380.[Medline]
14
Yoshimura S, Banno Y, Nakashima S, Takenaka K, Sakai H, Nishimura Y, et al. Ceramide formation leads to caspase-3 activation during hypoxic PC12 cell death. Inhibitory effects of Bcl-2 on ceramide formation and caspase-3 activation. J Biol Chem 1998;273:69217.
15
Ruvolo PP, Deng X, Ito T, Carr BK, May WS. Ceramide induces Bcl2 dephosphorylation via a mechanism involving mitochondrial PP2A. J Biol Chem 1999;274:20296300.
16 Shayman JA, Abe A. Glucosylceramide synthase: assay and properties. Methods Enzymol 2000;311:429.[Medline]
17
Abe A, Shayman JA. Purification and characterization of 1-O-acylceramide synthase, a novel phospholipase A2 with transacylase activity. J Biol Chem 1998;273:846774.
18
Jarvis WD, Fornari FA, Traylor RS, Martin HA, Kramer LB, Erukulla RK, et al. Induction of apoptosis and potentiation of ceramide-mediated cytotoxicity by sphingoid bases in human myeloid leukemia cells. J Biol Chem 1996;271:827584.
19 Lucci A, Han TY, Liu YY, Giuliano AE, Cabot MC. Modification of ceramide metabolism increases cancer cell sensitivity to cytotoxics. Int J Oncol 1999;15:5416.[Medline]
20 Toker A. Signaling through protein kinase C. Front Biosci 1998;3:D113447.[Medline]
21 Cuvillier O, Pirianov G, Kleuser B, Vanek PG, Coso OA, Gutkind S, et al. Suppression of ceramide-mediated programmed cell death by sphingosine-1-phosphate. Nature 1996;381:8003.[Medline]
22 Wang YM, Seibenhener ML, Vandenplas ML, Wooten MW. Atypical PKC zeta is activated by ceramide, resulting in coactivation of NK-kappaB/JNK kinase and cell survival. J Neurosci Res 1999;55:293302.[Medline]
23 Berra E, Diaz-Meco MT, Lozano J, Frutos S, Municio MM, Sanchez P, et al. Evidence for a role of MEK and MAPK during signal transduction by protein kinase C zeta. EMBO J 1995;14:615763.[Abstract]
24
Romanelli A, Martin KA, Toker A, Blenis J. p70 S6 kinase is regulated by protein kinase Czeta and participates in a phosphoinositide 3-kinase-regulated signalling complex. Mol Cell Biol 1999;19:29218.
25
Merrill AH Jr, Sereni AM, Stevens VL, Hannun YA, Bell RM, Kinkade JM Jr. Inhibition of phorbol ester-dependent differentiation of human promyelocytic leukemic (HL-60) cells by sphinganine and other long-chain bases. J Biol Chem 1986;261:126105.
26
Hannun YA, Loomis CR, Merrill AH Jr, Bell RM. Sphingosine inhibition of protein kinase C activity and of phorbol dibutyrate binding in vitro and in human platelets. J Biol Chem 1986;261:126049.
27 Schwartz GK, Ward D, Saltz L, Casper ES, Spiess T, Mullen E, et al. A pilot clinical/pharmacological study of the protein kinase C-specific inhibitor safingol alone and in combination with doxorubicin. Clin Cancer Res 1997;3:53743.[Abstract]
28 Shayman JA, Lee L, Abe A, Shu L. Inhibitors of glucosylceramide synthase. Methods Enzymol 2000;311:37387.[Medline]
29
Lee L, Abe A, Shayman JA. Improved inhibitors of glucosylceramide synthase. J Biol Chem 1999;274:146629.
30 Cabot MC, Giuliano AE, Volner A, Han TY. Tamoxifen retards glycosphingolipid metabolism in human cancer cells. FEBS Lett 1996;394:12931.[Medline]
31 Keshelava N, Seeger RC, Groshen S, Reynolds CP. Drug resistance patterns of human neuroblastoma cell lines derived from patients at different phases of therapy. Cancer Res 1998;58:5396405.[Abstract]
32 Anderson CP, Seeger RC, Satake N, Monforte-Munoz HL, Keshelava N, Bailey HH, et al. Buthionine sulfoximine and myeloablative concentrations of melphalan overcome resistance in a melphalan-resistant neuroblastoma cell line. J Pediatr Hematol Oncol. In press 2000.
33 Reynolds CP, Tomayko MM, Donner L, Helson L, Seeger RC, Triche TJ, et al. Biological classification of cell lines derived from human extra-cranial neural tumors. Prog Clin Biol Res 1988;271:291306.[Medline]
34 Reynolds CP, Biedler JL, Spengler BA, Reynolds DA, Ross RA, Frenkel EP, et al. Characterization of human neuroblastoma cell lines established before and after therapy. J Natl Cancer Inst 1986;76:37587.[Medline]
35 Fairchild CR, Ivy SP, Kao-Shan CS, Whang-Peng J, Rosen N, Israel MA, et al. Isolation of amplified and overexpressed DNA sequences from adriamycin-resistant human breast cancer cells. Cancer Res 1987;47:51418.[Abstract]
36
Liu YY, Han TY, Giuliano AE, Cabot MC. Expression of glucosylceramide synthase, converting ceramide to glucosylceramide, confers adriamycin resistance in human breast cancer cells. J Biol Chem 1999;274:11406.
37 Grant JL, Smith B. Bone marrow gas tensions, bone marrow blood flow, and erythropoiesis in man. Ann Intern Med 1963;58:8019.
38 Vaupel P, Schlenger K, Knoop C, Hockel M. Oxygenation of human tumors: evaluation of tissue oxygen distribution in breast cancers by computerized O2 tension measurements. Cancer Res 1991;51:331622.[Abstract]
39 Fragala T, Proffitt RT, Reynolds CP. A novel 96-well plate cytotoxicity assay based on fluorescence digital imaging microscopy [abstract]. Proc Am Assoc Cancer Res 1995;36:303.
40 Wang E, Merrill AH Jr. Ceramide synthase. Methods Enzymol 2000;311:1521.[Medline]
41 Dickson RC, Lester RL, Nagiec MM. Serine palmitoyltransferase. Methods Enzymol 2000;311:39.[Medline]
42 Chou TC, Hayball MP. Calcusyn manual: Windows Software for dose effect analysis. Cambridge (U.K.): Biosoft; 1996.
43 Chou TC. Drug combinations: from laboratory to practice. J Lab Clin Med 1998;132:68.[Medline]
44 Sundaram KS, Lev M. Inhibition of sphingolipid synthesis by cycloserine in vitro and in vivo. J Neurochem 1984;42:57781.[Medline]
45 Merrill AH Jr, Wang E. Enzymes of ceramide biosynthesis. Methods Enzymol 1992;209:42737.[Medline]
46 Stoffel W, Bister K. Stereospecificities in the metabolic reactions of the four isomeric sphinganines (dihydrosphingosines) in rat liver. Hoppe-Seylers Z Physiol Chem 1973;354:16981.[Medline]
47 Stoffel W, Bister K. Studies on the desaturation of sphinganine. Ceramide and sphingomyelin metabolism in the rat and in BHK 21 cells in tissue culture. Hoppe-Seylers Z Physiol Chem 1974;355:91123.[Medline]
48 Abe A, Radin NS, Shayman JA, Wotring LL, Zipkin RE, Sivakumar R, et al. Structural and stereochemical studies of potent inhibitors of glucosylceramide synthase and tumor cell growth. J Lipid Res 1995;36:61121.[Abstract]
49
Lavie Y, Cao Ht, Volner A, Lucci A, Han TY, Geffen V, et al. Agents that reverse multidrug resistance, tamoxifen, verapamil, and cyclosporin A, block glycosphingolipid metabolism by inhibiting ceramide glycosylation in human cancer cells. J Biol Chem 1997;272:16827.
50 Cinatl J Jr, Cinatl J, Kotchetkov R, Pouckova P, Vogel JU, Rabenau H, et al. Cytotoxicity of L-cycloserine against human neuroblastoma and medulloblastoma cells is associated with suppression of ganglioside expression. Anticancer Res 1999;19:534954.[Medline]
51 Merrill AH Jr, Wang E, Vales TR, Smith ER, Schroeder JJ, Menaldino DS, et al. Fumonisin toxicity and sphingolipid biosynthesis. Adv Exp Med Biol 1996;392:297306.[Medline]
52 Schmelz EM, Dombrink-Kurtzman MA, Roberts PC, Kozutsumi Y, Kawasaki T, Merrill AH Jr. Induction of apoptosis by fumonisin B1 in HT29 cells is mediated by the accumulation of endogenous free sphingoid bases. Toxicol Appl Pharmacol 1998;148:25260.[Medline]
53 Wong DT, Fuller RW, Molloy BB. Inhibition of amino acid transferases by L-cycloserine. Adv Enzyme Regul 1973;11:13954.[Medline]
54 Cornell NW, Zuurendonk PF, Kerich MJ, Straight CB. Selective inhibition of alanine aminotransferase and asparate aminotransferase in rat hepatocytes. Biochem J 1984;220:70716.[Medline]
55 Malashkevich VN, Strop P, Keller JW, Jansonius JN, Toney MD. Crystal structures of dialkylglycine decarboxylase inhibitor complexes. J Mol Biol 1999;294:193200.[Medline]
56
Perez-Sala D, Cerdan S, Ballesteros P, Ayuso MS, Parrilla R. Pyruvate decarboxylating action of L-cycloserine. The significance of this in understanding its metabolic inhibitory action. J Biol Chem 1986;261:1396972.
57 Hanada K, Nishijima M, Fujita T, Kobayashi S. Specificity of inhibitors of serine palmitoyltransferase (SPT), a key enzyme in sphingolipid biosynthesis, in intact cells. A novel evaluation system using an SPT-defective mammalian cell mutant. Biochem Pharmacol 2000;59:12116.[Medline]
58
Perry DK, Carton J, Shah AK, Meredith F, Uhlinger DJ, Hannun YA. Serine palmitoyltransferase regulates de novo ceramide generation during etoposide-induced apoptosis. J Biol Chem 2000;275:907884.
59
Garcia-Ruiz C, Colell A, Mari M, Morales A, Fernandez-Checa JC. Direct effect of ceramide on the mitochondrial electron transport chain leads to generation of reactive oxygen species. Role of mitochondrial glutathione. J Biol Chem 1997;272:1136977.
60
Quillet-Mary A, Jaffrezou JP, Mansat V, Bordier C, Naval J, Laurent G. Implication of mitochondrial hydrogen peroxide generation in ceramide-induced apoptosis. J Biol Chem 1997;272:2138895.
61 Suzuki S, Higuchi M, Proske RJ, Oridate N, Hong WK, Lotan R. Implication of mitochondria-derived reactive oxygen species, cytochrome C and caspase-3 in N-(4-hydroxyphenyl)retinamide-induced apoptosis in cervical carcinoma cells. Oncogene 1999;18:63807.[Medline]
62 Lemasters JJ, Qian T, Bradham CA, Brenner DA, Cascio WE, Trost LC, et al. Mitochondrial dysfunction in the pathogenesis of necrotic and apoptotic cell death. J Bioenerg Biomembr 1999;31:30519.[Medline]
63 Nicotera P, Leist M, Single B, Volbracht C. Execution of apoptosis: converging or diverging pathways? Biol Chem 1999;380:103540.[Medline]
64 Formigli L, Papucci L, Tani A, Schiavone N, Tempestini A, Orlandini GE, et al. Aponecrosis: morphological and biochemical exploration of a syncretic process of cell death sharing apoptosis and necrosis. J Cell Physiol 2000;182:419.[Medline]
65 Salinas M, Lopez-Valdaliso R, Martin D, Alvarez A, Cuadrado A. Inhibition of PKB/Akt1 by C2-ceramide involves activation of ceramide-activated protein phosphatase in PC12 cells. Mol Cell Neurosci 2000;15:15669.[Medline]
66
Zhou H, Summers SA, Birnbaum MJ, Pittman RN. Inhibition of Akt kinase by cell-permeable ceramide and its implications for ceramide-induced apoptosis. J Biol Chem 1998;273:1656875.
67
Wilson E, Olcott MC, Bell RM, Merrill AH Jr, Lambeth JD. Inhibition of the oxidative burst in human neutrophils by sphingoid long-chain bases. Role of protein kinase C in activation of the burst. J Biol Chem 1986;261:1261623.
68 Wooten MW. Function for NF-kB in neuronal survival: regulation by atypical protein kinase C. J Neurosci Res 1999;58:60711.[Medline]
69
Romanelli A, Martin KA, Toker A, Blenis J. p70 S6 kinase is regulated by protein kinase Czeta and participates in a phosphoinositide 3-kinase-regulated signalling complex. Mol Cell Biol 1999;19:29218.
70 Cuvillier O, Pirianov G, Kleuser B, Vanek PG, Coso OA, Gutkind S, et al. Suppression of ceramide-mediated programmed cell death by sphingosine-1-phosphate. Nature 1996;381:8003.[Medline]
71
Cuvillier O, Rosenthal DS, Smulson ME, Spiegel S. Sphingosine 1-phosphate inhibits activation of caspases that cleave poly(ADP-ribose) polymerase and lamins during Fas- and ceramide-mediated apoptosis in Jurkat T lymphocytes. J Biol Chem 1998;273:29106.
72
Jarvis WD, Fornari FA Jr, Auer KL, Freemerman AJ, Szabo E, Birrer MJ, et al. Coordinate regulation of stress- and mitogen-activated protein kinases in the apoptotic actions of ceramide and sphingosine. Mol Pharmacol 1997;52:93547.
73
Jarvis WD, Fornari FA Jr, Tombes RM, Erukulla R, Bittman R, Schwartz GK, et al. Evidence for involvement of mitogen-activated protein kinase, rather then stress-activated protein kinase, in potentiation of 1-beta-D-arabinofuranoscylcytosine-induced apoptosis by interruption of protein kinase C signaling. Mol Pharmacol 1998;54:84456.
74
Chalfant CE, Kishikawa K, Mumby MC, Kamibayashi C, Bielawska A, Hannun YA. Long chain ceramides activate protein phosphatase-1 and protein phosphatase-2A. Activation is stereospecific and regulated by phosphatidic acid. J Biol Chem 1999;274:203137.
75 Parchment RE, Jasti BR, Koracek TA, Wiegand RA, Kassab J, Wurster W, et al. Pharmacologic issues for fenretinide chemotherapy (4-HPR, NSC-374551) [abstract]. Clin Cancer Res 1999;5(Suppl):3800s.
76 Bagniewski PG, Reid JM, Villablanca JG, Reynolds CP, Ames M.M. A phase I pharmacokinetic study of fenretinide (HPR) in children with high-risk tumors [abstract]. Proc Am Assoc Cancer Res 1999;40:92.
77 Schwartz GK, Ward D, Saltz L, Casper ES, Spies T, Mullen E, et al. A pilot clinical/pharmacological study of the protein kinase C-specific inhibitor safingol alone and in combination with doxorubicin. Clin Cancer Res 1997;3:53743.[Abstract]
78 Berman E, McBride M, Lin S, Menedez-Botet C, Tong W. Phase I trial of high-dose tamoxifen as a modulator of drug resistance in combination with daunorubicin in patients with relapsed or refractory acute leukemia. Leukemia 1995;9:16317.[Medline]
79 Keshelava N, Groshen S, Reynolds CP. Cross-resistance of topoisomerase I and II inhibitors in neuroblastoma cell lines. Cancer Chemother Pharmacol 2000;45:18.[Medline]
Manuscript received January 19, 2000; revised September 7, 2000; accepted September 21, 2000.
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