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1 Division of Biological Sciences, University of Missouri, Columbia, Missouri; 2 Katholieke Universiteit Leuven, Departement Moleculaire Celbiologie, Afdeling Farmakologie, Leuven, Belgium; and 3 Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma
Requests for reprints: Stephen Alexander, Division of Biological Sciences, University of Missouri, 303 Tucker Hall, Columbia, MO 65211-7400. Phone: 573-882-6670; Fax: 573-882-0123. E-mail: alexanderst{at}missouri.edu
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Introduction |
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Cisplatin acts by causing DNA damage, which in turn initiates a cascade of events that result in cell death. Resistance to the drug can result from pre-damage events, such as changes in drug accumulation in the cell or drug inactivation. Alternatively, resistance can result from post-damage events, such as increased repair of the damaged DNA, or altering the signal transduction cascade leading to death (1, 2). Identifying the many pathways and molecules that are involved in the cellular response to cisplatin and other drugs will identify new molecular targets that may be exploited to increase the efficacy of the drugs. Given the difficulty and expense of producing new anticancer drugs, improving the usefulness of existing drugs is important.
A search for cisplatin-resistant mutants in the model organism Dictyostelium discoideum identified the gene that encodes the enzyme sphingosine-1-phosphate (S-1-P) lyase, which catalyzes the degradation of S-1-P to phosphoethanolamine and hexadecenal, as involved in modulating the response of cells to cisplatin (3). S-1-P is a bioactive lipid with multiple regulatory roles in the cell (4), and it has been suggested that it is the relative level of S-1-P to sphingosine or ceramide that determines a cell's fate: elevated levels of ceramide lead to cell death and elevated levels of S-1-P will result in cell proliferation (5, 6). We hypothesized that an alteration in the sphingolipid balance, as a result of inactivation of S-1-P lyase, was the cause for the increased resistance of the S-1-P lyase–null mutant cells to cisplatin.
Subsequent genetic studies in D. discoideum have firmly established the centrality of this pathway in controlling sensitivity to cisplatin in this organism (7, 8). As predicted, overexpression of the S-1-P lyase had the opposite effect of the null mutation and resulted in increased sensitivity to cisplatin. Disruption of the two sphingosine kinases (the enzymes that synthesize S-1-P from sphingosine and ATP) caused increased sensitivity to the drug, whereas overexpression of the sphingosine kinase enzyme resulted in decreased sensitivity. Pharmacologically, we have shown that addition of S-1-P resulted in increased resistance, whereas treatment with dimethylsphingosine, an inhibitor of sphingosine kinase (9), potentiated sensitivity to cisplatin, suggesting that cotreatment with both drugs may be useful clinically. Taken together, the results show that manipulation of the expression/activation of the S-1-P lyase and the sphingosine kinase enzymes directly influenced cisplatin efficacy.
The most surprising finding of all these studies in D. discoideum was that the effect of altering the S-1-P lyase and sphingosine kinase levels was specific for cisplatin and the related platinum drug carboplatin, whereas sensitivity to other chemotherapeutic drugs, such as etoposide, 5-fluoro-2'-deoxyuridine, or doxorubicin, was unchanged. S-1-P and ceramide have been shown to regulate opposing mitogen-activated protein kinases (MAPK). S-1-P up-regulates the survival MAPK/extracellular signal-regulated kinase (ERK) 1/2 and down-regulates the death kinase p38, whereas ceramide has the opposing effects on ERK1/2 and p38 (10). Thus, we asked whether the increase in sensitivity of the S-1-P lyase–overexpressing cells to cisplatin stems from the up-regulation of p38 or from the lack of up-regulation of the ERK1/2 and whether this can account for the preferential response to platinum compounds.
In the current study, we extend our previous studies in D. discoideum to human cells. We show that overexpression of the human S-1-P lyase (hSPL) results in increased sensitivity to the chemotherapeutic drug cisplatin and the related compound carboplatin as was shown for D. discoideum. In human cells, the increase in sensitivity also extends to doxorubicin but not to vincristine and chlorambucil. We further show that the increased sensitivity to cisplatin in the hSPL-expressing cells is mediated by the death MAPK p38 but not by the ERK1/2 or the phosphatidylinositol 3-kinase (PI3K) survival enzymes and that this accounts for the observed drug specificity.
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To test whether the specificity profile to the drugs was cell type specific, we studied the response of A549 lung cancer cell line transiently transfected with the hSPL gene to the same drugs (Fig. 3B). The results shows that the hSPL-overexpressing A549 cells are more sensitive to cisplatin, carboplatin, and doxorubicin but not to the other drugs. Although the A549 cells are generally less sensitive to the drugs, the effect of hSPL overexpression is not unique to one cell type.
Pharmacologic Modulation of S-1-P Metabolism in HEK293 Cells Alters Sensitivity to Cisplatin
Treatment of the cells with the sphingosine kinase inhibitor dimethylsphingosine was predicted to mimic the phenotype of the hSPL-overexpressing cells (7, 8). Figure 4A shows that dimethylsphingosine has a dose-dependent toxic effect on the control HEK293 cells. Moreover, when given together, dimethylsphingosine dramatically increases the sensitivity of the cells to cisplatin. The level of killing is greater than the sum of the cell killing from cisplatin alone and dimethylsphingosine alone. When hSPL-overexpressing cells are treated with dimethylsphingosine, both the effect of dimethylsphingosine alone and the effect of dimethylsphingosine plus cisplatin are even more pronounced. This is presumably because the cells already have reduced levels of S-1-P due to the increased hSPL activity. Indeed, a decrease in the basal levels of S-1-P and sphingosine in HEK293 cells overexpressing the hSPL has been documented (14).
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The effect of extracellular S-1-P likely is mediated through a G-protein-coupled receptor, because in the presence of pertussis toxin (an inhibitor of Gi subunits) S-1-P failed to increase the resistance to cisplatin in either control or hSPL-overexpressing cells (Fig. 4C). Interestingly, although pertussis toxin alone had no effect on the viability of the control cells, it increased the sensitivity of the cells to cisplatin. This may indicate that there is cycling of S-1-P out of the cell and that the presence of pertussis toxin blocks its binding to the S-1-P receptor. The effect of pertussis toxin is even more apparent in the hSPL-overexpressing cells.
Increase in Cisplatin Sensitivity Due to the hSPL Overexpression Is Mediated by p38
S-1-P is known to regulate the MAPKs that in turn regulate cell survival or cell death (10). To determine if the increased sensitivity of the overexpressing strain to cisplatin resulted from up-regulation of p38 or from the lack of up-regulation of the ERK1/2 kinase, we treated the control and hSPL-overexpressing cells with specific inhibitors of MAPKs and determined the response of the cells to cisplatin.
Inhibiting the survival enzymes ERK1/2 (Fig. 5A) did not result in a statistically significant increase in sensitivity to cisplatin in either the control or the hSPL-overexpressing cells. Similarly, inhibition of the survival enzyme PI3K did not alter the response of either cell line to cisplatin (Fig. 5B). The simultaneous inhibition of both ERK1/2 and PI3K enzymes (Fig. 5C) resulted in an increased sensitivity to cisplatin. However, because both the control and the hSPL-overexpressing cell lines responded to the same extent, it suggests that the effect may be the result of a general inhibition of cell survival (see also Fig. 5F and G below). It also suggests that these enzymes can compensate for each other.
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The stress-activated MAPK c-Jun NH2-terminal kinase (JNK) is also regulated by cellular levels of S-1-P (10). However, the inhibition of JNK (Fig. 5E) resulted in only a slight increase of resistance to cisplatin in both the control and the hSPL-overexpressing lines. These findings suggest that the primary mediator of cell death in response to cisplatin is p38.
To further test the specificity of the above findings for cisplatin, the effect of these inhibitors was examined with vincristine and chlorambucil, whose action was not influenced by the overexpression of S-1-P lyase (Fig. 3). As observed with cisplatin, simultaneous inhibition of both ERK and PI3K increased the sensitivity of both control and hSPL-overexpressing cells to vincristine (Fig. 5F) and chlorambucil (Fig. 5G) although to a lesser extent than with cisplatin. These data imply again that inhibiting both ERK1/2 and PI3K decreases the ability of a cell to survive drug toxicity in a nonspecific manner and that it was independent of the level of S-1-P lyase. Remarkably, inhibition of p38, which had a significant effect on the response to cisplatin, did not alter the sensitivity of the cell lines to either vincristine (Fig. 5H) or chlorambucil (Fig. 5I). This clearly shows that p38 does not modulate the response to all drugs and explains the observed specificity of the response to those drugs that function through p38.
Overexpression of hSPL Results in an Increase in the Level of Activated p38 in Response to Cisplatin
Pharmacologic inhibition of MAPKs using specific inhibitors indicated that the effect of hSPL expression on cisplatin sensitivity was mediated primarily through p38. This suggested that stimulation with cisplatin would result in a concomitant change in the level of activation of the p38 protein. Western analysis, using antibodies to both the total and the phosphorylated forms of several MAPKs, shows that cisplatin treatment of both control and hSPL-overexpressing HEK293 cells causes the level of phospho-p38 to increase. The increase occurs by 30 minutes and is maintained through 6 hours, with a greater increase in the hSPL-overexpressing cells (Fig. 6A). The level of total p38 is constant for both cell lines.
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In contrast, examination of ERK1/2 phosphorylation status (Fig. 6C) shows an opposing response where the phosphorylation of the 44- and 42-kDa forms of ERK is more pronounced in the control cells than in the hSPL-overexpressing cells. Indeed, in the overexpressing cells, the ERK1/2 phosphorylation seems to go down in the first 30 minutes and return to basal level at 6 hours. The level of total p44 and p42 ERK is constant for both cell lines. Western analyses of tubulin protein (Fig. 6D) confirmed equal protein loading for each time point.
Sensitivity to Cisplatin Correlates with Increased Enzyme Activity of p38
The pharmacologic studies and the MAPK phosphorylation data strongly support the idea that p38 plays a central role in modulating the effect of hSPL expression on cisplatin sensitivity. To measure this directly, we tested the kinase activity of p38 in control and hSPL-overexpressing cells after drug treatment. The results shown in Fig. 7 confirm that the increased sensitivity of the overexpressing cells correlates with increased p38 enzyme activity in both time- and concentration-dependent manner. The in vivo toxicity experiments in this study were done in the range of 1 to 10 µmol/L cisplatin, and these experiments are carried out for 3 days. To be able to measure the immediate, short-term, response in the in vitro p38 kinase assay, it is necessary to perform these experiments at higher doses of cisplatin. These conditions are based on the sensitivities of the assay and have been documented previously for in vitro p38 kinase assays (15, 16). Therefore, hSPL-overexpressing or control cells were treated with 50 and 250 µmol/L cisplatin for 30 minutes and 3 hours and were then analyzed for p38 activity by monitoring the phosphorylation of the p38 target protein activating transcription factor-2 (ATF-2). The level of active p38 enzyme activity in the control cells increased 1.03- and 1.3-fold over the basal level after 30 minutes of treatment with 50 or 250 µmol/L cisplatin, respectively, and 1.3- and 4.0-fold over the basal level after 3 hours of treatment. This agrees with the data in Fig. 6 and the studies of Losa et al. (16), showing that p38 is phosphorylated in response to cisplatin. In contrast, the level of p38 activity in the hSPL-overexpressing cells increased 1.15- and 1.65-fold over basal level after 30 minutes and 4.2- and 7.8-fold after 3 hours of treatment with cisplatin. Thus, it is clear that the phosphorylation of p38 (Fig. 6A) accurately reflects the activation of the enzyme and that p38 activity is greatly enhanced in the hSPL-overexpressing cells.
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Discussion |
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The level of S-1-P lyase in the HEK293 hSPL-overexpressing cells was increased 13-fold over the endogenous level and properly localized in the endoplasmic reticulum. These cells showed growth inhibition in minimal medium, which was reversed by addition of S-1-P. These results are consistent with those of Reiss et al. (14). The hSPL-overexpressing cells were more sensitive to cisplatin, carboplatin, and doxorubicin but not to vincristine or chlorambucil. Parallel studies on hSPL-overexpressing A549 lung cancer cells showed the same pattern of drug sensitivity, indicating that the effect is not specific to one cell type. The increase in sensitivity to doxorubicin seen in these studies is likely a reflection of the biology of human cells, as it was not observed in D. discoideum (7). Nevertheless, the overexpression of hSPL did not result in an across the board increase in drug sensitivity. Atomic absorption spectrophotometry measurements of platinum levels did not indicate altered cisplatin uptake as the proximal cause of the change in sensitivity. These results agree with those in D. discoideum where cisplatin uptake was normal in S-1-P lyase overexpressor and null cells and in sphingosine kinase overexpressor and null cells. Taken together, these results indicate that S-1-P acts through a mechanism not directly associated with plasma membrane transporters or pumps.
We proposed previously a model where the balance between ERK and p38, which reflects the balance between the basal levels of S-1-P and ceramide (10), ultimately would control sensitivity to cisplatin. The model was based on the genetic studies in D. discoideum (7, 8), where modulating the enzymes of S-1-P metabolism altered the response of cells to chemotherapeutic drugs in a specific manner, and on recent reports that suggest that cisplatin exerts its cytotoxic effect through the downstream activation of the MAPK p38 and the down-regulation of the ERK1/2 MAPK (16-18). Here, we present three types of evidence—genetic (through the use of stable and transient overexpression cell lines), pharmacologic (through the use of inhibitors), and biochemical (by directly measuring enzyme activity)—to support this model. These data show that p38 signaling seems to be the main pathway, which mediates cellular viability in response to changes in sphingolipid levels. We show that overexpression of the S-1-P lyase results in a significantly higher level of p38 enzyme activity, which in turn results in an increased sensitivity to cisplatin. Inhibition of p38 by siRNA in the overexpressor line reversed this increased sensitivity to that observed with the control cells. The model then argues that in normal cells the balance between the bioactive lipids allows for activation of p38 to a certain level in response to cisplatin, resulting in a corresponding level of cell death. In cells overexpressing the S-1-P lyase, where the basal level of S-1-P is much lower than in normal cells, the activation of p38 is more robust and the level of cisplatin-mediated cell death is correspondingly higher.
The study by Reiss et al. (14) showed that the overexpression of S-1-P lyase in HEK293 cells resulted in a diminished basal level of sphingosine and S-1-P as well as an increase in the intracellular levels of ceramide. These findings agree with the findings in this study because ceramide does activate p38 (19). Thus, the combination of lower S-1-P in the cells, as a result of S-1-P lyase overexpression, and a concomitant increase in ceramide result in shifting the balance between the opposing MAPKs, which ultimately influences the response of the cells to drugs that act through the activation of p38.
The involvement of p38 in the response of cells of nontumor origin to cisplatin raises the question of preferential toxicity of cisplatin for tumor cells (20) and suggests ways to protect nontumor cells from the drug. For example, cisplatin shows significant nephrotoxicity in humans and different mechanisms have been implicated (21-23). The specific inhibition of hSPL (and/or activation of sphingosine kinase) in the kidney might reduce the toxicity and allow cisplatin to be used at a higher dose that is more effective in killing tumor cells.
S-1-P levels in cells are regulated by the activity of the sphingosine kinases, S-1-P phosphatase and the S-1-P lyase. Previous studies suggested that the sphingosine kinase acts as an oncogene (24), and elevated levels of sphingosine kinase have been reported in a variety of tumors (25, 26). The S-1-P lyase is located on chromosome 10q21. Several cytogenetic studies have shown that this region of chromosome 10 is deleted or rearranged in a variety of different cancers (27-29). Direct evidence for a tumor-suppressing function for S-1-P lyase is still missing, although there is ample evidence that S-1-P modulates the immune response (30, 31) and as a consequence probably modulates sensitivity to cancer. The data in the current study linking the level of this enzyme to cisplatin sensitivity strengthen this correlation. Moreover, we suggest that deletion of this chromosomal region in some tumors may result in cells that are more resistant to cisplatin-based therapy. This information then could be used clinically to establish alternative chemotherapy regimens.
The next important step will be to test the usefulness of modulating sphingolipids in xenograft studies in animals. Local administration of enzyme inhibitors to tumors, or tumor-specific expression of the relevant enzymes, should show the usefulness of S-1-P lyase and sphingosine kinase as targets to improve the efficacy of cisplatin in chemotherapy.
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Recombinant hSPL
A recombinant clone was generated as follows. An amplicon, covering the 5' end of the hSPL cDNA (base –177 to 286), was obtained by PCR on a human liver 5' stretch plus 
gt11 cDNA library (Clontech, Palo Alto, CA; ref. 32). This was used as a primer together with hSPL15r (5'-GGAATTCTGTCTCATGGTCTATCTTGAG) and EST44070as template to generate a full-length hSPL cDNA. After a second PCR on this full-length cDNA with hSPL17f (5'-CGAGATCTCCTAGCACAGACCTTCTGATG) and hSPL15r as primers, the amplicon was treated with dATP/Taq DNA polymerase, ligated into pCRII-Topo (Invitrogen), and transfected in Top1OF' Escherichia coli. Plasmids from selected clones were treated with BglII and EcoRI and the insert was sublconed into BamHI/EcoRI–restricted pCMV-Tag2B (Stratagene, La Jolla, CA) to generate pVB003 vector (11).
Cell Lines and Transfection
The human embryonic kidney cell line (HEK293) or the lung cancer cell line (A549) were cultured at 37°C, 5% CO2 in DMEM, 10% fetal bovine serum or in serum-free Opti-MEM medium. For generating stable cell lines, cells were seeded in 75 mm2 flasks and transfected with 20 µg plasmid DNA of the recombinant hSPL cDNA pVB003 or pcDNA3 vector control using LipofectAMINE 2000 according to the manufacturer's recommendations and selected with 500 µg/mL geneticin (G418) added 48 hours after transfection. The transfected HEK293 cells were passed for 2 to 3 weeks and stocks were frozen. Each experiment was started from a frozen stock. For transient transfection, cells were seeded in 75 mm2 flask in DMEM, transfected according to the manufacturer's recommendations, incubated for 36 to 48 hours, and then used in assays of drug sensitivity. Transfection efficiencies were >95%.
Growth Measurements
Cells (5 x 103 per well) were plated in six-well plates in DMEM, 10% fetal bovine serum. After 24 hours, cells were washed twice with Opti-MEM and then grown in the indicated medium. At indicated times, cells were washed with PBS, trypsinized, counted three times in Beckman-Coulter Z1 Particle Counter (Hialeah, FL), and the results were averaged. The data represent the average of three wells per time point.
S-1-P Lyase Assay
Cells (1 x 107) were harvested and washed in cold lysis buffer (0.25 mol/L sucrose, 5 mmol/L MOPS, 1 mmol/L EDTA, 1 mmol/L DTT, protease inhibitors). Pellets were lysed in 80 µL lysis buffer, 0.5% Triton X-100 and diluted to 0.1% Triton X-100 with lysis buffer, and 40 µL were used per assay. Protein concentration was determined using BCA protein assay. The enzyme assay was done as described (33) using D-erythro-[4,5-3H]dihydrosphingosine-1-phosphate as substrate, and the activity is expressed as pmol/min/mg protein.
Drug Treatment and Cytotoxicity Assay
Exponentially growing cells were seeded 1 day before the experiment in Opti-MEM medium and then treated with various concentrations of indicated drugs. The use of Opti-MEM is necessary for drug assays because serum contains S-1-P (34) and because cisplatin binds to serum albumin (34, 35). Stock solutions were prepared in 3 mmol/L NaCl, 1 mmol/L NaPO4 (pH 6.5) or in DMSO (not to exceed a final concentration of 0.2% DMSO). For experiments using combinations of cisplatin and other reagents, cells were preincubated with the various agents 1 hour before the addition of cisplatin. Viability was determined using the modified 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, CellTiter 96 Aqueous One Solution Cell Proliferation Assay (MTS; Promega, Madison, WI) according to the manufacturer's instructions. Percent survival was calculated as the absorbance ratio of treated to untreated cells. In experiments where inhibitors were used, the effect of the inhibitor alone was compared with the untreated cells, but the effect of cisplatin in the presence of the inhibitor was compared with that of cells with inhibitor alone. Each experimental treatment was done in six replicates per point and each experiment was repeated twice. Results were expressed as mean ± SD. Data presented were analyzed with Student's t test. Ps < 0.05 were accepted as a statistically significant difference compared with controls (*, P 
0.05; **, P 0.001).
siRNA Inhibition of p38
A SignalSilence Pool p38 MAPK siRNA kit (Cell Signaling Technology) was used according to the manufacturer's specifications. p38 MAPK siRNAs (10 nmol/L) were transfected into stable hSPL-overexpressing and control cells. A target-specific anti-p38 antibody was used to confirm the silencing of p38 MAPK expression. A non-target-specific antibody to ATP citrate lyase was used to control for equal loading and to monitor the specificity of p38 MAPK siRNA. A fluorescein-labeled nontarget siRNA control was used to monitor the transfection efficiency.
p38 Enzyme Activity
hSPL-overexpressing and control cells were grown in six-well plates to 80% confluence and then treated with 50 and 250 µmol/L cisplatin for 30 minutes and 3 hours. Cultures were harvested in nondenaturing buffer, lysed by sonication, and centrifuged to remove cell debris. Protein concentration was determined by BCA. Extract (250 µg) was used for each assay of p38 activity by monitoring the phosphorylation of the p38 target protein AFT-2, using the Nonradioactive p38 MAPK Assay kit, according to the manufacturer's instructions. All assays were done using the same amount of protein extract, as determined by BCA, and was confirmed by probing a parallel set of samples with anti-tubulin antibodies.
Western Analysis
Cells were cultured in six-well plates, washed three times with ice-cold PBS, scraped into PBS, and collected by centrifugation. Pellets were washed three times in ice-cold PBS and lysed by sonication in buffer containing 62 mmol/L Tris-HCl (pH 6.8), 2% SDS, 5 mmol/L DTT, 1 mmol/L EDTA, and protease inhibitors. Protein concentration was determined by BCA, and protein (20 µg) was assayed as described (8). Loading of equal protein samples was confirmed by stripping the membranes and reprobing with anti-tubulin antibodies. All antibodies were used according to the manufacturer's instructions. Quantitation of Western analysis was done by scanning the blots in high resolution on a flat bed scanner and quantifying the bands using MetaMorph 4.6r.9 program.
Immunofluorescence Microscopy
Cells (3 x 105 per well) were grown in six-well plates for 48 hours on sterile glass coverslips. The cells were washed three times with ice-cold PBS, fixed in 3.7% paraformaldehyde for 30 minutes at 4°C, and washed three times with PBS. Cells were permeabilized with PBS, 0.1% Triton X-100 for 10 minutes at room temperature and blocked for 1 hour with PBS, 1% bovine serum albumin, and 5% normal goat serum. Cells were incubated for 1 hour at room temperature with a 1:200 dilution of mouse anti-FLAG monoclonal antibody and washed five times in PBS followed by 1-hour incubation with the appropriate anti-mouse IgG Alexa 488–conjugated secondary antibody. The cells were washed with PBS and examined with an Olympus 1X-70 microscope fitted with a MRC-600 confocal laser (Bio-Rad Laboratories, Hercules, CA).
Intracellular Platinum Concentration
Stably transfected HEK293 cells in six-well plates were exposed in triplicate to 0 to 100 µmol/L cisplatin for indicated times. After cisplatin treatment, cells were rapidly trypsinized, washed twice with ice-cold PBS, and pelleted at 4°C. The platinum concentration was determined as described using atomic absorption spectrophotometry (8).
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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: Current address for L. Zhang is New England Medical Center, Tufts University, Boston, MA.
4 P.P. Van Veldhoven, unpublished data. 
Received 12/ 2/04; revised 3/ 9/05; accepted 3/18/05.
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  N. Van Driessche, H. Alexander, J. Min, A. Kuspa, S. Alexander, and G. Shaulsky Global transcriptional responses to cisplatin in Dictyostelium discoideum identify potential drug targets PNAS, September 25, 2007; 104(39): 15406 - 15411. [Abstract] [Full Text] [PDF]
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B. Oskouian, P. Sooriyakumaran, A. D. Borowsky, A. Crans, L. Dillard-Telm, Y. Y. Tam, P. Bandhuvula, and J. D. Saba Sphingosine-1-phosphate lyase potentiates apoptosis via p53- and p38-dependent pathways and is down-regulated in colon cancer PNAS, November 14, 2006; 103(46): 17384 - 17389. [Abstract] [Full Text] [PDF]
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 R. L. Chisholm, P. Gaudet, E. M. Just, K. E. Pilcher, P. Fey, S. N. Merchant, and W. A. Kibbe dictyBase, the model organism database for Dictyostelium discoideum Nucleic Acids Res., January 1, 2006; 34(suppl_1): D423 - D427. [Abstract] [Full Text] [PDF]
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, medium with 10% fetal bovine serum; 
, Opti-MEM minimal medium; 
, Opti-MEM supplemented with 1 µmol/L S-1-P. Direct cell counts rather than the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assay allowed growth to be monitored over a longer period of time.
